Untitled - State Scientific Institution “Institute for Single Crystals”
Untitled - State Scientific Institution “Institute for Single Crystals”
Untitled - State Scientific Institution “Institute for Single Crystals”
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complete line venn diagram A4.indd 1 26/07/2011 17:16:10
Spectroscopy, Imaging and Probes<br />
Strasbourg, 11-14 September 2011<br />
Conference program<br />
&<br />
Book of abstracts<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 3
4 — MAF 12, Strasbourg, France, September 11-14, 2011 —
TABLE OF CONTENTS<br />
Welcome ________________________________________ p 7<br />
Committees _____________________________________ p 8<br />
Sponsors and exhibitors ________________________ p 9<br />
Conference program ____________________________ p 14<br />
Plenary lectures _________________________________ p 21<br />
Oral communications ___________________________ p 53<br />
Posters __________________________________________ p 87<br />
Poster main authors index ______________________ p 347<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 5
6 — MAF 12, Strasbourg, France, September 11-14, 2011 —
Welcome to the Attendants of MAF12<br />
It is our great pleasure to welcome you in Strasbourg, the seat of the European Parliament, <strong>for</strong> the<br />
12th Conference on Methods and Application of Fluorescence (MAF12) that focuses on<br />
spectroscopy, imaging and probes. Since their foundation in 1989, the biannual MAF conferences<br />
have become the largest meetings dedicated to studies concerning fluorescence, being highly<br />
interdisciplinary and covering areas that range from physics, chemistry, nanotechnology to biology<br />
and medicine.<br />
This year’s conference has attracted more than 360 attendees, coming from 40 countries all<br />
over the world. Due to the excellent work done by the Permanent Steering Committee (PSC),<br />
composed of 40 leading scientists in the fluorescence field, an exciting program has been<br />
elaborated with 25 top-level invited speakers and 28 speakers carefully selected from the list<br />
of posters. Furthermore, about 230 posters will be presented, mainly by young scientists. Finally,<br />
close to 30 companies will be presenting their products during the meeting. Special care was taken<br />
in order that the various aspects of the fluorescence field be covered in a balanced way, so that<br />
each attendee will necessarily find interesting new in<strong>for</strong>mation in his/her own research field.<br />
This book contains the abstracts of scientific contributions that will be presented at MAF12: plenary<br />
lectures, oral communications and poster presentations. Oral contributions are listed in<br />
chronological order, while posters have been grouped into 6 different categories (reflecting<br />
contributions from similar fields of study).<br />
To keep the very specific flavour of the MAF conference series, a number of social activities have<br />
been planned. The social programme includes a reception-buffet, a boat cruise around the<br />
Strasbourg city centre which has been listed as UNESCO World Heritage site, a concert in the<br />
Strasbourg Cathedral, and a gala dinner in the Maison Kammerzell, a renowned restaurant of<br />
Renaissance style, next to the Cathedral.<br />
We would like to express our deepest thanks to all those who have contributed to the organization<br />
and success of this conference: the members of the PSC (<strong>for</strong> their selection of the speakers), the<br />
speakers that agreed to give a lecture, the authors presenting a poster and all attendees <strong>for</strong> their<br />
active participation. We also thank the local organizing committee, and notably the members of our<br />
lab, the Laboratory of Biophotonics and Pharmacology, <strong>for</strong> their dedicated work (with a special<br />
mention <strong>for</strong> Ingrid Barthel, Marlyse Wernert and Ludovic Four). We are also grateful to the<br />
Université de Strasbourg and the Centre National de la Recherche Scientifique (CNRS) <strong>for</strong> their<br />
support and help. Last, but certainly not least, our thanks go to our public and private sponsors, as<br />
well as exhibitors, whose names are listed in the following pages. Their support has been critical<br />
<strong>for</strong> making this conference possible.<br />
Finally, on behalf of the Permanent Steering Committee, as well as the whole fluorescence<br />
community, we would like to thank Professor Otto Wolfbeis, the charismatic founder and leader<br />
of the MAF series, <strong>for</strong> his tireless and creative work in the organization and development of these<br />
conferences. After more than 20 years at the head of the PSC, Otto has decided to step down from<br />
his PSC chair position at MAF12. Thus, befittingly, we would like to dedicate MAF12 to him.<br />
Yves Mély and Guy Duportail (chair and co-chair of MAF12)<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 7
COMMITTEES<br />
International Program Committee<br />
Otto S. Wolfbeis (Germany) Yun-Bao Jiang (China)<br />
A. Ulises Acuña (Spain) Lennart B.Å. Johansson (Sweden)<br />
Manfred Auer (UK) Anita C. Jones (UK)<br />
Mario Berberan-Santos (Portugal) Paavo K.J. Kinnunen (Finland)<br />
David J.S. Birch (UK) Joseph R. Lakowicz (USA)<br />
John Birmingham (USA) Helge Lemmetyinen (Finland)<br />
Amilra P. de Silva (UK) János Matkó (Hungary)<br />
Alexander P. Demchenko (Ukraine) Yves Mély (France)<br />
Alberto Diaspro (Italy) Ute Resch-Genger (Germany)<br />
Jörg Enderlein (Germany) Catherine Royer (France)<br />
Hans C. Gerritsen (The Netherlands) Petra Schwille (Germany)<br />
Enrico Gratton (USA) Claus Seidel (Germany)<br />
Albin Hermetter (Austria) Trevor Smith (Australia)<br />
Martin Hof (Czech Republic) Hans J. Tanke (The Netherlands)<br />
Johan Hofkens (Belgium) Jacek Waluk (Poland)<br />
Totaro Imasaka (Japan) Jerker Widengren (Sweden)<br />
David M. Jameson (USA) Sergiy M. Yarmoluk (Ukraine)<br />
Local Organizing Committee<br />
Yves Mély (Chair)<br />
Guy Duportail (Co-chair)<br />
Cyril Favard<br />
Andrey Klymchenko<br />
Youri Arntz<br />
Frédéric Bolze<br />
Pascal Didier<br />
Marlyse Wernert (secretary)<br />
Ingrid Barthel (secretary)<br />
Ludovic Four (webmaster)<br />
8 — MAF 12, Strasbourg, France, September 11-14, 2011 —
Thanks to our Sponsors:<br />
Gold Sponsors<br />
Silver Sponsors<br />
HORIBA <strong>Scientific</strong><br />
www.horiba.com/scientific<br />
PCO<br />
www.pco.de<br />
Sanofi-Pasteur<br />
www.sanofipasteur.fr<br />
Becker & Hickl<br />
www.becker-hickl.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 9
Thanks to our sponsors and exhibitors:<br />
Agilent Technologies<br />
www.chem.agilent.com<br />
AUREA TECHNOLOGY<br />
www.aureatechnology.net<br />
CHROMA<br />
www.chroma.com<br />
DYOMICS<br />
www.dyomics.com<br />
Edinburgh Instruments<br />
www.edinst.com<br />
EuroPhoton<br />
www.europhoton.de<br />
FIANIUM<br />
www.fianium.com<br />
GILDEN Pλotonics<br />
www.gildenphotonics.com<br />
HAMAMATSU<br />
www.hamamatsu.fr<br />
IDIL Fibres Optiques<br />
Ocean Optics<br />
www.idil.fr/en<br />
IBA<br />
www.oligo-specialist.com<br />
ISS<br />
www.iss.com<br />
Leica MicroSystems<br />
www.leica-microsystems.com<br />
10 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
LEUKOS<br />
www.leukos-systems.com<br />
MICRO-CONTROLE<br />
Spectra-Physics<br />
www.newport.com<br />
NIKON<br />
www.nikoninstruments.eu<br />
NKT Photonics<br />
www.nkphotonics.com<br />
OLYMPUS<br />
www.olympus.co.uk<br />
OptoPhase<br />
www.optophase.com<br />
OPTOPRIM<br />
www.optoprim.com<br />
OXXIUS<br />
www.oxxius.com<br />
PicoQuant<br />
www.picoquant.com<br />
QIAGEN Lake Constance<br />
www.qiagen.com<br />
SPRINGER<br />
www.springer.com<br />
VISITRON Systems<br />
www.visitron.de<br />
ZEISS<br />
www.zeiss.com
Thanks to our Academic Sponsors:<br />
EPA<br />
European Photochemistry Association<br />
Fondation icFRC: International Center<br />
<strong>for</strong> Frontier Research in Chemistry<br />
GDR 2588 du CNRS<br />
SFB<br />
Société Française de Biophysique<br />
SFµ<br />
Société Française des Microscopies<br />
CNRS<br />
Université de Strasbourg<br />
Thanks to:<br />
Région Alsace<br />
Communauté Urbaine de Strasbourg<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 11
12 — MAF 12, Strasbourg, France, September 11-14, 2011 —
PROGRAM AT A GLANCE<br />
Sunday, 11 September 2011<br />
14:00 Registration & exhibition build-up Forum<br />
17:00 Opening ceremony Schuman<br />
Opening keynote lecture Schuman<br />
17:15<br />
Wolfbeis O.<br />
room<br />
18:15 Welcome cocktail Galerie<br />
Monday, 12 September 2011 Tuesday, 13 September 2011 Wednesday, 14 September 2011<br />
SESSION 1 ‐ High resolution microscopy SESSION 3 - Fluorescence microscopy & cell imaging SESSION 5 - <strong>Single</strong> molecule & FCS<br />
09:00<br />
Dahan M. Wouters F. Schmidt T.<br />
Schuman<br />
09:30 Eggeling C. Coppey-Moisan M. Millar D.<br />
room<br />
10:00 Diaspro A. König K. Clegg R.<br />
10:30 Coffee break Coffee break Coffee break Forum<br />
SESSION 2 - Nanomaterials & quantum dots SESSION 4 - Fluoresc. spectrosc. & fluoresc. sensing SESSION 6 - Fluorescent probes, labels & proteins<br />
11:00<br />
Suzuki K. Favaro G. Chudakov D.<br />
Schuman<br />
11:30 Scholes G. Anslyn E. Ziessel R.<br />
room<br />
12:00 Smith A. Hahn K. Blanchard-Desce M.<br />
12:30 Poster session 1 (red) - Lunch & coffee Poster session 2 (blue) - Lunch & coffee Poster session 3 (green) - Lunch & coffee Forum<br />
14:00 AGILENT presentation (Tivoli room) OLYMPUS presentation BECKER & HICKL presentation Schuman<br />
SESSION 1 SESSION 2 SESSION 3 SESSION 4 SESSION 5 SESSION 6<br />
14:30<br />
Flors C. Schuman Wilhelmsson M. Beaurepaire E. Schuman Ameer-Beg S. Seidel C. Schuman de Silva A. Tivoli<br />
15:00 Bastmeyer M. room Mattoussi H. Holst G. room Gertstenecker B. Heikal A. room Huppert D. room<br />
15:20 Johansonn S. Rampazzo E. Margineanu A. Tor Y. Wennmalm S. Rolinski O.<br />
15:40 Coffee break Coffee break Coffee break Forum<br />
16:20 Neely R. Schuman Brouwer A. Jones A. Schuman Nakatani K. Marguet D. Schuman Merola F. Tivoli<br />
16:40 Manley S. room Yao H. Kress A. room Park S.B. Ito S. Klymchenko A. room<br />
17:00 Smith T. Meixner A. Lisse D. Falke J. Closing ceremony Schuman<br />
Evening break<br />
17:20 Session of the steering committee<br />
18:30 Boat trip around Strasbourg<br />
19:00 Concert<br />
20:00 Gala dinner<br />
14 — MAF 12, Strasbourg, France, September 11-14, 2011 —
Sunday, 11 September 2011<br />
14:00<br />
—<br />
18:00<br />
Strasbourg, 11-14 September 2011<br />
CONFERENCE PROGRAM<br />
17:00 Opening ceremony<br />
17:15<br />
Registration & exhibition build-up Forum<br />
Opening keynote lecture<br />
Otto WOLFBEIS (Germany)<br />
Fluorescence spectroscopy: people, progress, perspectives<br />
18:15 Welcome cocktail<br />
Schuman<br />
room<br />
Schuman<br />
room<br />
Galerie<br />
de marbre<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 15
Monday, 12 September 2011<br />
09:00<br />
09:30<br />
SESSION 1 — High resolution microscopy<br />
Chairman: T. Smith<br />
Maxime DAHAN (France)<br />
Exploring the dynamic properties of molecular assemblies in live cells with single molecule<br />
and nanoparticle imaging<br />
Christian EGGELING (Germany)<br />
Observing nanoscale dynamics with far-field fluorescence STED microscopy<br />
16 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
Schuman<br />
room<br />
10:00 Alberto DIASPRO (Italy)<br />
Augmented two-photon excitation (2PE) microscopy<br />
10:30 Coffee break Forum<br />
11:00<br />
11:30<br />
SESSION 2 — Nanomaterials & quantum dots<br />
Koji SUZUKI (Japan)<br />
Needs-oriented fluorescent and chemiluminescent probes<br />
Chairman: M. Berberan-Santos<br />
Gregory SCHOLES (Canada)<br />
Semiconductor nanocrystal photophysics: from quantum coherence on the femtosecond<br />
timescale to photoluminescence up to microseconds<br />
Schuman<br />
room<br />
12:00 Andrew SMITH (USA)<br />
Next-generation quantum dots <strong>for</strong> molecular and cellular imaging<br />
12:30 Poster session 1 (red) — Lunch & coffee Forum<br />
14:00 AGILENT presentation<br />
14:30<br />
15:00<br />
15:20<br />
SESSION 1<br />
High resolution microscopy<br />
Schuman<br />
room<br />
�<br />
SESSION 2<br />
Nanomaterials & quantum dots<br />
Chairman: C. Eggeling Chairman: A. Demchenko<br />
Cristina FLORS (UK)<br />
High density labelling of DNA<br />
with photoswitchable dyes:<br />
implications in super-resolution<br />
microscopy<br />
Martin BASTMEYER (Germany)<br />
(ZEISS)<br />
Super-resolution microscopy: a<br />
direct comparison of SIM,<br />
dSTORM and CLSM<br />
Sofia JOHANSONN (Sweden)<br />
A modified FCCS procedure<br />
applied to Ly49A–MHC class I<br />
cis-interaction studies in cell<br />
membranes<br />
Marcus WILHELMSSON (Sweden)<br />
The first nucleic acid base analogue<br />
FRET-pair - A versatile tool <strong>for</strong> detailed<br />
biochemical and nanotechnological<br />
structure in<strong>for</strong>mation<br />
Hedi MATTOUSSI (USA)<br />
Polyethylene glycol-based<br />
multidentate oligomers <strong>for</strong> enhancing<br />
the biocompatiblity of semiconductor,<br />
gold and magnetic nanocrystals<br />
Enrico RAMPAZZO (Italy)<br />
Fluorescent avidinated silicacore/PEG-shell<br />
nanoparticles:<br />
brightness <strong>for</strong> labeling and imaging<br />
Tivoli<br />
room<br />
Tivoli<br />
room<br />
15:40 Coffee break Forum<br />
16:20<br />
16:40<br />
17:00<br />
Robert K. NEELY (Belgium)<br />
DNA fluorocode: a single<br />
molecule, optical map of DNA<br />
with nanometer resolution<br />
Suliana MANLEY (Switzerland)<br />
Super-resolution imaging and<br />
more with photoactivatable<br />
fluorescence<br />
Trevor A. SMITH (Australia)<br />
Applications of sub-diffraction<br />
fluorescence imaging in<br />
biological, botanical and<br />
materials sciences<br />
Schuman<br />
room<br />
�<br />
Albert M. BROUWER (Netherlands)<br />
Fluorescence microspectroscopy:<br />
direct observation of polymer<br />
dynamics<br />
17:20 Session of the steering committee<br />
18:30 Boat trip around Strasbourg<br />
Hiroshi YAO (Japan)<br />
Organic dye nanoparticles with<br />
intense fluorescence caused by a<br />
combined effect of intermolecular Haggregation<br />
and restricted<br />
intramolecular rotation<br />
Alfred MEIXNER (Germany)<br />
Probing the radiative transition and<br />
determining the fluorescence<br />
quantum yield of a single molecule<br />
with a tunable microresonator<br />
Tivoli<br />
room
Tuesday, 13 September 2011<br />
09:00<br />
09:30<br />
10:00<br />
SESSION 3 — Fluorescence microscopy & cell imaging<br />
Fred WOUTERS (Germany)<br />
Imaging neurodegenerative events in cells using FRET/FLIM<br />
Maïté COPPEY-MOISAN (France)<br />
Spatio-temporal fluctuations of supranucleosomal state regulating accessibility of<br />
bromodomain to acetylated H4 in living cells by high-resolution FLIM<br />
Karsten KÖNIG (Germany)<br />
High resolution imaging and nano-processing with femtosecond lasers<br />
Chairman: G. Krishnamoorthy<br />
Schuman<br />
room<br />
10:30 Coffee break<br />
SESSION 4 — Fluorescence spectroscopy & fluorescence sensing<br />
Forum<br />
11:00<br />
Gianna FAVARO (Italy)<br />
Chairman: D. Birch<br />
Luminescence techniques: a powerful non-invasive diagnostic tool <strong>for</strong> artwork materials<br />
Schuman<br />
Eric V. ANSLYN (USA)<br />
11:30<br />
Triggered reactions <strong>for</strong> producing optical responses<br />
Klaus HAHN (USA)<br />
12:00<br />
Peeking and poking at rapid GTPase signaling in living cells<br />
room<br />
12:30 Poster session 2 (blue) — Lunch & coffee Forum<br />
14:00 OLYMPUS presentation Schuman<br />
SESSION 3<br />
Fluorescence microscopy & cell imaging<br />
SESSION 4<br />
Fluorescence spectroscopy<br />
& fluorescence sensing<br />
14:30<br />
Chairman: F. Wouters<br />
Emmanuel BEAUREPAIRE (France)<br />
Chairman: E. Anslyn<br />
Simon AMEER-BEG (UK)<br />
Nonlinear microscopy of tissues<br />
Fluorescence lifetime and anisotropy<br />
and embryonic morphogenesis<br />
imaging <strong>for</strong> HCS of protein-protein<br />
Schuman interactions<br />
15:00<br />
Gerhard HOLST (PCO)<br />
FLI-Cam – a frequency-domain<br />
fluorescence lifetime imaging<br />
system based on a new directly<br />
room<br />
�<br />
Bernard GERTSTENECKER (QIAGEN)<br />
A miniaturized, highly sensitive<br />
fluorescence detector and its routine<br />
application in point-of-need<br />
Tivoli<br />
room<br />
modulatable CMOS image<br />
sensor<br />
instrumentation<br />
15:20<br />
Anca MARGINEANU (UK)<br />
Multiplexed time lapse imaging<br />
using homoFRET and timeresolved<br />
polarisation fluorescence<br />
microscopy<br />
Yitzhak TOR (USA)<br />
New fluorescent nucleosides <strong>for</strong><br />
exploring nucleic acid damage and<br />
recognition<br />
15:40 Coffee break Forum<br />
16:20<br />
Anita JONES (UK)<br />
Optically trapped microsensors<br />
<strong>for</strong> microfluidic temperature<br />
measurement by fluorescence<br />
lifetime imaging microscopy<br />
Kazuhiko NAKATANI (Japan)<br />
Ligand inducible fluorescence: tools<br />
<strong>for</strong> ligand discovery and PCR<br />
monitoring<br />
Seung Bum PARK (Korea)<br />
16:40<br />
Alla KRESS (France)<br />
Studies on lipid order in cell<br />
membranes using fluorescence<br />
polarimetric microscopy<br />
Schuman Discovery and biological application<br />
room of full-color tunable and predictable<br />
fluorescent core skeleton (Seoul-<br />
Fluor) and fluorescent glucose<br />
� bioprobes (GBs)<br />
Tivoli<br />
room<br />
17:00<br />
Domenik LISSE (Germany)<br />
Specific targeting of<br />
nanoparticles to proteins <strong>for</strong><br />
visualization of single protein<br />
dynamics inside living cells<br />
Joseph FALKE (USA)<br />
Kinase on-off switching in the<br />
functional, membrane-associated<br />
chemosensory signaling array of E.<br />
coli – structural changes detected by<br />
OS-FRET<br />
17:20 Evening break<br />
19:00 Concert in the Cathedral Notre-Dame<br />
20:00 Gala dinner in the Restaurant Kammerzell<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 17
Wednesday, 14 September 2011<br />
09:00<br />
09:30<br />
SESSION 5 — <strong>Single</strong> molecule & fluorescence correlation spectroscopy<br />
Thomas SCHMIDT (Netherlands)<br />
Imaging cellular signaling on molecule at a time<br />
David MILLAR (USA)<br />
Assembly of ribonucleoproteins required <strong>for</strong> HIV-1 trafficking: one molecule at a time<br />
18 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
Chairman: D. Marguet<br />
Schuman<br />
room<br />
10:00<br />
Robert CLEGG (USA)<br />
Chasing fluorescence lifetimes in complex biological systems.<br />
What can Fluorescence Lifetime Imaging Microscopy (FLIM) tell us?<br />
10:30 Coffee break Forum<br />
11:00<br />
11:30<br />
12:00<br />
SESSION 6 — Fluorescent probes, labels & proteins<br />
Chairman: A. de Silva<br />
Dmitry CHUDAKOV (Russia)<br />
Fluorescent proteins and tools: Moscow news<br />
Raymond ZIESSEL (France)<br />
Water-solubilisation and bio-conjugation of novel red emitting BODIPY markers<br />
Mireille BLANCHARD-DESCE (France)<br />
Molecular nanoprobes <strong>for</strong> multiphotonics as new tools <strong>for</strong> bioimaging. Design and applications<br />
Schuman<br />
room<br />
12:30 Poster session 3 (green) — Lunch & coffee Forum<br />
14:00 BECKER & HICKL presentation Schuman<br />
14:30<br />
15:00<br />
15:20<br />
SESSION 5<br />
<strong>Single</strong> molecule & FCS<br />
Schuman<br />
room<br />
�<br />
SESSION 6<br />
Fluorescent probes, labels & proteins<br />
Chairman: R. Clegg Chairman: U. Resch-Genger<br />
Claus SEIDEL (Germany)<br />
Con<strong>for</strong>mational dynamics and<br />
biomolecular structure studied by<br />
super-resolution FRET<br />
Ahmed HEIKAL (USA)<br />
Multiscale dynamics of single<br />
molecules in biomimetic crowding<br />
Stefan WENNMALM<br />
(Sweden)<br />
Inverse-Fluorescence Correlation<br />
Spectroscopy: label-free analysis<br />
of the absolute volume of<br />
biomolecules in solution<br />
Amilra P. de SILVA (UK)<br />
2011: a small space odyssey with<br />
luminescent molecules<br />
Dan HUPPERT (Israel)<br />
Excited-state intermolecular proton<br />
transfer of the firefly’s chromophore<br />
d-luciferin<br />
Olaf ROLINSKI (UK)<br />
Detecting early stages of<br />
neurodegenerative disease by<br />
intrinsic fluorescence – beta-amyloid<br />
aggregation in Alzheimer’s disease<br />
Tivoli<br />
room<br />
15:40 Coffee break Forum<br />
16:20<br />
16:40<br />
Didier MARGUET (France)<br />
Spot variable Fluorescence<br />
Correlation Spectroscopy reveals<br />
fast scouting of K-Ras at the<br />
plasma membrane of living cells<br />
Syoji ITO (Japan)<br />
Mechanical interaction between<br />
photons and<br />
macromolecules/nanoparticles as<br />
evaluated by FCS<br />
Schuman<br />
room<br />
�<br />
Fabienne MEROLA (France)<br />
Physical chemistry and photophysics<br />
of the cyan fluorescent protein<br />
Andrey KLYMCHENKO (France)<br />
Fluorescent probes <strong>for</strong> monitoring<br />
lipid order selectively at one bilayer<br />
leaflet<br />
17:00 Closing ceremony — announcement of MAF13<br />
Tivoli<br />
room<br />
Schuman<br />
room
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pco_edge_ad_1p_EN_20100915.indd 1 15.09.2010 07:41:31
20 — MAF 12, Strasbourg, France, September 11-14, 2011 —
PLENARY<br />
PLENARY<br />
PLENARY<br />
LECTURES<br />
LECTURES<br />
LECTURES<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 21
22 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L1 Fluorescence spectroscopy: people, progress, perspectives<br />
Otto S. Wolfbeis<br />
L2 Exploring the dynamic properties of molecular assemblies in live cells with single molecule<br />
and nanoparticle imaging<br />
Ignacio Izeddin, Samuel Clarke, Fabien Pinaud, Patrice Dionne, Davide Normanno,<br />
Vincent Recamier, Xavier Darzacq & Maxime Dahan<br />
L3 Observing nanoscale dynamics with far-field fluorescence STED microscopy<br />
Christian Eggeling, Veronika Mueller, Alf Honigmann, Debora Machado Andrade<br />
& Stefan W. Hell<br />
L4 Augmented two-photon excitation (2PE) microscopy<br />
Alberto Diaspro, Paolo Bianchini, Francesca Cella Zanacchi, Benjamin Harke,<br />
Giuseppe Vicidomini, Zeno Lavagnino, Silvia Galiani, Jenu Chacko, Emiliano Ronzitti,<br />
Mario Faretta, Laura Furia & Partha Pratim Mondal<br />
L5 Needs-oriented fluorescent and chemiluminescent probes<br />
Koji Suzuki<br />
L6 Semiconductor nanocrystal photophysics: from quantum coherence on the femtosecond<br />
timescale to photoluminescence up to microseconds<br />
Gregory D. Scholes, Daniel B. Turner, Cathy Y. Wong, Shun S. Lo & Marcus Jones<br />
L7 Next-generation quantum dots <strong>for</strong> molecular and cellular imaging<br />
Andrew Smith & Shuming Nie<br />
L8 High density labelling of DNA with photoswitchable dyes: implications in super-resolution<br />
microscopy<br />
Cristina Flors<br />
L9 The first nucleic acid base analogue FRET-pair - A versatile tool <strong>for</strong> detailed biochemical<br />
and nanotechnological structure in<strong>for</strong>mation<br />
L. Marcus Wilhelmsson<br />
L10 Imaging neurodegenerative events in cells using FRET/FLIM<br />
Angela de Gomes Dani, Matthias Gralle, Sebastian Deeg, Jochen Weishaupt , Pawel<br />
Kermer, Alessandro Esposito & Fred S. Wouters<br />
L11 Spatio-temporal fluctuations of supranucleosomal state regulating accessibility of<br />
bromodomain to acetylated H4 in living cells by high-resolution FLIM<br />
Nicolas Audugé, Sergi Padilla-Parra, Marc Tramier & Maïté Coppey-Moisan<br />
L12 High resolution imaging and nanoprocessing with femtosecond lasers<br />
Karsten König<br />
L13 Luminescence techniques: a powerful non-invasive diagnostic tool <strong>for</strong> artwork materials<br />
Aldo Romani, Catia Clementi, Costanza Miliani & Gianna Favaro<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 23
L14 Triggered reactions <strong>for</strong> producing optical responses<br />
Eric V. Anslyn<br />
L15 Peeking and poking at rapid GTPase signaling in living cells<br />
Klaus Hahn, Christopher Welch, Hui Wang, Andrei Karginov, Jason Yi & Pei-Hsuan Chu<br />
L16 Nonlinear microscopy of tissues and embryonic morphogenesis<br />
Emmanuel Beaurepaire<br />
L17 Fluorescence lifetime and anisotropy imaging <strong>for</strong> HCS of protein-protein interactions<br />
Daniel Matthews, Viput Visitkul, Gregory Weissman, Elena Ortiz-Zapater, Simon Poland,<br />
Gilbert Fruhwurth, Paul Barber, Melanie Keppler, Leo Carlin, Jody Barbeau, Anthony<br />
Coolen, Tony Ng & Simon M. Ameer-Beg<br />
L18 Imaging cellular signaling on molecule at a time<br />
Thomas Schmidt<br />
L19 Assembly of ribonucleoproteins required <strong>for</strong> HIV-1 trafficking: one molecule at a time<br />
Rajan Lamichhane, Rae Robertson-Anderson & David Millar<br />
L20 Chasing fluorescence lifetimes in complex biological systems. What can Fluorescence<br />
Lifetime Imaging Microscopy (FLIM) tell us?<br />
R. M. Clegg<br />
L21 Fluorescent proteins and tools: Moscow news<br />
Dmitry Chudakov<br />
L22 Water-solubilisation and bio-conjugation of novel red emitting BODIPY markers<br />
Raymond Ziessel<br />
L23 Molecular nanoprobes <strong>for</strong> multiphotonics as new tools <strong>for</strong> bioimaging. Design and<br />
applications<br />
Mireille Blanchard-Desce<br />
L24 Con<strong>for</strong>mational dynamics and biomolecular structure studied by super-resolution FRET<br />
Claus A. M. Seidel, Simon Sindbert, Stanislav Kalinin & Hayk Vardanyan<br />
L25 2011: a small space odyssey with luminescent molecules<br />
Gareth J. Brown, A. Prasanna de Silva, Kaoru Iwai, Gareth D. McClean, Bernadine<br />
O.F. McKinney, David C. Magri, Seiichi Uchiyama & Sheenagh M. Weir<br />
24 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L1 Lecture 1<br />
Fluorescence spectroscopy: people, progress, perspectives<br />
Otto S. Wolfbeis<br />
University of Regensburg, Institute of Analytical Chemistry, Chemo- and Biosensors, D-93040<br />
Regensburg, Germany<br />
The lecture will be divided into three sections. The first will be a short account on the history of<br />
luminescence until around 1990, and the most significant people involved. The second will cover the<br />
progress that has been made in the past 20 years following the establishment of the MAF conference<br />
series. The third section will be an attempt to identify future perspectives of fluorescence spectroscopy.<br />
Luminescence has been observed in early times as described by Aristotle. Pliny the Younger,<br />
Menardes on fluorescent waters in South America, etc. However, all these reports were kind of<br />
descriptive and not systematic. This has been reviewed by Harvey[1] and, more recently, by Acuña &<br />
Amat-Guerri[2]. The 19 th and 20 th century only brought the first truly scientific break-through. Sir Stokes<br />
coined the term fluorescence, Göppelsröder recognized the interaction of Al(III) ion with morin (to result<br />
in the first fluorescence-based analytical test), Jean and Francis Perrin reported on their fundamental<br />
studies on polarized fluorescence, Stern & Volmer were able to mathematically describe and interprete<br />
the process of dynamic quenching (the law bearing their names), Jablonski established his famous<br />
diagram, Gaviola was the first to determine a fluorescence decay time, Kautsky made the first oxygen<br />
sensor (and eventually discovered the Kautsky effect in photosynthesis), Lewis & Kasha defined the<br />
triplet state, Förster described FRET and photodissociation, Gregorio Weber per<strong>for</strong>med studies on the<br />
fluorescence of biomolecules, created the first EEMs, discovered the red-edge-effect, developed phase<br />
fluorometry), and so <strong>for</strong>th.<br />
By the year 1980, fluorescence spectroscopy was becoming (slowly) accepted by a wider<br />
community, mainly in biosciences. In 1976, Dick Haugland founded Molecular Probes to match the<br />
increasing need <strong>for</strong> fluorescent probes and labels, and Lakowicz, in 1984, wrote the first edition of<br />
Principles of Fluorescence. It was the time when chemists began to synthesize new (longwave)<br />
fluorescent probes <strong>for</strong> enzymes and ions, when laser-compatible labels <strong>for</strong> proteins and intercalators<br />
<strong>for</strong> DNA became available, when people recognized the potential of FRET as a molecular ruler to<br />
determine biomolecular distances, when the American Instrument Company (Aminco) realized that<br />
their fluorometers became bestsellers, when other user-friendly fluorometers <strong>for</strong> measurement of<br />
spectra, and the first instruments <strong>for</strong> measurement of lifetime and anisotropy became commercially<br />
available, when time-resolved fluorometers and respective (europium-derived) probes were developed<br />
and found their application in the widely used DELFIA assay, when fiber optics enabled remote<br />
fluorescent (micro)sensing, and when the MAF conference series was founded (www.maf-sip.com; the<br />
first in 1989).<br />
In my opinion, the largest progress made in the past 20 years relates to fluorescent methods of<br />
microscopy. The quality of confocal images and the resolution of images (down to
L2 Lecture 2<br />
Exploring the dynamic properties of molecular assemblies in live cells<br />
with single molecule and nanoparticle imaging<br />
Ignacio Izeddin 1 , Samuel Clarke 1 , Fabien Pinaud 1 , Patrice Dionne 1 , Davide Normanno 1 ,<br />
Vincent Recamier 2 , Xavier Darzacq 2 & Maxime Dahan 1<br />
1<br />
Laboratoire Kastler Brossel, Département de Physique et Institut de Biologie, Ecole normale<br />
supérieure, Paris, France<br />
2<br />
Functional imaging of transcription, Institut de Biologie, Ecole normale supérieure<br />
<strong>Single</strong> molecule techniques are becoming ubiquitous tools in in vitro and in vivo assays. In cell<br />
biology, they now can be used to count molecules, image with sub-diffraction resolution and track<br />
individual molecules as they move in their natural habitat. Thus, single molecule imaging is a tool<br />
of choice to address the dynamic, compisition and structural properties of supramolecular<br />
assemblies in live cells. In this talk, I will present our ef<strong>for</strong>t the develop and apply ultrasensitive<br />
fluorescence methods. I will discuss some recent developments in the design of efficient labeling<br />
methods using either organic molecules or inorganic nanoparticles. In particular, I will present a<br />
new approach based on complementation activation of split-GFP labeled proteins. I will also<br />
discuss novel optical methods based on adaptive optics that should prove useful to optimally use<br />
the limited “photon budget” provided by single fluorophores. All these techniques will be illustrated<br />
by experiments made on a variety of biological systems, such as post-synaptic membrane<br />
receptors, molecular motors or transcription factors.<br />
This work was supported by grants from FRM, HFSP, CNRS and ANR.<br />
Recent publications:<br />
1. F. Pinaud, M. Dahan, “Targeting, imaging and post-translational modification of single biomolecules in living<br />
cells by complementation activated light microscopy with split fluorescent proteins”, Proc. Natl. Acad. Sci.<br />
USA (2011) 108 (24) E201–E210.<br />
2. I. Izeddin, C. Specht, M. Leleck, X. Darzacq, A. Triller, C. Zimmer, M. Dahan, « Super-resolution dynamic<br />
imaging of dendritic spines using a low-affinity photoconvertible actin probe », PLoS ONE 6(1): e15611<br />
(2011).<br />
3. S. Clarke, F. Pinaud, O. Beutel, C. You, J. Piehler, M. Dahan, « Covalent monofunctionalization of peptidecoated<br />
semiconductor quantum dots <strong>for</strong> single molecule assays », Nanolett. 2010, 10 (6) 2147–2154<br />
4. F. Pinaud, S. Clarke, A. Sittner, and M. Dahan, “Probing cellular events, one quantum dot a time”, Nature<br />
Methods 7, 275-85 (2010).<br />
5. V. Roullier, S. Clarke, C. You, F. Pinaud, G. Gouzer, D. Schaible, V. Marchi-Artzner, J. Piehler, M. Dahan,<br />
“High-affinity quantum-dot labeling of histidine-tagged proteins <strong>for</strong> multiplexed targeting and single-molecule<br />
tracking in live cells”, Nanolett. 9, 1228-1234 (2009).<br />
6. P. Pierobon, S. Achouri, S. Courty, A. Dunn, J.A. Spudich, M. Dahan and G. Cappello, “Velocity, processivity<br />
and individual steps of single myosin V molecules in live cells", Biophys. J 96(10):4268-75 (2009).<br />
26 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L3 Lecture 3<br />
Observing nanoscale dynamics with far-field fluorescence<br />
STED microscopy<br />
Christian Eggeling*, Veronika Mueller, Alf Honigmann, Debora Machado Andrade<br />
& Stefan W. Hell<br />
Department of Nanobiophotonics, Max Planck Institute <strong>for</strong> Biophysical Chemistry, Am Fassberg<br />
11, 37077 Göttingen, Germany<br />
Fluorescence spectroscopy is a non-invasive and very sensitive analysis technique and allows the<br />
disclosure of complex dynamical processes. Tools such as fluorescence correlation spectroscopy<br />
(FCS) are usually combined with far-field (confocal) microscopy. However, prominent (biological)<br />
problems can often not be solved due to the limited resolution of >200nm of conventional optical<br />
microscopy. For example, the resolution limit impedes the direct and non-invasive observation of<br />
important cellular process, such as lipid-lipid or lipid-protein interactions in the cellular plasma<br />
membrane. We use the superior spatial resolution of stimulated emission depletion (STED) far-field<br />
microscopy [1] and combine tunable focal spot sizes of down to 30 nm (or even less) with FCS [2,3] .<br />
We demonstrate the per<strong>for</strong>mance of this novel STED-FCS tool in open volumes and on<br />
membranes [2] and obtain new details of molecular membrane dynamics in living cells [3] . For<br />
example, we highlight ms-long trapping of certain lipids and proteins on the nanoscale in<br />
cholesterol-mediated molecular complexes, while other membrane constituents diffuse free or<br />
render a hopping-like diffusion. STED-FCS is a highly sensitive and exceptional tool to study<br />
nanoscale membrane organization, which introduces a new approach to determine their cellular<br />
functionality and molecular inter-dependencies.<br />
References: [1] S.W. Hell, Science 316 (2007) 1153. [2] C. Ringemann, et al., New J. Physics 11 (2009)<br />
103054. [3] C. Eggeling, et al., Nature 457 (2009) 1159.<br />
______________<br />
* Corresponding author: e-mail: ceggeli@gwdg.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 27
L4 Lecture 4<br />
Augmented two-photon excitation (2PE) microscopy<br />
Alberto Diaspro 1,2,* , Paolo Bianchini 1 , Francesca Cella Zanacchi 1 , Benjamin Harke 1 ,<br />
Giuseppe Vicidomini 1 , Zeno Lavagnino 1,2 , Silvia Galiani 1,2 , Jenu Chacko 1,2 , Emiliano<br />
Ronzitti 1 , Mario Faretta 3 , Laura Furia 3 & Partha Pratim Mondal 4<br />
1 Department of Nanophysics, Istituto Italiano di Tecnologia, via Morego, 30, 16163 Genova<br />
2 Department of Physics, LAMBS-MicroScoBio, University of Genova, Italy<br />
3 Department of Experimental Oncology, IEO, IFOM-IEO Campus, Milano Italy<br />
4 Department of Instrumentation and Applied Physics, Indian Institute of Science, Bangalore, India<br />
The Two-photon excitation (2PE) fluorescence microscopy is a well established far-field<br />
fluorescence optical microscopy technique <strong>for</strong> the study of the three-dimensional (3D) and dynamic<br />
properties of biological systems from cellular compartments like membranes to thick specimens<br />
like brain slides or large cellular aggregates [1]. Since the excitation process is restricted by the<br />
diffraction limited spatial extension of the explored volume using near-infrared radiation it has some<br />
advantages and some drawbacks. The main advantages are given by better penetration in<br />
scattering samples and low overall photo-toxicity/bleaching coupled to intrinsic 3D optical<br />
sectioning properties, while the main drawback lies in the loss of resolution and signal efficiency<br />
with respect to the 1PE case. For such reasons we decided to couple 2PE with two, comparatively<br />
young, optical methods specially realized <strong>for</strong> resolution improvement and 3D imaging of large<br />
samples, i.e. STED microscopy (STimulated Emission Depletion) [2] and SPIM (Selective Plane<br />
Illumination Microscopy) [3]. In the <strong>for</strong>mer case we aim to get a better resolution <strong>for</strong> 2PE<br />
microscopy and at the same time to improve the deep penetration capability of the STED<br />
approach. In the latter we aim to get better penetration depth in thick scattering samples by shifting<br />
the light sheet wavelengths to the red. We show results and related characterizations as obtained<br />
by means of 2PE STED-CW and 2PE SPIM adapted architectures available at the Italian Institute<br />
of Technology. A critical discussion will be conducted including other extensions of 2PE within the<br />
framework of modern advanced optical microscopy methods [4, 5].<br />
This work was supported by grants from IIT, IFOM-IEO, MIUR PRIN 2008JZ4MLB, European<br />
Project SMD FP7-NMP 2800-SMALL-2 proposal no. CP-FP 229375-2 .<br />
References: [1] A.Diaspro, et al., Q.Rev.Biophys., 38 (2005) 97. [2] S.W. Hell, Science, 316 (2007) 1153. [3]<br />
J. Huisken, et al., Scienc,e 305 (2004) 1007. [4] A. Diaspro, (ed.), Optical Fluorescence Microscopy: From<br />
the Spectral to the Nano Dimension, (2010) Springer Verlag. [5]A. Diaspro, (ed.), Nanoscopy and<br />
Multidimensional Optical Fluorescence Microscopy, (2010) Taylor and Francis, CRC.<br />
______________<br />
* Corresponding author: e-mail: alberto.diaspro@iit.it<br />
28 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L5 Lecture 5<br />
Needs-oriented fluorescent and chemiluminescent probes<br />
Koji Suzuki *<br />
Department of Applied, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan<br />
The creation of original chemical sensors is one of the main concerns in our research group. In<br />
biological research fields, chemical sensors such as synthetic molecular probes play an important<br />
role in the investigation of biological functions, and in cellular or in vivo imaging.<br />
In the past, designed ion sensing ligand molecules (ionophores) have been successfully<br />
synthesized and are now in practical use in biomedical electrolyte analysis. Considering the design<br />
of ionophores with high selectivity over interfering ions, our original ionophores exhibit the highest<br />
selectivities <strong>for</strong> lithium, sodium, ammonium, calcium, magnesium, silver, and mercury ions. These<br />
ionophores are now commercially available (Aldrich-Sigma-Fluka, Dojindo, etc) and used by<br />
several companies in ion sensors <strong>for</strong> clinical serum electrolyte analysis.<br />
One of the recent research highlights was the creation of a magnesium-imaging probe (KMG<br />
series fluorescent molecules) <strong>for</strong> living cells. The KMG-series molecules <strong>for</strong> magnesium (Mg)<br />
imaging were designed and synthesized based on our prior knowledge of Mg ionophore design [1].<br />
While the first example (KMG-20) was based on a coumarin fluorophore, we later developed the<br />
fluorescein derivative KMG-104, which is the best magnesium fluorescent probe <strong>for</strong> imaging<br />
applications in the cytoplasm of living cells. More recently, KMG-301 relying on a rhodamine<br />
fluorophore was prepared, which is suitable <strong>for</strong> the magnesium imaging of mitochondria.<br />
To obtain bright fluorescent probes, we have developed a set of fluorescent dyes (BODIPYbased<br />
KFL series dyes), which have excellent optical properties like sharp fluorescence spectra<br />
with high quantum yields, and moreover, the wavelength is finely tunable over a wide spectral<br />
range including the NIR region by introducing proper electron-donating groups into the furan<br />
moieties of the chromophore [2]. By linking an ion recognizing ionophore with a fluorescent dye as<br />
a transducer, a chemical probe (fluorescent probe) is obtained, trans<strong>for</strong>ming a simple molecular<br />
recognition ligand into a sensor ligand. For instance, a KFL fluorescent dye combined with a<br />
BAPTA chelating group resulted in a fluorescent probe <strong>for</strong> calcium ions. In this case, the<br />
introduction of a suitable linker connecting the KFL dye moiety and the BAPTA chelating moiety<br />
allowed to realize an off/on fluorescent probe with high calcium sensitivity.<br />
We also have developed a set of chemiluminescent (CL) dyes (BODIPY-based KCL and KBI<br />
series dyes) with excellent CL properties [3]. KCL and KBI consist of two moieties; a CL causing<br />
subunit of luminol or luciferin and a fluorescent BODIPY dye. KCL dyes exhibit high brightness in<br />
CL that can be applied to labeling probes <strong>for</strong> trace level protein analysis. The luciferin-based CL<br />
probe KBI is a useful probe <strong>for</strong> highly sensitive detection of ROS such as O2-.<br />
As these examples demonstrate, chemical probes are useful <strong>for</strong> bioanalysis.<br />
References:<br />
[1] H. Komatsu, K. Suzuki, et al., J.Am.Chem.Soc. 116, 16353-16360 (2004); J.Am.Chem.Soc..117, 10798-<br />
10799 (2005).<br />
[2] K. Umezawa, D. Citterio, K. Suzuki, et al., J.Am.Chem.Soc., 124, 213-217 (2008); Chem. Eur. J.,15,<br />
1096-1106 (2009).<br />
[3] M Sekiya, D. Citterio, K. Suzuki, et al., Chem. Commun, 3047-3049 (2009).<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 29
L6 Lecture 6<br />
Semiconductor nanocrystal photophysics: from quantum coherence on<br />
the femtosecond timescale to photoluminescence up to microseconds<br />
Gregory D. Scholes 1,* , Daniel B. Turner 1 , Cathy Y. Wong 1,2 , Shun S. Lo 1,3 & Marcus Jones 4<br />
1<br />
Department of Chemistry, 80 St. George Street, Institute <strong>for</strong> Optical Sciences, and Centre <strong>for</strong> Quantum<br />
In<strong>for</strong>mation and Quantum, Control University of Toronto, Toronto, Ontario M5S 3H6 (Canada).<br />
2<br />
Present address : UC Berkeley, Dept of Chemistry, D64 Hildebrand Hall, Berkeley, CA 94720 (USA).<br />
3<br />
Present address : University of Notre Dame, Department of Chemistry and Biochemistry, Notre<br />
Dame, IN 46556-5670 (USA).<br />
4<br />
Department of Chemistry, University of North Carolina at Charlotte, Burson 200, 9201 University<br />
City Boulevard, Charlotte, NC 28223-0001 (USA).<br />
Nanocrystalline semiconductor quantum dots are often studied owing to their size tunable<br />
properties, but are also fascinating because their electronic states and photophysics can be<br />
manipulated by ‘heterostructure’ growth [1]. Challenges compared to molecular systems are to<br />
probe details of spectroscopy that are obscured by the particle size distribution and to differentiate<br />
properties, especially photoluminescence, that are intrinsic properties of the nanocrystal compared<br />
to those related to the surface. While advances in synthetic techniques have lowered the size<br />
distribution in colloidal samples to 5%, the resultant inhomogeneous line broadening still prohibits<br />
the study of electronic fine structure with most linear and non-linear spectroscopic techniques. In<br />
the first part of the talk I will report on the use of two-dimensional photon echo experiments to<br />
determine the identity of some excited state absorption features in CdSe nanocrystals [2]. This<br />
provides insight on the fine structure of the biexcitonic states of CdSe nanocrystals, and also<br />
highlights the power of this spectroscopy to shed light on electronic structure. More recently, we<br />
have used 2D spectroscopy to produce and probe electronic superposition states in these<br />
nanocrystals at ambient temperatures. In the remainder of the talk I will focus on<br />
photoluminescence measurements and methods we have developed <strong>for</strong> the quantitative analysis<br />
of photoluminescence decay curves [3,4].<br />
This work was supported by NSERC.<br />
References: [1] Shun S. Lo, et al., Adv. Mater. 23 (2011) 180–197. [2] Cathy Y. Wong and Gregory D.<br />
Scholes, J. Phys. Chem. A 115 (2011) 3797–3806. [3] Marcus Jones, et al., Proc. Natl. Acad. Sci. USA 106<br />
(2009) 3011–3016. [4] Marcus Jones and Gregory D. Scholes, J. Mater. Chem. 20 (2010) 3533–3538.<br />
______________<br />
*<br />
Corresponding author: e-mail: gscholes@chem.utoronto.ca<br />
30 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L7 Lecture 7<br />
Next-generation quantum dots <strong>for</strong> molecular and cellular imaging<br />
Andrew Smith & Shuming Nie<br />
Departments of Biomedical Engineering and Chemistry, Emory University and Georgia Institute of<br />
Technology, 101 Woodruff Circle Suite 2001, Atlanta, GA 30322, USA<br />
Semiconductor quantum dots (QDs) are tiny light-emitting particles that have emerged as a new<br />
class of fluorescent labels <strong>for</strong> biology and medicine. Compared with traditional fluorescent probes,<br />
QDs have unique optical and electronic properties such as size-tuneable light emission, narrow<br />
and symmetric emission spectra, and broad absorption spectra that enable the simultaneous<br />
excitation of multiple fluorescence colors. The semiconducting crystalline nature of these particles<br />
also gives rise to large absorption cross sections, which causes QDs to be 10–100 times brighter<br />
than organic dyes and fluorescent proteins. In addition, these particles are dramatically more<br />
resistant to photodegradation compared with traditional probes. However, after surface capping<br />
and water solubilization, the current QDs are an order of magnitude larger than organic dyes,<br />
which severely limit their use in biomedical applications that require very compact and<br />
noninterfering labels. In this talk, we discuss a new generation of size-minimized QDs, new tagging<br />
strategies, as well as methods <strong>for</strong> QD intracellular delivery and dynamic cellular imaging.<br />
Andrew M. Smith is a Distinguished Fellow of the NIH Center <strong>for</strong> Cancer Nanotechnology Excellence at<br />
Emory University. He received his BS degree in Chemistry and his PhD in Bioengineering, both from the<br />
Georgia Institute of Technology. His research focuses on nanomaterials engineering <strong>for</strong> molecular imaging of<br />
cancer and the exploration of the interactions between nanostructures and biological systems.<br />
Shuming Nie is Wallace H. Coulter Distinguished Chair Professor of Biomedical Engineering at Emory<br />
University and Georgia Institute of Technology. He received his BS degree from Nankai University, his MS<br />
and PhD degrees from Northwestern University, and did his postdoctoral training at both Georgia Tech and<br />
Stan<strong>for</strong>d University. His research interests are primarily in the areas of biomolecular engineering and<br />
nanotechnology, with a focus on bioconjugated nanoparticles <strong>for</strong> molecular imaging, molecular profiling,<br />
pharmacogenomics, and targeted therapy.<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 31
L8 Lecture 8<br />
High density labelling of DNA with photoswitchable dyes: implications<br />
in super-resolution microscopy<br />
Cristina Flors *<br />
EaStChem School of Chemistry, University of Edinburgh, Joseph Black Building, The King’s<br />
Buildings, West Mains Rd, EH9 3JJ Edinburgh (United Kingdom)<br />
With the dramatic expansion of super-resolution fluorescence microscopy techniques, the ability to<br />
label the structures of interest with appropriate fluorophores in high density is becoming more<br />
important. In particular, labelling of DNA with a high density of photoswitchable fluorophores is still<br />
a challenge. Intercalating and minor-groove binding cyanine dyes in combination with special<br />
buffers that induce photoblinking have been used to image isolated and cell DNA using singlemolecule<br />
localization microscopy. [1] In addition, it will be shown that the use of new modified DNA<br />
polymerases that allow the efficient incorporation of fluorophores into fragments of up to 1 kb [2] is a<br />
promising alternative to achieve controllable fluorescence photoswitching in these DNA fragments.<br />
Moreover, reversible and reproducible photoswitching in DNA also enables other advanced<br />
microscopy modalities such as optical-lock in detection imaging, which enhances the contrast in<br />
fluorescence imaging. [3] The combination the above techniques have great potential to study the<br />
structure of chromosomes at the nanoscale.<br />
This work was supported by The Royal Society.<br />
References: [1] C. Flors, Biopolymers, 95 (2011) 290. [2] N. Ramsay, et al., J. Am. Chem. Soc. 132 (2010)<br />
5096. [3] G. Marriott, et al., Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 17789.<br />
______________<br />
* Corresponding author: e-mail: cristina.flors@ed.ac.uk<br />
32 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L9 Lecture 9<br />
The first nucleic acid base analogue FRET-pair - a versatile tool <strong>for</strong><br />
detailed biochemical and nanotechnological structure in<strong>for</strong>mation<br />
L. Marcus Wilhelmsson *<br />
Department of Chemical and Biochemical Engineering/Physical Chemistry, Chalmers University of<br />
Technology, S-41296 Göteborg, Sweden<br />
Utilization of the tricyclic cytosine family, tC, tC O , and tCnitro, is rapidly increasing in biophysical,<br />
biochemical and (bio)nanotechnological applications. [1] The two fluorescent members of this family,<br />
tC and tC O , both have unique properties among fluorescent base analogues. [2,3] In contrast to other<br />
reported fluorescent base analogues tC and tC O have i) a high quantum yield (φf≈0.2) in duplex<br />
that is virtually insensitive to neighboring base combination, ii) an emission after incorporation into<br />
DNA being characterized by a single exponential decay in double stranded systems, and iii) an<br />
average luminescence brightness of the base analogues in duplex DNA being the highest reported<br />
so far and up to 50 times higher than the most commonly used fluorescent base analogue 2aminopurine.<br />
[2,3] Furthermore, the three tricyclic base analogues all <strong>for</strong>m stable base pairs with<br />
guanine and give minimal perturbations to the native structure of DNA. [1] Importantly, we have<br />
recently utilized tC O as a donor and developed tCnitro as an acceptor and, thus, established the first<br />
nucleic acid base analogue förster resonance energy transfer (FRET)-pair. [4] The FRET-pair<br />
successfully monitors distances covering up to more than one turn of the DNA duplex and, more<br />
importantly, we have shown that the rigid stacking of the two base analogues, and consequently<br />
excellent control of the their exact positions, results in very distinct FRET changes as the number<br />
of bases separating the donor and acceptor is varied. As a consequence of the exact positioning,<br />
this FRET-pair enables very high control of the orientation factor (κ 2 ). A set of DNA strands<br />
containing the FRET-pair at wisely chosen locations will, thus, make it possible to accurately<br />
distinguish distance- from orientation-changes using FRET. In combination with the good base<br />
analogue properties this points towards detailed studies of the inherent dynamics of nucleic acid<br />
structures. Moreover, the placement of FRET-pair chromophores inside the base stack will be a<br />
great advantage in studies where other (biomacro)molecules interact with the nucleic acid.<br />
This work was supported by grants from the Swedish Research Council (VR) and Olle Engkvist<br />
Byggmästare’s Foundation.<br />
References: [1] L.M. Wilhelmsson, Q. Rev. Biophys., 43 (2010) 159. [2] P. Sandin, et al., Nucleic Acids<br />
Res., 33 (2005) 5019. [3] P. Sandin, et al., Nucleic Acids Res. 36 (2008) 157. [4] K. Börjesson, et al., J. Am.<br />
Chem. Soc. 131 (2009) 4288.<br />
______________<br />
* Corresponding author: e-mail: marcus.wilhelmsson@chalmers.se<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 33
L10 Lecture 10<br />
Imaging neurodegenerative events in cells using FRET/FLIM<br />
Angela de Gomes Dani 1 , Matthias Gralle 1,3 , Sebastian Deeg 2 , Jochen Weishaupt 2, Pawel<br />
Kermer 2 , Alessandro Esposito 1,4 & Fred S. Wouters 1,*<br />
1Laboratory <strong>for</strong> Molecular and Cellular Systems, University Medicine Göttingen, Dept. of Neuro-<br />
and Sensory Physiology, Humboldtalee 23, 37073 Göttingen (Germany)<br />
2 Neurology Department, University Medicine Göttingen, Göttingen (Germany)<br />
3 Present address : Dept. of Medical Biochemistry, Federal University of Rio de Janeiro (Brazil)<br />
4 Present address: MRC Cancer Cell Unit – Hutchinson/MRC Research Centre, Cambridge (UK)<br />
The accumulation of misfolded and aggregated proteins lie at the basis of human<br />
neurodegenerative disease. How misfolding and aggregation of proteins constitute a toxic insult to<br />
the cell is largely unknown. The high load of aggregated proteins during neurodegeneration is<br />
thought to lead to the failure of detoxifying protein quality control (PQC) mechanisms to meet<br />
normal physiological demand, ultimately resulting in a pathological “de-compensated” condition<br />
from which the cell cannot recover.<br />
In order to study the involvement of PQC mechanisms in the cellular response to aggregated<br />
proteins, we developed FRET (Förster Resonance Energy Transfer) / FLIM (Fluorescence Lifetime<br />
Imaging)-based biosensors <strong>for</strong> the quantitative detection of various aspects of these protective<br />
responses and applied these in cellular model systems of human neurodegenerative conditions.<br />
Our sensors visualize protein ubiquitination [1] and degradation; proteasomal activity; foldase<br />
activity [2] using a folding-impaired GFP mutant which gains fluorescence conditional on the<br />
upregulation of chaperone activity; chaperone binding to unfolded proteins; and autophagosome<br />
<strong>for</strong>mation/lysosomal integrity via the targeted and sensitive FRET-based measurement of pH<br />
changes [4] . These sensors are employed in cellular model systems <strong>for</strong> Parkinson’s disease,<br />
Alzheimer’s disease, and Amyotrophic Lateral Sclerosis (ALS) to delineate the molecular pathway<br />
of cellular demise, to identify cellular stress checkpoints and to gain a mechanistic understanding<br />
of the toxicity of protein aggregates and the basis <strong>for</strong> the vulnerability of neurons.<br />
This work was supported by grants from DFG and BMBF.<br />
References: [1] S. Ganesan, et al. Proc. Natl. Acad. Sci. USA 103 (2006) 4089. [2] S. Deeg, et al. J. Cell<br />
Biol. 188 (2010) 505. [2] S.V. Avilov, et al., Biochemistry 48 (2009) 2422. [3] S. Ganesan et al., Cell Death<br />
Differ. 15 (2008) 312. [4] A. Esposito, et al. Biochemistry 47 (2008) 13115.<br />
______________<br />
* Corresponding author: e-mail: fred.wouters@gwdg.de<br />
34 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L11 Lecture 11<br />
Spatio-temporal fluctuations of supranucleosomal state regulating<br />
accessibility of bromodomain to acetylated H4 in living cells by highresolution<br />
FLIM<br />
Nicolas Audugé 1 , Sergi Padilla-Parra 1, 2 , Marc Tramier 1, 3 & Maïté Coppey-Moisan 1,*<br />
1<br />
Equipe Macromolecular Complexes in Living Cells, Institut Jacques Monod UMR 7592 CNRS,<br />
Université Paris-Diderot, 15 rue Hélène Brion, 75250 Paris (France).<br />
2<br />
Present adress : Emory University Department of Pediatrics, Infectious Diseases 2015 Uppergate<br />
Dr.Atlanta, GA 30322. U.S.A<br />
3<br />
Present adress : Institut de Génétique et Développement de Rennes, UMR 6061 CNRS -<br />
Université Rennes1, Faculté de Médecine 2 avenue du Professeur Léon Bernard CS 34317 35043<br />
Rennes Cedex, France<br />
We unveiled spatio-temporal fluctuations of the fluorescence lifetime of EGFP-histone H4 in living<br />
cells by using fast time-lapse FLIM measurement and two-photon excitation [1,2,3] . We showed that<br />
these fluctuations are likely due to the variations of the microenvironment of the EGFP-H4<br />
incorporated in chromatin, likely the refractive index and thus reflect the variations of a supranucleosomal<br />
state of chromatin domains of about 0.6 micron of diameter. Interestingly, the<br />
interaction between the double bromodomain of TAFII250 and the acetylated histone H4 exhibit<br />
similar spatio-temporal fluctuations. Furthermore, no interactions can be detected when chromatin<br />
is shifted to a supra-nucleosomal state characterized by short fluorescence lifetime of EGFP-H4,<br />
after ATP depletion. A detailed pixel by pixel analysis over 100 time-lapse FLIM images revealed<br />
the existence of a correlation between the temporal mean value and the amplitude of the<br />
fluctuations of fluorescence lifetime of EGFP-H4. Spatial mapping of this correlation unveiled a<br />
more dynamical chromatin at the nucleus periphery. We propose that these fluctuations of supranucleosomal<br />
state would modulate the accessibility of protein factors to nucleosomes and thus<br />
likely correspond to a switch between two states of chromatin domain.<br />
This work was supported by grants from FRM, CNRS and ANR.<br />
References: [1] S. Padilla-Parra, et al., Biophys. J., 95 (2008) 2976. [2] S. Padilla-Parra, et al.,<br />
Biophys. J. 97 (2009) 2368. [3] S. Padilla-Parra, et al., Biophys. Review in press<br />
______________<br />
* Corresponding author: e-mail: coppey.maite@ijm.univ-paris-diderot.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 35
L12 Lecture 12<br />
High resolution imaging and nanoprocessing with femtosecond lasers<br />
Karsten König 1,2<br />
1<br />
Department of Biophotonics and Laser Technology, Saarland University, Campus A5.1, 66123<br />
Saarbrücken (Germany)<br />
2<br />
Jenlab GmbH, Schillerstrasse 1, 07745 Jena, www.jenlab.de (Germany)<br />
Femtosecond laser microscopy was introduced by Denk, Strickler, and Webb in 1990. One<br />
decade later the first clinical femtosecond laser imaging systems were introduced by JenLab<br />
GmbH in Jena, Germany. These multiphoton tomographs are now working in leading cancer<br />
hospitals and research institutions of large cosmetic companies in Australia, Japan, Cali<strong>for</strong>nia, and<br />
Europe. The non-invasive certified laser tomographs provide optical biopsies with the best tissue<br />
resolution (0.3 µm lateral, 1 µm axial) of all existing state-of-the-art clinical imaging tools and may<br />
replace the current procedure of physically taken skin biopsies, slicing, staining, and observation<br />
by pathologists. The most recent development is a hybrid multiphoton/CARS tomograph which is<br />
currently working at the hospital Charite at Berlin. Major applications include melanoma detection,<br />
early diagnosis of dermatitis, wound healing management, testing of the biosafety of sunscreen<br />
nanoparticles ans well as evaluation of anti-aging products. The imaging procedure is per<strong>for</strong>med<br />
with transient GW/cm 2 light intensities at low picojoule pulse energies (80 MHz).<br />
When increasing the intensities by 3 or 4 orders (TW/cm 2 ) destructive effects based on multiphoton<br />
ionization and plasma <strong>for</strong>mation occur. These effects can be used <strong>for</strong> sub-100nm 3D<br />
nanomachining of glass, silicon, polymers, and metal layers. ITO nanowires of less than 50 nm can<br />
be produced. In addition, 3D nanolithography can be per<strong>for</strong>med. A furter application of<br />
femtosecond laser nanoprocessing is targeted transfection and optical cleaning.<br />
This talk provide an overview on 80 MHz femtosecond laser systems <strong>for</strong> medicine, material<br />
processing, cell biology, and nanobiotchnology.<br />
This work was supported by grants from the European Commission within the 7 th framework, the<br />
German ministry BMBF, and the German Science Foundation (DFG).<br />
References: [1] K. König, et al., Advanced Drug Delivery Reviews., 63 (2011) 388. [2] K. König, et al., Laser<br />
Phys Lett. 8 (2011) 465.<br />
______________<br />
Corresponding author: e-mail: info@jenlab.de<br />
36 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L13 Lecture 13<br />
Luminescence techniques: a powerful non-invasive diagnostic tool <strong>for</strong><br />
artwork materials<br />
Aldo Romani 1,2 , Catia Clementi 2 , Costanza Miliani 3 & Gianna Favaro 1<br />
1<br />
Dipartimento di Chimica, Università di Perugia, Via Elce di Sotto, 8, 06123 – Perugia, Italy<br />
2<br />
Centro SMAArt, c/o Dipartimento di Chimica, Università di Perugia, Perugia, Italy<br />
3<br />
Istituto CNR di Scienze e Tecnologie Molecolari (ISTM), c/o Dipartimento di Chimica, Università<br />
di Perugia, Perugia, Italy<br />
In the scientific approach to cultural heritage, which is a continuously expanding research field,<br />
light-based techniques occupy a relevant position, because of their non-invasive character<br />
combined with an exceptional temporal and spatial resolution. Coupled to absorption<br />
measurements, luminescence techniques often give integrative and precious results, being<br />
emission generally more sensitive and selective than absorption. It may provide basic in<strong>for</strong>mation<br />
about the materials composing artworks, which is an indispensable starting point <strong>for</strong> their<br />
restoration and conservation. [1,2]<br />
In this widely expanding research field we limit this report to polychromatic materials which<br />
are used in figurative arts, including paintings, tapestries and parchments. Methodologies followed<br />
in the research will be described. The first step develops in laboratory by investigating the material<br />
of interest in pure crystalline phase, solution and mock-up samples, which reproduce as closely as<br />
possible the original composition of artworks (painted canvas, murals, wood tables and papers).<br />
Once laboratory samples have been characterized from the point of view of their luminescent<br />
properties, the subsequent step is transferring measurement techniques on the original artworks.<br />
With the aim to guarantee their integrity the applied techniques must be non-invasive and to avoid<br />
transportation of master pieces spectroscopic methods in easily portable assemblages are<br />
desirable. In the case of luminescence, portable instrumentation has been realized <strong>for</strong> both<br />
stationary and time resolved measurements.<br />
The important role of laboratory experiments carried out in solution and in mock-up samples<br />
is highlighted in the photophysical characterization of real cases. Examples of investigations on<br />
original artworks will be described that spam over time from Roman age, through Middle Ages and<br />
Renaissance, to modern and contemporary art.<br />
References: [1] A. Romani et al., Acc. Chem.Res., 43 (2010) 837. [2] Degano et al., Appl. Spectrosc. Rev.,<br />
44 2009, 44, 363-410<br />
______________<br />
* Corresponding author: e-mail: favaro@unipg.it<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 37
L14 Lecture 14<br />
Triggered reactions <strong>for</strong> producing optical responses<br />
Eric V. Anslyn<br />
Department of Chemistry and Biochemistry, The University of Texas at Austin, NHB, A1590, Austin<br />
TX 78712<br />
The use of supramolecular chemistry principles in the design of optical sensors is now an<br />
established and flourishing field. A common approach to creating modulation of fluorescence upon<br />
a molecular recognition-binding event is photoinduced electron transfer (PET). This is most often<br />
accomplished by modulating the energy level of the lone pairs on an amine relative to a<br />
fluorophore HOMO and LUMO. Alternatively, our group has popularized the use of indicatordisplacement<br />
assays (IDAs), where a pH indicator is allowed to bind to a receptor in a reversible<br />
fashion, followed by displacement upon addition of an analyte. The presence of the target triggers<br />
a shift in equilibria between free and bound indicator. Further, the modular approach to an IDA<br />
allows several individual assays to be readily created in parallel within an array plat<strong>for</strong>m, as a<br />
means of creating pattern-based differential sensing routines. A third strategy <strong>for</strong> modulating<br />
fluorescence involves the initiation of a chemical reaction upon analyte addition. Sometimes this is<br />
a simple single step process involving selective removal of a protecting group on a flourophore by<br />
the target. Alternative, the fluorophore can be created in a multi-step reaction triggered by the<br />
target. All three of these methods, as well as differential sensing schemes, are being widely<br />
adopted by the supramolecular and physical organic chemistry communities to create optical<br />
sensors. This talk will present the three methods, along with differential sensing concepts, and then<br />
showcase the techniques in the context of sensors <strong>for</strong> heparin in blood, terpenes in perfumes, and<br />
NO in cellular imaging studies.<br />
38 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L15 Lecture 15<br />
Peeking and poking at rapid GTPase signaling in living cells<br />
Klaus Hahn*, Christopher Welch, Hui Wang, Andrei Karginov, Jason Yi & Pei-Hsuan Chu<br />
Department of Pharmacology, University of North Carolina - Chapel Hill. 120 Mason Farm Road,<br />
Chapel Hill, North Carolina, USA 27514.<br />
Cell motility requires the orchestration of many dynamic cellular systems, including the<br />
cytoskeleton, adhesion complexes and vesicle trafficking. Understanding the spatio-temporal<br />
coordination of molecules mediating motility requires quantitation of protein activity in living cells.<br />
This talk will describe new tools to visualize and manipulate rapid Rho family signaling in vivo,<br />
focused on understanding the coordination of adhesion, microtubules and actin to generate<br />
polarization. “Computational multiplexing” will be used to examine the coordination of three or more<br />
GTPases during cell protrusion. The role of specific GTPases will be dissected through<br />
development of novel methods to activate and inactivate proteins at specific times and places in<br />
living cells. Protein activity can be controlled either with light or through engineered allosteric<br />
activation. The talk will also describe new biosensors elucidating the roles of RhoG and RhoC in<br />
polarized motility.<br />
This work was supported by grants from the National Institutes of Health, the American Cancer<br />
Society and the Arthritis Foundation.<br />
______________<br />
* Corresponding author: e-mail: khahn@med.unc.edu<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 39
L16 Lecture 16<br />
Nonlinear microscopy of tissues and embryonic morphogenesis<br />
Emmanuel Beaurepaire<br />
Lab <strong>for</strong> Optics and Biosciences, Ecole Polytechnique, Palaiseau, France.<br />
http://www.lob.polytechnique.fr<br />
Understanding the dynamics at play in embryo morphogenesis requires tissue-scale<br />
measurements with subcellular resolution. Nonlinear microscopy is attractive <strong>for</strong> live tissue studies<br />
because it provides deep 3D imaging, and provides different complementary contrast mechanisms<br />
[1]. Coherent signals such as second- and third-harmonic generation (SHG, THG) provide<br />
structural in<strong>for</strong>mation from unstained tissues, and can be used in conjunction with fluorescence<br />
(2PEF) imaging. SHG signals are obtained from dense organized structures such as collagen<br />
fibrils, myofilaments, or polarized microtubules assemblies. THG signals are obtained from<br />
heterogeneities such as lipid/water interfaces and can provide 3D morphological images.<br />
Polarization-sensitive THG can detect anisotropy. We will discuss these principles and applications<br />
such as nonlinear human cornea imaging [2].<br />
We will then report on a strategy providing complete imaging of unstained zebrafish embryos<br />
<strong>for</strong> their first 10 cell division cycles [3]. Scanning patterns optimized to preferentially probe the<br />
innermost regions combined to harmonic imaging highlights mitotic spindles and cell boundaries<br />
through the entire embryo. Image quality is amenable to automated analysis, revealing the<br />
phenomenology of cell proliferation. These data reveal a progressive onset of asynchrony<br />
contrasting with classical descriptions, and should provide a reference framework <strong>for</strong> further<br />
studies.<br />
Finally, we point out that in toto observation of evolving embryos remains challenging. We<br />
will discuss ongoing ef<strong>for</strong>ts to improve the capabilities of nonlinear microscopy, such as wavefront<br />
control <strong>for</strong> aberration correction [4] and broadband pulse shaping <strong>for</strong> multiplexed addressing of<br />
several chromophores [5].<br />
References:<br />
[1] Supatto, Débarre, Moulia, Brouzés, Martin, Farge, Beaurepaire, In vivo modulation of morphogenetic<br />
movements in Drosophila embryos with femtosecond laser pulses, PNAS 102, 1047 (2005)<br />
[2] Olivier, Aptel, Plamann, Schanne-Klein, Beaurepaire, Harmonic microscopy of isotropic and anisotropic<br />
microstructure of the human cornea, Opt. Express (2010)<br />
[3] Olivier, Luengo-Oroz, Duloquin, Faure, Savy, Veilleux, Solinas, Débarre, Bourgine, Santos, Peyriéras,<br />
Beaurepaire, Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy,<br />
Science 339, 967 (2010)<br />
[4] Olivier, Débarre, Beaurepaire, Dynamic aberration correction <strong>for</strong> multiharmonic microscopy, Opt. Lett. 34,<br />
3145 (2009)<br />
[5] Labroille, Pillai, Solinas, Boudoux, Olivier, Beaurepaire, Joffre, Dispersion-based pulse shaping <strong>for</strong><br />
multiplexed two-photon fluorescence microscopy, Opt Lett 35, 3444 (2010)<br />
40 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L17 Lecture 17<br />
Fluorescence lifetime and anisotropy imaging <strong>for</strong> HCS of<br />
protein-protein interactions<br />
Daniel Matthews, Viput Visitkul, Gregory Weissman, Elena Ortiz-Zapater, Simon Poland,<br />
Gilbert Fruhwurth, Paul Barber, Melanie Keppler, Leo Carlin, Jody Barbeau, Anthony<br />
Coolen, Tony Ng & Simon M. Ameer-Beg<br />
Functional imaging can provide a level of quantification that is not possible in what might be termed<br />
traditional high-content screening. This is due to the fact that the current state-of-the-art highcontent<br />
screening systems take the approach of scaling-up single cell assays, and are there<strong>for</strong>e<br />
based on essentially pictorial measures as assay indicators. Such phenotypic analyses have<br />
become extremely sophisticated, advancing screening enormously, but this approach can still be<br />
somewhat subjective. Development, and validation, of a prototype high-content screening plat<strong>for</strong>m<br />
that combines steady-state fluorescence anisotropy imaging with fluorescence lifetime imaging will<br />
be described. This functional approach allows objective, quantitative screening of small molecule<br />
libraries and siRNAs in protein-protein interaction assays. The development of the instrumentation,<br />
the process by which in<strong>for</strong>mation on fluorescence resonance energy transfer (FRET) can be<br />
extracted from wide-field, acceptor anisotropy imaging and cross-checking and quantification of<br />
this modality using lifetime imaging by time-correlated single-photon counting will be discussed.<br />
We have validated this approach using a small-scale inhibitor screen of the Cdc42 variant of the<br />
so-called Raichu biosensor probe. This has been expressed in an endothelial cancer cell line<br />
(A431) and was prepared in a 96 well-plate <strong>for</strong>mat. The assay was able to demonstrate all the<br />
capabilities of the instrument, image processing and analytical techniques that have been<br />
developed. Direct correlation between acceptor anisotropy and donor FLIM is observed <strong>for</strong> FRET<br />
assays, providing an opportunity to screen proteins, interacting on the nano-meter scale, using<br />
wide-field imaging. Finally we show that these methodologies can be extended to microfluidic<br />
cytometric screening of protein-protein interactions using burst integrated fluorescence lifetime<br />
spectroscopy.<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 41
L18 Lecture 18<br />
Imaging cellular signaling on molecule at a time<br />
Thomas Schmidt<br />
Physics of Life Process, the University of Leiden, the Netherlands<br />
The interaction of G-protein-coupled receptors with G proteins is a key event in transmembrane<br />
signal transduction that leads to vital decision-making by the cell. Here, we applied single-molecule<br />
epifluorescence microscopy to study the mobility of both the Gβγ and the Gα2 subunits of the G<br />
protein heterotrimer in comparison with the cAMP receptor responsible <strong>for</strong> chemotactic signaling in<br />
Dictyostelium discoideum. Our experimental results suggest that ~30% of the G protein<br />
heterotrimers exist in receptor-precoupled complexes. Upon stimulation in a chemotactic gradient,<br />
this complex dissociates, subsequently leading to a linear diffusion and collision amplification of the<br />
external signal. We further found that Gβγ was partially immobilized and confined in an agonist-, Factin-<br />
and Gα2-dependent fashion. This led to the hypothesis that functional nanometric domains<br />
exist in the plasma membrane, which locally restrict the activation signal, and in turn, lead to<br />
faithful and efficient chemotactic signaling.<br />
______________<br />
Corresponding author: e-mail: schmidt@physics.leidenuniv.nl<br />
42 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L19 Lecture 19<br />
Assembly of ribonucleoproteins required <strong>for</strong> HIV-1 trafficking:<br />
one molecule at a time<br />
Rajan Lamichhane, Rae Robertson-Anderson & David Millar*<br />
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037 USA.<br />
Following cellular entry and genome integration of HIV-1, the viral genome must be expressed in<br />
the appropriate sequence and at the proper time to ensure successful completion of the viral life<br />
cycle. The HIV-1 Rev protein plays an essential role, orchestrating the transition between the early<br />
and late programs of viral gene expression. To do so, Rev mediates the nucleocytoplasmic export<br />
of the unspliced viral RNA genome and singly-spliced mRNAs encoding viral structural proteins.<br />
Rev interacts with a highly conserved element within the viral pre-mRNA known as the Rev<br />
response element (RRE). This is a complex interaction in which multiple Rev monomers assemble<br />
on the RRE, mediated by a combination of RNA-protein and protein-protein interactions. Moreover,<br />
the oligomerization of Rev on the RRE is essential <strong>for</strong> nuclear export. While Rev is the central<br />
player, a number of cellular proteins, including the transport receptor CRM1 and the DEAD box<br />
helicase DDX1, interact with Rev and are required <strong>for</strong> efficient Rev function. The mechanism of<br />
oligomeric Rev-RRE assembly and the role of cellular cofactors during this process are not well<br />
understood. We have developed a single-molecule fluorescence method to monitor oligomerization<br />
of Rev on the RRE in real-time and with single monomer resolution. Our experimental system<br />
utilizes a 351 nt RRE RNA, generated by in vitro transcription and immobilized on a quartz surface,<br />
and recombinant Rev protein labeled with Alexa-Fluor 555 at a unique cysteine residue. Binding of<br />
individual Rev monomers to the immobilized RRE is readily monitored over time by singlemolecule<br />
TIRF microscopy. Individual binding or dissociation events are detected as discrete and<br />
abrupt jumps in fluorescence intensity from the immobilized RNA. Hundreds of individual assembly<br />
reactions are monitored in parallel on an intensified CCD camera. From statistical analysis of jump<br />
size and dwell-time distributions, we obtained in<strong>for</strong>mation on the number of Rev monomers binding<br />
at each step of assembly as well as the associated rate constants. The results show that assembly<br />
is initiated by binding of a Rev monomer at stem loop IIB of the RRE and proceeds rapidly by<br />
sequential binding of single Rev monomers [1]. Notably, our results exclude previously proposed<br />
models in which pre<strong>for</strong>med Rev oligomers bind directly to the RRE. The single-molecule assay has<br />
also been used to visualize Rev-RRE assembly in the presence of DDX1, which is known to be<br />
required <strong>for</strong> efficient Rev function. The presence of DDX1 has a significant impact on Rev-RRE<br />
assembly, suppressing non-productive nucleation events and accelerating the binding of the first<br />
few Rev monomers. As a result, higher order Rev-RRE assembly states are observed more<br />
frequently in the presence of DDX1. Interestingly, our results suggest that DDX1 acts on the Rev<br />
protein rather than the RRE RNA, even though DDX1 is a putative RNA helicase [2]. Most recently,<br />
we have developed methods to label the RRE and selected cellular proteins with distinct<br />
fluorescent dyes and we are using two- or three-color single-molecule TIRF imaging to dissect<br />
RNP assembly in even greater detail. These studies are revealing the points at which specific<br />
cellular cofactors impact the RNP assembly pathway, as well as the temporal order and relative<br />
stoichiometries of Rev and cofactor binding events.<br />
This work was supported by the US National Institutes of Health.<br />
References: [1] S. J. Pond et al., Proc. Natl. Acad. Sci. USA, 106, 1404-1408 (2009). [2] R. Robertson-<br />
Anderson et al., J. Mol. Biol. in press (2011).<br />
______________<br />
*Corresponding author: e-mail: millar@scripps.edu<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 43
L20 Lecture 20<br />
Chasing fluorescence lifetimes in complex biological systems.<br />
What can Fluorescence Lifetime Imaging Microscopy (FLIM) tell us?<br />
R. M. Clegg<br />
Department of Physics, University of Illinois, Urbana-Champaign, Illinois, USA<br />
To resolve changes in different molecular components in highly complex intact, functional<br />
biological systems, which adapt rapidly to changing conditions, we have developed rapid<br />
nanosecond fluorescence lifetime imaging (FLI) to follow these processes in real time (sub-second<br />
to many minutes). We have used FLI to investigate non-photochemical quenching (NPQ)<br />
mechanisms used by plants to protect themselves from over exposure to light, which can produce<br />
destructive reactive oxygen species (ROS). Plants possess the two central mechanisms of NPQ,<br />
the xanthophyll and lutein cycles, which allow plants to react rapidly to variable light conditions.<br />
Lifetime-resolved fluorescence emission from chlorophyll “brings to light” underlying mechanisms,<br />
such as energy transfer, photochemical and non-photochemical quenching, photoinhibition, and<br />
state changes (shuttling of components of antennae systems between photosystems PSI and<br />
PSII). Some aspects of bioassays based on FRET and fluorescence lifetimes <strong>for</strong> measuring<br />
intracellular oxidation reduction potentials and the enzymatic activity of a matrix metalloproteinase<br />
will be discussed, as well as FLIM applications to protoporphyrin IX aggregation in vivo and in vitro.<br />
Fluorescent protein hybrids have played a central role in the development many of these bioassay<br />
probes. The overview and examples will emphasize some analysis techniques specific to FLIM that<br />
provide rapid and detailed multi-parameter analysis of complex systems.<br />
______________<br />
Corresponding author: e-mail: rclegg@illinois.edu<br />
44 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L21 Lecture 21<br />
Fluorescent proteins and tools: Moscow news<br />
Dmitry Chudakov<br />
Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,<br />
Moscow (Russia)<br />
We report development of several fluorescent protein-based molecular instruments:<br />
Far-red fluorescent proteins <strong>for</strong> whole body imaging. Fluorescent proteins which<br />
emission falls within infra-red are of high demand <strong>for</strong> the whole body imaging techniques. We<br />
report two enhanced far-red fluorescent proteins, eqFP650 and eqFP670, developed on the basis<br />
of Katushka. While eqFP650 is the brightest fluorescent protein ever reported with emission<br />
maximum above 635 nm and is currently the optimal genetically encoded probe <strong>for</strong> in vivo imaging,<br />
eqFP670 is the first fluorescent protein with an emission maximum peaked at 670 nm, which is<br />
characterized by low toxicity, high pH stability and unprecedented photostability.<br />
Low toxic “supermonomeric” red fluorescent protein. A number of red dimeric and<br />
tetrameric fluorescent proteins underwent artificial dimerization, mostly losing their brightness,<br />
maturation rate and fluorescence stability. Despite such wastages, resulting red monomers still are<br />
not able to per<strong>for</strong>m in fusions as good as natural green monomers. Artificial red monomers, such<br />
as popular mCherry and other mFruits, along with mKO, TagRFP, mKate and mKate2 developed<br />
in later years are suitable as fusion labels <strong>for</strong> many proteins of interest. Still, with these proteins<br />
one may face problems related to abnormal protein localization or toxic effects. Most likely such<br />
behavior is related to residual dimerization tendency and/or unpredictable interactions between<br />
newly exposed and modified hydrophobic surfaces of artificially monomerized fluorescent proteins.<br />
We report low toxic “supermonomeric” red fluorescent protein which per<strong>for</strong>mance in fusions is<br />
comparable to that of the best green variants.<br />
KillerRed <strong>for</strong> optogenetic studies. We have shown that genetically encoded<br />
photosensitizer KillerRed can be used as a powerful instrument <strong>for</strong> optogenetic studies of the role<br />
of ROS and modeling of ROS-associated pathological conditions in vivo. In zebrafish model,<br />
membrane-localized KillerRed expressed in cardiac muscle cells is effective tool <strong>for</strong> studies of the<br />
ROS-induced conditions, including pathological conditions associated with heart failure. Further,<br />
we demonstrated that irradiation of cells expressing KillerRed-H2B allows to block cell divisions in<br />
culture temporarily, <strong>for</strong> approximately 24 h. In transgenic Xenopus laevis embryos expressing H2BtKR<br />
under the control of tissue-specific promoters, we observed retardation of the development of<br />
these tissues in green-light-illuminated tadpoles. Thus we believe that H2B-tKR represents an<br />
efficient optogenetic tool, which can be used to study mitosis and meiosis progression, and to<br />
investigate the roles of specific cell populations in development, regeneration and carcinogenesis<br />
in vivo.<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 45
L22 Lecture 22<br />
Water-solubilisation and bio-conjugation of novel red emitting<br />
BODIPY markers<br />
Raymond Ziessel*<br />
Laboratoire de Chimie Organique et Spectroscopies Avancées (LCOSA, http://www-lmspc.ustrasbg.fr/lcosa/),<br />
Ecole Européenne de Chimie, Polymères et Matériaux, 25 rue Becquerel 67087<br />
Strasbourg Cedex, France<br />
Several sets of borondipyrromethene (BODIPY) dyes have been engineered in such a way that<br />
the central fluorescent unit bears one or more appended aromatic polycycles. [1] These latter units<br />
function as ancillary light-harvesters and channel incident photons to the BODIPY emitter. The<br />
detailed mechanisms of intra-molecular energy transfer have been elucidated on the basis of<br />
steady-state and time-resolved spectroscopic measurements and related to the geometry of the<br />
assembly. It is noteworthy that the attached polycycle serves to enhance the virtual Stokes shift. [2]<br />
The novelty of these new dyes relates to the substitution pattern and most notably the replacement<br />
of the usual fluorine atoms with aromatic polycycles. In the extreme case, multiple substitution<br />
leads to arrays bearing different polycycles that absorb over much of the visible spectral window<br />
and transfer photons to the BODIPY centre within a few picoseconds. [3]<br />
Separate synthetic protocols allows the tuning of colours of these dyes from yellow to green<br />
but also the fluorescence from green to red and to the NIR. Separate synthetic protocols were<br />
designed in order to solubilize the Bodipy dyes into water or biological media. [4] The difficulty to<br />
prepare soluble and fluorescent blue dyes emitting in the red part of the visible spectra was<br />
overcome by balancing the solubilizing groups on two distinguish region of the molecule.<br />
The central pseudo meso position was used to anchor an activated ester suitable <strong>for</strong> grafting<br />
to protein and to biological material. Several examples will be discussed in details. [5]<br />
This work was supported by grants from CNRS and ANR.<br />
References:<br />
[1] R. Ziessel, G. Ulrich, A. Harriman, New J. Chem. 2007, 31, 496 (Highlight article).<br />
[2] G. Ulrich, R. Ziessel, A. Harriman, Angew. Chem. Int. Ed. 2008, 47, 1184 (Review article).<br />
[3] R. Ziessel, A. Harriman Chem. Commun. (Feature article), 2011, 47, 611.<br />
[4] G. Ulrich, C. Goze, M. Guardigli, A. Roda, R. Ziessel Angew. Chem., Int. Ed. 2005, 44, 3694.<br />
[5] L. Niu, G. Ulrich, R. Ziessel, A. Kiss, P.-Y. Renard and A. Romieu, Org. Lett., 2009, 11, 2049.<br />
______________<br />
* Corresponding author: e-mail: ziessel@unistra.fr<br />
46 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L23 Lecture 23<br />
Molecular nanoprobes <strong>for</strong> multiphotonics as new tools <strong>for</strong> bioimaging.<br />
Design and applications<br />
Mireille Blanchard-Desce 1,2<br />
1 Chimie et Photonique Moléculaires, UMR 6510, Université de Rennes 1, Rennes (France)<br />
2 Institut des Sciences Moléculaires, UMR 5255, Université de Bordeaux 1, Talence (France)<br />
Mutiphotonics has attracted a lot of interest over recent years due to the many applications it offers<br />
both in biological imaging and in material science. These include 3D optical data storage,<br />
microfabrication, optical power limitation, localized photodynamic therapy as well as novel imaging<br />
techniques. In particular novel microscopies based on multiphoton phenomena such as two-photon<br />
excited fluorescence (TPEF) and second-harmonic generation (SHG) have gained overwhelming<br />
popularity in the biology community owing to the many advantages they provide in biological<br />
imaging. These include a capacity <strong>for</strong> a highly confined excitation and intrinsic three-dimensional<br />
resolution, increased penetration depth in living tissues and reduced photodamage.<br />
This has triggered the design of "multiphotonic" probes specifically engineered <strong>for</strong> these new<br />
microscopies and able to generate types and levels of contrast unattainable otherwise. In the case<br />
of laser-scanning two-photon fluorescence microscopy (LSTPM), more selective excitation and<br />
concomitant reduction of background fluorescence (hence higher sensitivity) can be achieved by<br />
using biphotonic probes having much larger TPEF cross-sections than endogenous chromophores.<br />
However, conventional fluorescent dyes, such as fluorescein, the rhodamines etc., do not meet this<br />
requirement.<br />
In this context, we have implemented molecular engineering approaches towards novel<br />
biphotonic fluorescent probes having giant two-photon absorption cross-sections in the visible red–<br />
NIR region, outper<strong>for</strong>ming standard fluorophores such as fluorescein by more than two orders of<br />
magnitude, [1] leading to biphotonic markers allowing non-damaging imaging of cells. Furthermore,<br />
“smart” biphotonic fluorescent probes showing marked dependence of their response and/or<br />
fluorescence properties on their environment have been developed. [2] These probes open a wide<br />
range of applications such as monitoring of micropolarity or pH in various media. Finally, the<br />
design of novel classes of "ultra-bright" fully organic nanoparticles will be presented. [3] These<br />
nanoparticles represent an attractive route towards biocompatible and eco-friendly substitutes <strong>for</strong><br />
semiconductor quantum dots. They have already been demonstrated of major interest as contrast<br />
agents <strong>for</strong> in vivo angiography in small animals or remote sensing of nitrated explosives. [4]<br />
This work was supported by grants from ANR, Italo-French University and Région Bretagne.<br />
References: [1] L. Ventelon, et al., Angew. Chem., 40 (2001) 2098; O. Mongin, et al., Chem. Eur. J. 13<br />
(2007) 1481; F Terenziani et al, Adv. Mater., 20 (2008) 4641. [2] M. H. V. Werts. et al., J. Amer. Chem. Soc.,<br />
126 (2004) 16294; M. Parent, et al., Chem. Comm. (2005) 2029; C. Le Droumaguet, et al., Chem. Comm.<br />
(2005) 2802. [3] O. Mongin, et al., Chem. Commun. (2006) 915; O. Mongin,et al. SPIE Proc. 7403 (2009),<br />
740303; V. Parthasarathy, et al., (2011) submitted. [4] T. R. Krishna, et al., Angew. Chem., Int. Ed. 45 (2006)<br />
4645; O. Mongin, et al., SPIE Proc. 7040 (2008) 704006; M. Guo, et al., J. Phys. Chem. A, 113 (2009), 4763.<br />
______________<br />
* Corresponding author: e-mail: mireille.blanchard-desce@univ-rennes1.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 47
L24 Lecture 24<br />
Con<strong>for</strong>mational dynamics and biomolecular structure studied by<br />
super-resolution FRET<br />
Claus A. M. Seidel, Simon Sindbert, Stanislav Kalinin & Hayk Vardanyan<br />
Chair <strong>for</strong> Molecular Physical Chemistry, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1,<br />
40225 Düsseldorf, Germany<br />
While X-ray crystallography reveals snapshots of proteins at atomic resolution, many biomolecules<br />
have an inherent dynamic character, which governs their functions in space and time. In several<br />
cases, low populated con<strong>for</strong>mational states are overlooked or simply not accessible using<br />
conventional structural-biology tools. FRET that is considered a low-resolution tool has not yet<br />
received much attention in the field. However, recent developments in my group have shown the<br />
ability to reconstruct biomolecular structures using super-resolution FRET [4,5]. Considering long<br />
dye linkers the key step is to apply a procedure that allows <strong>for</strong> very high accuracy of FRET based<br />
structure determination through proper consideration of the position distribution of the dye and of<br />
linker dynamics.<br />
Using a confocal fluorescence microscope the newly developed multiparameter fluorescence<br />
detection (MFD) enables us to simultaneously collect all fluorescence in<strong>for</strong>mation such as intensity,<br />
lifetime, anisotropy in several spectral ranges from picoseconds to seconds under in vitro (1-5) and<br />
in vivo conditions.<br />
MFD is applied to per<strong>for</strong>m single-molecule FRET studies on a various con<strong>for</strong>mationally<br />
flexible RNAs and various proteins, such GTPases and DNA enzymes. Thus, it is possible to<br />
circumvent the classical pitfalls of the FRET method in ensemble measurements. In addition, we<br />
developed a simple procedure <strong>for</strong> the proper consideration of the position distribution of the dye<br />
and of linker dynamics to achieve super-resolution and a very high accuracy of FRET based<br />
structure determination. These novel FRET-based detection and analysis methodologies allowed<br />
us to resolve several structural subpopulations with Ångström resolution und derived FRET based<br />
structural models <strong>for</strong> several species from the same measurement.<br />
References:<br />
1. Kalinin, S., Felekyan, S., Valeri, A. Seidel C. A. M.; Characterizing multiple molecular states in singlemolecule<br />
multi-parameter fluorescence detection by probability distribution analysis; J. Phys. Chem. B<br />
112, 8361-8374 (2008).<br />
2. Woźniak, A. K., Schröder, G. F., Grubmüller, H., Seidel, C. A. M.; Oesterhelt F.; <strong>Single</strong>-molecule<br />
FRET measures bends and kinks in DNA. Proc. Natl. Acad. Sci. USA. 105, 18773-42 (2008);<br />
3. Gansen, A., Valeri, A., Hauger, F., Felekyan, S., Kalinin, S., Tóth, K., Langowski, J., Seidel, C. A. M.;<br />
Nucleosome disassembly intermediates characterized by single-molecule FRET, Proc. Natl. Acad. Sci.<br />
USA. 106, 15308-13 (2009).<br />
4. Sisamakis, E., Valeri, A., Kalinin, S., Rothwell, P. J., Seidel, C. A. M.; Accurate single-molecule FRET<br />
studies using multiparameter fluorescence detection. Methods in Enzymology 475 Chapter 18, 455-<br />
514 (2010).<br />
5. Sindbert, S., Kalinin, S., Nguyen, H., Kienzler, A., Clima, L., Bannwarth, W., Appel, B., Müller, S.,<br />
Seidel, C. A. M. Accurate distance determination of nucleic acids via FRET: Implications of dye linker<br />
length and rigidity. J. Am. Chem. Soc. 133, 2463-2480 (2011).<br />
______________<br />
* Corresponding author: e-mail: c.seidel@hhu.de<br />
48 — MAF 12, Strasbourg, France, September 11-14, 2011 —
L25 Lecture 25<br />
2011: a small space odyssey with luminescent molecules<br />
Gareth J. Brown, 1 A. Prasanna de Silva, 1,* Kaoru Iwai, 2 Gareth D. McClean, 1 Bernadine O.F.<br />
McKinney, 1 David C. Magri, 1 Seiichi Uchiyama 3 & Sheenagh M. Weir 1<br />
1<br />
School of Chemistry and Chemical Engineering, Queen’s University, Belfast, Northern Ireland<br />
2<br />
Department of Chemistry, Nara Women's University, Kitauoya-Nishimachi, Nara, Japan<br />
3<br />
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku,<br />
Tokyo, Japan<br />
Designed supermolecules of nanometric size allow us enter the subject of in<strong>for</strong>mation handling.<br />
Chemical scientists have the skills to launch molecular vehicles to explore small inaccessible<br />
spaces and to send back raw or processed in<strong>for</strong>mation that we can act upon in the real world, e.g.<br />
in a medical context.<br />
The design of the supermolecules is as follows. Photoinduced electron transfer (PET) is the<br />
heart of photosynthesis and is a major channel of de-exciting excited states. Luminescence is<br />
another such channel. The controlled competition of luminescence with photoinduced electron<br />
transfer (PET) can switch the luminescence ‘on’ or ‘off’ by chemical means in an easy, predictable<br />
manner. The modular nature of ‘lumophore-spacer-receptor’ supramolecular systems is not only<br />
vital <strong>for</strong> the occurrence of PET, but also <strong>for</strong> the prediction of system characteristics such as colours<br />
of the optical signals and the concentration range of the analyte.<br />
The first-generation systems use a single chemical controller. These give rise to examples<br />
which monitor sodium levels in blood within millimeter-sized channels or the status of acidic<br />
compartments in micrometer-sized cells. Some of these even map proton distributions in<br />
nanometric spaces near membranes. 1 Notably, some of these serve wider society by operating in<br />
hospital intensive care units. 2<br />
The second-generation systems use multiple chemical controllers. These <strong>for</strong>m molecularscale<br />
in<strong>for</strong>mation processors 3 which employ chemical species as inputs, light as output and<br />
wireless interfacing to human operators. Some of these processors are self-assembled and they<br />
operate com<strong>for</strong>tably in nanometer-sized regions near membranes, These spaces are too small <strong>for</strong><br />
the tiniest silicon-based electronic devices to enter. Such molecular logic devices are continually<br />
growing in complexity. Some of these have potential as ‘lab-on-a-molecule’ systems <strong>for</strong> intelligent<br />
medical diagnostics. Others allow molecular computational identification (MCID) of<br />
nano/micrometric objects. 4 This is the first of such applications which addresses a problem that<br />
does not currently have solutions from semiconductor computing technology.<br />
References: [1] S. Uchiyama, et al., Angew. Chem. Int. Ed. Engl. 47 (2008) 4667. [2] See OPTI® blood gas<br />
and electrolyte analyzers at www.optimedical.com. [3] A.P. de Silva, Chem. Asian J. 6 (2011) 750. [4] A.P.<br />
de Silva, et al., Nature Mater. 5 (2006) 787.<br />
______________<br />
* Corresponding author: e-mail: a.desilva@qub.ac.uk<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 49
50 — MAF 12, Strasbourg, France, September 11-14, 2011 —
A4photonlines110802.indd 1 02/08/11 14:30
52 — MAF 12, Strasbourg, France, September 11-14, 2011 —
ORAL<br />
ORAL<br />
ORAL<br />
COMMUNICATIONS<br />
COMMUNICATIONS<br />
COMMUNICATIONS<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 53
54 — MAF 12, Strasbourg, France, September 11-14, 2011 —
OC1 Super-resolution microscopy: a direct comparison of structural illumination microscopy<br />
(SIM), direct stochastic reconstruction microscopy (dSTORM) and confocal laser scanning<br />
microscopy (CLSM)<br />
Martin Bastmeyer<br />
OC2 A modified FCCS procedure applied to Ly49A–MHC class I cis-interaction studies in cell<br />
membranes<br />
Sofia Johansson, Johan Strömqvist, Lei Xu, Yu Ohsugi, Katja Andersson, Hideki Muto,<br />
Masataka Kinjo, Petter Höglund & Jerker Widengren<br />
OC3 DNA fluorocode: a single molecule, optical map of DNA with nanometer resolution<br />
Robert K. Neely, Jochem Deen, Giedrė Urbanavičiūtė, Saulius Klimašauskas<br />
& Johan Hofkens<br />
OC4 Super-resolution imaging and more with photoactivatable fluorescence<br />
Julia Gunzenhaeuser, Dylan Burnette, Jennifer Lippincott-Schwartz & Suliana Manley<br />
OC5 Applications of sub-diffraction fluorescence imaging in biological, botanical and materials<br />
sciences<br />
Trevor A. Smith, Xiaotao Hao & Liisa M. Hirvonen<br />
OC6 Polyethylene glycol-based multidentate oligomers <strong>for</strong> enhancing the biocompatiblity of<br />
semiconductor, gold and magnetic nanocrystals<br />
Goutam Palui, Hyon Bin Na & Hedi Mattoussi<br />
OC7 Fluorescent avidinated silica-core/PEG-shell nanoparticles: brightness <strong>for</strong> labeling and<br />
imaging<br />
Enrico Rampazzo, Sara Bonacchi, Damiano Genovese, Riccardo Juris, Mara Mirasoli,<br />
Marco Montalti, Luca Prodi, Luisa Stella Dolci & Nelsi Zaccheroni<br />
OC8 Fluorescence microspectroscopy: direct observation of polymer dynamics<br />
Albert M. Brouwer, Tanzeela N. Raja & Joanna Sierkierzycka<br />
OC9 Organic dye nanoparticles with intense fluorescence caused by a combined effect of<br />
intermolecular H-aggregation and restricted intramolecular rotation<br />
Hiroshi Yao & Koji Ashiba<br />
OC10 Probing the radiative transition and determining the fluorescence quantum yield of a single<br />
molecule with a tunable microresonator<br />
Alfred J. Meixner, Alexey I. Chizhik, Anna M. Chizhik, Dmitry Khoptyar, Sebastian Bär<br />
& Jörg Enderlein<br />
OC11 FLI-Cam – a frequency-domain fluorescence lifetime imaging system based on a new<br />
directly modulatable CMOS image sensor<br />
Robert Franke & Gerhard Holst<br />
OC12 Multiplexed time lapse imaging using homoFRET and time-resolved polarisation<br />
fluorescence microscopy<br />
Anca Margineanu, Sean Warren, Dominic Alibhai, Romain Laine, Ian Munro, Yuriy<br />
Alexandrov, Christopher Kimberley, Clif<strong>for</strong>d Talbot, James McGinty, Gordon Kennedy,<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 55
Alessandro Sardini, Mark A.A. Neil, Christopher Dunsby, Matilda Katan & Paul M. W.<br />
French<br />
OC13 Optically trapped microsensors <strong>for</strong> microfluidic temperature measurement by fluorescence<br />
lifetime imaging microscopy<br />
Mathieu A. Bennet, Patricia R. Richardson , Jochen Arlt , Aongus McCarthy , Gerald S.<br />
Buller & Anita C. Jones<br />
OC14 Studies on lipid order in cell membranes using fluorescence polarimetric microscopy<br />
Alla Kress, Patrick Ferrand, Xiao Wang, Hubert Ranchon, Philippe Réfrégier, Hervé<br />
Rigneault, Sophie Brasselet, Tomasz Trombik, Hai-Tao He & Didier Marguet<br />
OC15 Specific targeting of nanoparticles to proteins <strong>for</strong> visualization of single protein dynamics<br />
inside living cells<br />
Domenik Liße, Verena Wilkens, Changijang You, Karin Busch & Jacob Piehler<br />
OC16 A miniaturized, highly sensitive fluorescence detector and its routine application in pointof-need<br />
instrumentation<br />
Bernhard Gerstenecker, Roman Gruler, Michael Eberhard & Klaus Haberstroh<br />
OC17 New fluorescent nucleosides <strong>for</strong> exploring nucleic acid damage and recognition<br />
Yitzhak Tor<br />
OC18 Ligand inducible fluorescence: tools <strong>for</strong> ligand discovery and PCR monitoring<br />
Fumie Takei, Masaki Hagihara, Shiori Umemoto, Jinhua Zhan, Takeo Fukuzumi, Asako<br />
Murata, Yasue Harada & Kazuhiko Nakatani<br />
OC19 Detecting early stages of neurodegenerative disease by intrinsic fluorescence – β-amyloid<br />
aggregation in Alzheimer’s disease<br />
Mariana Amaro, Karina Kubiak-Ossowska, David JS Birch & Olaf J. Rolinski<br />
OC20 Kinase on-off switching in the functional, membrane-associated chemosensory signaling<br />
array of E. coli – structural changes detected by OS-FRET<br />
Annette H. Erbse, Adam J. Berlinberg & Joseph J. Falke<br />
OC21 Multiscale dynamics of single molecules in biomimetic crowding<br />
Robb Welty, Jacob Bentley , Dhanushka Wickramasinghe & Ahmed Heikal<br />
OC22 Inverse-Fluorescence Correlation Spectroscopy: label-free analysis of the absolute<br />
volume of biomolecules in solution<br />
Tor Sandén, Romain Wyss, Christian Santschi, Olivier J.F. Martin, Stefan Wennmalm<br />
& Horst Vogel<br />
OC23 Spot variable Fluorescence Correlation Spectroscopy reveals fast scouting of K-Ras at<br />
the plasma membrane of living cells<br />
Tomasz Trombik, Verena Ruprecht, Sébastien Mailfert, Cyrille Billaudeau, Fabien<br />
Conchonaud, Gerhard J. Schütz, Yoav Henis, Stefan Wieser & Didier Marguet<br />
OC24 Mechanical interaction between photons and macromolecules/nanoparticles<br />
as evaluated by Fluorescence Correlation Spectroscopy<br />
Syoji Ito, Hiroaki Yamauchi & Hiroshi Miyasaka<br />
56 — MAF 12, Strasbourg, France, September 11-14, 2011 —
OC25 Excited-state intermolecular proton transfer of the firefly’s chromophore d-luciferin<br />
Dan Huppert, Itay Presiado, Yuval Erez & Rinat Gepshtein<br />
OC26 Discovery and biological application of full-color tunable and predictable fluorescent core<br />
skeleton (Seoul-Fluor) and fluorescent glucose bioprobes (GBs)<br />
Eunha Kim, Hyang Yeon Lee, Sanghee Lee, Jongmin Park & Seung Bum Park<br />
OC27 Physical chemistry and photophysics of the cyan fluorescent protein<br />
Fabienne Mérola, Hélène Pasquier, Marie Erard, Agathe Espagne, Asma Fredj, Luis<br />
Alvarez, Germain Vallverdu, Gabriella Jonasson, Isabelle Demachy, Jacqueline Ridard &<br />
Bernard Levy<br />
OC28 Fluorescent probes <strong>for</strong> monitoring lipid order selectively at one bilayer leaflet<br />
Andrey S. Klymchenko, Oleksandr A. Kucherak, Zeinab Darwich, Sule Oncul, Youri<br />
Arntz, Pascal Didier, Guy Duportail & Yves Mely<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 57
OC1 Oral communication 1<br />
Super-resolution microscopy: a direct comparison of structural<br />
illumination microscopy (SIM), direct stochastic reconstruction<br />
microscopy (dSTORM) and confocal laser scanning microscopy (CLSM)<br />
Martin Bastmeyer<br />
Karlsruher Institüt für Technologie, Zoologisches Institüt, Abteilung für Zell- und Neurobiologie,<br />
76131 Karlsruhe, Germany<br />
(oral communication sponsored by Carl ZEISS)<br />
______________<br />
Corresponding author: E-mail: bastmeyer@bio.uka.de<br />
58 — MAF 12, Strasbourg, France, September 11-14, 2011 —
OC2 Oral communication 2<br />
A modified FCCS procedure applied to Ly49A–MHC class I cisinteraction<br />
studies in cell membranes<br />
Sofia Johansson 1 , Johan Strömqvist 1 , Lei Xu 1 , Yu Ohsugi 2 , Katja Andersson 3 , Hideki Muto 2 ,<br />
Masataka Kinjo 2 , Petter Höglund 3 & Jerker Widengren 1+<br />
+<br />
Corresponding author<br />
1<br />
Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of<br />
Technology, AlbaNova University Center, S-10691 Stockholm, Sweden<br />
2<br />
Laboratory of Supramolecular Biophysics, R.I.E.S, Hokkaido University, N12W6, Kita-Ku, Sapporo<br />
060-0812, Japan<br />
3<br />
Department of Microbiology Tumor and cell Biology (MTC), Box 280, Karolinska Institute, S-171<br />
77 Stockholm, Sweden<br />
The activity of Natural Killer (NK) cells is regulated by a fine-tuned balance between activating and<br />
inhibitory receptors. Dual-colour fluorescence cross-correlation spectroscopy (FCCS) was used to<br />
directly demonstrate a so-called cis-interaction between a member of the inhibitory NK cell<br />
receptor family Ly49 (Ly49A), and its ligand, the Major Histocompatibility Complex (MHC) class I,<br />
within the plasma membrane of the same cell. A refined FCCS model was used to calculate the<br />
displacement of the two excitation laser foci and the cross-talk, calibrated by positive and negative<br />
control experiments on cells from the same lymphoid cell line. Based on this refined model,<br />
concentrations and diffusion coefficients of free and interacting proteins could be determined on a<br />
collection of cells. Using the intrinsic inter-cellular variation of their expression levels <strong>for</strong> titration, it<br />
was found that the fraction of Ly49A receptors bound in cis increases with increasing amounts of<br />
MHC class I ligand present on the same cell. This increase shows a tendency to be even more<br />
abrupt than <strong>for</strong> a diffusion-limited bimolecular reaction in solution, which most likely reflects that<br />
interactions are facilitated by the two-dimensional confinement of the membrane. The MHC class I<br />
concentration dependence <strong>for</strong> the percentage of Ly49A receptors bound in cis was unpredicted by<br />
previous studies of this interaction, and it may indicate a distinct regulation mechanism of NK cell<br />
activity, given that the concentrations of MHC class I and Ly49A is within a critical concentration<br />
range also in native NK cells. The project now moves on to elucidate these factors in freshly<br />
isolated NK cells from mice.<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 59
OC3 Oral communication 3<br />
DNA fluorocode: a single molecule, optical map of DNA with<br />
nanometer resolution<br />
Robert K. Neely 1 , Jochem Deen 1 , Giedrė Urbanavičiūtė 2 , Saulius Klimašauskas 2 and Johan<br />
Hofkens 1<br />
1 Departement Chemie, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Heverlee,<br />
Belgium. 2 Institute of Biotechnology, V.A. Graiciuno 8, LT-02241 Vilnius, Lithuania<br />
We present a new method <strong>for</strong> single-molecule optical DNA profiling using an exceptionally dense,<br />
yet sequence-specific coverage of DNA with a fluorescent probe. The method employs a DNA<br />
methyltransferase enzyme to direct the DNA labeling, followed by molecular combing of the DNA<br />
onto a polymer-coated surface and subsequent sub-diffraction limit localization of the fluorophores.<br />
The result is a ‘DNA fluorocode’; a simple description of the DNA sequence, with a maximum<br />
achievable resolution of less than 20 bases, which can be read and analyzed like a barcode [1] .<br />
We demonstrate the generation of a fluorocode <strong>for</strong> bacterial genomic DNA molecules. The<br />
resulting optical map of the DNA sequence has application in the assembly of genomic DNA<br />
sequences and in the detection of genomic copy number variations; repeating stretches of<br />
genomic DNA that are difficult to detect using conventional sequencing methods and are<br />
implicated in a range of diseases [2] . We will present recent advances in our DNA labeling<br />
technology; a click-chemistry-based approach which enables highly efficient labeling of large<br />
genomic molecules. We also discuss new analytical approaches to the assembly of the superresolution<br />
optical maps and their application in genomic studies.<br />
The fluorocode method is low cost, technically simple and has the potential to enable DNA<br />
mapping in unprecedented detail at the single molecule level.<br />
References: [1] R. K. Neely et al, Chem Sci, 1 (2010), 453. [2] R. Redon et al. Nature, 444 (2006), 444.<br />
______________<br />
* Corresponding author: e-mail: robert.neely@chem.kuleuven.be<br />
60 — MAF 12, Strasbourg, France, September 11-14, 2011 —
OC4 Oral communication 4<br />
Super-resolution imaging and more with photoactivatable fluorescence<br />
Julia Gunzenhaeuser 1 , Dylan Burnette 2 , Jennifer Lippincott-Schwartz 2 & Suliana Manley 1<br />
1Laboratory<br />
of Experimental Biophysics, IPSB, Ecole Polytechnique Fédérale de Lausanne,<br />
Switzerland<br />
2<br />
National Institute of Child Health and Human Development, National Institutes of Health,<br />
Bethesda, Maryland 20892, USA.<br />
Photoactivatable fluorescence has enabled several imaging methods that provide in<strong>for</strong>mation-rich,<br />
quantitative data <strong>for</strong> dissecting biological structure and dynamics. These include fluorescence<br />
pulse-chase imaging as well as super-resolution imaging approaches like photoactivated<br />
localization microscopy (PALM) [1] and sptPALM [2]. Three vignettes demonstrate insights these<br />
tools provide into the cytoskeleton, viruses, and the endoplasmic reticulum.<br />
Using pulse-chase imaging approaches, we demonstrate that the lamellipodial actin network<br />
at the edge of cells evolves into the lamella behind it [3]. This occurs during edge retraction, when<br />
myosin II redistributes to the lamellipodial actin and condenses it into an actin arc.<br />
Imaging with minimal perturbations is crucial to gain meaningful insights into processes such<br />
as the assembly of the HIV-1 Gag protein into virus-like particles (VLPs). The impact of the<br />
fluorescent label on VLP assembly is controversial due to their size, which are too small to resolve<br />
using diffraction-limited imaging. We address this by studying the morphology of VLPs with PALM<br />
imaging.<br />
The endoplasmic reticulum is a complex structure composed of membrane sheets and<br />
tubules. We use a combination of 3D and live cell PALM to dissect the organization of reticulons,<br />
proteins responsible <strong>for</strong> shaping the ER.<br />
This work was supported by the National Research Council, the National Institutes of Health, and<br />
the European Research Council.<br />
References:<br />
[1] Betzig E, Patterson GH et al., "Imaging intracellular fluorescent proteins at nanometer resolution,"<br />
Science. 2006 Sep 15;313(5793)<br />
[2] S. Manley, J. M. Gillette et al., “High-density mapping of single-molecule trajectories with photoactivated<br />
localization microscopy,” Nature Methods 5(2):155-157 (2008)<br />
[3] D.T. Burnette, S. Manley et al., “A role <strong>for</strong> actin arcs in the leading-edge advance of migrating cells,”<br />
Nature Cell Biology 13(4):371-382 (2011)<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 61
OC5 Oral communication 5<br />
Applications of sub-diffraction fluorescence imaging in biological,<br />
botanical and materials sciences<br />
Trevor A. Smith*, Xiaotao Hao & Liisa M. Hirvonen<br />
Ultrafast and Microspectroscopy Laboratories, School of Chemistry, University of Melbourne,<br />
Victoria, Australia, 3010<br />
Fluorescence microscopy is an invaluable tool in biological imaging, allowing non-destructive<br />
imaging of specific components inside living cells and tissues, and other disciplines including the<br />
botanical and materials sciences. Un<strong>for</strong>tunately, due to diffraction phenomenon, the resolution of<br />
an optical microscope is fundamentally limited to about 200 nm in the lateral plane. Various<br />
methods of optical microscopy capable of providing spatial detail beyond the diffraction limit have<br />
evolved in recent years. The ability provided by these “super-resolution” methods to “see” the<br />
cellular structures and the processes going on within them is central to our understanding of living<br />
systems. These microscopy techniques are already providing views of cellular components that<br />
were beyond imagining only a few years ago. They also have largely untapped applications in<br />
others fields.<br />
Of the methods developed to date, each shows advantages and drawbacks <strong>for</strong> given sample<br />
types or acquiring desired in<strong>for</strong>mation, and development is continuing to optimise the different<br />
approaches to certain imaging scenarios. In this presentation we will report on the development of<br />
a number of techniques capable of providing sub-diffraction fluorescence imaging, and their<br />
application to the study of biological, polymeric and botanical samples.<br />
We have developed structured illumination microscopy (SIM), where a fine grating is<br />
projected onto the sample, and the final image is reconstructed from a set of images taken at<br />
different grating positions. With this technique, resolution improvement by a factor of two can be<br />
achieved compared to conventional wide-field imaging without the need <strong>for</strong> scanning or special<br />
properties of the fluorophores. Scanning near field optical microscopy (SNOM) and excited-state<br />
depletion methods have also been developed and applied to cellular imaging and polymeric films.<br />
The resolution achieved from these various optical imaging methods can be used to complement<br />
other emerging modes such as coherent diffractive X-ray imaging <strong>for</strong> appropriate samples.<br />
This work was supported by the Australian Research Council Centre of Excellence <strong>for</strong> Coherent X-<br />
Ray Science (CXS: www.coecxs.org) and the Cellular Nano-Imaging Consortium (CNIC).<br />
______________<br />
* Corresponding author: e-mail: trevoras@unimelb.edu.au<br />
62 — MAF 12, Strasbourg, France, September 11-14, 2011 —
OC6 Oral communication 6<br />
Polyethylene glycol-based multidentate oligomers <strong>for</strong> enhancing the<br />
biocompatiblity of semiconductor, gold and magnetic nanocrystals<br />
Goutam Palui, Hyon Bin Na & Hedi Mattoussi*<br />
Florida <strong>State</strong> University, Department of Chemistry and Biochemistry, Chemical Science<br />
Laboratories, Tallahassee, FL 32306<br />
Interest in developing inorganic nanocrystals as optical and magnetic plat<strong>for</strong>ms <strong>for</strong> biological<br />
applications has steadily grown in the past decade. These materials range from fluorescent<br />
semiconductor quantum dots (QDs), plasmonic Au nanoparticles (AuNPs) to magnetic<br />
nanocrystals (MnNPs), and they promise great advances in understanding a variety of biological<br />
processes. The development of effective and reproducible strategies <strong>for</strong> preparing aggregate-free<br />
(monodispersed) hydrophilic QDs, AuNPs and MnNPs that are stable and compatible with<br />
common biological coupling chemistries is highly sought. Growth of high quality QDs and MnNPs<br />
relies on high temperature reduction of organometallic precursors and usually provides<br />
nanocrystals that are hydrophobic. Conversely, AuNPs are often made via citrate-reduction but<br />
require subsequent surface-functionalization to make them biocompatible. Thus, post-synthetic<br />
surface modification must be applied to render these nanocrystals stable in aqueous media and<br />
biologically compatible.<br />
We have developed a set of multifunctional compact ligands each made of an oligomer<br />
coupled to several copies of a short poly(ethylene glycol) (PEG)-appended thioctic acid (TA).<br />
Reduction of the TAs provides dihydrolipoic acid (DHLA)-appended OligoPEG ligands. Here the<br />
PEG segments promote water solubility, while TA and DHLA groups provide multidentate<br />
anchoring onto Au and ZnS-overcoated semiconductor QDs, respectively. If the TA is replaced<br />
with dopamine groups the oligoPEG exhibits strong affinity to iron oxide nanocrystals. Dispersions<br />
of nanoparticles that exhibit remarkable colloidal stability over a broad range of pHs and in the<br />
presence of added electrolytes and reducing agents have been prepared. Moreover, introducing<br />
different functional groups into the oligomer structure opens the opportunity <strong>for</strong> effective orthogonal<br />
coupling of these plat<strong>for</strong>ms to target biomolecules such as proteins and peptides. We will discuss<br />
the ligand design, capping of the various nanocrystals, coupling of the NPs to target biomolecules,<br />
and use in few specific biological investigations such as sensor design and imaging.<br />
______________<br />
* Corresponding author: e-mail: mattoussi@chem.fsu.edu<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 63
OC7 Oral communication 7<br />
Fluorescent avidinated silica-Core/PEG-shell nanoparticles: brightness<br />
<strong>for</strong> labeling and imaging<br />
Enrico Rampazzo 1* , Sara Bonacchi 1 , Damiano Genovese 1 , Riccardo Juris 1 , Mara Mirasoli 2 ,<br />
Marco Montalti 1 , Luca Prodi 1 , Luisa Stella Dolci 2 & Nelsi Zaccheroni 1<br />
1<br />
Dipartimento di Chimica “G. Ciamician”, Università di Bologna, via Selmi 2, 40126 Bologna (Italy).<br />
2<br />
Dipartimento di Scienze Farmaceutiche, Università di Bologna, Via Belmeloro 6, 40126 Bologna<br />
(Italy)<br />
Dye Doped Silica Nanoparticles (DDSNs) are versatile self-organized plat<strong>for</strong>ms with many useful<br />
features, such as long term colloidal stability in water and intrinsic low toxicity. Multiple dyes are<br />
embedded in the inert silica matrix and properly organized in space and energy to yield extremely<br />
bright and color-tunable nanosystems.[1] The topological control [2] allows to confine the dyes in<br />
the small and rigid silica core and to exploit the properties of PEG as a biological carrier: it is<br />
membrane permeable, non-toxic, transparent to the immune system and can be functionalized <strong>for</strong><br />
targeting of specific biomolecules. Such nanomaterials are thus extremely advisable <strong>for</strong> biomedical<br />
imaging and labeling applications.<br />
modified<br />
Pluronic F127<br />
Pluronic F127<br />
TEOS<br />
HCl/H 2O<br />
DYE(S)<br />
PEG<br />
SHELL<br />
FUNCTIONAL<br />
GROUPS ON THE<br />
NPs SURFACE<br />
64 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
10 nm<br />
25 nm<br />
SILICA<br />
In this contribution we present a versatile one-pot synthetic method to prepare extremely stable<br />
Silica-core/PEG-shell DDSNs equipped with functional groups on the surface. This strategy is<br />
based on the use of direct micelles of a triblock copolymer surfactant as template,[3] as<br />
schematically shown in figure.<br />
We will show how the preparation of a library of functionalized surfactants gives access to a<br />
virtually infinite set of multifunctional nanoparticles, whose emission color, brightness, functional<br />
groups <strong>for</strong> selective targeting can be tailored <strong>for</strong> any particular application. The preparation of<br />
these monodisperse materials is reliable and reproducible, af<strong>for</strong>dable, well-standardized and fast.<br />
In this presentation, in particular, we will describe the synthesis and characterization of fluorescent<br />
DDSNs, provided with surface amino-groups, obtained through the introduction of a modified<br />
surfactant in a one-pot synthesis. Such 25 nm diameter Silica−core/PEG−shell DDSNs were then<br />
bio-conjugated to avidin and used as probes in sandwich type immuno-assays, to validate the<br />
present method as an easy and functional approach.<br />
References:<br />
[1] S. Bonacchi, et al., Angew. Chem. Int. Ed., 50 (2011) 4056.<br />
[2] E. Rampazzo, et al, J. Phys. Chem. B, 114 (2010) 14605.<br />
[3] S. Zanarini, et al., J. Am. Chem. Soc., 131 (2009) 14208.<br />
______________<br />
* Corresponding author: e-mail: enrico.rampazzo@unibo.it
OC8 Oral communication 8<br />
Fluorescence microspectroscopy: direct observation of<br />
polymer dynamics<br />
Albert M. Brouwer*, Tanzeela N. Raja & Joanna Sierkierzycka<br />
van ‘t Hoff Institute <strong>for</strong> Molecular Sciences, University of Amsterdam, Amsterdam, The Netherlands<br />
Fluorescence microspectroscopy is of obvious importance in biology and medical sciences, but is<br />
less popular in materials science. [1] In this work we will present two applications related to polymer<br />
dynamics. The first one demonstrates how single molecule spectroscopy can be used to map out<br />
the glass transition in polymer films, both spatially and temporally. Below Tg essentially all<br />
molecules are fluorescent. As the temperature is increased, more and more individual molecules<br />
appear to be switched off in wide field images (Fig. 1). Confocal observation of molecules one-byone,<br />
however, shows that even molecules that are mostly “off” at temperatures > Tg, occasionally<br />
are turned on again. The working principle of the method is based on excited state electron<br />
transfer. This type of process requires a thermodynamic driving <strong>for</strong>ce, but also enough free volume<br />
to occur on the short timescale of the excited state lifetime. [2] The free volume experienced by the<br />
individual probe molecules apparently fluctuates on a timescale of tens of seconds.<br />
(a)<br />
Fig. 1. (a) Structure of fluorescent probe molecule. (b) Number of fluorescing probe molecules detected in an<br />
area of 26 µm × 26 μm in a poly(vinylacetate) film (left axis) as function of temperature together with part of<br />
the DSC curve (right axis). [2]<br />
A second area of application is film <strong>for</strong>mation of latex emulsions. In this case, labelling of individual<br />
latex particles allows the direct observation of the spreading of polymer particles across the<br />
interparticle boundaries in the last stage of film <strong>for</strong>mation. [3] It is anticipated that this simple<br />
technique <strong>for</strong> the evaluation of the crucial step in film <strong>for</strong>mation can become a standard method <strong>for</strong><br />
the coatings industry.<br />
The work on latex film <strong>for</strong>mation is part of the Dutch Polymer Institute (project #606). The singlemolecule<br />
experiments were supported by NanoNed, a national nanotechnology program<br />
coordinated by the Dutch Ministry of Economic Affairs.<br />
References: [1] H. Morawetz, Science 240, (1988) 172; P. Bosch et al., Chem. Eur. J. 11, (2005)<br />
4314; T. N. Raja, et al., Photochem. Photobiol. Sci. 9, (2010) 975; D. Wöll, et al., Chem. Soc. Rev.<br />
38, (2009) 313. [2] J. R. Siekierzycka, et al., J. Am. Chem. Soc. 132, (2010) 1240. [3] J.L. Keddie,<br />
Mat. Sci. Eng. R, 21, (1997) 101; M.A. Winnik, J. Coat. Technol. Res. 74, (2002) 49.<br />
______________<br />
* Corresponding author: e-mail: a.m.brouwer@uva.nl<br />
(b)<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 65
OC9 Oral communication 9<br />
Organic dye nanoparticles with intense fluorescence caused by a<br />
combined effect of intermolecular H-aggregation and restricted<br />
intramolecular rotation<br />
Hiroshi Yao * & Koji Ashiba<br />
Graduate School of Material Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo<br />
678-1297 (Japan)<br />
Recently developed fluorescent organic nanoparticles (FONs) are expected to play roles in a wide<br />
variety of applications such as nano-sized organic light emitting diodes and biologics. Such FONs are<br />
generally prepared with a simple reprecipitation method, and several approaches have attracted<br />
significant attention. [1] Meanwhile, we have developed a new route <strong>for</strong> the synthesis of organic<br />
nanoparticles based on an ion-association technique that has advantages in simplicity and versatility. [2]<br />
This method utilizes the <strong>for</strong>mation of water-insoluble ion-pair aggregates in aqueous phases by<br />
association of a chromophoric ion with a hydrophobic counterion to fabricate organic<br />
nanoarchitectures. In this paper, we report the ion-based synthesis of highly fluorescent organic dye<br />
nanoparticles with various particle sizes. The observed intense fluorescence is found to originate from<br />
a combined effect of restricted “intramolecular” rotation and “intermolecular” H-aggregation of the<br />
organic dye molecules.<br />
3,3’-Diethylthiacyanine (TC, chemical structure is shown in Fig. 1) was selected as the cationic<br />
dye (fluorophore) because radiationless deactivation of this dye has been accounted <strong>for</strong> in terms of<br />
rotational relaxation to a funnel state on the singlet potential surface, followed by partitioning of this<br />
intermediate between the two possible ground state isomers. [3] Aqueous-phase ion association<br />
between TC cations and tetrakis(4-fluorophenyl)borate (TFPB) anions, in the presence of<br />
poly(vinylpyrrolidone), produced the ion-based dye nanoparticles ranging from 15 to 90 nm in diameter.<br />
Particle size could be controlled by changing the molar ratio (ρ) of the loaded anion to the dye cation.<br />
In the absorption spectra of nanoparticles, we could<br />
observe a prominent second peak centered at higher<br />
energy as compared to the monomer peak position. This<br />
peak can be attributed to an H-type aggregate of TC. [4]<br />
Interestingly, we found that large ρ values facilitated the<br />
<strong>for</strong>mation of H-type aggregates. In comparison with TC<br />
monomer in water, the nanoparticles showed a significant<br />
increase in their fluorescence. The fluorescence intensity<br />
also increased with a decrease in the nanoparticle size (or<br />
increase in ρ), resulting in a very high quantum yield of<br />
about 0.8–0.9 (see Fig. 1). The fluorescence<br />
enhancement can be attributed to the synergetic effect of<br />
restriction of intramolecular rotation and intermolecular Htype<br />
aggregation in an inclined manner. The simple and<br />
versatile synthesis techniques based on ion-association<br />
will provide improvement in the fluorescence properties in<br />
a new type of organic nanoparticles.<br />
This work was supported by Grant-in-Aids <strong>for</strong> <strong>Scientific</strong><br />
Research (C: 22510104) from Japan Society <strong>for</strong> the<br />
Promotion of Science (JSPS).<br />
References: [1] (a) H.-B. Fu, et al., J. Am. Chem. Soc. 123 (2001) 1434; (b) D. Horn, et al., Angew. Chem. Int.<br />
Ed. 40 (2001) 4330. [2] (a) H. Yao, et al., Chem. Lett. 34 (2005) 1108; (b) H. Yao, et al., Langmuir 25 (2009) 1131.<br />
[3] M. S. Thomas, et al., Langmuir 26 (2010) 9756. [4] (a) C. Peyratout, et al., Phys. Chem. Chem. Phys. 4 (2002)<br />
3032; (b) U. Rösch, et al., Angew. Chem. Int. Ed. 45 (2006) 7026.<br />
______________<br />
* Corresponding author: e-mail: yao@sci.u-hyogo.ac.jp<br />
66 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
Fig. 1. Fluorescence spectra of TC<br />
nanoparticle samples prepared at ρ = 1, 2,<br />
and 4, along with that of TC monomer in<br />
water. The inset shows a photo of<br />
fluorescence taken under UV light<br />
irradiation.
OC10 Oral communication 10<br />
Probing the radiative transition and determining the fluorescence<br />
quantum yield of a single molecule with a tunable microresonator<br />
Alfred J. Meixner 1 , Alexey I. Chizhik 1,2 , Anna M. Chizhik 1 , Dmitry Khoptyar 1 , Sebastian Bär 1 ,<br />
& Jörg Enderlein 2<br />
1Institute of Physical and Theoretical Chemistry, Eberhard Karls University, 72076 Tübingen (Germany).<br />
2 III. Institute of Physics & Biophysics, Georg August University, 37077 Göttingen (Germany).<br />
Small optical microresonators are structures which confine light to volumes with dimensions on the<br />
order of one wavelength and hence have become increasingly important <strong>for</strong> controlling light-matter<br />
interaction in integrated optics [1]. The ultimate limit of miniaturization <strong>for</strong> generating laser radiation<br />
is e.g. a single quantum system enclosed in an optical microresonator [2]. While it is well known<br />
from quantum electrodynamics nowadays that the spontaneous emission is not an intrinsic process<br />
of an atom but can be modified by tailoring the electromagnetic environment, the situation <strong>for</strong> a<br />
molecule is more complex since the radiation rate and relaxation path of a molecule depend on the<br />
balance between the radiative (the far-field) and non-radiative (the near-field) relaxation paths, the<br />
later being sensitively dependent on the interaction of the molecule with the local chemical<br />
environment. Hence, by controlling the electromagnetic environment of a single molecule we select<br />
the vibronic transition where fluorescence will mostly occur [3] and we can tune its radiative<br />
transition and determine its fluorescence quantum yield [4]. Using a tunable optical microresonator<br />
with subwavelength spacing, we demonstrate controlled modulation of the radiative transition rate<br />
of a single molecule, which is measured by monitoring its fluorescence lifetime. Variation of the<br />
cavity length changes the local mode structure of the electromagnetic field, which modifies the<br />
radiative coupling of an emitting molecule to that field. By comparing the experimental data with a<br />
theoretical model, we extract both the pure radiative transition rate as well as the quantum yield of<br />
individual molecules. We observe a broad scattering of quantum yield values from molecule to<br />
molecule, which reflects the strong variation of the local interaction of the observed molecules with<br />
their host environment.<br />
References: [1] Yokoyama, H.; Ujihara, K. “Spontaneous emission and laser oscillation in microcavities”<br />
1995, CRC Press, Taylor and Francis Group. [2] McKeever et al. Nature 2003, 425, 268. [3] Chizhik A. I. et<br />
al. , J. Phys. Rev. Lett. 2009, 102, No. 073002. [4] Chizhik, A. I et al. J. Nano Lett. 2011, DOI:<br />
10.1021/nl200215v.<br />
______________<br />
* Corresponding author: e-mail: alfred.meixner@uni-tuebingen.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 67
OC11 Oral communication 11<br />
FLI-Cam – a frequency-domain fluorescence lifetime imaging system<br />
based on a new directly modulatable CMOS image sensor<br />
Robert Franke & Gerhard Holst *<br />
PCO AG, Donaupark 11, 93309 Kelheim (Germany)<br />
For many years, the benefit of the luminescence lifetime as an analytical parameter has been<br />
described and many instruments including the 2D measuring setups with cameras have been<br />
developed and applied. However, since the instrumentation to per<strong>for</strong>m either time- or frequencydomain<br />
lifetime measurements is rather complex, new developments in the technology of CMOS<br />
image sensors generated image sensors, which can be efficiently used <strong>for</strong> this purpose. While<br />
originally designed <strong>for</strong> distance measurements, the principle <strong>for</strong> these measurements shows a<br />
clear analogy to frequency-domain FLIM measurements, which also have been proven by<br />
researchers [1,2] . Based on this principle, a new CMOS image sensor has been developed and is<br />
investigated in line with a research project.<br />
The image sensor has a resolution of 1024 x 1024 pixels with a 5.6 µm pitch and can be<br />
modulated up to 50 MHz. The first measurements show an effective dynamic range of 1:1000 (10<br />
bit). The frame rate will be in the range of 40 frames/s. The camera system, which incorporates the<br />
sensor, also generates all required modulation signals from 1 kHz to 50 MHz (sinusoidal and<br />
rectangular). It includes enough memory to store reference images and do image processing, such<br />
that the user can read out either raw data, to test and improve custom algorithms, or images, which<br />
represent lifetime distributions of the sample (at a later stage). The modulation frequency can be<br />
freely adjusted and frequency sweep operation will be possible with up to 16 selectable<br />
frequencies.<br />
First per<strong>for</strong>mance results are shown and discussed with the help of the phasor approach [3] ,<br />
that has been established to provide a more global view to pixel-wise fluorescence lifetime data<br />
and compare time- and frequency-domain results. Based on these results and the experiences of<br />
the on-going tests, it can be expected, that the FLI-Cam will ease the introduction of luminescence<br />
lifetime imaging systems to broader applications.<br />
This work was supported by a grant of the German Federal Ministry <strong>for</strong> Education and Research<br />
(BMBF) <strong>for</strong> the project “FLI-Cam” in the Biophotonik III program.<br />
References: [1] H. Heß, et al., Proceedings Opto 2002, P7 (2002). [2] A. Esposito, et al., Optics Express, 13<br />
(2005) 9812. [3] Michelle A. Digman, et al., Biophysical Journal: Biophysical Letters (2007) L14.<br />
______________<br />
* Corresponding author: e-mail: gerhard.holst@pco.de<br />
68 — MAF 12, Strasbourg, France, September 11-14, 2011 —
OC12 Oral communication 12<br />
Multiplexed time lapse imaging using homoFRET and time-resolved<br />
polarisation fluorescence microscopy<br />
Anca Margineanu 1* , Sean Warren 1,2 , Dominic Alibhai 1,2 , Romain Laine 1,2 , Ian Munro 1 , Yuriy<br />
Alexandrov 1 , Christopher Kimberley 3 , Clif<strong>for</strong>d Talbot 1 , James McGinty 1 , Gordon Kennedy 1 ,<br />
Alessandro Sardini 4 , Mark A.A. Neil 1 , Christopher Dunsby 1 , Matilda Katan 3 & Paul M. W.<br />
French 1<br />
1Photonics<br />
Group, Department of Physics, Imperial College London, Prince Consort Road, London<br />
SW7 2AZ, UK<br />
2<br />
Institute of Chemical Biology, Imperial College London, South Kensington Campus, London SW7 2AZ, UK<br />
3<br />
Chester Beatty Laboratories, Institute <strong>for</strong> Cancer Research, 237 Fulham Road, London, SW3 6JB, UK<br />
4<br />
Clinical Sciences Centre, MRC, Imperial College London, Hammersmith Campus, London W12 0NN, UK<br />
The aim of our study is to follow the temporal and spatial interactions of proteins within signaling<br />
networks in live cells. For this purpose, Förster resonant energy transfer (FRET) between different<br />
fluorophores (heteroFRET) is currently the most widely applied technique, either in spectrallyresolved<br />
ratio imaging or in fluorescence lifetime imaging (FLIM). However, detecting more than<br />
two FRET pairs simultaneously <strong>for</strong> multiplexed read out of different signaling pathways using ratio<br />
imaging becomes challenging when the fluorescence spectra of pairs of fluorescent proteins cover<br />
practically the whole visible spectrum. We are developing multiplexed FLIM FRET technologies<br />
that can reduce the impact of spectral cross-talk, requiring only the donor fluorescence to be<br />
measured and taking advantage of dim or dark acceptors [1] . Another approach is to exploit energy<br />
transfer between identical fluorophores (homoFRET), which has mainly been used to demonstrate<br />
lipid and protein clustering [2] and to study con<strong>for</strong>mational changes related to protein activation [3] . In<br />
contrast to heteroFRET, there is no expected change of the fluorescence lifetime <strong>for</strong><br />
monoexponential fluorophores and the read out of homoFRET is based on polarisation<br />
measurements. We show that it is possible to extend the applications of homoFRET to follow<br />
protein-protein interactions and to multiplex the polarisation read out <strong>for</strong> different fluorescent<br />
biosensors expressed in living cells.<br />
We have implemented this homoFRET approach using a laser scanning confocal<br />
microscope (Leica SP5) equipped with dual channel time-correlated single photon counting<br />
(Becker & Hickl TCSPC). Excitation is provided by a spectrally filtered supercontinuum from a<br />
microstructured optical fibre pumped by a femtosecond Ti:Sapphire laser (Newport MaiTai) and the<br />
fluorescence is passed through a polarising beamsplitter to two external hybrid detectors.<br />
To demonstrate the multiplexed read out of homoFRET we have cotransfected COS7 cells<br />
with an intermolecular FRET pair, K-Ras-eGFP and its binding partner Raf-eGFP, and with a<br />
modified version of the H-Ras Raichu biosensor [4] where both CFP and YFP have been replaced<br />
with mCherry. We will present analysis based on both steady-state and time-resolved anistropy<br />
measurements. We note that the complex decay profile of fluorophores such as CFP and mCherry<br />
can complicate the analysis and we are working toward identifying fluorescent proteins with the<br />
favorable spectral characteristics and monoexponential decay profiles in order to increase the<br />
possibilities <strong>for</strong> multiplexed FRET readouts. We also aim to utilise time-resolved polarisation<br />
imaging of homoFRET to provide more in<strong>for</strong>mation about the biological system, e.g. concerning<br />
the number of proteins per cluster [3] and changes in the anisotropy decay due to changes in FRET<br />
or binding to membranes.<br />
This work was supported by the UK Biotechnology and Biological Sciences Research Council, the<br />
Engineering and Physical Sciences Research Council and the Wellcome Trust.<br />
References: [1] D. Grant et al., Biophys J, 95 (2008) L69; S. Kumar, et al., ChemPhysChem, 12 (2011) 609.<br />
[2] A.N. Bader, et al., ChemPhysChem, 12 (2011) 475. [3] C. Thaler, et al., Proc. Nat. Acad. Sci. USA, 106<br />
(2009) 6369. [4] N. Mochizuchi, et al., Nature, 411 (2001) 1065.<br />
______________<br />
Corresponding author: e-mail: a.margineanu@imperial.ac.uk<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 69
OC13 Oral communication 13<br />
Optically trapped microsensors <strong>for</strong> microfluidic temperature<br />
measurement by fluorescence lifetime imaging microscopy<br />
Mathieu A. Bennet 1,2 , Patricia R. Richardson 1,2 , Jochen Arlt 2,3 , Aongus McCarthy 4 , Gerald S.<br />
Buller 4 & Anita C. Jones * 1,2<br />
1 EaStCHEM School of Chemistry, King’s Buildings, The University of Edinburgh, EH9 3JJ<br />
2 Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre (COSMIC), King’s<br />
Buildings, The University of Edinburgh, EH9 3JZ<br />
3 SUPA, School of Physics, King’s Buildings, University of Edinburgh, EH9 3JZ<br />
4 School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, EH14 4AS<br />
With the emergence of ever new applications in microfluidic technology, innovative methods to<br />
measure environmental and chemical properties within microchannels are required. Precise control<br />
of temperature is a key requirement in many microfluidic applications, <strong>for</strong> example, in biology <strong>for</strong><br />
polymerase chain reaction amplification of DNA, in medicine <strong>for</strong> drug delivery, and in chemistry <strong>for</strong><br />
control of reaction rates, determination of phase transition temperatures and activation energies.<br />
However, the measurement of temperature on such a small scale is challenging.<br />
Fluorescence lifetime imaging microscopy (FLIM) has been used successfully to generate high<br />
spatial resolution maps of temperature distribution in microfluidic devices. 1-3 Use of a customised<br />
polymer based temperature-sensitive fluorescent probe allowed the temperature in a microfluidic<br />
device to be measured with a resolution of less than 0.1ºC. 4 (Graham EM et al.) While FLIM offers<br />
high spatial resolution and accurate mapping of the temperature in devices, the technique has the<br />
disadvantage that the fluorescent dye pervades the whole measurement volume. Although this may<br />
not be problematic <strong>for</strong> measurements on prototype devices during<br />
design and development, it precludes application in functioning<br />
microfluidic systems where the presence of the dye would interfere<br />
with the function.<br />
We have used the combination of optical tweezers and FLIM,<br />
facilitated by the use of a custom-built multi-modal microscope, to<br />
develop a new, non-invasive approach to microfluidic temperature<br />
measurement, based on the well known temperature-dependence<br />
of the Rhodamine B fluorophore. Temperature-sensitive<br />
fluorescence lifetime microprobes have been fabricated by<br />
encapsulating Kiton Red (Sul<strong>for</strong>hodamine B) within microdroplets<br />
which can be held and manipulated in the microfluidic flow using<br />
optical tweezers. The microdroplet is a double bubble in which an<br />
aqueous droplet of the fluorescent dye is surrounded by an oil shell<br />
which serves both to contain the fluorophore and to provide the refractive index differential required<br />
<strong>for</strong> optical trapping of the droplet in an external aqueous medium. The existence of Kiton Red in free<br />
solution in the inner water phase of the droplet preserves the response of its fluorescence lifetime to<br />
temperature. (The temperature response is lost when the dye is bound to a solid bead.)<br />
Microdroplets with a diameter of 5 μm have been trapped and manipulated by optical tweezers<br />
within a microfluidic flow. FLIM imaging of an optically trapped microdroplet held at selected positions<br />
across a temperature gradient within a microfluidic device has enabled the non-invasive<br />
measurement of local temperature in a microchannel, with a temperature resolution of 1 o C and<br />
micron-scale spatial resolution.<br />
This work was supported by the Engineering and Physical Sciences Research Council and the<br />
National Physical Laboratory.<br />
References: [1] D. A. Mendels et al, Microfluid. Nanofluid. 5 (2008) 603. [2] R. K. P. Benninger et al, Anal.<br />
Chem. 78 (2008) 2272. [3] F. Gielen et al, Anal. Chem. 82 (2010) 7509. [4] E. M. Graham et al, Lab Chip 10<br />
(2010) 1267.<br />
______________<br />
*Corresponding author email: a.c.jones@ed.ac.uk<br />
70 — MAF 12, Strasbourg, France, September 11-14, 2011 —
OC14 Oral communication 14<br />
Studies on lipid order in cell membranes using fluorescence<br />
polarimetric microscopy<br />
Alla Kress 1 , Patrick Ferrand 1 , Xiao Wang 1 , Hubert Ranchon, Philippe Réfrégier 1 , Hervé<br />
Rigneault 1 , Sophie Brasselet 1 , Tomasz Trombik 2 , Hai-Tao He 2 & Didier Marguet 2<br />
1 Aix-Marseille Université, Institut Fresnel, Campus de Saint Jérôme, F-13013 Marseille, France<br />
2 Centre d'Immunologie de Marseille-Luminy, INSERM, UMR-S 631, F-13009 Marseille, France<br />
Biomolecular orientational organization of lipids and proteins in the plasma membrane is a crucial<br />
factor in biological processes where functions can be closely related to orientation and ordering<br />
mechanisms. The concept of transient nanosized phase separations in ordered and disordered<br />
domains, called “lipid rafts” is now widely accepted. Furthermore, the ordered domains are<br />
enriched in signalling proteins, which highlights the crucial impact of phase separation during the<br />
signalling processes. While this field has been so far largely addressed by studying the<br />
translational diffusion behaviour of membrane proteins and lipid analogs by <strong>Single</strong> Molecule<br />
Tracking or Fluorescence Correlation Spectroscopy, only little is known about the orientational<br />
behaviour of signalling proteins and lipids in the plasma membranes.<br />
One-photon polarized fluorescence microscopy provides a convenient and powerful tool to<br />
study orientational order in very general cell membranes shapes. The absorption and emission of<br />
light are strongly dependent on the orientation of the fluorescence dipole with respect to the<br />
polarization of the excitation and emission fields. We have developed a fully polarization-resolved<br />
fluorescence imaging technique that relies on the analysis on the fluorescence image that is<br />
recorded <strong>for</strong> several directions of exciting polarizations. With this technique, the angular distribution<br />
of an ensemble of dipoles present in the confocal volume can be monitored without a priori<br />
knowledge on its average orientation [1], [2].<br />
We apply this technique to the measurement of quantitative orientational distribution using<br />
the fluorescent reporter di-8-ANEPPQ. We observe that di-8-ANEPPQ orientational order in the<br />
plasma membrane is highly affected by actin depolymerisation (upon Latrunculin A or Cytochalasin<br />
D treatment, or hypotonic shock) and cholesterol depletion. This indicates a structural change in<br />
the membrane morphology and membrane viscosity.<br />
We will discuss the principle of the method, the determining parameters that allow to improve the<br />
measurement duration and accuracy, as well as the statistical data processing methodology. The<br />
power of the method will be illustrated on living cells, presenting the orientational distribution<br />
heterogeneities within one cell and between several cells be<strong>for</strong>e and after pharmacological treatments.<br />
Fig. 1: Orientational distribution of di-8-ANEPPQ in plasma membrane of a COS7 cell. a) Fluorescence<br />
image, b) cartographies of angular position ρ, c) width Ψ and d) the distribution of Ψ in a histogram.<br />
References: [1] A. Gasecka, et al. Biophys. J., 97 (2009) 2854; [2] A. Kress, et al. "Probing orientational<br />
behavior of MHC Class I protein and lipid probes in cell membranes by fluorescence polarization-resolved<br />
imaging", Biophys. J., in press.<br />
______________<br />
Corresponding author: e-mail: sophie.brasselet@fresnel.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 71
OC15 Oral communication 15<br />
Specific targeting of nanoparticles to proteins <strong>for</strong> visualization of single<br />
protein dynamics inside living cells<br />
Domenik Liße 1 , Verena Wilkens 2 , Changijang You 1 Karin Busch 2 & Jacob Piehler 1*<br />
1 Division of Biophysics, University of Osnabrück, Babarastrasse 11, Osnabrück (Germany)<br />
2 Mitochondrial Dynamics, University of Osnabrück, Babarastrasse 11, Osnabrück (Germany)<br />
Individual proteins labeled with highly photostable fluorescent nanoparticles (FNP) can be<br />
monitored over long time periods with high spatial and temporal resolution, yielding powerful<br />
in<strong>for</strong>mation on the spatiotemporal dynamics and localization of proteins within living cells. Selective<br />
targeting of nanoparticles (NP) to proteins inside living cells, however, remains highly challenging<br />
because well established methods such as biotin/streptavidin, antibodies or transition metal ions<br />
cannot be applied in the cytoplasm of live cells. Intracellular NP targeting is furthermore<br />
challenging, because blocking of non-specific binding and washing out of non-bound FNP is not<br />
possible. Based on a bioorthogonal enzymatic labeling system the HaloTag [1] , we have<br />
established a highly specific and efficient approach <strong>for</strong> NP targeting inside live cells [2] , to overcome<br />
these particular challenges. We attempted functionalization of FNPs with commercial HaloTagligand<br />
(HTL) through maleimide/thiol-chemistry. However, neither no significant specific binding<br />
was observed to immobilized HaloTag® nor upon microinjection into live cells expressing<br />
HaloTag® fusion proteins. For this reason, we explored the structure-activity relationship of<br />
different derivatives of the HTL in more detail by using real-time surface-sensitive detection by<br />
simultaneous reflectance interference (RIf) and total internal reflection fluorescence spectroscopy<br />
(TIRFS) detection. Based on these results, we developed a HTL <strong>for</strong> surface functionalization of<br />
nanoparticles based on click chemistry. A ~10-fold faster reaction of the improved clickHTL<br />
compared to HTL-thiol was observed in vitro. Intracellular targeting of clickHTL functionalized-FNP<br />
after microinjection into live cells expressing Lifeact-EGFP-HaloTag as model protein yielded very<br />
good co-localization of the FNP with the actin cytoskeleton. In order to employ this labeling<br />
approach <strong>for</strong> tracking molecules within live cells, we functionalized FNP with clickHTL at a very low<br />
degree of functionalization (average DOF: 0.7 clickHTL moieties/FNP). The number of binding<br />
sites per FNP was estimated after reaction with purified HaloTag® fused to EGFP by using<br />
analytical size exclusion chromatography. These clickHTL-FNPs were efficiently targeted to the<br />
translocase of the mitochondrial outer membrane (TOM) through the subunit TOM20 fused to the<br />
HaloTag. Trajectories obtained from individual FNP nicely followed the shape of the mitochondria.<br />
<strong>Single</strong> FNP could be readily localized with precision of < 10 nm <strong>for</strong> more than 1000 frames, without<br />
significant photo bleaching. In summary, we here present a generic method <strong>for</strong> specific and<br />
efficient targeting of nanoparticles to fusion proteins in the cytoplasm of living cells.<br />
This work is supported by the DFG, SFB 944.<br />
References : [1] G. V. Los, et al. ACS Chem Biol., 3, 2008, 73.; [2] D. Liße, V. Wilkens, et al., Angewandte<br />
Chemie International Edition, in press.<br />
______________<br />
* Corresponding author: e-mail: piehler@uos.de<br />
72 — MAF 12, Strasbourg, France, September 11-14, 2011 —
OC16 Oral communication 16<br />
A miniaturized, highly sensitive fluorescence detector and its routine<br />
application in point-of-need instrumentation<br />
Bernhard Gerstenecker 1* , Roman Gruler 1 , Michael Eberhard 1 & Klaus Haberstroh 1<br />
1 QIAGEN Lake Constance, Jacques-Schiesser-Strasse 3, 78333 Stockach (Germany)<br />
QIAGEN Lake Constance has developed a miniaturized detector based on a confocal optical<br />
system <strong>for</strong> the simultaneous measurement of two fluorescence dyes with individual<br />
excitation/emission wavelengths. In addition to complete firmware <strong>for</strong> data acquisition and data<br />
processing, a digital interface is also embedded in the detector system. Through the use of specific<br />
excitation and emission filters, the optical system can be easily configured to suit the customer’s<br />
needs. For fine tuning of the detector, the dynamic range can be adjusted <strong>for</strong> OEM applications.<br />
The miniaturized detector provides very high sensitivity: less than 10 pM fluorescein can be<br />
detected. Due to its low power consumption, its close proximity to the sample without needing a<br />
light guide, its low excitation energy, and its high analytical sensitivity, the fluorescence detector<br />
meets the requirements of portable point-of-need instruments. The fluorescence detector is used in<br />
a variety of point-of-need instruments. The ESEQuant Lateral Flow System is the most elaborate<br />
point-of-need system <strong>for</strong> reading and interpreting rapid tests. It offers high flexibility to rapid test<br />
manufacturers and users (1). Together with a comprehensive set of software tools, the ESEQuant<br />
Lateral Flow System provides a complete OEM solution to rapid test manufacturers. The<br />
ESEQuant Lateral Flow System is applied in a variety of application areas: point-of-care<br />
diagnostics in clinics and physicians’ offices as well as in home-care settings, environmental<br />
testing, food safety, brand security, and animal health tests. The ESEQuant Tube Scanner<br />
represents a new generation of compact and easy-to-use fluorescence readers <strong>for</strong> a wide<br />
spectrum of applications (2). Most recently, the ESEQuant Tube Scanner has been used as a<br />
cutting edge real-time instrument <strong>for</strong> isothermal nucleic acid amplification on an OEM basis. In<br />
addition, homogeneous immunoassays can be processed with the ESEQuant Tube Scanner.<br />
References:<br />
1. Faulstich, K., Gruler, R., Eberhard, M., Haberstroh, K. (2007) Developing rapid mobile POC systems, Part<br />
1: Devices and applications <strong>for</strong> lateral-flow immunodiagnostics. IVD Technology, 13(6), 47.<br />
2. Faulstich, K., Gruler, R., Eberhard, M., Haberstroh, K. (2007) Developing rapid mobile POC systems, Part<br />
2: Nucleic acid-based testing plat<strong>for</strong>ms. IVD Technology, 13(7), 49.<br />
______________<br />
* Corresponding author: e-mail: bernhard.gerstenecker@qiagen.com<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 73
OC17 Oral communication 17<br />
New fluorescent nucleosides <strong>for</strong> exploring nucleic acid damage<br />
and recognition<br />
Yitzhak Tor*<br />
Department of Chemistry and Biochemistry, University of Cali<strong>for</strong>nia, San Diego, 9500 Gilman<br />
Drive, La Jolla, CA, 92093-0358 (USA)<br />
Nucleic acids experience a variety of perturbations while per<strong>for</strong>ming their essential everyday<br />
functions. These may include strand cleavage and ligation, local con<strong>for</strong>mational changes, base<br />
damage and flipping, as well as structural and environmental perturbations that are induced upon<br />
protein and low MW ligand binding. Fluorescent nucleoside analogues that are sensitive to their<br />
local environment are powerful tools <strong>for</strong> investigating nucleic acids structure, dynamics, recognition<br />
and damage. Our criteria <strong>for</strong> ideal fluorescent nucleoside analogues include: (a) high structural<br />
similarity to the native nucleobases to faithfully mimic their size and shape, as well as hybridization<br />
and recognition properties; (b) red shifted absorption bands to minimize overlap with the absorption<br />
of the natural bases; (c) red shifted emission bands (preferably in the visible); (d) reasonable<br />
emission quantum efficiencies; and, importantly, (e) sensitivity of their photophysical parameters to<br />
changes in the microenvironment. To maintain the H-bonding face of the natural nucleosides and<br />
to best preserve the overall dimensions of their Watson–Crick base pairs, two primary scaffolds<br />
were explored <strong>for</strong> emissive pyrimidines. Motif I relies on conjugating five-membered aromatic<br />
heterocycles to position 5 in the pyrimidines, and motif II is based on substituted quinazoline-<br />
2,4(1H,3H)-diones:<br />
Y<br />
Mot if I<br />
R<br />
X<br />
N<br />
U*<br />
O<br />
N H<br />
O<br />
H<br />
H<br />
N<br />
N<br />
74 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
N<br />
A<br />
N<br />
N<br />
R<br />
Y<br />
Mot if II<br />
Both motifs were found to be emissive, although the emission wavelength, quantum yield and<br />
responsiveness vary greatly. [1] Such fluorescent pyrimidine analogues have been utilized in assays<br />
exploring A-site–antibiotics binding. [2,3] Additional analogues have found utility in detecting the<br />
activity of ribosome inactivating proteins, such as ricin. [4] Recent developments include the<br />
discovery of fluorescent and minimally perturbing ribonucleosides that can be used as FRET<br />
partners with other synthetic fluorophores, or with tryptophan residues in RNA-binding proteins.<br />
The lecture will present the design and synthesis of new emissive nucleosides and their use <strong>for</strong> the<br />
fabrication of "real-time" fluorescence-based discovery and biophysical assays.<br />
R<br />
N<br />
O<br />
N H<br />
This work was supported by grants from the National Institutes of Health.<br />
References: [1] R. W. Sinkeldam, et al., Chem. Rev. 110 (2010) 2579. [2] S. G. Srivatsan, Y. Tor, J. Am.<br />
Chem. Soc. 129 (2007) 2044. [3] Y. Xie, et al. J. Am. Chem. Soc. 131 (2009) 17605. [4] S. G. Srivatsan et<br />
al., Angew. Chem. Int. Ed. 47 (2008) 6661.<br />
______________<br />
* Corresponding author: e-mail: ytor@ucsd.edu<br />
X<br />
U*<br />
O<br />
H<br />
H<br />
N<br />
N<br />
N<br />
A<br />
N<br />
N<br />
R
OC18 Oral communication 18<br />
Ligand inducible fluorescence: tools <strong>for</strong> ligand discovery and<br />
PCR monitoring<br />
Fumie Takei, Masaki Hagihara, Shiori Umemoto, Jinhua Zhan, Takeo Fukuzumi, Asako<br />
Murata, Yasue Harada & Kazuhiko Nakatani*<br />
Regulatory Bioorganic Chemistry, The Institute of <strong>Scientific</strong> and Industrial Research (SANKEN),<br />
Osaka University, 8-1 Mihogaoka, 567-0047, Japan<br />
We have studied small organic molecules binding to characteristic nucleic acid structures, and<br />
succeeded in the finding of the first molecule binding to a single nucleotide bulge and a<br />
mismatched base pair in double stranded DNA [1] . As well studied, fluorescence of molecules could<br />
be dramatically modulated upon binding to nucleic acids. The characteristic fluorescence could be<br />
used in indicating the presence of nucleic acids. We recently reported fluorescent-indicator<br />
displacement assay <strong>for</strong> discovering small molecules binding to RNA [2] .<br />
One of the molecule, 2,7-diamino-1,8-naphthyridine, that selectively binds to the cytosine bulge<br />
in dsDNA emits characteristic fluorescence upon binding. We have used this ligand inducible<br />
fluorescence to monitor the progress of polymerase chain reaction (PCR). PCR is one of the most<br />
fundamental technologies in current biology. We here describe the chemistry concept of non-covalent<br />
fluorescent DNA labelling by ligand binding to the secondary structure, which allows one to monitor<br />
PCR progress by measuring the change in fluorescence emitted from ligand–primer complexes in a<br />
homogeneous solution [3] . This concept is not only complementary to that using fluorescent dye bound<br />
selectively to PCR product duplexes, but also expands the possibility of real-time PCR.<br />
The concept of DNA labeling by secondary structure-inducible ligand fluorescence is shown in<br />
Figure 1. PCR primers are labeled with a hairpin tag containing cytosine bulges (C-bulges). The<br />
molecule 2,7-diamino-1,8-naphthyridine derivative (DANP) binds to a C-bulge as a protonated <strong>for</strong>m<br />
(DANPH + ) and emits fluorescence at 430 nm with a<br />
hairpin primer<br />
fluoresce!<br />
C<br />
G<br />
C<br />
C 5'<br />
C<br />
C<br />
3'<br />
5'<br />
3'<br />
N<br />
H<br />
N N<br />
DANP<br />
DANP<br />
Taq polymerase<br />
3'<br />
N<br />
H<br />
30 nm bathochromic shift from the fluorescence of<br />
free unbound DANP. We hypothesized that, as the<br />
PCR progresses, the hairpin tag will dissolve and be<br />
trans<strong>for</strong>med into a duplex, resulting in the loss of the<br />
DANP-binding site and a decrease in the<br />
fluorescence at 430 nm. Toward this end, we<br />
investigated the hairpin tags, which should identify the<br />
DANP-binding site without disturbing the fluorescence<br />
efficiency, should not interfere with the PCR, should<br />
be trans<strong>for</strong>med effectively into the duplex during<br />
PCR, and should be applicable to broad primers.<br />
The changes in DANP fluorescence from<br />
be<strong>for</strong>e to after PCR with primers labeled with the Cbulge<br />
hairpin tag were well correlated to the PCR<br />
progress, and can report the allele type when<br />
combined with an allele-specific PCR. The concept<br />
regarding the secondary structure-inducible ligand<br />
fluorescence described here would expand the<br />
chemistry of molecules binding to the specific DNA<br />
structure and emitting characteristic fluorescence.<br />
This work was supported by Grants-in-Aid <strong>for</strong> <strong>Scientific</strong> Research (S) <strong>for</strong> KN (18105006) from the<br />
Ministry of Education, Sports, Culture, Science and Technology, Japan.<br />
References: [1] For a review, see: Nakatani, K. Bull. Chem. Soc. Jpn. 2009, 82, 1055-1069. [2] Zhang, J. et<br />
al. J. Am. Chem. Soc. 2010, 132, 3660-3661. [3] Takei, F. et al. Angew. Chem. Int. Ed. 2009, 48, 7822-7824.<br />
______________<br />
* Corresponding author: e-mail: nakatani@sanken.osaka-u.ac.jp<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 75
OC19 Oral communication 19<br />
Detecting early stages of neurodegenerative disease by intrinsic<br />
fluorescence – β-amyloid aggregation in Alzheimer’s disease<br />
Mariana Amaro 1 , Karina Kubiak-Ossowska 2 , David J.S. Birch 1 & Olaf J. Rolinski 1 *<br />
1Photophysics<br />
group, Centre <strong>for</strong> Molecular Nanometrology, Department of Physics, Scottish<br />
Universities Physics Alliance, University of Strathclyde, 107 Rottenrow, Glasgow G4 0NG, UK<br />
2<br />
Department of Chemical and Process Engineering, University of Strathclyde, 75 Montrose Street,<br />
Glasgow G1 1XJ, UK<br />
A newly developed method <strong>for</strong> monitoring the aggregation of beta-amyloid (Aβ) peptides by using<br />
their intrinsic fluorescence will be presented. We have brought our research from a level of the<br />
hypothesis, that the fluorescence of the Aβ’s intrinsic fluorophore tyrosine (Tyr) is influenced by<br />
amyloid aggregation, to a fluorescence lab-based method <strong>for</strong> monitoring very early stages of<br />
aggregation, which have not been detected by any other methods.<br />
Developing sensitive and non-invasive methodology <strong>for</strong> monitoring early protein aggregation<br />
would make a big impact in molecular medicine and drug discovery, as aggregation and deposition<br />
of Aβ peptides onto neuronal cells has been demonstrated to play a central role in Alzheimer’s<br />
disease (AD). AD is the most common of the neurodegenerative diseases and is known to afflict<br />
5% men and 6% women over 60 years of age and currently there is no cure..If we start by<br />
considering AD as a paradigm of protein aggregation disease, then the scale of the problem rapidly<br />
becomes evident. Other protein that come to mind include τ-protein (Alzheimer’s), α-synuclein<br />
(Parkinson’s) and hungtingtin (Huntington’s).<br />
Here we present our recent studies [1,2] on mimicking physiological conditions leading to<br />
<strong>for</strong>mation of Aβ aggregates in vitro and monitoring the oligomerisation of single peptides by<br />
measuring the fluorescence decay time distribution of Tyr. Using fluorescence decay time methods<br />
enables monitoring interactions of individual biomolecules and, contrary to previously used<br />
techniques, reveals crucial in<strong>for</strong>mation on the early stages of protein aggregation and<br />
destabilisation. We will demonstrate that the complex fluorescence decay of Tyr in Aβ can be<br />
represented by the four-band decay time distribution gN(τ) [3] (Fig.1),<br />
a<br />
g N (τ)<br />
0.12<br />
0.08<br />
0.04<br />
0.00<br />
Aβ 40<br />
3<br />
0.27h<br />
2.13h<br />
32.33h<br />
125.12h<br />
1 10<br />
τ /ns<br />
76 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
Fig.1. Monitoring Aβ aggregation by<br />
detecting Tyr fluorescence decay<br />
time response. Shown are the Tyr<br />
decay time distributions gN(τ)<br />
measured <strong>for</strong> the 50 μM Aβ1-40<br />
solution in HEPES buffer of pH7.3 at<br />
37 °C, after ~0.3, 2, 32 and 125<br />
hours of aggregation.<br />
and will show how this distribution evolves during the aggregation, giving the opportunity <strong>for</strong> finding<br />
the relationship between the <strong>for</strong>mation of the Aβ oligomers and the observed Tyr photophysics.<br />
The full analysis of the excited-state kinetics, the output of the molecular dynamics simulations of<br />
the Aβ1-40 and Aβ1-42 amyloids aggregation and the resulting model of the Aβ oligomerisation, will<br />
be also presented.<br />
This work was supported by a grant from the EPSRC.<br />
2<br />
1<br />
References: [1] O.J.Rolinski, et al., Biosens.Bioelectron., 25 (2010) 2249. [2] M.Amaro, et al., Phys.Chem.<br />
Chem.Phys., 13 (2011) 6434. [3] O.J.Rolinski, et al., Phys.Rev.E, 79(5) (2009) Art No. 050901.<br />
______________<br />
* Corresponding author: e-mail: o.j.rolinski@strath.ac.uk<br />
4
OC20 Oral communication 20<br />
Kinase on-off switching in the functional, membrane-associated<br />
chemosensory signaling array of E. coli – Structural changes detected<br />
by OS-FRET<br />
Annette H. Erbse, Adam J. Berlinberg & Joseph J. Falke*<br />
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Campus Box 215,<br />
Boulder, CO 80309, USA<br />
All motile bacteria possess a chemosensory signaling array, <strong>for</strong>med by a hexagonal lattice of<br />
transmembrane receptors in the cell membrane and by kinases and coupling proteins stably<br />
docked to the receptor cytoplasmic domains. Chemical attractants and repellents bind to the<br />
receptors and generate transmembrane signals that switch the associated kinase components on<br />
and off. The lattice architecture enhances ultrasensitivity and ultrastability by allowing cooperative<br />
interactions between multiple components in the same lattice region. The chemosensory array of<br />
E. coli is best studied and has become the standard model system <strong>for</strong> bacterial 2-component<br />
signaling pathways – the predominant type of signaling pathway in bacteria and an important target<br />
<strong>for</strong> revolutionary, new generation broad spectrum antibiotics. The present study focuses on the<br />
signaling array <strong>for</strong>med by the transmembrane serine receptor, the histidine kinase CheA, and the<br />
coupling protein CheW. Progress has been made in understanding the molecular mechanism of<br />
receptor transmembrane signaling, but the mechanism of receptor-mediated CheA kinase on-off<br />
switching remains an enigma.<br />
To probe structure and con<strong>for</strong>mational changes in this and other systems, we have<br />
developed a novel One-Sample FRET (OS-FRET) method [1]. The method employs a new,<br />
reversibly coupled non-fluorescent acceptor, Non-Fluorescent Quencher 1 (NFQ1) coupled via a<br />
disulfide bond. When donor fluorescein is coupled irreversibly to the CheA kinase and NFQ1 is<br />
coupled to CheW in the functional, membrane-associated signalling array, the quenched donor<br />
fluorescence is measured, then NFQ1 is released into solution by reduction, enabling<br />
measurement of the unquenched donor fluorescence. The ability to make all necessary<br />
measurements in just one sample improves the accuracy and precision of FRET, while decreasing<br />
sample and time requirements.<br />
In the present study, application of OS-FRET to the functional, membrane-associated<br />
chemosensory signalling array has, <strong>for</strong> the first time, enabled detection and analysis of the<br />
structural changes in CheA kinase triggered by receptor signals during kinase on-off switching. The<br />
results reveal a large structural rearrangement of the interface between the P5 regulatory domain<br />
of CheA and the CheW coupling protein. Moreover, the FRET efficiencies observed in the<br />
membrane-bound signalling array shed new light on the architecture of the bacterial signalling<br />
array. Overall, these initial findings indicate that OS-FRET will be useful in analyzing the structure,<br />
structural changes, and dynamics of the bacterial chemosensory signalling lattice, and likely other<br />
important biological systems as well.<br />
This work was supported by NIH R01 GM-040731.<br />
References: [1] Annette H. Erbse, Adam J. Berlinberg, Ching-Ying Cheung, Wai-Yee Leung, and Joseph J.<br />
Falke, OS-FRET: A new one-sample method <strong>for</strong> improved FRET measurements Biochemistry 50 (2011)<br />
451-7.<br />
______________<br />
*Corresponding author e-mail: joseph.falke@colorado.edu<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 77
OC21 Oral communication 21<br />
Multiscale dynamics of single molecules in biomimetic crowding<br />
Robb Welty 1 , Jacob Bentley 1 , Dhanushka Wickramasinghe 1 & Ahmed Heikal 1,2 *<br />
1 The Department of Chemistry and Biochemistry, Swenson College of Science and Engineering,<br />
2 The Department of Pharmacy Practice and Pharmaceutical Sciences, College of Pharmacy, The<br />
University of Minnesota Duluth, 246 Chem, 1039 University Drive, Duluth, MN 55812 (USA)<br />
Molecular crowding in living cells influences diffusion and intracellular transport, which regulates a<br />
number of cellular processes such as signal transduction, macromolecular assembly, and<br />
intermolecular interactions. In this contribution, we investigate the size effect of both the crowding<br />
agents as well as solute molecules on multiscale molecular process to elucidate the role of nonspecific<br />
binding in a crowded environment. Ficoll-70, bovine serum albumin (BSA), and ovalbumin<br />
were used as agents <strong>for</strong> biomimetic crowding and compared with glycerol as a control. The<br />
rotational (ps - ns) and translational (μs-s) diffusion of rhodamine green and EGFP were<br />
investigated as a function of crowding using time-resolved fluorescence anisotropy and<br />
fluorescence correlation spectroscopy. Our single-molecule and ensemble studies suggest that<br />
molecular crowding effects on diffusion depend on the size of crowding agent, the hydrodynamic<br />
volume of the solute, and the temporal resolution of the technique used. The results also highlight<br />
the importance of spatial confinement and non-specific binding in regulating molecule-molecule<br />
interactions in a crowded environment.<br />
This work was supported in part by grants from NSF, GIA-UMN, and NIH.<br />
______________<br />
*Corresponding author: e-mail: aaheikal@d.umn.edu<br />
78 — MAF 12, Strasbourg, France, September 11-14, 2011 —
OC22 Oral communication 22<br />
Inverse-Fluorescence Correlation Spectroscopy: label-free analysis of<br />
the absolute volume of biomolecules in solution<br />
Tor Sandén 1 , Romain Wyss 1 , Christian Santschi 2 , Olivier J.F. Martin 2 , Stefan Wennmalm 3,*<br />
& Horst Vogel 1<br />
1<br />
Laboratory of Physical Chemistry of Polymers and Membranes, Swiss Federal Institute of<br />
Technology, 1015 Lausanne, Switzerland<br />
2<br />
Nanophotonics and Metrology Laboratory, Swiss Federal Institute of Technology, 1015<br />
Lausanne, Switzerland<br />
3<br />
Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of<br />
Technology, Stockholm, Sweden<br />
Several methods can estimate the relative size of biomolecules in solution, however, they all<br />
estimate the size indirectly via the biomolecules’ diffusion coefficient. In contrast, inverse-<br />
Fluorescence Correlation Spectroscopy, iFCS, measures the volume of individual biomolecules<br />
directly, from the amount of solution they displace, in line with Archimedes’ principle (Wennmalm S<br />
et al., 2009; Wennmalm S,Widengren J, 2010; Wennmalm S,Widengren J, 2011). It is the first<br />
method that accurately can determine the absolute volume of biomolecules in solution, without any<br />
assumptions about molecular shape. As a result of this ability, iFCS can also detect significantly<br />
smaller size-changes compared to methods that rely on estimating the diffusion coefficient.<br />
A separate property of iFCS is that unlabeled and labeled biomolecules can be measured<br />
simultaneously. This allows <strong>for</strong> example direct measurement of the fraction of biomolecules that<br />
carry a labeled ligand, measuring the success of post-translational labeling of proteins, determining<br />
the exact fraction of virus particles that carry an antibody, etc.<br />
Here, the ability to accurately predict the volume in solution of a well known protein,<br />
Allophycocyanin, is demonstrated.<br />
References:<br />
(1) Wennmalm, S.; Thyberg, P.; Xu, L.; Widengren, J. Anal Chem 2009, 81, 9209.<br />
(2) Wennmalm, S.; Widengren, J. Anal Chem 2010, 82, 5646.<br />
(3) Wennmalm, S.; Widengren, J. Frontiers in Bioscience 2011, S3, 385.<br />
______________<br />
*Corresponding author: e-mail: stewen@kth.se<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 79
OC23 Oral communication 23<br />
Spot variable Fluorescence Correlation Spectroscopy reveals fast<br />
scouting of K-Ras at the plasma membrane of living cells<br />
Tomasz Trombik 1 , Verena Ruprecht 2 , Sébastien Mailfert 1 , Cyrille Billaudeau 1 , Fabien<br />
Conchonaud 1 , Gerhard J. Schütz 2 , Yoav Henis 3 , Stefan Wieser 1,2 & Didier Marguet 1 *<br />
1<br />
Centre d’Immunologie de Marseille-Luminy, CNRS - INSERM - Université de la Méditerranée,<br />
Marseille, France<br />
2<br />
Biophysics Institute, Johannes-Kepler-University Linz, Austria<br />
3<br />
Department of Neurobiology, George S Wise Faculty of Life Sciences, Tel Aviv University, Israel<br />
Among the membrane associated proteins, the Ras family, which is lipid-anchored G protein, plays<br />
a key role in a large range of physiological processes and, more importantly, is deregulated in a<br />
large variety of cancer. In this context, plasma membrane heterogeneity appears as a central<br />
concept since it ultimately tunes the specification and regulation of Ras-dependent signaling<br />
processes. There<strong>for</strong>e, to investigate the dynamic and complex membrane lateral organization in<br />
living cells, we have developed an original approach based on molecule diffusion measurements<br />
per<strong>for</strong>med by fluorescence correlation spectroscopy at different spatial scales (spot variable FCS,<br />
svFCS) [1] . We have shown in a variety of cell types that lipid-based nanodomains are instrumental<br />
<strong>for</strong> cell membrane compartmentalization. We have also observed that these nanodomains are<br />
critically involved in the activation of signaling pathways and are essential <strong>for</strong> physiological<br />
responses [2-3] . More recently, we extend the application of svFCS to characterize the dynamics of<br />
K-Ras protein at the plasma membrane. As major result, we demonstrated that the rate of K-Ras<br />
association/dissociation from the membrane is fast but vary as a functional of the activation state of<br />
the molecule as well as of specific intracellular protein interactions.<br />
This work is supported by institutional grants from the CNRS and INSERM and by specific grants<br />
from Région PACA, INCA, ANR (ANR-08-PCVI-0034-02, ANR 2010 BLAN 1214 01) & FRM.<br />
References: [1] He HT & Marguet D Annu Rev Phys Chem 62 (2011) 417. [2] Lasserre R et al Nat Chem<br />
Biol 4 (2008) 538. [3] Guia S et al Science Signaling 4 (2011) ra21.<br />
______________<br />
* Corresponding author: e-mail: marguet@ciml.univ-mrs.fr<br />
80 — MAF 12, Strasbourg, France, September 11-14, 2011 —
OC24 Oral communication 24<br />
Mechanical interaction between photons and<br />
macromolecules/nanoparticles as evaluated by Fluorescence Correlation<br />
Spectroscopy<br />
Syoji Ito 1,2,* , Hiroaki Yamauchi 1 & Hiroshi Miyasaka 1*<br />
1Division of Frontier Materials Science, Graduate School of Engineering Science and Center <strong>for</strong><br />
Quantum Science and Technology under Extreme Conditions, Osaka University, 1-3<br />
Machikaneyama-cho, Toyonaka, Osaka 560-8531 (Japan)<br />
2 PRESTO, Japan Science and Technology Agency<br />
The radiation pressure of laser beams offers microscopic manipulation methods that can control the<br />
motion and the position of small objects ranging from atoms in a free space to 100-micrometer-sized<br />
particles in fluid medium. The optical manipulation in the solution, so called optical tweezer, has been<br />
applied to various processes as important methods, such as the sorting of particles, fabrication of<br />
colloidal structures, analysis of interparticle <strong>for</strong>ces, manipulation of DNA con<strong>for</strong>mation, growth control of<br />
neurons, and manipulation of living cells. Recently, the targets of the micromanipulation method in<br />
solution have been downsizing to nanometer scale systems; the trapping and patterning of<br />
nanoparticles [1] and macromolecules [2] have been demonstrated.<br />
An estimation based on the Rayleigh approximation predicts that trapping potential acting on<br />
particles with ca. 10-20 nm diameters is comparable to the averaged thermal energy, kT, of particles in<br />
solution at room temperature. The trapping kinetics of such small objects < 20 nm, however, have not<br />
been well understood because of the following reasons; 1) it is difficult in general to detect nanometersized<br />
particles at the optical trapping potential in solution at single particle/molecule level, and 2) the<br />
con<strong>for</strong>mation of macromolecules varies depending on their<br />
environmental conditions, leading to different effective volumes<br />
under different conditions. In the present study, we have<br />
combined fluorescence correlation spectroscopy (FCS) and<br />
Brownian dynamics simulation (BDS) in order to quantitatively<br />
evaluate the radiation pressure acting on macromolecules.<br />
The number density of single-stranded DNA (ss-DNA)<br />
with a fluorescent dye at the trapping point of optical tweezer<br />
with near infrared (NIR, at 1064 nm) trapping light source was<br />
determined by FCS. As shown in Fig.1, the number of the DNA<br />
increased with an increase in the NIR laser power. To<br />
quantitatively evaluate this trapping effect, we have simulated<br />
the translational motion of the DNA in the optical trapping<br />
potential and the detection of fluorescent photons from the dye<br />
attached to the DNA. [3] A series of calculations taking into<br />
account the effect of localized heating due to the solvent<br />
absorption of the NIR light revealed that, in relatively shallow<br />
optical <strong>for</strong>ce potential up to 1.0 kTR (k is the Boltzmann constant<br />
and TR is a room temperature), the conventional theoretical model<br />
without strict consideration of the optical gradient <strong>for</strong>ce could<br />
Fig. 1. The average residence time<br />
and the number density of a<br />
macromolecule in the presence of<br />
optical trapping potential up to<br />
1.0kTR.<br />
evaluate the experimental results. On the other hand, there was large deviation between the simulated<br />
fluorescence correlation curve and the theoretical model under the potential depth > 1.0kTR. It was also<br />
deduced that the temperature elevation does not seriously affect the average number of molecules in the<br />
confocal volume, while the average residence time is more sensitively affected by temperature change.<br />
The present approach also succeeded in experimentally estimating the polarizability of the ss-DNA.<br />
References: [1] S. Ito, et al., Appl. Phys. Lett. 78, 2566-2568 (2001); S. Ito, et al., Appl. Phys. Lett. 80, 482-<br />
484 (2002). [2] J. Hofkens, et al., J. Am. Chem. Soc. 119, 2741-2742 (1997); S. Masuo et al., J. Phys. Chem.<br />
B, 109, 6917-6921 (2005). [3] S. Ito, N. Toitani, H. Yamauchi, and H. Miyasaka, Phys. Rev. E 81, 061402<br />
(2010).<br />
______________<br />
* Corresponding authors: e-mails: sito@chem.es.osaka-u.ac.jp; miyasaka@chem.es.osaka-u.ac.jp<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 81
OC25 Oral communication 25<br />
Excited-state intermolecular proton transfer of the firefly’s<br />
chromophore d-luciferin<br />
Dan Huppert 1 *, Itay Presiado 1 , Yuval Erez 1 & Rinat Gepshtein 1<br />
1Raymond<br />
and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv<br />
University, Tel Aviv 69978, Israel<br />
D-Luciferin is the chromophore found in Lampyridae, a family of winged insects. D-Luciferin is<br />
responsible <strong>for</strong> the bioluminescence that gives these lampyridae their more familiar name, fireflies.<br />
Fireflies are known to make crepuscular use of bioluminescence, believed to play a role in sexual<br />
attraction between the adults. The light is produced by organs located on the underside of the<br />
abdomen.<br />
For many years intermolecular excited-state-proton-transfer (ESPT) to a solvent or to a base in a<br />
liquid solution, and more recently in ice, has been widely researched. In the past decades we<br />
extensively studied the reversible and irreversible photo-protolytic cycle of a photoacid. We used a<br />
proton transfer model that accounts <strong>for</strong> the diffusion assisted geminate recombination of the transferred<br />
proton with the deprotonated <strong>for</strong>m of the photoacid.<br />
Steady-state absorption and emission as well as time-resolved emission spectroscopies were<br />
employed to study the photophysics and photochemistry of d-luciferin. 1 In aqueous solution the<br />
electronically excited-state protonated d-luciferin compound undergoes an efficient process of proton<br />
10 -1<br />
transfer to the solvent, with a rate constant, k PT = 3.0× 10 s . We found a kinetic isotope effect of<br />
about 2.5 <strong>for</strong> this process. The deprotonated <strong>for</strong>m of d-luciferin in the excited-state recombines<br />
irreversibly with the geminate proton. Hence, the fluorescence decay of the deprotonated <strong>for</strong>m is<br />
nonexponential and the fluorescence quantum yield is low.<br />
Moreover, we found that in water methanol solutions in the concentration range of 0 0.8 the proton transfer (PT) rate constant decreases with an even steeper slope. The KIE<br />
maintains a constant value of 2.4 ± 0.2 at all the mixture's compositions. 2<br />
In another study 3 we found that in 1 M aqueous solutions of acetate or higher a PT process to the<br />
acetate takes place within 30 ps in both H2O and D2O solutions. The time-resolved emission signal is<br />
composed of three components. We found that the short-time component decay time is 300 fs and 600<br />
fs in H2O and D2O respectively. This component is attributed to either a PT process via the shortest<br />
water bridged complex available, ROH H2O Ac −<br />
⋅⋅ ⋅⋅ , or to PT taking place within a contact ion pair. The<br />
second time component of 2000 fs and 3000 fs <strong>for</strong> H2O and D2O respectively is attributed to ROH*<br />
acetate complex, whose proton wire is longer by one water molecule. The decay rate of the third, longtime<br />
component is proportional to the acetate concentration. We attribute it to the diffusion-assisted<br />
reaction as well as to PT process to the solvent.<br />
The emission spectrum of d-luciferin in a 20 mM HCl aqueous solution or higher has an<br />
additional emission band at 590 nm red-shifted with respect to the strongest emission band positioned<br />
at 530 nm of the deprotonated NRO - * <strong>for</strong>m in a pH-neutral aqueous solution. 4 We attribute this<br />
emission band to the zwitterion <strong>for</strong>m designated as + HNRO - . The time-resolved emission signals show<br />
that the NRO - * emission band at 530 nm and the zwitterion emission band at 590 are strongly<br />
quenched by a recombination process with a proton in an acidic solution and in ice. In ice the<br />
quenching rate is 10 times faster than in the liquid state. We attribute the fast quenching rate to the<br />
high value of the proton diffusion constant in ice.<br />
This work was supported by grants from the Israel Science Foundation and from the James-Franck<br />
German-Israeli Program in Laser-Matter Interaction.<br />
References: [1] Erez, Y.; Huppert, D. J. Phys. Chem. A 2010, 114, 8075. [2] Presiado, I. et al J. Phys. Chem. A<br />
2010, 114, 9471. [3] ] Presiado, I. et al J. Phys. Chem. A 2010, 114, 13337. [4] Erez, Y. et al J.Phys Chem A. 115,<br />
1617, 2011.<br />
______________<br />
* Corresponding author: e-mail: huppert@tulip.tau.ac.il<br />
82 — MAF 12, Strasbourg, France, September 11-14, 2011 —
OC26 Oral communication 26<br />
Discovery and biological application of full-color tunable and<br />
predictable fluorescent core skeleton (Seoul-Fluor) and fluorescent<br />
glucose bioprobes (GBs)<br />
Eunha Kim 1 , Hyang Yeon Lee 1 , Sanghee Lee 1 , Jongmin Park 1 & Seung Bum Park 1,2*<br />
1Department 2<br />
of Chemistry and Department of Biophysics and Chemical Biology, Seoul National<br />
University, Seoul 151-747, Korea<br />
A study of 9-aryl-1,2-dihydropyrrolo[3,4-b]indolizin-3-one, we name it Seoul-Fluor, were presented<br />
in this presentation. During our continuous ef<strong>for</strong>ts on the construction of drug-like small-molecule<br />
libraries using diversity-oriented synthesis (DOS) strategy, we identified a novel fluorescent core<br />
skeleton, 1,2-dihydropyrrolo[3,4-b]indolizin-3-one. Guiding with computational simulation, 24<br />
fluorescent compounds library were constructed in combinatorial fashion, and which covers the<br />
full-color range. Further bioapplication of these fluorescent compounds was successfully<br />
demonstrated in the immunofluorescent staining. After discovery of Seoul-Fluor, we analyzed the<br />
fluorescent core skeleton more systematically and extensively. Using a concise and practical onepot<br />
synthetic procedure, a 44-member library of new fluorescent compounds was synthesized. The<br />
systematic perturbation of electronic densities on the specific positions of Seoul-Fluor, guided with<br />
the Hammett constant, allows tuning the emission wavelength in full-color range. Furthermore, on<br />
the basis of these observations and a computational analysis, we extracted a simple first-order<br />
correlation of emission wavelength with the theoretical calculation and accurately predicted the<br />
emission wavelength of Seoul-Fluors prior to their synthesis. There<strong>for</strong>e, we clearly demonstrated<br />
that Seoul-Fluor could provide a powerful gateway <strong>for</strong> the generation of desired fluorescent probes<br />
without the need <strong>for</strong> tiresome synthesis and trial-and-error process.<br />
In addition, we developed a novel fluorescent glucose bioprobe. Glucose is the most<br />
important energy source <strong>for</strong> cell growth, there<strong>for</strong>e fast-growing cancer cell requires more glucose<br />
than normal cell. Based on this phenomenon, diagnosis of various cancers has been per<strong>for</strong>med by<br />
PET(Positron Emission Tomography) in these days. FDG(2-fluoro-2-deoxy-D-glucose), which can<br />
be detected by PET, allows us imaging exact positions of tumors in our body. Another Nglycosylated<br />
glucose analog, 2-NDBG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-Dglucose),<br />
is a glucose-mimicking bioprobe which can be detected by fluorescence instead of using<br />
radioisotope. To address this issue, we synthesized α and β anomers of D-glucose labeled with<br />
Cy3 dye. This novel glucose-based fluorescent bioprobes were examined <strong>for</strong> the application of<br />
bioassay system and high-throughput screening through the measurement of glucose uptake of<br />
cells by CLSM (confocal laser scanning microscope). Behaviors of our bioprobes are superb to<br />
previous glucose analogs, most importantly, it does not require the glucose starvation of media,<br />
which is critical to observe glucose metabolism in cell’s normal physiology. With these results, we<br />
tried to establish assay system <strong>for</strong> the evaluation of bioactive small molecules by the measurement<br />
of glucose uptake in cancer cells. For instance, cancer cells were pre-treated with anticancer agent<br />
and measured the reduced uptake of our bioprobes. We expect that our assay can be used <strong>for</strong><br />
HTS (High Throughput Screen) and bioresearch on glucose uptake-related disease.<br />
References:<br />
[1] Kim, E.; Koh, M.; Lim, B.J.; Park, S.B.* J. Am. Chem. Soc., 133 (2011) 6642–6649<br />
[2] Kim, E.; Ryu, J.; Koh, M.; Park, S.B.* J. Am. Chem. Soc., 130 (2008) 12206–12207<br />
[3] Tian, Y. S.; Lee. H.Y.; Lim, C.S.; Park, J.; Kim, H.M.; Shin, Y.N.; Kim, E.S.; Jeon, H.J.; Park, S.B.*; Cho,<br />
B.R.* Angew. Chem. Int. Ed. 48 (2009) 8027–8031<br />
[4] Lee, H.-Y.; Cho, M.-H.; Park, S. B.* Angew. Chem. Int. Ed. 46 (2007) 2018–2022<br />
[5] Lee, H.Y.; Lee, J.J.; Park, J.; Park, S.B.* Chem. Eur. J. 2011, 17(1), 143–150.<br />
______________<br />
* Corresponding author: e-mail: sbpark@snu.ac.kr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 83
OC27 Oral communication 27<br />
Physical chemistry and photophysics of the cyan fluorescent protein<br />
Fabienne Mérola * , Hélène Pasquier, Marie Erard, Agathe Espagne, Asma Fredj, Luis Alvarez,<br />
Germain Vallverdu, Gabriella Jonasson, Isabelle Demachy, Jacqueline Ridard & Bernard<br />
Levy<br />
Laboratoire de Chimie Physique, UMR8000 CNRS, Université Paris Sud 11, Bat 349 Centre<br />
Universitaire, 91405 Orsay<br />
Proteins of the GFP family allow the design of genetically encoded fluorescent constructs<br />
combining in less than 100 Å the three essential functions of a biological sensor : functional<br />
targeting, chemical detection and optical conversion [1] . These biosensors, which are massively<br />
used in biotechnology and biomedical protocols, have a nearly unlimited potential of evolution<br />
through genetic engineering. Optimization of GFPs <strong>for</strong> chemical sensing requires a predictive<br />
understanding of their stucture-photophysics relationships, as well as a careful control of their<br />
molecular interactions, chemical reactivity and environmental sensitivity. In the recent years, we<br />
have adressed these questions in the cyan fluorescent protein (CFP), which, together with the<br />
yellow fluorescent protein (YFP), remains by far the most widely used variant in sensing and<br />
imaging techniques based on FRET. Native CFP has a very complex photophysics, characterized<br />
by spectral heterogeneity and strongly non-exponential fluorescence decays [2] . We have studied<br />
the responses of CFP fluorescence to factors such as temperature and pH [2] , reactive oxygen<br />
species (ROS) [3] , or to more overlooked parameters, such as protein concentration [4-5] or the<br />
excitation regime. With the help of single point mutations and molecular simulations [6-7] , our results<br />
start to bring some insights into the possible structural and dynamic determinants of CFP<br />
fluorescence, and provide some rationale <strong>for</strong> the mechanisms at work in its recently improved<br />
variants Cerulean and mTurquoise.<br />
Our work is supported by grants from CNRS, MENESR, Paris Sud 11 University, and ANR.<br />
References: [1] F. Merola et al., in Springer Series "Advanced Fluorescence Reporters in Chemistry and<br />
Biology" Vol 1 (2010) 347. [2] A. Villoing et al., Biochemistry 47 (2008) 12483. [3] L. Alvarez, et al.,<br />
Photochem. Photobiol. 86 (2010) 55. [4] R. Grailhe, et al., ChemPhysChem 7 (2006) 1442. [5] A. Espagne,<br />
et al., Biochemistry 50 (2011) 437. [6] Vallverdu, G. et al., Proteins 78 (2010) 1040. [7] G. Jonasson, et al., J.<br />
Chem. Theory Comput. 7 (2011) 1990.<br />
______________<br />
* Corresponding author: e-mail: fabienne.merola@u-psud.fr<br />
84 — MAF 12, Strasbourg, France, September 11-14, 2011 —
OC28 Oral communication 28<br />
Fluorescent probes <strong>for</strong> monitoring lipid order selectively at one<br />
bilayer leaflet<br />
Andrey S. Klymchenko, Oleksandr A. Kucherak, Zeinab Darwich, Sule Oncul, Youri Arntz,<br />
Pascal Didier, Guy Duportail & Yves Mely<br />
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg,<br />
Faculté de Pharmacie, 74, Route du Rhin, 67401 ILLKIRCH (France)<br />
Biological membranes present a remarkable asymmetry of the lipid distribution at the two leaflets.<br />
Sphingomyelin, which is a key component of liquid ordered phase of lipid membranes, locates<br />
specifically at the outer leaflet of cellular membranes. There<strong>for</strong>e, monitoring lipid organization<br />
selectively at the outer leaflet of biological membranes is of particular interest. For this purpose, a<br />
solvatochromic dye Nile Red was modified with an amphiphilic anchor group. To evaluate the flipflop<br />
of the obtained probe (NR12S), we developed a methodology of reversible redox switching of<br />
its fluorescence at one leaflet using sodium dithionite. This method showed that, NR12S, in<br />
contrast to parent Nile Red, binds exclusively the outer membrane leaflet of model lipid vesicles<br />
and living cells with negligible flip-flop in the time scale of hours. In model lipid vesicles, NR12S<br />
showed strong blue shift in the ordered phase (shingonyelin-cholesterol mixture) as compared to<br />
liquid disordered phase (DOPE-cholesterol). There<strong>for</strong>e, these two phases could be clearly<br />
distinguished in NR12S-stained giant vesicles by fluorescence microscopy images of intensity ratio<br />
between the blue and red parts of the probe emission spectrum. Being added to living cells,<br />
NR12S binds exclusively their plasma membranes, showing an emission spectrum intermediate<br />
between those in the ordered and disordered phases of model membranes. Decrease in<br />
cholesterol content by methyl-β-cyclodextrin and cholesterol oxidize as well as enzymatic cleavage<br />
of sphingomyelin in cell plasma membranes produced red shifts in the emission of the probe,<br />
indicating the decrease in their lipid order. Finally, apoptosis resulted in a similar red shifted<br />
emission of NR12S, suggesting that the apoptotic decrease in transmembrane asymmetry also<br />
leads to the decrease in the lipid order at the outer membrane leaflet. Thus, the new probe based<br />
on solvatochromic Nile Red is a useful tool <strong>for</strong> monitoring lipid order and transmembrane<br />
asymmetry in biomembranes.<br />
Chemical structure of NR12S and its application <strong>for</strong> fluorescence imaging of separate ordered and<br />
disordered phase domains in giant vesicles (A) and changes in the lipid order in cell membranes<br />
on cholesterol depletion by methyl-β-cyclodextrin (B and C).<br />
This work was supported by Conectus Alsace.<br />
References: [1] Kucherak et al., J. Am. Chem. Soc. 132 (2010) 4907. [2] V.V. Shynkar, et al., J. Am. Chem.<br />
Soc., 129 (2007) 2187.<br />
______________<br />
Corresponding author: e-mail: andrey.klymchenko@unistra.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 85
86 — MAF 12, Strasbourg, France, September 11-14, 2011 —
POSTERS<br />
POSTERS<br />
POSTERS<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 87
88 — MAF 12, Strasbourg, France, September 11-14, 2011 —
New Technical Approaches & Microfluidics<br />
P1 Make invisible visible: a Diversity Oriented Fluorescence Library Approach (DOFLA)<br />
Young-Tae Chang<br />
P2 New perspective in background suppression using Matrix Isopotential Synchronous<br />
Fluorymetry<br />
János Erostyák, Géza Makkai<br />
P3 Spectral phasor analysis allows rapid and easy unmixing of fluorescence microscopy<br />
spectral images<br />
Arjen N. Bader, Farzad Fereidouni, Hans C. Gerritsen<br />
P4 Analysis of time-gated FLIM data by means of the phasor approach<br />
Farzad Fereidouni, Dave J. van den Heuvel, Jarno Voortman, Erik Hofman, Hans C.<br />
Gerritsen<br />
P5 Fluorescence and FRET of mass-selected gaseous biomolecules<br />
Rebecca A. Jockusch, Francis O. Talbot, Martin F. Czar, Sandeep K. Sagoo, Huihui Yao<br />
P6 A comparative time-resolved cuvette system study of two Calcium FRET sensors: towards<br />
making lifetime easier<br />
Romain Laine, Daniel W. Stuckey, Christopher Dunsby, Alessandro Sardini, David<br />
Carling, Paul M. W. French<br />
P7 Rapid analysis of peptides and isomeric dipeptides using miniaturized CE with deep UV<br />
native fluorescence detection<br />
Qiang Li, Stefan Seeger<br />
P8 A nanosecond-gate-mode-driven silicon-avalanche-photodiode and its application to<br />
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Tsuyoshi Miyata, Tetsuo Iwata, Susumu Nakayama Tsutomu Araki<br />
P9 Phase-modulation fluorometry using a phase-modulated light source<br />
Takahiko Mizuno, Yasuhiro Mizutani, Tetsuo Iwata<br />
P10 On the spectral sensitivity calibration of fluorescence spectrometers: Extension to the NIR,<br />
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Sebastian Tannert, Peter Kapusta, Alexander Glatz, Alexander Goschew, Uwe Ortmann,<br />
Rainer Erdmann<br />
P11 Bimolecular Photochemistry under Microfluidic Conditions<br />
K.-H. Feller, S. Harz, M. Schimmelpfennig<br />
P12 Picosecond fluorescence of out-of-equilibrium biomolecular complexes <strong>for</strong>med in<br />
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Sacha Maillot, Alain Carvalho, Jean-Pierre Vola, Stefan Haacke, Jérémie Léonard<br />
P13 Fluorescent chemical sensing in microfluidic miniaturised analytical plat<strong>for</strong>ms<br />
Leonid Gitlin, Stefan Jezierski, Georg Jochem, Christian Höra, Stefan Nagl<br />
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Marina Sierra-Rodero, Juan Manuel Fernández-Romero, Agustina Gómez-Hens<br />
Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule<br />
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Jianhua Chen, Yves Kellner, Marta Zagrebelsky, Martin Korte, Peter Jomo Walla<br />
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Meike Kloster, Gaétan Herbomel, Yves Usson, Irène Wang, Claire Vourc'h, Catherine<br />
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P17 Anomalous diffusion in lipid membranes and associated proteins: Experiments and<br />
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Jean Louis Meunier, Pierre François Lenne, Hervé Rigneaul, Cyril Favard<br />
P19 Evaluation of Radiation Force under the Resonance Optical Trapping Condition using<br />
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Hiroaki Yamauchi, Masataka Taguchi, Syoji Ito, Hiroshi Miyasaka<br />
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Christian Spies, Michael Vester, Gregor Jung<br />
P21 Self-assembly of the Immuno-Active GTPase hGBP1 as monitored by ensemble time<br />
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Y. Ajaj, Peulen, C. S. Hengstenberg, T., M. Richrt, A. Valeri, C. Herrmann, C. A. Seidel<br />
P22 Binding of Organic Dyes with Human Serum Albumin: A <strong>Single</strong> Molecule Study<br />
Dibyendu Kumar Das, Tridib Mondal, Amit Kumar Mandal, Kankan Bhattacharyya<br />
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Björn Finkler, Anh-Minh Huynh, Gregor Jung<br />
P24 Protein distribution on liposomes by detection of single fluorescent diffusing particles: a<br />
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Ouided Friaa, Melissa Furukawa, Radhika Voleti, Sanjeevan Shivakumar, Aisha Shamas-<br />
Din, Cécile Fradin<br />
P25 Binding kinetics of the HIV-1 NCp7 on oligonucleotides at the single-molecule level<br />
Julien Godet, Pascal Didier, Armelle Jouonang, Youri Arntz, Guy Duportail, Yves Mély<br />
P26 Con<strong>for</strong>mations and con<strong>for</strong>mational dynamics of calmodulin detected by single-molecule<br />
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Carey K. Johnson, E. Shane Price, Mihailo Backović, Matthew S. DeVore, John P.<br />
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P27 <strong>Single</strong>-molecule fluorescence dynamics of perylene diimides cyclic arrays<br />
Ji-Eun Lee, Jaesung Yang, Hyejin Yoo, Dongho Kim<br />
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Konstantinos Lymperopoulos, Alejandro Macias-Torre, William Godinez, Stefan<br />
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Frank Schleifenbaum, Christian Blum, Martijn Stopel, Sebastien Peter, Marcus Sackrow,<br />
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P30 Investigation of chemical reactions with <strong>Single</strong>-Molecule Fluorescence Spectroscopy<br />
Arina Rybina, Alexander Kiel, Nicole Bach, Birgit Thaler, Arnd Sprödefeld, Daniel Hack,<br />
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Anne Seefeld, Konstantinos Lymperopoulοs, Dirk-Peter Herten<br />
P32 Low-temperature spectral dynamics of single impurity molecules in ultrathin polymer film<br />
Yaroslav I. Sobolev, Yuri G. Vainer, Andrei V. Naumov, Lothar Kador<br />
P33 Stability of terrylenediimide (TDI) single molecules in various polymer films<br />
Adam Sokolowski, Jacek Waluk<br />
P34 First computational determination of four-way RNA junction’s structure using simplified<br />
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FRET spectroscopy experiment, rigid body modeling and predicted alternative<br />
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Tomasz Sołtysiński, Stanislav Kalinin, Michał Boniecki, Simon Sindbert, Hayk<br />
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P35 Polymer matrix dependence of con<strong>for</strong>mational dynamics within π-stacked perylenediimide<br />
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Hyejin Yoo, Heewon Bahng, Michael R. Wasielewski, Dongho Kim<br />
P36 Real-time observation of platinum anticancer drug–DNA interactions at the singlemolecule<br />
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Yuko Yoshikawa, Seiji Komeda, Kenichi Yoshikawa, Tadayuki Imanaka<br />
Fluorescence Microscopies and Cell Imaging<br />
P37 Skin diagnosis by non-invasive observation of dermal collagen with a second harmonic<br />
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Tsutomu Araki, Ryosuke Tanaka, Takeshi Yasui<br />
P38 Determination of Protein-Protein-Interactions in Microfluidic Droplets Using Fluorescence<br />
Lifetime Microscopy<br />
Christian Benz, Heiko Retzbach, Stefan Nagl, Detlev Belder<br />
P39 Probing protein homodimerization by coupling time-resolved Fluorescence Anisotropy<br />
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Viviane Devauges, Catherine Marquer, Sandrine Lécart, Marie-Claude Potier, Emmanuel<br />
Fort, Sandrine Lévêque-Fort<br />
P40 Intracellular dynamics of HIV-Gag: Activation of phospholipase C and virus-like particles<br />
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Andrea Gramatica, Roland Schwarzer, Andreas Herrmann<br />
P41 DNA Biosensors <strong>for</strong> <strong>Single</strong> Molecule Transcription Factor Detection with ALEX-TIRF<br />
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Kristin Grußmayer, Tanja Ehrhard, Anton Kurz, Michael Schwering, Jessica Balbo,<br />
Konstantinos Lymperopoulos, Dirk-Peter Herten<br />
P42 Combining fluorescence techniques to unravel ligand binding mechanisms at G-protein<br />
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Brigitte Ilien, Sandrine Daval, Dominique Bonnet, Pascal Didier, Marcel Hibert, Yves<br />
Mély, Jean-Luc Galzi<br />
P43 B cells can communicate through membrane nanotubes: Imaging studies on the<br />
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Emese Izsepi, Anna Csala, Janos Matko<br />
P44 Investigation by high resolution microscopy of the internalization of the neuropeptide Y<br />
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Noémie Kempf, Pascal Didier, Ludovic Richert, Viktoriia Postupalenko, Andrey<br />
Klymchenko, Hugues de Rocquigny, Bernard Bucher, Yves Mély<br />
P45 Bi-plane Calibration in Super-resolution Microscopy<br />
Hagai Kirshner, Thomas Pengo, Nicolas Olivier, Daniel Sage, Suliana Manley, Michael<br />
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P46 3D PSF Models <strong>for</strong> Fluorescence Microscopy in ImageJ<br />
Hagai Kirshner, Daniel Sage, Michael Unser<br />
P47 Fluorescence imaging of living hybrid materials<br />
Alexander M. Macmillan, Dylan M. Owen, Katharina Gaus, David J. S. Birch, Jan Karolin<br />
P48 Mechanisms of increased amyloid peptide production by membrane cholesterol loading:<br />
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Catherine Marquer, Viviane Devauges, Jack-Christophe Cossec, Géraldine Liot,<br />
Sandrine Lécart, Frédéric Saudou, Charles Duyckaerts, Sandrine Lévêque-Fort, Marie-<br />
Claude Potier<br />
P49 IR excitation intravital imaging: Role of Hemodynamics in the development of zebrafish<br />
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H. Mojzisova, J. Goetz, J. Vermot<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 91
P50 Laser-induced DNA damage in nonlinear imaging: Biosafety study<br />
Oleg Nadiarnykh, Giju Thomas, Johan Van Voskuilen, Henricus J. C. M. Sterenborg,<br />
Hans C. Gerritsen<br />
P51 The stereological distribution of Langerhans islets studied by selective plane illumination<br />
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Nils Norlin, Jim Swoger, Anna Eriksson, Jürgen Mayer, Ulf Ahlgren, James Sharpe<br />
P52 Modelisation of the Imaging Process <strong>for</strong> the Fluorescence Macroscope<br />
Praveen Pankajakshan, Elie Maalouf, Bruno Colicchio, Jean-Christophe Olivo-Marin,<br />
Alain Dieterlen<br />
P53 Imaging the methylglyoxal-induced changes in the peripheral sensory neurons from dorsal<br />
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Beatrice Mihaela Radu, Adela Marin, Diana Ionela Rotaru, Adina Daniela Iancu, Cosmin<br />
Mustaciosu, Dorel Radu, Maria-Luisa Flonta, Mihai Radu<br />
P54 The HIV-1 Gag induces Vpr oligomers accumulation in the viral particles : analysis by<br />
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Hugues de Rocquigny, Denis Dujardin, Tania Steffan, Pascal Didier, Yves Mély<br />
P55 Determining the cellular uptake and localisation of novel anti-cancer drugs using<br />
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Laura Rowley, Nikolas J. Hodges, Josephine Bunch, Ela Claridge, Michael J. Hannon<br />
P56 Two-photon excitation and stimulated emission depletion by a single wavelength<br />
Teodora Scheul, Ciro D’Amico, Jean-ClaudeVial, Iréne Wang<br />
P57 FIDSAM – a novel fluorescence microscopy approach <strong>for</strong> quantitative and highly sensitive<br />
life-cell and FRET-imaging<br />
Frank Schleifenbaum, Kirstin Elgass, Marcus Sackrow, Sebastien Peter, Klaus Harter,<br />
Alfred J. Meixner<br />
P58 FLIM-FRET as a tool to study protein partitioning in plasma membrane microdomains of<br />
living cells. Identifying raft localisations signals of the HIV glycoprotein gp41<br />
Roland Schwarzer, David Reismann, Andreas Herrmann<br />
P59 CyDNA: A versatile photoswitchable biopolymer <strong>for</strong> advanced fluorescence microscopy<br />
applications<br />
Darren A. Smith, Philipp Holliger, Cristina Flors<br />
P60 Monitoring the oxygen concentration and redox state of living cells using long-lived<br />
transient states of fluorophores<br />
Thiemo Spielmann, Sofia Johansson, Jerker Widengren<br />
P61 Fluorescence lifetimes, anisotropy and FRAP recovery curves measured simultaneously in<br />
living cells<br />
James A. Levitt, Pei-Hua Chung, Klaus Suhling<br />
P62 On/Off Switching of a Genomic DNA Caused by Nonspecific Environmental Parameter, as<br />
is Evidenced by Real-Time Observation with Fluorescence Microscopy<br />
Kenichi Yoshikawa, Akihiko Tsuji, Ayako Kato, Yuko Yoshikawa<br />
P63 Scarless Regeneration of the Newt Heart<br />
Tanja Piatkowski, Thilo Borchardt, Thomas Braun<br />
Polymers, Materials & Nanomaterials, Quantum Dots<br />
P64 Fluorescence and Electrical Measurements <strong>for</strong> Studying Internal Morphology of Imprinted<br />
and Random PNIPA Gels<br />
Esra Alveroglu Durucu, Cagatay İleten, Yasar Yilmaz<br />
P65 Schiff bases containing phenothiazine units with fluorescence properties<br />
Castelia Cristea, Emese Gál, Larisa Mataranga-Popa, Luiza Găină, Luminiţa Silaghi-<br />
Dumitrescu<br />
92 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P66 Nanofilamentous molecularly imprinted polymers in a miniaturized fluorescence-optical<br />
sensor<br />
S. Harz, Aude Cordin, K. Haupt, K.-H. Feller<br />
P67 Photophysical investigation of a fluorophore sensor and using its self-assembly <strong>for</strong> the<br />
detection of environmental changes in polyesters<br />
M. Amine Fourati, C. Géraldine Bazuin, Robert E. Prud’homme<br />
P68 Luminescent Light-Harvesting Pendant PPV Polymers<br />
Kenneth Ghiggino, Andrew Tilley, Jonathan White, Ming Chen<br />
P69 Time Evolution of Multiple Point Interactions of Pyranine Fluoroprobe with Polyacrylamide<br />
Chains During the Polymerization: Experiment and Simulation<br />
Alptekin Yildiz, Ali Gelir, Yasar Yilmaz<br />
P70 Comparative study of the sensitized luminescence of terbium(III) ions and Tb4O7<br />
nanoparticles as derivatising reagents in ultrafast liquid chromatography<br />
M.L. Castillo-García, M.P. Aguilar-Caballos, A. Gómez-Hens<br />
P71 Fluorescence Spectroscopy of Phosphor Powders – Difficulties and Challenges<br />
Peter Barnekow, Sven Brüninghoff, Robert Schiwon, Holger Winkler<br />
P72 Polymer nanoparticles <strong>for</strong> the controlled production and release of singlet oxygen<br />
Sofia Martins, José P. S. Farinha, Carlos Baleizão, Mário N. Berberan-Santos<br />
P73 Plasmon induced fluorescence enhancement in LH2 complexes<br />
Łukasz Bujak, Tatas H.P. Brotosudarmo, Nikodem Czechowski, Maria Olejnik Radek<br />
Litvin, Richard J. Cogdell, Wolfgang Heiss, Sebastian Maćkowski<br />
P74 Versatile multilayered structure <strong>for</strong> the generation of Metal Enhanced Fluorescence<br />
Eleonora V. Canesi, Martina Capsoni, Mirella Del Zoppo, Andrea Lucotti, Chiara Bertarelli<br />
P75 Absorption enhancement of light-harvesting complexes through plasmonic silver island<br />
film<br />
Kamil K. Ciszak, Maria Olejnik, Janusz Strzelecki, Eckhard Hofmann, Sebastian<br />
Mackowski<br />
P76 One-pot Synthesis of Pegylated Fluorescent Nanoparticles by RAFT Miniemulsion<br />
Polymerization using a Phase Inversion Process<br />
Chloé Grazon, Rachel Méallet-Renault, Jutta Rieger, Bernadette Charleux, Gilles Clavier<br />
P77 Fluorescence microscopy of corrole-single silver nanowire hybrid structures<br />
Nikodem Czechowski, Maria Olejnik, Bartosz Krajnik, Agnieszka Nowak-Król, Dawid<br />
Piątkowski, Wolfgang Heiss, Daniel T. Gryko, Sebastian Maćkowski<br />
P78 Delivery of luminescent gold nanoparticles into human platelets using a pH controlled<br />
strategy<br />
Amy Davies, Steve Watson, Ela Claridge, Steve Thomas, Zoe Pikramenou<br />
P79 Preparation and optical properties of new hybrid materials: silica gels doped with a<br />
[Eu(2NTBD)3 ][PPhenDCN] complex<br />
N. Danchova, T. Deligeorgiev, N. Lesev, S. Kaloyanova, S. Stoyanov, S. Gutzov<br />
P80 Few-atom clusters of silver in organic solvents: synthesis and properties<br />
Isabel Díez, Robin H. A. Ras, Mykola I. Kanyuk, Alexander P. Demchenko<br />
P81 Luminescent Silica Nanoparticles <strong>for</strong> Enrofloxacin Detection via FRET Signaling<br />
Clara Somoza, Ana B. Descalzo, M. Cruz Moreno-Bondi, Guillermo Orellana<br />
P82 Fluorescence spectroscopy and spectral modeling as a tool <strong>for</strong> study of interaction of<br />
nanoparticles with biomacromolecules. Plant cell walls<br />
Daniela Djikanović, Aleksandar Kalauzi, Milorad Jeremić, Jianmin Xu, Miodrag Mićić,<br />
Roger Leblanc, Ksenija Radotić<br />
P83 Fluorescent organic ion pairs: An original approach <strong>for</strong> the preparation of nanomaterials<br />
Suzanne Fery-Forgues, Abdelhamid Ghodbane, Joe Chahine, Martine Cantuel<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 93
P84 Fast FRET events in dye-doped nanoparticles provide functionalities: high brightness and<br />
tunable emission<br />
Damiano Genovese, Sara Bonacchi, Riccardo Juris, Marco Montalti, Enrico Rampazzo,<br />
Nelsi Zaccheroni, Luca Prodi<br />
P85 Upconverting luminescent nanoparticles (UCLNPs) enable background-free cellular<br />
imaging, immunoassays and ratiometric encoding<br />
Hans H. Gorris, Raphaela Liebherr, Sayed M. Saleh, Reham Ali, Otto S. Wolfbeis<br />
P86 Effect of rare earth elements on the photoluminescence of CaGa2S4:Eu<br />
Chiharu Hidaka, TakeoTakizawa<br />
P87 The influence of silver nanostructures <strong>for</strong>med in-situ in silica sol-gel derived films on the<br />
rate of Förster resonance energy transfer<br />
Gary McDowell, Marion Toury, David McLoskey, Graham Hunger<strong>for</strong>d, A. Sheila Holmes-<br />
Smith<br />
P88 In-situ <strong>for</strong>mation of silver nanostructures within a polysaccharide film and its application as<br />
a potential biocompatible fluorescence sensing medium<br />
Nicole Donaldson, Marion Toury, David McLoskey, A. Sheila Holmes-Smith, Graham<br />
Hunger<strong>for</strong>d<br />
P89 ESR study of Mn 2+ and Ce 3+ ions in single crystals of calcium thiogallate phosphors<br />
Ittetsu Kitajima, Takeo Takizawa, Chiharu Hidaka, Shigetaka Nomura<br />
P90 Metal-enhanced fluorescence studied with SIL – based microscopy<br />
Bartosz Krajnik, Dawid Piatkowski, Nikodem Czechowski, Eckhard Hofmann, Wolfgang<br />
Heiss, Sebastian Mackowski<br />
P91 Nature of SYBRGreen fluorescence quenching on DNA matrix under Au nanoparticles<br />
action<br />
Ekaterina S. Lisitsyna, Sergey G. Skuridin, Yuriy M. Evdokimov, Victor M.Rudoy, Olga V.<br />
Dementieva, Vladimir A. Kuzmin<br />
P92 Photophysical properties of a clinical photosensitiser in pegylated silica nanoparticles<br />
Alexandra Mackenzie, Josephine Woodhams, Iria Echevarria, Fabrizio Mancin,<br />
Alexander MacRober<br />
P93 Photoinduced Size-Controlled Generation of Silver Nanoparticles Coated with<br />
Carboxylate-Derivatized Thioxanthones<br />
Jean-Pierre Malval, Ming Jin, Lavinia Balan, Raphaël Schneider, Davy-Louis Versace,<br />
Hélène Chaumeil, Albert Defoin, Olivier Soppera<br />
P94 Enhancement of Acid Photogeneration Through a Para-to-Meta Substitution Strategy in a<br />
Sulfonium-based Alkoxystilbene Designed <strong>for</strong> Two-Photon Polymerization<br />
Rongjie Xia, Jean-Pierre Malval, Ming Jin, Arnaud Spangenberg, Decheng Wan,<br />
Hongting Pu, Fabrice Morlet-Savary, Hélène Chaumeil, Patrice Baldeck, Olivier Poizat,<br />
Olivier Soppera<br />
P95 Synthesis and fluorescence imaging of solid-state amorphous films or particles from new<br />
BODIPY derivatives<br />
Thanh Truc Vu, Elena Senotrusova, Robert B. Pansu, Pierre Audebert, Elena Yu Schmidt,<br />
Boris A.Trofimov, Gilles Clavier, Rachel Méallet-Renault<br />
P96 Photolumiscent Properties of CoMoO4 Nanorods quickly synthesized and annealed in a<br />
domestic microwave owen<br />
Ana Paula de Moura, Larissa Helena de Oliveira, Ieda Lúcia Viana Rosa, Máximo Siu Li,<br />
Elson Longo, José Arana Varela.<br />
P97 Study of the annealing temperature effect on the structural and luminescent properties of<br />
SrWO4: Eu 3+ phosphors prepared by a non-hydrolytic sol-gel process<br />
Ieda Lúcia Viana Rosa, Paula Fabiana Santos Pereira, Ana Paula de Moura Içamira<br />
Costa Nogueira, Márcia Valéria Lima, Elson Longo, Paulo César de Sousa Filho, Osvaldo<br />
Antônio Serra, Eduardo José Nassar<br />
94 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P98 Investigation of the photodynamics of fluorescent dendronized perylenediimide<br />
P99<br />
nanoparticles combining single particle and ultrafast spectroscopy<br />
Michel Sliwa, Ryohei Yasukuni, Bruno Debus, Cyril Ruckebusch, Tom Vosch, Johan<br />
Hofkens, Tsuyoshi Asahi<br />
Concentration dependence of the enhancing effect of REE on Mn 2+ red emission in<br />
CaGa2S4: Mn 2+ , REE<br />
Akihiro Suzuki, Chiharu Hidaka, Takeo Takizawa, Shigetaka Nomura<br />
P100 Ratio of anti-Stokes and Stokes fluorescence intensities as a parameter sensitive to the<br />
ionic strength in the environment of fluorophore-conjugated organically modified silica<br />
nanoparticles<br />
Valentyna Tokar, Tymish Ohulchanskyy, Atcha Kopwitthaya, Paras N Prasad<br />
P101 Adsorption of 5,5’-disulfopropyl-3,3’-dichlorothyocyanine and fluorescence quenching in<br />
the gold nanoparticle assembly<br />
Vesna Vodnik, Ana Vujačić, Miroslav Dramićanin, Sofija P. Sovilj, Vesna Vasić<br />
P102 Gold nanorods <strong>for</strong> fluorescence imaging and sensing in biology<br />
Yinan Zhang, David J. S. Birch, Yu Chen<br />
P103 Characterization of highly fluorescent candidate glass reference standards<br />
Katrin Hoffmann, Axel Engel, Monika Bäumle, Ute Resch-Genger<br />
P104 Targeting of Quantum Dots with Adaptor Protein Nck<br />
Ruta Araminaite, Vitalijus Karabanovas, Marija Ger, Mindaugas Valius, Simona<br />
Steponkiene, Ricardas Rotomskis<br />
P105 Preparation of size controllable highly luminescent CdSe quantum dots in AOT reverse<br />
micelles using polyselenide precursors under mild conditions<br />
Arlindo M. Fontes Garcia, Paulo J.G. Coutinho<br />
P106 Delivery of quantum dots to cytosol by chlorin e6-mediated photochemical internalization<br />
Vitalijus Karabanovas, Jurga Valanciunaite, Mindaugas Valius, Ricardas Rotomskis<br />
P107 Receptor mediated targeting of quantum dots to kidney podocytes<br />
Klaus Pollinger, Robert Hennig, Joerg Tessmar, Miriam Breunig, Ralph Witzgall, Achim<br />
Goepferich<br />
P108 A Novel Highly Sensitive Nanobiosensor <strong>for</strong> Detection of Helicobacter Pilori Based on<br />
Fluorescence Resonance Energy Transfer<br />
Mojtaba Shamsipur, Maryam Shanehsaz, Afshin Mohsenifar, Sadegh Hasannia, S.H.<br />
Kazemi, Nazanin Pirooznia<br />
P109 Spectroscopic study on complex <strong>for</strong>mation between different amphiphilic coating bearing<br />
quantum dots and photosensitizer chlorin e6<br />
Jurga Valanciunaite, Artiom Skripka, Reda Jarmalaviciene, Ricardas Rotomskis<br />
P110 Photosensitizing properties and accumulation in cancer cells of non-covalent quantum dotchlorin<br />
e6 complex<br />
Jurga Valanciunaite, Simona Steponkiene, Artiom Skripka, Margarita Chernych, Giedre<br />
Streckyte, Ricardas Rotomskis<br />
Miscellaneous, Micelles & Biopolymers, Fluorescence<br />
Spectroscopy applied to Biology, Biomembranes<br />
P111 Investigation on Romanian icons – chromatic changes following gamma irradiation<br />
Maria Geba, Ana Maria Vlad, Daniela Salajan, Corneliu Catalin Ponta, Constantin-Daniel<br />
Negut<br />
P112 Tracing non-specific riverine pollution caused by discharge of industrial effluents: Use of a<br />
recalcitrant fluorescent component of chromophoric dissolved organic matter (CDOM)<br />
Mikhail Borisover, Yael Laor, Ibrahim Saadi, Marcos Lado, Nadezhda Bukhanovsky<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 95
P113 1,2-Dioxetane-based chemiluminescent uphill energy conversion: Principle and future<br />
perspectives<br />
Luiz Francisco M. L. Ciscato, Erick L. Bastos, Josef Wilhelm Baader<br />
P114 Host-guest complexes of styryl dye with cucurbit[8]uril<br />
Denis A. Ivanov, Nikolai Kh. Petrov, Sergey P. Gromov<br />
P115 Pluronic F127-β-cyclodextrins derivatives supramolecular inclusion complexes studied by<br />
fluorescence probe method<br />
Marilena Vasilescu, Daniel G. Angelescu<br />
P116 Fluorescence Dynamics in Supercooled Molten Mixtures<br />
Biswajit Guchhait, Ranjit Biswas<br />
P117 Modulation of Ground and Excited <strong>State</strong> Dynamics of [2,2′-bipyridyl]-3,3′-diol by Micelles<br />
Dipanwita De, Anindya Datta<br />
P118 Fluorescence probes and microrheology with fluorescent particles in investigation of<br />
aggregation behavior in ionic surfactant/non-ionic surfactant/polyelectrolyte system<br />
Tereza Halasová, Dominika Pihíková, Jana Szewieczková, Filip Mravec<br />
P119 Interaction between amphiphilic fluorescent probes and biopolymer<br />
Filip Mravec, Hana Střondalová, Tereza Halasová, Jakub Mondek<br />
P120 I1/I5 ratio of pyrene monomer fluorescence as a new polarity scale <strong>for</strong> solvents and<br />
colloidal systems<br />
Kenichi Nakashima, Dian Liu, Yusuke Yonemura<br />
P121 Role of Solvation Dynamics in the Kinetics of Solvolysis Reactions in Microreactors<br />
Pramod Kumar Verma, Abhinanadan Makhal, Rajib Kumar Mitra, Samir Kumar<br />
P122 Labeling o-dihydroxyphenyl compounds with biocompatible fluorogenic reactions<br />
A. Ulises Acuña, Marta Liras, Francisco Amat-Guerri<br />
P123 Novel Fluorescent Capture Compound <strong>for</strong> Mild Labeling and Visualization of Functional<br />
Subproteomes – Physicochemical Characterization and Biochemical Application<br />
Matthias Baranowski, Thomas Lenz, Marion Herrmann, Oliver Klein, Maik Berg 3 , Mathias<br />
Dreger, Joachim Klose, Hans-Gerd Löhmannsröben, Michael<br />
P124 A fluorescence spectroscopic investigation of the self-assembling properties of the<br />
tyrocidines, a group of antimicrobial cyclic decapeptides<br />
Bhaswati Bhattacharya, Marina Rautenbach<br />
P125 A fluorescence study of the structure and kinetics of eumelanin <strong>for</strong>mation<br />
Jens Sutter, Tereza Bidláková, Vlastimil Fidler, Jan Karolin, David J.S. Birch<br />
P126 Fluorescence investigation of the nucleic acid annealing properties of the HIV-1 Tat protein<br />
Christian Boudier, Roman Storchak, Kamal K. Sharma, Pascal Didier, Nicolas Humbert,<br />
Florian Moser, Jean-Luc Darlix, Yves Mély<br />
P127 Quantification of amino acids and vitamins in cell culture media using Excitation-Emission<br />
Matrix fluorescence spectroscopy and chemometry<br />
Amandine Calvet, Alan G. Ryder<br />
P128 Characterisation and optimisation of fluorescent labelled bacterial glucose-binding protein<br />
in glucose sensing<br />
Jonathan Coulter, Faaizah Khan, Dalibor Panek, Tania Saxl, John Pickup, David Birch<br />
P129 Biophysical Study of Transport Proteins from SLC11 (SoLute Carrier 11) Family<br />
Iva Doležalová, Věra Ňuňuková, Eva Urbánková, Ondřej Novák, Roman Chaloupka<br />
P130 Time-resolved Fluorescence Spectroscopy: A Tool <strong>for</strong> the Design and Evaluation of a<br />
Homogeneous Immunoassay <strong>for</strong> the Detection of GnRH-1<br />
Peter D. Dowd, Jan Karolin, Carol Trager-Cowan, David J.S. Birch, William H. Stimson<br />
P131 Self-association of the hsRad51 recombinase on ssDNA. Studies by combined high<br />
pressure and fluorescence methods<br />
G. Schay, M. Fekete, J. Kardos, M.S.Z. Kellermayer, Cs. Pongor, Cs., J. Fidy<br />
96 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P132 Measurement of Trp-Trp distance using homo-FRET and photobleaching: observation of<br />
RTX toxin folding<br />
Lucia Motlová, Eliška Doktorová, Radovan Fišer, Ladislav Bumba, Ivo Konopásek<br />
P133 Fluorescence properties of new phenothiazinyl-porphyrine derivatives<br />
Emese Gál, Balázs Brém, Luiza Gãina, Tamás Lovász, Castelia Cristea, Luminiţa Silaghi-<br />
Dumitrescu<br />
P134 Front-surface long-wavelength fluorescence immunoassay <strong>for</strong> monensin determination<br />
J. Godoy-Navajas, M. P. Aguilar-Caballos, A. Gómez-Hens<br />
P135 Effect of the medium pH on the fluorescence of betalains<br />
Letícia Christina Pires Gonçalves, Nathana Barbosa Lopes, Bruno Martorelli Di<br />
Genova, Luiz Francisco Monteiro Leite Ciscato, Erick Leite Bastos<br />
P135B Fluorescence polarization approaches to high-throughput screening (HTS) <strong>for</strong><br />
P136<br />
development of natural kinase inhibitors<br />
Nam Joo Kang, Sohee Baek, Ki Won Lee<br />
Fluorescence Lifetime Based Glucose Sensing in Diagnostic Test Strips<br />
Alexa von Ketteler, Dirk-Peter Herten, Wolfgang Petrich<br />
P137 Quantitative Analysis of Yeastolate and eRDF in model cell culture media using<br />
Fluorescence Spectroscopy<br />
Bridget Kissane, Boyan Li, Alan G. Ryder<br />
P138 Mechanistic Insights on Protein Unfolding from Time-Domain Fluorescence<br />
Saswata S. Sarkar, Santosh Jha, Deepak Dhar, Jayant B. Udgaonkar, G.<br />
Krishnamoorthy<br />
P139 Determination of honey’s botanical origin by synchronous fluorescence spectroscopy<br />
Lea Lenhardt, Ivana Zeković, Tatjana Dramićanin, Živoslav Tešić, Dušanka Milojković-<br />
Opsenica, Miroslav D. Dramićanin<br />
P140 Prediction of bioprocess per<strong>for</strong>mance by multidimentional fluorescence spectroscopy<br />
analysis of reactor broths<br />
Boyan Li, Alan G. Ryder<br />
P141 Characterization of P. abyssi NucS-PCNA complex <strong>for</strong>mation with branched DNA<br />
structures using Fluorescence Anisotropy, Fluorescence Resonance Energy Transfer and<br />
SAXS<br />
Alessio Ligabue, Sergey P. Laptenok, Christophe Creze, Joelle Kuhn, Marten H. Vos,<br />
Ursula Liebl, Didier Flament, Hannu Myllykallio<br />
P142 The interplay between terbium/calcium binding and con<strong>for</strong>mational changes in self-splicing<br />
module of FrpC protein<br />
Petra Lišková, Radovan Fišer, Ivo Konopásek<br />
P143 A New Method <strong>for</strong> Long-distance FRET Analysis<br />
Vincenzo Manuel Marzullo, Piotr Bojarski, Leszek Kulak, Katarzyna Walczewska-Szewc,<br />
Anna Synak, Alberto Luini, Sabato D’Auria<br />
P144 Analysis of citrate in low-volume seminal fluid samples using a time-gated measurement of<br />
europium luminescence<br />
Robert Pal, Andrew Beeby, David Parker<br />
P145 Short-distance FRET applied to the polypeptide chain<br />
Ana Rei, Indrajit Gohosh, Roy D'Souza, Amir Norouzy, Maik H. Jacob, Werner M. Nau<br />
P146 Investigation of MADS box intrinsic fluorophores<br />
Barbora Řezáčová, Yves-Marie Coïc, Christian Zentz, Pierre-Yves Turpin, Josef Štěpáne<br />
P147 Medium viscosity effects on the fluorescence of betalains<br />
Ana Clara Beltran Rodrigues, Letícia Christina Pires Gonçalves, and Erick Leite Bastos<br />
P148 Excited state lifetimes of riboflavine derivatives in solution<br />
Yvonne Schmitt, Madina Mansurova, Wolfgang Gärtner, Markus Gerhards<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 97
P149 Fluorescence imaging of (phospho)lipase patterns using differential activity-based gel<br />
electrophoresis (DABGE)<br />
Maria Morak, Bojana Stojčić, Albin Hermetter<br />
P150 Con<strong>for</strong>mational transitions in the Mg 2+ -dependent 7S11 deoxyribozyme, a fluorescence<br />
study<br />
Elisa Turriani, Claudia Höbartner, Thomas M. Jovin<br />
P151 The heart tissues differentiation by time resolved fluorescence spectroscopy<br />
Jonas Venius, Edvardas Žurauskas, Ričardas Rotomskis<br />
P152 Organization of ECF and ABC Transporter Modules During Substrate Translocation<br />
Joanna Ziomkowska, Johanna Heuveling, Daniela Weidlich, Franziska Kirsch, Thomas<br />
Eitinger, Erwin Schneider, Andreas Herrmann<br />
P153 The influence of polymer structure on the cationic polymer – DNA complexes revealed by<br />
time-resolved fluorescence studies<br />
Tiia-Maaria Ketola, Martina Hanzlíková, Linda Cameron, Arto Urtti, Helge Lemmetyinen,<br />
Marjo Yliperttula, Elina Vuorimaa<br />
P154 Poly(β-amino ester) - DNA complexes: time-resolved fluorescence and cellular<br />
transfection studies<br />
Elina Vuorimaa-Laukkanen, Tiia-Maaria Ketola, Jordan J. Green, Martina Hanzlíková,<br />
Helge Lemmetyinen, Robert Langer, Daniel G. Anderson, Arto Urtti, Marjo Yliperttula<br />
P155 Modulation of raft-dependent immune functions by a monoclonal cholesterol-specific<br />
antibody: a biophysical study on the mechanism of action<br />
Andrea Balogh, Emese Izsépi, Anikó Molnár, László Cervenák, George Füst, Zoltán<br />
Beck, György Vámosi, Gábor Mocsár, Glória László, János Matk<br />
P156 The action of chlorogenic acid on erythrocyte and model membranes<br />
Dorota Bonarska-Kujawa, Hanna Pruchnik, Halina Kleszczyńska<br />
P157 Fluorescence studies on new potential antitumoral 1,3-diarylurea derivatives of thieno[3,2b]pyridines<br />
in solution and in nanoliposomes<br />
Elisabete M. S. Castanheira, Cátia N. C. Costa, Ana Rita O. Rodrigues, Daniela Peixoto,<br />
Maria-João R. P. Queiroz<br />
P158 Study of morphology, hydration and mobility of cationic lipophosphoramidates liposomes<br />
by fluorescent solvent relaxation<br />
Damien Loizeau, Piotr Jurkiewicz, Laure Deschamps, Paul-Alain Jaffrès, Martin Hof,<br />
Philippe Giamarchi<br />
P159 Characterization by steady-state and time resolved fluorescence spectroscopy of the lipid<br />
and protein organization in HBsAg particles<br />
Vanille J. Greiner, Caroline Egelé, Sule Oncul, Frédéric Ronzon, Catherine Manin,<br />
Andrey Klymchenko, Yves Mély<br />
P160 Spectroscopic studies of the novel, cationic gluconamide-type surfactants/DNA and lipid<br />
interactions<br />
Teresa Kral, Hanna Pruchnik, Martin Hof, Renata Skrzela, Kazimiera A. Wilk<br />
P161 Effects of supplementation with L-proline or inositol on yeast membrane fluidity and<br />
ethanol tolerance<br />
Safri Ishmayana, Ursula Kennedy, Robert Learmonth<br />
P162 Study of the phase transition of mixtures of original cationic phospholipids by fluorescence<br />
anisotropy<br />
Sélim Mahfoudhi, Damien Loizeau, Laure Deschamps, Paul-Alain Jaffrès,Tristan<br />
Montier, Pierre Lehn, Philippe Giamarchi<br />
P163 Dynamic organization of HIV co-receptors in the membrane of immune cells revealed by<br />
single quantum dot tracking<br />
Patrice Mascalchi, Anne-Sophie Lamort, Evert Haanapel, Fabrice Dumas, Bernard<br />
Lagane, Fernando Arenzana-Seisdedos, André Lopez, Laurence Salomé<br />
P164 Lipid raft detecting in membranes of live erythrocytes<br />
Ilya Mikhalyov, Andrey Samsonov<br />
98 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P165 Domains II and III of Bacillus thuringiensis Cry1Ab toxin remain exposed to the solvent<br />
after Insertion of part of domain I into the membrane<br />
Liliana Pardo-Lopez, Luis Enrique Zavala, Emiliano Cantón, Isabel Gómez, Mario<br />
Soberón, Alejandra Bravo<br />
P166 Quantification of Local Water Concentration in Biomolecules Using Dual-Fluorescence<br />
Labels<br />
Vasyl G. Pivovarenko, Oleksandr M. Zamotaiev, Volodymyr V. Shvadchak, Viktoriia Y.<br />
Postupalenko, Andrey S. Klymchenko, Yves Mély<br />
P167 Biophysical state of plasma membrane and intrinsic efficiency of δ-opioid receptors;<br />
fluorescence spectroscopy studies of HEK293 cells expressing DOR-Giα1 fusion protein<br />
Lenka Roubalova, Pavel Ostasov, Jan Sykora, Jana Brejchova, Martin Hof, Petr<br />
Svoboda<br />
P168 Interactions of α-synuclein with membranes<br />
Volodymyr V. Shvadchak, Lisandro J. Falomir-Lockhart, Dmytro A. Yushchenko,<br />
Thomas M. Jovin<br />
P168B Trans-membrane potential affects lateral microdomain organization of the yeast plasma<br />
membrane<br />
Jaroslav Vecer, Petra Vesela, Jan Malinsky, Petr Herman<br />
P169 Photo-induced modifications of model membranes<br />
Georges Weber, Maurício Baptista, Adjaci F. Uchoa, Rosângela Itri, Carlos M. Marques,<br />
André P. Schröder, Thierry Charitat<br />
Fluorescent Proteins, Fluorescent Probes & Labels<br />
P170 GFP—quantification by fluorescent lifetime measurements<br />
Dagmar Auerbach, Benjamin Hötzer, Gregor Jung<br />
P171 A photoswitching chromoprotein <strong>for</strong> use in pcFRET applications<br />
Craig Don Paul, Daouda AK Traore, Csaba Kiss, Alexander May, Giuseppe Lucarelli,<br />
Rodney J Devenish, Andrew Bradbury, Matthew CJ Wilce, Mark Prescott<br />
P172 Photophysical properties of the Cyan Fluorescent Protein<br />
Asma Fredj, Marie Erard, Agathe Espagne, Germain Vallverdu, Isabelle Demachy,<br />
Jacqueline Ridard, Bernard Levy, Fabienne Mérola, Hélène Pasquier<br />
P173 AmCyan100 - new fluorescent protein <strong>for</strong> cell flow applications<br />
Oleg Guryev, Marta Matvienko, Barny Abrams, Tim Dubrovsky<br />
P174 Pyrimidine derivatives as fluorescent sensors<br />
Sylvain Achelle, Caroline Hadad, Joaquin C. García-Martinez, Julián Rodríguez-López,<br />
Nelly Plé<br />
P175 New long-wave excitable, covalently coupled sensors <strong>for</strong> pH based on photoinduced<br />
electron transfer<br />
Daniel Aigner, Sergey M Borisov, Ingo Klimant<br />
P176 Absorption and fluorescence spectroscopic study of Genistein isoflavone : Towards a<br />
natural hormone replacement therapy<br />
Saadia Ait Lyazidi, M. Haddad, K. Benthami, B. Benneta, S. Shinkaruk<br />
P177 The novel terbium(III) chelate probe <strong>for</strong> hemoglobin determination using resonance energy<br />
transfer<br />
D. Aleksandrova, А.Yegorova, I.Leonenko, V. Antonovich<br />
P178 Spectroscopic investigations of fluorescent dyes and their chemical reactions<br />
Nicole Baltes, Marcel Wirtz, Gudrun Nürenberg, Gregor Jung<br />
P179 Photofluorescent systems based on chromones <strong>for</strong> three-dimensional archival optical<br />
memory<br />
Valery Barachevsky, Olga Kobeleva, Tatyana Valova, Anton Ayt, Igor Martynov,<br />
Konstantin Levchenko, Vladimir Yarovenko, Mikhail Krayushkin<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 99
P180 A molecular-size thermometer<br />
Sara Bonacchi, Daniele Cauzzi, Roberto Pattacini, Massimiliano Delferro, Marco Montalti,<br />
Luca Prodi, Nelsi Zaccheroni, Matteo Calvaresi, Francesco Zerbetto<br />
P181 Phosphorescent platinum(II) and palladium(II) complexes with donor-acceptor Schiff bases<br />
– new red light-excitable indicators <strong>for</strong> oxygen sensing<br />
Sergey M. Borisov, Robert Saf, Roland Fischer, Ingo Klimant<br />
P182 Imaging intracellular viscosity of live cells by fluorescent molecular rotors<br />
Pei-Hua Chung, James A. Levitt, Marina K. Kuimova, Gokhan Yahioglu, Klaus Suhling<br />
P183 Studies of apoptosis and lipid order of cell membranes using new fluorescent probes<br />
Zeinab Darwich, Oleksandr A. Kucherak, Youri Arntz, Pascal Didier, Andrey S.<br />
Klymchenko and Yves Mély<br />
P184 A Novel PET Probe <strong>for</strong> Fluorescent Detection and Cellular Imaging of Hydrogen Peroxide,<br />
and <strong>for</strong> Oxidase-based Enzymatic Assays<br />
Axel Duerkop, Dominik B. M. Groegel, Martin Link, Joachim Wegener, Otto S. Wolfbeis<br />
P185 Excited states of multichromophoric compounds based on 2-aminoanthracene<br />
Miroslav Dvořák, Numan Almonasy, Martin Michl, Miloš Nepraš<br />
P186 The Homodimeric Nucleic Acid Dyes 6-ChloroYOYO-1 and 6-ChloroTOTO-1<br />
John J. Naleway, Ying Jiang, Fiona K. Harlan, Todor Deligeorgiev, Nikolay Gadje, Stefka<br />
Kaloyanova, Nedyalko Lesev, Aleksey Vasilev, Iliana Timcheva, Vera Maximov<br />
P187 Fluorescent Probes <strong>for</strong> Studying the Interactions of Cryoprotective Organic Substances<br />
with Biomembranes<br />
Tatyana S. Dyubko<br />
P188 Synthesis and spectral properties of modified nucleobases bearing multiparametric and<br />
environment sensitive 3-hydroxychromones<br />
Dmytro Dziuba, Iuliia Karpenko, Benoit Michel, Marie Spada<strong>for</strong>a, Rachid Benhida,<br />
Victoria Y. Postupalenko, Volodymyr V. Shvadchak, Andrey S. Klymchenko, Yves Mély,<br />
Alain Burger<br />
P189 Profiling of functional group: an approach to develop a bio-sensitive quencher <strong>for</strong> the<br />
development of turn-on fluorescent probes<br />
Coraline Egloff, Denis Weltin, Alain Wagner<br />
P190 Fluorescent sensor <strong>for</strong> Zn(II) based on coumarin Schiff base derivative<br />
Amel F. El Husseiny, Elham S. Aazam, Huda M. Al Amri<br />
P191 Kinetic analysis of phagosomal production of reactive oxygen species<br />
Asma Tlili, Sophie Dupré-Crochet, Marie Erard, Oliver Nüße<br />
P192 Fluorescence Properties of Thioflavin-T and modeling its nonradiative processes<br />
Yuval Erez, Dan Huppert, Nadav Amdursky<br />
P193 Highly solvatochromic fluorophores based on 7-aryl-3-hydroxychromones<br />
Luciana Giordano, Dmytro A. Yushchenko, Volodymyr Shvadchak, Jonathan A.<br />
Fauerbach, Elizabeth A. Jares-Erijman, Thomas M. Jovin<br />
P194 Influence of Cu + and Cu 2+ cations on luminescent properties of new 3-cyano-4dicyanomethylene-5-oxo-4,5-dihydro-1H-pyrrol-2-olate<br />
anion<br />
Stanislav I. Gurskiy, Viktor A. Tafeenko, Andrey N. Baranov<br />
P195 Synthesis, fluorescence and solvatochromism of novel 3-aminoderivatives of<br />
P196<br />
benzanthrone<br />
Irena D. Ivanova, Natalja Orlova, Elena M. Kirilova<br />
Discovery of full-color tunable and predictable fluorescent core skeleton, 9-aryl-1,2dihydropyrrolo[3,4-b]indolizin-3-one<br />
(Seoul-Fluor)<br />
Eunha Kim, Minseob Koh, Byung Joon Lim, Seung Bum Park<br />
P197 Aminobenzanthrone derivatives as novel fluorescent probes <strong>for</strong> membrane studies<br />
Valeriya M. Trusova, Inta Kalnina, Pavel Fedorov, Elena Kirilova, Georgiy Kirilov, Galyna<br />
P. Gorbenko<br />
100 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P198 Application of Amino Benzanthrone Based Fluorescent Probes <strong>for</strong> Studying of<br />
Erythrocytes under Ozonetherapy Procedures<br />
Tatyana S. Dyubko, Elena Kirilova, Inta Kalnina, Oksana A. Sokolyk, Yuriy Kozin, Nikita<br />
V. Pereverzev<br />
P199 SeTau-647: The brightest label currently available <strong>for</strong> the Kr-ion laser line<br />
Oleksii P. Klochko, Yuliia O. Klochko, Yevgen A. Povrozin, Ewald A. Terpetschnig,<br />
Leonid D. Patsenker<br />
P200 Impact of the rotaxane-based protection on the properties of fluorescent squaraine dyes<br />
Yuliia O. Klochko, Oleksii P. Klochko, Inna Yermolenko, Yevgen A. Povrozin, Ewald A.<br />
Terpetschnig, Leonid D. Patsenke<br />
P201 Functionalized phosphorescent Ir III - and Pt II -porphyrins – <strong>for</strong> bioconjugation and polymer<br />
coupling<br />
Klaus Koren, Sergey M. Borisov, Ruslan Dmitriev, Dmitri B. Papkovsky, Ingo Klimant<br />
P202 Trans<strong>for</strong>mation of chromones into fluorescent ones <strong>for</strong> archival optical memory<br />
M.M. Krayushkin, V.N.Yarovenko, M.A. Kalik, K.S. Levchenko, A. G. Devyatko, G. E.<br />
Adamov, V. A. Barachevsky, T.M. Valova, O.I. Kobeleva<br />
P203 New environment-sensitive fluorescent dyes <strong>for</strong> biological applications<br />
Oleksandr Kucherak, Ludovic Richert, Youri Arntz, Pascal Didier, Yves Mely and Andrey<br />
Klymchenko<br />
P204 ATP sensitive fluorescence turn-on probe based on a pyrene-bipyridinium complex<br />
Thomas Lang, Michael Schäferling<br />
P205 Discovery of new LD (Lipid Dropet) staining fluorescent compounds<br />
Eunha Kim, Sanghee Lee, Seung Bum Park<br />
P206 Turn-on fluorescent probe with activatable internal control<br />
Geoffray Leriche, Ghyslain Budin, Zeinab Darwich, Yves Mély, Andrey Klymchenko, Alain<br />
Wagner<br />
P207 Synthesis of fluorescent probes <strong>for</strong> the non-invasive imaging of cancer hallmarks<br />
Martha Mackay, Mark Bradley<br />
P208 Covalent labeling of His-tagged protein with a “turn-on” fluorescent probe utilizing a<br />
quencher-conjugated histidine peptide<br />
Atsushi Murata, Satoshi Arai, Shinji Takeoka<br />
P209 Phosphorescence based intracellular oxygen sensing probes and assays<br />
Dmitri B. Papkovsky, Alexander V. Zhdanov, Ruslan I. Dmitriev, Andreas Fercher<br />
P210 Impact of hydrophilic groups on fluorescent properties of long-wavelength squaraine dyes<br />
Larysa I. Markova, Sania U. Khabuseva, Yevgen A. Povrozin, Ewald A. Terpetschnig,<br />
Leonid D. Patsenker<br />
P211 Comparison of the reaction rates of strain-mediated click-chemistry reagents<br />
Anatoliy Tatarets, Yevgen A. Povrozin, Leonid D. Patsenker, Ewald A. Terpetschnig<br />
P212 A new strategy towards fluorescent cyclotriveratrylenes<br />
L.Peyrard, S. Chierici, S. Pinet, G. Jonusauskas, P. Meyrand, I. Gosse<br />
P213 Novel nonlinear octupolar merocyanine chromophores<br />
Yevgen Poronik, Mireille Blanchard-Desce, Daniel Gryko<br />
P214 Ratiometric fluorescent probes <strong>for</strong> sensing peptide interactions with nucleic acids and<br />
membranes<br />
Viktoriia Postupalenko, Aleksandr Stryzhak, Volodymyr Shvadchak, Guy Duportail,<br />
Andrey Klymchenko, Vasyl Pivovarenko, Yves Mély<br />
P215 Optical Stimulation and Imaging of Cell Activity with new Optogenetic Probes<br />
Matthias Prigge, Franziska Schneider, Satoshi P. Tsunoda, Peter Hegemann<br />
P216 Novel cationic near infrared fluorescent probes possessing ester, hydroxyl and amino<br />
functional groups in different combinations<br />
Bachu R. Raju, A. Luísa S. Costa, Paulo J. G. Coutinho, M. Sameiro T. Gonçalves<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 101
P217 New fluorescence standards <strong>for</strong> the NIR<br />
Thomas Behnke, Martin Hübner, Christian Würth, Jutta Pauli, Katrin Hoffmann, Angelika<br />
Hoffmann, Ute Resch-Genger<br />
P218 Targeted Luminescent NIR Nanoprobes <strong>for</strong> In Vivo Imaging of Tumor Hypoxia<br />
Ute Resch-Genger, Thomas Benke, Joanna Napp, C. Würth, Lorenz Fischer, Frauke<br />
Alves, Michael Schäferling<br />
P219 Hydroxyacridizinium Ions: A Novel Class of Water-Soluble Photoacids<br />
Katy Schäfer, Heiko Ihmels<br />
P220 Luminescent Properties of Green Emitting Cyclometalated Platinum(II) Complexes<br />
Michael Schäferling, Lorenz Fischer, Cüneyt Karakus, Elisabeth Holder<br />
P221 Long-wavelength Benzodipyrrolenine Based Bis-Cyanine and Bis-Styryl Dyes<br />
Olga Semenova, Oleksii Klochko, Iryna Fedyunyayeva, Saniya Khabuseva, Leonid<br />
Patsenker<br />
P222 2- and 4-[2-(4-Dimethylaminophenyl)-1-ethenyl]-1-alkylpyridinium Tosylates (DSP) as the<br />
Fluorescent Probes <strong>for</strong> Cells<br />
Oksana O. Sokolyk, Tatyana S. Dyubko, Yevgenia I. Smolyaninova, Leonid D. Patsenker<br />
P223 Charge Separation and Charge Recombination Excited <strong>State</strong>s in Pentacoordinated<br />
Complex of Zinc Tetraphenylporphyrin and Axially Bounded Isoquinoline N-oxide Ligand<br />
K.Oberda, I. Deperasińska, Y.Nizhnik, L. Jerzykiewicz, A.Szemik-Hojniak<br />
P224 The charge transfer nature of the emissive state of 3-Aminoflavone<br />
Łukasz Wiśniewski, Irena Deperasińska, Bogusława Żurowska, Anna Szemik-Hojnia<br />
P225 Controllable C=N Isomerization <strong>for</strong> Rational Design of Fluorescent Sensors<br />
Jiasheng Wu, Ruilong Sheng, Weimin Liu, Pengfei Wang<br />
P226 New samarium (III) chelates as an efficient donors <strong>for</strong> fluorescence resonance energy<br />
transfer<br />
А.Yegorova, D. Aleksandrova, I.Leonenko, D.Patsenker, A.Tatarets<br />
P227 Two-color genetically targetable and switchable fluorescent probes<br />
Dmytro Yushchenko, Sujung Kim, Ming Zhang, Qi Yan, Brigitte Schmidt, Christopher<br />
Szent-Gyorgyi, Alan Waggoner, Marcel Bruchez<br />
102 — MAF 12, Strasbourg, France, September 11-14, 2011 —
New Technical Approaches<br />
&<br />
Microfluidics<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 103
104 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P1 New Technical Approaches & Microfluidics Poster 1<br />
Make invisible visible: a Diversity Oriented Fluorescence Library<br />
Approach (DOFLA)<br />
Young-Tae Chang 1,2<br />
1 Department of Chemistry & Med Chem Program, Life Sciences Institute, National University of<br />
Singapore, 3 Science Drive 3, Singapore, 117543, Indonesia<br />
2 Lab of Bioimaging Probe Development, Singapore Bioimaging Consortium (SBIC), 11 Biopolis Way,<br />
# 02-02 Helios, Agency <strong>for</strong> Science, Technology and Research (A*STAR), Biopolis, 138667, Indonesia<br />
With the successful result of Human Genome Project, we are facing the problem of handling<br />
numerous target genes whose functions remain to be studied. In chemical genetics, instead of<br />
using gene knock-out or overexpression as in conventional genetics, a small molecule library is<br />
used to disclose a novel phenotype, eventually <strong>for</strong> the study of gene function. While a successful<br />
chemical genetics work will identify a novel gene product (target protein) and its on /off switch, the<br />
small molecule complement, and thus chemical genetics promises an efficient “two birds with one<br />
stone” approach, the most serious bottleneck of modern chemical genetics is the step of target<br />
identification. The currently popular affinity matrix technique is challenging because the<br />
trans<strong>for</strong>mation of the lead compound into an efficient affinity molecule without losing the biological<br />
activity is not easy, requiring intensive SAR studies. To surrogate the well known problem, our<br />
group has developed a linker tagged library and has successfully identified multiple target proteins<br />
so far. While successful, the affinity matrix technique requires a breakdown of the biological system<br />
to pool the proteins into one extract, which inherently introduce a lot of artifacts, such as dilution<br />
and abolishing the biological environment, etc.<br />
As the next generation of tagged library, we are currently developing fluorescence tagged<br />
libraries <strong>for</strong> in situ target identification and a visualization of the biological events using Diversity<br />
Oriented Fluorescence Library Approach (DOFLA). The basic hypothesis is DOFLA of the same<br />
fluorescence scaffold, but with various diversity elements directly attached around the core, may<br />
selectively respond to a broader range of target proteins in intact biological system and facilitate<br />
the mechanism elucidation and target identification. The high throughput strategy using colorful<br />
chemical genetics <strong>for</strong> stem cell study will be discussed.<br />
References:<br />
1. A fluorescent rosamine compound selectively stains pluripotent stem cells, Im, C. N.; Kang, N. Y.; Ha, H.<br />
H.; Bi, X.; Lee, J. J.; Park, S. J.; Lee, S. Y.; Vendrell, M.; Kim, Y. K.; Lee, J. S.; Li, J.; Ahn, Y. H.; Feng,<br />
B.; Ng, H. H.; Yun, S. W.; Chang, Y. T. Angew. Chem., Int. Ed. Engl., 2010, 49, 7497-7500.<br />
2. Control of muscle differentiation by a mitochondria-targeted fluorophore, Kim, Y. K.; Ha, H. H.; Lee, J. S.;<br />
Bi, X. Z.; Ahn, Y. H.; Hajar, S.; Lee, J. J.; Chang, Y. T. J. Am. Chem. Soc. 2010, 132, 576-579.<br />
3. Diversity-oriented Fluorescence Library Approaches <strong>for</strong> Probe Discovery and Development, Vendrell, M.;<br />
Lee, J. S.; Chang, Y. T. Curr. Opin. Chem. Biol. 2010, 14, 383-389.<br />
4. Synthesis of a bodipy library and its application to the development of live cell glucagon imaging probe,<br />
Lee, J. S.; Kang, N. Y.; Kim, Y. K.; Samanta, A.; Feng, S.; Kim, H. K.; Vendrell, M.; Park, J. H.; Chang,<br />
Y. T. J. Am. Chem. Soc. 2009, 131, 10077.<br />
______________<br />
* Corresponding author: e-mail: chmcyt@nus.edu.sg<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 105
P2 New Technical Approaches & Microfluidics Poster 2<br />
New perspective in background suppression using Matrix Isopotential<br />
Synchronous Fluorymetry.<br />
János Erostyák 1,* & Géza Makkai 1<br />
1<br />
Department of Experimental Physics, Faculty of Sciences, University of Pécs, Ifjúság u. 6., H-<br />
7632 Pécs (Hungary)<br />
Matrix Isopotential Synchronous Fluorimetry (MISF) is a sophisticated data mining method used in<br />
fluorescence spectroscopy. It works on selected subdomains of Excitation-Emission Matrices<br />
(EEM).<br />
MISF is often combined with derivative technique (DMISF), and thus able to eliminate the<br />
contribution of the background emission [1,2]. Un<strong>for</strong>tunately, DMISF is sensitive to noises and<br />
fluctuations, thus the concentration region where it can be efficiently used is limited. Our aim was<br />
to reduce the influences of measurement errors and to increase the sensitivity by at least more<br />
than one order of magnitude.<br />
MISF spectra are constructed using isopotential trajectories in the EEM of background<br />
sample. An interpolating routine is needed to extract the proper excitation and emission<br />
wavelength data of isopotential trajectories from the measured EEM [3]. After differentiating a<br />
MISF spectrum, a background free DMISF spectrum can be got, which is still suffering from the<br />
unavoidable fluctuations of fluorescence intensity data taken from consecutive points of MISF<br />
spectrum. We stepped over this problem calculating intensity differences from not neighbouring<br />
points of MISF spectrum. At the selected points, the fluorescence intensity of the wanted<br />
component is significantly different and this difference is much higher than the intensity fluctuations<br />
of the MISF spectrum.<br />
In this work we show how this technique can be extended to three component analysis,<br />
where the wanted component can be precisely find in the presence of two, independently varying<br />
background components.<br />
At the final <strong>for</strong>m of the evaluation process, when the investigation of a certain complex<br />
sample has been done, only two spectral points are to be selected from the interpolated EEM. It<br />
means, that a very sensitive, extremely fast, almost realtime routine measurement can be<br />
established <strong>for</strong> measuring samples<br />
which have complex fluorescence<br />
spectra.<br />
The Figure shows the 3D positions of<br />
isopotential trajectories of two<br />
background components, named G and<br />
B (simulating Rhodamine G and B). The<br />
two blue lines represent the trajectories’<br />
spectral intersections which are proper<br />
<strong>for</strong> DMISF analysis of the wanted<br />
component.<br />
It was found, that the method is<br />
capable to discriminate fluorophores<br />
being in less than 1:500 fluorescence<br />
intensity ratio compared to each other<br />
even at higher noise level (1–2 %).<br />
This work was supported by the Hungarian National Development Agency, grant No. TÁMOP-<br />
4.2.1.B-10/2/KONV-2010-0002.<br />
References: [1] D.-L. Lin et al., Clinical Chemistry 50:10, (2004) 1797. [2] J.A. Murillo et al., Anal. Chim.<br />
Acta 583 (2007) 55. [3] G. Makkai G, et al., J. Fluoresc. 20 (2010) 87.<br />
______________<br />
* Corresponding author: e-mail: erostyak@fizika.ttk.pte.hu<br />
106 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P3 New Technical Approaches & Microfluidics Poster 3<br />
Spectral phasor analysis allows rapid and easy unmixing of<br />
fluorescence microscopy spectral images<br />
Arjen N. Bader 1 , Farzad Fereidouni 1 & Hans C. Gerritsen 1<br />
1 Department of Molecular Biophysics, Debye Institute, Utrecht University, Utrecht, The Netherlands<br />
Global analysis algorithms based on the phasor representation have been demonstrated to be very<br />
powerful <strong>for</strong> the analysis of lifetime imaging data. Here, we present the analogous approach <strong>for</strong><br />
spectral imaging data. In spectral phasor analysis the fluorescence spectrum of each pixel in the<br />
image is Fourier trans<strong>for</strong>med. Next, the real and imaginary components of the first harmonic of the<br />
trans<strong>for</strong>m are employed as X and Y coordinates in a scatter (spectral phasor) plot. Importantly, the<br />
spectral phasor representation allows <strong>for</strong> rapid (real time) semi-blind spectral unmixing of up to<br />
three components in the image. This is demonstrated on a specimen containing fluorescently<br />
labeled DNA, actin and tubilin. In addition the method is used to analyze autofluorescence of grass<br />
halm cells. The spectral phasor approach can be used on spectral imaging data recorded with 8 or<br />
more spectral channels.<br />
______________<br />
* Corresponding author: e-mail:f.fereidouni@uu.nl<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 107
P4 New Technical Approaches & Microfluidics Poster 4<br />
Analysis of time-gated FLIM data by means of the phasor approach<br />
Farzad Fereidouni 1 , Dave J. van den Heuvel 1 , Jarno Voortman 2 , Erik Hofman 2 & Hans C.<br />
Gerritsen 1<br />
1 Molecular Biophysics, Science Faculty, Utrecht University, Utrecht, the Netherlands<br />
2 Cellular Dynamics, Science Faculty, Utrecht University, Utrecht, the Netherlands<br />
Fluorescence lifetime imaging is a versatile tool which can be utilized to distinguish or identify the<br />
molecular environment. Both time correlated single photon counting (TCSPC) and time gating<br />
methods are used <strong>for</strong> lifetime imaging but to obtain high accuracies high signal-to-noise ratios are<br />
required.<br />
The phasor approach is a graphical global analysis method that increases the S/N ratio of<br />
the analysis. This method simplifies the analysis of FLIM data and avoids difficulties of nonlinear<br />
regression fitting.<br />
It has been successfully employed <strong>for</strong> analyzing both frequency domain and time domain<br />
lifetime images. Time gating detection methods run at very high count rates (~10 MHz) but use of<br />
the phasor approach to analyze the data is complicated by truncation and under sampling of the<br />
decay curve due to the limited number of gates. In this paper we present a modification to the<br />
phasor analyses method that takes into account the cut-off and sampling problem. This approach<br />
is tested on both simulated lifetime images and on real data. We demonstrate that this method can<br />
be applied to retrieve two lifetimes from time gating data that cannot be resolved using standard<br />
(non global) fitting techniques.<br />
______________<br />
* Corresponding author: e-mail:f.fereidouni@uu.nl<br />
108 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P5 New Technical Approaches & Microfluidics Poster 5<br />
Fluorescence and FRET of mass-selected gaseous biomolecules<br />
Rebecca A. Jockusch 1 , Francis O. Talbot 1 , Martin F. Czar 1 , Sandeep K. Sagoo 1 & Huihui Yao 1<br />
1Chemistry<br />
Department, University of Toronto, 80 St. George Street, Toronto, Ontario, M5S 3H6<br />
(Canada)<br />
Characterization of biomolecules and biomolecular complexes in a highly-controlled gas-phase<br />
environment allows valuable simplification of complex biological systems and provides a route to<br />
elucidate the effects of specific non-covalent interactions. We have recently built a flexible interface<br />
<strong>for</strong> doing optical spectroscopic experiments on gaseous molecular ions and clusters, <strong>for</strong>med by<br />
electrospray ionization (ESI), mass selected and stored in a quadrupole ion trap mass<br />
spectrometer (QIT-MS). [1] Here, I present recent results from my laboratory using fluorescence<br />
and photodissociation action spectroscopy to probe the intrinsic photophysics of several small ionic<br />
dye molecules, isolated in the gas phase. In addition, I will present the first spectra and lifetime<br />
measurements showing the occurrence of FRET in gaseous peptides [2].<br />
This work was supported by grants from NSERC (Canada), CFI (Canada) and the Ontario Ministry<br />
of Research and Innovation.<br />
References: [1] Q. Bian, et al., Phys. Chem. Chem. Phys., 12 (2010) 2590. [2] F. O. Talbot, et al., J. Am.<br />
Chem. Soc. 132 (2010) 16156.<br />
______________<br />
* Corresponding author: e-mail: rebecca.jockusch@utoronto.ca<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 109
P6 New Technical Approaches & Microfluidics Poster 6<br />
A comparative time-resolved cuvette system study of two Calcium FRET<br />
sensors: towards making lifetime easier<br />
Romain Laine 1,2,3 , Daniel W. Stuckey 3 , Christopher Dunsby 1 , Alessandro Sardini 3 , David<br />
Carling 3 & Paul M. W. French 1<br />
1 Photonics Group, Physics department, Imperial College London, Prince Consort road, SW7 2AZ, UK<br />
2 Institute of Chemical Biology (ICB), Imperial College London, Exhibition road, SW7 2AZ, UK<br />
3 Medical Research Council (MRC), Clinical Sciences Centre, Faculty of Medicine, Imperial<br />
College, Hammersmith Hospital Campus, Du Cane road, London w12 0NN, UK<br />
We present a study of two FRET (Förster resonance energy transfer) Calcium sensors based on<br />
the non-ubiquitous Troponin C protein [1]. First we applied an analysis, originally developed by<br />
Visser et al. [2], <strong>for</strong> evaluating the distance and the angular separation between two fluorophores in<br />
an established eCFP/Citrine-based calcium FRET sensor and a new purposely created<br />
mTFP/Citrine-based calcium FRET sensor. Our approach directly estimates the fluorophore<br />
orientation factor, rather than assuming random donor-acceptor orientations. A time-resolved<br />
Calcium titration of the two FRET sensors was also carried out. A global analysis was applied to<br />
the obtained dataset, assuming a two population model (one population involved in FRET and one<br />
not involved in FRET).<br />
We conclude that replacing CFP with teal fluorescent protein (mTFP), that exhibits a monoexponential<br />
fluorescence decay, proved to significantly simplify the data analysis by reducing the<br />
complexity of the models and there<strong>for</strong>e increasing the confidence in the outcome. The global<br />
analysis also appears to be an appropriate approach with mTFP there<strong>for</strong>e validating a two<br />
population model.<br />
In order to make samples <strong>for</strong> such cuvette measurements, we also developed a novel<br />
protocol <strong>for</strong> cytosol extraction from mammalian cells. This provides a biologically relevant system<br />
in solution phase that can be readily and rapidly made compared to protein purification.<br />
Experimental measurements were undertaken using a home-built multidimensional<br />
fluorometer resolving fluorescence lifetime, spectrum and polarization [3]. The detection system<br />
combines time-correlated single photon counting (TCSPC, Becker & Hickl, SPC 730) <strong>for</strong> precision<br />
lifetime measurements with a scanning monochromator and automated polarisers <strong>for</strong> spectrally-<br />
and polarization-resolved fluorescence emission analysis. The excitation source is a picosecond<br />
fibre laser-pumped supercontinuum source that is spectrally filtered to provide the optimum<br />
excitation wavelength. Data analysis was per<strong>for</strong>med with discrete exponential models (single,<br />
double or triple exponential models) or the full polarized decay model using the TRFA (SSTC)<br />
software package.<br />
References:<br />
[1] Griesbeck et al. (2004), Journal of Biophysical Chemistry.<br />
[2] Visser et al. (2008), Biophysical Journal.<br />
[3] Manning et al (2008), Journal of Biophotonics.<br />
______________<br />
* Corresponding author: e-mail: paul.french@imperial.ac.uk<br />
110 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P7 New Technical Approaches & Microfluidics Poster 7<br />
Rapid analysis of peptides and isomeric dipeptides using miniaturized<br />
CE with deep UV native fluorescence detection<br />
Qiang Li & Stefan Seeger *<br />
Institute of Physical Chemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich,<br />
Switzerland<br />
Label free detection based on native fluorescence excited at UV region shows great potential <strong>for</strong><br />
the life sciences. It offers simple, low-cost and fast method <strong>for</strong> sensitive detection of important<br />
biological analytes without any modification. [1] In this contribution we present a deep UV<br />
fluorescence lifetime imaging microscopy system (DUV-FLIM) based on a picosecond deep UV<br />
laser using time-correlated single-photon counting method, a miniaturized CE system connected<br />
with this DUV-FLIM system has designed and constructed. [2-4] In order to show the applicability of<br />
this new system <strong>for</strong> biological applications of ultrasensitive detection, several peptides and<br />
isomeric dipeptides have been investigated.<br />
The separation conditions <strong>for</strong> the analysis of five peptides has been validated, separation<br />
was per<strong>for</strong>med using 10 mmol/L phosphate buffer, pH = 2.5, containing 40 μmol/L cationic<br />
surfactant hexadecyl-trimethylammonium bromide (CTAB) and at +4 kV voltage. The analysis time<br />
<strong>for</strong> peptide separation was shorten to 80s by using miniaturized CE system and the detection limit<br />
was 20-40 nmol/L (Fig. 1a). Further, the separation of isomeric dipeptides which having the same<br />
amino acid composition and molecular weight, but different sequences, by miniaturized CE system<br />
has been developed using native fluorescence detection (Fig. 1b). Four pairs of isomeric dipeptide<br />
(Trp-Tyr and Tyr-Trp, Trp-Phe and Phe-Trp, Trp-Val and Val-Trp, Trp-Leu and Leu-Trp) were<br />
investigated, excellent separation selectivity <strong>for</strong> these isomeric dipeptide pairs have been<br />
achieved. It has been demonstration that the pH value of running buffers is key factor which affect<br />
the separation behaviour under selected condition. To explore the potential of using miniaturized<br />
CE with native fluorescence detection <strong>for</strong> peptide analysis, a trpytic digest of BSA was prepared<br />
and separated, over 15 peaks were observed in electropherogram, the number is similar to the 18<br />
peptides containing Trp and/or Tyr expected from a tryptic digest of BSA.<br />
Fluorescence Intensity<br />
4x10 5<br />
3x10 5<br />
2x10 5<br />
1x10 5<br />
5x10 4<br />
4x10 4<br />
3x10 4<br />
2x10 4<br />
1.75x10 4<br />
1.50x10 4<br />
1.25x10 4<br />
1.00x10 4<br />
(a)<br />
A Trp-Gly: 5μg/ml<br />
B Val-Trp: 5μg/ml<br />
C Trp-Tyr: 5μg/ml<br />
D D-Trp-Ala-Trp-D-Phe: 2.5μg/ml<br />
E Trp-His-Trp-Leu-Gln-Leu: 5gμ/ml<br />
10x diluted<br />
100x diluted<br />
A<br />
B C E D<br />
0 20 40 60 80 100<br />
Time (s)<br />
Fluorescence Intensity<br />
pH 3.5<br />
pH 4.0<br />
pH 4.5<br />
Trp-Tyr<br />
Tyr-Trp<br />
Tyr-Trp<br />
Tyr-Trp<br />
20 40 60 80<br />
Time (s)<br />
100 120 140<br />
Figure 1 (a) Separation electropherogram of five peptides. (b) Effect of pH on the separation of dipeptide.<br />
This work was supported by grants from Swiss National Science Foundation.<br />
(b)<br />
pH 3.0<br />
References: [1] Q. Li, S. Seeger, Appl. Spectrosc. Rev. 45 (2010) 12. [2] Q. Li, et al., J Phys. Chem. B 108<br />
(2004) 8324. [3] Q.Li, S. Seeger, Anal. Chem. 78 (2006) 2732. [4] G. K. Belin, et al., Electrophoresis 30<br />
(2009) 2565.<br />
______________<br />
* Corresponding author: e-mail: sseeger@pci.uzh.ch<br />
Tyr-Trp<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 111<br />
Trp-Tyr<br />
Trp-Tyr<br />
Trp-Tyr
P8 New Technical Approaches & Microfluidics Poster 8<br />
A nanosecond-gate-mode-driven silicon-avalanche-photodiode and its<br />
application to measuring fluorescence lifetimes of Ce-doped YAG<br />
ceramics<br />
Tsuyoshi Miyata 1* , Tetsuo Iwata 2 , Susumu Nakayama 3 & Tsutomu Araki 4<br />
1<br />
Department of Mechanical Engineering, Kochi National College of Technology, 200-1 Monobe-<br />
Otsu, Nankoku, Kochi 783-8508, Japan<br />
2<br />
Division of Energy System, Institute of Technology and Science, The University of Tokushima,<br />
2-1 Mimani-jyosanjima, Tokushima 770-8506, Japan<br />
3<br />
Department of Applied Chemistry and Biotechnology, Niihama National College of Technology,<br />
7-1 Yagumo-cho, Niihama, Ehime 792-8580, Japan<br />
4<br />
Division of Bioengineering, Graduate School of Engineering Science, Osaka University,<br />
1-3Machikaneyama, Toyonaka, Osaka 560-8531, Japan<br />
In order to obtain fluorescence lifetime values, we usually use a high-speed and high-gain<br />
photomultiplier tube (PMT) as a photodetector. We however sometimes encounter a situation that<br />
such a high per<strong>for</strong>mance PMT is not necessary. This is the case when measuring fluorescence<br />
samples whose quantum yields are moderately high and whose lifetime values are several tens of<br />
nanoseconds or more. However, use of a versatile photodiode, <strong>for</strong> an example, results in lack in<br />
sensitivity and speed <strong>for</strong> the fluorescence detection. A light detector with intermediate per<strong>for</strong>mance<br />
between the PMT and the photodiode is required.<br />
We think that use of a silicon avalanche photodiode (Si-APD) is suitable <strong>for</strong> such a<br />
requirement. Because the APD is less affected by an external magnetic field than the PMT, it can<br />
be used in a magnetic-resonance-tomography (MRT) environment <strong>for</strong> a purpose of bio-optical<br />
diagnostics. In the previous paper, we reported a pseudo-lock-in light detection system, in which<br />
we operated the Si-APD at a gate mode [1] . With applying a direct-current (dc) reverse-bias voltage<br />
Vr to the APD, the value of which was somewhat below the breakdown voltage Vb, we<br />
superimposed a gate-pulse train of an amplitude Vg with a duty ratio 50 % on the Vr so that the<br />
peak voltage was nearly equal to Vb but so as not to exceed it. Then the current multiplication<br />
factor Mi of the APD was enhanced instantaneously by several tens of times with respect to that of<br />
the conventional dc reverse-biased APD. By using the gated APD, we were able to detect weak<br />
signal light varied in the large background light, otherwise not able to because of unstable<br />
behaviour of the APD. The gate mode drive was carried out easily by using a normal transistortransistor-logic<br />
(TTL) circuit. Recently, we have found that the multiplication factor Mi can be<br />
enhanced further by decreasing the duty ratio less than 50 % or by narrowing the duration tw of the<br />
gate pulse to a nanosecond scale [2] . Because the gated Si-APD plays a role of a sampling unit as<br />
well as a photodetector <strong>for</strong> the repeatable signal light incident on the APD, the proposed scheme<br />
has an advantage in signal-to-noise ratio in measurements in principle.<br />
With these as background, the aim of the present paper is to propose a simple scheme of a<br />
boxcar integrator that incorporates the gated Si-APD that works in a nanosecond region. In order<br />
to demonstrate the basic per<strong>for</strong>mance of the scheme, we have measured fluorescence lifetimes of<br />
cerium-doped yttrium-aluminium-garnet (Ce:YAG) ceramics, in which concentration of Ce is varied.<br />
The Ce:YAG ceramics have been used as materials <strong>for</strong> an ultraviolet-to-visible wavelength<br />
converter in a scintillator [3] and recently as one of the fluorophers in a white LED. They also have<br />
been utilized in a thermo-luminescence sensor by making use of temperature dependency of<br />
fluorescence lifetimes [4] . Thus a simple scheme <strong>for</strong> obtaining fluorescence lifetime values is<br />
required.<br />
References: [1] T. Miyata, et al., Meas. Sci. Technol. 16 (2005) 2453. [2] T. Miyata, et al., Proc. of Optics &<br />
Photonics Japan 2008, 6pP4. [3] E. Mihokova, et al., J. Luminescence 126 (2007) 77.[4] V. Bachmann, et<br />
al., Chem. Mater. 21(2009) 2077.<br />
______________<br />
* Corresponding author: e-mail: miyata@me.kochi-ct.ac.jp<br />
112 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P9 New Technical Approaches & Microfluidics Poster 9<br />
Phase-modulation fluorometry using a phase-modulated light source<br />
Takahiko Mizuno, Yasuhiro Mizutani & Tetsuo Iwata<br />
Division of Energy System, Institute of Technology and Science, The University of Tokushima,<br />
2-1, Minami-Jyosanjimacho, 770-8506 Tokushima (Japan)<br />
We propose a phase-modulation fluorometer (PMF) that incorporates a phase-modulated (PM) excitation<br />
light source. By employing the PM light source, we can obtain frequency sidebands that spread over<br />
around a carrier frequency fc with an interval of a modulation frequency fm. Such an excitation light source<br />
enables us to obtain fluorescence lifetime values of a multi-component fluorescent sample at a time in<br />
line with the conventional procedure used in phase modulation fluorometry.<br />
Fluorescence lifetime measurements are important in a light of enhancing capability of<br />
discriminating samples in fluorescence analysis. By introducing the fluorescence lifetime values, we are<br />
often able to discriminate two fluorescent samples whose spectral shapes are similar to each other,<br />
otherwise not able to. A method <strong>for</strong> obtaining fluorescence lifetime values is divided into two categories: a<br />
time-domain method and a frequency-domain method. Although the time-domain method using a pulsed<br />
excitation light source enables us to obtain a fluorescence decay wave<strong>for</strong>m directly, safety considerations<br />
are necessary <strong>for</strong> biological samples. On the contrary, although the frequency-domain method has<br />
difficulties in obtaining the decay wave<strong>for</strong>m, it seems promising <strong>for</strong> use <strong>for</strong> biological samples. It however<br />
usually requires a high-repetition frequency light source, resulting in somewhat sophistication in<br />
constructing and adjusting the measurement system.<br />
Among the frequency-domain method, however, the conventional phase-modulation fluorometer<br />
(PMF) but employing a recently-available ultraviolet (UV) or a blue light-emitting diode (LED) or a laser<br />
diode (LD) as the excitation light source is easy in construction and simple in operation. Such a PMF<br />
might be useful especially <strong>for</strong> a screening purpose of biological samples. The application of the PMF is<br />
however limited to fluorescent samples whose quantum efficiency are moderately high. This limitation<br />
might be alleviated by a photon-counting PMF [1] . Another limitation is that a fluorescence decay curve<br />
should be expressed by a single exponential function. When the decay curve is expressed by a<br />
summation of multiple exponential ones, we have to use plural modulation frequencies. Even if the decay<br />
curve is expressed by the single exponential function, we have to know the fact in advance. As the result,<br />
we have to use plural modulation frequencies. As one of the solutions <strong>for</strong> such a requirement, we have<br />
proposed a Fourier-trans<strong>for</strong>m PMF [2,3] and a frequency-multiplexed PMF [4,5] . Although the two PMF’s work<br />
well, there still remains a problem in generating excitation wave<strong>for</strong>ms, which is somewhat troublesome.<br />
In the present report, in order to alleviate the problem, we propose a concept of phase-modulation<br />
fluorometry basing on an another idea, that is, to use a phase-modulated (PM) excitation light source. By<br />
introducing the PM excitation light source, we can obtain many sidebands that spread over at the upper<br />
and the lower side of a carrier frequency fc with an interval of a modulation frequency fm, depending upon<br />
a maximum phase deviation Δ φ . The PM wave<strong>for</strong>m as a function of time t is given by<br />
{ 2πf<br />
t + φ cos(<br />
2πf<br />
t)<br />
}<br />
f ( t)<br />
= Acos<br />
c Δ<br />
m .<br />
In order to show the effectiveness of the proposed PMF, we have carried out some numerical<br />
simulations. In order to demonstrate the basic per<strong>for</strong>mance of the PMF, we have constructed the PMF<br />
and have obtained fluorescence lifetime values of fluorescent glasses and that of YAG fluorescent<br />
materials. The proposed PMF is also useful <strong>for</strong> confirming that the fluorescence decay profile is really<br />
expressed by a single exponential function.<br />
This work was supported in part by a Grant-in-Aid <strong>for</strong> <strong>Scientific</strong> Research (B) No. 21300167 from Japan<br />
Society <strong>for</strong> the Promotion of Science (JSPS).<br />
References: [1]T. Iwata, et al., Opt. Rev., 8 (2001) 326. [2] T. Iwata, et al., Opt. Rev., 10 (2003) 31. [3] T.<br />
Iwata, et al., Meas. Sci. Technol., 16 (2005) 2351. [4] T. Iwata, et al., Appl. Spectrosco., 61 (2007) 950. [5] T.<br />
Iwata, et al., Appl. Spectrosco., 63 (2009) 1256.<br />
______________<br />
* Corresponding author: e-mail: iwata@me.tokushima-u.ac.jp<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 113
P10 New Technical Approaches & Microfluidics Poster 10<br />
On the spectral sensitivity calibration of fluorescence spectrometers:<br />
extension to the NIR, polarization and grating effects<br />
Sebastian Tannert, Peter Kapusta, Alexander Glatz, Alexander Goschew, Uwe Ortmann<br />
& Rainer Erdmann<br />
PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, (Germany), info@picoquant.com<br />
Reliable and traceable correction of recorded emission spectra is an important and un<strong>for</strong>tunately<br />
often overlooked procedure. For example measuring quantum yields, calculating Stokes shifts, or<br />
discussing the shape of emission bands is meaningless without taking into account the wavelength<br />
and polarization plane dependent sensitivity of the complete detection system.<br />
Recording correct emission spectra without excitation and emission polarizers assumes<br />
completely depolarized emission. However, this is a strong assumption, not always valid <strong>for</strong> fluid<br />
solutions and certainly invalid <strong>for</strong> solid samples. There<strong>for</strong>e, to record the true emission spectra the<br />
grating polarization bias has to be avoided by the proper use of polarizers.<br />
We present data illustrating that correction factors vary widely <strong>for</strong> different emission<br />
polarization states, and different gratings in the same monochromator. There<strong>for</strong>e, a single<br />
sensitivity correction curve is not applicable <strong>for</strong> all measurements. With the existing emission<br />
standards <strong>for</strong> the visible range obtaining all required correction curves is feasible.<br />
However, with the advent of new, far-red sensitive photomultipliers (sensitive up to 900nm), new<br />
calibration standards are necessary.<br />
114 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P11 New Technical Approaches & Microfluidics Poster 11<br />
Bimolecular photochemistry under microfluidic conditions<br />
K.-H. Feller 1* , S. Harz 1 & M. Schimmelpfennig 1<br />
1University<br />
of Applied Sciences Jena, Instrumental Chemical Analysis Group, D-07745 Jena<br />
(Germany)<br />
Aim of the presented work is to investigate photochemical and photophysical conversion processes<br />
under microfluidic conditions with an ultrasensitive optical measurement system <strong>for</strong> fluorescence<br />
detection.<br />
The system consists of two inlets, a micromixer <strong>for</strong> homogenizing the fluids, a reaction<br />
volume (delay segment), excitation as well as detection windows and an outlet (Figure 1). In case of<br />
photochemical conversion the mixture is excited within the mixing chamber (in our case a 405 nm,<br />
190 mW blue diode laser is used as excitation source).<br />
Various photochemical processes can be<br />
investigated under microanalytical conditions. After<br />
excitation with a blue diode laser the photochemistry<br />
in the sample is checked at one detection spot with<br />
a mini-spectrometer. Under these conditions the<br />
temporal evolution of the photochemical conversion<br />
can be followed by different means. The main<br />
advantage of the system is that the delay time<br />
between excitation and detection can be tuned by<br />
means of changing flow speed. In this case the<br />
photo-product efficiency of the bimolecular<br />
photochemical reaction (e.g. in 4-dimethylamino-4’nitrostilbene,<br />
DANS) is combined with the efficiency<br />
of the mixing process bringing the two components<br />
efficiently together (micro mixer).<br />
After excitation with the blue diode laser the<br />
excimer fluorescence of DANS was shown in the<br />
700 nm region under the fluorescence spectrum of<br />
the all-trans DANS monomer. This excimer<br />
fluorescence should be efficiently quenched by the<br />
tri-molecular interaction with dimethyl-fumarate<br />
trans<strong>for</strong>ming the stilbene molecule in the excited state into an oxetane photoproduct.<br />
all-trans DANS* � all-trans DANS + hν‘<br />
Figure: Plat<strong>for</strong>m <strong>for</strong> photochemical analysis of<br />
bimolecular photochemical reactions<br />
405 nm<br />
all-trans DANS* + all-trans DANS � (all-trans DANS)2*<br />
Alternatively, the dimethyl-fumarate reacts within sub-seconds with the DANS monomer. So, the<br />
photo product <strong>for</strong>mation with the excimer should be the more effective process by adding (mixing)<br />
the dimethyl-fumarate within milliseconds after the creation of the excimer. Our results show that<br />
the presented measurement system is suitable <strong>for</strong> photochemical reaction excitation, control and<br />
detection.<br />
______________<br />
* Corresponding author: e-mail: feller@fh-jena.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 115
P12 New Technical Approaches & Microfluidics Poster 12<br />
Picosecond fluorescence of out-of-equilibrium biomolecular complexes<br />
<strong>for</strong>med in microfluidic devices<br />
Sacha Maillot, Alain Carvalho, Jean-Pierre Vola, Stefan Haacke & Jérémie Léonard *<br />
Institut de Physique et Chimie des Matériaux de Strasbourg, CNRS - Université de Strasbourg<br />
UMR 7504, 23, Rue du Loess, 67034 Strasbourg (France).<br />
In this project, microfluidic mixers are used to measure the kinetics of chemical reactions by<br />
producing out-of-equilibrium initial conditions in water-in-oil droplets of typically few 100 pL in<br />
microchannels. The technique permits a rapid mixing (≤ 1 ms) of reagents and a very low material<br />
consumption [1] . Microfluidic devices are made of Poly-(dimethylsiloxane) (PDMS) and are<br />
fabricated by rapid prototyping [2] . Microfluidic chips of typical cross-sectional dimensions 50 µm ×<br />
50 µm or 100 µm × 50 µm are fabricated.<br />
The reagent mixing time in the droplets limits the time resolution that can be achieved to<br />
investigate relaxation kinetics. To characterize it, we mix fluorescein with iodide quencher ions and<br />
we monitor the fluorescence quenching on a picosecond timescale with a streak camera, along the<br />
microfluidic flow (see Figure). A mixing time of 6 milliseconds is obtained in this example. The<br />
measurement of molecular complex <strong>for</strong>mation kinetics, which are slower than the diffusion limited<br />
mixing, is in progress. In this case, fluorescence lifetime measurement will reveal structural<br />
relaxation of the (bio)molecular complex along propagation in the microchannels.<br />
Figure – (a) Fluorescein emission is quenched by iodide ions in water-in-oil droplets. The<br />
fluorescence lifetime measurements along droplets propagation reveals the mixing time. A<br />
horizontal laser beam is sent onto the microfluidic device and intersects it in several parts of the<br />
microchannel, showing a different fluorescence decay time (b). The first trace (c) is the<br />
fluorescence decay signal of non-quenched fluorescein (4.1 ns decay time). The mean decay time<br />
from the fourth trace on is ~160 ps. Knowing the flow velocity (83 mm/s), we infer a 6 ms mixing<br />
time in these channels of 50 × 35 µm 2 cross section.<br />
Supported by the CNRS call “Interface Physique, Chimie, biologie : soutien à la prise de risque” –<br />
2009<br />
References:<br />
[1] H. Song, et al., Angew. Chem. Int. Ed., 42 (2003) 767; H. Song, et al., Appl. Phys. Lett., 83 (2003) 4664.<br />
[2] D.C. Duffy, et al., Anal. Chem. 70 (1998) 4974.<br />
______________<br />
* Corresponding author: e-mail: leonard@ipcms.u-strasbg.fr<br />
116 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P13 New Technical Approaches & Microfluidics Poster 13<br />
Fluorescent chemical sensing in microfluidic miniaturised<br />
analytical plat<strong>for</strong>ms<br />
Leonid Gitlin, Stefan Jezierski, Georg Jochem, Christian Höra & Stefan Nagl*<br />
Institute of Analytical Chemistry, University of Leipzig, Johannisallee 29, 04103 Leipzig (Germany)<br />
The integration of fluorescent chemical sensors and biosensors into microfluidic analytical systems<br />
("lab on a chip") is a very promising approach since it allows simultaneous exploitation of the<br />
advantages of both areas. Such microdevices are capable of fast and continuous on-line analyte<br />
monitoring avoiding elaborate off-line analysis steps.<br />
We are currently working on the application of fluorescent oxygen sensors in microfluidic<br />
reaction plat<strong>for</strong>ms. We integrated polystyrene-based oxygen sensing layers containing a platinum<br />
porphyrin dye into a microreaction layout displayed in Fig. 1 and demonstrated its per<strong>for</strong>mance<br />
using oxygen-consuming chemical reactions. In the first step, a star shaped structure was wet<br />
etched into a glass slide utilising a photoresin as mask material. After the introduction of a<br />
polymeric oxygen sensor layer into the glass grooves, a fluidic structure was fabricated from an<br />
acrylic photosensitive polymer on top of it. Finally, by means of a photocure bonding technique it<br />
was sealed with a glass slide containing fluidic connections. As a model <strong>for</strong> biosensing,<br />
immobilisation of glucose oxidase was carried out and the chips were used <strong>for</strong> glucose monitoring.<br />
Another area of interest is in the combination of fluorescent chemical sensing and micro free-flow<br />
electrophoresis (μ-FFE), which is a mild separation technique frequently used <strong>for</strong> preparative<br />
fractionation. However, since its separation efficiency is only moderate, precise monitoring of its<br />
parameters is required. We developed a multistep liquid phase lithography method <strong>for</strong> μ-FFE chip<br />
fabrication that allows straight<strong>for</strong>ward integration of sensing polymer structures. Using<br />
photodefinable polyethyleneglycol acrylates, microfluidic devices with hydrophilic channels of only<br />
25 µm in height and ion-permeable segregation walls were generated. Acrylic layers containing<br />
pH-sensitive dyes were integrated and first results in μ-FFE isoelectric focusing with on-line pH<br />
monitoring are presented.<br />
This work is supported by grants from the Deutsche Forschungsgemeinschaft (DFG) and the<br />
European social fund (ESF) of the European Union.<br />
______________<br />
* Corresponding author: e-mail: nagl@chemie.uni-leipzig.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 117
P14 New Technical Approaches & Microfluidics Poster 14<br />
Chemiluminescence determination of amino acids and aminoglycoside<br />
antibiotics using an on-chip µ-fluidic device<br />
Marina Sierra-Rodero, Juan Manuel Fernández-Romero & Agustina Gómez-Hens<br />
Departament of Analytical Chemistry. Institute of Fine Chemistry and Nanochemistry (IUIQFN-<br />
UCO) Campus de Rabanales. Marie Curie Building (Annex) University of Córdoba<br />
E-14071-Córdoba, Spain. Phone: 34-957218645, Fax: 34-957218644, email: q22sirom@uco.es.<br />
Web: http://www.uco.es/investiga/grupos/FQM-303.<br />
Microfluidic devices have become of interest due to their special features such as low reagent and<br />
sample consumptions, simple manipulations, low analysis time and suitability <strong>for</strong> miniaturized<br />
systems [1]. Chemiluminescence (CL) is a highly sensitive detection approach, characterized by<br />
simple optical systems and extremely low background noise. These features have given rise to the<br />
development of several on-chip analytical systems with CL detection [2]. A new on-chip µ-flow<br />
injection (µFI) approach <strong>for</strong> the determination of amino acids and aminoglycoside antibiotics using<br />
CL detection is described.<br />
The method is based on the inhibition of the CL emission occurred from the reaction between<br />
luminol (3-aminophthalhydrazide) and H2O2, catalyzed by Cu(II). This CL depletion was caused by<br />
the presence of amino acids or aminoglycoside antibiotics, which <strong>for</strong>m complexes with the Cu(II)<br />
decreasing the catalytic effect [3]. The degree of CL suppression was proportional to the analyte<br />
concentration. The optimal conditions <strong>for</strong> the indirect CL detection were determined with regard to<br />
chemical, physical and hydrodynamical variables. The delivery of sample and reagents are driven<br />
flow-through the microfluidic device using syringes pump as convenience. The CL emission was<br />
collected using an X-Y-Z focused optic fibre bundler connected to a luminescence detector.<br />
L + S: luminol + sample<br />
C: carrier<br />
CS: cleaning solution<br />
CLD: chemiluminescence<br />
detection<br />
OF: optic fibre<br />
SDS: syringe driven system<br />
118 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
The analytical usefulness of<br />
the system has been assessed by its<br />
application to the determination of<br />
cysteine (cys) which was chosen as<br />
analyte model. The dynamic range of<br />
the calibration graph was 0.88 –<br />
16.51 µmol L -1 , <strong>for</strong> cysteine with a<br />
detection limit of 0.26 µmol L -1 . The<br />
precision of the method, expressed<br />
as relative standard deviation (RSD)<br />
was 0.20 %. The features of the<br />
method <strong>for</strong> monitoring<br />
aminoglycosides (i.e. neomycin and<br />
amikacin) on the system have been<br />
also studied, showing the following<br />
dynamic ranges of the calibration<br />
graphs, limits of detection (LOD) and<br />
precision data: 0.32 – 3.30 µmol L -1<br />
(LOD of 0.01 µmol L -1 , RSD of 0.05 %) <strong>for</strong> neomycin and 0.82 – 8.50 µmol L-1 (LOD of 0.24 µmol<br />
L-1, RSD of 0.15 %) <strong>for</strong> amikacin.<br />
This work has been supported from the Spanish Ministerio de Ciencia e Innovación, MICINN<br />
(Grant No. CTQ2009-08621/BQU) and from the Junta de Andalucía and the FEDER-FSE Program<br />
(Grant No. PO9-FQM-4433).<br />
References: [1] G.M. Whitesides., Nature, 442 (2006) 368. [2] B. Kuswandi, et al., Anal. Chim. Acta, 601<br />
(2007) 141. [3] S.Y. Liao, et al., J. Chromatogr. A, 736 (1996) 247.
Fluorescence Correlation Spectroscopy<br />
and<br />
<strong>Single</strong> Molecule<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 119
120 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P15 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 15<br />
Actin dynamics within dendritic spine investigated by two photon<br />
fluorescence correlation spectroscopy<br />
Jianhua Chen 1 , Yves Kellner 2 , Marta Zagrebelsky 2 , Martin Korte 2 & Peter Jomo Walla 1, 3*<br />
1<br />
AG Biomolecular Spectroscopy and <strong>Single</strong>-Molecule Detection, Max Planck-Institute <strong>for</strong><br />
Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany<br />
2<br />
Division of Cellular Neurobiology, Zoological Institute, TU Braunschweig, 38106 Braunschweig,<br />
Germany<br />
3<br />
Department of Biophysical Chemistry, Institute <strong>for</strong> Physical and Theoretical Chemistry, Technical<br />
University of Braunschweig, Hans-Sommer-Strasse 10, 38106 Braunschweig, Germany<br />
Fluorescence correlation spectroscopy (FCS) combined with fluorescence microscopy to monitor<br />
both the temporal and spatial in<strong>for</strong>mation is necessary. FCS also known as fluorescence<br />
fluctuation spectroscopy is a technique with single molecule sensitivity [1]. Some biophysical<br />
properties, such as diffusion time, concentration and particle numbers could be derived by autocorrelating<br />
or cross correlating the fluctuation of the fluorescence molecules within the detection<br />
focal volume.<br />
Dendritic spines are tiny membrane protrusions that <strong>for</strong>m the postsynaptic part of most<br />
excitatory synapses on neurons in the nervous system. Activity induced changes in the shape and<br />
sizes of dendritic spines are correlated with changes in the strength of excitatory synaptic<br />
connections [2] and are believed to play a critical role in learning and memory processes. Dendritic<br />
spines are highly enriched in actin [3] and their <strong>for</strong>ms as well as their function are critically<br />
dependent on the actin cytoskeleton. In this study we used FCS combined with fluorescence<br />
microscopy to analyze the actin dynamics within single spines upon chemically induced changes in<br />
synaptic activity. Two photon excitation was used as the light source <strong>for</strong> FCS in our setup (Fig.1) to<br />
get a well-confined observation area with the same volume as single spine head. In order to<br />
monitor the morphological changes of single spines, a full field fluorescence microscope was built<br />
to enable the simultaneous acquisition of image and spectroscopy.<br />
568 nm<br />
laser<br />
BE<br />
2 P-FCS<br />
References: [1] K. Bacia, et al., Nat Method 3 (2006) 83. [2] A. Holtmaat, et al., Nature Rev Neurosci 10<br />
(2009) 647. [3] K. Okamoto, et al., Nature Neurosci 7 (2004) 10.<br />
______________<br />
* Corresponding author: e-mail: pwalla@gwdg.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 121<br />
CCD<br />
IF<br />
IF<br />
1 P-FM
P16 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 16<br />
Multi-confocal fluorescence correlation spectroscopy to study heat<br />
shock response of living cells<br />
Meike Kloster 1 , Gaétan Herbomel 2 , Yves Usson 3 , Irène Wang 1 , Claire Vourc'h 2 , Catherine<br />
Souchier 2 & Antoine Delon 1,*<br />
1 LIPhy, UMR 5588 CNRS, Université de Grenoble 1, BP 87, 38402 Saint Martin d'Hères<br />
2 Equipe 10, CRI U823 INSERM, Université de Grenoble 1, IAB, BP 170, 38042 Grenoble Cedex 9<br />
3 TIMC-IMAG, UMR5525 CNRS, Université de Grenoble 1, 38706 La Tronche Cedex (France)<br />
Fluorescence correlation spectroscopy (FCS) is a common tool <strong>for</strong> studying concentrations,<br />
mobility and dynamic interactions of molecules in living cells. This method is taking advantage of<br />
the intensity fluctuations caused by a small number of fluorescent particles crossing the<br />
observation volume of a confocal microscope. However, it is usually limited to a single confocal<br />
volume, so that in<strong>for</strong>mation can only be obtained from one position at a time. A technique that<br />
allows simultaneous FCS measurements in different locations within a cell is of great interest <strong>for</strong><br />
the study of dynamical properties of cellular proteins. We present a multi-confocal FCS setup that<br />
takes advantage of a Spatial Light Modulator (SLM) to create multiple excitation spots in an<br />
adjustable spatial configuration [1] . Parallel detection is per<strong>for</strong>med using an Electron-multiplied CCD<br />
camera. Each pixel of the camera acts as a standard pinhole, enabling confocal detection in<br />
parallel. This multiplexed confocal strategy is applied to study the cellular response to thermal<br />
stress [2] , by labelling the transcription factor HSF1 (see Fig. 1).<br />
G(τ)<br />
2,0<br />
1,8<br />
1,6<br />
1,4<br />
1,2<br />
1,0<br />
10 1<br />
Be<strong>for</strong>e heat shock (25 spots)<br />
10 2<br />
122 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
10 3<br />
After heat shock (34 spots)<br />
τ (μs)<br />
Fig. 1: averaged autocorrelation functions, corresponding to ca. 300 acquisitions measurements per<strong>for</strong>med<br />
over 3 Hela cells (HSF1-eGFP).<br />
This work was supported by grant from ANR under contract ANR-08-PCVI-0004-01 and by the<br />
Région Rhône-Alpes (CIBLE 2009).<br />
References: [1] R. Galland, et al., Front. Biosci., 3 (2011) 476. [2] A. Eymery, et al., Exp. Cell Res. 316<br />
(2010) 1845.<br />
10 4<br />
______________<br />
* Corresponding author: e-mail: antoine.delon@ujf-grenoble.fr<br />
10 5<br />
10 6
P17 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 17<br />
Anomalous diffusion in lipid membranes and associated proteins:<br />
experiments and simulations using FRAP and FCS at different radii<br />
Jean Louis Meunier 2 , Pierre François Lenne 3 , Hervé Rigneault 1 & Cyril Favard 1*<br />
1 Institut Fresnel, CNRS UMR 6133, Marseille (France)<br />
2 Institut Non Linéaire de Nice, CNRS UMR 6618, Sophia Antipolis (France)<br />
3 Institut de Biologie du Développement de Marseille Luminy, CNRS UMR 6216, Marseille (France)<br />
Anomalous diffusion is widely used to describe and analyze molecular motions in and on the<br />
surface of living cells. It has been shown <strong>for</strong> more than thirty years now that lipids and proteins of<br />
the plasma membrane exhibit non-Brownian motion. Indeed, in the mid 90’s, Feder et al. 1 , first fit<br />
their FRAP experiments using anomalous diffusion to interpret qualitatively the deviation to the<br />
Brownian behavior. A lot of work has been done until now, both numerically and experimentally<br />
with the help of different techniques such as FRAP, FCS and SPT. Nevertheless, if any of the<br />
experimental data can be correctly analyzed using anomalous diffusion approach, no structural<br />
in<strong>for</strong>mation can be obtained.<br />
An alternative approach is to use FRAP or FCS at different radii in order to explore the<br />
molecular motions. This approach gives “diffusion laws” that have been shown to be different<br />
depending on the deviation to the Brownian motion 2,3,4 .<br />
In this study, we have tried to gather these approaches and we have analyzed the variation<br />
of the anomalous diffusion characteristic exponent (α) as a function of the explored radii. If theory<br />
predicts that this exponent is constant at different space scale, here it is shown that both on FCS<br />
simulations and experiments, using a lipid mixture known to exhibit fractal dimensions at the phase<br />
transition, α decreases linearly with the inverse of the explored radii. This is confirmed on pure<br />
numerical simulations mimicking FRAP.<br />
More interestingly, experimental results obtained by FRAP on a PH domain (EFA6) in living<br />
cells strangely show an opposite behavior <strong>for</strong> α. Hence, its dynamics is better explained by an<br />
“open domains” model as described in Salomé et al. 2 instead of anomalous diffusion.<br />
References:<br />
[1] Feder TJ et al., Biophys J., 70 (1996) 2767.<br />
[2] Salomé L et al., Eur Biophys J. 27, (1998), 391.<br />
[3] Wawrezinieck L et al., Biophys J. 89, (2005), 4029.<br />
[4] Favard C et al., Biophys J., 100, (2011), 1242.<br />
______________<br />
* Corresponding author: e-mail: cyril.favard@fresnel.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 123
P19 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 19<br />
Evaluation of radiation <strong>for</strong>ce under the resonance optical trapping<br />
condition using Fluorescence Correlation Spectroscopy<br />
Hiroaki Yamauchi 1 , Masataka Taguchi 1 , Syoji Ito 1 & Hiroshi Miyasaka 1,*<br />
1 Division of Frontier Materials Science, Graduate School of Engineering Science and Center <strong>for</strong><br />
Quantum Science and Technology under Extreme Conditions, Osaka University,<br />
1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan<br />
Optical trapping is a powerful tool <strong>for</strong> manipulating microscopic particles in solution without<br />
mechanical contact. In recent years, the targets of optical trapping have been downsized from<br />
micrometer-scaled objects to nanoparticles [1,2]. However, the difficulty arises with decreasing size<br />
of trapped objects because the radiation <strong>for</strong>ce is in proportion with the volume of a particle. To<br />
overcome this difficulty, it has been proposed that the radiation <strong>for</strong>ce is enhanced under the<br />
resonance condition [3]. With an aim to experimentally confirm this prediction, we have applied<br />
fluorescence correlation spectroscopy (FCS) <strong>for</strong> the elucidation of translational diffusion<br />
behaviours of molecules under the resonance optical trapping condition. From the average number<br />
of molecules in the confocal volume, we can estimate the potential depth of the radiation <strong>for</strong>ce and<br />
evaluate the effect of the resonance optical trapping.<br />
We prepared aqueous solutions of a double-stranded DNA with a green-dye (Alexa488) and<br />
a red dye (Alexa647) at each end as shown in scheme1. The green dye was used as a probe<br />
molecule <strong>for</strong> FCS measurement, while the red one with a relatively large absorption cross-section<br />
was applied <strong>for</strong> the resonant optical trapping through the absorption under the irradiation of a red<br />
laser beam. Two CW lasers at 488 and 633 nm were respectively used <strong>for</strong> the excitation of the<br />
green dye and the resonant trapping of the red dye.<br />
scheme1<br />
A series of FCS measurement revealed that the local concentration of DNA molecules<br />
increased with increasing power of the red laser, whereas the photon-counting-rate decreased with<br />
an increase in the red laser power. Picoseconds transient absorption measurements revealed that<br />
the decrease of the photon-counting-rate was due to the stimulated emission of a green dye under<br />
the irradiation of red laser beam. Because the stimulated emission is not taken into consideration<br />
in the conventional FCS analysis, we examined the effect of the stimulated emission on an<br />
autocorrelation curve by Brownian Dynamics Simulation (BDS) [4]. The result of numerical<br />
simulation showed that the local concentration of molecules in the detection volume of FCS<br />
increased apparently even if the trapping potential was not taken into consideration. By integrating<br />
these result, we evaluated the effect of resonance trapping, in terms of the concentration of<br />
molecules at the trapping point.<br />
At the conference site, we will discuss the enhancement of the radiation <strong>for</strong>ce under the<br />
resonance optical trapping condition by introducing the experimental and computational results.<br />
References: [1] S. Ito, et al., Appl. Phys. Lett., 78 (2001) 2566. [2] S. Ito. et al., Appl. Phys. Lett., 80 (2002)<br />
482. [3] R. R. Agayan, et al., Appl. Opt. 41 (2002) 2318. [4] S. Ito, et al., Phys. Rev. E. 81 (2010) 061402.<br />
______________<br />
* Corresponding author: e-mail: miyasaka@chem.es.osaka-u.ac.jp<br />
124 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P20 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 20<br />
Photophysics of photoacids based on pyrene<br />
Christian Spies * , Michael Vester & Gregor Jung<br />
Biophysical Chemistry, Saarland University, Saarbrücken, Germany<br />
The transfer of a proton to a base is one of the most important chemical reactions. Especially,<br />
photoexcitation of aromatic alcohols, leading to an enhanced acidity, served as paradigm <strong>for</strong> this<br />
kind of reaction [1]. One of the most widely used photoacids is Hydroxypyrene-trisulfonate (HPTS)<br />
and its even more acidic sulfonamide derivative (HPTA) [2]. Because of the low photostability of<br />
HPTS, which is its main drawback, we are searching <strong>for</strong> new alternatives based on pyrene by<br />
means of solvatochromism [3, 4]. Photostability, which is also important <strong>for</strong> single-molecule<br />
spectroscopy, is investigated by fluorescence correlation spectroscopy (FCS) [4]. The visualization<br />
of the proton transfer reaction on the single molecule level is the long-term goal in our group.<br />
Solvatochromism studies were expanded towards further photoacids in this presentation. We<br />
determined the solvent dependence of the excited-state proton transfer (ESPT) of several<br />
photoacids. Two different models <strong>for</strong> considering of different solvent effects were compared, i.e.<br />
Kamlet-Taft and Catalán analyses. The kinetics of this fundamental reaction was also investigated<br />
by triggering it with picosecond laser pulses. DFT-calculations using a combined explicit/implicitsolvent<br />
model offer deeper insight in the solvatochromic behaviour. In this model the solute and a<br />
few solvent molecules are treated quantum mechanically and are located in a cavity of a<br />
polarizable continuum, which comprises all the other solvent molecules.<br />
The rate constants of protonation and deprotonation on the ground state of the anionic<br />
species were determined by FCS with two approaches. The dependence of the rate constants on<br />
the buffer concentration as well as the pH-value of the solution was investigated and compared to<br />
known reaction rate constants. An alternative analytical model which takes the lowered<br />
photobasicity of the anionic chromophore into account is presented.<br />
References: [1] O.F. Mohammed, et al., Angew. Chem. Int. Ed., 46 (2007) 1458. [2] D. Spry, M. Fayer,<br />
J.Chem.Phys. 127 (2007) 204501. [3] G. Jung, et al., PhysChemChemPhys 11 (2009) 1416. [4] B.<br />
Hinkeldey, et al., ChemPhysChem 9 (2008) 2019.<br />
______________<br />
* Corresponding author: e-mail: c.spies@mx.uni-saarland.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 125
P21 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 21<br />
Self-assembly of the immuno-active GTPase hGBP1 as monitored by<br />
ensemble time correlated single photon counting and single molecule<br />
FRET<br />
Y. Ajaj 1 , Peulen 1 , C. S. Hengstenberg 2 , T., M. Richrt 1 , A. Valeri 1 , C. Herrmann 2 & C. A. Seidel 1<br />
1<br />
Heinrich-Heine-Universitaet Duesseldorf, Institut fuer Physikalische Chemie II, Lehrstuhl fuer<br />
Molekulare Physikalische Chemie, Universitaetstr. 1, 40225 Duesseldorf, Germany<br />
2<br />
Ruhr-Universitaet Bochum, Physikalische Chemie I -AG Proteininteraktionen, Universitaetstr. 150,<br />
44787 Bochum, Germany<br />
The human guanylate binding protein-1 (hGBP1) belongs to the family of dynamin-related large<br />
GTP-binding proteins. The common property of hGBP’s is the ability to undergo oligomerization as<br />
a function of GTP binding and hydrolysis. hGBP1 consists of an LG (Large G domain) and an<br />
elongated α-helical domain. Studies of in vitro self-assembly of hGBP1 have shown different<br />
oligomeric structures. In addition, it was proposed that the self-assembly of hGBP1 may involve the<br />
movement of helix α12, and α13 with respect to the middle/LG-domain.<br />
Here we address the nucleotide-dependent structural changes of full-length hGBP1 in the<br />
presence of analogue nucleotides (GppNHp/GTPγS or GDP AlFx). First, cysteines were introduced<br />
at distinct positions (N18C, Q344C, T481C, A496C, Q525C, V540C and D577C) by site-directed<br />
mutagenesis <strong>for</strong> maleimide labelling. Each double mutant was labelled with a FRET dye pair<br />
(Alexa 488 as a donor dye and Alexa 647 as an acceptor dye) <strong>for</strong> intra-FRET measurements.<br />
<strong>Single</strong> mutants where also labelled with either of the dyes <strong>for</strong> resolving the oligomeric states of<br />
hGBP1. Ensemble time correlated single photon counting and single molecule FRET were<br />
employed to obtain inter- and intra- distances of fluorophores. With the measured distance<br />
constraints a structural model of the protein self-assembly is presented.<br />
References: [1] Vöpel T., Kunzelmann1 S. and Herrmann C., FEBS Lett., 583 (2009) 1923.; [2] Prakash B.,<br />
Prakash B., Praefcke G. J., Renault L., Wittinghofer A. and Herrmann C., Nature, 403 (2000) 567.; [3] Kalinin<br />
S., Valeri A., Antonik M, Felekyan S. and Seidel C. A., J Phys Chem B, 114 (2010) 7983.; [4] Sindbert S.,<br />
Kalinin S., Kienzler A., Clima L., Bannwarth W., Nguyen H., Appel B., Müller S. and Seidel C. A., J Am Chem<br />
Soc., 133 (2011) 2463.<br />
126 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P22 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 22<br />
Binding of organic dyes with Human Serum Albumin: a single molecule<br />
study<br />
Dibyendu Kumar Das 1 , Tridib Mondal 1 , Amit Kumar Mandal 1 & Kankan Bhattacharyya 1,*<br />
1<br />
Physical Chemistry Department, Indian Association <strong>for</strong> the Cultivation of Science,<br />
Jadavpur, Kolkata 700 032, India<br />
Kinetics of binding of dyes at different sites of human serum albumin (HSA) has been studied by<br />
single molecule spectroscopy. The protein was immobilized on a glass surface. In order to probe<br />
different binding sites (hydrophobic and hydrophilic) two dyes, coumarin 153 (C153, neutral) and<br />
rhodamine 6G (R6G, cationic) were chosen. For both the dyes, a major (~96-98%) binding site and<br />
a minor (~3%) binding site were detected. [1] Rate constants of association and dissociation were<br />
simultaneously determined from directly measuring fluctuations in fluorescence intensity (τoff and<br />
τon) and from this the equilibrium (binding) constants were calculated. Fluorescence lifetimes at<br />
individual sites were obtained from burst integrated lifetime analysis. Distributions of lifetime<br />
histograms <strong>for</strong> both the probes (C153 and R6G) exhibit two maxima indicating presence of two<br />
binding domains in the protein. Unfolding of the protein has been studied by adding guanidinium<br />
hydrochloride (GdnHCl) to the solution. It is observed that addition of GdnHCl affects the<br />
dissociation and association kinetics and hence, binding equilibrium of the association of C153.<br />
However, the effect of binding of R6G is not affected much. It is proposed that GdnHCl affects the<br />
hydrophobic binding sites more than the hydrophilic site. [2] Recently we have also studied the effect<br />
of ionic liquid on the unfolding dynamics of the HSA protein by Fluorescence Correlation<br />
Spectroscopy Techniques. [3]<br />
Number of events<br />
This work was supported by grants from DST and CSIR.<br />
References: [1] D.K. Das, et al., Chem. Phys Chem., 12 (2011) 814. [2]. D.K. Das, et al., Chem. Asian. J.,<br />
(In Press). [3] D.K. Das, et al.,(unpublished).<br />
______________<br />
* Corresponding author: e-mail: pckb@iacs.res.in<br />
8<br />
6<br />
4<br />
2<br />
4<br />
3<br />
2<br />
1<br />
0<br />
C153_HSA<br />
R6G_HSA<br />
2 4 6 8 10<br />
Lifetime (ns)<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 127
P23 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 23<br />
Synthesis of wavelength-ratiometric substrates <strong>for</strong> fluorimetric assay of<br />
hydrolases <strong>for</strong> single-molecule investigations<br />
Björn Finkler 1 , Anh-Minh Huynh 1 & Gregor Jung 1,*<br />
1Biophysical Chemistry, Saarland University, Campus B2.2, D-66123 Saarbruecken.<br />
Several esters of HPTS (1-Hydroxypyrene-3,6,8-trisulfonicacid) are known to be hydrolyzed to<br />
HPTS by certain hydrolases. [1] Furthermore, HPTS (λexc= 404 nm, λem = 510 nm) exhibits a<br />
bathochromic shifted emission in aqueous solvents compared to its esters (λem = 390 / 410 nm)<br />
due the excited state proton transfer (ESPT) of HPTS. [2] Based on this feature, a wavelengthratiometric<br />
detection of the hydrolysis can be established. In principle, such ratiometric systems<br />
which can change their fluorescence color are appropriate <strong>for</strong> single-molecule studies.<br />
Un<strong>for</strong>tunately, HPTS is rather photolabile. There<strong>for</strong>e, our main request is the development of new<br />
fluorimetric substrates derivated from HPTS, which is suitable <strong>for</strong> single molecule detection. HPTA<br />
(8-hydroxy-N 1 ,N 1 ,N 3 ,N 3 ,N 6 ,N 6 -hexamethylpyrene-1,3,6-trisulfonamide), which is a derivate of<br />
HPTS, shows a higher photostability. By esterification of the hydroxyl group of HPTA, however, the<br />
water solubility was found to decrease enormously so that no enzymatic hydrolysis is observable.<br />
We present a modified HPTA-system with sufficient water solubility circumventing the low<br />
hydrophilicity.<br />
Electrophilic fluorination by Selectfluor TM is aimed at further improving the photostability of<br />
HPTA. The strong electron withdrawing effect of the introduced fluorine atom changes the<br />
electrochemical and optical properties of fluorescent dyes. [3] Especially the photostability will be<br />
increased after the electrophilic introduction of fluorine atoms. [4] We also inventing whether the<br />
acidity of the hydroxygroup is changed upon fluorination of HPTA.<br />
References: [1] O.S. Wolfbeis, E. Koller, Analytical Biochemistry, 129 (1983) 365. [2] Th. Foerster,<br />
Zeitschrift für Elektrochemie, 54 (1950) 42, [3] Y. Sakamoto et al., Journal of the American Chemical Society,<br />
126 (2004) 8138, [4] B. Renikuntla et al., Organic Letters, 6 (2004) 909.<br />
______________<br />
*Corresponding author: e-mail: b.finkler@mx.uni-saarland.de<br />
128 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P24 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 24<br />
Protein distribution on liposomes by detection of single fluorescent<br />
diffusing particles: a coincidence method based on confocal imaging<br />
Ouided Friaa 1* , Melissa Furukawa 1 , Radhika Voleti 1 , Sanjeevan Shivakumar 2 , Aisha Shamas-<br />
Din 2 & Cécile Fradin 1,2*<br />
1<br />
Department of Physics & Astronomy, Mc Master University, Hamilton, Ontario (Canada).<br />
2<br />
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario<br />
(Canada).<br />
The core molecular mechanism regulating the distribution and the interactions of proteins with<br />
cellular membranes still need to be understood. To improve our understanding of proteins<br />
interactions with cellular membranes, we have used a method based on confocal fluorescence<br />
microscopy that enables us to correlate the number of proteins bound to a lipid liposome with the<br />
liposome properties (membrane integrity, membrane potential, aggregation…).<br />
This method consists in the simultaneous recording of confocal images in two different<br />
channels, and in the coincidental detection of single mobile particles in these channels. This allows<br />
quantifying the particle concentration and the fluorescence emission of single particles at two<br />
different wavelengths, from which a study of protein distribution can be made by “counting” how<br />
many proteins are attached to each particular liposome. To demonstrate the usefulness of this<br />
method, we have used a model system consisting of small unilamellar liposomes rendered<br />
fluorescent by the addition of a small percentage of fluorophore and interacting with fluorescently<br />
labeled proteins.<br />
To optimize the detection of single diffusing liposomes, we systematically varied a number of<br />
experimental parameters (fluorophore to liposome ratio, excitation intensity, confocal pixel size and<br />
dwell time) as well as a number of analysis parameters (binsize and threshold level), in order to<br />
detect a maximum of photons from particles with independent positions. We compared the results<br />
of this single particle detection method with results obtained by fluorescence fluctuation methods<br />
such as fluorescence correlation spectroscopy (FCS), and fluorescence intensity distribution<br />
analysis (FIDA). Finally, using liposomes labeled with two different fluorophores, we measured the<br />
probability that a liposome detected in one channel would also be detected in the second channel,<br />
and compared our results with those obtained by fluorescence cross-correlation spectroscopy<br />
(XCS).<br />
We expect this method to be useful <strong>for</strong> studies of protein or peptide insertion and pore<br />
<strong>for</strong>mation into lipid membranes.<br />
This work was supported by grants from CIHR IRSC, NSERC CRSNG.<br />
______________<br />
* Corresponding authors: e-mail: : friaao@mcmaster.ca ; fradin@physics.mcmaster.ca<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 129
P25 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 25<br />
Binding kinetics of the HIV-1 NCp7 on oligonucleotides at the singlemolecule<br />
level<br />
Julien Godet 1,2,* , Pascal Didier 1 , Armelle Jouonang 1 , Youri Arntz 1 , Guy Duportail 1 & Yves<br />
Mély 1<br />
1Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg,<br />
Faculté de Pharmacie, 74, Route du Rhin, 67401 Illkirch (France).<br />
2 Département d’In<strong>for</strong>mation Médicale et Biostatistiques, Hôpitaux Universitaires de Strasbourg, 1<br />
place de l’Hôpital, 67000 Strasbourg (France).<br />
The nucleocapsid protein (NCp7) of the human immunodeficiency virus type 1 (HIV-1) is a small<br />
basic protein which plays key functions in the viral life cycle. The activity of NCp7 mainly relies on<br />
its potent RNA- and DNA-chaperone properties that direct the rearrangement of numerous nucleic<br />
acid molecules into their most stable con<strong>for</strong>mation. Two main features of NCp7 chaperone activity<br />
are its abilities to aggregate and destabilize nucleic acids. On the basis of a correlation between an<br />
indirect measurement of the nucleic acid dissociation kinetics of NCp7 and its overall chaperone<br />
activity, the rapid kinetics of interaction with nucleic acids was recently proposed as an additional<br />
component of NCp7 chaperone functions. But so far, no direct measurement of the on/off rates of<br />
NCp7 binding to oligonucleotides was per<strong>for</strong>med. In the present work, we realized single molecule<br />
fluorescence measurements to probe the transient interactions between one labeled NCp7<br />
molecule and a short labeled DNA oligonucleotide confined into nanovesicles. The binding kinetics<br />
constants are discussed in the context of the DNA-chaperoning activity of NCp7.<br />
This work was supported by grants from ANRS, Sidaction and ANR.<br />
______________<br />
* Corresponding author: e-mail: julien.godet@unistra.fr<br />
130 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P26 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 26<br />
Con<strong>for</strong>mations and con<strong>for</strong>mational dynamics of calmodulin detected by<br />
single-molecule fluorescence<br />
Carey K. Johnson 1,* E. Shane Price, 3 Mihailo Backović, 2 , Matthew S. DeVore 1 & John P.<br />
Ralston 2<br />
1 Department of Chemistry, University of Kansas, Lawrence, KS 66045 (USA)<br />
2 Department of Physics, University of Kansas, Lawrence, KS 66045 (USA).<br />
3 Department of Chemistry, William Jewell College, 500 College Hill, Liberty, MO 64068 (USA)<br />
In response to calcium signals the calcium binding protein calmodulin (CaM) regulates multiple<br />
signalling pathways in cells. Typically calmodulin changes global con<strong>for</strong>mational as it binds<br />
enzymes that it regulates. Using single-molecule fluorescence methods, we have shown that even<br />
in the absence of a target CaM explores a wide con<strong>for</strong>mational space.(Johnson CK, 2006;<br />
Slaughter BD et al., 2004) Exploration of multiple con<strong>for</strong>mations may be essential to the<br />
mechanism by which CaM recognizes and binds target enzymes. However, the time scale of<br />
con<strong>for</strong>mational interchange has been unclear. We have there<strong>for</strong>e carried out experiments to<br />
characterize the dynamics of con<strong>for</strong>mational change in calmodulin. To probe dynamics on the<br />
millisecond time scale, we entrapped CaM in lipid vesicles that were then tethered to a surface.<br />
Con<strong>for</strong>mational transitions were observed on time scales from milliseconds to tens of milliseconds.<br />
We analysed the con<strong>for</strong>mational dynamics by hidden Markov methods and by a new iterative<br />
Bayesian approach that describes the propagation of the probability distribution of FRET<br />
states.(Backović M et al., 2011) Dynamics on the microsecond time scale were detected by<br />
fluorescence correlation spectroscopy. Global fitting of the autocorrelation functions of the donor<br />
and acceptor fluorophores together with the cross-correlations of donor with acceptor and acceptor<br />
with donor revealed dynamics on the 100-μs time scale <strong>for</strong> CaM with one FRET label in the Nterminal<br />
domain and the other in the C-terminal domain.(Price ES et al., 2010) Further FCS<br />
experiments were carried out with both fluorescence labels in either the N-terminal and C-terminal<br />
domains to detect intra-domain dynamics. Dynamics were detected on the time scale of 10s of<br />
microseconds. For the N-terminal domain, the amplitude of dynamics decreased with addition of<br />
Ca 2+ , suggesting that the dynamics are coupled to the Ca 2+ -binding (EF-hand) domains. We also<br />
report a new maximum-entropy approach <strong>for</strong> analysis of single-molecule fluorescence burst<br />
measurements. The method, based on “classic” maximum entropy analysis, predicts the joint<br />
probability distribution of FRET efficiency and total fluorescence, P(E,F). Marginalization yields the<br />
underlying FRET efficiency distribution. We report characterization of the approach with simulated<br />
data and application of then method to the CaM FRET efficiency distribution. Cumulatively, the<br />
reported results support a dynamic picture where, even in the absence of target binding domains,<br />
CaM explores the range of con<strong>for</strong>mational states needed <strong>for</strong> function.<br />
This work was supported by grants from the NSF and the American Heart Association.<br />
References: [1] C.K. Johnson, Biochemistry 45 (2006) 14233. [2] B.D. Slaughter, et al., J. Phys. Chem. B<br />
108 (2004) 10388. [3] M. Backović, et al., J Chem Phys 134 (2011) 145101. [4] E.S. Price, et al., J. Phys.<br />
Chem. B 114 (2010) 5895.<br />
______________<br />
* Corresponding author: e-mail: ckjohnson@ku.edu<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 131
P27 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 27<br />
<strong>Single</strong>-molecule fluorescence dynamics of perylene diimides cyclic arrays<br />
Ji-Eun Lee 1 , Jaesung Yang 1 , Hyejin Yoo 1 & Dongho Kim 1,*<br />
1<br />
Spectroscopy Laboratory <strong>for</strong> Functional π–Electronic Systems and Department of Chemistry,<br />
Yonsei University, Seoul 120-749 (Korea)<br />
We have investigated single-molecule fluorescence dynamics of perylene diimide (PDI) cyclic<br />
arrays C3, C4, C5, and C6 (Figure 1), with a particular focus on the influences of the overall<br />
structural rigidity as the ring size of PDI arrays increases. By recording single-molecule<br />
fluorescence intensity trajectories (FITs) and polarization, we observed stepwise photobleaching<br />
behaviors in the FITs <strong>for</strong> all PDI cyclic arrays, and the change in polarization according to<br />
sequential photobleaching of constituent PDI units. These features indicate that each PDI units<br />
maintain monomeric character due to weak interaction, and con<strong>for</strong>mational heterogeneities<br />
increase with the increase of the number of PDI units, because neighboring PDI moieties are<br />
linked by the phenyl-butadiyne-phenyl linkage. PDI cyclic arrays thus exhibit through-space energy<br />
transfer between the PDI units. Consequently, we expect that PDI cyclic arrays show efficient<br />
excitation energy hopping processes between the PDI chromophores.<br />
Figure 1. PDI cyclic arrays.<br />
This work was supported by World Class University Program (R32-2010-000-10217) from the<br />
Ministry of Education, Science, and Technology (MEST) and the Fundamental R&D Program <strong>for</strong><br />
Core Technology of Materials funded by the Ministry of Knowledge Economy, Republic of Korea.<br />
The sample has been synthesized by F. Würthner group.<br />
References: [1] H. Yoo, et al., J. Am. Chem. Soc., 132 (2010) 3939.<br />
______________<br />
* Corresponding author: e-mail: dongho@yonsei.ac.kr<br />
132 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P28 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 28<br />
Experimental and modeling studies on EpoR-EPO signaling on the<br />
single-molecule and single-cell level<br />
Konstantinos Lymperopoulos 1 , Alejandro Macias-Torre 1 , William Godinez 2,3 , Stefan<br />
Kallenberger 2,3 , Agustin Rodriquez 4 , Michael Jarsch 5 , Roland Eils 2,3 , Karl Rohr 2,3 , Ursula<br />
Klingmueller 4 & Dirk Peter Herten 1<br />
1<br />
Cellnetworks Cluster of Excellence/Institute of Physical Chemistry, University of Heidelberg,<br />
Heidelberg, Germany<br />
2<br />
IPMB, Dept. Bioin<strong>for</strong>matics and Functional Genomics, University of Heidelberg, Heidelberg, Germany<br />
3<br />
Division of Theoretical Bioin<strong>for</strong>matics, German Cancer Research Center (DKFZ), Heidelberg,<br />
Germany<br />
4<br />
Division Systems Biology of Signal Transduction, German Cancer Research Center (DKFZ),<br />
Heidelberg, Germany<br />
5<br />
Roche Diagnostics GmbH, Penzberg, Germany<br />
Cells sense changes and adapt to their highly dynamic environment by utilising cell surface<br />
receptors. Their affinity to specific ligands and activation, their subsequent internalisation,<br />
intracellular localisation and downstream signalling are all part of a well-orchestrated mechanism,<br />
perturbation of which can lead to diseases. We choose to study the interaction between the<br />
erythropoietin receptor (EpoR) and its ligand erythropoietin (Epo) on the single-molecule and<br />
single-cell level, which enable us to discern properties that would otherwise be masked due to the<br />
averaging effect on the cellular and cell culture level respectively.<br />
<strong>Single</strong>-particle tracking experiments were per<strong>for</strong>med in living HeLa cells that over-express<br />
the hEpoR fused with EGFP and incubated with labelled Epo. Both EpoR and EPO were tracked<br />
simultaneously and interacting particles were identified based on their respective trajectories.<br />
Particles were classified according to their type of motion and speed into three distinct categories.<br />
We observed a significant alteration of the frequency of each type of motion after cell stimulation<br />
with EPO. Similar studies are to be extended in H838 human lung carcinoma cells. On the singlecell<br />
level the total concentration of EpoR and uptake of Epo were simultaneously measured in<br />
living cells as well as their respective concentration in different subcellular compartments. Similar<br />
experiments were per<strong>for</strong>med in H838 cells and the above data were compared with a simple<br />
mathematical model that describes the EpoR-Epo interaction and their internalisation, resulting in a<br />
good agreement.<br />
This work was supported by the BMBF (SBCancer) and the DFG (Cellnetworks Cluster, EXC 81)<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 133
P29 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 29<br />
<strong>Single</strong> molecule photoluminescence excitation spectroscopy at room<br />
temperature<br />
Frank Schleifenbaum 1* Christian Blum 2 , Martijn Stopel 2 , Sebastien Peter 1 , Marcus<br />
Sackrow 3# , Vinod Subramaniam 2 & Alfred J. Meixner 3<br />
1Center<br />
<strong>for</strong> Plant Molecular Biology, Biophysical Chemistry, University of Tuebingen, Tuebingen,<br />
Germany.<br />
2<br />
Nanobiophysics, Faculty of Science and Technology and MESA+ Institute <strong>for</strong> Nanotechnology,<br />
University of Twente, Enschede, The Netherlands<br />
3<br />
Institute of Physical and Theoretical Chemistry, University of Tuebingen, Tuebingen, Germany.<br />
#<br />
current address: Picoquant GmbH, Berlin, Germany<br />
During the last two decades, single molecule spectroscopy has experienced tremendous advances<br />
and a variety of spectral parameters is accessible by now, offering fascinating insight into<br />
molecular properties. However, at room temperatures the frequency dependent coupling of an<br />
individual emitter to an external electromagnetic field has remained an elusive experimental<br />
observable. We developed a new single molecule detection modality that enables<br />
photoluminescence excitation spectroscopy of single molecules or nanoparticles at room<br />
temperature. We demonstrate the potential of single molecule photoluminescence excitation<br />
spectroscopy by recording excitation spectra, spanning a wide spectral range of 100 nm, of single<br />
CdSe nanocrystals. As a characteristic of single molecules, the recorded spectra exhibit<br />
intermittent emission. Moreover, we find strong indications <strong>for</strong> the existence of specific excitation<br />
wavelengths <strong>for</strong> which the probability of a transition to a long lived dark state is increased. The<br />
recorded excitation spectra show variations in the spectral position of the excitonic peak, reflecting<br />
the distribution of quantum dot sizes within the measured ensemble.<br />
On the basis of these investigations of quantum dots, we expect single molecule excitation<br />
spectroscopy to become a valuable addition to established singe molecule spectroscopy methods.<br />
As one prerequisite <strong>for</strong> single molecule spectroscopy is spatially resolution, the technique can be<br />
directly applied to spectroscopic imaging. We demonstrate this capability by investigation plant<br />
chloroplasts in a living cell context. Within the chloroplast, complex energy transfer processes take<br />
place, which cannot be addressed by conventional techniques in vivo. Our fluorescence excitation<br />
spectroscopy approach allows <strong>for</strong> monitoring the single energy transfer steps <strong>for</strong> the first time. Our<br />
data reveal that different mutant plants exhibit a reduced ability to adopt to changed light<br />
conditions.<br />
______________<br />
* Corresponding author: e-mail: frank.schleifenbaum@uni-tuebingen.de<br />
134 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P30 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 30<br />
Investigation of chemical reactions with <strong>Single</strong>-Molecule Fluorescence<br />
Spectroscopy<br />
Arina Rybina 1 , Alexander Kiel 1 , Nicole Bach 3 , Birgit Thaler 2 , Arnd Sprödefeld 2 , Daniel Hack 1 ,<br />
Elias Rüdiger 1 , Anton Kurz 1 , Kristin Grußmayer 1 , Michael Schwering 1 , Gregor Jung 3 , Roland<br />
Krämer 2 & Dirk-Peter Herten 1<br />
1<br />
Bioquant & Physikalisch-Chemisches Institut, Universität Heidelberg, Im Neuenheimer Feld 267,<br />
D-69120 Heidelberg (Germany)<br />
2<br />
Anorganisch-Chemisches Institut, Universität Heidelberg, Im Neuenheimer Feld 270, D-69120<br />
Heidelberg (Germany)<br />
3<br />
Biophysikalische Chemie, Universität des Saarlandes, Gebäude B2.2, D-66041 Saarbrücken<br />
(Germany)<br />
Chemical trans<strong>for</strong>mations play a key role in synthesis of new materials as well as in many<br />
biological processes. In contrast to bulk experiments, <strong>Single</strong>-Molecule-Fluorescence<br />
Spectroscopy (SMFS) serves as tool <strong>for</strong> studying chemical reactions on the molecular level and<br />
bears the potential to resolve elementary reaction steps in real time with a high time resolution up<br />
to microseconds. However, a specific probe design is necessary that links changes in molecular<br />
structure with changes in fluorescence signal. We work on design of molecular probes and<br />
experimental approaches to study different chemical reactions 1 like metal ion coordination 2 and<br />
catalytic reactions 3 each using different mechanisms to sense molecular processes. The sensing<br />
of metal ions is based on the decrease of fluorescence intensity of a fluorophore, placed in<br />
proximity to a chelating ligand, due to electron transfer. To study redox-reactions we coupled a<br />
fluorescent dye directly to hydroquinone. Here, oxidation of the hydroquinone subunit leads to<br />
fluorescence quenching. Aiming at the catalytic cycle of the metathesis we present fluorescent<br />
substrates undergoing a shift in the emission wavelength upon metathesis reaction due to the<br />
structural change of the chromophore.<br />
This work was supported by DFG.<br />
References: [1] A. Kiel et al., Angew. Chem. Int. Ed., 46, 3363–3366 (2007); [2] R. M. Kierat et al., Bioorg.<br />
Med. Chem. Lett., 20(4), 1457-1459 (2010); [3] A. Schmitt et al., J. Phys. Org. Chem., 22, 1233–1238 (2009)<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 135
P31 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 31<br />
Studying dynamics of biomolecules in living cells using single molecule<br />
spectroscopy: fluorescent probes <strong>for</strong> RNA detection<br />
Anne Seefeld, Konstantinos Lymperopoulοs & Dirk-Peter Herten<br />
<strong>Single</strong>-Molecule Spectroscopy, Bioquant/CellNetworks, University of Heidelberg, Germany<br />
Application of single-molecule and high-resolution fluorescence methods to monitor gene<br />
expression in living cells increase the demand on novel probes. For quantitative real-time detection<br />
of certain mRNAs probes with high sensitivity and specificity are needed, especially <strong>for</strong> low<br />
abundance genes and with low copy numbers.<br />
We implement hairpin probes[1], oligodeoxynucleotides (ODNs), which contain a fluorophore<br />
at one end and unpaired guanosines on the other. Depending on the redox potential of the<br />
fluorophor and the designed hairpin structure of the probe, the fluorophore is quenched upon<br />
contact <strong>for</strong>mation with guanosine and in absence of a complementary target. Hybridisation with the<br />
target sequence opens the hairpin, thus separating the fluorophore from its quenching guanosine<br />
nucleotides, allowing a fluorescent signal to be emitted upon excitation. Thus, they can provide<br />
detailed in<strong>for</strong>mation about RNA dynamics, like expression, localization and degradation levels in<br />
living cells with a reduced background and without any amplification.<br />
A critical aspect is the selection of the mRNA target region. In theory any sequence within an<br />
mRNA transcript can be chosen as a binding site <strong>for</strong> hairpin probes. As secondary and tertiary<br />
structures can mask many of these target binding sites, we correlate several known prediction<br />
algorithms in order to model target-probe behaviour.<br />
Here we present a systematic method and criteria <strong>for</strong> designing functional probes. We test<br />
their functionality and specificity with in vitro and in vivo experiments using fixed cells. Furthermore,<br />
we used and compared the efficiency of different transfection methods to deliver the probes in<br />
living eukaryotic cells targeting mRNAs and confirmed their specificity by FISH experiments. [2]<br />
Currently, we try to combine fluorescence correlation spectroscopy (FCS) and photon<br />
counting histogram (PHC) to gain a higher spatial and temporal resolution and <strong>for</strong> direct<br />
quantitative investigation of probe dynamics, which in turn should reflect the dynamics of gene<br />
expression under different stimuli and conditions. This new in<strong>for</strong>mation can be extremely useful in<br />
cancer detection and <strong>for</strong> studying the response of cancer cells to biological and therapeutic<br />
reagents.<br />
This work was supported by the BMBF (LungSys) and the DFG (Cellnetworks Cluster, EXC 81)<br />
References: [1] K. Stöhr, et al, Anal Chem (2005)7195 [2] K. Lymperopoulos, et al, ChemPhysChem 11<br />
(2010) 43<br />
______________<br />
* Corresponding author: e-mail: anne.seefeld@bioquant.uni-heidelberg.de<br />
136 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P32 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 32<br />
Low-temperature spectral dynamics of single impurity molecules in<br />
ultrathin polymer film<br />
Yaroslav I. Sobolev 1,* , Yuri G. Vainer 1,2 , Andrei V. Naumov 1,2 & Lothar Kador 3<br />
1 Institute <strong>for</strong> Spectroscopy, Russian Academy of Sciences, Troitsk, Moscow Reg., 142190, Russia<br />
2 Moscow Institute of Physics and Technology, Dolgoprudny, Russia<br />
3 Institute of Physics and BIMF, University of Bayreuth, D-95440 Bayreuth, Germany<br />
Behaviour of amorphous solids at low temperatures is generally explained in terms of a few simple<br />
phenomenological models [1-2] that simplify the multidimensional potential surface of a disordered<br />
solid to a set of elementary excitations: tunnelling two-level systems (TLS) and quasilocalized lowfrequency<br />
vibrational modes (LFM). Tunnelling TLSs, which are believed to be groups of atoms or<br />
molecules undergoing tunnelling transitions between their local potential minima, are mainly<br />
responsible <strong>for</strong> the glass dynamics at temperatures below 2 K. At higher temperatures (few K – few<br />
tens of K) contribution due to LFM begins to prevail. It has been widely accepted, that this concept<br />
remains valid <strong>for</strong> all amorphous media (with few rare exceptions) and does not qualitatively depend<br />
on chemical composition or macroscopic features of material. The powerful way to observe these<br />
dynamical processes is to study temporal evolution of fluorescence spectra of dye molecules<br />
embedded into (transparent) disordered solid under study (see <strong>for</strong> review [3] and references<br />
therein). As electronic spectrum of a single impurity molecule is sensitive to its local nano-scale<br />
environment, changes in this environment modify the spectrum. If spectroscopy is done on a given<br />
single molecule repeatedly, then through observed changes in single-molecule spectrum one<br />
obtains in<strong>for</strong>mation on processes that took place in the vicinity of this molecule during the time of<br />
experiment. In present work we used this approach to investigate the effect of surface and<br />
confinement on internal dynamics in ultrathin polymer films at low temperatures. To achieve this,<br />
we observed spectral dynamics of single dye molecules embedded in ultrathin films of<br />
polyisobutylene, and dependence of this dynamics on film thickness was studied. It was found that<br />
certain percent of molecules shows spectral evolution inconsistent with TLS model: their spectral<br />
lines were subject to irreversible jumps and continuous shifting, reflecting complex and irregular<br />
local dynamics in the sample. Percent of such “non-standard” molecules increased with decrease<br />
of sample thickness at fixed excitation laser power. Moreover, it increased with increasing of<br />
excitation laser power at fixed sample thickness. Characteristic thickness at which the effect<br />
becomes prominent is ~100 nm. Possible mechanisms are discussed.<br />
The work was supported by the Deutsche Forschungsgemeinschaft, Russian Foundation of Basic<br />
Research (10-02-00609, 10-02-90047, 11-02-00816), Grant of the President of Russia <strong>for</strong> leading<br />
scientific schools НШ-3470.2010.2, and state contract №02.740.11.0431.<br />
References: [1] W.A. Phillips, Springer (1981). [2] U. Buchenau et al., Phys. Rev. Lett. 77, 659 (1996). [3]<br />
A.V. Naumov, Yu.G. Vainer, Physics-Uspekhi, 52, 298-304 (2009).<br />
______________<br />
* Corresponding author: e-mail: yaroslav.sobolev@phystech.edu<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 137
P33 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 33<br />
Stability of terrylenediimide (TDI) single molecules in various<br />
polymer films<br />
Adam Sokolowski 1 & Jacek Waluk *<br />
1<br />
Institute of Physical Chemistry of the Polish Academy of Sciences, Kasprzaka 44/52, 01-224<br />
Warsaw (Poland)<br />
The goal of the present investigations is to find an optimal microenviroment <strong>for</strong> single molecule<br />
studies. Various experimental environments, such as different polymer matrices can provide quite<br />
extensive material <strong>for</strong> characterizing photostability of single molecules. The lifetime of an isolated<br />
molecule can be influenced by many parameters specific <strong>for</strong> a particular polymer matrix, such as<br />
oxygen diffusion coefficient or polymer morphology, polymer molecular composition and chemical<br />
structure. Changing the excitation light intensity can also provide in<strong>for</strong>mation about the bleaching<br />
mechanism of a studied molecule embedded in a polymer matrix.<br />
We define single molecule lifetime as a time from the start of an experiment to the total<br />
destruction of a studied molecule (bleaching). Another phenomenon, called blinking (sequential<br />
switching between fluorescent and non-fluorescent states) is also observed in all cases.<br />
Four different polymers: polycarbonate, poly(vinyl butyral), poly(methyl metacrylate), and poly(vinyl<br />
chloride) (PVC) were used as matrices doped with terrylenediimide molecules with the<br />
concentration of 10 -9 mol/dm 3 . The samples, about 30 nm thick polymer layers, were obtained by<br />
spin-coating.<br />
After pre-scanning of a certain area (20 by 20 μm) of the sample, single molecules were<br />
located. After focusing on each of them (sequentially), fluorescence was collected <strong>for</strong> the time<br />
period (single experiment time) ranging from 150 seconds to 30 minutes, with the channel<br />
collection time of 15 ms.<br />
In most cases, the shortest time <strong>for</strong> a single experiment was sufficient to completely destroy<br />
(bleach) the TDI molecule, but in the case of PVC matrix, elongation of experiment time was<br />
necessary to observe the destruction of TDI. This may - considering low permeability and oxygen<br />
diffusion coefficient in PVC - support the theory of oxygen-dependent bleaching mechanism.<br />
Also, different blinking character was observed in the investigated polymers. Depending on<br />
the laser power used, we could distinguish matrices with short and long fluorescent and nonfluorescent<br />
(on/off) periods during the experiment.<br />
In order to compare the effects of matrix influence on TDI photostability, histograms of<br />
lifetimes and blinking characteristics were constructed. They clearly demonstrate the merits of PVC<br />
as a stability-supporting matrix <strong>for</strong> the study of single TDI molecules, in agreement with previous<br />
suggestions [1]. Further experiments on another compound, porphycene, known as less stable<br />
than TDI, are under way.<br />
References:<br />
[1] F. Vargas, O. Hollricher, O. Marti, G. de Schaetzen, G. Tarrach, J. Chem. Phys. 117 (2002) 866.<br />
______________<br />
* Corresponding author: e-mail: jwaluk@ichf.edu.pl<br />
138 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P34 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 34<br />
First computational determination of four-way RNA junction’s structure<br />
using simplified representation of RNA and statistical potential enriched<br />
by constraints from single-molecule FRET spectroscopy experiment,<br />
rigid body modeling and predicted alternative secondary structures<br />
Tomasz Sołtysiński 1 , Stanislav Kalinin 2 , Michał Boniecki 1 , Simon Sindbert 2 , Hayk<br />
Vardanyan 2 , Juliusz Stasiewicz 1 , Irina Tuszyńska 1 , Claus Seidel 2 & Janusz M.<br />
Bujnicki 1<br />
1<br />
International Institute of Molecular & Cell Biology in Warsaw, Ks. Trojdena 4, 02-109 Warsaw, Poland<br />
2<br />
Institute of Molecular Physical Chemistry, Heinrich-Heine Universität, Universitätsstrasse 1, 40225<br />
Düsseldorf, Germany<br />
For the first time, novel methods <strong>for</strong> RNA computational modeling are used to model 4-way<br />
junction RNA system restrained either by experimental data obtained by multiparameter<br />
fluorescence detection and predicted secondary structure. Based on the output of well established<br />
bioin<strong>for</strong>matics tools (MetaRNA server; RNA centrafold, RNAsubopt, RNAfold) and the recently<br />
developed method of Monte-Carlo based scheme <strong>for</strong> browsing the con<strong>for</strong>mational space and<br />
energy landscape of nucleid acids, SimRNA, a novel study is per<strong>for</strong>med to investigate the<br />
variability of spatial configurations of structure of interest. Using the mentioned methods and<br />
starting from sequence we have made a study on numerical folding of 4-way junction RNA<br />
structure restrained by given secondary structure and a set of 40 smFRET-derived distances<br />
between labeled nucleotides. There has been common dyes used; Alexa488, donor and Cy5,<br />
acceptor. Monte Carlo scheme of folding along with the replica exchange method have been<br />
implemented and the reduced representation of a nucleid acid is used to make the method fast and<br />
efficient enough to find energetically the best con<strong>for</strong>mers within created statistical <strong>for</strong>ce field.<br />
Evolution of structures has been constrained by three, the most probable, predicted secondary<br />
structures under assumption of Watson-Crick base pairing. Selected con<strong>for</strong>mers were used to fully<br />
relax the structures. Resulting trajectories has been subsequently filtered by limits determined<br />
through rigid body initial modeling of general con<strong>for</strong>mation including the accessible volumes of<br />
dyes in the vicinity of RNA’s grooves. Such a pipeline producing an ensemble of alternative<br />
con<strong>for</strong>mations <strong>for</strong> 4-way junction RNA is presented and discussed along with their energy<br />
landscapes. The reduced con<strong>for</strong>mers fulfilling the experimental limits the best are further selected<br />
to be rebuilt into full atom representation using novel RebuildRNA tool and finally optimized by<br />
QRNA, a new multipurpose tool to refine and locally optimize the structure of interest.<br />
______________<br />
Correspondence author: e-mail; tsoltysinski@genesilico.pl<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 139
P35 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 35<br />
Polymer matrix dependence of con<strong>for</strong>mational dynamics within πstacked<br />
perylenediimide dimer and trimer as revealed by singlemolecule<br />
fluorescence spectroscopy<br />
Hyejin Yoo 1 , Heewon Bahng 1 , Michael R. Wasielewski 2,* & Dongho Kim 1,*<br />
1<br />
Spectroscopy Laboratory <strong>for</strong> Functional π-Electronic Systems and Department of Chemistry,<br />
Yonsei University, Seoul 120-749 (Korea).<br />
2<br />
Department of Chemistry, and Argonne-Northwestern Solar Energy Research (ANSER) Center,<br />
Northwestern University, Evanston, Illinois 60208-3113 (USA).<br />
Probing local nano-environments in polymers is important to understand the properties of<br />
polymers, such as strength, flexibility and conductivity, especially <strong>for</strong> practical applications.<br />
Particularly, single molecule spectroscopic methods have been utilized to visualize local sites of<br />
the polymer matrix by monitoring rotational diffusion and fluctuating fluorescence of the probe<br />
molecule. Here, we have per<strong>for</strong>med single molecule spectroscopic experiments on a π-stacked<br />
perylenediimide (PDI) dimer and trimer, in which enhanced π-π interaction in π-stacked PDIs<br />
makes the fluorescence lifetime longer embedded in two different polymers, namely poly<br />
(methylmethacrylate) (PMMA) and poly (butylmethacrylate) (PBMA) to reveal their roles as<br />
molecular probes of nano-environments. The fluorescence lifetimes of π-stacked PDIs are<br />
influenced by polymer surroundings because their molecular con<strong>for</strong>mations are dependent on their<br />
interaction with the polymer. Furthermore, from an in-depth analysis of autocorrelation functions of<br />
fluorescence intensity trajectories, we could assign that the autocorrelation decay times of probe<br />
molecules are about 1 s. In addition, the first autocorrelation value (lag 1) is larger as the intensity<br />
trace becomes more fluctuating. Thus, we expect that π-stacked PDIs, a model system <strong>for</strong> the<br />
<strong>for</strong>mation of PDI excimer-like states, can be utilized as a molecular probe to unveil the<br />
heterogeneities of the polymer matrix.<br />
This work was supported by World Class University Program (R32-2010-000-10217) from the<br />
Ministry of Education, Science, and Technology (MEST) and the Fundamental R&D Program <strong>for</strong><br />
Core Technology of Materials funded by the Ministry of Knowledge Economy.<br />
References: [1] H. Yoo, et al., J. Am. Chem. Soc., 132 (2010) 3939.<br />
______________<br />
* Corresponding author: e-mail: dongho@yonsei.ac.kr<br />
140 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P36 Fluorescence Correlation Spectroscopy and <strong>Single</strong> Molecule Poster 36<br />
Real-time observation of platinum anticancer drug–DNA interactions at<br />
the single-molecule level<br />
Yuko Yoshikawa 1,* , Seiji Komeda 2 , Kenichi Yoshikawa 3 & Tadayuki Imanaka 1<br />
1 Laboratory of Environmental Biotechnology, Research Organization of Science and Engineering,<br />
Ritsumeikan University, Kusatsu 525-8577 (Japan)<br />
2 Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka 513-8670 (Japan)<br />
3 Department of Physics, Graduate School of Science, Kyoto University, Kyoto 606-8502 (Japan)<br />
Genomic DNA is an important molecular target of chemotherapeutic drugs in cancer treatments. Much<br />
attention has been focused on studying the interaction of drugs with DNA and developing new DNAtargeted<br />
drugs. As <strong>for</strong> the research of DNA-drug interactions, X-ray crystallography and NMR techniques<br />
are useful <strong>for</strong> defining the detail local binding mode of drug-DNA complex, but require the crystallization<br />
of DNA or encounter the limitation of size of DNA. Thus, these techniques are not adequate to investigate<br />
the effect of drugs on the overall morphology of a large DNA. Since genomic DNA is a very long polymer,<br />
studying the change of the higher-order structure of large DNA induced by drugs may provide additional<br />
insights <strong>for</strong> understanding the mechanism of their activities in living cells.<br />
Platinum compounds, including cisplatin, are now among the most commonly used anticancer<br />
drugs. Many studies have been conducted to understand the mechanism of action of cisplatin. It is<br />
generally accepted that cisplatin <strong>for</strong>ms coordinative adducts with genomic DNA, such as 1,2-intrastrand<br />
cross-links, to interfere with transcription and/or DNA replication, which eventually leads to apoptotic cell<br />
death. Thus, the Pt–DNA binding modes and kinetics seem to be closely related to its anticancer activity.<br />
Despite the high potential anticancer activity of cisplatin, its clinical use is often limited by acquired drug<br />
resistance and undesirable side effects. Much ef<strong>for</strong>t has been devoted to the development of new<br />
platinum-based drugs which circumvent cross-resistance to cisplatin.<br />
We recently found through single DNA observations in solution using fluorescence microscopy that<br />
long duplex DNA molecules with a size larger than<br />
several tens of kilo base-pairs exhibit a discrete<br />
con<strong>for</strong>mational transition from a coil state to a<br />
folded compact state upon the addition of various<br />
condensing agents, but that short DNA fragments<br />
behave like rigid rods and cannot undergo such a<br />
folding transition. We also found that an anticancer<br />
drug, daunomycin, can loosen the packing of DNA<br />
and induce double-strand breaks .[1]<br />
In the present, we will show a dose- and<br />
time-dependent effect of platinum coordinative<br />
compounds on the higher-order structure of a large<br />
DNA, T4 phage DNA (166 kbp), by adapting singlemolecule<br />
observation with fluorescence<br />
microscopy. [2,3] The right figure exemplifies the<br />
histograms of the long-axis length distributions of<br />
T4 DNA molecules together with an assignment of<br />
the con<strong>for</strong>mational characteristics of fluorescent<br />
DNA images in solution. [3] We classified the DNA<br />
Time-dependent con<strong>for</strong>mational change of<br />
DNA induced by a dinuclear Pt(II) complex<br />
con<strong>for</strong>mation into the coil, partial globule, and globule states on the basis of the inspection of successive<br />
movie frames. From the inspection of the time-dependent structural changes, it is concluded that<br />
dinuclear Pt(II) complex acts on DNA through both electrostatic interaction and coordination binding. [3,4]<br />
References: [1] Y. Yoshikawa et al., Chem. Phys. Lett., 366 (2002) 305. [2] Y. Katsuda et al., Chem. Phys.<br />
Lett., 473 (2009) 155. [3] N. Kida et al., J. Biol. Inorg. Chem. 15 (2010) 701. [4] S. Komeda et al.,<br />
ChemMedChem 6 (2011) 987.<br />
______________<br />
* Corresponding author: e-mail: yyoshi@fc.ritsumei.ac.jp<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 141
142 — MAF 12, Strasbourg, France, September 11-14, 2011 —
Fluorescence Microscopies<br />
and<br />
Cell Imaging<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 143
144 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P37 Fluorescence Microscopies and Cell Imaging Poster 37<br />
Skin diagnosis by non-invasive observation of dermal collagen with a<br />
second harmonic generation microscope<br />
Tsutomu Araki 1, * , Ryosuke Tanaka 1 & Takeshi Yasui 2<br />
1<br />
Division of Bioengineering, Graduate School of Engineering Science, Osaka University,<br />
Toyonaka, Osaka 560-8531, Japan<br />
2<br />
Division of Energy System, Institute of Technology and Science, The University of Tokushima,<br />
Tokushima 770-8506, Japan<br />
Non-invasive methods <strong>for</strong> in situ inspection of dermal collagen are essential <strong>for</strong> diagnosis in<br />
dermatology. Among the various methods, that utilizes an optical probe is attractive because this is<br />
capable of non-contact and real-time measurement. When ultra-short pulse light is applied to<br />
collagen-rich tissues, a second-harmonic-generation (SHG) light that is a half wavelength light of<br />
the incident light wavelength is generated. Such the SHG light provides unique imaging modality:<br />
high specificity <strong>for</strong> collagen, high spatial resolution, optical sectioning, and low invasion. Based on<br />
this concept, we have constructed a SHG microscope that is sensitive to collagen structure <strong>for</strong> skin<br />
diagnosis.<br />
For safety measurement, wavelength of light source should be longer than that of<br />
Ti:Sapphire laser (enter wavelength ~880nm) that is commonly used <strong>for</strong> SHG microscope. In this<br />
experiment, we have employed a mode-locked Cr:Forsterite laser (center wavelength 1250 nm,<br />
pulse width 100 fs, repetition 73MHz) as the light source. The laser light irradiated onto a sample<br />
via an optical attenuator and an objective lens (NA 0.9, oil immersion). To construct optical 3D<br />
sectioning image of SHG signal, the laser beam was scanned in two dimensions by a pair of<br />
galvano mirrors and also scanned in Z direction by a piezo stage. The backscattered SHG light<br />
component in the sample was detected by a photomultiplier tube with Peltier cooling <strong>for</strong> photon<br />
counting operation. The laser power and measurement time <strong>for</strong> one spot were set to be 40 mW<br />
and 3.1×10 -5 sec/(μm) 2 , respectively. Irradiation power on the sample was 1.2×10 -6 J/(μm) 2 , and<br />
this value was much lower than the reported values [1-3] with which safety measurements were<br />
done <strong>for</strong> vital cell and tissue. In the present microscope, measurement area and time of one frame<br />
were set to 600 μm x 600 μm and 2 sec, respectively. A larger area SHG image was constructed<br />
by connecting this frame successively. Penetration of the infrared laser light up to 300 μm enables<br />
direct probing of the dermis layer across the epidermis layer without risk of the damage.<br />
We have applied this microscope <strong>for</strong> in vivo observation of the dermal collagen fiber in cheek<br />
of volunteers, under permission of Bioethics Committee <strong>for</strong> Human Experiment at Osaka<br />
University. We found that concentration of dermal collagen becomes lower by aging and sunburn.<br />
Sunburn causes photo-aging, resulting in increase of wrinkles. We have also investigated influence<br />
of UV exposure to the collagen orientation and wrinkle direction on mouse skin using polarization<br />
SHG measurement [4] .<br />
The SHG light is resulted from non-centrosymmetric herical structure in the collagen<br />
molecule. When the collagen molecule denatures and loses the helical structure by burn, resultant<br />
SHG signal decreases. Following this concept, degree of burn was investigated on rat skin by the<br />
measurement SHG signal. Experimental protocol of burn <strong>for</strong> animal skin was approved by the<br />
Committee <strong>for</strong> Animal Experiment at Osaka University. Resultant SHG images clearly showed the<br />
structural change of collagen fiber by the burn. For example, SHG light signals corresponding fiber<br />
structure of collagen was well observed in the control skin but the signal disappeared in the burned<br />
skin when the skin was immersed in hot water at temperature of 98 o C <strong>for</strong> 10 sec that causes deep<br />
burn. Resultant SHG light intensity significantly relates to the burn temperature, suggesting SHG<br />
light measurement is applicable to diagnosis of skin burn.<br />
References: [1] I.-H. Chen, et al., Optical and Quantum Electronics, 34, 1251-1266 (2002).; [2] Chi-Kuang<br />
Sun, et al., Structural Biology, 147, 19-30 (2004).; [3] Shin-Peng Tai, et al, Optics Express, 14, 6178-6187<br />
(2006).; [4] T. Yasui, et al, Optics Express, 17, 912-923 (2009).<br />
______________<br />
* Corresponding author: e-mail: araki@me.es.osaka-u.ac.jp<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 145
P38 Fluorescence Microscopies and Cell Imaging Poster 38<br />
Determination of protein-protein-interactions in microfluidic droplets<br />
using Fluorescence Lifetime Microscopy<br />
Christian Benz 1,2* , Heiko Retzbach 2 , Stefan Nagl 2 & Detlev Belder 2<br />
1 LIFE – Leipzig Research Center <strong>for</strong> Civilization Diseases, Universität Leipzig<br />
2 Institute of Analytical Chemistry, Universität Leipzig, Johannisallee 29, 04103 Leipzig (Germany)<br />
Microfluidic chips aim at the miniaturization of chemical and biochemical processes in tiny<br />
microchannels and feature high reaction speeds due to short diffusion lengths accompanied by<br />
decreased sample consumption. Especially biochemical procedures are addressed by<br />
miniaturization since frequently only very small sample amounts of precious samples like proteins<br />
and antibodies are available. Monodisperse droplet systems are <strong>for</strong>med in microchips by<br />
dispersing a solvent into an immiscible liquid in a suitable microstructure such as a T junction.<br />
Droplets on the scale of femtoliters to nanoliters are created allowing very small sample amounts<br />
to be investigated. By transfer of different reagents into discrete droplets, various chemical<br />
environments may be realized sequentially. Protein-protein interactions under various conditions<br />
can be investigated and stabilities of complexes between disease-related proteins with artificial<br />
binding partners may be determined in microfluidic droplets. Fluorescence lifetime is an attractive<br />
parameter since it is sensitive to the environment and may be used <strong>for</strong> interactions studies but has<br />
not been applied to microdroplet protein studies so far.<br />
Microfluidic droplets of aqueous solutions of two fluorescent samples dispersed in perfluorinated solvent<br />
Microfluidic structures were generated in biocompatible PDMS (polydimethylsiloxane) by<br />
softlithographic molding of a master structure. The initial structure was created in polyester by laser<br />
ablation and then hot embossed into PMMA (acrylic glass). After copying the structure into PDMS<br />
and bonding to a plain PDMS substrate a ready-to-use microfluidic chip was obtained.<br />
Protein-protein interactions were investigated using fluorescence lifetime measurements of<br />
Förster resonance energy transfer (FRET-FLIM) in individual picoliter microdroplets. Biotinstreptavidin<br />
and antibody-antigen interactions were investigated using model proteins and<br />
detection was accomplished using time-correlated single photon counting (TCSPC) in the donor<br />
and acceptor channel on a confocal microscope setup. Fluorescence lifetime detection was<br />
per<strong>for</strong>med and the results compared to intensity-based and intensity ratio measurements in both<br />
channels.<br />
This publication is supported by LIFE – Leipzig Research Center <strong>for</strong> Civilization Diseases,<br />
Universität Leipzig. This project was funded by means of the European Social Fund and the Free<br />
<strong>State</strong> of Saxony.<br />
______________<br />
* Corresponding author: e-mail: christian.benz@uni-leipzig.de<br />
146 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P39 Fluorescence Microscopies and Cell Imaging Poster 39<br />
Probing protein homodimerization by coupling time-resolved<br />
Fluorescence Anisotropy Imaging on a TIRF Microscope<br />
Viviane Devauges 1,2,3 , Catherine Marquer 4 , Sandrine Lécart 3 , Marie-Claude Potier 4 ,<br />
Emmanuel Fort 5 & Sandrine Lévêque-Fort 1,3<br />
1 Institut des Sciences Moléculaires d’Orsay, UMR 8214, Orsay (France)<br />
2 Laboratoire Charles Fabry Institut d'Optique CNRS UMR 8501, Palaiseau (France)<br />
3 Centre de Photonique Biomédicale, CLUPS/LUMAT FR2764, Orsay (France)<br />
4 CRICM, UPMC/Inserm UMR-S975/CNRS UMR7225, Hôpital de la Pitié-Salpêtrière, Paris (France)<br />
5 Institut Langevin, ESPCI ParisTech, UMR 7587, Paris (France)<br />
Forster Resonance Energy Transfer (FRET) imaging is a precise tool to follow proteins<br />
interactions. In the case of proteins homodimerization, it is more efficient to use HomoFRET which<br />
only requires one labeling and can be evidenced thanks to time-resolved Fluorescence Anisotropy<br />
Imaging (tr-FAIM). This technique not only enables to follow proteins homodimerization but it also<br />
permits to probe intracellular viscosity [1] . In order to have a dynamic follow-up of the interaction of<br />
proteins from the plasma membrane to the inside of the cell, we implemented a polarizationresolved<br />
imager (dualview, Cairn) on our versatile microscope. Thus, we can excite the sample<br />
with angle from epifluorescence to evanescent wave, leading to a Total Internal Reflection<br />
Fluorescence Lifetime Imaging Microscope with a sub-wavelength axial resolution [2] . To preserve<br />
the wide-field approach <strong>for</strong> time-domain fluorescence lifetime measurements, the fluorescence<br />
decay was sampled thanks to a High Rate Imager (Kentech Ltd). The excitation is per<strong>for</strong>med with<br />
a supercontinuum source which permits to excite a large range of fluorophores and to measure<br />
long fluorescence lifetimes or anisotropy decays (20 MHz repetition rate). First, in order to measure<br />
the rotational correlation time of molecules and probe their environment viscosity, calibration<br />
measurements were per<strong>for</strong>med on fluorescein solutions with different viscosities under<br />
epifluorescent excitation, and using different objectives. By inducing a linearly polarized excitation<br />
light, we acquired simultaneously fluorescent lifetime images <strong>for</strong> parallel and perpendicular<br />
polarization components. We could then deduce the anisotropy decay which gives us in<strong>for</strong>mation<br />
about the rotational diffusion of fluorophores and about the viscosity of their local environment.<br />
Since we want to study proteins interactions at the plasma membrane, we need to excite our<br />
samples in TIRF configuration, which requires objectives with high numerical aperture (NA). We<br />
show that high NA objectives used in our wide field configuration induce a depolarization in the<br />
detection pathway [3] , hence modifying the anisotropy decays as predicted by simulations. Then,<br />
experiments of Homo-FRET were pursued on HEK-293 cells. We used GFP since its rotational<br />
correlation time is very long compared to its fluorescence lifetime, so the observed depolarization<br />
will only be due to energy transfer. HEK-293 cells expressed cytosolic GFP or a constitutively<br />
dimerizing GFP tandem as a positive control. Measurements were also made on HEK-293 cells<br />
expressing wild-type Amyloid Precursor Protein (APP), a key protein in Alzheimer's disease, or a<br />
mutated APP which <strong>for</strong>ms covalent dimers. All results regarding anisotropy calibration using<br />
fluorescein solutions or cells will be presented. First results of time-resolved fluorescence<br />
anisotropy in TIRF configuration will also be shown.<br />
References: [1] K. Suhling et al., Opt. Lett.,29 (2004).[2] P. Blandin, et al., Appl. Opt. 48 (2009) 553, [3]<br />
J.Fisz et al., J. Phys.Chem. 111, (2007), 8606-8621.<br />
______________<br />
* Corresponding author: e-mail: sandrine.leveque-<strong>for</strong>t@u-psud.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 147
P40 Fluorescence Microscopies and Cell Imaging Poster 40<br />
Intracellular dynamics of HIV-Gag: activation of phospholipase C and<br />
virus-like particles release<br />
Andrea Gramatica, Roland Schwarzer & Andreas Herrmann<br />
Institute of Biology/Biophysics, Humboldt-University of Berlin, Invalidenstrasse 42, 10115 Berlin,<br />
(Germany)<br />
Assembly of the human immunodeficiency virus type 1 (HIV-1) is determined by the single<br />
structural polyprotein Pr55 Gag, and may occur at the plasma membrane or within late<br />
endosomes/multivesicular bodies (LE/MVB). Independently of the site where the assembly occurs,<br />
the <strong>for</strong>med particles are released from the plasma membrane of the host cell. The Gag protein is<br />
the only viral component necessary and sufficient <strong>for</strong> the assembly of virus-like particles (VLPs) [1] .<br />
It is well known that participation of host cell components is particularly required <strong>for</strong> any of<br />
the many Gag-encoded functions [2] . In particular, previous studies have shown that the activation<br />
of the phospholipase C (PLC) and the inositol-(1,4,5)-triphosphate receptor (IP3R), resulting in an<br />
increase of intracellular calcium concentration, are both required <strong>for</strong> efficient Gag trafficking and<br />
VLPs release [3] . It is still unclear which cellular or viral factor might activate this signalling pathway<br />
<strong>for</strong> the virus release process. For delivery of calcium at spatially and temporally appointed events<br />
in the VLPs release process, the Gag protein itself or some other of the proteins that Gag recruits<br />
may serve as a trigger of PLC and IP3R activation. We are there<strong>for</strong>e interested to find out how this<br />
pathway is activated in Gag-expressing cells.<br />
Different cell lines transfected with Gag fused with a fluorescent protein (e.g. EGFP, EYFP)<br />
are our principal tool to investigate the intracellular localization of the viral protein upon activation<br />
or inhibition of the PLC signalling pathway. Chemical inhibitors and RNAi technology were utilized<br />
to turn off this pathway at different steps and time points. Total Internal Reflection Fluorescence<br />
microscopy (TIRFM), Förster Resonance Energy Transfer (FRET) methods and coimmunoprecipitation<br />
(Co-IP) were then applied to study the localization of Gag at the plasma<br />
membrane and interactions with specific binding partners of the host cell.<br />
This work is part of the Virus Entry Project – Marie Curie Actions (EUROPEAN COMMISSION 7th<br />
Framework Programme Initial Training Networks).<br />
References: [1] P.D. Bieniasz, et al., Cell Host and Microbe, 5 (2009) 550. [2] S.P. Goff, Nature<br />
Microbiology, 5 (2006) 253. [3] L.S. Ehrlich, et al., Journal Of Virology. 84 (2010) 6438.<br />
______________<br />
* Corresponding author: e-mail: andrea.gramatica@hu-berlin.de<br />
148 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P41 Fluorescence Microscopies and Cell Imaging Poster 41<br />
DNA biosensors <strong>for</strong> single molecule Transcription Factor detection with<br />
ALEX-TIRF microscopy<br />
Kristin Grußmayer 1 , Tanja Ehrhard 1 , Anton Kurz 1 , Michael Schwering 1 , Jessica Balbo 1 ,<br />
Konstantinos Lymperopoulos 1 & Dirk-Peter Herten 1*<br />
1<strong>Single</strong><br />
Molecule Spectroscopy, Bioquant/ CellNetworks, University of Heidelberg, Germany, Im<br />
Neuenheimer Feld 267 69120<br />
Transcription factors (TFs) are sequence-specific DNA binding proteins that play an important role<br />
in the control of gene expression. In a cell, they regulate fundamental processes such as the cell<br />
cycle and DNA repair and in multicellular organisms many TFs are involved in organism<br />
development. Maintaining appropriate TF levels is crucial and alterations often indicate or lead to a<br />
disease.<br />
Thus, it is of great interest to accurately determine TF levels both <strong>for</strong> research and as a<br />
diagnostic tool. Traditional methods are the electrophoretic mobility shift assay (EMSA), the<br />
luciferase reporter assay or the enzyme-linked immunosorbent assay (ELISA) to name only a few.<br />
These ensemble methods often lack sensitivity or require signal amplification steps or are not<br />
compatible with in vivo measurements. <strong>Single</strong> molecule fluorescence spectroscopy is ideally suited<br />
to overcome these limitations. It allows measurements in living cells and can quickly provide<br />
results in a diagnostic setting.<br />
Here, we use alternating-laser excitation (ALEX) on a single molecule sensitive total internal<br />
reflection fluorescence microscope (TIRFM) to detect TFs in vitro down to pM levels using a DNA<br />
biosensor and custom-made Matlab analysis software.<br />
The biosensor consists of the TF DNA target site cut into two half-sites (H) termed H1 and<br />
H2 with single-stranded overhangs. The half-sites are labeled with fluorophores and single<br />
molecules of H1 are immobilized on the surface of a Lab-Tek chambered glass coverslide (Nunc).<br />
H2 and TF are in solution and the TF is sensed through TF-dependent coincidence of the<br />
fluorescently labeled DNAs, similar to as it was described be<strong>for</strong>e [1] . We extended the previously<br />
developed model that uses two coupled reaction equilibria (H1 and H2 association and TF binding<br />
to the fully-assembled TF target site) that relates the fraction of DNA with TF bound to the<br />
concentration of TF present in solution, in order to account <strong>for</strong> the fact that one of the half-sites is<br />
immobilized. The model is validated through experiments with known concentrations of E. coli TF<br />
Lactose Repressor (LacR, control of lactose metabolism). Using biosensor measurements, we<br />
compare our TIRFM-based assay with in solution single molecule confocal microscopy detection<br />
as previously established. TF detection with our assay is similar in sensitivity and the TIRFM-based<br />
approach allows rapid sampling of hundreds of molecules to quickly accumulate statistics needed<br />
<strong>for</strong> concentration determination. Together with the possibility of multiplexing through spatially<br />
controlled immobilization of different half-sites, this approach is well suited <strong>for</strong> TF detection in a<br />
diagnostic setting. As a step towards robust measurements in biological fluids or even in cells, we<br />
tested PTO-modified half-sites <strong>for</strong> protection against nucleases.<br />
References: [1] K. Lymperopoulos, et al., Angew. Chem. Int. Ed., vol. 49 (7) (2010) 1316<br />
______________<br />
* Corresponding author: e-mail: dirk.herten@bioquant.uni-heidelberg.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 149
P42 Fluorescence Microscopies and Cell Imaging Poster 42<br />
Combining fluorescence techniques to unravel ligand binding<br />
mechanisms at G-protein coupled receptors : the cholinergic<br />
muscarinic M1 receptor as an example<br />
Brigitte Ilien 1,* , Sandrine Daval 1 , Dominique Bonnet 2 , Pascal Didier 3 , Marcel Hibert 2 , Yves<br />
Mély 3 & Jean-Luc Galzi 1<br />
1 Laboratoire de Biotechnologie et Signalisation Cellulaire, UMR 7242 CNRS, Université de<br />
Strasbourg, Ecole Supérieure de Biotechnologie de Strasbourg, Bvd S. Brant, 67412 Illkirch (France)<br />
2 Laboratoire d’Innovation Thérapeutique, UMR 7200 CNRS, Université de Strasbourg, Faculté de<br />
Pharmacie, 74 route du Rhin, 67401 Illkirch (France)<br />
3 Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg,<br />
Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch (France)<br />
G protein-coupled receptors (GPCRs) are involved in a number of physio-pathological processes and<br />
are attractive therapeutic targets. These transmembrane proteins oscillate between various<br />
con<strong>for</strong>mational and functional states, depending on the cellular context, differentially stabilized by<br />
ligands and discriminated by downstream protein partners. Muscarinic receptors (M1-M5 subtypes) are<br />
such allosterically regulated GPCRs that bind acetylcholine and a variety of ligands (orthosteric or<br />
allosteric) according to poorly understood molecular mechanisms, most often interpreted from<br />
radioligand binding studies.<br />
We report here on a combination of fluorescence techniques to address the initial ligand<br />
recognition and binding step at M1 receptors : kinetic description, relationship with relevant receptor<br />
con<strong>for</strong>mational states and sensitivity to allosteric modulators. Binding of the commercial fluorescent<br />
muscarinic antagonist Bodipy-Pirenzepine (BoPz) to EGFP-fused muscarinic M1 receptors, within their<br />
cellular context, was first examined through fluorescence resonance energy transfer (FRET), under<br />
steady-state conditions. A robust extinction of EGFP fluorescence (FRET signal) was obtained through<br />
shortening the distance separating the EGFP donor from the Bodipy fluorophore of the ligand in the<br />
receptor-bound state. This was achieved using a receptor construct with a deleted N-terminus that<br />
retained native pharmacological and functional properties [1] . It also perfectly localized at the cell plasma<br />
membrane, as shown by confocal microscopy. This EGFP(Δ17)hM1 construct proved to be an ideal<br />
tool to search <strong>for</strong> new fluorescent or dye ligands that could further improve FRET sensitivity [2,3] . It<br />
allowed us to follow BoPz binding in real-time, as a function of ligand concentration, and to obtain a fine<br />
description of association and dissociation kinetics. Time-resolved fluorescence measurements, such<br />
as fluorescence lifetime imaging and time-correlated single photon counting, allowed to visualize the<br />
binding event at the cell plasma membrane level and, most interestingly, to identify discrete EGFP<br />
lifetime species with a typical redistribution upon ligand binding. The comparison of several binding<br />
models <strong>for</strong> their pertinence in reconciling quantitative in<strong>for</strong>mation provided by kinetic and time-resolved<br />
studies led us to propose a three-step mechanism (with four identified receptor con<strong>for</strong>mational states)<br />
driving ligand-receptor complexes towards a high affinity dimeric state. Most importantly, such a<br />
prediction was experimentally validated by fluorescence correlation spectroscopy, with EGFP<br />
brightness analysis on cells : at the plasma membrane level, EGFP-fused muscarinic M1 receptors<br />
predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding [4] . Ongoing<br />
research is devoted to allosteric modulation of M1 receptors, the search <strong>for</strong> fluorescent allosteric<br />
tracers and the definition of their binding domains. This study is based on equilibrium and kinetic<br />
binding studies per<strong>for</strong>med under FRET conditions or using a tritiated orthosteric tracer. A series of<br />
fluorescent derivatives of the orthosteric antagonist pirenzepine [2] or of the allosteric agonist AC-42<br />
have been found to display a bitopic binding behaviour in bridging the acetylcholine site to an allosteric<br />
site (overlapping with or distinct from known allosteric domains).<br />
This work was supported by the CNRS, INSERM, Université de Strasbourg, grants from MESR,<br />
CIFRE, ANR, Région Alsace and Communauté Urbaine de Strasbourg.<br />
References : [1] B. Ilien et al., J. Neurochem. 85 (2003) 768. [2] C. Tahtaoui et al., J. Med. Chem. 47 (2004)<br />
4300. [3] Tahtaoui et al., J. Med. Chem. 48 (2005) 7847. [4] B.Ilien et al., J. Biol. Chem. 284 (2009) 19533<br />
______________<br />
* Corresponding author : Email : brigitte.ilien@unistra.fr<br />
150 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P43 Fluorescence Microscopies and Cell Imaging Poster 43<br />
B cells can communicate through membrane nanotubes: imaging<br />
studies on the background of a tunnel <strong>for</strong>mation mystery<br />
Emese Izsepi 1 , Anna Csala & Janos Matko 1,*<br />
1 Eötvös Lorand University, Budapest, Hungary<br />
Membrane nanotubes are transient long-distance connections between cells that can facilitate<br />
intercellular communication. Beside neuronal cells several kinds of immune cells are also known to<br />
<strong>for</strong>m nanotubes, like T; NK and myeloid cells [1] . These tethers can be <strong>for</strong>med in two ways: an actindriven<br />
protrusion from one cell can be driven out to connect to a nearby cell and cells might come<br />
into contact and draw out nanotubes as they subsequently move apart. Cells can create also open,<br />
and closed-ended tunnels and they always contain F-actin. They can traffic bacteria on their<br />
surfaces; vesicles; calcium flux; viral protein; cytoplasmic molecules; membrane anchored proteins<br />
either inside (thin nanotubes) or along their surface (thick nanotubes) [1] .<br />
The mechanism of <strong>for</strong>mation and the role of nanotube <strong>for</strong>mation between lymphocytes,<br />
however, still remained largely unclear and controversial. There<strong>for</strong>e we decided to investigate the<br />
major factors involved in <strong>for</strong>mation of such nanotubes between lymphocytes with particular<br />
attention to B cells.<br />
We noticed, that 15-20% of primary B cells of spleen origin, as well as B cells modelling<br />
different differentiation stages also <strong>for</strong>m long and frequently branching nanotube networks with<br />
each other. These tubes are rather thick, long and mostly arise from one cell towards to a nearby<br />
one.<br />
Our aims here were to investigate the mechanism and regulation of nanotube <strong>for</strong>mation<br />
between B cells in different maturation status, there<strong>for</strong>e we analysed the role of actin integrity;<br />
myosin function and cholesterol using inhibitor molecules of their function or contribution<br />
(Latrunculin B; Cytochalasin B; Blebbistatin; Methyl-beta-cyclodextrin, MCD), respectively. Actin<br />
integrity and membrane cholesterol content seemed to be indispensable factors in the nanotube<br />
<strong>for</strong>mation. Treatment of the B cells with Blebbistatin, a myosin-2 motor protein inhibitor,<br />
interestingly decreased nanotube <strong>for</strong>mation, but resulted in occurance of a more branching<br />
connection network in the cell culture.<br />
We tested what kind of molecule(s) can induce and direct <strong>for</strong>mation of these nanotubes? To<br />
answer this question we are testing the effect of chemokine injection into the cell culture and the<br />
effect of membrane depolarization, as two effects controlling the dendritic growth and coupling in<br />
neuronal cell cultures.<br />
Furthermore we are planning to analyse what kind of molecules (costimulators, MHC,<br />
tetraspans, etc.) appear along these membrane nanotube tethers between the B cells, together<br />
with the question how the apoptosis of B cells affects nanotube <strong>for</strong>mation.<br />
This work was supported by grants from OTKA-NFÜ CK 80935, The European Union and the<br />
European Social Fund have provided financial support to the project under the grant agreement<br />
no. TÁMOP 4.2.1./B-09/1/KMR-2010-0003.<br />
References: [1] D. M. Davis, S Sowinski, Nat Rev Mol Cell Biol., 2008 Jun; 9(6):431-6<br />
______________<br />
* Corresponding author: e-mail: matko@elte.hu<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 151
P44 Fluorescence Microscopies and Cell Imaging Poster 44<br />
Investigation by high resolution microscopy of the internalization of the<br />
neuropeptide Y bound to its Y1 receptor<br />
Noémie Kempf, Pascal Didier, Ludovic Richert, Viktoriia Postupalenko, Andrey Klymchenko,<br />
Hugues de Rocquigny, Bernard Bucher & Yves Mély *<br />
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg,<br />
Faculté de Pharmacie, 74, Route du Rhin, 67401 Illkirch (France).<br />
The human neuropeptide Y (NPY) modulates numerous physiological processes, including<br />
regulation of cardiovascular and renal functions, intestinal motility, memory, anxiety, seizure,<br />
feeding, circadian rhythms and nociception. The NPY G-protein coupled receptor (GPCR) Y1 is<br />
thought to mediate most of the physiological and physiopathological actions of NPY. Thus, the<br />
study of the mechanisms involved in the regulation of the Y1 receptors should contribute to a better<br />
understanding of the mechanism and functions of NPY. Previous fluorescence studies have shown<br />
that activated GPCR Y1 rapidly internalize through clathrin-coated pits, and subsequently recycle<br />
from early and recycling endosomes 1 . Furthermore, the C-terminal cytoplasmic tail of the Y1<br />
GPCR was found to play a role <strong>for</strong> NPY induced internalization 2,3 . In an ef<strong>for</strong>t to characterize the<br />
NPY intracellular pathway, the Y1 receptor was tagged with eGFP, while NPY was fluorescently<br />
labeled at its N-terminus by two different probes. These probes were selected <strong>for</strong> per<strong>for</strong>ming<br />
stochastic optical reconstruction microscopy (STORM), which enables imaging of small objects<br />
with a lateral resolution well below the diffraction limit. The first of these fluorescent probes is a<br />
derivative of Nile Red (NR) and its fluorescence can be reversibly switched by sodium dithionite, a<br />
chemical compound that does not enter into the cells 5 . The second dye is Cy5 which was used<br />
together with an oxygen scavenger. An overlap was observed between the TIRF images of the<br />
EGFP-labeled Y1 receptors and the STORM images of both NR-NPY and Cy5-NPY, confirming<br />
that NPY peptides bind to the receptors. Quenching of the fluorescence of the extracellular and<br />
membrane bound NR-NPY peptides by sodium dithionite, revealed an intracellular distribution of<br />
small fluorescent spots, whose size is consistent with that of endosomes. This intracellular<br />
distribution and the spot sizes were found to change with time. Work is in progress to further<br />
characterize and quantify the NPY internalization process.<br />
References: [1] H. Gicquiaux, et al., J. Biol. Chem., 227 (2002) 6645. [2] M. Ouedraogo, et al., Traffic 9<br />
(2008) 305. [3] S. Lecat, et al., Cellular Signaling 23 (2011) 228. [4]. R. Henriques, et al., Biopolymers 95<br />
(2011) 322. [5]. O.A. Kucherak, et al., J. Am. Chem. Soc 132 (2010) 4907.<br />
______________<br />
* Corresponding author: e-mail: yves.mely@unistra.fr<br />
152 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P45 Fluorescence Microscopies and Cell Imaging Poster 45<br />
Bi-plane calibration in super-resolution microscopy<br />
Hagai Kirshner 1, * , Thomas Pengo 2 , Nicolas Olivier 2 , Daniel Sage 1 , Suliana Manley 2<br />
& Michael Unser 1<br />
1 Biomedical Imaging Group, EPFL, Lausanne<br />
2 Laboratory of Experimental Biophysics, EPFL, Lausanne<br />
We consider the task of aligning the imaging detectors<br />
of a bi-plane microscope <strong>for</strong> super-resolution<br />
applications [1]. Such a microscope consists of two<br />
separate focal planes and the optical misalignment is<br />
modelled as an affine trans<strong>for</strong>m. In particular,<br />
r<br />
u 1 = A r<br />
u 2 + r<br />
b accounts <strong>for</strong> translation, rotation and<br />
scaling operations (see illustration <strong>for</strong> an example of<br />
misaligned data). Here, r<br />
u 1, r<br />
u 2 describe a single point<br />
in space in terms of the two coordinate systems, A is a<br />
3x3 matrix, and r<br />
b is a translation vector. There are 12 misalignment parameters that need to be<br />
found, i.e. the matrix A and the vector r b.<br />
The calibration data consists of two z-stacks, one <strong>for</strong> each plane. The microscopic sample is<br />
composed of fixed fluorophores beads that are located at unknown lateral and axial positions. The<br />
algorithm has two main stages: particle localisation and affine trans<strong>for</strong>m estimation.<br />
In the localisation stage, the two stacks are processed separately. Local maxima are<br />
assumed to originate from fluorophore beads, and a threshold value is then used to keep the<br />
prominent ones only [2]. Each one of these local maxima is then fitted with a PSF model, yielding<br />
the lateral and the axial coordinates of each fluorophore bead. The fitting error is further compared<br />
with a threshold value <strong>for</strong> obtaining reliable localisation results. The algorithm fits the local maxima<br />
with the Gibson and Lanni PSF [3]. This model considers aberrations that are due to refractive<br />
indices mismatches at the sample - cover slip - immersion interfaces, as well as <strong>for</strong> the thickness<br />
of these layers. In particular, axial stage displacements correspond to different values of the<br />
immersion layer thickness. The output of the localisation stage is two lists of 3D beads locations.<br />
The next stage of the algorithm consists of a least square estimation of the matrix A and the<br />
vector r<br />
b<br />
min<br />
A, r<br />
r n r r 2<br />
n ∑ u 2 − Au 1 − b ,<br />
b<br />
l 2<br />
n<br />
where the n-th subscript is a running index <strong>for</strong> the two localisation lists. The minimum least square<br />
solution is analytically described by a set of linear equations. It assumes, however, that the two<br />
lists are properly ordered in the sense that r Camera 1 Camera 2<br />
n r n<br />
u and 1 u correspond to the same fluorophore bead.<br />
2<br />
This does not hold true in practice and the algorithm uses the nearest neighbour criterion <strong>for</strong><br />
ordering the two lists as a preliminary operation. The nearest neighbour criterion works well in this<br />
case as the affine trans<strong>for</strong>m introduces small discrepancies between the two coordinate systems.<br />
Once the 12 parameters of the affine trans<strong>for</strong>m are estimated, the algorithm interprets them in<br />
terms of translation, scaling and rotation operations that need to be applied to one of the imaging<br />
planes. Alternatively, the calibration parameters can be used in localization algorithms that<br />
mathematically overcome misalignment.<br />
We developed an ImageJ plugin and validated it <strong>for</strong> both synthetic and real data. It consists<br />
of fast multi-threaded calculations of the PSF and it posseses a simple user interface.<br />
References: [1] M. Juette et al., “Three-dimensional sub–100 nm resolution fluorescence microscopy of<br />
thick samples”, Nature Methods - 5, 527 – 529, 2008 [2] Eric Betzig et al., “Imaging Intracellular Fluorescent<br />
Proteins at Nanometer Resolution”, Science, Vol. 313, no. 5793 pp. 1642-1645, 2006. [3] S. F. Gibson and<br />
F. Lanni, “Experimental test of an analytical model of aberration in an oil-immersion objective lens used in<br />
three-dimensional light microscopy,” J. Opt. Soc. Am. A, vol. 8, no. 10, pp. 1601–1613, 1991.<br />
______________<br />
* Corresponding author: e-mail: hagai.kirshner@epfl.ch<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 153
P46 Fluorescence Microscopies and Cell Imaging Poster 46<br />
3D PSF models <strong>for</strong> fluorescence microscopy in ImageJ<br />
Hagai Kirshner, Daniel Sage & Michael Unser<br />
Biomedical Imaging Group, EPFL, Lausanne<br />
We introduce an ImageJ application that allows one to generate various<br />
3D PSF models. Keeping the biological practitioner in mind, few input<br />
parameters are required only (Figure 1). Our application can generate zstacks<br />
at any size and at any lateral and axial resolution. Our<br />
implementation utilizes a multi-thread design that allows <strong>for</strong> parallel and<br />
fast computations.<br />
The current version allows <strong>for</strong> five different PSF models: The<br />
Gaussian model simulates a blurring effect by setting three different<br />
variance values. These values characterize the width of the PSF at<br />
various axial positions along the z-stack. The defocus model is simulated<br />
in the Fourier domain by a modulated Gaussian where the sinc<br />
modulation depends on the axial defocusing distance. Another<br />
defocusing model is due to Koehler, which also uses the Gaussian<br />
function in the Fourier domain. In this model, however, the sinc<br />
modulation is replaced by a linear term <strong>for</strong> the variance.<br />
The Born & Wolf model provides yet another defocusing model <strong>for</strong><br />
which the observed fluorophore particle is located at the focal plane of the<br />
objective lens, right beneath the coverslip. The slices of the output z-stack<br />
correspond then to different values of the microscope’s stage. The<br />
Gibson & Lanni PSF model can be seen as a generalization of Born &<br />
Wolf in the sense that the fluorophore particle can be located at any depth<br />
within the sample. It also considers three optical layers (sample-coverslipimmersion)<br />
instead of two (glass-immersion). This, in turn allows <strong>for</strong> nonsymmetric<br />
PSF models that originate from refractive indices mismatch<br />
(Figure 2). Both models use Kirchhoff’s diffraction integral <strong>for</strong>mula,<br />
where I is the pixel value located at a distance r from the centre of the<br />
image, NA is the numerical aperture of the microscope, k is the wave<br />
number of the fluorophore, and OPD is the optical path difference<br />
described by each model. We implemented this <strong>for</strong>mula by means of<br />
iterative Riemann sums, which allows one to set the accuracy of the<br />
integral approximation a-priori. The software design is modular and<br />
additional PSF models can be easily incorporated. Relevant works on this<br />
topic are the Diffraction PSF 3D ImageJ plugin [1] and the PSF LAB<br />
Matlab-based application [2]. The <strong>for</strong>mer relies on a simplified fourth<br />
power model <strong>for</strong> modelling spherical aberrations, and the latter relies on a<br />
detailed vectorial model in providing 2D images that may take several<br />
minutes each.<br />
The models of the ImageJ plugin were successfully applied to fluorescence microscopy<br />
applications such as de-convolution [3], fluorescent particles tracking [4], extended depth of field<br />
estimation [5] and super-resolution 3D PALM localization. These models can also be used <strong>for</strong> validating<br />
experimental PSF measurements and to further find optimal model parameters <strong>for</strong> a given experimental<br />
data set. The plugin is available at http://bigwww.epfl.ch/algorithms/psfgenerator/.<br />
References: [1] http://www.optinav.com/Diffraction-PSF-3D.htm [2] M. J. Nasse and J. C. Woehl ”Realistic modeling<br />
of the illumination point spread function in confocal scanning optical microscopy” Journal of the Optical Society of<br />
America A 27, (2010) [3] A. Griffa et al. "Comparison of Deconvolution Software in 3D Microscopy. A User Point of<br />
View - Part 1", G.I.T. Imaging & Microscopy, vol. 1, 2010. [4] D. Sage et al. "Automatic Tracking of Individual<br />
Fluorescence Particles: Application to the Study of Chromosome Dynamics," IEEE Transactions on Image<br />
Processing, 14(9), 2005. [5] F. Aguet et al. "Model-based 2.5-D deconvolution <strong>for</strong> extended depth-of-field in<br />
brightfield microscopy," IEEE Trans. Image Process, 17(7), 2008.<br />
______________<br />
* *Corresponding author: e-mail: hagai.kirshner@epfl.ch<br />
154 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
Figure 1: User interface. The<br />
“Output” section is common to all<br />
models. A detailed description of<br />
each model is provided by the<br />
interface, too, along with additional<br />
required parameters.<br />
Figure 2: Gibson & Lanni 3D PSF<br />
model. Shown here is a nonsymmetric<br />
z-stack due to refractive<br />
indices mismatch.
P47 Fluorescence Microscopies and Cell Imaging Poster 47<br />
Fluorescence imaging of living hybrid materials<br />
Alexander M. Macmillan 1† , Dylan M. Owen 2 , Katharina Gaus 2 , David J. S. Birch 3<br />
& Jan Karolin 3<br />
1 Biomedical Imaging Facility, University of New South Wales, Sydney, Australia<br />
2 Centre <strong>for</strong> Vascular Research, University of New South Wales, Sydney, Australia<br />
3 Centre <strong>for</strong> Molecular Nanometrology, University of Strathclyde, Glasgow, United Kingdom<br />
†To whom correspondence should be addressed: Alex Macmillan, Biomedical Imaging Facility,<br />
University of New South Wales, Sydney, Australia<br />
Living hybrid materials may be designed to drive biochemical reactions in highly controlled<br />
environments. In this contribution we investigate the application of silica sol-gels based on a<br />
tetramethoxy orthosilicate (TMOS) precursor to live cell encapsulation and immobilization. Cells<br />
are introduced in the sol phase which allows pores to be <strong>for</strong>med that closely match their<br />
dimensions. The encapsulated cells remain accessible through interconnecting channels with<br />
typical radius of a few nanometers allowing <strong>for</strong> the diffusion of analytes through the gel network.<br />
Typical sol-gel preparation requires harsh conditions which are not biocompatible and conducive to<br />
protein or cell viability. These detriments can be overcome by closely monitoring the time required<br />
<strong>for</strong> hydrolysis and subsequent methanol removal using the fluorescent probe 6-propionyl-2-(N,Ndimethylamino)naphthalene<br />
(PRODAN)(Macmillan AM et al., 2009). Furthermore, PRODAN<br />
fluorescence provides in<strong>for</strong>mation regarding the gel solvent phase allowing <strong>for</strong> guidance in the<br />
development of improved biocompatible materials. We investigate the cell membrane order of the<br />
entrapped cells using fluorescence dye di-4-ANEPPDHQ which displays differing emission spectra<br />
and fluorescence lifetimes when in ordered or disordered phase within the cell membrane.<br />
Fluorescence lifetime imaging (FLIM) of di-4-ANEPPDHQ provides enhanced contrast, in<br />
comparison with spectral shift, when imaging live cells(Owen DM et al., 2006). We further apply a<br />
panopoly of fluorescence imaging techniques to investigate the effect of encapsulation.<br />
References:<br />
1. Macmillan, A.M., et al., Improved biocompatibility of protein encapsulation in sol-gel materials. Journal of<br />
Sol-Gel Science and Technology, 2009. 49(3): p. 380-384.<br />
2. Owen, D.M., et al., Fluorescence lifetime imaging provides enhanced contrast when imaging the phasesensitive<br />
dye di-4-ANEPPDHQ in model membranes and live cells. Biophysical Journal, 2006. 90(11): p.<br />
L80-L82.<br />
______________<br />
Corresponding author: alex.macmillan@unsw.edu.au<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 155
P48 Fluorescence Microscopies and Cell Imaging Poster 48<br />
Mechanisms of increased amyloid peptide production by membrane<br />
cholesterol loading: relevance <strong>for</strong> Alzheimer's disease development<br />
Catherine Marquer 1 , Viviane Devauges 2,3 , Jack-Christophe Cossec 1 , Géraldine Liot 4 ,<br />
Sandrine Lécart 2 , Frédéric Saudou 4 , Charles Duyckaerts 1 , Sandrine Lévêque-Fort 2,3 & Marie-<br />
Claude Potier 1*<br />
1<br />
CRICM, UPMC/Inserm UMR-S975/CNRS UMR7225, Hôpital de la Pitié-Salpêtrière, Paris (France)<br />
2<br />
CLUPS, Université Paris-Sud 11, Orsay (France)<br />
3<br />
Institut des Sciences Moléculaires d’Orsay, CNRS FRE3363, Orsay (France)<br />
4<br />
Unité Signalisation, neurobiologie et cancer, Institut Curie/CNRS UMR3306/INSERM U1005,<br />
Orsay (France)<br />
Senile plaques in the brains of Alzheimer's disease patients result from the deposition of amyloid<br />
peptide (Aβ), produced by the sequential processing of the amyloid precursor protein (APP) by two<br />
enzymes, β-secretase (Bace1) and the γ-secretase complex. Recent converging data point to an<br />
important role <strong>for</strong> cholesterol in AD pathogenesis [1] . We wanted to explore whether the higher Aβ<br />
production observed in cells with enriched membrane cholesterol [2] resulted from (i) increased<br />
activity of Bace1 or (ii) from increased access of Bace1 to its substrate, APP. We showed that the<br />
catalytic activity of Bace1 did not significantly vary with increasing membrane cholesterol<br />
concentration. We thus tested the second hypothesis that Bace1 may have greater access to APP,<br />
using two different microscopy techniques in live hippocampal neurons. Proximity (
P49 Fluorescence Microscopies and Cell Imaging Poster 49<br />
IR excitation intravital imaging: role of Hemodynamics in the<br />
development of zebrafish vasculature<br />
H.Mojzisova, J. Goetz & J. Vermot<br />
1 Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue L. Fries, 67404 Illkirch (France)<br />
Intravital imaging of the early stages embryo development is particularly sensitive to light induced<br />
toxicity which may perturb these physiological processes and harm the observed organism. Using<br />
multiphoton microscopy and excitation wavelengths in the near infrared region reduces the risk of<br />
photo-damage compared to confocal microscopy. At these wavelengths, the biological samples<br />
absorb significantly less photons than in the visible part of the spectrum, increasing thus the depth<br />
of light penetration. Since multiphoton excitation occurs exclusively in the focal spot, a detector<br />
pinhole is not necessary. Moreover the photobleaching is reduced in the out-of -focus surrounding<br />
structures. Two- photon microscopy using near IR fluorescent proteins is thus a suitable technique<br />
<strong>for</strong> long time lapse observations of the embryonic development as well <strong>for</strong> imaging the morphology<br />
of adult tissue.<br />
Here we characterized a multiphoton Ti:Sapphire-OPO excitation system to address whether<br />
far red multiphoton microscopy can be used to study the role of the hemodynamic parameters<br />
during zebrafish cardiovascular development. In particular, we analyzed blood flow patterns and<br />
the cardiovascular architecture at several embryonic stages. For optimal excitation, we measured<br />
the excitation spectra of several red shifted fluorescent proteins in multiple cellular compartements<br />
using an array of transgenic fish and several fusion proteins. Overall our results suggest that infrared<br />
excitation is a powerful approach <strong>for</strong> fast, multicolor intravital imaging and open new<br />
applications <strong>for</strong> multiphoton microscopy.<br />
______________<br />
* Corresponding author: e-mail: hmojzi@igbmc.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 157
P50 Fluorescence Microscopies and Cell Imaging Poster 50<br />
Laser-induced DNA damage in nonlinear imaging: biosafety study<br />
Oleg Nadiarnykh 1* , Giju Thomas 2 , Johan Van Voskuilen 1 , Henricus J. C. M. Sterenborg 2<br />
& Hans C. Gerritsen 1<br />
1<br />
Debye Institute, Molecular Biophysics, Utrecht University, Princetonplein 5, 3508 TA Utrecht (The<br />
Netherlands)<br />
2<br />
Center <strong>for</strong> Optical Diagnostics and Therapy, Erasmus Medical Center, POB 2040, NL-3000 CA<br />
Rotterdam (The Netherlands)<br />
Nonlinear optical imaging modalities (multi-photon excited fluorescence, second and third<br />
harmonic generation) applied in vivo are increasingly promising <strong>for</strong> clinical diagnostics and<br />
monitoring of cancer and other disorders, as they can probe tissue with high diffraction-limited<br />
resolution (0.3μm) at near-IR wavelengths (700-1000nm). Contrast in tissue is provided by<br />
autofluorescence of NADH, FAD, melanin and various lipoproteins, while non- centrosymmetric<br />
protein arrays (collagen, myosin) give rise to second harmonic generation. However, high peak<br />
intensity of femtosecond laser pulses required <strong>for</strong> two-photon processes can cause <strong>for</strong>mation of<br />
cyclobutane-pyrimidin-dimers (CPDs) in cellular DNA similar to damage from exposure to solar UV<br />
light. Inaccurate repair of subsequent mutations increases the risk of carcinogenesis.<br />
In this study, we investigated CPD damage introduced in Chinese Hamster Ovary cells in<br />
vitro by imaging them with two-photon excited fluorescence. The resultant CPD levels were<br />
quantified by immunofluorescent staining. We further evaluated the extent of CPD damage with<br />
respect to varied laser parameters: wavelength between 690 and 810nm, pulsewidth at focal plane<br />
varied with custom-built grating pair, and pixel dwell time as compared to more pronounced<br />
damage from solar simulator UV source. While CPD damage has been expected to result from<br />
three-photon absorption, our results reveal that CPDs are induced by competing two- and threephoton<br />
absorption processes, where the <strong>for</strong>mer accesses UVA absorption band. This finding is<br />
independently confirmed by nonlinear dependencies of damage on laser power, wavelength and<br />
pulsewidth. Based on our analysis relatively safe regimes are identified as damage is undetectable<br />
below 0.3 TW/cm 2 with pixel dwell times still sufficiently long <strong>for</strong> imaging.<br />
Different distribution of CPDs within the nuclei was observed resulting from two-photon<br />
excitation as compared to UVC source.<br />
Finally, we estimated the risk of carcinogenesis due to an annual nonlinear optical biopsy at<br />
reasonable peak intensities to that from a routine outdoor exposure to sunlight.<br />
This work was supported by grant from STW (The Netherlands).<br />
______________<br />
* Corresponding author: e-mail: o.nadyarnykh1@uu.nl<br />
158 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P51 Fluorescence Microscopies and Cell Imaging Poster 51<br />
The stereological distribution of Langerhans islets studied by selective<br />
plane illumination microscopy and optical projection tomography<br />
Nils Norlin 1,2* , Jim Swoger 1 , Anna Eriksson 2 , Jürgen Mayer 1 , Ulf Ahlgren 2 & James Sharpe 1*<br />
1 Centre <strong>for</strong> Genomic Regulation (CRG), PRBB, Barcelona, Spain<br />
2 Centre <strong>for</strong> Molecular Medicine, (UCMM), Umeå University, Umeå, Sweden<br />
Selective plane illumination microscopy (SPIM) [1] has during recent years been established as a<br />
technique that enables high-resolution three-dimensional imaging with low levels of photo<br />
bleaching. As such, the technique is especially well suited <strong>for</strong> developmental studies and <strong>for</strong> whole<br />
organ imaging [2] . The islets of Langerhans are scattered by the thousand throughout the murine<br />
pancreas and play a key role in maintaining blood glucose homeostasis. In<strong>for</strong>mation about the<br />
mass, number and distribution of the islets is key to many areas of diabetes research.<br />
Un<strong>for</strong>tunately, a stereological assessment of these parameters is a tedious, time-consuming task<br />
that relies on the extrapolation of two-dimensional data. In this study, we have addressed the<br />
possibility to use SPIM as a tool to investigate the spatial (and quantitative) distribution of the<br />
pancreatic islets of Langerhans in the mouse. The intact gastric lobe [3] was stained <strong>for</strong> insulin,<br />
cleared in BABB [4] and subjected to SPIM imaging. The study shows that SPIM can be used with<br />
advantage <strong>for</strong> retrieving high-resolution data about the stereological distribution of islets and beta<br />
(insulin)-cell mass in situ. The method is likely to enable also studies of other features such as of<br />
islet composition, interaction with neighbouring cell-types (e.g. auto-immune cell), the screening <strong>for</strong><br />
rare events or cell niches etc. with unprecedented resolution in the intact pancreatic lobe.<br />
We hope that the developed methods/protocols will find use in research on the pancreas during<br />
various physiological and genetic conditions including type-1 and type-2 diabetes disease models<br />
but also in islet transplantation studies ex vivo. Current work also aims to improve spectral and<br />
spatial resolution in SPIM and optical projection tomography (OPT) <strong>for</strong> better visualisation and<br />
monitoring of the disease progression affecting the Langerhans islets.<br />
Figure:<br />
This work was supported by grants from: Foundation Olle Engkvist Byggmästare, and VIBRANT.<br />
References: [1] Huisken et al., Science 305 (2004) 1007. [2] J. Swoger et al., Biophotonics 4 (2011) 122. [3]<br />
A. Hörnblad et al., Islets Accepted 3 (2011). [4] T. Alanentalo, et al., Nature methods, 4 (2007) 31.<br />
______________<br />
* Corresponding authors: e-mail: james.sharpe@crg.es, nils.norlin@ucmm.umu.se<br />
Maximum value projection of a SPIM<br />
generated stack through a murine<br />
pancreatic gastric lobe. The bright<br />
punctuate structures are beta-cells stained<br />
with an Alexa594 dye and surrounding<br />
tissue is visible due to autofluorescence,<br />
excited at 488nm.<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 159
P52 Fluorescence Microscopies and Cell Imaging Poster 52<br />
Modelisation of the imaging process <strong>for</strong> the fluorescence macroscope<br />
Praveen Pankajakshan 1 , Elie Maalouf 2 , Bruno Colicchio 2 , Jean-Christophe Olivo-Marin 1<br />
& Alain Dieterlen 2<br />
1 Quantitative Image Analysis Unit, Institut Pasteur, 75015 Paris (France)<br />
2 Laboratoire MIPS-Lab.El, Université de Haute Alsace, 68093 Mulhouse (France)<br />
In the last decade a number of innovative technologies were deployed in the market to produce<br />
images of specimens at cellular and tissular levels. In spite of these recent advances at micron<br />
levels, it is only recently that the fluorescent MACROscopes were commercialized that enables the<br />
best of the micro-macro imaging world using a single setup. In addition to a low magnification<br />
objective lens, a MACROscope is also equipped with an apochromatic zoom lens that can work at<br />
different magnification levels <strong>for</strong> the same setup. This auxiliary zoom system helps to observe<br />
large fields and to work at large distances. However, we can show that fluorescence MACROscope<br />
cannot guarantee spatial-invariance <strong>for</strong> all zoom conditions. So, <strong>for</strong> large specimens, under low<br />
zoom settings, the field aberrations become prominent and the MACROscope’s point-spread<br />
function (PSF) varies in the lateral field proportional to the distance from the optic centre.<br />
Variation of 2.5µm bead images with lateral position in the object field. Left: The five lateral positions are<br />
shown on the lateral field while the bead images are the maximum intensity projections along their axial<br />
direction. Right: Stack XZ projection<br />
As the experimental PSF is difficult to obtain <strong>for</strong> all positions, the PSF is calculated by using the<br />
Stokseth’s model. The pupil function in the back focal plane is modeled by chopping it based on<br />
the relative position between the two overlapping apertures. A Fourier trans<strong>for</strong>m of this function<br />
gives the PSF at every location. Once the MACROscope and its aberrations are characterized, we<br />
can calculate its practical working zoom modes. To exploit low zoom conditions, we present our<br />
ongoing computational approach to aberration correction and near distortion-free imaging.<br />
This work was supported by grants from ANR DIAMOND Project.<br />
The authors gratefully acknowledge Dr. D. Hentsch and J.L. Vonesch (IGBMC) <strong>for</strong> helpful<br />
discussions.<br />
______________<br />
* Corresponding author: e-mail: alain.dieterlen@uha.fr<br />
160 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P53 Fluorescence Microscopies and Cell Imaging Poster 53<br />
Imaging the methylglyoxal-induced changes in the peripheral sensory<br />
neurons from dorsal root ganglia<br />
Beatrice Mihaela Radu 1 , Adela Marin 1 , Diana Ionela Rotaru 1 , Adina Daniela Iancu 2 , Cosmin<br />
Mustaciosu 3 , Dorel Radu 2 , Maria-Luisa Flonta 1 & Mihai Radu 3,*<br />
1 Department of Anatomy, Animal Physiology and Biophysics, Faculty of Biology, University of<br />
Bucharest, Splaiul Independentei, 91-95, 050095, Bucharest (Romania)<br />
Laboratory of Cell Immunology, Cantacuzino National Institute <strong>for</strong> Immunology and Microbiology,<br />
Bucharest (Romania)<br />
3 Department of Health and Environmental Physics, ‘Horia Hulubei’ National Institute <strong>for</strong> Physics<br />
and Nuclear Engineering, Magurele (Romania)<br />
The <strong>for</strong>mation of advanced glycation endproducts is one of the major factors implying in diabetic<br />
neuropathy, aging and neurodegenerative diseases [1] . Reactive carbonyl compounds (i.e.<br />
methylglyoxal) are either products of glucose metabolism or products of oxidative damage to lipids.<br />
They contribute to protein cross-linking and thus present a toxic burden to a variety of cells,<br />
especially neurons, which have a high rate of oxidative metabolism and a reduction in glyoxalase-I<br />
expression / activity [1] . Our aim is to investigate the local effects of MG on sensory neurons (from<br />
dorsal root ganglia), and to evaluate the MG-effect in an autoimmune diabetic mice model. We<br />
have used BALB/c mice and TCR-HA +/- /Ins-HA +/- type 1 diabetic mice (16 weeks aged). Primary<br />
cultures from dorsal root ganglia (DRG) neurons were obtained. Cells were incubated <strong>for</strong> 24h with<br />
MG (0.05 - 0.75 mM). Cell viability was evaluated by MTS assay and apoptosis was evaluated by<br />
acridine orange/ethidium bromide fluorescence microscopy. Time-lapse fluorescence microscopy<br />
(Olympus IX 71) by Fura-2 was used <strong>for</strong> monitoring the intracellular calcium changes (ΔF / F0)<br />
upon depolarization (high KCl Ringer solution). The excitation was per<strong>for</strong>med using a Xe lamp with<br />
monochromator Polychrome V (Till Photonics), and images were acquired by an iXon+ Andor<br />
camera. iQ software was used to analyse the images. Neurite outgrowth was followed in cell<br />
culture upon 24h of MG treatment both in normal and diabetic mice. After one week from the<br />
treatment, neurons are para<strong>for</strong>maldehyde-fixed and immunostained with anti–beta(III)-tubulin<br />
antibody FITC-conjugated (1:500, Abcam), a pan-neuronal marker labeling all cell bodies and<br />
neurites as previously described [2] . Our results indicate that MG is metabolised at low doses (up to<br />
150 microM) and at high doses (250 – 750 microM) it reduces neuronal excitability and viability,<br />
becoming neurotoxic. The neurite outgrowth is differently affected by MG treatment in normal and<br />
diabetic conditions. A reduction in neurite branching is observed upon MG treatment. A recent<br />
study has proved in a streptozotocin-induced diabetes model that glycation (MG) of laminin and<br />
fibronectin causes a reduction in neurotrophin-stimulated neurite outgrowth and preconditioned<br />
neurite outgrowth [3] . Our study is one of the first that takes into account the effect of MG against<br />
sensory neurons in primary culture and indicates the importance of this compound in diabetic<br />
conditions.<br />
This work was supported by grants from Romanian Ministry of Research (41-074/2007 and<br />
POSDRU/89/1.5/S/58852).<br />
References: [1] T.H. Fleming , et al., Gerontology (2010). DOI: 10.1159/000322087S. [2] N.J. Gardiner, et<br />
al., Mol Cell Neurosci 35(2007) 249. [3] B. Duran-Jimenez, et al., Diabetes 58(2009) 2893.<br />
______________<br />
* Corresponding author: e-mail: mradu@nipne.ro<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 161
P54 Fluorescence Microscopies and Cell Imaging Poster 54<br />
The HIV-1 Gag induces Vpr oligomers accumulation in the viral<br />
particles : analysis by Fluorescence Imaging Microscopy<br />
Hugues de Rocquigny * , Denis Dujardin, Tania Steffan, Pascal Didier & Yves Mély<br />
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg,<br />
Faculté de Pharmacie, 74, Route du Rhin, 67401 Illkirch (France)<br />
The human immunodeficiency virus type 1 (HIV-1) encodes the Vpr protein which influences the<br />
survival of the infected cells by causing a G2/M arrest and apoptosis (Planelles V,Benichou S,<br />
2009). This protein is encapsidated in the nascent particles though the interaction with the p6 part<br />
of Gag polyproteins. To determine their sub-cellular distribution and monitor the <strong>for</strong>mation of Gag-<br />
Vpr complexes, we used two photon fluorescent lifetime microscopy (FLIM), fluorescence<br />
correlation spectroscopy (FCS) and time laps microscopy on HeLa cells. There<strong>for</strong>e, transfection of<br />
plasmids expressing eGFP-labelled Vpr and tetracystein-tagged Gag proteins detected by the<br />
biarsenical ReAsH labelling reagent was carried out. Using these fluorescent microscopy<br />
approaches, we confirmed that Vpr accumulates at the level of the nuclear envelop and we found<br />
that Vpr self associates as dimers and trimers (Fritz JV et al., 2008). In presence of Gag, a direct<br />
interaction was imaged between Gag and Vpr resulting to the transfer of Vpr from the nuclear<br />
envelop to the plasma membrane (Fritz JV et al., 2010). Vpr oligomerization was found critical <strong>for</strong><br />
both its interaction with Gag proteins and its transfer to the plasma membrane while mutations that<br />
disrupt Gag oligomerization have no effect on Gag-Vpr complex <strong>for</strong>mation. Finally, high resolution<br />
PALM microscopy was used to image individual Gag-EOS containing viral particles. Using this<br />
approach, we were able to establish that the Gag-Vpr complexes observed at the plasma<br />
membrane correspond to individual viral like particles. This work strengthens the regulatory role of<br />
Vpr oligomerisation <strong>for</strong> its localisation in budding particles.<br />
This work was supported by grants from ANRS, Sidaction and MCER Luxembourg.<br />
References: [1] V. Planelles et al, Current topics in microbiology and immunology, 339 (2009) 177-200.<br />
[2] J.V. Fritz et al, Retrovirology 5 (2008) 87. [3] J.V. Fritz, J Virol 84 (2010) 1585-1596.<br />
______________<br />
* Corresponding author: e-mail: hderocquigny@unistra.fr<br />
162 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P55 Fluorescence Microscopies and Cell Imaging Poster 55<br />
Determining the cellular uptake and localisation of novel anti-cancer<br />
drugs using fluorescence microscopy<br />
Laura Rowley 1 , Nikolas J. Hodges 2 , Josephine Bunch 1,3 , Ela Claridge 4 & Michael J. Hannon 1,3<br />
1 PSIBS Doctoral Training Centre, 2 School of Biosciences, 3 School of Chemistry, 4 School of<br />
Computer Science, University of Birmingham, Edgbaston, Birmingham, B15 2TT (U.K.)<br />
Novel triple-helicate metallo-drugs have been<br />
shown to bind at the centre of a DNA three-way<br />
junction – something which no other reported<br />
compound has the ability to do. Our compounds<br />
contain 3 ligands (C25H20N4) wrapped around two<br />
metal centres such as iron, nickel or ruthenium.<br />
These compounds (known as ‘cylinders’) have<br />
been shown to possess exciting anti-cancer<br />
properties, showing toxicity comparable with<br />
current clinically used drugs, but without the<br />
genotoxic or mutagenic side effects associated<br />
with drugs such as cisplatin. The next step in our<br />
work is to discover both their cellular uptake and<br />
mode of action within cells. To answer these<br />
questions, particular attention has been paid to the<br />
ruthenium cylinder which enjoys an inherent<br />
fluorescence due to a metal-ligand charge transfer<br />
at 484nm. Emission from this transition is<br />
observed at 700nm, far removed from any cellular<br />
auto-fluorescence, making it suitable <strong>for</strong> imaging<br />
using fluorescence confocal microscopy. Imaging within MDA-MB-231 breast cancers cells has<br />
revealed small vesicles of fluorescence within the cells but it has proved difficult to determine the<br />
localisation of the compound. However further co-localisation studies with nuclear stain Hoechst<br />
34580 have shown that the intensity of the Hoechst decreases following addition of the cylinder,<br />
suggesting quenching or displacement of Hoechst by the ruthenium cylinder. Due to the low<br />
quantum yield of the cylinder, recent work has led to the development of a copper based cylinder,<br />
which contains anthracene tagged at the end of each ligand. The addition of this well known<br />
fluorophore to the cylinder has led to a much improved quantum yield, and imaging using an<br />
integrated UV/Visible spectrometer and bright field microscope system has clearly shown the<br />
accumulation of this cylinder within MDA-MB-231 cells.<br />
This work was supported by the EPSRC-funded PSIBS Doctoral Training Centre.<br />
References: [1] A.C.G. Hotze, et al., Chemistry & Biology, 15 (2008) 1258. [2] G.I. Pascu, et al., Angew.<br />
Chem. Int. Ed 46 (2007) 4374.<br />
______________<br />
* Corresponding author: E-mail: ler825@bham.ac.uk<br />
Figure 1: Minor groove side view of a cylinder<br />
binding to a DNA three-way junction.<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 163
P56 Fluorescence Microscopies and Cell Imaging Poster 56<br />
Two-photon excitation and stimulated emission depletion by a<br />
single wavelength<br />
Teodora Scheul 1 , Ciro D’Amico 2 , Jean-ClaudeVial 1 & Iréne Wang 1<br />
1 LIPhy, UMR 5588 CNRS, Université de Grenoble 1, BP 87, 38402 Saint Martin d'Hères, France<br />
2 Institut de Physique de Rennes (IPR), UMR6251, Rennes, France<br />
Super-resolved optical microscopy using stimulated emission depletion (STED) [1] is now a mature<br />
method <strong>for</strong> imaging fluorescent samples at scales beyond the diffraction limit. Nevertheless the<br />
practical implementation of STED microscopy is complex and costly, especially since it requires<br />
laser beams with different wavelengths <strong>for</strong> excitation and depletion. In this paper, we propose<br />
using a single wavelength to induce both processes. We studied stimulated emission depletion of<br />
4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM) dye with a femtosecond<br />
laser delivering a single wavelength in the near infrared. Fluorescence was excited by two photon<br />
absorption and depleted by one photon stimulated emission with a femtosecond pulse and<br />
respectively a stretched pulse. Time-resolved fluorescence decay measurements were per<strong>for</strong>med<br />
to determine the depletion efficiency and to prove that fluorescence quenching is not affected by<br />
side effects. Numerical simulations show that this method can be applied to super-resolved<br />
microscopy.<br />
Fluorescence (Arb. Un.)<br />
0 1 2 3 4 5 6 7<br />
Time (ns)<br />
excitation STED<br />
This work was supported by Nanosciences Fondation, Grenoble<br />
References: [1] S. W. Hell and J. Wichmann, Optics Letters 19, 780-782 (1994).<br />
______________<br />
* Corresponding author: E-mail: ascheul@liphy.ujf-grenoble.fr<br />
II<br />
164 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
I<br />
Figure 1: Fluorescence decay curves showing the two-photon excited fluorescence quenched<br />
by a STED pulse delayed with 0.5 ns – results obtained using a single wavelength from the<br />
same laser source<br />
I) Two-photon excited fluorescence by the fs beam<br />
II) One photon depletion of two photon excited fluorescence
P57 Fluorescence Microscopies and Cell Imaging Poster 57<br />
FIDSAM – a novel fluorescence microscopy approach <strong>for</strong> quantitative<br />
and highly sensitive life-cell and FRET-imaging<br />
Frank Schleifenbaum 1* , Kirstin Elgass 3 , Marcus Sackrow 2# , Sebastien Peter 1 , Klaus Harter 1<br />
& Alfred J. Meixner 2<br />
1<br />
Center <strong>for</strong> Plant Molecular Biology, Biophysical Chemistry, University of Tuebingen, Tuebingen,<br />
Germany.<br />
2<br />
Institute of Physical and Theoretical Chemistry, University of Tuebingen, Tuebingen, Germany.<br />
3<br />
Biochemistry Department, Physical Science 4, La Trobe University, Bundoora, Australia.<br />
#<br />
current address: Picoquant GmbH, Berlin, Germany<br />
Biological fluorescence studies often suffer from a strong autofluorescent background contribution<br />
which strongly reduces the obtainable dynamic contrast. Accordingly, fluorescence-based<br />
investigations ranging from sensor applications to life-cell imaging are often impossible <strong>for</strong> those<br />
samples or require high marker concentrations which possible affect the native cellular function.<br />
We present a novel technique (FIDSAM, fluorescence intensity decay shape analysis<br />
microscopy) to enhance the dynamic contrast of fluorescence studies of at least one order of<br />
magnitude by discrimination of autofluorescence from target signal. The method bases on the<br />
analysis of the shape of the fluorescence intensity decay (fluorescence lifetime curve) and benefits<br />
from the fact that the decay patterns of typical fluorescence label dyes strongly differ from emission<br />
decay curves of autofluorescent sample areas. By recording a fluorescence lifetime microscopy<br />
(FLIM) image and determining the deviation of the spatially recorded intensity decays from the<br />
decay shape of the pure label dye, which typically exhibits a monoexponential decay. This way, we<br />
obtain a robust coefficient, which describes the fraction of autofluorescent background to the<br />
recorded signal. This value, which corresponds to the error-value of a monoexponential fit-function,<br />
can be used to weight the intensity image, resulting in the suppression of background emission.<br />
We demonstrate the capability of our technique by studying Arabidopsis thaliana plant cells,<br />
which are GFP-labelled at the cell membranes. Whereas in a conventional confocal fluorescence<br />
image the membranes cannot be optically resolved due to the strong autofluorescence of the cellwall,<br />
our method allows <strong>for</strong> a contrast enhancement of one order of magnitude, rendering imaging<br />
of the isolated GFP-labeled cell membranes possible [1].<br />
Moreover, we show that FIDSAM can also be applied <strong>for</strong> qualitative and quantitative<br />
fluorescence resonance energy transfer (FRET) studies. As the fluorescence decay of the donor<br />
deviates from a monoexponential decay in dependence of the degree of energy transfer, the error<br />
value obtained in the FIDSAM analysis can be used as measure <strong>for</strong> the FRET efficiency.<br />
Compared to conventional FLIM-FRET studies, the application of FIDSAM is more sensitive to<br />
slight changes in FRET efficiency and thus enables the robust determination of even remote<br />
protein-protein interactions [2].<br />
References: [1] F. Schleifenbaum. et al. Mol. Plant 3: (2010) 55. [2] K. Caesar, Plant J 66(3) (2011) 528<br />
______________<br />
* Corresponding author: e-mail: frank.schleifenbaum@uni-tuebingen.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 165
P58 Fluorescence Microscopies and Cell Imaging Poster 58<br />
FLIM-FRET as a tool to study protein partitioning in plasma membrane<br />
microdomains of living cells. Identifying raft localisations signals of the<br />
HIV glycoprotein gp41<br />
Roland Schwarzer 1 , David Reismann 1 & Andreas Herrmann 1<br />
1<br />
Molecular Biophysics, Institute of Biology, Humboldt-University, Invalidenstrasse 42, 10115 Berlin,<br />
Germany<br />
The Human Immunodeficiency Virus (HIV) is a member of the group of enveloped viruses. Its only<br />
transmembrane protein, the glycoprotein gp41 mediates, in concert with the glycoprotein gp120,<br />
the host cell infection by binding cellular receptors and later on triggering membrane fusion,.<br />
Hitherto, our knowledge about the role of gp41 during assembly and budding of newly synthesized<br />
virus particles is incomplete.<br />
For interaction with the cellular transport machinery and additional viral proteins, several<br />
gp41 amino acid motifs were found to be responsible and different partners have been identified.<br />
However, the exact routes of synthesized gp41 proteins in infected cells still needs to be<br />
elucidated. Furthermore, it has been reported that membranes of HIV-infected cells as well as<br />
virions are enriched in cholesterol and virus proteins were found in detergent resistant membrane<br />
fractions [1] of infected cells. Both findings point to an important function of lipid microdomains, so<br />
called rafts, in the late virus lifecycle. Hence, it was suggested that HIV may take advantage of<br />
those highly ordered lateral subcompartments in the host cell plasma membrane <strong>for</strong> efficient virus<br />
assembly and budding.<br />
This work focuses on the detection of Förster Resonance Energy Transfer (FRET) between a<br />
Glycosylphospatidylinositol-anchored cyan fluorescent protein (CFP) as a raft marker and gp41<br />
fusion proteins labeled with yellow fluorescent proteins (YFP) to elucidate raft clustering. Since<br />
energy transfer is highly dependent on the distance between the participating molecules, efficient<br />
FRET can be considered as a strong indication <strong>for</strong> close proximity of raft marker and fusion<br />
proteins and, there<strong>for</strong>e <strong>for</strong> colocalization in lipid microdomains [2] . Fluorescence lifetime imaging<br />
microscopy (FLIM) was employed to accurately investigate FRET in living cells. In combination<br />
with acceptor fluorescence analysis the ascertained energy transfer efficiencies provide reliable<br />
in<strong>for</strong>mation about clustering independent of expression level and fluorophore concentration.<br />
Several gp41 chimera were produced to address the role of different protein domains <strong>for</strong> raft<br />
association but also intracellular distribution and trafficking. The impact of truncations of the<br />
cytoplasmic tail as well as mutations of the cholesterol recognition amino acid consensus (CRAC)<br />
domain, intrinsic trafficking signals and a palmitoylation site were studied in this context.<br />
References: [1] Pickl WF et al.. Lipid rafts and pseudotyping. J. Virol. 2001;75:7175–7183. [2] Scolari, S.,et<br />
al. 2009. Lateral distribution of the transmembrane domain of influenza virus hemagglutinin revealed by timeresolved<br />
fluorescence imaging. J. Biol. Chem. 284:15708-15716.<br />
______________<br />
Corresponding author: roland-schwarzer@gmx.de<br />
166 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P59 Fluorescence Microscopies and Cell Imaging Poster 59<br />
CyDNA: a versatile photoswitchable biopolymer <strong>for</strong> advanced<br />
fluorescence microscopy applications<br />
Darren A. Smith 1 , Philipp Holliger 2 & Cristina Flors 1,*<br />
1 EaStChem School of Chemistry, University of Edinburgh, Edinburgh, EH9 3JJ, United Kingdom<br />
2 MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, United Kingdom<br />
CyDNA is the product of the controlled synthesis of a DNA fragment with a high density of Cy3 or<br />
Cy5 dyes incorporated into its structure. [1] This novel material can be up to 1kb and is constructed<br />
by a modified DNA polymerase that allows substitution of dC bases by their fluorescent dyelabelled<br />
analogue, Cy3- or Cy5-dC. The resulting biopolymer displays hundreds of fluorophores<br />
and is brightly coloured and fluorescent. The high dye content can, however, result in dye-dye<br />
interactions that affect the overall brightness of CyDNA. This may hinder the application of this<br />
material in microarray and microfluidic applications. We will present a bulk and single-molecule<br />
photophysical study that reveals several quenching mechanisms occurring in CyDNA, namely, the<br />
<strong>for</strong>mation of non-fluorescent H-aggregates of the dyes as well as energy hopping and transfer to<br />
lower energy, non-fluorescent traps. We investigate the optimal substitution patterns to enhance<br />
CyDNA brightness.<br />
Furthermore, it has previously been shown that, in the presence of a thiol and an enzymatic<br />
oxygen scavenging system, proximal Cy3 and Cy5 fluorophores <strong>for</strong>m an optical switch. [2] We have<br />
used this property to trans<strong>for</strong>m CyDNA into an efficient photoswitchable biopolymer by hybridizing<br />
complementary single stranded Cy3- and Cy5-substituted CyDNA. Photoswitching has been<br />
applied in localization-based super-resolution fluorescence microscopy to study CyDNA topology in<br />
nanoscale detail. Moreover, we have used CyDNA photoswitching in Optical Lock-in Detection<br />
imaging, [3] which is a technique capable of greatly enhancing image contrast in fluorescence<br />
microscopy. The combination of CyDNA and the above techniques has enormous potential in the<br />
study of the structure of chromosomes at the nanoscale. [4]<br />
This work was supported by a Universitas21 PhD Scholarship and The Royal Society.<br />
References: [1] N. Ramsay et al., J. Am. Chem. Soc. 132 (2010) 5096. [2] M. Bates et al., Phys. Rev. Lett.<br />
94 (2005) 108101. [3] G. Marriott et al., P. Natl. Acad. Sci. U.S.A. 105 (2008) 17789. [4] C. Flors,<br />
Biopolymers 95 (2011) 290.<br />
______________<br />
* Corresponding author: cristina.flors@ed.ac.uk<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 167
P60 Fluorescence Microscopies and Cell Imaging Poster 60<br />
Monitoring the oxygen concentration and redox state of living cells<br />
using long-lived transient states of fluorophores<br />
Thiemo Spielmann 1 , Sofia Johansson 1 & Jerker Widengren 1,*<br />
1<br />
Experimental Biomolecular Physics, Applied Physics, Albanova University Center, Royal Institute<br />
of Technology, Stockholm (Sweden)<br />
Due to their long lifetime, triplet, redox and other transient states of fluorophores (Fig. A) are highly<br />
sensitive to the micro-environment. Imaging the spatial distribution of their populations in biological<br />
samples can thus help answer interesting questions about the metabolism of living cells. However,<br />
as these states are at best weakly luminescent, they have up to now only been used to a limited<br />
extend in life sciences. In transient state (TRAST) imaging, the characteristic build up of transient<br />
states is instead monitored via fluorescence, as the excitation is modulated. [1,2] When the illumination<br />
pulse width is step-wise increased (Fig. B), transient states are progressively populated. The<br />
resulting depletion of the singlet excited state can be monitored via the time-averaged fluorescence.<br />
This fluorescence decay (Fig. C) is characteristic <strong>for</strong> the transient state kinetics of the given<br />
fluorophore in a given environment. Traditional fluorescence parameters can only be influenced<br />
within the lifetime of the fluorophore. In contrast, TRAST imaging can monitor photo-induced states<br />
with 10 3 -10 6 times longer lifetimes and is there<strong>for</strong>e far more sensitive to sparse quencher molecules,<br />
such as dissolved oxygen. Transient state kinetics can also be studied using fluorescence correlation<br />
spectroscopy (FCS). In contrast to FCS, transient state imaging circumvents the need of time<br />
resolution in the fluorescence detection, thereby allowing simultaneous readout over a large number<br />
of pixels using a CCD. It can be applied over a broader range of concentrations and does not require<br />
a strong fluorescence brightness of the sample molecules.<br />
Previously, the sensitivity of the fluorophore triplet state to oxygen has been exploited in a<br />
widefield microscope to monitor oxygen consumption during the contraction of smooth muscle<br />
cells. [3] The method has also been applied in a total internal reflection fluorescence microscope to<br />
monitor the redox reactions of fluorescent dyes in solution. [4] In this work we show the recent<br />
application of transient state imaging to the measurement of the redox state of living cells. Healthy<br />
cells contain millimolar concentrations of reduced glutathione (GSH) which help to neutralize<br />
reactive oxygen species (ROS). GSH can influence the population dynamics of photo-induced dark<br />
states of many organic fluorophores which can be monitored using the presented method.<br />
A) Simplified Jablonski diagram of a rhodamine; B) Laser modulation schemes with varying pulse widths w<br />
and periods T; C) Measured and fitted time-averaged fluorescence <strong>for</strong> different pulse widths and excitations<br />
intensities.<br />
This work was supported by grants from EU FP7 (FLUODIAMON, 201 837) and from the “Fond<br />
national de la Recherche (Luxembourg)”.<br />
References: [1] Tor Sandén, et al., Anal. Chem., 79 (2007), 3330. [2] Tor Sandén, et al. Anal. Chem., 80,<br />
9569. [3] Matthias Geissbuehler, et al., Biophysical J., 98 (2010), 339. [4] Thiemo Spielmann, et al., J. Phys.<br />
Chem. B, 114, (2010), 4035.<br />
______________<br />
* Corresponding author: e-mail: jerker@biomolphysics.kth.se<br />
168 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P61 Fluorescence Microscopies and Cell Imaging Poster 61<br />
Fluorescence lifetimes, anisotropy and FRAP recovery curves<br />
measured simultaneously in living cells<br />
James A. Levitt, Pei-Hua Chung & Klaus Suhling<br />
Department of Physics, King’s College London, Strand, London, WC2R 2LS, UK<br />
It is advantageous to exploit the many properties of fluorescence in imaging experiments.[1-3] We<br />
demonstrate a novel experimental arrangement <strong>for</strong> measurements of intracellular dynamics by<br />
simultaneous acquisition of fluorescence recovery curves (FRAP), fluorescence lifetime imaging<br />
(FLIM) and fluorescence anisotropy imaging (FAIM). We have used this set-up to obtain the<br />
translational and rotational diffusion properties of green fluorescent protein (GFP)-labelled proteins<br />
in living cells. This method allows extraction of fluorescence lifetimes, rotational correlation times<br />
and diffusion characteristics simultaneously and thus avoids excessive photobleaching or artefacts<br />
due to cell movement. It can also measure phenomena that each method on its own cannot<br />
measure, e.g. diffusing homo-dimers.<br />
Fig 1: Schematic images of our experimental arrangement. FRAP image series consisting of FLIM images at<br />
polarizations parallel (║) and perpendicular (┴) to that of the excitation.<br />
References:<br />
[1] J. A. Levitt, et al., Current Opinion in Biotechnology, 20 (2009) 28<br />
[2] F. Festy, et al., Molecular Biosystems, 3 (2007) 381<br />
[3] K. Suhling, et al., Photochemical & Photobiological Sciences, 4 (2005) 13<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 169
P62 Fluorescence Microscopies and Cell Imaging Poster 62<br />
On/off switching of a genomic DNA caused by nonspecific<br />
environmental parameter, as is evidenced by real-time observation with<br />
fluorescence microscopy<br />
Kenichi Yoshikawa 1 , * Akihiko Tsuji 1 , Ayako Kato 1 & Yuko Yoshikawa 2<br />
1 Department of Physics, Kyoto University, Kyoto 606-8502 (Japan)<br />
2 Laboratory of Environmental Biotechnology, Research Organization of Science and Engineering,<br />
Ritsumeikan University, Kusatsu 525-8577 (Japan)<br />
Currently, it has become clear that giant DNAs above the size of several tens of kilo base pairs<br />
(kbp) exhibit much different properties from short DNAs [1,2] : i) Individual DNA molecule undergoes<br />
large discrete transition between elongated coil and folded compact states accompanied by the<br />
density difference on the order of 10 4 -10 5 , whereas such characteristic is failed on short oligomeric<br />
DNA. The discrete character is rather general<br />
regardless the chemical nature of the condending<br />
agents, such as polyamine, hydrophilic polymer,<br />
cationic surfactant. Interesingly, the characteristics on<br />
ensemble average of the DNAs seem always<br />
continuous, i.e, cooperative transition. ii) Bulky<br />
polycations such as histone H1 causes intramolecular<br />
segregation between elongated and compact parts<br />
along a single giant DNA molecule, i.e., pearling<br />
structure is generated. iii) The folding transition of<br />
DNA is classified as first-order phase transition<br />
between disordered coil and ordered compact states.<br />
Thus, similar to the usual crystalization process,<br />
week nonequlibricity in solution causes regular<br />
structure such as toroid and rod without<br />
entanglement, through the kinetics of nucleationgrowth.<br />
In the present paper, we will report the<br />
observation on the discrete on/off switching of the<br />
genetic activity, such as transcription and expression,<br />
of genomic DNA, as the result of discrete transition of<br />
its higher order structure. Especially, we will show the<br />
unique effect on the structure and fuction of genomic<br />
DNA confined in a cell-sized sphere covered by lipid<br />
membrane. [3],[4] 1) In a crowding environment with<br />
negatively charged proteins, DNA molecules are<br />
depressed onto the membrane surface by keeping<br />
elongated con<strong>for</strong>mation. 2) Accompanied by the micro<br />
phase segregation, DNA is specifically attached solely<br />
onto a certain domain. 3) By the change of the<br />
environmental factor such as concentration of<br />
magnesium ion, genomic DNA exhibits discrete<br />
transition inducing all-or-none switching of the<br />
transcriptional activity (See Figure).<br />
ON OFF ON<br />
Transcription<br />
References: [1] K. Yoshikawa, et al., Phys. Rev. Lett. 76(1996)3029. [2] K. Yoshikawa, et al., 'Compaction<br />
and Condensation of DNA', in Pharmaceutical Perspectives of Nucleic Acid-based Therapeutics, edt. Mahato<br />
& Kim, Taylor & Francis, New York (2002). [3] A. Kato, et al., J. Phys.Chem.Lett., 1(2010)3391. [4] A. Tsuji,<br />
et al., J.Am.Chem.Soc., 132(2010)12464.<br />
______________<br />
* Corresponding author: e-mail: yoshikaw@scphys.kyoto-u.ac.jp<br />
170 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
Figure. Top: FRET design <strong>for</strong> the detection<br />
of transcripts. Middle: Fluorescence<br />
Microscopy (Left) DNA molecules inside the<br />
artificial model cell, (Right) Transcripts on<br />
individual DNA molecule as detected by<br />
FRET. Bottom: On/Off switchig of<br />
transcription reaction, where the middle<br />
pictures correspond to (a).
P63 Fluorescence Microscopies and Cell Imaging Poster 63<br />
Scarless regeneration of the newt heart<br />
Tanja Piatkowski, Thilo Borchardt & Thomas Braun<br />
Max Planck Institute <strong>for</strong> Heart and Lung Research, Bad Nauheim, Germany<br />
Salamanders possess extraordinary regenerative capacities. Notophthalmus viridescens, the red<br />
spotted newt can regenerate entire appendages after amputation, as well as the lens and retina<br />
and parts of central nervous system [1]. Even more remarkable, studies in the 1970s discovered<br />
the regenerative potential of newt hearts. Upon tissue resection, remaining cardiomyocytes and<br />
other cell types respond with massive proliferation [2] [3]. However, amputation of ventricular tissue<br />
also lead to a deposition of extracellular matrix within 1 month despite newly <strong>for</strong>med myocardium<br />
[2]. It was there<strong>for</strong>e assumed, that newts only possess a limited capacity to regenerate cardiac<br />
tissue.<br />
To readdress the old debate, whether newt hearts possess the capability to fully regenerate<br />
cardiac tissue without scar <strong>for</strong>mation, we established three different models of heart injury: (i)<br />
amputation (based on the resection of one-eighth of the ventricle) (ii) necrosis (based on freezing<br />
of one half of the ventricle using dry ice) and (iii) mechanical injury (based on repeated squeezing<br />
of half of the ventricle with <strong>for</strong>ceps). Regenerative responses in the newt heart were monitored <strong>for</strong><br />
up to 200 days after injury by immunostaining of regenerating cardiac tissue using different<br />
antibodies.<br />
Interestingly, we did not observe marked differences between the three injury models<br />
indicating that the process of regeneration utilizes similar mechanisms. We found that newt hearts<br />
regenerated the myocardium completely without scar <strong>for</strong>mation. Deposition of extracellular matrix<br />
proteins occurred only transiently, indicating an impressing capability of newt tissue to remodel<br />
damaged myocardium. Irrespective of the type of injury, regeneration was initiated at the<br />
undamaged upper part of the ventricle. Regeneration proceeded in an inside to outside pattern<br />
towards the apex. We found evidence <strong>for</strong> <strong>for</strong>mation of a blastema-like structure and interaction of<br />
different cell types during the remodeling process. Damaged tissue was first colonized by trabecula<br />
supporting cells, which <strong>for</strong>med thin trabeculae. Later on, trabeculae were successively filled up by<br />
cardiomyocytes. Absence of extracellular matrix deposition and presence of regularly shaped<br />
cardiomyocytes, expressing the full repertoire of muscle specific markers, indicated completion of<br />
regeneration at 200 days after injury.<br />
Taken together, we demonstrated that heart regeneration in newts is based on a complex<br />
interaction of different cell types, which are able to replace damaged or even lost myocardial<br />
tissue. The lack of scar <strong>for</strong>mation after amputation indicates that newts are able to replace not only<br />
missing appendicle structures such as limbs and tails but also parts of internal organs.<br />
References: [1] Brockes et al. Current Biology (2005) 15(2); [2] Oberpriller et al. J Exp Zool, (1974) 187(2);<br />
[3] Mc Donnell et al. Tissue Cell, (1983) 15(3)<br />
______________<br />
Corresponding author e-mail: thomas.braun@mpi-bn.mpg.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 171
172 — MAF 12, Strasbourg, France, September 11-14, 2011 —
Polymers,<br />
Materials & Nanomaterials,<br />
Quantum Dots<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 173
174 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P64 Polymers, Materials & Nanomaterials, Quantum Dots Poster 64<br />
Fluorescence and electrical measurements <strong>for</strong> studying internal<br />
morphology of imprinted and random PNIPA gels<br />
Esra Alveroglu Durucu 1,* , Cagatay İleten 1 & Yasar Yilmaz 1<br />
1<br />
Istanbul Technical University, Faculty of Science and Letters, Department of Physics Engineering,<br />
34469, Istanbul (Turkey)<br />
The specially designed gels to study the molecular recognition processes are called as “imprinted<br />
gels”. In this study we prepared imprinted and random N-izopropilakrylamid (NIPA) gels.<br />
Metakrilamidopropiltrimetil amonyum klorür (MAPTAC) and 8-Hidroksi-1,3,6-pirentrisülfonik acid<br />
trisodyum (Pyranine-fluorescent molecule) were used to <strong>for</strong>m a complex. Imprinted PNIPA gels<br />
were synthesized with presence of this complex as explained in ref.[1]. For synthesizing random<br />
gel, same molecules used <strong>for</strong> complex were added separately to pre-gel solution without <strong>for</strong>ming a<br />
complex. After gelation fluorescence and electrical measurements were per<strong>for</strong>med on these gels<br />
<strong>for</strong>med cylindrical in shape. Samples were excited at 350 nm wavelength of light and the emission<br />
spectra were collected at 90°. Florescence spectrum <strong>for</strong> imprinted and non-imprinted (random)<br />
gels is clearly different from each other as shown in Figure. In imprinted gels the number of the<br />
free pyranine is considerably smaller than random gel indication that most of the pyranines are<br />
trapped to <strong>for</strong>m templates. In addition the scattered intensity <strong>for</strong> imprint gels is considerably bigger<br />
than that of random one showing that the imprinted gel is more heterogeneous. Current vs. time<br />
plots <strong>for</strong> both random and imprinted gels were also evaluated by using our recent theory on the<br />
heterogeneity of the hydrogels. [2] Both methods used showed that random gels are more<br />
homogeneous than that of imprinted gels.<br />
References: [1] K. Ito, et al., Prog. Polym. Sci., 28 (2003) 1489. [2] E. Alveroglu and Y. Yilmaz, Macromol.<br />
Chem. and Phys., in early wiev.<br />
______________<br />
* Corresponding author: e-mail: alveroglu@itu.edu.tr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 175
P65 Polymers, Materials & Nanomaterials, Quantum Dots Poster 65<br />
Schiff bases containing phenothiazine units with<br />
fluorescence properties<br />
Castelia Cristea, Emese Gál, Larisa Mataranga-Popa, Luiza Găină & Luminiţa Silaghi-<br />
Dumitrescu<br />
Babeş-Bolyai University, Faculty of Chemistry and Chemical Engineering, 11, Arany Janos str.,<br />
400028 Cluj-Napoca (Romania)<br />
Schiff bases characterized by diverse structural properties can be obtained by relatively simple<br />
preparation procedures based on condensation reaction of aromatic amines with carbonyl<br />
derivatives. This synthetic flexibility enabled the design of molecular structures with tunable<br />
properties and numerous representative derivatives found a wide variety of applications in many<br />
fields such as: biology, analytical chemistry, as well as in materials science investigations.<br />
The favorable electronic properties of phenothiazine core (low and highly reversible first<br />
oxidation potential and a pronounced tendency to <strong>for</strong>m stable cation-radicals [1]) has given rise to<br />
applications such as: polyazomethine-type conjugated polymers with alternating phenothiazine and<br />
azomethine units with electroluminescent properties as single- and double-layer polymer lightemitting<br />
diode PLEDs [2]. <strong>Single</strong>-layered and double-layered PLEDs made with copolymer poly[N-<br />
(2-ethylhexyl)phenothiazine-alt-6-Ph phenanthridine] as an emitting layer exhibited emission at 572<br />
nm [3].<br />
The target of our work was to synthesize and fully characterize new Schiff bases containing<br />
phenothiazine units, which may develop interesting unconventional physical properties due to the<br />
combination of the electronodonor effects of the phenothiazine nucleus with those of an extended<br />
π conjugated system. The Schiff bases were obtained either by condensation of aminophenothiazine<br />
derivatives with aromatic-aldehydes or by condensation of phenothiazinylcarboxaldehyde<br />
with aromatic primary amines. Their structural characterization was based on high<br />
resolution NMR, UV-Vis and Luminescence spectroscopy. Emission maxima of these fluorophores<br />
were situated in the range 520-590 nm showing large Stokes shifts (6000-8600 cm -1 ), a fact in<br />
agreement with literature data stating that great Stokes shifts can be assigned to large geometrical<br />
changes upon excitation from nonplanar ground state to an essentially planarized excited state of<br />
the phenothiazine moiety [4].<br />
UV-visible absorption spectra were recorded on a UV/VIS PERKIN ELMER LAMBDA 35<br />
spectrometer and emission spectra were recorded on a PERKIN ELMER Model LS 55 instrument.<br />
This work was supported by Romanian Ministry of Education Research, Youth and Sports, grant<br />
PCCE 140/2008.<br />
References: [1] M. Sainsbury, in Comprehensive Heterocyclic Chemistry (Eds.: A. R. Katritzky, C. W.<br />
Rees), Pergamon Press, Ox<strong>for</strong>d, New York, Toronto, Sydney, Paris, Frankfurt, 1984, vol. 3, p. 995. [2] W.<br />
Jeon et all. J. Nonlinear Opt. Phys. Mater., 14(4), (2005), 545. [3] Y. S. Han; et all , Mol. Cryst. Liq. Cryst.,<br />
459 (2006) 119. [4] L. Yang et all , J. Org. Chem. 70, (2005), 5987.<br />
______________<br />
* Corresponding author: e-mail: castelia@chem.ubbcluj.ro<br />
176 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P66 Polymers, Materials & Nanomaterials, Quantum Dots Poster 66<br />
Nanofilamentous molecularly imprinted polymers in a miniaturized<br />
fluorescence-optical sensor<br />
S. Harz 1 , Aude Cordin 2 , K. Haupt 2 & K.-H. Feller 1<br />
1 University of Applied Sciences Jena, Germany<br />
2 Technical University of Compiègne, France<br />
This paper reports on the sensitive detection of biologically relevant molecules with nanostructured<br />
molecularly imprinted polymers (MIPs) as biomimetic recognition elements in a miniaturized<br />
fluorescence-based microfluidic sensor. Due to its modularity the sensor setup is adaptable to a<br />
variety of analytes in order to analyse biofluids <strong>for</strong> point-of-care diagnostics.<br />
Molecularly imprinted polymers (MIPs) are synthetic receptors that can be synthesized <strong>for</strong> a<br />
variety of target molecules. The advantage of MIPs in comparison to other synthetic detection<br />
systems using guest-host-molecule-interactions is their high selectivity. Compared to biological<br />
receptor molecules such as antibodies and enzymes, MIPs can be tailor made inexpensively <strong>for</strong><br />
various targets, and their physical and chemical stability and durability is much higher [1].<br />
In the present sensor setup MIPs were integrated in the <strong>for</strong>m of surface-bound nanofilaments<br />
(nanograss, Figure 1 A) in order to increase the surface area of the sensitive layer and the<br />
accessibility of the binding sites by the analyte. We have used a nanomolding technique combined<br />
with photolithography <strong>for</strong> MIP nanofilament synthesis on the sensor surface (Figure 1 B). Different<br />
aspect ratios of the filaments are achievable and their binding properties are superior compared to<br />
porous films [2].<br />
Fluorescence measurements were per<strong>for</strong>med on MIPs specific <strong>for</strong> the fluorescence-labeled<br />
amino acid dansyl-L-phenylalanine as a model target. The detection of analytes with fluorescence<br />
allows <strong>for</strong> a very sensitive and specific method to analyze the interaction between MIP and analyte.<br />
Thus, the high selectivity of MIPs is combined with the high sensitivity of fluorescence detection.<br />
In the presented work the MIP nanofilaments are analyzed under microfluidic conditions. We<br />
designed microfluidic structures with 100 µm optical light paths (Figure 1 B and C) which allow<br />
achieving a very high signal coming from the fluorescent MIP with a very low signal from the<br />
background (sample solution). The investigation of analytes under microfluidic conditions allows<br />
online measurements and uses only small amounts of analyte, which is an advantage <strong>for</strong><br />
measurements of biological samples in particular <strong>for</strong> diagnostic purposes.<br />
Additionally, the design and build-up <strong>for</strong> a reproducible fluorescence measuring system is<br />
described [3]. The imprinting factor and binding kinetics are compared between nanostructured<br />
and plain porous MIP dots. Our results show that nano-filamentous MIPs in combination with<br />
fluorescence detection and microfluidic devices are a very sensitive tool <strong>for</strong> the detection of<br />
biologically relevant analytes.<br />
Figure 1. Nanofilament MIPs (A) on a chip surface (B) as part of a microfluidic chip with miniaturised optical<br />
detection (C).<br />
References:<br />
[1] L. Ye, K. Haupt, Anal Bioanal Chem, 2004, 378, 1887–1897<br />
[2] A.V. Linares et al., Adv Funct Mater, 2009, 19, 1-5<br />
[3] S. Harz et al., Eng Life Sci, accepted, DOI: 10.1002/elsc.201000222<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 177
P67 Polymers, Materials & Nanomaterials, Quantum Dots Poster 67<br />
Photophysical investigation of a fluorophore sensor and using its selfassembly<br />
<strong>for</strong> the detection of environmental changes in polyesters<br />
M. Amine Fourati 1 , C. Géraldine Bazuin 1,* & Robert E. Prud’homme 1,*<br />
1 Département de Chimie, Centre de recherche sur les matériaux auto-assemblés<br />
(CRMAA/CSACS), Université de Montréal, CP 6128, Succ. Centre-Ville, Montréal, Québec, H3C<br />
3J7, Canada<br />
The incorporation of a fluorescent aromatic molecule, 4,4’-bis(2-benzoxazolyl)stilbene (BBS), in<br />
semi-crystalline polyesters, polylactide (PLA) and poly(1,4-butylene succinate) (PBS), by meltprocessing<br />
can lead to in<strong>for</strong>mation on the fluorophore con<strong>for</strong>mation. The emission characteristics<br />
of these doped polymeric films depend on the BBS concentration. Indeed, a well-defined excimer<br />
band at 500 nm is observed by adding more than 0.5 wt % of BBS, conferring to the film a green<br />
luminescence. The observed increase in ratio of fluorescence peak intensities (IE/IM), related to the<br />
molecularly dispersed dye (IM) and the excimer (IE), is accompanied by a reversible colour change<br />
from blue to green that occurs as a result of temperature or annealing time increase in quenched<br />
blends. This change is related to the <strong>for</strong>mation of BBS aggregates favouring the excimer. Thus, the<br />
optical behaviour of the quenched blends provides films characterized by a sensitive temperaturedependent<br />
luminescence response. Indeed, quenching dye/polymer blends below their Tg<br />
kinetically traps the dyes in a dispersed state exhibiting monomer fluorescence, whereas, upon<br />
annealing above the Tg, self-assembly of the dye molecules into aggregates occurs allowing<br />
excimer <strong>for</strong>mation, concomitant with permanent and pronounced fluorescence colour change from<br />
blue to green. A similar phenomenon occurs as a function of annealing time leading to an increase<br />
in the IE/IM ratio until reaching a plateau. Polymer stretching destroys the BBS excimers, leading to<br />
the reverse colour change. On the other hand, BBS was found to exhibit consistently high<br />
fluorescence, with absolute quantum yields (Φfl) ≥ 0.8 in solution, and ≥ 0.5 in polymeric films, with<br />
a BBS concentration up to 2 wt %. There<strong>for</strong>e, this food-grade commercial stilbene derivative can<br />
act as an internal probe of temperature, de<strong>for</strong>mation and annealing time in polyesters.<br />
This work was supported by grants from Centre <strong>for</strong> Self-Assembled Chemical Structures, Natural<br />
Sciences and Engineering Research Council of Canada, and Fonds Québecois de la recherche<br />
sur la nature et les technologies. AF thanks the Tunisian Government <strong>for</strong> the scholarship of<br />
excellence.<br />
______________<br />
* Corresponding authors: e-mail: re.prudhomme@umontreal.ca ; geraldine.bazuin@umontreal.ca<br />
178 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P68 Polymers, Materials & Nanomaterials, Quantum Dots Poster 68<br />
Luminescent light-harvesting pendant PPV polymers<br />
Kenneth Ghiggino 1, * , Andrew Tilley 1 , Jonathan White 1 & Ming Chen 2<br />
1 School of Chemistry, University of Melbourne, Victoria, 3010 (Australia)<br />
2 CSIRO Materials Science and Engineering, Clayton, Victoria, 3168 (Australia)<br />
Conjugated polymers based on phenylene vinylene (PPV) have been widely studied as the active<br />
materials in both light emitting diodes and photovoltaic devices due their useful semi-conducting<br />
properties. However structural and con<strong>for</strong>mational defects disrupt the main-chain conjugation of<br />
these polymers leading to heterogeneity in chromophore type and complexities in understanding<br />
and controlling polymer photophysics. To address these issues we have synthesized a series of<br />
novel luminescent light harvesting polymers that contain PPV-based chromophores of defined<br />
conjugation length (2-4 units) as pendant groups attached to an appropriately functionalised<br />
polystyrene backbone (Fig. 1). The polymer backbone was synthesized by the RAFT controlled<br />
free radical polymerization procedure to provide highly monodisperse polymers. The luminescence<br />
properties of these polymers have been studied in both solution and films and at the single<br />
molecule level. As the conjugation length of the pendant chromophores increases, the polymers<br />
display red-shifting of absorption and emission maxima as well as a decrease in fluorescence<br />
quantum yield (0.40 to 0.18) and fluorescence lifetime (Fig. 1). Fluorescence polarisation<br />
measurements of the homopolymers demonstrate that efficient energy migration occurs between<br />
pendant chromophores along a single polymer chain. For copolymers containing a mixture of the<br />
chromophore types, energy transfer from the highest energy to lowest energy pendant groups<br />
occurs confirming the usefulness of these polymers as light harvesting materials. Variable<br />
temperature photophysical experiments on the polymers and related oligomers [1] are compared<br />
and show that emission occurs from a restricted set of torsional configurations compared to the<br />
ground state which gives rise to the structured emission observed. In solid films, chain aggregation<br />
plays an additional role in the excited state relaxation pathways. The relevance of the results to the<br />
application of these materials in luminescent devices will be presented.<br />
Fig. 1. Structures of the PPV-based side-chain polymers and their fluorescence spectra in chloro<strong>for</strong>m.<br />
The provision of a Julius Career Award to M.C. by the CSIRO OCE Science Team is<br />
acknowledged. This research is supported by funding from the Australian Research Council.<br />
References: [1] A.J. Tilley, et al., J. Org. Chem., 76 (2011) 3372.<br />
______________<br />
* Corresponding author: e-mail: ghiggino@unimelb.edu.au<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 179
P69 Polymers, Materials & Nanomaterials, Quantum Dots Poster 69<br />
Time evolution of multiple point interactions of pyranine fluoroprobe<br />
with polyacrylamide chains during the polymerization:<br />
experiment and simulation<br />
Alptekin Yildiz 1 , Ali Gelir 1 & Yasar Yilmaz 1<br />
1 Department of Physics, Istanbul Technical University, Maslak, 34469 Istanbul, Turkey<br />
Multiple point interactions of pyranine (8-hydroxypyrene-1, 3,6-trisulfonic acid, trisodium salt)<br />
fluoroprobe with the polymer chains during the free-radical polymerization of acrylamide (AAm)<br />
was studied using the steady state fluorescence measurements and also computer simulation<br />
based on Monte Carlo method.<br />
In recent literature it has been shown that [1] a considerable blue-shift, from 515 to 406 nm,<br />
occurs in the emission spectra when OH group on the pyranine binds covalently to a vinyl group of<br />
−<br />
the growing polymer chains. At the same time, three SO 3 groups on the probe molecule interact<br />
electrostatically with protonated amide groups of macroradicals, and thus cause a gradual red-shift<br />
in the maximum of the short-wavelength-peak, between 406 and 430 nm. As a result of these<br />
spectroscopic behaviors of pyranine, it may be an excellent candidate <strong>for</strong> monitoring the<br />
polymerization of PAAm [1-3] .<br />
Time evolution of multiple point interactions of pyranine with polyacrylamide chains during<br />
the polymerization was compared with the simulation results. It was observed that the time<br />
evolution obtained from simulation is very similar to the results of the fluorescence measurements.<br />
References:<br />
[1] Y. Yilmaz, et al., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 72 (2009) 332.<br />
[2] N. Kızıldereli, et. al., Journal of Applied Polymer Science, 115 (2010) 2455.<br />
[3] Y. Yilmaz, et al., Physical Review E, 77 (2008) 051121.<br />
______________<br />
* Corresponding author: e-mail: alyildiz@itu.edu.tr<br />
180 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P70 Polymers, Materials & Nanomaterials, Quantum Dots Poster 70<br />
Comparative study of the sensitized luminescence of terbium(III) ions<br />
and Tb4O7 nanoparticles as derivatising reagents in ultrafast<br />
liquid chromatography<br />
M.L. Castillo-García 1 , M.P. Aguilar-Caballos 1 & A. Gómez-Hens 1*<br />
1 Analytical Chemistry Department, Institute of Fine Chemistry and Nanochemistry (IAQFN).<br />
Campus of Rabanales. Marie Curie Building (Annex). University of Cordoba. 14071-Cordoba.<br />
Spain<br />
Terbium-sensitized luminescence is a useful option to improve the sensitivity and spectral<br />
selectivity compared to some conventional chromatographic approaches described <strong>for</strong> this<br />
purpose. Terbium(III) ions have been previously described to achieve the luminescent detection of<br />
several organic compounds, such as aromatic aldehydes and ketones, carboxylic compounds and<br />
nucleotides, among others, using LC columns with conventional particle sizes [1]. The use of<br />
Tb4O7 nanoparticles as luminescent reagents has been scarcely reported up to date, so a<br />
systematic study in order to demonstrate their usefulness as analytical reagents is desirable. Two<br />
structurally related compounds, the ionophore antibiotic lasalocid and the antimicrobial salicylic<br />
acid, have been chosen as model analytes. The influence of different physicochemical variables,<br />
such as reagent concentration, the use of synergetic agents and surfactants, has been assayed <strong>for</strong><br />
both analytes using Tb4O7 and terbium(III) ions <strong>for</strong> comparative purposes.<br />
Preliminary studies per<strong>for</strong>med in order to couple the luminescence detection system with<br />
ultrafast liquid chromatography have shown that pre-column derivatization is the most suitable<br />
option to achieve the long-wavelength luminescence detection of these analytes. The usefulness of<br />
either terbium(III) ions or Tb4O7 nanoparticles depends on the analyte structure and the<br />
experimental conditions assayed.<br />
References: [1] A. Gómez-Hens, M.P. Aguilar-Caballos, Trends Anal. Chem., 21 (2002) 131.<br />
______________<br />
* Corresponding author: e-mail: qa1gohea@uco.es<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 181
P71 Polymers, Materials & Nanomaterials, Quantum Dots Poster 71<br />
Fluorescence spectroscopy of phosphor powders – difficulties<br />
and challenges<br />
Peter Barnekow 1 , Sven Brüninghoff 1 , Robert Schiwon 1 & Holger Winkler 2<br />
1<br />
Department <strong>for</strong> Central Analytical Services, Merck KGaA, Frankfurter Strasse 250, 64293<br />
Darmstadt, Germany<br />
2<br />
Department <strong>for</strong> Solid <strong>State</strong> Lighting, Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt,<br />
Germany<br />
LEDs become increasingly important as economical general lighting or back-lighting <strong>for</strong> monitors<br />
and TVs. In order to generate white light with LEDs or to adjust the colour of LEDs, phosphors are<br />
necessary and are presently developed and optimised. The most important properties concerning<br />
this matter are the chromaticity and the quantum efficiency. There<strong>for</strong>e the fluorescence<br />
spectroscopic characterisation of phosphor powders is an important topic, but, especially <strong>for</strong><br />
powder samples, the determination of the chromaticity and the quantum efficiency of phosphors<br />
with a high accuracy is challenging. Since very small differences in fluorescence spectra result in<br />
significantly different values <strong>for</strong> chromaticity, the spectral calibration of instruments is of great<br />
importance. Un<strong>for</strong>tunately, a verification of the calibration is difficult due to the lack of certified<br />
phosphor powders.<br />
In this study the influence of different calibration methods will be shown. The measurements<br />
are accomplished with a fluorescence spectrometer composed of a white light lamp,<br />
monochromators, an integrating sphere, and a photomultiplier as detector. The focus of the<br />
calibration has been the measurement of the chromaticity x, y, and z (CIE 1931) with a high<br />
precision and correctness. For the calibration different light sources with different constructive<br />
arrangements have been used: i) a standard set up <strong>for</strong> coupling the light of the certified white light<br />
lamp into the optical path of the spectrometer, ii) a specially designed LED <strong>for</strong> the calibration of the<br />
spectrometer, and iii) a white light calibration lamp with a specially designed housing. Every set-up<br />
has advantages and disadvantages. However, in order to achieve a good correctness <strong>for</strong> the<br />
chromaticity, the parameters influencing the spectrum of the calibration light source have to be<br />
considered and quantified. Altogether, it is important to have an independent method to check the<br />
quality of the calibration. In this study a certified, phosphor converted LED has been used.<br />
The results of the different calibration methods will be presented.<br />
______________<br />
* Corresponding author: e-mail: peter.barnekow@merck.de<br />
182 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P72 Polymers, Materials & Nanomaterials, Quantum Dots Poster 72<br />
Polymer nanoparticles <strong>for</strong> the controlled production and release<br />
of singlet oxygen<br />
Sofia Martins 1 , José P. S. Farinha 1 , Carlos Baleizão 1 & Mário N. Berberan-Santos 1, *<br />
1 Centro de Química-Física Molecular and IN-Institute of Nanoscience and Nanotechnology,<br />
Instituto Superior Técnico, Universidade Técnica de Lisboa, 1049-001 Lisboa (Portugal).<br />
<strong>Single</strong>t oxygen ( 1 O2) is a reactive <strong>for</strong>m of O2 that plays an important role in many oxidation<br />
processes, e. g. photooxidation, DNA damage, and photodynamic therapy [1]. Owing to its<br />
relatively short lifetime and to the often low local concentration of ground state dioxygen, a<br />
controlled production and release of 1 O2 is difficult to achieve. In this work we describe a new way<br />
of producing singlet oxygen, by thermolysis of 9,10-diphenylanthracene (DPA) endoperoxides<br />
incorporated within polymer nanoparticles. DPA is known to react reversibly with singlet oxygen,<br />
the oxidation product of DPA being an endoperoxide which by thermolysis can regenerate the<br />
initial aromatic compound and release excited state oxygen [2]:<br />
Nanoparticles of polystyrene and polybutylmethacrylate were prepared by mini-emulsion<br />
polymerization in the presence of DPA functionalized monomers. The size distribution of<br />
nanoparticles was obtained by dynamic light scattering and the respective photophysical properties<br />
were studied by steady-state and time-resolved fluorescence spectroscopy. The endoperoxides<br />
were produced within the nanoparticles by photoirradiation in the presence of a photosensitiser,<br />
methylene blue or the fullerene C70. The photooxidation was monitored by the disappearance of<br />
the characteristic blue fluorescence of DPA. The nanoparticles were subsequently heated, leading<br />
to singlet oxygen release. The respective kinetics of thermolysis was followed by steady-state<br />
fluorescence.<br />
Sofia Martins was supported by a Ph. D. grant from FCT- Fundação para a Ciência e Tecnologia<br />
(SFRH/BD/47660/2008)<br />
References: [1] DeRosa, M. C., Crutchley, R. J., Coord. Chem. Rev. 233 (2002) 351<br />
[2] Turro, N. J., Chow, M. F., Rigaudy, J., J. Am. Chem. Soc. 103 (1981) 7218<br />
______________<br />
* Corresponding author: e-mail: berberan@ist.utl.pt<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 183
P73 Polymers, Materials & Nanomaterials, Quantum Dots Poster 73<br />
Plasmon induced fluorescence enhancement in LH2 complexes<br />
Łukasz Bujak 1* , Tatas H.P. Brotosudarmo 2 , Nikodem Czechowski 1 , Maria Olejnik 1 , Radek<br />
Litvin 1 , Richard J. Cogdell 1 , Wolfgang Heiss 3 & Sebastian Maćkowski 1<br />
1 Institute of Physics, Nicolaus Copernicus University, Grudziadzka 5, 87-100 Torun (Poland)<br />
2 Glasgow Biomedical Research Centre, University of Glasgow, Glasgow (Great Britain)<br />
3 Institute fuer Halbleiterphysik, Johannes Kepler Universitaet, Linz (Austria)<br />
Sun is almost endless source of energy and nature have made use of it in efficient way by the<br />
process of photosynthesis. It would be highly attractive to mimic and perhaps make better light<br />
harvesting devices to collect as much as possible of sunlight energy. One of the routes is goes<br />
through constructing hybrid devices composed of natural photosynthetic pigment - protein<br />
complexes that would surpass per<strong>for</strong>mance of natural<br />
systems. The efficiency of light harvesting of natural<br />
photosynthetic complexes can be achieved by<br />
coupling the natural complexes with metallic or<br />
semiconducting nanoparticles [1,2].<br />
In this work we study the optical properties of<br />
hybrid nanostructures composed of LH2 membrane<br />
antenna complexes from purple bacteria (Rps.<br />
palustris) and either gold spherical or elongated<br />
nanoparticles. We attempt to optimize the effect of<br />
plasmon induced fluorescence enhancement byy<br />
varying the separation between the two constituents.<br />
Peripheral antenna complex LH2 shows major<br />
absorption bands in the near-infrared and are due to<br />
strongly coupled bacteriochlorophylls arranged in two<br />
rings, B800 and B850. The complex features also<br />
carotenoid absorption between 430 nm and 560 nm.<br />
The samples were prepared by spin-coating gold<br />
nanoparticles on glass substrate. Then SiO2 layer was evaporated with thickness from 4 nm to 40<br />
nm. Finally, we spin-coated LH2 in a PVA polymer solution on top of SiO2 layer.<br />
Firstly we study the structure composed of LH2 complexes coupled with 5 nm gold spherical<br />
nanoparticles with plasmon resonance at 530 nm. Fluorescence measurements show that <strong>for</strong> a 12<br />
nm thick spacer the intensity is 5 times higher (see Fig. 1) than <strong>for</strong> a reference sample (d = 40 nm).<br />
For shorter distances fluorescence quenching is dominant. Importantly, fluorescence decay time<br />
show little dependence upon the spacer thickness. In next step we used gold nanorods (15 nm<br />
thickness and 50 nm length) with plasmon resonances at 530nm and 808nm. In this case<br />
fluorescence measurements show that <strong>for</strong> d = 10 nm intensity is twofold increased as compared to<br />
the reference sample (d = 30 nm).<br />
These results indicate that by careful design of hybrid nanostructures it is possible to<br />
optimize plasmon induced effects on complex biomolecules and thus enhance their functionality -<br />
in this case – light harvesting. Planned single molecule experiments will clear up processes<br />
responsible <strong>for</strong> the observed effects.<br />
Financial support from the WELCOME program “Hybrid nanostructures as a stepping-stone<br />
towards efficient artificial photosynthesis” awarded by the Foundation <strong>for</strong> Polish Science is<br />
gratefully acknowledged.<br />
References: [1] S. Mackowski, J. Phys.: Condens. Matter, 22 (2010) 193102, [2] I. Carmeli, et al. Nano Lett.<br />
10 (2010) 2069.<br />
______________<br />
* Corresponding author: e-mail: bujak@fizyka.umk.pl<br />
184 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
Fig. 2 Fluorescence Enhancement factor <strong>for</strong><br />
LH2 with gold nanospheres (dashed line) and<br />
gold nanorods (solid line).
P74 Polymers, Materials & Nanomaterials, Quantum Dots Poster 74<br />
Versatile multilayered structure <strong>for</strong> the generation of Metal<br />
Enhanced Fluorescence<br />
Eleonora V. Canesi 1* , Martina Capsoni 2 , Mirella Del Zoppo 2 , Andrea Lucotti 2<br />
& Chiara Bertarelli 1,2<br />
1<br />
Center <strong>for</strong> Nano Science and Technology @Polimi, Istituto Italiano di Tecnologia, via Pascoli 70/3,<br />
20133 Milano (Italy)<br />
2<br />
Dipartimento di Chimica, Materiali e Ing. Chimica ’’G. Natta’’, Politecnico di Milano, piazza<br />
Leonardo da Vinci 32, 20133 Milano (Italy)<br />
Metal Enhanced Fluorescence, the increase of the emission intensity of a fluorophore when<br />
interacting with a surface plasmon, is strongly dependent on the distance between the fluorophore<br />
itself and the metal nanostructure. Several strategies have been reported in the literature to control<br />
such distance, mainly involving interlayers that work as spacers: examples are silica coatings [1],<br />
or organic films deposited through layer by layer [2] or Langmuir Blodgett [3] techniques, that<br />
require complex, multistep depositions to obtain films of increasing thickness.<br />
In this contribution, we present an all solution processed multilayered structure <strong>for</strong> the<br />
generation of metal enhanced fluorescence, in which the three different layers, even the metallic<br />
one, are deposited by means of spincoating. Interlayers of different thickness are obtained with the<br />
same single processing step, by tuning the deposition conditions. Suitable strategies have been<br />
developed to avoid the dissolution or damage of the previously deposited layers while processing<br />
the others.<br />
The typical curve of enhancement versus the interlayer distance [4] has been detected.<br />
Analogous results have been obtained when the measure of fluorescence has been carried out by<br />
exciting with a laser beam focused on areas of few μm 2 (by means of a microscope), thus<br />
highlighting a good uni<strong>for</strong>mity of the multilayer response across the substrate surface.<br />
Despite its simplicity, this system shows several advantages:<br />
- it is applicable both to oligomeric and polymeric fluorophores, the only requirement being a<br />
solubility of few mg/ml in common organic solvents;<br />
- the plasmonic band of the metal nanostructure can be tuned as well, by acting on the<br />
dimensions of the metal nanoparticles during their synthetic procedure;<br />
- multilayers can be grown in principle on any substrate, including transparent and/or<br />
conducting ones, without functionalization;<br />
- being spincoating the processing technique commonly exploited <strong>for</strong> the deposition of active<br />
layers <strong>for</strong> organic electronics and optoelectronics, the system here proposed can be directly<br />
integrated in the processing production of organic devices.<br />
This versatility opens the way to several applications, ranging from sensing plat<strong>for</strong>ms to solar<br />
concentrators to be combined with organic solar cells.<br />
References: [1] K. Aslan, et al., J. Am. Chem. Soc., 129 (2007) 1524. [2] N. Ma, et al., Macromol. Rapid<br />
Commun. 32 (2011) 587. [3] K. Ray, et al., Langmuir 22 (2006) 8374. [4] J.R. Lakowicz, Analytical<br />
Biochemistry. 298 (2001) 1.<br />
______________<br />
* Corresponding author: e-mail: eleonora.canesi@iit.it<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 185
P75 Polymers, Materials & Nanomaterials, Quantum Dots Poster 75<br />
Absorption enhancement of light-harvesting complexes through<br />
plasmonic silver island film<br />
Kamil K. Ciszak a,* , Maria Olejnik a , Janusz Strzelecki a , Eckhard Hofmann b<br />
& Sebastian Mackowski a<br />
a Nicolaus Copernicus University, 87-100 Torun, Poland<br />
b Department of Biology and Biotechnology, Ruhr-University Bochum, D-44780 Bochum, Germany<br />
Silver island film (SIF) has been recently applied to efficiently tune the optical properties of organic<br />
dyes [1], semiconductor nanocrystals [2], and light-harvesting complexes [3]. Relatively easy<br />
fabrication method and straight<strong>for</strong>ward ways to assemble hybrid structure render the SIF an<br />
excellent geometry to investigate plasmon induced effects on biomolecules. While most of the work<br />
has focused on radiative lifetime engineering, it would be appealing to use metallic nanoparticles to<br />
improve the absorption of light-harvesting complexes.<br />
In this work, we fabricate hybrid nanostructures composed of light-harvesting complexes<br />
deposited on SIF with varied morphology. The SIF was obtained on clean glass surfaces by<br />
chemical synthesis based on reduction of AgNO3 using NaOH and D-glucose. In order to change<br />
the density of silver islands, we prepared several SIF substrates by varying the reaction time. The<br />
obtained samples were analyzed using atomic <strong>for</strong>ce microscopy technique and absorption<br />
spectroscopy to determine plasmon resonance frequency. For coverslips kept in solution <strong>for</strong> less<br />
than 5 minutes we obtained a semi-transparent SIF characterized with plasmon resonance at 400<br />
nm. For longer times a corrugated silver surface was obtained. On such prepared substrates, we<br />
deposit thin PVA polymer layers containing peridinin-chlorophyll-protein (PCP) light-harvesting<br />
complexes. The PCP complexes are peripheral photosynthetic units responsible <strong>for</strong> light<br />
absorption in algea Amphidinium cartera. Its absorption matches well the range of plasmon<br />
resonance of the SIF.<br />
The impact of the plasmon resonance in the SIF on the optical properties of the PCP<br />
complexes was investigated by means of fluorescence excitation spectroscopy and time-correlated<br />
single photon counting technique. An example of time traces measured <strong>for</strong> the PCP complexes on<br />
the SIF substrate is shown in Fig.1. In contrast to the reference sample of PCP complexes<br />
deposited on glass substrate that feature monoexponential decay with a characteristic time of<br />
about 3.7 ns, the fluorescence decay of PCP complexes deposited on the SIF is characterized by<br />
biexponential decay. Importantly, the overall fluorescence intensity is increased by a factor of 30<br />
<strong>for</strong> the hybrid structure.The results show that by<br />
controlling the morphology of the SIF substrates we can<br />
tune the influence of the optical properties of the complex<br />
light-harvesting biomolecules.<br />
Fig.1. Fluorescence decay of PCP in PVA layer on glass (dots)<br />
and PCP in PVA on the surface of the SIF (solid). Exposure<br />
time <strong>for</strong> PCP complexes on glass substrate was 100 seconds,<br />
<strong>for</strong> PCP complexes on SIF substrate was 20 seconds.<br />
Support from the WELCOME program “Hybrid nanostructures as a stepping-stone towards efficient<br />
artificial photosynthesis” awarded by the Foundation <strong>for</strong> Polish Science is acknowledged.<br />
References: [1] J. R. Lakowicz Plasmonics 1, 5 (2006), [2] K. Ray et al. J. Am. Chem. Soc. 128 (28), 8998<br />
(2006), [3] S. Mackowski, J. Phys.: Condens. Matter, 22, 193102 (2010)<br />
______________<br />
* Corresponding author: e-mail: 210574@fizyka.umk.pl<br />
186 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P76 Polymers, Materials & Nanomaterials, Quantum Dots Poster 76<br />
One-pot synthesis of pegylated fluorescent nanoparticles by RAFT<br />
miniemulsion polymerization using a phase inversion process<br />
Chloé Grazon 1 , Rachel Méallet-Renault 1 , Jutta Rieger 2 , Bernadette Charleux 3<br />
& Gilles Clavier 1,*<br />
1<br />
PPSM (UMR8531), ENS Cachan, CNRS, UniverSud, 61 av President Wilson, F-94230 CACHAN,<br />
France<br />
2<br />
Laboratoire de Chimie des Polymères (UMR7610), Université Pierre et Marie Curie, 3, rue<br />
Galilée, 94200 Ivry, France<br />
3<br />
Laboratoire C2P2, Equipe LCPP (UMR5265), Université de Lyon 1, CPE Lyon, Bat 308F, 43 Bd<br />
du 11 novembre 1918, 69616 Villeurbanne, France<br />
In the last decade, fluorescent nanoobjects have received increasing interest <strong>for</strong> their high potential<br />
in biology and biochemistry. They are especially attractive <strong>for</strong> sensing, imaging and biomedical<br />
applications. Recent works in controlled radical polymerization (CRP), allows the use of<br />
amphiphilic macromolecular RAFT agents and miniemulsion in order to both stabilise the<br />
nanoparticles and control the polymerisation of hydrophobic monomers.<br />
Based on this methodology we developed a new strategy to design fluorescent nanoobjects<br />
where the fluorophore is copolymerised in the hydrophobic centre of the polymeric micelle. The<br />
approach to obtain fluorescent nanoparticles of well-defined chain lengths and sizes, via a<br />
miniemulsion process in water is based on a phase inversion, without the use of surfactants and<br />
hydrophobic cosolvent. In our study, we chose a hydrophilic PEO-b-PAA-TTC-C12 (poly(ethylene<br />
oxide)-b-poly(acrylic acid)-trithiocarbonate) macroRAFT agent in order to reach pegylated<br />
biocompatible and pH-sensitive auto-stabilized fluorescent nanoparticles. It is used to control the<br />
copolymerization of styrene with a fluorescent BODIPY (BODPY-methacrylate, BDPMA) based<br />
monomer in miniémulsion (see scheme). BODPY was chosen as fluorophore since it exhibits<br />
attractive spectroscopic characteristics such as emission spectra tuneable from green to red and<br />
high fluorescence quantum yields.<br />
The particles present a 60nm diameter and a narrow size distribution and are stable at pH>5. Their<br />
fluorescence colour is close to that of the monomer. Timed resolved fluorescence anisotropy<br />
reveals a fast interchormophore energy hoping. The presence of carboxylic groups at the exterior<br />
of the nanoobjects allows <strong>for</strong> their trans<strong>for</strong>mation into nanosensors by reaction with various<br />
molecules and macromolecules bearing an amine. The first results of such functionalisations with<br />
pH sensitive chromophores and proteins will be presented. The short distance between those<br />
peripheral groups and the hydrophobic heart of the nanoparticle allows <strong>for</strong> a modulation of<br />
fluorescence of the BODIPY by a FRET process which depends on the colour of the pH sensitive<br />
molecule.<br />
References: [1] C. Grazon, et al., Macromol. Rapid Commun. 32 (2011) 699.<br />
______________<br />
* Corresponding author: e-mail: gclavier@ppsm.ens-cachan.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 187
P77 Polymers, Materials & Nanomaterials, Quantum Dots Poster 77<br />
Fluorescence microscopy of corrole-single silver nanowire<br />
hybrid structures<br />
Nikodem Czechowski 1* , Maria Olejnik 1 , Bartosz Krajnik 1 , Agnieszka Nowak-Król 2 , Dawid<br />
Piątkowski 1 , Wolfgang Heiss 3 , Daniel T. Gryko 2 & Sebastian Maćkowski 1<br />
1 Institute of Physics, Nicolaus Copernicus University, Grudziadzka 5, 87-100 Torun, Poland<br />
2 Institute of Organic Chemistry, Polish Academy of Science, Kasprzaka 44/52, 01-224 Warsaw, Poland<br />
3 Institute of Solid <strong>State</strong> Physics Johanes Kepler University, Altenbergerstrasse 69, A-4040 Linz, Austria<br />
Corroles are synthetic, aromatic macrocycles of tetrapyrrolic family that also includes porphyrins,<br />
phthalocyanine sans vitamin B12. These dyes were synthesized in 1965 [1], but the interest in<br />
these compounds has grown recently as these molecules could be potentially used <strong>for</strong> antitumor<br />
therapy, catalytic and optical sensor devices [2], and artificial light-harvesting systems. It is also<br />
well known that plasmon-fluorophore interaction can lead to substantial changes in fluorescence<br />
intensity [3]. One of the critical parameters influencing this interaction is the distance between the<br />
dye and the metallic nanoparticle. It is there<strong>for</strong>e important to develop ways to control this<br />
separation and investigate its impact on the optical properties of a plasmon hybrid nanostructure.<br />
In this work we study fluorescence properties of corroles deposited on single silver<br />
nanowires. The nanowires were spin-coated on a glass substrate and coated with dielectric SiO2<br />
spacer with thickness varying from 5 nm to 30 nm. Next, 5,10,15-tris(pentafluorophenyl)corrole<br />
molecules in PMMA matrix were deposited. Importantly, the absorption spectrum of the corrol is<br />
well matched with plasmon resonance of silver nanowires, located around 400 nm.<br />
It is important to correlate the position of a nanowire with the fluorescence image obtained <strong>for</strong><br />
corroles. We achieve it using a confocal scanning fluorescence microscope. First, using reflected<br />
laser light we locate the nanowires, and<br />
subsequently, we scan the same region and<br />
detect the corrole fluorescence, as seen in<br />
Fig. 1. In the case of thick spacers (30 nm)<br />
we observe weak, twofold fluorescence<br />
enhancement. In contrast, <strong>for</strong> 5 nm thick<br />
spacers the enhancement of the corrole<br />
fluorescence reaches factor of 15.<br />
Fig 1. Left: reflection image of single nanowire.<br />
Right: fluorescence image of corroles.<br />
188 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
Importantly, our experimental setup allows<br />
<strong>for</strong> subsequent measurement of both<br />
fluorescence spectra and fluorescence<br />
lifetimes from any given location on the<br />
map. In this way a complete in<strong>for</strong>mation regarding the interactions present in our system can be<br />
elucidated.<br />
These results demonstrate convincingly that by proper design of a plasmonic hybrid<br />
nanostructure we should be able to optimize the influence of plasmon excitation in metallic<br />
nanoparticle on the optical properties of synthetic molecules.<br />
Financial support from the Foundation <strong>for</strong> Polish Science under WELCOME program “Hybrid<br />
nanostructures as a stepping-stone towards efficient artificial photosynthesis” is gratefully<br />
acknowledged.<br />
References:<br />
[1] A. W. Johnson et al., J. Chem. Soc., 1620 (1965), [2] B. Ventura et al., New J. Chem., 29, 1559 (2005),<br />
[3] S. Mackowski J. Phys. Condens. Matter 22, 193102 (2010).<br />
______________<br />
* Corresponding author: e-mail: nikodem@fizyka.umk.pl
P78 Polymers, Materials & Nanomaterials, Quantum Dots Poster 78<br />
Delivery of luminescent gold nanoparticles into human platelets using a<br />
pH controlled strategy<br />
Amy Davies 1 , Steve Watson 2 , Ela Claridge 3 , Steve Thomas 2 & Zoe Pikramenou 4,*<br />
1<br />
PSIBS Doctoral Training Centre, University of Birmingham, Edgbaston, Birmingham, B15 2TT (UK)<br />
2<br />
Centre <strong>for</strong> Cardiovascular Sciences, Institute of Biomedical Research, University of Birmingham,<br />
Edgbaston, Birmingham, B15 2TT (UK)<br />
3<br />
School of Computer Science, University of Birmingham, Edgbaston, Birmingham, B15 2TT (UK)<br />
4<br />
School of Chemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT (UK)<br />
Gold nanoparticles (AuNP) are attractive <strong>for</strong> use in biological<br />
and biomedical applications due to their optical properties and<br />
low toxicity [1] . Their high electron density makes them useful as<br />
probes <strong>for</strong> electron microscopy and they can be utilised as<br />
scaffolds <strong>for</strong> delivery of multiple probes.<br />
Luminescent lanthanide complexes are ideal optical<br />
probes <strong>for</strong> imaging in biomedical systems due to their distinctive<br />
emission profiles in the visible or near-infra red, large Stokes<br />
shift and long lifetimes. The coating of gold nanoparticles with<br />
surface active lanthanide complexes and the insertion into cells<br />
has been previously demonstrated in the Pikramenou group [2, 3] .<br />
Further functionalisation of gold nanoparticles using peptides<br />
which are known to bind specific cellular proteins has also been<br />
achieved [4] .<br />
Herein, we present a novel pH controlled strategy to<br />
enable rapid uptake of gold nanoparticles into human platelets. We have developed gold<br />
nanoparticles coated with peptides and lanthanide complexes and investigated the delivery into<br />
platelets with transmission electron microscopy and light microscopy techniques, including<br />
luminescence imaging.<br />
This work was supported by PSIBS EPSRC Doctoral Training Centre and BHF.<br />
References: [1] P. Ghosh, et al., J. American Chemical Society 132 (2010) 2642. [2] D.J. Lewis, et al.,<br />
Chemical Communications 13 (2006) 1433. [3] D.J. Lewis, et al., Nanomedicine 5 (2010) 1547. [4] A.C.<br />
Savage and Z Pikramenou, Chemical Communications 47 (2011) 6431.<br />
______________<br />
* Corresponding author: e-mail: z.pikramenou@bham.ac.uk<br />
Schematic representation of<br />
Peptide•EuL•AuNP<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 189
P79 Polymers, Materials & Nanomaterials, Quantum Dots Poster 79<br />
Preparation and optical properties of new hybrid materials: silica gels<br />
doped with a [Eu(2NTBD)3 ][PPhenDCN] complex<br />
N. Danchova, T. Deligeorgiev, N. Lesev, S. Kaloyanova, S. Stoyanov & S. Gutzov<br />
University of Sofia, Faculty of Chemistry, J. Bourchier Blvd. 1, 1164 Sofia, Bulgaria<br />
Lanthanide chelates have found applications in a variety of areas - as probes in bioanalytical<br />
assays, as optical signal amplifier and electroluminescent devices. It is a common practice to <strong>for</strong>m<br />
complexes of the lanthanide ions with organic ligands that strongly absorb light and transfer the<br />
energy to the metal ion (antenna effect, donor-acceptor transfer) [1-2].<br />
The present contribution deals with the preparation and optical properties of transparent<br />
silica gels, doped with a new red emitting Eu(III) complex: [Eu(2NTBD)3 ][PPhenDCN] obtained<br />
from Europium(III)tris[4,4,4-trifluoro-1-(naphthalene-2-yl)-1,3-butandione] - Eu(2NTBD)3 and<br />
pyrazino[2,3-f][1,10-phenathroline-2,3-dicarbonitrile - PPhenDCN [3]. The new complex is<br />
incorporated in silica gels at room temperature using a sol-gel scheme with acid catalyzed<br />
hydrolysis and pH of gelation about 7 [1,2]. The doped amorphous silica sol-gel materials are<br />
characterized by room temperature luminescence / excitation spectroscopy, UV/Vis transmission<br />
and reflectance spectroscopy, NMR, IR and X-Ray diffraction. The hybrid sol-gel materials display<br />
a strong red f-f Eu(III) luminescence even at low doping levels nEu/nSi ≈ 3·10 -4 with a maximum at<br />
615 nm coming from the 5 D0→ 7 F2 transition. The luminescence spectra of the solid [Eu(2NTBD)3<br />
][PPhenDCN] complex and of doped silica gels suggest a non-centrosymmetrical environment of<br />
the europium Eu(III) ion, the site symmetry of the Eu(III) ion in the new complex and is C2v, C2 or Cs<br />
which allows a high luminescence intensity. Two strong absorption maxima at about 270 nm and<br />
340 nm are detected by UV/Vis transmission and reflectance measurements of [Eu(2NTBD)3<br />
][PPhenDCN] solutions and doped silica gels. The excitation spectra of the doped gels display a<br />
broad maximum at 345 nm ( 5 D0→ 7 F2 nm monitoring), they are completely different from the well<br />
known Eu(III) excitation spectra in inorganic matrixes.<br />
A blue emission at 410 nm (335 nm excitation) is detected depending on the doping<br />
concentration which disappear at higher europium doping. The red Eu(III) emission completely<br />
disappears after the samples are heated at 200 o C and the luminescence spectrum consists only a<br />
blue emission. DTA/TG measurements supports the hypothesis, that the blue emission is caused<br />
by decomposition of [Eu(2NTBD)3 ][PPhenDCN] at about 260 o C. Heating at 400 o C leads to a<br />
quenching of the blue luminescence.<br />
The work is supported by the Bulgarian National Science Fund, grant TK 02/26-2009.<br />
References :<br />
[1]. S.Gutzov, et al., J. Non-Cryst. Solids 354, ( 2008) 3438. [2]. G. Ahmed, et al., J. Incl. Phenom. Macro.,<br />
59 (2007) 167. [3]. C. Vancaeyzeele, et al., J. Am. Chem. Soc. 129 (2007) 13653.<br />
190 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P80 Polymers, Materials & Nanomaterials, Quantum Dots Poster 80<br />
Few-atom clusters of silver in organic solvents: synthesis<br />
and properties<br />
Isabel Díez 1 , Robin H. A. Ras 1 , Mykola I. Kanyuk 2 & Alexander P. Demchenko 2,*<br />
1<br />
Helsinki University of Technology/Aalto University, Department of Applied Physics,<br />
Puumiehenkuja 2, 02150 Espoo (Finland)<br />
2<br />
Laboratory of Nanobiotechnologies, Palladin Institute of Biochemistry, Leontovicha 9, Kiev 01030<br />
(Ukraine)<br />
Few-atom silver clusters are fluorophores with a set of attractive of properties including subnanometer<br />
size, high quantum yield and large Stokes shift. [1-3] Sharing high photostability with<br />
semiconductor quantum dots but being of much smaller size, lacking blinking and with expected<br />
lack of toxicity, they are especially attractive <strong>for</strong> biological imaging, down to single molecules. No<br />
less promising are their applications in chemical sensing and biosensing as well as <strong>for</strong> molecular<br />
optic and electronic devices on a single molecular level.<br />
Synthesis of silver clusters is very simple and can be achieved by chemical reduction and<br />
photoreduction in solutions, so the greatest problem is the selection of scaffolds that can provide<br />
their long-term stability. Different water-soluble polymers, dendrimers, peptides and DNA<br />
oligonucleotides were suggested as such scaffolds <strong>for</strong> use in aqueous solutions, but with a limited<br />
success. In this research we tried to achieve production of stable fluorescent clusters in a series of<br />
organic solvents using an amphiphilic polystyrene-block-poly(methacrylic acid) block copolymer<br />
(PS-b-PMAA). The PMAA homopolymer was already reported to be an excellent template <strong>for</strong> silver<br />
clusters in aqueous medium, (Díez I et al., 2010; Díez I et al., 2009) and in the current work the PS block was<br />
selected to extend the solubility to polar and apolar organic solvents. We clearly show that<br />
photoreduction of silver ions can be achieved successfully in different organic solvents, including<br />
those of high and low polarity, demonstrating that it is the matrix that matters much stronger than<br />
the major solvent. Meantime, strong variations in stability and fluorescence properties (positions of<br />
spectra, quantum yields and lifetimes) <strong>for</strong> clusters synthesized in different solvents are observed.<br />
The clusters <strong>for</strong>med in dimethyl<strong>for</strong>mamide (DMF) were characterized by the lowest light<br />
scattering and highest stability (no change of properties during more than one year of storage).<br />
Their detailed studies allowed identifying three types of clusters differing in positions of their<br />
excitation and emission bands that could be attributed to difference in the cluster composition. In<br />
addition, each band exhibits characteristic features of inhomogeneous broadening - strong<br />
dependences of excitation spectra on emission wavelength and of emission spectra on excitation<br />
wavelength. High anisotropy of their fluorescence emission witness <strong>for</strong> the absence of rotational<br />
mobility during fluorescence lifetime.<br />
Thus, we demonstrate that it is not a unique property of water that can provide the <strong>for</strong>mation<br />
and stability of silver clusters. Photoreduction can be a universal method <strong>for</strong> production of these<br />
clusters in different organic solvents using the same polymeric template. A long-term stability of<br />
these clusters producing bright fluorescence emission can be achieved.<br />
References: [1] I. Díez, R.H.A. Ras, Nanoscale 3 (2011) 1963. [2] I. Díez, R.H.A. Ras, in “Advanced<br />
Fluorescence Reporters in Chemistry and Biology II” (Demchenko A.P., ed.) Springer Series on<br />
Fluorescence 9 (2010) 307. [3] T. Vosch et al., PNAS, 104 (2007) 12616. [4] I. Díez et al., Angew. Chem. Int.<br />
Ed. 48 (2009) 2122.<br />
______________<br />
* Corresponding author: e-mail: alexdem@ukr.net<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 191
P81 Polymers, Materials & Nanomaterials, Quantum Dots Poster 81<br />
Luminescent silica nanoparticles <strong>for</strong> enrofloxacin detection<br />
via FRET signaling<br />
Clara Somoza 1 , Ana B. Descalzo 1 ,* M. Cruz Moreno-Bondi 2 & Guillermo Orellana 1<br />
Chemical Optosensors and Applied Photochemistry Group (GSOLFA). Departments of 1 Organic<br />
Chemistry and 2 Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid<br />
(UCM), Av. Complutense s/n, 28040 Madrid (Spain)<br />
Developing quick and cheap methods <strong>for</strong> selective detection of enrofloxacin, –a broad-spectrum<br />
antibiotic of the fluoroquinolone family–, is of interest due to its widespread use in human and<br />
veterinary medicine. [1] One of the strategies currently being followed in our group is the<br />
development of an optical sensor system using FRET (Förster Resonance Energy Transfer)<br />
process as the signal transduction method. Since the efficiency of FRET depends on the distance<br />
between two chromophores, –a luminescent FRET donor (D) and a suitable acceptor molecule<br />
(A)–, it can be employed <strong>for</strong> monitoring binding of two species labelled with appropriate D–A pairs.<br />
These D–A pairs are chosen on the basis of the spectral overlap between Dem and Aabs. In this<br />
work we have selected a cyanine-labelled enrofloxacin with λabs/λem = 705/793 nm and τL = 0.92 ns,<br />
as the FRET acceptor, and a Ru(bpy)3 2+ complex (bpy: 2,2’-bipyridine), with λabs/λem = 450/584 nm<br />
and τL = 0.40 μs (under air), as the FRET donor. Besides the detection in the near-infrared (NIR)<br />
window,–enabled by the cyanine label–, an advantage of this D–A pair is that the Ru(II) complex<br />
emission decays in several hundred nanoseconds. This feature allows <strong>for</strong> a proper discrimination<br />
of the original FRET-sensitized cyanine emission at 800 nm, from that arising from direct excitation<br />
of the unbound cyanine. In order to maximize signal output [2] and minimize the Ru(II) quenching by<br />
dissolved oxygen, [3] FRET donor molecules were encapsulated into silica nanoparticles (NPs) of<br />
ca. 150 nm diameter. Since these NPs are rather large compared to the FRET scale (typical FRET<br />
occurs within 1 < dD-A < 10 nm), [4] it is convenient to have the maximum amount of D luminophores<br />
located on the external shell of the NPs. Interestingly, the distribution of Ru(II) complexes can be<br />
finely tuned by controlling the time at which Ru(bpy)3 2+ is added during the Stöber synthesis of the<br />
silica NPs. [5] In this communication we will show how the Ru(II) distribution affects the FRET<br />
efficiency on assays per<strong>for</strong>med with the luminescent NPs and the NIR-labelled enrofloxacin.<br />
FRET<br />
NIR labelled enrofloxacin<br />
Ligand<br />
Ru2+ doped silica NPs<br />
192 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
A norm<br />
FRET<br />
Exc Em<br />
D A<br />
400 500 600<br />
λ / nm<br />
700 800<br />
Figure 1. Left: schematic<br />
representation of the<br />
novel NP-based Ru(II)cyanine<br />
FRET system<br />
described herein and,<br />
right: absorption and<br />
emission spectra of the<br />
D–A pair.<br />
This work was supported by the Spanish Ministry of Science and Innovation (Ramón y Cajal<br />
Program and CTQ2009-14565-C03-01), a Marie-Curie European Reintegration Grant (NANOLUM)<br />
and Complutense University of Madrid (GR35/10-A).<br />
References: [1] E. Benito-Peña, et al., Anal. Bioanal. Chem. 393 (2009) 235. [2] S. Zhu, et al., Top. Curr.<br />
Chem. 300 (2011) 51. [3] G. López-Gejo, et al., Langmuir 26 (2010) 2144. [4] N.L. Vekshin, “Energy Transfer<br />
in Macromolecules”, SPIE Optical Engineering Press, Bellingham, Washington (1997). [5] D. Zhang, et al.,<br />
Langmuir 26 (2010) 6657.<br />
______________<br />
* Corresponding author e-mail: ab.descalzo@quim.ucm.es<br />
F norm
P82 Polymers, Materials & Nanomaterials, Quantum Dots Poster 82<br />
Fluorescence spectroscopy and spectral modeling as a tool <strong>for</strong> study of<br />
interaction of nanoparticles with biomacromolecules. Plant cell walls<br />
Daniela Djikanović a , Aleksandar Kalauzi a , Milorad Jeremić a , Jianmin Xu b , Miodrag Mićić c,d ,<br />
Roger Leblanc b & Ksenija Radotić a<br />
a<br />
Institute <strong>for</strong> Multidisciplinary Research, University of Belgrade, Bul. Despota Stefana 142, 11000<br />
Belgrade, Serbia<br />
b<br />
Department of Chemistry, University of Miami, Coral Gables, FL 33124, USA;<br />
mmicic@mpbio.com<br />
c<br />
MP Biomedicals, LLC, 3 Hutton Center, Santa Ana, CA 92707, USA;<br />
d<br />
BioMEMS Lab, Dept of Mechanical and Aerospace Engineering, University of Cali<strong>for</strong>nia –<br />
Irvine,4200 Engineering Gateway, Irvine, CA92697-3975, USA.<br />
Quantum dots (QDs) are increasingly applied in plant science, as markers <strong>for</strong> the cells or their cell<br />
walls. In a plant, the cell wall is a first target place <strong>for</strong> external agents. We studied interaction of<br />
CdSe quantum dots (QDs) with cell walls isolated from a conifer - Picea omorika (Panč) Purkynĕ<br />
branch. Fluorescence and FT-IR spectroscopy and epifluorescence microscopy have been<br />
per<strong>for</strong>med in order to study interaction of the QDs with the whole cell wall, as well as with its<br />
individual constituent polymers: cellulose, lignin and hemicellulose. The isolated cell wall is an<br />
appropriate object <strong>for</strong> study of the interactions with nanoparticles. The aim of the study was to see<br />
whether the QDs induce structural changes in the cell wall, as well as to find out which kind of<br />
interaction between QDs and cell wall’s occurs. We also investigated affinity of cell wall polymers<br />
<strong>for</strong> binding quantum dots. The results show that in the cell wall, CdSe quantum dots predominantly<br />
binds to cellulose, through OH groups and to lignin, through the conjugated C=C/C-C chains. The<br />
differences in interaction of wet and dry CWs with QDs/chloro<strong>for</strong>m were also per<strong>for</strong>m. We treated<br />
cell wall with water as hydrophilic and chloro<strong>for</strong>m as hydrophobic solvent. In the reaction of the dry<br />
cell wall sample with QDs/chloro<strong>for</strong>m, hydrophobic interactions are dominant. When water was<br />
added after QDs/chloro<strong>for</strong>m, hydrophilic interactions enable a partial reconstruction of the C=C<br />
chains. The results have an implication on the employment of the QDs in plant bio-imaging.<br />
This work was supported by grants 173017 and 45012 from the Ministry of education and science<br />
of the Republic of Serbia.<br />
______________<br />
* Corresponding author: e-mail: danielle@imsi.rs<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 193
P83 Polymers, Materials & Nanomaterials, Quantum Dots Poster 83<br />
Fluorescent organic ion pairs: an original approach <strong>for</strong> the preparation<br />
of nanomaterials<br />
Suzanne Fery-Forgues 1* , Abdelhamid Ghodbane 1 , Joe Chahine 1 & Martine Cantuel 2<br />
1<br />
Laboratoire IMRCP, UMR CNRS 5623, Université Paul Sabatier, F-31062 Toulouse cedex 9<br />
(France)<br />
2<br />
Institut des Sciences Moléculaires, UMR CNRS 5255, Université de Bordeaux 1, 351 Cours de la<br />
Libération, 33405 Talence cedex (France)<br />
Fluorescent organic nanocrystals [1] and nanofibers [2] are presently attracting increasing attention<br />
due to the variety of potential applications. For example, they could be used as new materials in the<br />
field of submicron-sized optoelectronics, light emitting diodes, chemical and biochemical sensors,<br />
and biological imaging. However, their development is still a challenge <strong>for</strong> chemists. Their<br />
morphology is difficult to control and their fluorescence properties closely depend on the numerous<br />
intermolecular associations that take place in the solid state.<br />
We show here that organic salts composed of an aromatic fluorophore and an aliphatic bulky<br />
counter-ion display original self-association and optical properties, close to aggregation-induced<br />
emission enhancement (AIEE). For instance, the phenolate of 4-hydroxy-7-nitrobenzoxadiazole<br />
(NBDO - ) was associated to the tetrabutylammonium cation (TBA + ). Conversely, the aromatic<br />
berberine cation (Ber + ) was associated to the palmitate anion. In both cases, the fluorescence of the<br />
aromatic ion was quenched by water and the dissolved salts were virtually not fluorescent in this<br />
medium. However, the salts readily crystallized and transition to the solid state was accompanied by<br />
a strong increase in fluorescence intensity. This phenomenon can be explained by two reasons. The<br />
aromatic ions are protected from water molecules, and above all, the presence of the bulky counterions<br />
prevents parallel-stacking of the fluorophores, thus leading to an original molecular arrangement<br />
that is favourable to fluorescence. Using a simple preparation method based on solvent exchange,<br />
the TBA + NBDO - ion pair (1) spontaneously gave nano- and microcrystals [3], while the Ber + Pal - (2)<br />
derivative generated nanofibers that were stable <strong>for</strong> months in water but evolved upon drying towards<br />
reticulated material [4]. The nature of the organic counter-ions was easily changed, showing the<br />
versatility of the system. This concept is very promising <strong>for</strong> the design of new organic micro- and<br />
nanoparticles that must be fluorescent in the solid state, possibly in an aqueous environment.<br />
O<br />
O<br />
N +<br />
CH 3 (CH 2) 14 COO -<br />
(2)<br />
OCH 3<br />
OCH3<br />
Figure 1. Berberine palmitate: Chemical structure , (a) fluorescence microscopy image of aqueous<br />
suspensions of 2 (6.17 × 10 -5 10 µm<br />
M) after reprecipitation in water with 1.2% v/v ethanol and (b) scanning<br />
electron microscopy image of the solid <strong>for</strong>med after drying the sample.<br />
References: [1] H. Masuhara, H. Nakanishi, K. Sasaki, K. <strong>Single</strong> Organic Nanoparticles (2003) Springer-<br />
Verlag Berlin.[2] S. Fery-Forgues, C. Fournier-Noël, “Organic fluorescent nanofibers and sub-micrometer<br />
rods”. Nanofibers, A. Kumar (Ed.) In-Techweb, ISBN 978-953-7619-86-2, (2010) pp. 383-404. Available <strong>for</strong><br />
free at: http://www.sciyo.com/books/show/title/nanofibers. [3] J.-F. Lamère, N. Saffon, I. Dos Santos, S. Fery-<br />
Forgues, Langmuir, 26 (2010) 10210-10217. [4] J. Chahine, N. Saffon, M. Cantuel, S. Fery-Forgues,<br />
Langmuir 27 (2011) 2844-2853.<br />
______________<br />
* Corresponding author: e-mail: sff@chimie.ups-tlse.fr<br />
194 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P84 Polymers, Materials & Nanomaterials, Quantum Dots Poster 84<br />
Fast FRET events in dye-doped nanoparticles provide functionalities:<br />
high brightness and tunable emission<br />
Damiano Genovese a , Sara Bonacchi a , Riccardo Juris a , Marco Montalti a , Enrico Rampazzo a ,<br />
Nelsi Zaccheroni a & Luca Prodi a<br />
a<br />
Dipartimento di Chimica “G. Ciamician”, Università di Bologna, Via Selmi 2, 40126 BOLOGNA,<br />
ITALY<br />
Dye-doped nanoparticles display, compared to molecular dyes, a wide set of additional<br />
functionalities: many dyes can be embedded and combined to tune the overall absorption and<br />
emission spectra, and a surface is available <strong>for</strong> functionalization, allowing <strong>for</strong> tunable solubility and<br />
(bio)molecular target recognition. Such features make dye-doped nanoparticles an extremely<br />
versatile and desirable label <strong>for</strong> fluorescence imaging, particularly in biomedical and bioanalytical<br />
fields.<br />
We designed dye-doped nanoparticles with a core-shell architecture: the PEG shell (with<br />
possible surface functionalization to directly target biomolecules) and the small size (25 nm) allow<br />
use <strong>for</strong> in-vivo and in-vitro assays.<br />
We discuss in this contribution our strategy to obtain bright and multicolor core-shell NPs, based<br />
on interactions between different kinds of dyes embedded in the same NPs.<br />
Compared to reference samples doped with only one kind of dyes, the NPs here discussed present<br />
increased brightness and tunable emission owing to fast FRET events, which kinetically compete<br />
with either self-quenching processes or fluorescence.<br />
Such FRET processes are fully characterized by means of steady-state and time-resolved<br />
spectroscopic techniques, as well as by Montecarlo simulations. The orthogonality of resulting<br />
emissions was also checked by spectrally resolved confocal microscopy.<br />
Additional features such as increased pseudo-Stokes Shift and possibility of external<br />
functionalization will be discussed. We envisage application of this material in biological imaging as<br />
well as in sensing and in other fluorescence-based analytical techniques.<br />
The present silica-core/PEG-shell NPs, owing to the careful design, represent a valuable<br />
alternative to traditional labels such as molecular dyes or QDs, since they address many of their<br />
traditional issues (environment-dependent emission, solubility, toxicity).<br />
References: [1] S. Bonacchi et al., Angew. Chem. Int. Ed., 50 (2011) DOI: 10.1002/anie.201004996. [2] S.<br />
Bonacchi et al., Top. Curr. Chem. 300 (2011) 93-138. [3] E. Rampazzo et al., J. Phys. Chem. B., 114 (2010)<br />
14605.<br />
______________<br />
* Corresponding author: damiano.genovese2@unibo.it<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 195
P85 Polymers, Materials & Nanomaterials, Quantum Dots Poster 85<br />
Upconverting luminescent nanoparticles (UCLNPs) enable backgroundfree<br />
cellular imaging, immunoassays and ratiometric encoding<br />
Hans H. Gorris * , Raphaela Liebherr, Sayed M. Saleh, Reham Ali & Otto S. Wolfbeis<br />
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg,<br />
Universitätsstraße. 31, 93040 Regensburg, Germany<br />
Nanoparticles consisting of lanthanide-doped NaYF4 crystals exhibit unique luminescent<br />
properties. Their ability to convert near infrared (NIR, 980 nm) light into shorter visible wavelengths<br />
(anti-Stokes emission) minimizes autofluorescence and light scattering from biological materials 1 .<br />
We have developed a new strategy to render hydrophobic UCLNPs water soluble and modify them<br />
with thiol-reactive maleimide groups. The background-free detection mode of the surface modified<br />
UCLNPs provides the basis <strong>for</strong> a range of analytical applications: First, we conjugated UCNLPs to<br />
small molecule ligands that bind to receptors on the surface of cells. This approach is used <strong>for</strong><br />
labeling a cyclic peptide specific <strong>for</strong> αVβ3 integrin, a tumor marker of glioblastoma cells. Second,<br />
UCNLPs have been conjugated to antibodies as a detection label in a competitive immunoassay<br />
<strong>for</strong> the pollutant 2,4-dichlorophenoxyacetic acid (2,4-D). As the concomitant background<br />
fluorescence of environmental samples is avoided under NIR excitation, very low detection limits<br />
are envisioned. In addition to the anti-Stokes emission, UCLNPs also feature dual and narrow<br />
emission bands in the visible and NIR. This feature has been exploited to implement a ratiometric<br />
encoding strategy <strong>for</strong> multiplexed bioanalytical<br />
tasks 2 . The codes are generated by adjusting<br />
one of the dual emission spectra of UCLNPs in<br />
ten increments with selective filter dyes,<br />
whereas the second unfiltered emission band<br />
serves as a reference signal (Figure). The ratio<br />
of both emission bands defines the identifier<br />
code. Combining several types of UCLNPs -<br />
each exhiting distinct intensity levels - increases<br />
the coding capacity exponentially. Ratiometric<br />
optical measurements are largely indpendent of<br />
the concentration, the light source intensity and<br />
the photocodetector sensitivity. This encoding<br />
strategy thus enables the identification of<br />
analytes in a highly robust and reproducible<br />
way. For example, polystyrene beads carrying<br />
different types of DNA-probes or antibodies can<br />
be labeled with the encoded UCLNPs and<br />
loaded onto femtoliter arrays <strong>for</strong> miniaturized<br />
genotyping, screening combinatorial libraries,<br />
medical diagnosis, and environmental<br />
monitoring 3 .<br />
196 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
Figure: Adjusting the dual emission spectra of<br />
UCLNPs (NaYF4:Yb,Er; solid line) <strong>for</strong> ratiometric<br />
multiplexed encoding. Either the green emission<br />
band (543 nm) is selectively filtered by using<br />
rhodamine B (hatched line) or the red emission<br />
band (657 nm) is filtered by the long-wavelength<br />
absorbing dye S 0378 (dotted line) 4 . The second<br />
emission band, by contrast, serves as a constant<br />
reference signal.<br />
References: [1] H.S. Mader, et al.. Upconverting luminescent nanoparticles <strong>for</strong> use in bioconjugation and<br />
bioimaging. Curr. Opin. Chem. Biol. 14 (2010) 1-15. [2] H.H. Gorris, et al. Tuning the dual emission of<br />
photon-upconverting nanoparticles <strong>for</strong> ratiometric multiplexed encoding. Adv. Mater. 23 (2011) 1652-1655.<br />
[3] H.H. Gorris, D.R. Walt. Analytical chemistry on the femtoliter scale. Angew. Chem. Int. Ed. 49 (2010)<br />
3880-3895. [4] H.H. Gorris, et al. Long-wavelength absorbing and fluorescent chameleon labels <strong>for</strong> proteins,<br />
peptides and amines. Bioconj. Chem. in revision.<br />
______________<br />
* Corresponding author: e-mail: Hans-Heiner.Gorris@chemie.uni-r.de
P86 Polymers, Materials & Nanomaterials, Quantum Dots Poster 86<br />
Effect of rare earth elements on the photoluminescence of CaGa2S4:Eu<br />
Chiharu Hidaka 1* & TakeoTakizawa 1<br />
1 Department of Physics, College of Humanities and Sciences, Nihon University, Sakura-josui<br />
3-25-40, Setagaya-ku, Tokyo 156-8550 (Japan)<br />
Europium doped CaGa2S4 compounds show green emission with high effciency. When they are<br />
co-doped with other rare earth elements(REEs), afterglow has emerged where the longest one is<br />
reported in a Ho co-doped case [1-2]. The origin of this afterglow is surely the co-doping of REEs,<br />
but its mechanism has not been clarified yet. The elucidation of the role of REE co-doping<br />
definitely leads to the easy designing of phosphors with various decay time. Here we present the<br />
results of our study of Eu emissions and their decay times in CaGa2S4:Eu, REEs and will discuss<br />
the effect of REE co-doping on optical<br />
properties.<br />
The compounds of CaGa2S4:Eu are<br />
prepared co-doped with REEs (La, Ce, Pr,<br />
Nd, Sm, Gd, Tb, Dy, Ho, Er, Tm, Yb) and<br />
photoluminescence (PL) spectra of the<br />
compounds are measured. Figure 1<br />
shows PL spectra of CaGa2S4:Eu codoped<br />
with REEs together with that of an<br />
only Eu doped compound. The peaks of<br />
Eu emissions (560nm) do not shift and the<br />
emission intensities decrease in all codoped<br />
cases. We have reported PL<br />
spectra of Eu and Nd, Dy, Ho co-doped<br />
compounds, and concluded that the<br />
energy transfer from Eu to Nd, Dy, Ho is<br />
occurring under UV light excitation [3].<br />
Similar energy transfer is also observed<br />
between Eu and the other REEs. We have<br />
also measured decay times of Eu<br />
emissions of these compounds. Their<br />
decay curves have two long terms<br />
depending on REEs. The results explain<br />
that a part of electrons is transferred from<br />
Eu to the excited states of REEs and<br />
another part moves to be trapped in the<br />
electron reservoir being generated by<br />
REEs co-doping. These results will be<br />
discussed in view of the relations of their<br />
decay properties together with the 5d and 4f levels of divalent and trivalent REE ions in the band<br />
diagrams of CaGa2S4 as reported by Bessière et al.[4].<br />
This work is partly supported by grant <strong>for</strong> private universities <strong>for</strong> 2009-2013 from ministry of<br />
education, sports, science, culture and technology, Japan.<br />
PL intensities [arb.units]<br />
500 600 700<br />
Wave length [nm]<br />
References: [1] C. Guo et al., J. Lumin. 126 (2007) p.333 [2] C. Guoet al., J. Phys. Chem. Sol. 68 (2007)<br />
p.217 [3] C.Hidaka et al., Phys. Status.Solid C 6 (2009) p.1166 [4] Bessière et al., J.Electrochem.Soc. 151<br />
(2004) H254<br />
______________<br />
* Corresponding author: e-mail: komaz@phys.chs.nihon-u.ac.jp<br />
300<br />
400<br />
Only Eu doping<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 197<br />
800<br />
La<br />
Ce<br />
Pr<br />
Nd<br />
Sm<br />
Gd<br />
Tb<br />
Dy<br />
Ho<br />
Er<br />
Tm<br />
Yb<br />
900<br />
Fig.1 PL spectra of CaGa2S4 co-doped with Eu and<br />
REEs together with that of only Eu doped case.
P87 Polymers, Materials & Nanomaterials, Quantum Dots Poster 87<br />
The influence of silver nanostructures <strong>for</strong>med in-situ in silica sol-gel<br />
derived films on the rate of Förster resonance energy transfer<br />
Gary McDowell 1 , Marion Toury 2 , David McLoskey 2 , Graham Hunger<strong>for</strong>d 2 & A. Sheila Holmes-<br />
Smith 1,*<br />
1<br />
Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens<br />
Road, Glasgow G4 0BA, UK<br />
2<br />
HORIBA Jobin Yvon IBH Ltd, 45 Finnieston Street, Glasgow G3 8JU, UK<br />
There is growing interest in the detection of target analytes in biological systems which can be<br />
achieved by the development of suitable biosensors and lab-on-a-chip devices [1] . Media produced<br />
using the sol-gel method are suitable host materials <strong>for</strong> such biological based applications [2] . These<br />
robust glass-like materials are highly porous and exhibit large internal surface areas and previously<br />
we have reported upon the incorporation of enzymes entrapped within sol-gel derived hosts. This<br />
made use of fluorescent dyes to characterise the sol-gel manufacture and help elucidate protein<br />
con<strong>for</strong>mation, along with guest-host interactions [3] . Recently there has been increased research in<br />
the use of metal surfaces in order to influence the fluorescence emission, which can be<br />
advantageous as the radiative decay rate can be enhanced. We have previously put this to use to<br />
show the influence of silver nanostructures <strong>for</strong>med in-situ using a compact time-resolved<br />
fluorescence microscope system on the fluorescence of a tagged protein adsorbed onto a sol-gel<br />
derived film [4] .<br />
In this work we will demonstrate a model biosensor system employing enhanced Förster<br />
resonance energy transfer (FRET) in the presence of locally created silver colloids. The system is<br />
based on a sol-gel film containing silver salts, from which we can produce localised silver colloids<br />
in-situ via light irradiation. The presence of these silver nanostructures was ascertained via UV-vis<br />
and atomic <strong>for</strong>ce microscopy (AFM). Silane molecules with an NH2 group were attached to the<br />
surface of the sol-gel film. A fixed distance between a fluorescent donor molecule (Rhodamine<br />
6G), which was located within the sol-gel matrix, and an acceptor was there<strong>for</strong>e achieved by<br />
covalently binding the acceptor molecule to the NH2 group of the silane spacer. The effect of the<br />
localised silver colloids on FRET was monitored. Our model system employs Texas Red as the<br />
acceptor molecule, however in future applications this could be a biomolecule such as a protein or<br />
DNA, thus demonstrating the potential application of this methodology to developing a molecular<br />
recognition system.<br />
References: [1] W. Zhong, Anal. Bioanal. Chem., 394, (2009), 47. [2] J. Livage, et al., J. Phys.:Condens.<br />
Matter., 13 (2001) R673. [3] G. Hunger<strong>for</strong>d, et al., Progr. React. Kinetics. Mech., 34 (2009) 289. [4] G.<br />
Hunger<strong>for</strong>d, et al., Phys. Chem. Chem. Phys., 12 (2010) 4696.<br />
______________<br />
* Corresponding author: e-mail: a.s.smith@gcu.ac.uk<br />
198 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P88 Polymers, Materials & Nanomaterials, Quantum Dots Poster 88<br />
In-situ <strong>for</strong>mation of silver nanostructures within a polysaccharide film<br />
and its application as a potential biocompatible fluorescence sensing<br />
medium<br />
Nicole Donaldson 1 , Marion Toury 2 , David McLoskey 2 , A. Sheila Holmes-Smith 1<br />
& Graham Hunger<strong>for</strong>d 2,*<br />
1<br />
Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens<br />
Road, Glasgow G4 0BA, UK<br />
2<br />
HORIBA Jobin Yvon IBH Ltd, 45 Finnieston Street, Glasgow G3 8JU, UK<br />
There has been increased interest of the use of metal surfaces in conjunction with fluorescence<br />
molecules employing a plasmon effect, sometimes referred to as metal enhanced fluorescence [1] .<br />
This can be advantageous as some photophysical properties, such as the emission intensity of the<br />
fluorophore, can be enhanced. We have previously shown that it is possible to <strong>for</strong>m, in-situ, silver<br />
nanostructures in silica sol-gel derived media making use of a semiconductor diode laser on a<br />
compact time-resolved fluorescence microscope, which showed the effect of these particles on the<br />
fluorescence of FITC tagged BSA [2] . The use of light irradiation to <strong>for</strong>m the silver particles allows<br />
better particle localisation, which can be advantageous <strong>for</strong> sensing applications.<br />
In this work we investigate simple to manufacture polysaccharide films containing a silver<br />
salt, from which silver nanostructures can be produced via light irradiation. This is illustrated in a<br />
schematic manner from a microscope measurement in the figure below.<br />
20 μm<br />
Effect of irradiation time on a silver ion containing gellan gum film made using a compact<br />
time-resolved fluorescence microscope, illustrating the area affected <strong>for</strong> seven different laser<br />
irradiation times. These ranged from 15 seconds (top left) to 5 minutes (bottom right)<br />
The films were produced using gellan gum, which consists of a linear tetrasaccharide repeating<br />
unit [→4)-L-rhamnopyranosyl-(α-1→3)-D-glucopyranosyl-(β-1→4)-D-glucuronopyranosyl-(β-1→4)-<br />
D-glucopyranosyl-(β-1→]). This versatile polysaccharide has many applications, ranging from the<br />
food industry to drug delivery and tissue engineering applications. We have previously reported the<br />
use of fluorescence lifetime probes to monitor viscosity changes in gellan gum [3] and also make<br />
use of this in film characterisation. The presence of silver structures produced in-situ was<br />
confirmed using UV-vis, AFM and SEM techniques. The influence of the silver nanostructure<br />
containing film on the fluorescence of a protein label was monitored using fluorescence<br />
microscopy, thus helping to elucidate the potential of these films as biocompatible sensing media.<br />
References: [1] J.R. Lakowicz, Plasmonics, 1 (2006) 5. [2] G. Hunger<strong>for</strong>d, et al., Phys. Chem. Chem. Phys.,<br />
12 (2010) 14720. [3] G. Hunger<strong>for</strong>d, et al., J. Phys. Chem. B., 113 (2009) 12067.<br />
______________<br />
* Corresponding author: e-mail: graham.hunger<strong>for</strong>d@horiba.com<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 199
P89 Polymers, Materials & Nanomaterials, Quantum Dots Poster 89<br />
ESR study of Mn 2+ and Ce 3+ ions in single crystals of calcium<br />
thiogallate phosphors<br />
Ittetsu Kitajima 1 , Takeo Takizawa 1* , Chiharu Hidaka 1 & Shigetaka Nomura 2<br />
1 Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo (Japan).<br />
2 Tokyo University of Science, 1-14-6 Kudankita, Chiyoda-ku, Tokyo (Japan)<br />
1. Introduction<br />
Transition metals and rare earth elements (REE) doped to a calcium thiogallate show various<br />
luminescence under UV-light excitation. Especially, blue and green light emissions are obtained by<br />
doping Ce 3+ and Eu 2+ , respectively. The Mn 2+ ion is a good candidate <strong>for</strong> the remaining red light<br />
emission in the primary three colors. The weak red emission due to the <strong>for</strong>bidden d-d transition of Mn 2+<br />
is significantly affected by the local symmetry and the species of surrounding ligand ions. Actually it is<br />
enhanced about 20 times by co-doping REE [1], but the enhancing mechanism has not been clarified<br />
yet. The precise study of the local environment around dopant ions is now necessary. The calcium<br />
thiogallate has a very large and complicated unit cell, belonging to the space group Fddd (D2h 24 ). The<br />
unit cell contains three independent decahedron Ca 2+ sites and two tetrahedron Ga 3+ sites [2]. In order<br />
to investigate the substitution sites of Mn 2+ ions, we have carried out ESR measurements of CaGa2S4<br />
doped with Mn 2+ and Eu 2+ ions at room temperature. This result indicates that Mn 2+ and Eu 2+ ions<br />
replace the three Ca 2+ sites. However, some doubts <strong>for</strong> the possibility <strong>for</strong> Mn 2+ ions to replace the Ga 3+<br />
sites are still remaining, especially <strong>for</strong> Ce 3+ co-doped samples [3]. For the purpose of solving this<br />
problem, we report here the results of detailed investigation on the substitution sites of Mn 2+ and Ce 3+<br />
ions through ESR at various temperatures.<br />
2. Results<br />
ESR measurements have been carried out on CaGa2S4 single crystals doped with Mn 2+ or Ce 3+ using<br />
JEOL JES-FA300 ESR spectrometer. Temperature range is from liq. He to room temperature. Figure 1<br />
shows these spectra below ca. 20K. In the Mn 2+ doped samples, the whole spectra are similar to those<br />
at room temperature, but the intensity increases drastically and resonance field slightly shifts on<br />
lowering temperature. Temperature dependence of the ESR intensity has been confirmed different <strong>for</strong><br />
three Ca 2+ sites substituted by Mn 2+ ions [3]. Two of the Ca 2+ sites named as C1 and C2 obey Curie’s<br />
law, but the other sites named as B shows anti-ferromagnetic like behavior with increasing<br />
temperature. This result suggests that Mn 2+ ions show different magnetic properties according to their<br />
substitution sites. On the other hand, in the case of Ce 3+ doping, several resonances have been<br />
observed at 4.2K and they disappear above 40K. Their angular variation gives us in<strong>for</strong>mation that there<br />
would be at least five kinds of substitution sites of Ce 3+ . More detailed analyses are in progress.<br />
This work is partly supported by grant <strong>for</strong> private universities <strong>for</strong> 2009-2013 from ministry of education,<br />
sport, science, culture and technology, Japan.<br />
ESR intensity [arb.unit]<br />
2500 3000 3500<br />
Magnetic field [G]<br />
Figure 1. ESR spectra of Mn<br />
References<br />
[1] F. Boitier, et al., J. Lumin. 129 (2009) 554. [2] B. Eisenmenn, et al., Rev. Chimie Minérale, 20 (1983) 225.<br />
[3] T. Takizawa, et al., Jpn. J. Appl. Phys. 50 (2011) 05FG01.<br />
______________<br />
*<br />
Corresponding author: e-mail: takiz@phys.chs.nihon-u.ac.jp<br />
2+ and Ce 3+ ions in CaGa2S4 single crystals at low temperatures.<br />
200 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
2000<br />
CaGa 2 S 4 :Mn 21.8-22.0K<br />
CaGa 2 S 4 :Mn 4.2K<br />
CaGa 2 S 4 :Ce 4.2K<br />
4000<br />
4500
P90 Polymers, Materials & Nanomaterials, Quantum Dots Poster 90<br />
Metal-enhanced fluorescence studied with SIL – based microscopy<br />
Bartosz Krajnik 1* , Dawid Piatkowski 1 , Nikodem Czechowski 1 , Eckhard Hofmann 2 , Wolfgang<br />
Heiss 3 & Sebastian Mackowski 1<br />
1 Institute of Physics, Nicolaus Copernicus University, Torun, Poland<br />
2 Department of Biology and Biotechnology, Ruhr-University Bochum, Bochum, Germany<br />
3 Institute of Semiconductor and Solid <strong>State</strong> Physics, Johannes Kepler University, Linz, Austria<br />
The optical properties of light-harvesting complexes can be modified by plasmonic interactions.<br />
Precise tuning of the spectral properties and geometry of a hybrid structure composed of lightharvesting<br />
complexes and metallic nanoparticles should provide a way to improve absorption<br />
efficiency of the sunlight energy. Recent reports have shown that fluorescence intensity of<br />
peridinin-chlorophyll-protein (PCP) light-harvesting complex can be significantly increased by<br />
coupling to silver island film [1]. On the other hand, <strong>for</strong> photosystem I conjugated to metallic<br />
nanoparticles an opposite effect appeared [2].<br />
In this work we investigate the optical properties of PCP complex coupled to a monolayer of<br />
gold spherical nanoparticles in order to describe the distance dependence of metal-enhanced<br />
fluorescence <strong>for</strong> a multichromophoric system. Separation between the PCP complexes and Au<br />
nanoparticles was achieved using SiO2 spacer layer deposited with e-beam technique the<br />
thickness of which was changed from 4 nm to 40 nm. We used homochlorophyllous PCP<br />
reconstituted with two chlorophyll a molecules and heterochlorophyllous PCP reconstituted with<br />
one chlorophyll a and one chlorophyll b. In order to achieve high homogeneity of the sample, PCP<br />
molecules were spincoated on the spacer surface.<br />
Fig. 1. Histogram of (a) fluorescence intensity and (b) fluorescence lifetime of PCP reconstituted with two<br />
chlorophyll a molecules measured <strong>for</strong> substrates with different spacer thickness.<br />
In Fig. 1. we show the distribution of fluorescence lifetimes and fluorescence intensities <strong>for</strong><br />
homochlorophyllous PCP placed on substrates with spacer thickness of 4 nm, 12 nm, and 40 nm.<br />
The excitation wavelength was 485 nm, corresponding to the plasmon resonance of the gold<br />
nanoparticles. For the spacer of 12 nm, a 4-fold fluorescence enhancement is observed.<br />
Complementary time-resolved experiment shows a 20% fluorescence lifetime shortening. We<br />
conclude that observed increase of the emission intensity is predominantly due to absorption<br />
enhancement. Further work will focus on single molecule experiments, which should both<br />
contribute new in<strong>for</strong>mation on the mechanism of metal-enhanced fluorescence as well as help us<br />
optimize interactions between biomolecules and metal nanoparticles.<br />
Financial support from the WELCOME program "Hybrid nanostructures as a stepping-stone<br />
towards efficient artificial photosynthesis" awarded by the Foundation <strong>for</strong> Polish Science is<br />
acknowledged.<br />
References:<br />
[1] S. Mackowski et al., Nano Letters, 8 (2008) 558. [2] Carmeli et al., Nano Letters, 10 (2010) 2069.<br />
______________<br />
* Corresponding author: e-mail: bakrag@phys.uni.torun.pl<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 201
P91 Polymers, Materials & Nanomaterials, Quantum Dots Poster 91<br />
Nature of SYBRGreen fluorescence quenching on DNA matrix under<br />
Au nanoparticles action<br />
Ekaterina S. Lisitsyna 1,* , Sergey G. Skuridin 2 , Yuriy M. Evdokimov 2 , Victor M.Rudoy 3 , Olga V.<br />
Dementieva 3 & Vladimir A. Kuzmin 1<br />
1<br />
Laboratory of Photosensibilization Processes, Emanuel Institute of Biochemical Physics RAS,<br />
Kosigyna st., 4, 119334 Moscow (Russia)<br />
2<br />
Laboratory of Condensed <strong>State</strong> of Nucleic Acids, Engelhardt Institute of Molecular Biology RAS,<br />
Vavilova st., 32, 119991 Moscow (Russia)<br />
3<br />
Laboratory of surface phenomena in polymeric systems, Frumkin Institute of Physical Chemistry<br />
and Electrochemistry RAS, Leninsky pr., 31, 119991 Moscow (Russia)<br />
Nowadays, due to the development of many biological and medical applications of metal<br />
nanoparticles (Au NPs), their toxicity and their affect on cell structures, in particular DNA, attract<br />
much attention [1]. Energy transfer phenomenon is widely used as nanosize ruler in the biological<br />
sciences due to its ability to reveal changes in molecular separation and con<strong>for</strong>mation. Thus, this<br />
study aims to investigate Au nanoparticles influence on cell components, in particular DNA, using<br />
energy transfer as method of visualization. The properties of molecular organized DNA structures<br />
are very interesting at present because these DNA structures represent a model of DNA structural<br />
ordering in some biological objects [2]. Consequently, it is necessary to investigate different effects<br />
on this DNA ordering system. The study of previously mentioned molecular organized DNA<br />
structure has been carried out by a fluorescent method using a well known dye SYBRGreen (λem =<br />
525 nm) as a fluorescent label <strong>for</strong> the biomolecular identification. The method is founded on the<br />
fact that the fluorescence of the dye, which is negligible in the solution, is dramatically increased<br />
when it is bound to the nucleic acids [3].Spectral kinetic behavior of the complex of dye<br />
SYBRGreen with both double strand DNA and molecular organized DNA structure (liquid crystal) in<br />
the presence of gold nanoparticles (d = 2 nm) have been probed fluorimetrically. An addition of Au<br />
nanoparticles with a diameter of about 2 nm to the complex of SYBRGreen and the molecular<br />
organized DNA structure results in the quenching of fluorescence intensity of SYBRGreen<br />
intercalated between the pairs of bases in DNA molecules. The dramatic fluorescence decay of<br />
SYBRGreen intercalated between the base pairs of DNA is attributed to three mechanisms: (i)<br />
energy transfer from excited molecules of SYBRGreen dye to nanosize Au clusters; (ii) selfquenching<br />
of the SYBRGreen fluorescence by FRET mechanism, mediated by the rearrangement<br />
process of DNA layers in the molecular organized DNA structure, and (iii) intersystem crossing<br />
acceleration under the influence of the external heavy atom (Au cluster) effect.<br />
References: [1] S.G. Skuridin, et al., Doklady Biochemistry and Biophysics, 432, (2010), 6, 838. [2] Yu.M.<br />
Evdokimov, et al., Moscow: Radiotekhnika, (2008). [3] H. Zipper, et al., Nucl. Acids Res., 32, 2004, 12, e103<br />
(doi: 10.1093/nar/gnh101).<br />
______________<br />
* Corresponding author: e-mail: fox333@inbox.ru ; lisitsyna@sky.chph.ras.ru<br />
202 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P92 Polymers, Materials & Nanomaterials, Quantum Dots Poster 92<br />
Photophysical properties of a clinical photosensitiser in pegylated<br />
silica nanoparticles<br />
Alexandra Mackenzie 1 , Josephine Woodhams 1 , Iria Echevarria 2 , Fabrizio Mancin 2<br />
& Alexander MacRobert 1,*<br />
1 National Medical Laser Centre, Charles Bell House, University College London, London, UK<br />
2 Department of Chemical Science, Universita degli Studi di Padova, 35131 Padova, Padova, Italy<br />
mTHPC (meta-tetrahydroxyphenylchlorin) is a potent photosensitising drug currently in clinical use<br />
<strong>for</strong> photodynamic therapy (PDT) of head and neck cancer. Since mTHPC is hydrophobic it is<br />
difficult to administer in the photoactive monomeric <strong>for</strong>m using conventional water soluble<br />
<strong>for</strong>mulations. Nanocarriers hold potential to increase PDT efficacy while reducing side effects such<br />
as skin sensitivity [1], and in this work PEGylated Organically Modified Silica nanoparticles (NPs)<br />
were investigated as a carrier <strong>for</strong> mTHPC. The mTHPC moiety was covalently linked to the silica<br />
matrix using silane functionalisation of the hydroxy groups in two ways: either via one hydroxy<br />
group or all four hydroxy groups, as mono and tetra silane-mTHPC derivatives, i.e. s-mTHPC and<br />
4s-mTHPC, where s refers to the silane linker. The mean NPs diameter was 20nm with ~1% w/w<br />
mTHPC loadings.<br />
We investigated alterations of mTHPC photophysical properties in the silica NPs suspended<br />
in a range of solvents to determine potential therapeutic efficacy of the NPs. In methanol, a small<br />
red shift (1.5 nm) in the Soret-band peak was observed <strong>for</strong> both NPs with respect to pure mTHPC.<br />
In water, the 4s-mTHPC exhibits a larger shift than s-mTHPC (9 and 5.5 nm, compared to mTHPC<br />
in methanol). Fluorescence lifetimes were recorded using TCSPC, and singlet oxygen yields with<br />
time-resolved NIR photon counting at 1270 nm. In methanol, the fluorescence lifetime was<br />
shortened in both NPs, but the effect was greater in the 4s-mTHPC (mTHPC 8.3 ns, 4s-mTHPC<br />
7.4 ns, s-mTHPC 8.1 ns). In water the average lifetime of 4s-mTHPC was further reduced (4smTHPC<br />
2.3 ns, 1s-mTHPC 8.2 ns).<br />
In deuterated methanol the singlet oxygen quantum yield of both NPs was similar to pure<br />
mTHPC: both NPs achieved 81 % yield <strong>for</strong> mTHPC. However, in deuterated water the 4s-mTHPC<br />
exhibited a quantum yield 5 times lower than the s-mTHPC. In both solvents, singlet oxygen<br />
lifetimes were of the expected magnitude from the literature. However, in the presence of 10 %<br />
foetal calf serum (FCS) the singlet oxygen lifetime was reduced from 23 to 9 µs <strong>for</strong> both NPs in<br />
deuterated solution.<br />
In methanol, the photophysical properties of both NPs are similar to pure mTHPC. However,<br />
in water, there are significant differences in the photophysical properties of the mono and tetracoordinated<br />
photosensitisers: the tetra-coordinated 4s-mTHPC has a shorter fluorescence lifetime<br />
and much lower singlet oxygen and fluorescence yields, which indicates quenching of the excited<br />
singlet state occurs when the mesoporous silica NP is suspended in water. Since the 4s-mTHPC<br />
moiety is rigidly attached to the matrix via four linkers, it may be more susceptible to hydrogen<br />
bonding interactions of water with the silica matrix, which could distort the planarity of the porphyrin<br />
ring [2]. In contrast, the s-mTHPC has only one covalent linker to the matrix and so can then rotate<br />
to maintain planarity, thereby maintaining its higher singlet oxygen yield. The reduction of singlet<br />
oxygen lifetime in the presence of FCS is attributed to quenching via interactions of singlet oxygen<br />
with the serum proteins. Experimental tumour model studies are in progress with these<br />
nanoparticles so we may then be able to correlate the PDT efficacy with the photophysical<br />
measurements.<br />
References: [1] E. Paszko, et al., Photodiagnosis Photodyn. Ther., 8 (2011) 14. [2] N.C. Maiti, et. al.,<br />
Journal of the Chemical Society, Faraday Transactions., 92 (1996) 1095.<br />
______________<br />
* Corresponding author: e-mail: rmap007@live.ucl.ac.uk<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 203
P93 Polymers, Materials & Nanomaterials, Quantum Dots Poster 93<br />
Photoinduced size-controlled generation of silver nanoparticles coated<br />
with carboxylate-derivatized thioxanthones<br />
Jean-Pierre Malval* § , Ming Jin # , Lavinia Balan § , Raphaël Schneider † , Davy-Louis Versace ∀ ,<br />
Hélène Chaumeil ‡ , Albert Defoin ‡ & Olivier Soppera §<br />
§ Institut de Sciences des Matériaux de Mulhouse, LRC CNRS 7228. Mulhouse, France.<br />
# Institute of Functional Polymer Materials, School of Materials Science and Technology, Tongji<br />
Shanghai, China.<br />
† Département de Chimie Physique des Réactions, UMR CNRS 7630. Nancy, France<br />
∀ Département de Photochimie Générale.Mulhouse, France<br />
‡ Laboratoire de Chimie Organique, Bioorganique et Macromoléculaire. Mulhouse, France<br />
The development of synthetic routes devoted to the size- and shape-control of silver nanoparticles<br />
continues to be the subject of an intense research focus. Such morphological requirements<br />
constitute the key issues <strong>for</strong> optical, electronic, magnetic and catalytic properties which are clearly<br />
size-dependent 1 Chemical methods as opposed as photochemical ones have been extensively<br />
described. The photochemical method should constitute an original route <strong>for</strong> a fast light-triggered<br />
generation of nanoparticles. For instance, the photoreduction of silver cations by citrate can be<br />
catalyzed by silver seeds and lead to the generation of disk-shaped silver nanoparticles 2 . In this<br />
plasmon-mediated synthesis, the irradiation wavelength constitutes a key parameter which<br />
determines the final shape of the particles and<br />
offers a flexible tool to generate anisotropic<br />
nanostructures such as triangles, prisms, cubes<br />
or bipyramids(Zhang J et al., 2009). An other<br />
photoinduced method which does not required<br />
the preliminary addition of any metal<br />
nanaparticles seeds corresponds to the direct<br />
reduction of silver cation by an excited<br />
chromophore or by an intermediate reactant<br />
which is ‘in situ’ generated during excitation 3 .<br />
The homogeneous growth of nanoparticles is<br />
then regulated by the presence of stabilizers<br />
similar as those used <strong>for</strong> chemical methods.<br />
However such a photoinduced approach<br />
generally suffers from a deficiency in the<br />
nanoparticles size-control since the stabilizers<br />
372 nm<br />
concentration can not be modulated as conveniently as <strong>for</strong> chemical methods due to an inhibiting<br />
role of the stabilizer towards the photoreduction of silver precursors.<br />
We report on a new strategy <strong>for</strong> a rapid photoinduced synthesis of monodisperse ligandcoated<br />
silver nanoparticles 4 . Such nanoparticles are produced through a very fast reduction of Ag +<br />
by α-aminoalkyl radicals which are first generated from hydrogen abstraction towards an aliphatic<br />
amine by the excited triplet state of 2-substituted thioxanthone series (TX-O-CH2-COO - and TX-S-<br />
CH2-COO - ). The quantum yield of this prior reaction is tuned by substituent effect on thioxanthones<br />
and leads to a kinetic control of the conversion of Ag + to Ag(0). Combined with the capping role of<br />
a carboxylate function linked to the chromophores, a size-regulation of the growing nanoparticles is<br />
both promoted and optimized due to a concomitant kinetics adjustment between the<br />
photoreduction process and the subsequent functionalization of the nanoparticles. We demonstrate<br />
that the optimal adjustment is then obtained with TX-S-CH2-COO - .<br />
References: (2) Narayanan, R.; El-Sayed, M. A. J. Phys. Chem. B 2005, 109, 12663-12676 (2) Jin, R, et al<br />
Science 2001, 294, 1901-1903. (3) Balan, L.; Jin, M.; Malval, J.-P.; Chaumeil, H. Defoin, A.; Vidal, L,<br />
Macromolecules 2008, 41, 9359-9365. (4) Malval, J.-P. ; et al. J. Phys. Chem. C, 2010, 114, 10396-10402<br />
______________<br />
* Corresponding author: e-mail: jean-pierre.malval@uha.fr<br />
DH<br />
204 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
O<br />
S<br />
D●<br />
eT<br />
Radicals<br />
Ag + Ag +<br />
Ag 0<br />
O<br />
X O<br />
Reactivity<br />
Modulator<br />
Capping<br />
function
P94 Polymers, Materials & Nanomaterials, Quantum Dots Poster 94<br />
Enhancement of acid photogeneration through a para-to-meta<br />
substitution strategy in a sulfonium-based alkoxystilbene designed <strong>for</strong><br />
two-photon polymerization<br />
Rongjie Xia # , Jean-Pierre Malval § * , Ming Jin # * , Arnaud Spangenberg § , Decheng Wan # ,<br />
Hongting Pu # , Fabrice Morlet-Savary ¥ , Hélène Chaumeil ‡ , Patrice Baldeck ∂ , Olivier Poizat ¢<br />
& Olivier Soppera §<br />
# Institute of Functional Polymer Materials, Tongji University, Shanghai, China<br />
§ Institut de Sciences des Matériaux de Mulhouse, CNRS 7228, Mulhouse, France<br />
¥ Département de Photochimie Générale, Mulhouse, France<br />
‡ Laboratoire de Chimie Organique et Bioorganique, Mulhouse, France<br />
∂ Laboratoire de Spectrométrie Physique, CNRS 5588, Grenoble, France<br />
¢ Laboratoire de Spectrochimie Infrarouge et Raman, CNRS 8516, Lille, France<br />
Two-photon-initiated polymerization (TPIP) is gaining increasing interest as an advanced method<br />
<strong>for</strong> the production of intricate three-dimensional microstructures that cannot be obtained with<br />
conventional lithography techniques(Cumpston BH et al., 1999; Kawata S et al., 2001). The main<br />
advantage of TPIP relies on an excellent spatial control due to the confinement of the<br />
photoinduced reaction to the focal volume where both excitation and two-photon absorption (2PA)<br />
process occur. In addition, photoacid generators (PAGs) constitute an important class of cationic<br />
photoinitiators(Shirai M,Okamura H, 2009) which occupy a strategic position in the field of the<br />
photoresists. The applications of PAGs have been extensively developed in various research<br />
domains such as micro-electronics, photosensitized resins, 3D microfabrication and high density<br />
optical data storage. In this context, the molecular integration of PAGs into a two-photon activable<br />
structure clearly opens a relevant strategy to extend the use of these cationic photoinitiator <strong>for</strong><br />
TPIP(Zhou W et al., 2002).<br />
This contribution reports on the<br />
photochemical behavior of two new<br />
sulfonium-based photoacid generators<br />
(PAGs). We demonstrate that a parato-meta<br />
substitution of a methyl (pcyanobenzyl)<br />
sulfonium group in a 4alkoxystilbene<br />
core strongly influences<br />
the photodissociation efficiency of the<br />
PAGs and leads to an increase of the<br />
quantum yield <strong>for</strong> acid generation by a<br />
factor 2.4. This substantial effect<br />
which was also corroborated by a<br />
reactivity enhancement in cationic<br />
σ∗ π∗ σ∗ π∗ δ 2PA<br />
π<br />
para<br />
RO<br />
photopolymerization is assigned to the modulation of the electronic interaction between two low<br />
lying excited states whose energy gap is monitored by this substitution effect. Moreover it was<br />
found that the position of the sulfonium moiety hardly affects the two-photon absorption properties<br />
of these push-pull chromophores. By two-photon fabrication of microstructures, we finally<br />
demonstrate the potential use of the PAGs as efficient two-photon initiators.<br />
We there<strong>for</strong>e extended the potential applications of these high-sensitive initiators <strong>for</strong> two-photon<br />
patterning of acid-activated materials.<br />
References: (1) Cumpston, B. H., et al Nature 1999, 398, 51-54.(2) Kawata, S., et al Nature 2001, 412,<br />
697.(3) Shirai, M., et al Prog. Org. Coat. 2009, 64, 175-181.(4) Zhou, W., et al Science 2002, 296, 1106-<br />
1109.<br />
______________<br />
* Corresponding authors: jean-pierre.malval@uha.fr or mingjin@tongji.edu.cn<br />
Φ +<br />
H<br />
meta<br />
m<br />
p<br />
S<br />
+<br />
10 µm<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 205
P95 Polymers, Materials & Nanomaterials, Quantum Dots Poster 95<br />
Synthesis and fluorescence imaging of solid-state amorphous films or<br />
particles from new BODIPY derivatives<br />
Thanh Truc Vu 1 , Elena Senotrusova 1,2 , Robert B. Pansu 1 , Pierre Audebert 1 , Elena Yu<br />
Schmidt 2 , Boris A.Trofimov 2 , Gilles Clavier 1 & Rachel Méallet-Renault 1<br />
1 PPSM, ENS Cachan, CNRS, UniverSud, 61 av. President Wilson, F-94230 CACHAN, France<br />
2 A. E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of<br />
Sciences, 1 Favorsky Str. Irkutsk 664033, Russian Federation<br />
Our aim is to develop fluorescent organic nanocrystals (in sol-gel or suspensions) as sensors <strong>for</strong><br />
biological or environmental purposes. Nanocrystals have intermediate properties between<br />
molecules and microcrystals. They are extremely bright objects whose fluorescence can be<br />
modulated through energy transfer [1] [2] . In the past years we have developed a redox based<br />
oxygen titration with rubrene nanocrystals [1] and a sensitive copper (II) ion sensor based upon latex<br />
particles [2] . In order to improve the response of our systems, we need to get a better sensitivity<br />
designing highly fluorescent molecules in the solid state. BODIPY chromophores have a high<br />
absorption cross section [3] . Those dyes are hindered in order to avoid π-π stacking which is a<br />
known major cause of fluorescence inhibition in the solid state.<br />
The results of fluorescence lifetime (FLIM) imaging will demonstrate the ability of such molecules<br />
to emit efficiently in solid-state [3],[4] . We will first focuss on 4 new hindered BODIPY dyes (see<br />
schemes above). The chromophores were designed to shift their absorption and emission<br />
wavelengths to the red. [2.2]paracyclophane (PcP) was grafted on the BODIPY core as well as<br />
alkyl chains of various length to <strong>for</strong>ce the molecules away one from each other. The spectroscopic<br />
and electrochemical properties in solution will be presented.<br />
R 2<br />
R 1<br />
BODIPY<br />
N N<br />
B<br />
F F<br />
R 1<br />
R 2<br />
R 1 = [2.2]paracyclophanyl, Adamantyl<br />
R 2 = H, Methyl, Ethyl, n-Propyl<br />
Area (x10 3<br />
)<br />
40<br />
30<br />
20<br />
10<br />
0<br />
0.00<br />
Spectroscopy of PcP BODIPY<br />
in amorphous solid state<br />
0.15 0.20<br />
A 495<br />
206 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
PrPcP<br />
0.05<br />
0.10<br />
EtPcP<br />
MePcP<br />
0.25<br />
HPcP<br />
0.30<br />
0.35<br />
Fluorescence (a.u.)<br />
10 4<br />
10 3<br />
10 2<br />
10 1<br />
10 15<br />
Delay(ns)<br />
Absorption, emission and FLIM were done on drop-casted films of the fluorophores. The addition of<br />
alkyl substituents (-Me, -Et, n-Pr) next to PcP group clearly affects the emission properties in solid<br />
state : the dyes display sharper emission bands than in solution and their relative fluorescence<br />
quantum yield increases with the number of carbons in the alkyl chain. FLIM analysis reveals<br />
complex decay profiles.<br />
We will also introduce recent progresses in the <strong>for</strong>mulation of nanoparticles from another<br />
Bodipy derivative. Indeed using a microfluidic device we managed to produce size-controlled<br />
nanoparticles with luminescent properties. Those properties will be discussed after detailed<br />
spectroscopic analysis of PMMA films doped with the Bodipy molecule.<br />
References: [1] F. Treussard, et al., ChemPhysChem 4 (2003) 757. [2] R. Méallet-Renault, et al.,<br />
Chem.Commun. (2004) 2344. [3] A.B. Zaitsev, et al., Tetrahedron 61 (2005) 2683 - B.A. Trofimov, et al.,<br />
Tetrahedron Lett. 49 (2008) 4362. [4] E.Y. Schmidt, et al., Chem. Eur. J. 15 (2009) 5823 - T. T. Vu, et al., J.<br />
Phys. Chem. C 113 (2009) 11844.<br />
______________<br />
* Corresponding author: e-mail : rachel.meallet@ens-cachan.fr<br />
0<br />
PrPcP<br />
EtPcP<br />
HPcP<br />
MePcP<br />
EtPcP<br />
5<br />
20<br />
25<br />
30
P96 Polymers, Materials & Nanomaterials, Quantum Dots Poster 96<br />
Photolumiscent properties of CoMoO4 nanorods quickly synthesized<br />
and annealed in a domestic microwave owen<br />
Ana Paula de Moura 1 , Larissa Helena de Oliveira 2 , Ieda Lúcia Viana Rosa 2 , Máximo Siu Li 3 ,<br />
Elson Longo 1 & José Arana Varela 1<br />
1 UNESP, Instituto de Química, CEP 14800-900, Araraquara, SP, Brazil<br />
2 UFSCar, Departamento de Química, CEP 13565-905, São Carlos, SP, Brazil<br />
3 USP, Instituto de Física de São Carlos, CEP 13560-970, São Carlos, SP, Brazil<br />
Materials belonging to the molybdate family have a long history of practical applications due to<br />
their excellent optical properties in phosphors, laser materials, and scintillation detectors[1]. Cobalt<br />
molybdate (CoMoO4) is one of the most important components of industrial catalysts <strong>for</strong> the partial<br />
oxidation of hydrocarbons and precursors in the synthesis of hydrodesulphurization catalysts[2].<br />
Moreover, CoMoO4 can be also applied in electroindustry and bioscience due to their structural,<br />
magnetic, electronic, and antibacterial properties. In this paper, CoMoO4. xH2O precursors were<br />
prepared by microwave assisted hydrothermal method and then heat treated using microwave<br />
radiation to <strong>for</strong>m the desired schelite-type crystalline α and β-CoMoO4 phases. X-ray diffraction<br />
(XRD) patterns and Raman spectroscopy indicated that these powders present a schelite-type α<br />
and β-CoMoO4 monoclinic structure with space group C2/m. The emission scanning electron<br />
microscopy (FE-SEM) images showed that α and β-CoMoO4 powders present morphology like<br />
nanorods. The nanorods average thicknesses were evaluated as 100–300 nm and the nanorods<br />
lengths were determined as around 1–3 μm. The optical band gap energy (Eg) of the α and β-<br />
CoMoO4 nanorods are around 2.0, in accordance to ref [3]. Photoluminescence spectrum obtained<br />
at room temperature <strong>for</strong> α and β-CoMoO4 particles present maximum component around the blue<br />
light emission. In conclusion, these results show us that the domestic microwave oven (MO) has<br />
been successfully employed to obtain these α and β-CoMoO4 nanoparticles.<br />
Figure 1: XRD patterns of the all powders prepared by thermal decomposition of the CoMoO4. xH2O<br />
precursors at 600 °C <strong>for</strong> 10 minutes. Insert: FEG-SEM micrograph image.<br />
This work was supported by CNPq, CAPES and FAPESP.<br />
References: [1] A. P. A. Marques, V. M. Longo, D. M.A. de Melo, P. S. Pizani, E. R. Leite, J. A. Varela, E.<br />
Longo, Journal of Solid <strong>State</strong> Chemistry 181 (2008) 1249. [2] JE Miller, NB Jackson, L Evans, AG Sault, MM<br />
Gonzales, Catal Lett (1999) 58:147. [3] V P.K. Pandey, N.S. Bhave, R.B.Kharat, Indian Journal of Pure e<br />
Applied Physics, 44 (2006) 52.<br />
______________<br />
* Corresponding author: e-mail: apdemoura@gmail.com<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 207
P97 Polymers, Materials & Nanomaterials, Quantum Dots Poster 97<br />
Study of the annealing temperature effect on the structural and<br />
luminescent properties of SrWO4: Eu 3+ phosphors prepared by a<br />
non-hydrolytic sol-gel process<br />
Ieda Lúcia Viana Rosa 1 *, Paula Fabiana Santos Pereira 1 , Ana Paula de Moura 1 , Içamira Costa<br />
Nogueira 1 , Márcia Valéria Lima 1 , Elson Longo 1 , Paulo César de Sousa Filho 2 , Osvaldo<br />
Antônio Serra 2 & Eduardo José Nassar 3,1<br />
1<br />
Universidade Federal de São Carlos/ UFSCar, Av. Washington Luís, Km 235, ZIP 13565-905,<br />
São Carlos-SP, Brasil<br />
2<br />
Universidade de São Paulo/ FFCLRP-USP, Av. Bandeirante, 3900, ZIP 14040-901, Ribeirão<br />
Preto-SP, Brasil<br />
3<br />
Universidade de Franca/ UNIFRAN, Av. Dr. Armando Salles de Oliveira, 201, ZIP 14404-600,<br />
Franca – SP, Brasil<br />
The optical properties of trivalent rare earth ions (RE 3+ ) in tungstate materials, with scheelite structure,<br />
have been widely investigated [1] . There has been significant interest in studying the interactions between<br />
rare earth ions in solids. This is attributed in part to the importance of rare earth doped materials in optical<br />
systems applications and also to the advances in experimental techniques which allow the investigation<br />
of the properties of the interactions in greater detail than previously possible [2] . Some Eu 3+ activated<br />
tungstates and molybdates with scheelite structure were shown to have efficient red-light emission in the<br />
near – UV irradiation [3,4] . The sol–gel method using metal alkoxide has been investigated to prepare<br />
strontium tungstate oxide (SrWO4). In this paper, SrWO4 powders doped with Eu 3+ were synthesized by<br />
the non-hydrolytic sol-gel route. The material was synthesized using a modified version of the method<br />
described elsewere. In this synthesis one gram of tungsten chloride (WCl6) and 200 mL of methanol<br />
(MeOH), used as the solvent and oxygen donor, was mixed and acetylacetone (acac) was added to<br />
prevent the precipitation. Then strontium chloride in a molar ratio of 1:1 as well as 1.0% of EuCl3 was<br />
added to the reactional mixture. This mixture remains in reflux <strong>for</strong> 4 h. In this paper we investigate the<br />
influence temperature in the synthesis of the Eu 3+ -doped strontium tungstate oxide (SrWO4). The<br />
obtained powders were analyzed by X-ray diffraction (XRD) and Raman spectroscopy. The images were<br />
analyzed by field-emission gun scanning electron microscopy (FEG-SEM). A qualitative analysis of the<br />
obtained powders was per<strong>for</strong>med by using EDS.<br />
The optical properties were investigated by<br />
Ultraviolet–visible (UV–vis) absorption spectroscopy<br />
and photoluminescence (PL) measurements at<br />
room and liquid N2 temperature. X-Ray diffraction,<br />
vibrational and optical studies showed that the<br />
structure of the synthesized Sr0,99Eu0,01WO4<br />
compounds scheelite type were <strong>for</strong>med in a unique<br />
phase at 900 and 1000 o C <strong>for</strong> 2 h. The Raman<br />
spectra indicated only one type of [WO4] 2-<br />
tetrahedron. The morphology indicated that the<br />
increase of annealing temperature contributed to<br />
the coalescence process, which promoted the<br />
growth of aggregated particles of polydisperse<br />
nature. According to the PL study the 5 D0→ 7 F2<br />
electric dipole transition is dominant when Eu 3+<br />
occupies a noncentrosymmetric environment, such as<br />
is normally observed in the scheelite framework.<br />
References: [1] C. A. Kodaira, et. al., J. Braz. Chem. Soc., 15 (2004) 890. [2] J. K. Tyminski, et. al., J.<br />
Chem. Phys., 77 (1982) 4318. [3] S. Shi, et. al. Opt. Mater., 30 (2008) 1616. [4] S. Shi, et. al. Spectrochimica<br />
Acta Part A, 69 (2008) 396.<br />
______________<br />
* Corresponding author: e-mail: paulaufscar@hotmail.com<br />
208 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
Figure 1: Emission spectra of the SrWO4:Eu 3+<br />
treated at (a) 600, (b) 800, (c) 900 and (d) 1000<br />
°C <strong>for</strong> 2 h, with excitation at 393 nm ( 5 L6) at 77 K.
P98 Polymers, Materials & Nanomaterials, Quantum Dots Poster 98<br />
Investigation of the photodynamics of fluorescent dendronized<br />
perylenediimide nanoparticles combining single particle and<br />
ultrafast spectroscopy<br />
Michel Sliwa 1* , Ryohei Yasukuni 2 , Bruno Debus 1 , Cyril Ruckebusch 1 , Tom Vosch 3 , Johan<br />
Hofkens 4 & Tsuyoshi Asahi 5<br />
1<br />
LASIR, UMR 8516 CNRS, Université Lille Nord de France, 59655 Villeneuve d’Ascq (France)<br />
2<br />
ITODYS, UMR 7086 CNRS, Université Paris 7, 15, rue Jean Antoine de Baïf Bâtiment<br />
LAVOISIER, 15 rue Jean Antoine de Baïf 75205 Paris (France)<br />
3<br />
Nano-Science Center / Department of Chemistry, University of Copenhagen, Universitetsparken<br />
5, 2100 Copenhagen (Denmark)<br />
4<br />
Department of Chemistry and Institute <strong>for</strong> Nanoscale Physics and Chemistry, Katholieke<br />
Universiteit Leuven, Celestijnenlaan 200F, 3001 HeVerlee (Belgium)<br />
5<br />
Graduate School of Science and Engineering, Ehime University, 3 Bunkyo, Matsuyama, Ehime<br />
790-8577 (Japan)<br />
The number of fluorescence related applications has been increasing with the development of new<br />
fluorescent materials. Fluorescent nanoparticles are a new and very promising class of fluorescent<br />
material with regard to their applications such as fluorescence bioimaging and single molecular<br />
spectroscopy. Even though the photostability of some organic dyes is not so good compared to<br />
inorganic Q dots, their versatility (the creation and design of tailored nanoparticles with different<br />
physical properties) is seen as an advantage <strong>for</strong> specific applications. Perylenediimide (PDI) dyes<br />
have attracted a great deal of attention since they possess exceptional chemical, thermal,<br />
photochemical and photophysical stability in combination with high extinction coefficient and<br />
fluorescence quantum yield. We have succeeded previously in fabrication of fluorescent<br />
nanoparticles (≈ 100 nm) using a first generation dendronized perylenediimide compound (G1PDI)<br />
by laser ablation methods[1]. Our preliminary studies showed that the bulky dendron prevents<br />
strong intermolecular interaction between each chromophore inside a nanoparticle although the<br />
excited singlet state migrates in a nanoparticle via energy hopping and is quenched at the surface,<br />
leading to the size-dependent fluorescence quantum yield and a smaller fluorescence quantum<br />
yield <strong>for</strong> small nanoparticles [2]. In this presentation we would like to discuss on detailed<br />
fluorescence properties (fluorescence decaytime, fluorescence intensity trajectories, emission<br />
spectra) and ultrafast energy and electron transfer (femtosecond transient absorption<br />
spectroscopy) of G1PDI inside nanoparticles compared with molecule in solution. Our result<br />
demonstrate that G1PDI molecules inside nanoparticles have a more homogeneous fixed<br />
con<strong>for</strong>mation and are less sensitive to the local environment like is the case <strong>for</strong> single molecules.<br />
Fluorescence intensity trajectories reveal almost no blinking and a higher photo-stability compare<br />
to single molecules. Stepwise change of fluorescent intensity, fluorescence decaytimes and<br />
femtosecond transient absorption spectra confirm the exciton migration and the existence of<br />
quenching in the nanoparticles with the <strong>for</strong>mation of PDI radical anion. Also singlet-singlet<br />
annihilation, resulting in single photon emission even when multiple chromophores are excited,<br />
was demonstrated by antibunching experiments on single nanoparticles.<br />
This work was supported by the Ministères des Affaires étrangères et européennes (MAEE) et de<br />
l’Enseignement supérieur et de la Recherche (MESR) through Programme Hubert Curien<br />
Tournesol (20372WL) and the CNRS Interdisciplinary Programs grant.<br />
References: [1]R. Yasukuni, et al., Appl. Phys. A. 93(2008) 5. [2]R. Yasukuni, et al. Jpn. J. Appl. Phys.<br />
48(2009) 065002<br />
______________<br />
* Corresponding author: e-mail: michel.sliwa@univ-lille1.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 209
P99 Polymers, Materials & Nanomaterials, Quantum Dots Poster 99<br />
Concentration dependence of the enhancing effect of REE on Mn 2+ red<br />
emission in CaGa2S4: Mn 2+ , REE<br />
Akihiro Suzuki 1 , Chiharu Hidaka 1 , Takeo Takizawa 1* & Shigetaka Nomura 2<br />
1 Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo, Japan<br />
2 Tokyo University of Science, 1-14-6 Kudankita, Chiyoda-ku, Tokyo, Japan<br />
Introduction<br />
The ternary CaGa2S4 has been studied as a candidate <strong>for</strong> host materials of phosphors. Especially,<br />
it is well known that the compound doped with Ce 3+ or Eu 2+ shows blue or green emission with high<br />
intensity, respectively [1, 2]. Only the red emission is lacking to achieve three primary colors using<br />
CaGa2S4 as a host material. For this purpose, Mn 2+ ions are tried to be used. They emit weak red<br />
emission which can be enhanced by co-doping with a suitable rare earth element (REE) [3, 4].<br />
Recently, it is found that the enhancing effect on the Mn red emission has already occurred at a<br />
low-concentration below 0.1 mol% REE. However, the measurement in this concentration range<br />
has not been carried out so far. In this study, to clarify the interaction between Mn 2+ and REE ions,<br />
photoluminescence (PL) spectra of CaGa2S4:Mn 2+ co-doped with REE of lower concentrations<br />
below 0.1 mol% are reported. Discussion will be given on the enhancing mechanism.<br />
Results and discussion<br />
The PL spectra of polycrystalline samples synthesized by the solid-state reaction were measured<br />
at room temperature. Figure 1 shows the intensity of the Mn red emission as a function of Ce<br />
concentration, while that of Mn 2+ was kept at 0.2, 0.5 and 1.0mol%. The Mn red emission<br />
increased with increasing the Ce 3+ concentration, and then being saturated. Moreover, the<br />
saturation point shifted toward the high concentration side as that of Mn 2+ increased. This result<br />
cannot be explained by the idea that the enhancing effect would depend on the distance between<br />
Mn 2+ and Ce 3+ ions, but rather suggesting that Ce 3+ ions would be selectively located close to Mn 2+<br />
ions as pairs or clusters. The detailed results together with those of other REE will be presented.<br />
Fig.1 Effect of Ce concentration on the intensity of the Mn 2+ red emission.<br />
References:<br />
[1] T.E.Peters, et al., J. Electrochem. Soc. 119 (1972) 230-236<br />
[2] C.Chartier, et al., J. Lumin, 111, (2005), 147-158<br />
[3] F.Boitier, et al., J. Lumin 129 (2009) 554-562<br />
[4] T.Obonai, et al., Phys. Status Solid A, 206 (2009) 1026-1029<br />
______________<br />
*Corresponding author: e-mail: takiz@phys.chs.nihon-u.ac.jp<br />
210 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P100 Polymers, Materials & Nanomaterials, Quantum Dots Poster 100<br />
Ratio of anti-Stokes and Stokes fluorescence intensities as a parameter<br />
sensitive to the ionic strength in the environment of fluorophoreconjugated<br />
organically modified silica nanoparticles<br />
Valentyna Tokar 1* , Tymish Ohulchanskyy 2 , Atcha Kopwitthaya 2 & Paras N Prasad 2<br />
1<br />
Electronic and Optical Processes Laboratory, Physics Faculty at Taras Shevchenko National<br />
University of Kyiv, 64 Volodymyrska str., Kyiv (Ukraine)<br />
2<br />
Institute <strong>for</strong> Lasers, Photonics and Biophotonics, University at Buffalo, <strong>State</strong> University of New<br />
York, Buffalo, NY 14260 (USA)<br />
Organically modified silica nanoparticles (ORMOSIL) are promising nanomaterials <strong>for</strong> biomedical<br />
applications. The number of their advantageous features such as chemical inertness and colloidal<br />
stability together with optical transparency and controllable surface chemistry provides flexible<br />
nanoplat<strong>for</strong>m <strong>for</strong> combining bioimaging and biosensing modalities with a therapeutic one [1-4]. In<br />
this work we demonstrate the applicability of the covalently dye-linked ORMOSIL as sensor of the<br />
ionic strength of the surrounding.<br />
We have used Rhodamine 6G – conjugated ORMOSIL nanoparticles of 30 nm in size<br />
(synthesized as reported in [4-6] with slight modifications). The ratio of anti-Stokes (excitation at<br />
633nm) to Stokes (excitation at 514nm) fluorescence intensities was used to probe the ionic<br />
strength of the nanoparticles' environment. Measurements were per<strong>for</strong>med <strong>for</strong> colloidal<br />
suspensions of nanoparticles in buffers (PBS, pH=7.4 and TRIS, pH=8.2), with changing<br />
concentrations of salts. We observed an increase in the ratio of anti-Stokes to Stokes Rhodamine<br />
fluorescence intensities in response to increase of salts' concentration. The Stokes fluorescence<br />
intensity appears to be less sensitive to the ionic strength of the environment. In addition, it can be<br />
also sensitive to the other factors, including pH and polarity of the environment. The use of the<br />
anti-Stokes to Stokes excited fluorescence comparing measurements would allow <strong>for</strong> more<br />
accurate probing of the ionic strength of the environment. Moreover, nanoparticles embedded with<br />
this optically traceable property can be used to monitor the specific processes in living cells<br />
accompanied with the ion concentration changes.<br />
References: [1] I.Roy et al, J. Am. Chem. Soc., 125 (26) (2003) 7860–7865. [2] I.Roy, et al, Proc.Nat. Acad.<br />
Sci. of USA, 102 (2) (2005) 279-284. [3] S.Kim, et al., Chem. Commun., 19 (2006) 2071-2073. [4] T.Y.<br />
Ohulchanskyy, et al., Nano Lett., 7(9) (2007) 2835–2842. [5] R.Kumar et al, ACS Nano, 2(3) (2008) 449–<br />
456. [6] Rajiv Kumar, et al., ASC Nano, 4(2) (2010) 699-708.<br />
______________<br />
*Corresponding author: e-mail: valyatokar@gmail.com<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 211
P101 Polymers, Materials & Nanomaterials, Quantum Dots Poster 101<br />
Adsorption of 5,5’-disulfopropyl-3,3’-dichlorothyocyanine and<br />
fluorescence quenching in the gold nanoparticle assembly<br />
Vesna Vodnik 1,* , Ana Vujačić 1 , Miroslav Dramićanin 1 , Sofija P. Sovilj 2 & Vesna Vasić 1<br />
1 Vinča Institute of Nuclear Sciences, University of Belgrade, P.O. Box 522, 11001 Belgrade (Serbia)<br />
2 Faculty of Chemistry, University of Belgrade, P.O. Box 118, 11158 Belgrade, (Serbia)<br />
As one increasingly important class of nanostructures, the adsorbed fluorophores on the gold<br />
nanoparticles have captured recently interest in exploiting their optical properties <strong>for</strong> chemical<br />
sensing and biological applications. [1,2] Since cyanine dyes are highly fluorescent, [3] the<br />
measurements of fluorescence properties of the particle–dye assemblies can provide an important<br />
insight to understanding the electromagnetic interactions between metal core and dye shell. In this<br />
work we have investigated the adsorption and the fluorescence quenching upon the adsorption of<br />
5,5’-disulfopropyl-3,3’-dichlorothyocyanine dye (TC) on citrate and borate capped gold<br />
nanoparticles with mean diameters from 6-30 nm. The characterization of gold nanoparticles in the<br />
presence and absence of TC dye was per<strong>for</strong>med by measurements of the absorption spectra,<br />
fluorescence spectra, dynamic light scattering, TEM, FTIR and zeta potential. Furthermore, since<br />
the fluorescence quenching was shown to be quantitatively related to the surface coverage of the<br />
dye molecules on the Au nanoparticle surface, we also evaluated the sorption mechanism and<br />
parameters. The obtained results showed that metallic surface induce strong quenching of<br />
molecular fluorescence due to electromagnetic coupling between the metal and the dye molecules.<br />
Significant increase of quenching efficiency was noticed when nanoparticle size increased, keeping<br />
the concentration of nanoparticles of various size constant. In our calculations we assumed that<br />
the fluorescence quenching occurred only <strong>for</strong> those molecules that had direct contact with<br />
nanoparticle surface. Based on the comparison of experimentally obtained values <strong>for</strong> fluorescence<br />
quenching with the calculated ones, the conclusion can be drown that the maximum of quenched<br />
TC dye was restricted to a full monolyer coverage of TC on the nanoparticle surface which was<br />
dependent on the dye orientation. Moreover, as the experimentally obtained results showed that<br />
quenching of TC dye, at constant colloid concentration, was at least two times higher than the<br />
1,4<br />
calculated values, it seems that<br />
dimmers were adsorbed on the<br />
nanoparticle surface.<br />
Fluorescence intensity x 10 6<br />
1,2<br />
1,0<br />
0,8<br />
0,6<br />
0,4<br />
0,2<br />
0,0 3,0x10 -7<br />
0,0<br />
6,0x10 -7<br />
5 4 3 2<br />
9,0x10 -7<br />
1,2x10 -6<br />
TC dye concentration<br />
1,5x10 -6<br />
1,8x10 -6<br />
2,1x10 -6<br />
212 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
Dependence of fluorescence intensity<br />
at 485 nm vs. TC dye concentration <strong>for</strong><br />
gold colloids with different particles<br />
sizes, in the presence of 5×10 11<br />
nanoparticles with average diameter 1)<br />
30 nm, 2) 17 nm, 3) 10 nm, 4) 6 nm; 5)<br />
TC dye.<br />
This work was supported by grants from the Ministry of Science and Technological Development of<br />
the Republic of Serbia (research projects number: 172056 and 172023).<br />
References: [1] Y. Li, et al., Gold Bulletin 43 (2010) 29. [2] J. J. Hickman, et al., Science 252 (1991) 688. [3]<br />
I.I.S. Lim, et al., J. Phys. Chem. B 110 (2006) 6673.<br />
______________<br />
* Corresponding author: e-mail: vodves@vinca.rs<br />
1
P102 Polymers, Materials & Nanomaterials, Quantum Dots Poster 102<br />
Gold nanorods <strong>for</strong> fluorescence imaging and sensing in biology<br />
Yinan Zhang 1 , David J. S. Birch 1 & Yu Chen 1*<br />
1 Photophysics Group, Centre <strong>for</strong> Molecular Nanometrology, Department of Physics, SUPA,<br />
University of Strathclyde, John Anderson Building, 107 Rottenrow, Glasgow, G4 0NG, UK<br />
Two-photon luminescence (TPL) from gold nanorods (GNRs) shows considerable potential in<br />
biological imaging and sensing. [1-2] We study the imaging of gold nanorods in Madin-Darby canine<br />
kidney (MDCK) cells using fluorescence lifetime imaging microscopy (FLIM). FLIM provides<br />
images with better contrast and sensitivity than intensity imaging. [3] The characteristic fluorescence<br />
lifetime of gold nanorods is found to be less than 100 ps, which can be used to distinguish gold<br />
nanorods from other fluorescent labels and endogenous fluorophores in lifetime imaging. [4] We<br />
have also demonstrated energy transfer between 4'-6-Diamidino-2-phenylindole (DAPI), a<br />
commonly used DNA label, and gold nanoparticles under two-photon excitation in solution using<br />
FLIM. With comparable size and concentration, gold nanorods are shown to provide more efficient<br />
energy transfer than gold nanospheres (GNSs). We attribute this transfer enhancement effect to<br />
the longitudinal surface plasmon mode of GNRs overlapping with the incidence excitation<br />
wavelength. Energy transfer under two-photon excitation between GNRs and DAPI has also been<br />
observed in cell culture and found to be in accord with the solution phase results. The study shows<br />
the possibility of exploiting both the TPL imaging and energy transfer process to trace gold<br />
nanoparticles and their interaction with bio-molecules within cells.<br />
This work was supported by an EPSRC Science and Innovation Award, and a Pump Priming<br />
Award from SPIBS University of Strathclyde.<br />
References: [1] S. Link and M. El-Sayed, Int. Rev. Phys. Chem. 19(3) (2000) 409. [2] H. Wang, et al., Proc.<br />
Natl. Acad. Sci. 102(44) (2005) 15752. [3] M. Y. Berezin and S. Achilefu, Chem. Rev. 110 (2010) 264. [4] Y.<br />
Zhang, et al., Biomed. Opt. 15 (2010) 020504-3.<br />
______________<br />
* Corresponding author: e-mail: y.chen@strath.ac.uk<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 213
P103 Polymers, Materials & Nanomaterials, Quantum Dots Poster 103<br />
Characterization of highly fluorescent candidate glass<br />
reference standards<br />
Katrin Hoffmann 1 , Axel Engel 2 , Monika Bäumle 3 & Ute Resch-Genger 1<br />
1 BAM Federal Institute <strong>for</strong> Material Research and Testing, D-12489 Berlin (Germany)<br />
2 Schott AG, Hattenbergstr. 10, D-55014 Mainz<br />
3 Sigma-Aldrich Production GmbH / Industriestrasse 25 / CH-9470 Buchs / Switzerland<br />
Well-known in the community of users of fluorescence techniques is the dilemma of<br />
incommensurable, relative fluorescence signals, which are not only related to analyte-specific<br />
signals but also affected by instrument-specific distortions and time dependent fluctuations. [1]<br />
Numerous fluorescence applications are being increasingly used in environmental monitoring,<br />
material analysis, pharmaceutical research, and medical and clinical diagnostics with strict<br />
regulatory requirements. The acceptance of these techniques calls <strong>for</strong> simple and internationally<br />
accepted fluorescence standards <strong>for</strong> instrument qualification and method validation.<br />
To accommodate these developments, a first set of liquid fluorescence standards that<br />
enables the determination of a broad variety of fluorescence parameters was certified by BAM and<br />
distributed by Sigma-Aldrich. [1] Recently, also solid, certified, broad-band emitting reference<br />
materials reached the market that meet the upcoming demand <strong>for</strong> fluorescence intensity standards<br />
applicable without any preparation<br />
step. [2]<br />
In order to provide reference<br />
materials <strong>for</strong> the characterization and<br />
per<strong>for</strong>mance validation of various<br />
fluorescence measurement systems,<br />
we currently study highly fluorescent,<br />
narrow-band emitting glasses in<br />
different shapes doped with rare earth<br />
(RE) ions. The emission pattern of<br />
these glasses between 400 nm and<br />
720 nm (Figure 1) can be used to<br />
validate the accuracy of the<br />
wavelength scale and the<br />
wavelength-dependent spectral<br />
responsivity on a day-to-day basis<br />
and to consider changes of these<br />
parameters. A minimum emission<br />
anisotropy, an excellent long-term<br />
photostability, and a good spatial<br />
homogeneity of the fluorescence<br />
properties including the emission<br />
0.0<br />
300 400 500<br />
214 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
absorption<br />
1.5<br />
1.0<br />
0.5<br />
LEX-AbsEM.opj<br />
wavelength/ nm<br />
400 500 600 700<br />
wavelength/ nm<br />
Fig. 1: Photostable, narrow band emitting glass, to e.g. monitor<br />
the per<strong>for</strong>mance of fluorescence measuring systems.<br />
pattern provide the basis <strong>for</strong> many future applications of the RE-ion-doped glasses as ready-to-use<br />
solid-state fluorescence intensity and wavelength standards <strong>for</strong> a broad variety of instrumentation.<br />
Financial Support by the Federal Ministry of Education and Research 13N8850 and 13N8849<br />
(Biophotonic II) are gratefully acknowledged.<br />
References: [1] U. Resch-Genger, et al., Reviews in fluorescence 2007, Springer Science+Business Media<br />
(2009) 1-33. [2] Certificate of Analysis, Standard Reference Material SRM 2942/2941/2940 - Relative<br />
Intensity Correction Std <strong>for</strong> Fluorescence Spectroscopy (Blue/Green/Orange Emission). National Institute of<br />
Standards and Technology:Gaithersburg, MD, 200 . [3] K. Hoffmann, et al., Luminescence – The Journal of<br />
Biological and Chemical Luminescence 23 (2008), 229.<br />
______________<br />
* Corresponding author: e-mail: katrin.hoffmann@bam.de<br />
emission
P104 Polymers, Materials & Nanomaterials, Quantum Dots Poster 104<br />
Targeting of quantum dots with adaptor protein Nck<br />
Ruta Araminaite 1,* , Vitalijus Karabanovas 1,2 , Marija Ger 2 , Mindaugas Valius 2 , Simona<br />
Steponkiene 1 & Ricardas Rotomskis 1,3<br />
1<br />
Biomedical Physics Laboratory, Institute of Oncology, Vilnius University, Baublio 3b, LT-08406,<br />
Vilnius (Lithuania).<br />
2<br />
Developmental Biology Department, Institute of Biochemistry, Vilnius University, Mokslininku 12,<br />
LT-08662 Vilnius (Lithuania).<br />
3<br />
Biophotonics group of Laser Research Center, Vilnius University, Sauletekio 9, bldg. 3, LT-10222<br />
Vilnius (Lithuania).<br />
Nowadays, semiconductor nanoparticles, also known as quantum dots (QD), have caught great<br />
interests of scientists in biophysics, medicine and cellular biology. QD have been covalently linked<br />
to various biomolecules such as antibodies, peptides, nucleic acids and other ligands <strong>for</strong><br />
fluorescence probing applications. QD have been used to target and image tumor cells [ 1 ].<br />
Understanding the role of adaptor proteins in cellular biology could provide additional strategies <strong>for</strong><br />
cancer and diabetes prevention and treatment. One of the main adaptor proteinas which mediates<br />
protein-protein interactions and regulates cellular responses is oncogenic protein Nck. Recently<br />
Nck attracted attention not only as mediator in signal transduction and cellular proliferation but also<br />
as regulator cellular migration and actin cytoskeleton dynamics [2]. The application of QD as<br />
fluorescent markers revealed new aspects of Nck dynamics and the biological processes it<br />
regulate in living cells.<br />
In this research work, we developed an aqueous synthesis method, which can be used to<br />
produce new and high quality glutathione (GSH) modified CdSe QD. In recent investigations were<br />
found that QD modified with GSH to be more biocompatible than other water-soluble QDs [ 3 ]. We<br />
are characterized synthesized DQ: i) determined the particle size, ii) PL properties, iii) estimated<br />
the fluorescence quantum yields, iv) exhibit stability in aqueous solution, v) photostability QD in<br />
phosphate buffer solution (pH 7.4). CdSe-GSH may have potential using in bio-imaging <strong>for</strong><br />
biological objects. The second the subject of research is GST (glutathione S-transferase) high<br />
specificity and affinity the conjugation reactions of GSH. We means, of the GSH selectively bind<br />
the GST-targged protein, attached to the QD via electrostatic/hydrophobic self-assembly, to like<br />
the inorganic particle with protein Nck.<br />
By applying different conjugation methods adaptor protein Nck, it mutant Nck-0 (with an<br />
inactivating point mutations in all SH3 and SH2 domains) were covalently coupled to commercially<br />
available CdSe/ZnS QD (λPL=605 nm) with external carboxyl groups and GST-targged Nck coupled<br />
to ours synthesized CdSe-GSH QD (λPL=510 nm). We ascertained the best quantum dots<br />
conjugation with Nck method.<br />
In our study we investigated intracellular distribution and cytoskeleton changes after<br />
internalization of Nck- and mutant Nck0- QD biconjugates in mouse embryonic fibroblast NIH3T3<br />
and pancreatic adenocarcinoma (Mia-PaCa-2) cells lines.<br />
This work was supported by European Union Structural Funds project “Postdoctoral Fellowship<br />
Implementation in Lithuania”.<br />
References: [] M.A. Walling, et al., Int.J.Mol.Sci. 10 (2009) 441. [2] G.M. Rivera, et al., Current Biology 14<br />
(2004) 11. [ 3 ] Y. Zheng, et al., Adv. Mater. 19 (2007) 376.<br />
______________<br />
* Corresponding author: e-mail: ruta.araminaite@vuoi.lt<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 215
P105 Polymers, Materials & Nanomaterials, Quantum Dots Poster 105<br />
Preparation of size controllable highly luminescent CdSe quantum dots<br />
in AOT reverse micelles using polyselenide precursors under mild<br />
conditions<br />
Arlindo M. Fontes Garcia & Paulo J.G. Coutinho *<br />
Centre of Physics (CFUM), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal<br />
Semiconductor nanocrystals, also called quantum dots (QDs), are an emerging new class of<br />
powerful and versatile biomedical imaging probes. Their fluorescence is unique compared with that<br />
from traditional organic fluorophores. QDs exhibit high quantum yields, high photostability, large<br />
absorption coefficients, continuous absorption bands <strong>for</strong> multicolor capability, narrow and<br />
symmetric emissions, and many biofunctionalisation strategies. Most of the preparation techniques<br />
used a tri-n-octylphosphine (TOPO) based system in which the reagents are injected into a hot<br />
coordinating solvent at elevated temperature (200–400 ◦C) under nitrogen and moisture free<br />
atmosphere [1]. We started using polyselenide as a selenium precursor and managed to produce<br />
CdSe QDs with narrow band-edge emission through an AOT reverse micelle route both at normal<br />
atmosphere or with a previous slight nitrogen purge at mild temperatures (~80ºC) [2]. As there is<br />
no covalently linked capping layer, it was easy to couple these QDs with TiO2 and use the<br />
resulting nanocomposites in photodegradation studies [3].<br />
In this work we show the effect of water pool size and type of polyselenide (Sen 2- ). In the<br />
figure it can be seen that both this factors influence the size and quality of CdSe QDs.<br />
wo=2.9, Se3 2-<br />
wo=3.7, Se3 2-<br />
wo=4.3, Se6 2-<br />
wo=4.3, Se3 2-<br />
wo=5, Se3 2-<br />
This study was funded by FCT-Portugal and FEDER through CFUM.<br />
References: [1] J. Hambrock, et al, J. Mater. Chem. 11 (2001) 3197 . [2] A.G. Fontes Garcia, P.J.G.<br />
Coutinho, submited. [3] A. M. Fontes Garcia, et al., Nanoscale Research Letters 6 (2011) 426.<br />
______________<br />
* Corresponding author: e-mail: pcoutinho@fisica.uminho.pt<br />
216 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P106 Polymers, Materials & Nanomaterials, Quantum Dots Poster 106<br />
Delivery of quantum dots to cytosol by chlorin e6-mediated<br />
photochemical internalization<br />
Vitalijus Karabanovas 1,2* , Jurga Valanciunaite 2 , Mindaugas Valius 1 & Ricardas Rotomskis 2,3<br />
1<br />
Proteomics Centre, Vilnius University Institute of Biochemistry, Mokslininku str. 12, Vilnius LT-<br />
08662, Lithuania<br />
2<br />
Biomedical physics laboratory, Institute of Oncology Vilnius University, Baublio 3b, LT-08406,<br />
Vilnius (Lithuania)<br />
3<br />
Biophotonics group, Laser research center, Vilnius University, Sauletekio 9, Bldg. 3, LT-10222<br />
Vilnius (Lithuania)<br />
Exceptional photochemical properties, such as a broad and efficient absorption, photostability and<br />
bright photoluminescence (PL), make quantum dots (QDs) superior fluorophores over organic<br />
dyes. QDs can be easily chemically modified to create multifunctional and molecular targetdirected<br />
nanoparticles. Applications of QDs <strong>for</strong> multicolor imaging, site-specific targeting in vivo,<br />
cell tracking, drug delivery and other biological applications have been previously demonstrated in<br />
numerous reports [1, 2]. However, our previous study has revealed that under normal conditions<br />
QDs enter cells via endocytosis, remain entrapped in the endosomes and are not released into the<br />
cytoplasm [3]. This endocytic intracellular route of QDs dramatically minimizes their potential to use<br />
them <strong>for</strong> the intracellular molecular target visualization and other applications. There<strong>for</strong>e, additional<br />
manipulations should be applied to overcome the issue of lipid barrier.<br />
Here we demonstrate photochemical internalization of QDs entrapped into endosomes of<br />
immortalized mouse embryonic fibroblast NIH-3T3 cells by applying photosensitizing agent chlorin<br />
e6 (Ce6). Cell analysis by confocal microscopy shows that after 24 h of incubation QDs internalize<br />
into NIH-3T3 cells and concentrate in vesicular-like structures that were located near the<br />
perinuclear region. The overlapped fluorescence images after the additional 2 h incubation with<br />
Ce6 show co-localization of the Ce6 with QDs in the same vesicular compartment that<br />
morphologically resembles multivesicular bodies. Exposure of NIH-3T3 cells with 488 nm laser<br />
light resulted in photobleaching of Ce6 in the entire cytoplasm and membrane structures;<br />
meanwhile, PL intensity of QDs entrapped in the endosomes significantly increases. Interestingly,<br />
further irradiation of cells causes the morphological changes of vesicular structures and<br />
presumably leads to the disruption of endosomes. This coincides with the decrease of PL of QDs<br />
inside of the vesicles possibly due to the rapid escape of QDs into the cytoplasm. In parallel, the<br />
increase in PL intensity of QDs in the periphery of endosomes is observed, providing further<br />
evidence that laser irradiation of QDs/Ce6 -trapped cells effects photochemical internalization of<br />
QDs. Our findings highlight photochemical internalization of QDs mediated by Ce6 as a powerful<br />
technique useful <strong>for</strong> intracellular molecular imaging and photodynamic therapy applications.<br />
This work was supported by the project “Postdoctoral Fellowship Implementation in Lithuania"<br />
funded by the European Union Structural Funds.<br />
References: [1] A. M. Derfus, et al., Adv.Matter, 16 (2004) 961. [2] J. B. Delehanty, et al., Anal.<br />
Bioanal.Chem. 393 (2009) 1091. [3] L. Damalakiene, et al., IEEE NANO, 562 (2009) 465.<br />
______________<br />
* Corresponding author: e-mail: vitalijus.karabanovas@vuoi.lt<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 217
P107 Polymers, Materials & Nanomaterials, Quantum Dots Poster 107<br />
Receptor mediated targeting of quantum dots to kidney podocytes<br />
1 1 1 1 2<br />
Klaus Pollinger , Robert Hennig , Joerg Tessmar , Miriam Breunig , Ralph Witzgall<br />
1, *<br />
& Achim Goepferich<br />
1 Department of Pharmaceutical Technology, University of Regensburg, 93053 Regensburg, Germany<br />
2 Institute <strong>for</strong> Molecular and Cellular Anatomy, University of Regensburg, 93053 Regensburg, Germany<br />
The site-specific delivery of nanoparticles to specific cells of a tissue is of major interest in current<br />
nanomedical research. Such nanoparticles could be advantageous <strong>for</strong> diagnostic as well as <strong>for</strong><br />
drug delivery applications. Unequivocal cell identification can, thereby, be achieved by attachment<br />
of receptor ligands on the nanoparticle surface, giving the nanoparticles the ability to interact with<br />
cells by a highly specific ligand receptor mediated interaction.<br />
Up to now the kidney has been neglected in the field of receptor mediated nanoparticle<br />
targeting. However, it is a well-accepted fact that nanoparticles such as quantum dots (Qdots) are<br />
eliminated via the kidney and can there<strong>for</strong>e pass the renal filter [1,2]. This passage through the<br />
renal filter opens a completely new cellular target <strong>for</strong> nanoparticles, namely podocytes. These cells<br />
<strong>for</strong>m the slit membrane in the kidney glomeruli and there<strong>for</strong>e play a crucial role in renal function.<br />
Un<strong>for</strong>tunately, various inherited and acquired diseases can cause podocyte dysfunction that often<br />
leads to renal failure and permanent loss of kidney function. Nanoparticles could serve as tools <strong>for</strong><br />
a targeted delivery of drugs or nucleic acids to kidney podocytes and provide new therapeutic<br />
options <strong>for</strong> a number of kidney diseases.<br />
The present study aims to investigate receptor-mediated interaction of nanoparticles with<br />
podocytes. There<strong>for</strong>e, nanoparticle binding studies were carried out on isolated primary podocytes<br />
and freshly isolated glomeruli in vitro. As target receptor the αvβ3 integrin and the corresponding<br />
highly specific ligand cyclo(RGDfC) were chosen to direct the nanoparticles to the target cells.<br />
Qdots were used as model nanoparticles.<br />
In flow cytometry (FACS) experiments, the cyclo(RGDfC) modified Qdots showed an<br />
extensive binding to podocytes, which could be displaced using a surplus of free cyclo(RGDfC)<br />
peptide. This shows that the peptide modified Qdots exhibit a receptor mediated specific binding to<br />
the cells. In confocal laser scanning microscopy (CLSM) investigations, the binding and<br />
displacement experiments could be also confirmed. Furthermore, the distribution of the<br />
cyclo(RGDfC) Qdots throughout the cells was investigated by taking z-stack imaging. Here the<br />
nanoparticles showed a distribution over the complete cytosol and additional accumulation in<br />
vesicular structures throughout the cells. These findings suggest that the nanoparticles are taken<br />
up into podocytes, which would be essential <strong>for</strong> future intracellular drug delivery.<br />
Further experiments <strong>for</strong> nanoparticle targeting were per<strong>for</strong>med on whole glomeruli hosting<br />
podocytes in their natural 3-D environment. Here CLSM analysis showed a strong binding of the<br />
cyclo(RGDfC) Qdots, which was located in the podocyte specific regions of the glomeruli.<br />
In summary, these experiments show that a receptor mediated targeting of nanoparticles to<br />
kidney podocytes is possible. This provides the opportunity to design specifically targeted<br />
nanoparticles <strong>for</strong> future diagnostic and therapeutic applications within the kidney.<br />
This work was supported by DFG, Graduiertenkolleg 760 and DFG grant No GO565/17-1.<br />
References: [1] H. S. Choi, et al., Nat. Biotech. 25 (2007) 1165. [2] C. Zhou, et al., Angew. Chem. Ed. Engl.<br />
50 (2011) 3168.<br />
______________<br />
* Corresponding author: e-mail: achim.goepferich@chemie.uni-regensburg.de<br />
218 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P108 Polymers, Materials & Nanomaterials, Quantum Dots Poster 108<br />
A novel highly sensitive nanobiosensor <strong>for</strong> detection of helicobacter<br />
pilori based on Fluorescence Resonance Energy Transfer<br />
Mojtaba Shamsipur 1 , Maryam Shanehsaz 2* , Afshin Mohsenifar 3 , Sadegh Hasannia 4 , S.H.<br />
Kazemi 5 & Nazanin Pirooznia 4<br />
1<br />
Department of Chemistry, Razi University, Kermanshah, Iran<br />
2<br />
Department of Chemistry, Tarbiat Modares University, Tehran, Iran<br />
3<br />
Department of Toxicology, Tarbiat Modares University, Tehran, Iran<br />
4<br />
Department of Biology, Guilan University, Rasht, and NIGEB, Iran<br />
5<br />
Department of Chemistry, Institute <strong>for</strong> Advanced Studies in Basic Sciences (IASBS), zanjan,<br />
45137-66731, Iran<br />
Luminescent semiconductor quantum dots have been intensely studied due to their unique optical<br />
properties. In particular, semiconductor QDs are very attractive as biological labels because of<br />
their small size, emission tunability, superior photostability and longer photoluminescence (PL)<br />
decay times in comparison with conventional organic dyes [1,2] . In this study a nanobiosensor<br />
based on fluorescence resonance energy transfer (FRET) <strong>for</strong> sensitive detection of helicobacter<br />
using two specified oligonucleotides conjugated with quantum dot and Tamra was fabricated. First,<br />
the water-soluble CdTe QDs were prepared [3] and, subsequently, two specific oligonucleotides <strong>for</strong><br />
helicobacter were attached to the QDs and the Tamra, in order to <strong>for</strong>m functional QDs- and Tamraoligonucleotide<br />
conjugates. Along with the addition of DNA targets to a solution containing QDs<br />
and Tamra-oligonucleotide conjugates, hybridization was effectively carried out. The resulting<br />
assembly brings the Tamra fluorophoreas an acceptor and the QDs as a donor into the proximity of<br />
each other, leading to fluorescence emission transfer from the acceptor by means of FRET<br />
phenomenon on illumination of the donor. In the absence of target DNA, the detection probes were<br />
not ligated and no Tamra emission was produced due to the lack of FRET. The feasibility of the<br />
proposed method was also demonstrated in the detection of synthetic 210-mer nucleotide derived<br />
from helicobacter with a sensitivity of 5×10 -8 M. Thus, based on the results obtained thus far, the<br />
proposed homogeneously DNA detection method which is simple, rapid and efficient, due to the<br />
elimination of the washing and separation steps, will be useful in fabrication of a highly sensitive<br />
nanobiosensor <strong>for</strong> the detection of helicobacter pylori.<br />
References: [1] Stefania Impellizzeri, et al., J. Phys. Chem. C 2010, 114, 7007–7013. [2] I. Yildiz, et al., J.<br />
Mater. Chem., 2008, 18, 5577–5584. [3] Juan Li, et al., Spectrochimica Acta Part A 70 (2008) 811–817.<br />
______________<br />
*Corresponding author: e-mail: maryam_shaneh@yahoo.com<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 219
P109 Polymers, Materials & Nanomaterials, Quantum Dots Poster 109<br />
Spectroscopic study on complex <strong>for</strong>mation between different<br />
amphiphilic coating bearing quantum dots and photosensitizer<br />
chlorin e6<br />
Jurga Valanciunaite 1 , Artiom Skripka 1 , Reda Jarmalaviciene 1 & Ricardas Rotomskis 1,2<br />
1<br />
Biomedical Physics Laboratory of Institute of Oncology, Vilnius University, Baublio 3b., LT-08406,<br />
Vilnius, Lithuania<br />
2<br />
Faculty of Physics, Vilnius University, Sauletekio 9, bldg. 3, LT-10222 Vilnius, Lithuania<br />
Due to the broad absorption, size-dependent efficient photoluminescence (PL) and great<br />
photostability, semiconductor quantum dots (QDs) might be beneficial as energy donors <strong>for</strong><br />
conventional porphyrin-type photosensitizers (PS) used in photodynamic therapy of cancer (PDT)<br />
[1]. An improved excitation of PS via energy transfer from QDs would subsequently lead to<br />
enhanced efficiency of PDT. In our previous study we have demonstrated the <strong>for</strong>mation of noncovalent<br />
complex between QDs and second generation photosensitizer, chlorin e6 (Ce6), with the<br />
ability of efficient energy transfer [2] and singlet oxygen generation. We found that QD-Ce6<br />
complex was <strong>for</strong>med due to hydrophobic interaction resulted in insertion of non-polar moiety of Ce6<br />
to lipid part of QD coating.<br />
Here we compare two types of commercially available QDs, which coatings are based either<br />
on amphiphilic polymer (A-QDs) (Invitrogen Corp. (USA)) or lipids (L-QDs) (eBioscience Inc.<br />
(USA)), as potential candidates <strong>for</strong> stable complex <strong>for</strong>mation with Ce6.<br />
The initial value of quantum yield (QY) of A-QDs PL was about two times higher than L-QDs. A<br />
slight decrease in PL QY over time was observed <strong>for</strong> both types of QDs. Increasing the<br />
concentration of Ce6 resulted in gradual decrease in PL intensity of both types of QDs. Moreover,<br />
similar spectral changes of Ce6 were observed in A-QDs and L-QDs solutions. Binding to QDs<br />
produced a bathochromic shift of Ce6 fluorescence band from 660 nm to 670 nm, similar to that<br />
observed in non-polar solvents. In the presence of both types of QDs, the intensity of Ce6<br />
fluorescence was ten times higher than in aqueous solution. The excitation spectrum registered at<br />
a maximum fluorescence band (670 nm) of bound Ce6 showed the significant contribution of QDs<br />
spectra. These findings confirm the fact of energy transfer from QDs to Ce6. The efficiency of<br />
energy transfer from A-QDs to Ce6 was about two times higher than from L-QDs, which reveals<br />
that A-QDs are more efficient energy donors. The highest efficiency of energy transfer from L-QDs<br />
to Ce6 was reached at QD:Ce6 molar ratio 1:10, while in the case of A-QDs, the molar ratio was<br />
1:20. Additionally, the intensity of Ce6 bands in excitation spectrum registered at 670 nm was<br />
higher <strong>for</strong> L-QD-Ce6 complex. This indicates the stronger binding affinity of Ce6 to QDs bearing<br />
lipid based coating.<br />
The results of this comparison can provide a basis <strong>for</strong> effective selection of optimal structure<br />
of QD surface coating <strong>for</strong> non-covalent QD-photosensitizer complex <strong>for</strong>mation.<br />
This work was supported by the project “Postdoctoral Fellowship Implementation in Lithuania"<br />
funded by European Union Structural Fund and a grant (No. MIP-095/2011) from the Research<br />
Council of Lithuania.<br />
References: [1] A. C. Samia, et al., J. Am. Chem. Soc, 125 (2003) 15736. [2] J. Valanciunaite, et al., SPIE<br />
Proc., 7376 (2010).<br />
______________<br />
* Corresponding author: e-mail: artiom.skripka@ff.stud.vu.lt<br />
220 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P110 Polymers, Materials & Nanomaterials, Quantum Dots Poster 110<br />
Photosensitizing properties and accumulation in cancer cells of noncovalent<br />
quantum dot-chlorin e6 complex<br />
Jurga Valanciunaite 1* , Simona Steponkiene 1 , Artiom Skripka 1 , Margarita Chernych 1 , Giedre<br />
Streckyte 2 & Ricardas Rotomskis 1,2<br />
1<br />
Biomedical Physics Laboratory of Institute of Oncology, Vilnius University, Baublio 3b, LT-08406,<br />
Vilnius (Lithuania)<br />
2<br />
Biophotonic group of Laser Research Center, Vilnius University, Sauletekio 9, bldg. 3, LT-10222<br />
Vilnius (Lithuania)<br />
Semiconductor quantum dots (QDs) have been gaining much attention due to their unique sizedependent<br />
optical properties leading to potential applications in many biological fields including<br />
medicine. Recently it has been suggested that QDs could be used in the photodynamic therapy<br />
(PDT) of cancer as photosensitizing agents alone or as resonant energy donors <strong>for</strong> conventional<br />
porphyrin type photosensitizers [1]. Though numerous studies on the primary photophysical<br />
properties of QDs and various photosensitizers’ complexes in aqueous solutions assembled mainly<br />
by the electrostatic interaction have been per<strong>for</strong>med until now [2,3], further investigations of such<br />
complexes in biological media are still missing.<br />
Here we summarize our results obtained by studying a non-covalent complex between<br />
quantum dots and second generation photosensitizer, chlorin e6 (Ce6), in aqueous medium and in<br />
live cancer cells. The spectral changes of Ce6 observed upon binding to QDs have revealed that<br />
the <strong>for</strong>mation of QD-Ce6 complex is driven by the hydrophobic interaction between the lipid part of<br />
QDs coating and non-polar moiety of Ce6 [4]. Close localization of Ce6 to the core of QD resulted in<br />
an efficient energy transfer from QD to Ce6. The <strong>for</strong>mation, stability and efficiency of energy<br />
transfer within QD-Ce6 complex were highly dependent on the type of amphiphilic coating of QDs.<br />
Using similarly coated QDs of different size, the efficiency of energy transfer from QDs to bound<br />
Ce6 molecules correlated well with the amount of spectral overlap between emission of QDs and<br />
absorption of Ce6. The spectral results of singlet oxygen generation indicated that QD-Ce6 complex<br />
activated by light was able to produce singlet oxygen more effectively than QDs or Ce6 alone.<br />
Moreover, Ce6 molecules bound to QDs were found to be more photostable than free Ce6.<br />
Spectral measurements per<strong>for</strong>med on human pancreatic carcinoma cell line (MIA PaCa-2<br />
from American Type Culture Collection (ATCC)) using confocal microscope after 2 hours<br />
incubation with QD-Ce6 complex showed clear co-localization of QD and Ce6 in cellular<br />
membranes and endocytic vesicles. However, the fluorescence spectrum of free Ce6 in cytoplasm<br />
displayed a similar maximum as from the co-localization area with QDs. Thus the stability of QD-<br />
Ce6 complex within live cells still needs further proof.<br />
The perspectives of using such QD-Ce6 complex in practice will be discussed in the context of<br />
presented results.<br />
This work was supported by the project “Postdoctoral Fellowship Implementation in Lithuania"<br />
funded by European Union Structural Fund and by the Lithuanian Science Council Student<br />
Research Fellowship Award (A.S).<br />
References: [1] A. C. Samia, et al., J. Am. Chem. Soc., 125 (2003) 15736. [2] P. Juzenas, et al., Adv. Drug<br />
Deliv. Rev. 60 (2008) 1600. [3] E. Yaghini, et al., Nanomedicine 4 (2009) 353. [4] J. Valanciunaite, et al.,<br />
SPIE Proc., 7376 (2010).<br />
______________<br />
* Corresponding author: e-mail: jurga.valanciunaite@vuoi.lt<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 221
222 — MAF 12, Strasbourg, France, September 11-14, 2011 —
Miscellaneous,<br />
Micelles & Biopolymers,<br />
Fluorescence Spectroscopy<br />
applied to Biology,<br />
Biomembranes<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 223
224 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P111 Fluorescence Spectroscopy Poster 111<br />
Investigation on Romanian icons – chromatic changes following<br />
gamma irradiation<br />
Maria Geba 1* , Ana Maria Vlad 1 , Daniela Salajan 1 , Corneliu Catalin Ponta 2<br />
& Constantin-Daniel Negut 2<br />
1<br />
”Moldova” National Complex of Museums, Research and Restoration Conservation Center,<br />
1, Palat Street, Iasi, 700028, Romania<br />
2<br />
IRASM, ”Horia Hulubei” National Institute of Physics and Nuclear Engineering, Bucharest,<br />
Romania<br />
A study aiming at the identification of the mineral pigments used on a lot of icons of XVIII -XX<br />
century detained by the “Moldova” National Complex of Museums was occasioned by the Project<br />
DELCROM.<br />
The non-destructive x–ray fluorescence spectroscopy was an adequate analysis method due to its<br />
non-destructibility.<br />
Also the impact of irradiation conservation treatment on colors was investigated on artificially<br />
prepared samples and on some icons, by MiniScan XE Plus – HunterLab portable reflexion<br />
spectrophotocolorimeter.<br />
The analyses allowed the identification of many pigments traditionally used in icons painting, along<br />
with some modern ones.<br />
Icon „Virgin and Child; Resurrection; St. Nicolas”<br />
XRF spectrum of the red color – mixture of red lead and red ochre<br />
This study intended a characterization of painting materials, in order to determine the pigments<br />
behavior following a conservation treatment by gamma irradiation.<br />
This work was supported by grants from ANCS, CNMP, DELCROM: 92-086/2008.<br />
References: [1] N. Civici, , Science Meets Archaeology and Art History – Balkan Symposium on<br />
archaeometry Ohrid, Republic of Macedonia, 18th–20th September, 31 (2008). [2] E. West Fitzhugh, Artists’<br />
pigments. A Handbook of their history and characterisation. New York, Ox<strong>for</strong>d: National Gallery of Art:<br />
Washington, Ox<strong>for</strong>d University Press (1987-1997). [3] C.D. Negut, et al., Proc. SPIE (2007), 6618. [4] M.M.<br />
Rizzo, et al., Rad.Phys.Chem. 63 (2002), (3-6).<br />
______________<br />
* Corresponding author: e-mail: mariageba@yahoo.com<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 225
P112 Fluorescence Spectroscopy Poster 112<br />
Tracing non-specific riverine pollution caused by discharge of industrial<br />
effluents: Use of a recalcitrant fluorescent component of chromophoric<br />
dissolved organic matter (CDOM)<br />
Mikhail Borisover 1,* , Yael Laor 2 , Ibrahim Saadi 2 , Marcos Lado 3 & Nadezhda Bukhanovsky 1<br />
1<br />
Institute of Soil, Water and Environmental Sciences, Agricultural Research Organization, the<br />
Volcani Center, POB 6, Bet Dagan, 50250 (Israel).<br />
2<br />
Institute of Soil, Water and Environmental Sciences, Agricultural Research Organization, Newe<br />
Ya’ar Research Center, POB 1021, Ramat Yishay, 30095 (Israel)<br />
3<br />
Area of Soil Science, Faculty of Sciences, University of A Coruña, A Zapateira s/n, 15071 A<br />
Coruña, (Spain)<br />
Excitation-emission matrix (EEM) fluorescence spectroscopy has found important applications in<br />
characterizing contamination of multiple aqueous bodies, e.g., rivers, estuaries, lakes and<br />
groundwater. For example, riverine pollution caused by sewage effluents, land leachates and<br />
xenobiotics can be detected by means of EEM fluorescence spectroscopy [1,2]. Importantly, water<br />
pollution may be monitored based on fluorescence of certain (but not necessarily unique)<br />
constituents, such as components of chromophoric dissolved organic matter (CDOM). The present<br />
work demonstrated an application of EEM fluorescence spectroscopy <strong>for</strong> examining riverine pollution<br />
caused by severe and long-term discharge of industrial effluents into the lower Kishon River [3]. The<br />
Kishon River is one of the largest rivers in Israel and typifies streams that are affected by seawater<br />
tidal intrusion. In the year of the study, a volume of industrial effluents discharged into the river was<br />
almost half of the annual fresh water flow of the Kishon River. The industrial effluents may contribute<br />
about 90%, in terms of biochemical oxygen demand, of the total organic carbon discharged into the<br />
lower Kishon River. Yet, due to the dilution of the river water with intruding seawater, the effect of<br />
industrial effluent discharges on distribution of dissolved organic matter (DOM), CDOM components<br />
and riverine contaminants along the river can be masked. There<strong>for</strong>e, EEM fluorescence<br />
spectroscopy, combined with parallel factor analysis (PARAFAC) and measurements of UV<br />
absorbance and dissolved organic carbon (DOC) concentrations, was used to investigate the<br />
footprints of industrial effluents discharged into the lower Kishon River. During a period of 11 months,<br />
water samples were collected along the river, including the points of effluent discharge from industrial<br />
plants. Based on PARAFAC, concentration scores in water samples were determined <strong>for</strong> two major<br />
fluorescent CDOM components: (1) humic-like matter I and (2) component II spectrally similar to the<br />
matter associated with biological productivity [but differing, yet, from typical tryptophan-like<br />
fluorophore]. These fluorescent components and other constituents that absorbed light at 254 nm<br />
contributed to the significant DOC pool that was found stable against riverine microbial degradation<br />
under laboratory conditions, and that makes up to 70% of the overall riverine DOC. The variations in<br />
DOC concentration, UV absorbance at 254 nm, and concentration of humic-like matter<br />
(characterized by component I) correlated with the distance from the sea and the water electrical<br />
conductivity, and were linked to seawater tidal intrusion. There<strong>for</strong>e, due to the seawater inland<br />
penetration and associated dilution of riverine water, these indicators were hardly in<strong>for</strong>mative of the<br />
impact of industrial effluent discharge. However, the increased concentration of component II as well<br />
as its enlarged fraction in the overall riverine DOC pool was found to be clearly associated with the<br />
location of major inputs of the industrial effluents. These findings support the use of this fluorescent<br />
component as an indicator of non-specific river pollution caused by massive discharge of industrial<br />
effluents. The use of this fluorescent tracer may expand the potential of EEM fluorescence<br />
spectroscopy <strong>for</strong> monitoring various types of water pollution.<br />
This work was supported by Israel Ministry of Environmental Protection (Project No. 161/03).<br />
References: [1] A. Baker, Water, Air and Soil Pollution, 163 (2005) 229. [2] A. Baker and R. Inverarity,<br />
Hydrological Processes, 18 (2004) 2927. [3] M. Borisover, et al., Water, Air and Soil Pollution, in press<br />
(2011).<br />
______________<br />
* Corresponding author: e-mail: vwmichel @volcani.agri.gov.il<br />
226 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P113 Fluorescence Spectroscopy Poster 113<br />
1,2-Dioxetane-based chemiluminescent uphill energy conversion:<br />
principle and future perspectives<br />
Luiz Francisco M. L. Ciscato 1,* , Erick L. Bastos 2 & Josef Wilhelm Baader 1<br />
1<br />
Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, 05508-900 São<br />
Paulo / SP (Brazil)<br />
2<br />
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Rua Santa Adélia, 166,<br />
09210-170, Santo André / SP (Brazil)<br />
Photon upconversion is a photophysical process in which two photons combine in a nonlinear<br />
medium to produce one photon of higher energy. 1 Inorganic rare-earth-doped crystals (referred to<br />
as upconverting phosphors) are frequently used to produce upconversion luminescence using a<br />
single near-infrared excitation wavelength, and the mechanism of upconversion usually involves<br />
processes like sequential energy transfer, excited state absorption, and phonon interaction;<br />
alternatively, efficient photon upconversion systems can be based on sensitized triplet-triplet<br />
annihilation (TTA), which generally possess energy gaps of up to 0.8 eV.<br />
1,2-Dioxetanes are four-membered cyclic peroxides whose unimolecular or catalyzed<br />
decomposition results in chemiluminescence emission. 2 The thermal decomposition of those fourmembered-ring<br />
peroxides has sufficient energy to produce one of the two carbonyl fragments in its<br />
electronic excited triplet or singlet state. The methodology proposed here consists in irradiation of a<br />
methylene blue solution with low-energy red light in the presence of an appropriated olefin. 1,2-<br />
Cycloaddition reaction between the generated singlet oxygen and the olefin produces a 1,2dioxetane<br />
derivative; cleavage of this in situ produced 1,2-dioxetane leads to high-energy blue light<br />
emission. The overall effect of this procedure is the conversion of red excitation light into blue<br />
emission light, constituting an uphill energy conversion. 3 The observed emission half-life times<br />
obtained with this chemiluminescence method are four orders of magnitude higher than that<br />
achieved in TTA, enabling a much longer acquisition time. Moreover, the chemiluminescence<br />
method can be per<strong>for</strong>med in the presence of oxygen, which is actually required. Contrarily, careful<br />
degassing is necessary in the TTA based uphill energy emission, in order to increase the lifetime of<br />
the triplet species. These two aspects of our uphill energy conversion method are the most<br />
advantageous when compared to conventional photon upconversion methodologies like TTA.<br />
The quantum yields <strong>for</strong> this chemiluminescence-based energy upconversion are not yet<br />
known, as a complex reaction sequence is involved, making the exact determination of quantum<br />
yields extremely difficult. However, a rough estimate indicates that the efficiency should be around<br />
1 to 10%. These estimated quantum yields should be compared to those obtained in TTA based<br />
uphill energy conversion methods, which show similar, also somewhat lower values of around<br />
0.6%. Moreover, the anti-stokes shift of this new process can be as high as 1.1 V, meaning that the<br />
analytical application using red light activation and blue light measurement can have much higher<br />
values <strong>for</strong> the signal-to-noise ratio, lowering the detection limit of substances of interest. 4<br />
Additionally, the much longer emission lifetime and the possibility to utilize this method in airsaturated<br />
solutions makes it much more versatile and prone to the development of analytical and<br />
(bio)technological applications such as biolabels and optical oxygen sensors, in vitro and in vivo<br />
imaging based on resonance energy transfer detection, and security and/or cryptographic labeling.<br />
This work was supported by grants from CAPES, AiF, DAAD and FAPESP.<br />
References:<br />
[1] T. Soukka, et al., Ann. N.Y. Acad. Sci., 188 (2008) 1130<br />
[2] W. J. Baader, et al., in Chemistry of Peroxides (Wiley) 2 (2006) 1211<br />
[3] L. F. M. L. Ciscato, et al., J. Org. Chem. 75 (2010) 6574<br />
[4] L. F. M. L. Ciscato, et al., New J. Chem. 35 (2011) 773<br />
______________<br />
* Corresponding author: e-mail: ciscato@iq.usp.br<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 227
P114 Fluorescence Spectroscopy Poster 114<br />
Host-guest complexes of styryl dye with cucurbit[8]uril<br />
Denis A. Ivanov * , Nikolai Kh. Petrov & Sergey P. Gromov<br />
Photochemistry Centre RAS, Ul. Novatorov 7A, 119421 Moscow, Russia<br />
Pumpkin-shaped macrocycle of cucurbit[8]uril (CB[8]) has a rigid cavity (the internal diameter is 8.8<br />
Å, the portal diameter, 6.5 Å, the height, 9.1 Å) that allows producing 1:1 and 2:1 complexes with<br />
styryl dye as the guest-molecule [1].<br />
Figure 1. Fluorescence titration of styryl dye by cucurbit[8]uril.<br />
Photophysical properties of aqueous solution of styryl dye (12 μM) was studied in the presence of<br />
CB[8] by means of fluorescence spectroscopy. The complexation of dye by CB[8] results in the<br />
variation of dye-fluorescence quantum yields. There is a decrease in the fluorescence upon<br />
increasing the CB[8] concentration in the range from 0 to 50 µM. A further increase in CB[8]<br />
concentration leads to a significant fluorescence enhancement, in a manner similar to the influence<br />
of producing 1:1 complexes with CB[7] [2]. One can rationalize it assuming that 2:1 complexes<br />
largely exist <strong>for</strong> low CB[8] concentration and 1:1 complexes, <strong>for</strong> high CB[8] concentration. This<br />
model is in agreement with results on fluorescence anisotropy and lifetime measurements.<br />
Support from the Russian Foundation <strong>for</strong> Basic Research and the Russian Academy of Sciences is<br />
gratefully acknowledged.<br />
References: [1] S. P. Gromov, et al., Eur. J. Org. Chem., 13 (2010) 2587–2599. [2] D.A. Ivanov, et al., J.<br />
Phys. Chem. A, 115 (2011) 4505–4510.<br />
______________<br />
* Corresponding author: e-mail: ivanovd@photonics.ru<br />
228 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P115 Fluorescence Spectroscopy Poster 115<br />
Pluronic F127-β-cyclodextrins derivatives supramolecular inclusion<br />
complexes studied by fluorescence probe method<br />
Marilena Vasilescu* & Daniel G. Angelescu<br />
“Ilie Murgulescu” Institute of Physical Chemistry, Romanian Academy, Splaiul Independentei 202,<br />
060021 Bucharest Romania<br />
Pluronics are non-ionic polyoxyethylene(PEO)-polyoxypropylene(PPO)- polyoxyethylene(PEO)<br />
triblock copolymers that have many applications, especially pharmaceutical applications; Pluronic<br />
F127 is one of the most widely used of these copolymers. Cyclodextrins, by the central cavity of<br />
their molecules, can <strong>for</strong>m guest/host-type inclusion complexes. This work aimed at gaining insight<br />
into the complexation of F127 with hydrophilic derivatives of β-cyclodextrin (βCD) using the<br />
fluorescence probe method. The aqueous solubility of βCD is limited and the complexes resulting<br />
from the interaction of lipophiles with βCD can easily precipitate from water. The substitution of any<br />
of the hydrogen bonds <strong>for</strong>ming hydroxyl groups results in an improvement in their solubility. The<br />
water-soluble cyclodextrin derivatives studied in this work were : methyl-β-cyclodextrin,<br />
heptakis(2,6-di-O-methyl) β-cyclodextrin and heptakis(2,3,6-tri-o-methyl)- β-cyclodextrin. Our<br />
investigation focuses on the supramolecular host-guest inclusion complexes resulting from the<br />
interaction between βCD derivatives and Pluronic F127 micelles (the F127 concentration being 3%<br />
w/w). The influence of the methylation of βCD, [CD] /[F127] molar ratio and temperature (range 20-<br />
60 o C) on the Pluronic self-assembly was investigated using the pyrene and 1,10-bis-(1pyrene)decane<br />
fluorescence probes. The variation of their fluorescence parameters I1/I3 and IE/IM,<br />
respectively, provided in<strong>for</strong>mation on the micropolarity and microviscosity variation at the PPO-<br />
PEO interface, and thus highlighted the interaction of cyclodextrines-polymeric chains of micelles.<br />
As the micellization process of F127 is temperature-dependent [1], all measurements on F127-CD<br />
systems have been made comparatively with F127 aqueous solutions. The results were consistent<br />
with a temperature sensitive interaction between F127 micelles and βCD derivatives, greater at<br />
higher temperature. The interaction also depends on the [CD]/ [F127] molar ratio. Complexes have<br />
a good solubility in water, which confers important applications of these supramolecular structures,<br />
especially as drug delivery systems. The fluorescence measurements also evidenced that the<br />
nature of βCD derivatives plays an important role in the complexation and micellization processes.<br />
These features are in line with the reported results obtained by the SANS technique on similar<br />
systems [2,3] .<br />
This work was supported by the National University Research Council (CNCSIS) (Grant PN2 IDEI,<br />
Project code 1317).<br />
References: [1] M.Vasilescu et al Rev. Roum. Chim.,56 (1) (2011) 57-64.[2] C. Dreiss,et al.,Soft Matter, 5<br />
(2009)1888-1896. [3] Julie Joseph et al., Langmuir 23 (2007) 460-466.[3]<br />
______________<br />
*Corresponding author e-mail: vasilescu.marilena@gmail.com<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 229
P116 Fluorescence Spectroscopy Poster 116<br />
Fluorescence dynamics in supercooled molten mixtures<br />
Biswajit Guchhait & Ranjit Biswas<br />
S. N. Bose National Centre <strong>for</strong> Basic Sciences, JD Block, Sector III, Salt Lake, Kolkata,<br />
700 098 (India)<br />
Recently, we have explored the dynamics in several supercooled molten mixtures by following the<br />
fluorescence response of a dissolved solute probe in order to examine the existence of colossal<br />
static dielectric constant ( ε 0 ) and extremely slow time constant reported earlier in the dielectric<br />
relaxation measurements. [1] Our Steady state and time-resolved measurements [2-4] have revealed<br />
no such extreme medium polarity and slow relaxation times present in these molten mixtures. Both<br />
the average solvation ( τ s ) and rotation ( τ r ) times measured by using a probe, C153, in these<br />
p<br />
mixtures exhibit fractional power law dependence on medium viscosity ( τ x ∝ η , x being solvation<br />
or rotation) with p significantly differing from unity. In addition, strong decoupling between rotation<br />
and translation has also been observed. Subsequent applications of a semi-molecular theory<br />
reveal both the solute-medium dipole-dipole and ion-dipole interactions contribute significantly to<br />
the measured Stokes’ shifts and dynamics. Calculations in the underdamped and overdamped<br />
limits of frictional solvent response agree semi-quantitatively with those from time-resolved<br />
measurements. Subsequent simulation studies have revealed strong temporal heterogeneity<br />
present in these molten mixtures. Some of these results will be presented in this poster and<br />
discussed.<br />
We thank SNBNCBS, DST and CSIR <strong>for</strong> financial support; B. G. thanks CSIR <strong>for</strong> research<br />
fellowship.<br />
References: [1] G. Berchiesi, J. Mol. Liq. 83 (1999) 271. [2] H. Gazi, et al., Chem. Phys. Lett., 501 (2011)<br />
358. [3] B. Guchhait, et al., J. Phys. Chem. B., 114 (2010) 5066. [4] B. Guchhait, et al., 2011 (submitted).<br />
______________<br />
* Corresponding author: e-mail: ranjit@bose.res.in<br />
230 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P117 Fluorescence Spectroscopy Poster 117<br />
Modulation of ground and excited state dynamics of [2,2′-bipyridyl]-3,3′diol<br />
by micelles<br />
Dipanwita De 1 & Anindya Datta 1,*<br />
1 Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, (India)<br />
The molecule [2,2’-bipyridyl]-3,3’-diol (BP(OH)2) with a high C2h symmetry is found to exhibit<br />
excited state intramolecular double proton transfer (ESIDPT). [1] Its spectral behaviour and binding<br />
propensity towards hydrophobic centres have been exclusively studied in biological environments<br />
like cyclodextrins (CDs) and human serum albumin (HSA). [2] The strong green fluorescence in<br />
BP(OH)2 is due to the diketo <strong>for</strong>m <strong>for</strong>med after double proton transfer in the excited state. In this<br />
present investigation, the binding of BP(OH)2 with ionic and neutral surfactants like<br />
cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulphate (SDS) and triton X-100 (TX-<br />
100) has been studied by steady state and time resolved fluorescence spectroscopy. The<br />
absorption as well as emission spectra of BP(OH)2 are highly sensitive towards the variation of<br />
surfactant concentration and hydrophobicity of the environment. The fluorescent state of diketo<br />
<strong>for</strong>m gains stability in surfactant assemblies leading to a red shifted emission spectra. A sharp<br />
increase in the fluorescence quantum yield near critical micellar concentration (CMC) is<br />
encountered followed by saturation. This indicates a complete encapsulation of BP(OH)2 in the<br />
micelles. The maximum fluorescence quantum yield in anionic SDS is rationalized by the <strong>for</strong>mation<br />
of highly emissive cationic fluorophore at the Stern layer. The increase in quantum yield in neutral<br />
TX-100 is attributed to higher microviscosity experienced by the fluorophore in the palisade layer.<br />
A direct support in favour of this argument in TX-100 is provided by the viscosity dependence<br />
exhibited by the probe in different concentrations of sucrose solutions. CTAB exhibiting only<br />
hydrophobic effect shows least increase in qunatum yield of BP(OH)2 among all the surfactants.<br />
Time resolved fluorescence study of BP(OH)2 in micelles is used as a tool to monitor the extent of<br />
micellization in the lipophilic cavity. An increase in fluorescence quantum yield as well as lifetime of<br />
BP(OH)2 upon micellization indicates an enhanced extent of ESIDPT in hydrophobic medium.<br />
This work was supported by grants from the SERC, DST.<br />
References: [1] K. Rurack, et al., J. Phys. Chem. B 106 (2002) 9744. [2] O. K. Abou-Zied, J. Phys. Chem. B<br />
111 (2007) 9879.<br />
______________<br />
* Corresponding author: e-mail: anindya@chem.iitb.ac.in<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 231
P118 Fluorescence Spectroscopy Poster 118<br />
Fluorescence probes and microrheology with fluorescent particles in<br />
investigation of aggregation behavior in ionic surfactant/non-ionic<br />
surfactant/polyelectrolyte system<br />
Tereza Halasová, Dominika Pihíková, Jana Szewieczková & Filip Mravec<br />
Brno University of Technology, Faculty of Chemistry, Centre <strong>for</strong> Materials Research<br />
CZ.1.05/2.1.00/01.0012, Purkyňova 464/118, Brno, CZ-61200 (Czech Republic)<br />
This work is based on fluorescence and microrheology investigation of aggregation behavior of<br />
complex system, which is consists of non-ionic surfactant (Triton X-100), cationic surfactant<br />
(dioctadecyldimethylammonium chloride, DDAC), and polyanionic polymer (sodium<br />
polystyrenesulphonate, PSS). Aggregates and their aggregation processes are primarily<br />
investigated by the different fluorescence probes and techniques. Fluorescent probes pyrene,<br />
perylene, and nile red were used <strong>for</strong> the investigation. Pyrene, well known and the most exploited<br />
fluorescent probe [1] , were used to determine critical micelle concentration (CMC) as well as<br />
aggregation number of aggregates through the steady-state quenching experiments with quencher<br />
cetylpyridinium chloride [2] . Values CMC of surfactants and their mixtures, and the influence of PSS<br />
on the system Triton-DDAC has been determined. For comparison perylene and nile red were<br />
used to confirm pyrene results in the case of CMC. Also CMC of the CPC (cetylpyridinium<br />
chloride), which was used <strong>for</strong> fluorescence quenching in the determination of aggregate numbers<br />
of sugar surfactants, has been determined.<br />
Passive microrheology, as video particle tracking [3] , was used to compare results obtained<br />
from fluorescence probes data. As particles, fluorescently labeled polystyrene sulphonate was<br />
used to determine changes in solution, which were indicated by fluorescence probe method.<br />
Fluorescently labeled particles were selected because of increasing of measurement sensitivity.<br />
Results brings an interesting comparison between these two “insight probe techniques”.<br />
This work was supported by the project "Centre <strong>for</strong> Materials Research at FCH BUT" No.<br />
CZ.1.05/2.1.00/01.0012 from ERDF.<br />
References: [1] J. Aguiar, et al., J Colloid Interf Sci , 258 (2003) 116. [2] M.Wolszczak, J. Miller, J Photoch<br />
Photobio A 147 (2002) 45. [3] N. Willenbacher, C. Oelschlaeger, Curr Opin Colloid In 12 (2007) 43.<br />
______________<br />
* Corresponding author: e-mail: xchalasova@fch.vutbr.cz<br />
232 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P119 Fluorescence Spectroscopy Poster 119<br />
Interaction between amphiphilic fluorescent probes and biopolymer<br />
Filip Mravec, Hana Střondalová, Tereza Halasová & Jakub Mondek<br />
Brno University of Technology, Faculty of Chemistry, Centre <strong>for</strong> Materials Research<br />
CZ.1.05/2.1.00/01.0012, Purkyňova 464/118, Brno, CZ-61200 (Czech Republic)<br />
This paper is focused on the interaction of amphiphilic fluorescent probes with model biopolymer –<br />
hyaluronan. Main aim of this work is studying the interaction between hyaluronan and surfactants,<br />
promising system in the targeting drug delivery. Authors focused on the interaction between<br />
hyaluronan and amphiphilic fluorescent probe dodecyl acridine orange (DOA). This experiment is<br />
based on the hypothesis that DOA <strong>for</strong>ms dimers as well as acridine orange [1] itself and the wellknown<br />
derivative nonyl acridine orange [2] . Experiments with constant amount of the probe and<br />
increasing concentration of hyaluronan bring interesting results. Authors observed fluorescence<br />
intensity changes, directly related to the dimer <strong>for</strong>mation of probe which condensate on polyanionic<br />
chain, in wide concentration range of hyaluronan. In terms “P/D”, polymer binding site<br />
concentration (negatively charged carboxylic groups) to probe concentration, work covers range<br />
from 10 -1 to 10 3 . Obtained results had no simple trend, on contrary 5 different P/D regions had<br />
been found.<br />
At first, behavior of DOA corresponds to the expected behavior of acridine orange in the<br />
presence of polyanion [3] . First decreasing is, after the point of equivalency reaching, following by<br />
slightly increasing of fluorescence intensity. This is in agree with the model, which handle with the<br />
saturation of polymer binding sites by dimer and subsequent dimer breaking with increasing<br />
amount of these binding sites, presented in system. Expected increasing is followed by the next<br />
decreasing, next local minimum is reached and from the P/D = 3000 only sharp increasing is<br />
observed. This behavior is probably directly related to the behavior of hyaluronan and its<br />
con<strong>for</strong>mation behavior in this concentration range.<br />
As the secondary result was found point of equivalence, which indicate all accessible binding<br />
sites <strong>for</strong> amphiphiles are saturated and we have physically labeled hyaluronan. System at this<br />
state was selected to experiment with cationic surfactant cetyltrimethyammonium bromide (CTAB).<br />
To the system with constant value of P/D increasing amounts of CTAB were added. It was found<br />
that in a system of hyaluronan-DOA, the probe was pushed out of the complex by CTAB bromide,<br />
which was bounded instead.<br />
This work was supported by the project "Centre <strong>for</strong> Materials Research at FCH BUT" No.<br />
CZ.1.05/2.1.00/01.0012 from ERDF.<br />
References: [1] H. Yao, et al., J Colloid Interf Sci 307 (2007) 272. [2] P. Kaewsuya, et al., Anal Bioanal<br />
Chem 387 (2007) 2775. [3] R. Sjoback, et al., J Photoch Photobio A 142 (2001) 51.<br />
______________<br />
* Corresponding author: e-mail: mravec@fch.vutbr.cz<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 233
P120 Fluorescence Spectroscopy Poster 120<br />
I1/I5 ratio of pyrene monomer fluorescence as a new polarity scale <strong>for</strong><br />
solvents and colloidal systems<br />
Kenichi Nakashima*, Dian Liu & Yusuke Yonemura<br />
Department of Chemistry, Graduate School of Science and Engineering, Saga University, 1 Honjomachi,<br />
Saga 840-8502, Japan<br />
It is well known that pyrene monomer fluorescence shows a vibrational fine structure composed of<br />
five prominent bands [1]. Thomas group [2] and Winnik group [3] found that the intensity ratio<br />
between the first prominent band at approximately 375 nm (the band 1) and the third prominent<br />
band at approximately 385 nm (the band 3) is very sensitive to solvent polarity. They proposed that<br />
the intensity ratio (so-called I1/I3 ratio) can be employed as a polarity parameter <strong>for</strong> solvents and<br />
microheterogeneous systems including colloids, polymers and biomaterials. After the pioneering<br />
works of Thomas’ group [2] and Winnik’s group [3], the I1/I3 ratio has been widely used as a polarity<br />
scale <strong>for</strong> microenvironments of colloids, polymers, biomolecules, and other microheterogeneous<br />
systems. In spite of many ef<strong>for</strong>ts [1-3], however, there has never been a clear-cut explanation <strong>for</strong><br />
the sensitivity of the I1/I3 ratio to the polarity of solvents and microenvironments. One of the<br />
possible reasons <strong>for</strong> the sensitivity is that the band 1 is a <strong>for</strong>bidden 0-0 band of S1( 1 B3u) → S0( 1 Ag)<br />
electronic transition whereas the band 3 is a vibronic transition which is partially allowed by the<br />
vibronic coupling between S1( 1 B3u) and S2( 1 B2u) through b1g mode vibration [1, 2]. There<strong>for</strong>e, the<br />
band 1 is easily perturbed by the polar surroundings whiles the band 3 is not. As a result, the I1/I3<br />
ratio is strongly affected by the polar solvents. It is also reported that weak complexes are <strong>for</strong>med<br />
between pyrene and some polar solvents, and the complex <strong>for</strong>mation violates the selection rule <strong>for</strong><br />
electronic transition of pyrene resulting in the enhancement of the 0-0 band [4].<br />
In spite of the usefulness of the I1/I3 ratio, however, this parameter sometimes cannot be<br />
obtained with a fluorescence spectrophotometer having low band resolution, because the band 3 is<br />
heavily overlapped with the nearest band (the band 2) if measured with a low resolution<br />
instrument. It is necessary to use an instrument with the band resolution higher than 1.5 nm to<br />
obtain fine data of the I1/I3 ratio. There<strong>for</strong>e it is desirable to find an alternative parameter which can<br />
be measured with low resolution instruments.<br />
From a view point that the fifth prominent band (band 5) also belongs to a b1g mode [2], it is<br />
expected that the intensity ratio between the bands 1 and 5 (I1/I5 ratio) is sensitive to the polarity of<br />
surroundings as well. In this study, we observed pyrene monomer fluorescence at high and low<br />
band resolutions in various polar and nonpolar solvents, and examined the sensitivity of the I1/I5<br />
ratio to solvent polarity. Then, we tried to determine a critical micelle concentrations (CMC) of<br />
conventional surfactants by using the I1/I5 ratio. We revealed that the I1/I5 ratio shows a significant<br />
dependence on the solvent polarity even if measured with a low resolution instrument. We have<br />
succeeded in determining the CMCs of typical surfactants such as sodium dodecylsulfate (SDS)<br />
and cetyltrimethylammonium chloride (CTAC) in terms of the I1/I5 ratio.<br />
References: [1] A. Nakajima, Bull. Chem. Soc. Jpn. 44 (1971) 3272. [2] K. Kalyanasundaram and J. K.<br />
Thomas, J. Am. Chem. Soc. 99 (1977) 2039. [3] D. C. Dong and M. A. Winnik, Photochem. Photobiol. 35<br />
(1982) 17. [4] K. Nakashima and M. Koyanagi, Photochem. Photobiol. 44 (1986) 169.<br />
______________<br />
* Corresponding author: e-mail: nakashik@cc.saga-u.ac.jp<br />
234 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P121 Fluorescence Spectroscopy Poster 121<br />
Role of solvation dynamics in the kinetics of solvolysis reactions<br />
in microreactors<br />
Pramod Kumar Verma 1 , Abhinanadan Makhal 1 , Rajib Kumar Mitra 1 & Samir Kumar Pal 1,*<br />
Unit <strong>for</strong> Nano Science & Technology, Department of Chemical, Biological & Macromolecular<br />
Sciences, S.N. Bose National Center <strong>for</strong> Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata<br />
700098, India<br />
Reverse micellar (water pools stabilized in bulk organic phase by surfactants) systems have<br />
evolved as a potential reaction medium to carry out various chemical reactions otherwise difficult to<br />
control in homogeneous medium due to highly structured water molecules present in the system.<br />
The most extensively used surfactant is bis(2-ethylhexyl)-sulfosuccinate (AOT) because it can <strong>for</strong>m<br />
spherical Reverse micelles (RMs) in many nonpolar solvents over a wide range of w0 (w0 =<br />
[water]/[surfactant] and radius of water pool (r in Å) is empirically defined as, r ≈ 2×w0) values<br />
(Nave S et al., 2000). Solvent reorganization with fluorescent probes in RMs(Zhang J,Bright FV,<br />
1991) reveals two different solvation rates inside the RMs and these time scales are attributed to<br />
water bound to the polar head groups of AOT and bulk like water. While ordinary water molecules<br />
relax in the sub-picosecond time scale, the solvation dynamics of interfacial water molecules are<br />
several times slower and occur in the nanosecond time scale(Sarkar N et al., 1996). These slow<br />
solvation dynamics plays an important role in many natural biological processes e.g. electron<br />
transfer, ion transport, molecular recognition in hydrophilic cavities of proteins and membrane lipid<br />
bilayers. The origin of the slow component is associated with a dynamical equilibrium between<br />
bound and free type of water molecules. The transition of bound water to free type at the micellar<br />
interface with temperature is governed by an Arrhenius type of activation energy barrier crossing<br />
model (Nandi N,Bagchi B, 1997). Despite the large number of attempts made to study on the<br />
structure and dynamics of water inside RMs and their effect on the ordinary chemical reactions and<br />
the enzymatic activities, no such attempts has been made to show the reflection of bound to free<br />
water transition directly on a chemical reaction occurring inside RMs. It is there<strong>for</strong>e interesting to<br />
investigate how a temperature induced modification of the dynamics of interfacial water affects the<br />
reaction kinetics at the RM interface. In this contribution we attempt to correlate the dynamical<br />
states of water molecules in reverse micelle with a solvolysis reaction in accordance with the<br />
activation energy barrier crossing model at the micellar interface. Precise measurement of the<br />
different dynamical states of water molecules at the reverse micellar interface with various degrees<br />
of hydration is achieved through temperature dependent solvation dynamics (measurement of the<br />
time-dependent fluorescence of a solute dye molecule) of Coumarin 523. The fluorescence<br />
rotational anisotropy studies along with a wobbling-in-cone analysis show that the probe residing at<br />
the micellar interface pointing towards the core water experiences less microviscosity at elevated<br />
temperature. The consequences of the dynamical freedom of the water at elevated temperature in<br />
the solvolysis reaction of benzoyl chloride have also been explored. The accelerated rate of<br />
solvolysis has been correlated with the increased solvation dynamics, both of which are associated<br />
with a temperature induced transition of bound to free type water molecules at the micellar<br />
interface.<br />
We thank DST <strong>for</strong> financial grant (SR/SO/BB-15/2007). P.K.V thanks CSIR <strong>for</strong> research fellowship.<br />
References:[1] S. Nave, et al., Langmuir. 2000, 16, 8741-8748. [2] J. Zhang, et al., J. Phys. Chem. 1991,<br />
95, 7900-7907. [3] N. Sarkar, et al., J. Phys. Chem. 1996, 100, 10523-10527. [4] N. Nandi, B. Bagchi J.<br />
Phys. Chem. B. 1997, 101, 10954-10961.<br />
______________<br />
* Corresponding author: e-mail: skpal@bose.res.in<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 235
P122 Fluorescence Spectroscopy Poster 122<br />
Labeling o-dihydroxyphenyl compounds with biocompatible<br />
fluorogenic reactions<br />
A. Ulises Acuña 1 , Marta Liras 2 & Francisco Amat-Guerri 3<br />
1 Instituto de Química-Física «Rocasolano», CSIC, Serrano 119, 28006-Madrid (Spain)<br />
2 Instituto de Ciencia y Tecnología de Polímeros, CSIC, Juan de la Cierva 1, 28006-Madrid (Spain)<br />
3 Instituto de Química Orgánica General, CSIC, Juan de la Cierva 1, 28006-Madrid (Spain)<br />
Organic compounds containing the o-dihydroxyphenyl group (catechol) are widespread in Nature.<br />
Simple o-diphenol molecules, such as 3,4-dihydroxyphenylalanine (DOPA) and derivatives of 3,4dihydroxycinnamic<br />
acid, as well as more complex structures as those of many antioxidant stilbenes<br />
and flavonoids, are frequent constituents of vegetables. In vertebrates, DOPA and its metabolites<br />
(catecholamines) are involved as hormones or neurotransmitters in the regulation of many<br />
important functions. Detection of these compounds in complex media using the intrinsic<br />
fluorescence of the dihydroxyphenyl group is rarely possible, because the emission, if any,<br />
appears in the UV range (as expected), unless the catechol group is <strong>for</strong>ming part of a larger<br />
aromatic scaffold, as in the case of some flavonoids. Finding ways of attaching strongly emitting<br />
flurophores to simple catechols and catecholamines, excitable in the VIS range, would facilitate<br />
recording, <strong>for</strong> example, functional (concentration) images of these compounds in tissues and<br />
organismstaking advantage of the high sensitivity and lateral resolution of current fluorescence<br />
microscopy methods. There has been in the past several attempts in this direction [1] , but none of<br />
them has been completely successful in the imaging of samples of living tissues. We present here<br />
a set of novel fluorogenic reactions that may be developed <strong>for</strong> that specific purpose. These<br />
reactions are based on the oxidative coupling between simple resorcinol compounds and the<br />
corresponding o-dihydroxyphenyl group [2],[3] , and take place in water solution at room temperature.<br />
The coupling between resorcinol and hydroxytyrosol to yield a highly fluorescent oxacine shown in<br />
Scheme 1 illustrates these reactions. In general, the only reaction product is a rigid, strongly<br />
emitting four-ring structure, characterized by a large absorption coefficient in the VIS range and an<br />
intense fluorescence in the blue-green spectral domain, with a quantum yield in the 0.9-1.0 range.<br />
Examples of the fluorogenic reaction as applied to o-dihydroxyphenyl compounds of biological<br />
relevance would be presented, as well as the chemical and spectral properties of the emitting<br />
reaction products.<br />
OH<br />
O<br />
+<br />
OH<br />
HO OH<br />
OH HO O<br />
O<br />
OH<br />
resorcinol hydroxytyrosol<br />
fluorescent oxacine<br />
Scheme 1<br />
Work supported by Project CTQ2010/16547 from the Spanish MCINN.<br />
References<br />
[1] J.D.N. Kerr and W. Denk, Nature Rev. Neuroscience 9 (2008) 195; H. Nohta et al., Anal. Chim. Acta 267<br />
(1992) 137.<br />
[2] O. Crescenzi et al. Tetrahedron 47 (1991) 6243; A.U.Acuña et al. Org.Lett 11 (2009) 3022.<br />
[3] A.U. Acuña et al. (2011) to be published.<br />
______________<br />
* Corresponding author: e-mail roculises@iqfr.csic.es<br />
236 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P123 Fluorescence Spectroscopy Poster 123<br />
Novel fluorescent capture compound <strong>for</strong> mild labeling and visualization of<br />
functional subproteomes – physicochemical characterization and<br />
biochemical application<br />
Matthias Baranowski 1 , Thomas Lenz 1 , Marion Herrmann 2 , Oliver Klein 2 , Maik Berg 3 , Mathias<br />
Dreger 1 , Joachim Klose 2 , Hans-Gerd Löhmannsröben 3 & Michael Sefkow 1*<br />
1<br />
Caprotec bioanalytics GmbH, Volmerstrasse 5, 12489 Berlin, Germany<br />
2<br />
Institut für Humangenetik, Charité Universitätsmedizin Berlin, Campus Virchow-Klinikum,<br />
Augustenburger Platz 1, 13353 Berlin, Germany<br />
3<br />
Universität Potsdam, Institut für Chemie, Physikalische Chemie, Karl-Liebknecht-Str. 25, 14476<br />
Potsdam-Golm, Germany<br />
Nowadays, answering the question of small-molecule/protein interactions is one of the most<br />
important tasks in biochemistry. Though, in principle, it is possible to identify all proteins by 2D gel<br />
electrophoresis, in practice it is important to reduce the sample complexity to detect proteins with<br />
low abundance. One possibility to address just a defined class of proteins in a complex<br />
biochemical system offers “chemical proteomics”. In this approach, proteins that bind to small<br />
molecules, such as drugs, cofactors, inhibitors or second messengers, are addressed by chemical<br />
derivatives of these small molecules exhibiting additional functionalities, such as chemical or<br />
photo-crosslinking, sorting, or detection.<br />
Capture Compound Mass Spectrometry (CCMS) [1-4] is a novel technology <strong>for</strong> chemical<br />
proteomics that allows <strong>for</strong> functional reduction sample complexity and <strong>for</strong> isolation of target<br />
proteins. The core of this technology is a tri-functional molecule, the Capture Compound (CC),<br />
comprising the small molecule binding to the target proteins (the selectivity function), a photo-<br />
labile group <strong>for</strong> irreversibly attaching the CC to the reversibly bound proteins (the reactivity<br />
function), and a sorting or detection function. With these molecules, even low abundant but specific<br />
proteins (i.e. those binding the small molecule of interest) can be addressed, isolated and<br />
analyzed. Herein, we report novel CCs comprising a fluorophore as detection/sorting function.<br />
These compounds allow <strong>for</strong> detecting selectivity function-dependent specific subproteomes on 2D<br />
gels using otherwise standard fluorescence equipment. Interestingly, CCs having a phenyl azide<br />
moiety as reactivity function can be photo-activated at unprecedented long wavelengths. This<br />
finding has been biochemically employed and the mechanism physicochemically characterized.<br />
Typically, UV light is used <strong>for</strong> activation of the reactivity function; however, the UV irradiation may<br />
also damage the captured proteins by photochemical modification of aromatic amino acid side<br />
chains. There<strong>for</strong>e, in an application of the novel finding, activation of the present CC at long<br />
wavelengths (visible light) has been examined that provide extraordinarily mild functional labeling<br />
of a subproteome. The chemical and biochemical results will be reported.<br />
This work was supported by BMWi (ZIM program, KF2077501UL8), and Senator für Wirtschaft,<br />
Technologie und Frauen (Transferbonus program, 1096 and 1150)<br />
References: [1] H. Köster, et al. Assay Drug Dev. Technol. 5 (2007) 381. [2] Y. Luo, et al. Mol. Cell.<br />
Proteomics 8 (2009) 2843. [3] J. J. Fischer, et al. J. Proteome Res. 9 (2010) 806. [4] T. Lenz, et al.<br />
http://www.jove.com/details.stp?id=2264 doi: 10.3791/2264. J. Vis. Exp. 46 (2010).<br />
______________<br />
*Corresponding author: e-mail: michael.sefkow@caprotec.com<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 237
P124 Fluorescence Spectroscopy Poster 124<br />
A fluorescence spectroscopic investigation of the self-assembling<br />
properties of the tyrocidines, a group of antimicrobial<br />
cyclic decapeptides<br />
Bhaswati Bhattacharya 1 & Marina Rautenbach 1,*<br />
1 Department of Biochemistry, Stellenbosch University, Private Bag XI, 7602 Matieland (South Africa)<br />
The mode of action/activity of the tyrocidines (Trcs), a group of antimicrobial cyclic decapeptides, is<br />
influenced by their self-assembling behaviour. To address this issue, the aggregation/assembly<br />
behaviour of selected Trcs was studied using steady-state fluorescence spectroscopy in aqueous<br />
and membrane-mimetic environments. The critical aggregation/assembly concentrations (cac)<br />
were determined <strong>for</strong> the Trcs exploiting the fluorescence of an extrinsic fluorophore, 1,8anilinionaphthalenesulfonamide<br />
in aqueous environment. The trend in cac is found to follow the Trc<br />
activity trend towards Gram positive bacteria, Micrococcus luteus and strains of Listeria<br />
monocytogenes B73 and B73-MR1 (B. M. Spathelf, M. Rautenbach,, Bioorg. Med. Chem ., 17<br />
(2009) 5541). The assembly behaviour of Trcs containing one or two tryptophan residues were<br />
studied further. It was observed that their Trp residues redistribute into different environments with<br />
the increase in 2,2,2-trifluoroethanol (TFE) and deuterated TFE which provides a membranemimetic<br />
environment. The presence of divalent metal ions such as calcium was found to<br />
antagonise this assembly process. The fluorescence quenching constants determined in the<br />
membrane-mimicking environment, using acrylamide as fluorescence quencher, further supported<br />
the redistribution of the tryptophan residues in membrane-like environments and there<strong>for</strong>e<br />
indicates peptide con<strong>for</strong>mational changes. A con<strong>for</strong>mational change upon interaction with the Trc<br />
target membrane may <strong>for</strong>m part of Trc membrane-dependent mode of action towards living cells.<br />
This work was supported by grants from BIOPEP Peptide Fund and National Research<br />
Foundation, South Africa.<br />
______________<br />
* Corresponding author: e-mail: mra@sun.ac.za<br />
238 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P125 Fluorescence Spectroscopy Poster 125<br />
A fluorescence study of the structure and kinetics of eumelanin <strong>for</strong>mation<br />
Jens Sutter 1 , Tereza Bidláková 2 , Vlastimil Fidler 2 , Jan Karolin 1 & David J S Birch 1,*<br />
1 Photophysics Group, Dep’t of Physics, SUPA, University of Strathclyde, Glasgow, G4 0NG<br />
(Scotland). 2 Faculty of Biomedical Engineering, Czech Technical University in Prague, Kladno 2<br />
(Czech Republic)<br />
Melanin is an important pigment present in skin, hair, the eye and the brain. Although the<br />
constituents of melanin are quite well-known, its detailed structure has so far eluded all types of<br />
spectroscopy. Here we use Thioflavin T (ThT, figure 1a) fluorescence to provide evidence <strong>for</strong> a sheet<br />
like structure and reveal <strong>for</strong> the first time eumelanin’s synthesis kinetics. Synthesized in vertebrates<br />
by the enzyme tyrosinase catalyzing the oxidation of tyrosine and 3, 4-dihydroxy-L-phenylalanine,<br />
the latter readily auto-oxidises in the laboratory to <strong>for</strong>m structures such as 5,6- dihydroxyindole and<br />
5,6-dihydroxyindole-2-carboxylic acid. The oxidation of such primary structures is then thought to<br />
give rise to oligomers composed of 4 or 5 monomers. Despite widespread ef<strong>for</strong>t, the non-repeating<br />
nature of this heterogeneous biopolymer has so far defied attempts to identify a secondary molecular<br />
structure <strong>for</strong> melanin. Nevertheless progress is being made. Evidence from x-ray scattering, SEM<br />
and AFM measurements have suggested that a protomolecule is <strong>for</strong>med, which then assembles into<br />
planar sheets that stack due to π bonding. [1] More recently the protomolecule structure has been<br />
compared with porphyrins and the broad absorption spectra and other properties of melanin, such as<br />
its metal-ion binding and conductivity, explained in terms of the stacking of tetrameric porphyrins<br />
(figure 1b), three or four high, analogous to graphite. [2]<br />
As eumelanin is <strong>for</strong>med its tyrosine-like absorption (< 300 nm) and fluorescence (> 300 nm) give way<br />
to complex spectra that finally give a broad band absorption continuum throughout the visible and<br />
increasing towards the ultra-violet that characterises the photo-protection melanin provides.<br />
Concomitant with this polymerization melanin develops an unworkably low fluorescence quantum<br />
yield, at some excitation wavelengths ~ 10 -6 [3] . We have thus used an extrinsic probe, Thioflavin T<br />
(ThT), to monitor eumelanin synthesis and found its fluorescence to be described by a sigmoidal<br />
temporal dependence, reflecting the assembly of sheet structures (see figure 1c). This mirrors the<br />
use of ThT in detecting the <strong>for</strong>mation of β-sheets of beta–amyloid in fibrils associated with<br />
Alzheimer’s disease [4] and reveals a time-lag consistent with protomolecule <strong>for</strong>mation. In solution ThT<br />
has a low fluorescence quantum yield that is greatly enhanced by intercalating within sheet-like<br />
structures, in this case facilitated by ThT’s structural similarity to the sides of the proposed tetramer<br />
structure (figure 1a,b) <strong>for</strong> the melanin protomolecule. [2]<br />
This work was supported by grants from the EPSRC, SFC and the ERASMUS scheme.<br />
References: [1] C. M. R. Clancy and J. D. Simon, Biochemistry, 40 (2001) 13353. [2] E. Kaxiras et al., Phys.<br />
Rev. Letts. 97 (2006) 218102. [3] S. P. Nighswander-Rempel et al., J. Chem. Phys. 123 (2005) 194901. [4]<br />
M. Amaro et al., Phys. Chem. Chem.Phys. 13 (2011) 6434.<br />
______________<br />
* Corresponding author: e-mail: djs.birch@strath.ac.uk<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 239
P126 Fluorescence Spectroscopy Poster 126<br />
Fluorescence investigation of the nucleic acid annealing properties of<br />
the HIV-1 Tat protein<br />
Christian Boudier 1* , Roman Storchak 1 , Kamal K. Sharma 1 , Pascal Didier 1 , Nicolas Humbert 1 ,<br />
Florian Moser 1 , Jean-Luc Darlix 2 & Yves Mély 1<br />
1<br />
Laboratoire de Biophotonique et Pharmacologie, UMR-CNRS 7213, Faculté de Pharmacie,<br />
Université de Strasbourg, 74 route du Rhin, 67401 Illkirch-Cedex, France<br />
2<br />
LaboRetro, Unité de Virologie Humaine INSERM #758, Ecole Normale Supérieure de Lyon, 46<br />
allée d’Italie, 69364 Lyon, France<br />
The main function of the HIV-1 trans-activator of transcription (Tat protein) is to promote the<br />
transcription of the proviral DNA by the host RNA polymerase leading to the synthesis of large<br />
quantities of full length viral RNA. Tat is also thought to be involved in the reverse transcription<br />
reaction (RTion) by a still unknown mechanism. The recently reported nucleic acid annealing<br />
activity of Tat [1] might explain, at least in part, its role in RTion. To further investigate this possibility,<br />
we carried out a fluorescence study of the mechanism by which the full length Tat protein (Tat(1-<br />
86)) and the basic peptide(44-61) direct the annealing of complementary viral DNA sequences<br />
representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, essential<br />
<strong>for</strong> the early steps of RTion. Though both Tat(1-86) and the Tat(44-61) peptide were unable to melt<br />
the lower half of the cTAR stem, they strongly promoted cTAR/dTAR annealing through nonspecific<br />
attraction between the peptide-bound oligonucleotides. Using cTAR and dTAR mutants,<br />
we found that this Tat promoted-annealing proceeds through the thermally frayed 3’/5’ termini,<br />
resulting in an intermediate with 12 intermolecular base pairs, which slowly converts into the final<br />
extended duplex [2] , a mechanism rather similar to that already reported <strong>for</strong> NCp7 a major nucleic<br />
acid chaperone of HIV-1. We also found that Tat(1-86) was as efficient as NCp7, in promoting<br />
cTAR/dTAR annealing and that both proteins could act cooperatively during the annealing reaction.<br />
Taken together, our data are consistent with a role of Tat in the stimulation of the obligatory strand<br />
transfers during RTion.<br />
This work was supported by grants from ANRS<br />
References: [1] M. Kuciak et al., Nucleic Acids Research, 36 (2008) 3389. [2] C. Boudier et al., J. Mol. Biol.,<br />
400 (2010) 487.<br />
______________<br />
* Corresponding author: christian.boudier@unistra.fr<br />
240 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P127 Fluorescence Spectroscopy Poster 127<br />
Quantification of amino acids and vitamins in cell culture media using<br />
Excitation-Emission Matrix fluorescence spectroscopy and chemometrics<br />
Amandine Calvet 1, * & Alan G. Ryder 1,*<br />
1<br />
Nanoscale Biophotonic Laboratory, School of Chemistry, National University of Ireland, Galway,<br />
University Road, Galway (Ireland)<br />
Industrial mammalian cell culture uses complex media, such as eRDF, to support cell growth and<br />
product <strong>for</strong>mation as an integral part of the process and so the analysis of media variance/quality is<br />
of critical importance [1, 2] . The use of fluorescence Excitation-Emission Matrix (EEM) spectroscopy<br />
as a potential tool <strong>for</strong> the rapid, in-situ, and routine quantitative analysis of industrial cell culture<br />
media was investigated. There is a need to analyse the medium in its prepared state, and thus the<br />
presence of multiple chromophores in the sample leads to high absorbance values, causing large<br />
Inner Filter Effects (IFE). This introduces an intrinsic non-linearity into the EEM data which has to<br />
be addressed using chemometrics. Each fluorophore is affected differently by IFE, which requires<br />
that we adapt the chemometrics methods <strong>for</strong> each analyte. The first step was to use PARAFAC2<br />
on an EEM dataset collected from eRDF media of different concentrations, to determine the<br />
number of extractable components. These components are related to the various quantifiable<br />
fluorescent analytes, but are distorted by the various medium matrix effects induced by the high<br />
fluorophore and chromophore concentrations present. These results indicated that the<br />
quantification of tryptophan (Trp), tyrosine (Tyr), pyridoxine (Py), riboflavin (RF) and folic acid (FA))<br />
in the prepared eRDF media was possible. The various analytes were grouped together <strong>for</strong><br />
quantitative analysis according to fluorescence characteristics, with the stronger emitting Trp and<br />
Tyr being considered together. A Modified Standard Addition Method (MSAM) [3] coupled with<br />
NPLS (N-way Partial Least Square) was used in order to deal with the intrinsic IFE. Trp and Tyr<br />
were successfully quantified using the NPLS-2 algorithm with prediction errors of 3.1 and 4.3 %.<br />
The weaker fluorophores Py, RF and FA, could also be quantified using NPLS with errors of 3.2,<br />
11.5 and 2.8 %. This methodology represents an element of our long-term goal to develop a<br />
comprehensive and robust cell culture media characterisation method <strong>for</strong> routine industrial use.<br />
This work was carried out with the financial support of IRCSET (Irish Research Council <strong>for</strong><br />
Science, Engineering and Technology).<br />
References: [1] E. Read, et al., Biotechnol. Bioeng., 105 (2010) 285. [2] P.W. Ryan, et al., Anal. Chem., 82<br />
(2010), 1311. [3] V. Lozano, Anal. Chim. Acta, 610 (2008) 186.<br />
______________<br />
* Corresponding author: e-mail: a.calvet2@nuigalway.ie , alan.ryder@nuigalway.ie<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 241
P128 Fluorescence Spectroscopy Poster 128<br />
Characterisation and optimisation of fluorescent labelled bacterial<br />
glucose-binding protein in glucose sensing<br />
Jonathan Coulter 1 , Faaizah Khan 2 , Dalibor Panek 1 , Tania Saxl 2 , John Pickup 2 & David Birch 1<br />
1<br />
Photophysics Group, Department of Physics, University of Strathclyde, Glasgow G4 0NG, UK<br />
2<br />
Diabetes Research Group, King’s College London School of Medicine, Guy’s Hospital Campus,<br />
London SE1 1UL, UK<br />
Diabetes mellitus is an increasingly-common metabolic condition affecting some 250 million people<br />
worldwide. Effective treatment of diabetes is dependent on accurate blood-glucose monitoring: an<br />
ideal unattainable with current technology. An implanted continuous glucose sensor could satisfy<br />
an urgent clinical need if it provides rapid, safe and reliable detection of the glucose molecule. One<br />
approach we have been investigating is glucose detection using a bacterial glucose binding protein<br />
(GBP), where we have engineered a mutant (H152C/A213R/L238S) which shows a promising<br />
response to glucose in the human physiological range [1] . Attachment of an environmentallysensitive<br />
fluorophore (BADAN) near the glucose binding site has allowed characterisation of the<br />
glucose-response which increases the fluorescence emission and lifetime [2] . Recent published<br />
work has shown effective immobilisation of the GBP-BADAN on functionalised agarose and<br />
polystyrene beads [3] , creating a promising plat<strong>for</strong>m <strong>for</strong> a viable glucose fluorescence biosensor.<br />
Here we use fluorescence anisotropy decay to demonstrate the con<strong>for</strong>mational change<br />
occurring in GBP, when it closes upon binding glucose. We also report alternative dye labels such<br />
as Texas Red that are helping to increase our understanding of, and thereby optimise, the<br />
mechanisms involved.<br />
Acknowledgments: The authors acknowledge grant support from an EPSRC Science and<br />
Innovation Award and from the Diabetes Foundation.<br />
References: [1] Faaizah Khan, et al., Anal Biochem 2010, 399, 39-43. [2] Faaizah Khan et al. Biochem<br />
Biophys Res Commun. 2008 Jan 4;365(1):102-6. [3] Tania Saxl et al., Analyst, 2011, 136, 968<br />
242 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P129 Fluorescence Spectroscopy Poster 129<br />
Biophysical study of transport proteins from SLC11<br />
(SoLute Carrier 11) family<br />
Iva Doležalová, Věra Ňuňuková, Eva Urbánková, Ondřej Novák & Roman Chaloupka 1<br />
1<br />
Charles University, Faculty of Mathematics and Physics, Institute of Physics, Ke Karlovu 5,<br />
121 16 Prague 2, Czech Republic<br />
In this work, we have used fluorescence spectroscopy (also in combination with inductively<br />
coupled plasma mass spectroscopy) and different molecular fluorescent probes in order to study<br />
the functional properties of a bacterial membrane transport protein. We have studied the transport<br />
stoichiometry of prokaryotic protein MntH (Proton-dependent Manganese transporter) belonging to<br />
the SLC11/Nramp (Natural resistence-associated macrophage protein) family to find out more<br />
about the function of this membrane protein family. We determined the stoichiometric ratio using<br />
the pH-sensitive <strong>for</strong>m of Green Fluorescent Protein to measure the amount of protons transported<br />
into the cell and mass spectroscopy to measure the amount of transported divalent metal ions [1] .<br />
Furthermore, peptides corresponding to the selected transmembrane segments (TMS3 and TMS6)<br />
of the bacterial MntH adopt helical con<strong>for</strong>mation and are able to <strong>for</strong>m ion channels in the model<br />
membranes [2] . To study these ion fluxes through membranes in more details, we have used the<br />
fluorescent probes sensitive to membrane potential- and pH- changes such as diS-C3(3) or HPTS<br />
(pyranine), respectively and liposomes as suitable model membranes.<br />
Several previous studies indicated that the SLC11/Nramp membrane protein family including<br />
membrane proteins transporting the divalent metal ions together with protons into a cell presented<br />
somewhat unusual functional properties. For instance, the eukaryotic members of the family<br />
mediate the transport with variable stoichiometry (from 1:1 to 1:18 depending on experimental<br />
conditions) [3] , metal-independent proton transport and proton-independent metal transport were<br />
also described under specific conditions [4,5] . A spontaneous single-point mutation could convert<br />
eukaryotic Nramp into a calcium channel [6] . Furthermore, divalent metal ions play a major role in<br />
the cell metabolism and participate in many important intracellular processes, thus the study of<br />
function of proteins belonging to this family can advance knowledge not only of transport<br />
mechanism. The knowledge regarding mammalian members of the family contrasts with the<br />
relative paucity of data about bacterial Nramp prototypes. Hence, our work focuses on functional<br />
properties of bacterial homolog MntH from E. coli as a model <strong>for</strong> studying transport stoichiometry,<br />
uncoupled proton flux and the effect of calcium ions on coupled and uncoupled proton flux<br />
mediated by the transporter, moreover the functional properties of channels <strong>for</strong>med by the selected<br />
transmembrane segments of MntH in model membranes of liposomes<br />
More specifically, we have studied the wild type MntH from E. coli and the protein with singlepoint<br />
mutation N401G which possibly differs in function from the wild type. We have discovered the<br />
effect of external pH on the variability of the transport stoichiometry, the inhibiton of uncoupled<br />
proton flux caused by an addition of calcium under the experimental conditions, where uncoupled<br />
proton flux mediated by MntH was already observed. Interestingly, we have observed the<br />
activation of the coupled proton flux after the addition of calcium ions. Surprisingly, the transport<br />
stoichiometry of neither the wild type nor the protein with mutation N401G is affected by the<br />
addition of calcium or the change of the external medium.<br />
First experiments on liposomes indicated, that the peptides corresponding to the selected<br />
transmembrane segments of MntH possibly <strong>for</strong>m a channel, which allows proton passage.<br />
References: [1] R.Chaloupka, et al., Biochemistry 44 (2005) 726-733. [2] V.Ňuňuková, et al., Biopolymers<br />
93 (2010) 718-726. [3] X.Z.Chen, et al., J. Biol. Chem. 274 (1999) 35089-35094. [4] H.Gunshin, et al., Nature<br />
388 (1997) 482-488. [5] B.Mackenzie, et al., Pflugers Arch. 451 (2006) 544-558. [6] H.Xu, et al., PLoS Biol. 2<br />
(2004) 378-386.<br />
______________<br />
Corresponding authors: e-mail: roman.chaloupka@mff.cuni.cz ; ; iva.dolezalova@mff.cuni.cz<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 243
P130 Fluorescence Spectroscopy Poster 130<br />
Time-resolved fluorescence spectroscopy: a tool <strong>for</strong> the design and<br />
evaluation of a homogeneous immunoassay <strong>for</strong> the detection of GnRH-1<br />
Peter D. Dowd 1* , Jan Karolin 2 , Carol Trager-Cowan 2 , David J.S. Birch 2 & William H. Stimson 3<br />
1<br />
Department of Bioengineering, Bioengineering Unit, Wolfson Centre, University of Strathclyde,<br />
106 Rottenrow, Glasgow G4 0NW, UK<br />
2<br />
Department of Physics, University of Strathclyde, Glasgow G4 0NG, UK<br />
3<br />
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow<br />
G4 0RE, UK<br />
The homogeneous assay plat<strong>for</strong>m has been identified as having the potential to make a dramatic<br />
impact in the field of medical diagnostics, especially point-of-care testing (POCT), by eliminating<br />
multiple washing steps and thus greatly simplifying the pre-measurement method [1] . Time-resolved<br />
fluorescence techniques are ideally suited to the study of homogeneous solutions and there<strong>for</strong>e as<br />
a method to aid in the evaluation and design of certain immunoassays <strong>for</strong> POCT. To demonstrate<br />
the practicality of this method we apply these techniques to the evaluation of an assay <strong>for</strong> the<br />
detection of Gonadotropin-releasing Hormone, type-1, (GnRH-1) which is found in cancers of the<br />
breast, uterus and prostate.<br />
In this instance, a synthetic labelled 9-amino acid ‘fragment’, [des-pGlu 1 ]-LH-RH-Acp-FITC<br />
(LP), is introduced to compete with GnRH-1 <strong>for</strong> the two binding sites on the GnRH-1 specific<br />
antibody, 7B10.1D10 [2] (Ab). LP provides an extrinsic fluorescence measurand <strong>for</strong> time-correlated<br />
single photon counting experiments. Fluorescence lifetime decay and anisotropy decay<br />
measurements [3] are used to simultaneously describe the presence of free LP and the bound LP in<br />
a solution of LP and Ab (Figure 1). Furthermore, by taking into account both the situation where<br />
energy migrates between fluorophores when LP is bound to both Ab sites and the “Hook Effect”,<br />
we are able to evaluate potential initial conditions <strong>for</strong> the practical immunoassay. We also<br />
demonstrate the effect of disruption to the system (initial condition LP/Ab sites equal to 0.01) by the<br />
addition of GnRH-1.<br />
A B<br />
0 4 8 12 16<br />
Time (ns)<br />
r(t)<br />
0.2<br />
244 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
0.4<br />
0.0<br />
0.01<br />
0.2<br />
0.4<br />
0.6<br />
1.00<br />
1.59<br />
LP<br />
Figure 3: The time-resolved fluorescence anisotropy decays <strong>for</strong> LP in solution with Ab are compared with<br />
the anisotropy of LP only in solution (plot A). The time-resolved fluorescence lifetime decays <strong>for</strong> all sets are<br />
recorded in table B.<br />
This work was supported by the EPSRC.<br />
LP/Ab<br />
sites<br />
f1 (%) τ1<br />
(ns)<br />
References: [1]. St-Louis P., Clinical Biochemistry, 33(6) (2000) 427-440. [2]. Kahn M.A.H., et al., AJRI 49<br />
(2003) 239-248 [3]. Apperson K., et al., Meas. Sci. Technol. 20 (2009) 025310.<br />
______________<br />
*Corresponding author: e-mail: pd.dowd@strath.ac.uk<br />
f2<br />
(%)<br />
τ2 (ns) χ 2<br />
0.01 94.4 4.06 5.5 1.04 1.16<br />
0.20 94.1 4.01 5.9 1.03 1.18<br />
0.30 93.0 3.94 7.0 1.00 1.18<br />
0.40 92.0 3.82 8.0 0.85 1.20<br />
0.60 90.1 3.67 9.8 0.95 1.28<br />
0.80 88.7 3.53 11.0 0.96 1.29<br />
1.00 89.2 3.43 10.8 0.80 1.29<br />
1.30 90.0 3.30 10.1 0.66 1.40<br />
1.59 89.3 2.85 10.7 0.58 1.17<br />
LP 95.1 2.93 8.5 0.61 1.16
P131 Fluorescence Spectroscopy Poster 131<br />
Self-association of the hsRad51 recombinase on ssDNA. Studies by<br />
combined high pressure and fluorescence methods<br />
G. Schay 1 , M. Fekete 1 , J. Kardos 2 , M.S.Z. Kellermayer 1 , Cs. Pongor 1 & J. Fidy 1,3,*<br />
1 Dept. of Biophysics and Radiation Biology, Semmelweis University, Budapest<br />
2 Institute of Biochemistry, Eötvös Loránd University, Budapest<br />
3 Biomembrane Research Group, HAS, Budapest<br />
HsRad51 is a protein that plays key role in the homologous recombinational repair (HRR) of double<br />
strand DNA breaks (dsb) in human cells. It was reported that the presence of Rad51 homologues<br />
is essential <strong>for</strong> cell viability in higher eukaryotes. It is supposed that in vivo, the recombination<br />
process is initiated by the <strong>for</strong>mation of 3’ single stranded (ss)DNA overhangs which are then<br />
recognized by Rad51 with the help of other interacting proteins like RPA. Rad51 then <strong>for</strong>ms a<br />
helical mucleoprotein filament around ssDNA. In concert with other key players, the nucleoprotein<br />
filament then binds the dsDNA substrate, recognizes homology and exchanges the DNA strands in<br />
an ATP dependent manner. It was shown that in higher eukaryotes HRR requires the presence of<br />
the breast cancer associated protein BRCA2. Based on cocrystallization and structure<br />
determination of the complex of BRCA2 and the ATP-binding domain of HsRad51 it was suggested<br />
that the self-association of Rad51 is regulated by the repeats of BRCA2 intruding into the interface<br />
of polymer <strong>for</strong>mation (1). Such results suggest that the interface of homo-polymerization may be an<br />
important site of the regulation of HRR. The present study was based on the reported finding that<br />
in the absence of DNA, Rad51 <strong>for</strong>ms ring-like self-aggregates in solution by the same interfaces as<br />
on DNA. In the experiments, HsRad51 was produced by bacterial expression and purification in the<br />
lab. Fluorescence spectra, lifetime and anisotropy decay measurements were used to characterize<br />
the protein-protein association, and DNA-protein filament <strong>for</strong>mation. The fluorescence of ANS<br />
bound to the protein, the Tyrosine emission of the wild type protein, FRET pairs of fluorescence<br />
labels bound to the protein and to DNA were used. The topology of ring-like protein self-associates<br />
and DNA-filaments was characterized by dynamic light scattering and AFM. Fluorescence label<br />
studies combined with high pressure were used to determine the dissociation constant of the<br />
interface (2) and the effect of nucleoprotein <strong>for</strong>mation on the protein interface – interaction.<br />
This work was supported by OTKA (Hung.Science Foundation) grant no. K 84271<br />
References: [1] L. Pellegrini et al. Nature, 420, (2002) 287-293), [2] G. Schay et al., JBC 281, (2006),<br />
25972-25983<br />
______________<br />
*Corresponding author: e-mail: judit.fidy@eok.sote.hu<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 245
P132 Fluorescence Spectroscopy Poster 132<br />
Measurement of Trp-Trp distance using homo-FRET and<br />
photobleaching: observation of RTX toxin folding<br />
Lucia Motlová 1 *, Eliška Doktorová 1 , Radovan Fišer 1 , Ladislav Bumba 2 & Ivo Konopásek 1<br />
1<br />
Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague,<br />
Viničná 5, 128 44 Prague 2, Czech Republic<br />
2<br />
Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague 4, Czech<br />
Republic<br />
The homo-FRET studies are usually used <strong>for</strong> the measurement of the distance of two fluorophores<br />
of the same kind. The decrease of steady state fluorescence anisotropy (or polarization) is almost<br />
the exclusive measure of the homo-FRET [1], although time-resolved approach is sometimes used.<br />
The increased anisotropy value if fluorophore is excited at the red-edge of its absorption<br />
spectrum is used to estimate the contribution of molecule rotation to the fluorescence<br />
depolarization, since under these conditions the FRET efficiency is suppressed. The ratio of<br />
polarizations obtained at the red-edge and “normal” excitation wavelengths could be used to<br />
estimate the homo-FRET efficiency [2]. But this approach may be difficult because of low<br />
fluorescence intensities after red-edge excitation especially if proper blank samples are not<br />
available (e.g. protein with only one fluorophore).<br />
Another approach uses intended photobleaching or chemical inactivation of the fluorophores<br />
thus reducing the number of molecules capable of energy transfer [3]. We developed a simple<br />
model describing change of fluorescence anisotropy during photobleaching of the two fluorophores<br />
(e.g. tryptophans) on one protein molecule with different susceptibility to the photodegradation<br />
and/or influenced by different segmental motion. This model takes into account possible<br />
distributions of distances and dihedral angles of the tryptophan fluorophores.<br />
We analyzed the fluorescence properties of two Trp residues of 150AA long protein “CCyaA”<br />
derived from RTX toxin (CyaA) produced by bacterium Bordetella pertussis. CCyaA structure is not<br />
known but we obtained 3D model using homology modeling. CCyaA is folded in presence of Ca 2+<br />
ions into the <strong>for</strong>m of “β-sheet helix”, where individual loops between β-sheets are responsible <strong>for</strong><br />
Ca 2+ chelation. The transition from unfolded to folded state of CCyaA is accompanied by relatively<br />
small increase of anisotropy (r295nm 0.03→0.07) and considerable blue shift of Trp emission<br />
spectrum, as expected. During the Ca 2+ induced folding the Trp-Trp distance measured by homo-<br />
FRET is shortened to ~1 nm in accordance with the 3D model. Interestingly, in the folded state<br />
CCyaA shows double-exponential bleaching kinetics, the effect not observed with the singletryptophan<br />
CCyaA mutant. This suggests that each of Trp residues occupies different<br />
microenvironment in the protein - the observation also supported by the spectra decomposition.<br />
Moreover, the two Trp residues seems to be stacked to an adjacent arginine (Arg) residue, since<br />
the substitution of Arg to other amino-acids leads to the enhanced segmental motion of Trp and<br />
destabilization of the whole CCyaA structure. These three amino-acids (Trp, Arg, Trp) probably<br />
play crucial role in the folding and stability of the whole CCyaA protein.<br />
This work was supported by projects of the Ministry of Education, Youth, and Sports of the Czech<br />
Republic No. LC06034 and MSM 0021620858.<br />
References: [1] Weber, G. (1954). Trans Faraday Soc 50, 552-555. [2] Moens et al. (2004) Protein J 23, 79-<br />
83. [3] Sharma et al. Cell (2004) 116, 577-589.<br />
______________<br />
* Corresponding author: e-mail: motlovalucinka@seznam.cz<br />
246 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P133 Fluorescence Spectroscopy Poster 133<br />
Fluorescence properties of new phenothiazinyl-porphyrine derivatives<br />
Emese Gál, Balázs Brém, Luiza Gãina, Tamás Lovász, Castelia Cristea & Luminiţa Silaghi-<br />
Dumitrescu<br />
Faculty of Chemistry and Chemical Engineering, Babes-Bolyai University, 400028, Cluj-Napoca,<br />
Arany János street 11, Romania<br />
Porphyrins comprise an important class of molecules that serve nature in a variety of ways. The<br />
metalloporphyrin ring is found in a variety of important biological systems where it is the active<br />
component of the system or in some ways intimately connected with the activity of the system.<br />
Many of these synthesized porphyrins are similar to the basic structure of biological porphyrins<br />
which are the active sites of numerous proteins, whose functions range from oxygen transfer and<br />
storage (hemoglobin and myoglobin) to electron transfer (cytochrome c, cytochrome oxidase) to<br />
energy conversion (chlorophyll). They also have been proven to be efficient sensitizers and<br />
catalyst in a number of chemical and photochemical processes especially photodynamic therapy<br />
(PDT) [1] . Porphyrins are well known <strong>for</strong> their intense colors, which is a consequence of extended<br />
conjugation of the macrocycle, and their applications key. The role of porphyrins in photosynthetic<br />
mechanisms indicates a good attitude of these molecules to mediate visible photon – electron<br />
energy transfer processes. In the UV-Vis spectra of porphyrins are two distinct areas: the purple<br />
region, a very intense absorption band known as the Soret band extinction coefficient of about<br />
1x10 5 M -1 cm -1 , while variations of the peripheral substituents on the porphyrin ring often cause<br />
minor changes to the intensity and wavelength of the absorption features, protonation of two of the<br />
inner nitrogen atoms or the insertion/change of metal atoms into the macrocycle usually strongly<br />
change the visible absorption spectrum. The electronic absorption spectrum of a typical porphyrin<br />
consists of a strong transition to the second excited state (S0 → S2) at about 400 nm (the Soret or<br />
B band) and a weak transition to the first excited state (S0 → S1) at about 550 nm (the Q band).<br />
Internal conversion from S2 to S1 is rapid so fluorescence is only detected from S1 [2] . The<br />
fluorescence of free porphyrins is observed at room temperature. A minor complication observed is<br />
that some of these compounds are exposed to photodegradation (in dark purple color changes<br />
to green), compounds should be kept in the dark, except <strong>for</strong> measurements The fluorescence<br />
quantum yield (ΦF) of the synthesized phenothiazinyl-porphyrine derivative was calculated by a<br />
comparison of the area below the corrected emission spectrum in dichloromethane with that of<br />
TPP, as a fluorescence standard, exciting at λex =520 nm [3] , by using the steady-state comparative<br />
method [4] . UV-visible spectra were recorded on a UV/VIS PERKIN ELMER, LAMBDA 35<br />
spectrometer, fluorescence spectra were recorded on a PERKIN ELMER Model LS 55 apparatus<br />
in a 1 cm cuvette.<br />
This work was supported by grants from Romanian Ministry of Education Research, Youth and<br />
Sports, PCCE 140/2008.<br />
References: [1] K. M. Smith, Porphyrins and Metalloporphyrins, Ed. (Elsevier, Amsterdam, 1975), M.<br />
Boulton et al., J. Photochem. & Photobio. B: Biology, 64 (2001)144, E.I. Sagun et al., Chem. Phys. 275<br />
(2002) 211. [2] D. F. Marsh and L. M. Mink, J. Chem. Ed., 73 (1996) 1181. [3] J. N. Demas, G. A. Crosby, J.<br />
Phys. Chem. 75 (1971) 991. [4] D. Tatman et. al Photochem. Photobio. 68 (1998) 459.<br />
______________<br />
* Corresponding author: e-mail: gal_emese@yahoo.com<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 247
P134 Fluorescence Spectroscopy Poster 134<br />
Front-surface long-wavelength fluorescence immunoassay <strong>for</strong><br />
monensin determination<br />
J. Godoy-Navajas 1 , M. P. Aguilar-Caballos 1 & A. Gómez-Hens 1*<br />
1 Analytical Chemistry Department, Institute of Fine Chemistry and Nanochemistry (IAQFN).<br />
Campus of Rabanales. Marie Curie Building (Annex). University of Cordoba. 14071-Cordoba,<br />
Spain<br />
A long-wavelength heterogeneous fluoroimmunoassay <strong>for</strong> monensin determination in food samples<br />
using nile-blue doped silica nanoparticles (NPs) is described. A luminescent tracer is synthesized<br />
by conjugating anti-monensin antibodies to amino-group functionalized nile blue-doped silica NPs,<br />
which have been previously synthesized using a reverse-micelle method [1]. Two different <strong>for</strong>mats,<br />
with antigen and antibody capture, have been assayed using black and shallow Proxy-plate 96-well<br />
microplates as solid supports. The first assay relies on the immobilization of anti-sheep IgG<br />
previously to the incubation of sheep anti-monensin antibodies. Then, a mixture of monensin and<br />
tracer (monensin bound to nile blue-doped silica NPs) is added and, after subsequent incubation<br />
and washing steps, the fluorescence of the bound tracer fraction is measured onto the dry surface<br />
of the well. The second assay <strong>for</strong>mat relies on the competition of the monensin present in the<br />
samples with a monensin-BSA conjugate, which has been previously immobilised onto the well<br />
surface, <strong>for</strong> the active sites of anti-monensin antibodies. In both instances, the fluorescence signal<br />
obtained can be correlated to the analyte concentration. The best results have been achieved with<br />
the antibody capture heterogeneous immunoassay, which general scheme is depicted in the<br />
Figure:<br />
sample + tracer<br />
incubation washing<br />
measurement<br />
After the optimization of the variables involved, the method features a detection limit of 0.014 ng<br />
mL -1 and a dynamic range from 0.1 to 5 ng mL -1 . The precision of the method, at two different<br />
analyte concentrations, 0.2 and 1 ng mL -1 , and expressed as relative standard deviation, has given<br />
values between 4.0 and 5.9%. The method has been satisfactorily applied to the analysis of food<br />
samples, which requires a simple extraction step in order to remove the proteins from samples.<br />
This work has been supported by the Spanish MICINN (Grant No. CTQ2009-08621) and by the<br />
Junta of Andalucia (Grant No. P09-FQM4933) and from the FEDER-FSE Program (Grant No. P09-<br />
FQM4933). J. Godoy Navajas thanks the Junta of Andalucia (Grant No. P09-FQM4933) <strong>for</strong> the<br />
financial support of his pre-doctoral fellowship.<br />
References: [1] J. Godoy-Navajas et al., J. Fluoresc., 20 (2010) 171.<br />
______________<br />
* Corresponding author: e-mail: qa1gohea@uco.es<br />
λ ex 620<br />
248 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
λ em 680 nm
P135 Fluorescence Spectroscopy Poster 135<br />
Effect of the medium pH on the fluorescence of betalains<br />
Letícia Christina Pires Gonçalves 1 , Nathana Barbosa Lopes 1 , Bruno Martorelli Di Genova 1 ,<br />
Luiz Francisco Monteiro Leite Ciscato 2 & Erick Leite Bastos 1,*<br />
1 Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, SP (Brazil)<br />
2 Instituto de Química, Universidade de São Paulo, São Paulo, SP (Brazil)<br />
Betalains are water-soluble natural pigments found in some species of Caryophyllales plants and<br />
basidiomycete fungi. [1] The interest in these natural pigments was recently renewed due to both<br />
their antioxidant properties and its occurrence in fluorescent flowers. [2, 3] Betalains have been<br />
classified as red-violet betacyanins or yellow betaxanthins according to their chemical structure.<br />
The betacyanins (λabs ≈ 540 nm) are non-fluorescent glycosylated derivatives of betanidin, which is<br />
the iminium adduct of betalamic acid and cyclo-DOPA. Conversely, betaxanthins are green<br />
fluorescent pigments (λabs ≈ 470 nm, λem ≈ 520 nm) originating from the condensation of betalamic<br />
acid and amino acids or amines.<br />
The medium pH is an important factor influencing the properties of betalains. [4] There<strong>for</strong>e, in this<br />
work, we report the effect of pH on the fluorescence properties of betanin (Bn, betanidin 5-Oglucoside),<br />
indicaxanthin (BtP, L-Pro betaxanthin), and of an artificial semisynthetic betalain (BtPh,<br />
phenolic betaxanthin).<br />
The study was carried out using the Britton-Robinson (BR) buffer over the pH range 2 to 10.<br />
Betanin was purified from fresh beetroot extract using RP-HPLC and used in the semisynthesis of<br />
BtP and BtPh. All compounds were purified and characterized by HRMS and NMR spectroscopy.<br />
The maximum fluorescence wavelengths are 615 nm (λexc = 520 nm) <strong>for</strong> Bn, 520 nm (λexc = 475<br />
nm) <strong>for</strong> BtP and 560 nm (λexc = 500 nm) <strong>for</strong> BtPh and do not depend on the medium pH. The<br />
fluorescence quantum yield of BtP is about two orders of magnitude higher than that of Bn and<br />
BtPh. In acidic media (pH < 4) all three betalains show a similar decrease in the fluorescence<br />
intensity with the increase of medium acidity (Figure 1). On the other hand, Bn and BtPh show<br />
significant decrease in fluorescence intensity in alkaline media (pH > 8), whereas BtP does not.<br />
These results can be rationalized considering the following factors: i) the role of electron donating<br />
groups on the charge transfer character of the π-π* transition of betalains, and ii) the relative<br />
stability of betacyanins and betaxanthins (1 o /2 o ) to alkaline hydrolysis.<br />
Figure 1 – Effect of the medium pH on the normalized<br />
fluorescence intensity of Bn, BtP, and BtPh.<br />
This work was supported by grants from<br />
FAPESP, CAPES, CNPq, and UFABC.<br />
References: [1] D. Strack, et al., Phytochemistry, 62 (2003) 247. [2] J. Escribano, et al., Phytochem. Anal. 9<br />
(1998) 124. [3] F. Gandia-Herrero, et al., Nature 437 (2005) 334. [4] K. M. Herbach, et al., J. Food Chem., 71<br />
(2006) R41.<br />
______________<br />
*Corresponding author: e-mail: erick.bastos@ufabc.edu.br<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 249
P135B Fluorescence Spectroscopy Poster 135 B<br />
Fluorescence polarization approaches to high-throughput screening<br />
(HTS) <strong>for</strong> development of natural kinase inhibitors<br />
Nam Joo Kang 1 , Sohee Baek 2 & Ki Won Lee 2,3<br />
1<br />
School of Food Science and Biotechnology, Kyungpook National University, Daegu (Republic of Korea)<br />
2<br />
Food Science and Biotechnology Program, Department of Agricultural Biotechnology, Seoul National<br />
University, Seoul (Republic of Korea)<br />
3<br />
Advanced Institutes of Convergence Technology, Seoul National University, Suwon (Republic of Korea)<br />
Target-based therapies are widely considered to be the future of cancer treatment. As many of the<br />
genetic alterations found in cancers involve genes whose products are regulators of signal<br />
transduction [1] , much of the focus in the development of novel therapeutics has involved inhibitors<br />
of signal transduction molecules, in particular protein kinases [2] . The human kinome is comprised of<br />
over 518 protein kinases, with disease associations reported <strong>for</strong> over 150 kinases. Presently, the<br />
Food and Drug Administration (FDA) has approved 10 protein kinase inhibitors and over 100<br />
kinase-targeted agents are currently undergoing clinical evaluation.<br />
Mitogen-activated protein kinase (MAPK) cascades are key signaling pathways involved in the<br />
regulation of normal cell proliferation, survival and differentiation. Aberrant regulation of MAPK<br />
cascades contribute to cancer and other human diseases. In particular, the MAPK/ERK kinase<br />
(MEK) and c-Jun amino-terminal kinases (JNKs) have been the subject of intense research<br />
scrutiny leading to the development of pharmacologic inhibitors <strong>for</strong> the treatment of cancer. Here,<br />
we focused on developing high-throughput screening (HTS) method <strong>for</strong> discovering MEK or JNK<br />
natural inhibitors. IMAP is a technology based on the specific, covalent-coordinate, high-affinity<br />
interaction of trivalent metal containing nanoparticles with phosphogroups. Addition of the<br />
Immobilized metal ion affinity-based fluorescence polarization (IMAP) Binding System stops the<br />
kinase reaction and specifically binds the phosphorylated substrates. Though IMAP systems are<br />
very simple and useful, method development of HTS is missing <strong>for</strong> screening of natural kinase<br />
inhibitors. To address this, IMAP-based FP assay was carried out and established reaction<br />
conditions <strong>for</strong> virtual screening. Based on the developed method, we selected natural inhibitors of<br />
MEK or JNK. These are caffeic acid and quercetagetin.<br />
This work was supported by grants from the Basic Science Research Program (2010-0011345),<br />
World Class Institute Program, and Advanced Institutes of Convergence Technology, Republic of<br />
Korea.<br />
References: [1] Hanahan D and Weinberg RA., Cell 100 (2000) 57-70. [2] Arslan MA et al., Curr Cancer<br />
Drug Targets 6 (2006) 623–634.<br />
______________<br />
* Corresponding authors: e-mail: njkang@knu.ac.kr ; kiwon@snu.ac.kr<br />
250 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P136 Fluorescence Spectroscopy Poster 136<br />
Fluorescence lifetime based glucose sensing in diagnostic test strips<br />
Alexa von Ketteler 1,2,* , Dirk-Peter Herten 3 & Wolfgang Petrich 1,2<br />
1<br />
Kirchhoff Institute <strong>for</strong> Physics, Heidelberg University, Im Neuenheimer Feld 227, 69210<br />
Heidelberg (Germany)<br />
2<br />
Roche Diagnostics, Sandhofer Straße 116, 68305 Mannheim (Germany)<br />
3<br />
Cellnetworks Cluster and Institute <strong>for</strong> Physical Chemistry, Heidelberg University, Im Neuenheimer<br />
Feld 267/ BQ0007, 69210 Heidelberg (Germany)<br />
We present a new technique <strong>for</strong> the determination of glucose concentration in diagnostic test<br />
strips, which is based on measuring the fluorescence lifetime of NADH rather than the<br />
fluorescence intensity. We utilize a reaction system, in which NAD is reduced to NADH by<br />
Glucosedehydrogenase (GlucDH) while glucose is oxidized to Gluconolacton. The average<br />
fluorescence lifetime of NADH τav is composed of the two short lifetimes of unbound NADH<br />
(τ1=0.27ns and τ2=0.60ns) and the protein-bound lifetime of the GlucDH-NADH-complex<br />
(τ3=2.9ns). At a given enzyme concentration varying amounts of NADH shift the ratio between<br />
protein-bound and free NADH, whereby the total amount of NADH is determined by the glucose<br />
concentration. If the NADH concentration is much lower than the enzyme concentration, the<br />
majority of NADH molecules are protein-bound. Consequently τav converges to the protein-bound<br />
lifetime. If the coenzyme concentration dominates, the majority of NADH molecules are unbound.<br />
There<strong>for</strong>e τav approximates the mean fluorescence lifetime of free NADH τav,free=0.42(2)ns. First<br />
results in test solutions as well as test strips are described.<br />
______________<br />
* Corresponding author: e-mail: alexa.ketteler@kip.uni-heidelberg.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 251
P137 Fluorescence Spectroscopy Poster 137<br />
Quantitative analysis of yeastolate and eRDF in model cell culture<br />
media using fluorescence spectroscopy<br />
Bridget Kissane * , Boyan Li & Alan G. Ryder<br />
Nanoscale BioPhotonics Laboratory, School of Chemistry, National University of Ireland Galway,<br />
Galway, Ireland<br />
Complex cell culture media components, eRDF and Yeastolate (YE) are key ingredients <strong>for</strong><br />
industrial fermentations. YE is a highly filtered, aqueous extract of Baker’s or brewer’s yeast<br />
generated via autolysis, which is used as a nutritional supplement.(D.a.C. B, 2009) eRDF is a<br />
basal medium, composed of amino acids, inorganic salts and saccharides which maintains the<br />
basal metabolism of the cells.(Shinmoto H,Dousako S, 1996) The quantitative analysis of these<br />
components in complex blended cell culture media is time-consuming and expensive, yet very<br />
necessary <strong>for</strong> good manufacturing practices. YE and eRDF both contain aromatic amino acids that<br />
produce an intrinsic fluorescence signal and multi-dimensional fluorescence (MDF) spectroscopy<br />
offers a convenient method <strong>for</strong> their analysis. We are investigating the efficacy of MDF <strong>for</strong> both<br />
monitoring variations in YE and eRDF quality and to measure their concentration in model cell<br />
culture media. The model cell culture media were prepared using various aqueous mixtures of five<br />
components: YE (0.1 to 1.72g/L), eRDF(0.2 to 1.28g/L), D-Glucose (6.2g/L), L-Glutamine (0.8g./L),<br />
and D-Galactose (2.5g/L). For YE and eRDF concentration analysis, the concentration of the other<br />
analytes were fixed. MDF spectra were recorded in both Excitation Emission Matrix (EEM) and<br />
Total Synchronous Fluorescence Scan (TSFS) modes. EEM spectra covered wide emission (270-<br />
600 nm) and excitation (230-520 nm) ranges while TSFS covered an excitation range between 230<br />
and 520 nm and a ∆λ of 20-200 nm. MDF data was pre-treated to remove Rayleigh scatter, and<br />
then deconvoluted to a two way (IxJK) structure prior to chemometric analysis using PLS. The<br />
calibration models <strong>for</strong> eRDF using full region EEM/TSFS data gave correlation coefficients of<br />
0.993/0.997 and RMSECV of 0.41/0.31 g/L. For YE the correlation coefficients were 0.965/0.959<br />
and the RMSECV values were 0.17/0.18g/L g/L. The results indicate that TSFS and EEM methods<br />
are very similar in terms of quantitative per<strong>for</strong>mance, however, TSFS data avoids the collection of<br />
Rayleigh scatter and thus may be the better option <strong>for</strong> industrial use.<br />
References:<br />
1. D.a.C., B., Difco & BBL Manual of Microbiological Culture Media. 2009.<br />
2. Shinmoto, H. and S. Dousako, Serum free culture medium. 1996, Google Patents.<br />
______________<br />
* Corresponding author: e-mail: b.kissane1@nuigalway.ie; alan.ryder@nuigalway.ie<br />
252 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P138 Fluorescence Spectroscopy Poster 138<br />
Mechanistic insights on protein unfolding from time-domain<br />
fluorescence<br />
Saswata S. Sarkar 1 , Santosh Jha 2 , Deepak Dhar 3 , Jayant B. Udgaonkar 2<br />
& G. Krishnamoorthy 1<br />
1 3<br />
Dept. of Chemical Sciences and Dept. of Theoretical Physics, Tata Institute of Fundamental<br />
Research, Homi Bhabha Road, Mumbai 400005, India<br />
2<br />
National Centre <strong>for</strong> Biological Sciences, Tata Institute of Fundamental Research,Bangalore<br />
560065, India<br />
Temporal in<strong>for</strong>mation on structural trans<strong>for</strong>mations occurring during folding and unfolding reactions<br />
of proteins is of great importance <strong>for</strong> elucidating the mechanism of protein folding and unfolding.<br />
Time-resolved fluorescence techniques coupled to Maximum Entropy Method (MEM) of data<br />
analysis has been used in our laboratory to address several aspects of unfolding process.<br />
The unfolding of a small protein monellin was probed by measurement of changes in the<br />
distributions of 4 different intramolecular distances using a multisite time-resolved fluorescence<br />
resonance energy transfer methodology coupled to data analysis by the MEM. Our measurements<br />
during the course of unfolding showed that the protein undergoes slow and continuous diffusive<br />
swelling. The swelling process was modeled as a slow diffusion of a Rouse-like chain with intraresidue<br />
interactions. This complexity which remains hidden in several steady-state probes-based<br />
studies gets revealed in our studies.<br />
In general, the high level of heterogeneity associated with the fluorescence lifetime of<br />
tryptophan imposes restrictions on its use as the energy donor in several proteins. A search <strong>for</strong> a<br />
tryptophan analog having reduced lifetime heterogeneity when compared to tryptophan led us to 5fluorotryptophan<br />
(5F-Trp). A single tryptophan-containing mutant <strong>for</strong>m of barstar, a small protein,<br />
has multiple lifetime components in its various structural <strong>for</strong>ms. Biosynthetic incorporation of 5F-<br />
Trp in place of Trp in barstar resulted in a significant decrease in the level of heterogeneity of<br />
fluorescence decay when compared to Trp-barstar. This is expected to enable an unambiguous<br />
estimation of intra-molecular distance distributions during protein folding and unfolding.<br />
In<strong>for</strong>mation on solvent accessibility of protein core during folding and unfolding of proteins is<br />
expected to provide us vital clues in understanding the mechanism of folding. The question<br />
whether the ‘molten globule’ intermediates postulated in folding and unfolding processes are fully<br />
hydrated (wet) or not (dry) has become a central issue in the field of protein folding. We have<br />
addressed this question by taking the model protein barstar which has a natural fluorescent sidechain<br />
tryptophan (Trp) at the core of the protein. Using a pico-second laser system coupled to a<br />
stopped-flow set-up, we measured the time evolution of solvent accessibility of the protein core<br />
from fluorescence quenching of Trp by several quenchers. Our initial experiments have shown the<br />
presence of a ‘wet’ molten globule during the unfolding process.<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 253
P139 Fluorescence Spectroscopy Poster 139<br />
Determination of honey’s botanical origin by synchronous<br />
fluorescence spectroscopy<br />
Lea Lenhardt 1 , Ivana Zeković 1 , Tatjana Dramićanin 1 , Živoslav Tešić 2 , Dušanka Milojković-<br />
Opsenica 2 & Miroslav D. Dramićanin 1,*<br />
1 Institute of Nuclear Sciences “Vinča”, University of Belgrade, PO Box 522, 11001 Belgrade, Serbia<br />
2 Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia<br />
Development of rapid and sensitive technologies <strong>for</strong> food analysis is one of important responses to<br />
growing public interest in food quality and safety [1] . Potential of fluorescence used in food research<br />
has increased during the last years with the propagated application of chemometrics and with<br />
technical and optical developments of spectrofluorometers. Synchronous fluorescence<br />
spectroscopy coupled with chemometrics is used as a tool <strong>for</strong> classification of honey samples<br />
according to their botanical origins (acacia, sunflower and linden). Synchronous fluorescence<br />
spectra of honey samples were obtained in constant wavelenght mode (simoultaneous scanning of<br />
both emission and excitation wavelengths while keeping the interval of wavelenghts<br />
constant).Clear difference in emission between groups was showed due to variances of intrinsic<br />
fluorophore concentrations and their microenviroments. Various statistical methods [2] were used <strong>for</strong><br />
analysis of collected data and the results showed that synchronous fluorescence spectroscopy is<br />
suitable <strong>for</strong> determination of botanical origin of honey. Characteristic synchronous fluorescence<br />
landscapes of acacia and linden honey are given on figure (a) and (b) respectively.<br />
References: [1] R.Karoui, et al., Food Chemistry, 101 (2007) 314-323. [2] A. Smilde, et al., Multi-way<br />
Analysis, (2004) 35-86.<br />
______________<br />
* Corresponding author: e-mail: dramican@vinca.rs<br />
254 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P140 Fluorescence Spectroscopy Poster 140<br />
Prediction of bioprocess per<strong>for</strong>mance by multidimentional fluorescence<br />
spectroscopy analysis of reactor broths<br />
Boyan Li & Alan G. Ryder *<br />
Nanoscale Biophotonics Laboratory, School of Chemistry, National University of Ireland, Galway,<br />
Galway, Ireland<br />
Protein production by Chinese Hamster Ovary (CHO) cell-based processes is fast becoming one of<br />
the dominant industrial methods <strong>for</strong> the production of biological active pharmaceutical ingredients<br />
(BAPIs). A typical bioprocess involves multiple steps and scales, staring with a small vial of<br />
material from a cell bank that progresses through to bioreactors in the kL volume range. Process<br />
per<strong>for</strong>mance and product quality are significantly related to a wide variety of individual physical and<br />
chemical parameters in the bioreactor. To ensure optimal productivity requires detailed<br />
understanding of the complex interactions taking place during the bioreactor phase of the<br />
production process.<br />
However, the liquid broths in the bioreactors are very complex mixtures comprising of the<br />
feed media (amino acids, carbohydrates, vitamins, proteins, etc.), whole cells and cell debris,<br />
product protein, and metabolites, and thus comprehensive analysis is generally not feasible from a<br />
cost and/or time standpoint. In this study, we present a multidimensional fluorescence (MDF)<br />
spectroscopy method [1] <strong>for</strong> the quantitative and qualitative analysis of these types of complex broth<br />
mixtures over a complete bioprocess sampled at 12 different timepoints. This rapid, holistic method<br />
can identify compositional changes and also predict bioreactor productivity via a range of<br />
chemometric methods such as multiway robust principal component analysis (MROBPCA), n-way<br />
partial least squares regression (NPLS) implemented on various fluorescence excitation-emission<br />
matrix datasets. [2] For example, a typical NPLS model (Figure 1) shows the productivity prediction<br />
model in the final phase of the bioprocess. These methods have the potential to become an<br />
important part of upstream biopharmaceutical quality control and analysis.<br />
Figure 1: Plot of predicted versus measured<br />
productivity (in terms of final yield) extracted from EEM<br />
data collected at the final bioprocess stage. Calibration<br />
model used a set of 28 randomly selected samples and<br />
validated with the 5 test samples.<br />
0.6<br />
0.55<br />
2<br />
Rcal = 1<br />
2<br />
Rtest = 0.99<br />
0.5<br />
LVs = 9<br />
RMSEC = 0.005<br />
0.45<br />
0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8<br />
RMSEP = 0.007<br />
0.85 0.9 0.95<br />
This work was funded by the Irish Industrial Development Authority (IDA) and Bristol-Myers Squibb<br />
under the Centre <strong>for</strong> Bioanalytical Sciences (CBAS), collaborative research programme involving<br />
the National University of Ireland, Galway, Dublin City University, and Bristol-Myers Squibb.<br />
References: [1] K.A. Bakeev. Process analytical technology: spectroscopic tools and implementation<br />
strategies <strong>for</strong> the chemical and pharmaceutical industries. Blackwell Pub. Ox<strong>for</strong>d, 2005. [2] P.W. Ryan, et al.,<br />
Anal. Chem. 82 (2010) 1311.<br />
______________<br />
* Corresponding author: e-mail: alan.ryder@nuigalway.ie<br />
Predicted productivity titre<br />
0.95<br />
0.9<br />
0.85<br />
0.8<br />
0.75<br />
0.7<br />
0.65<br />
Calibration (28 points)<br />
Test (5 points)<br />
1:1 Diagonal line<br />
x-axis<br />
y-axis<br />
Measured productivity titre<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 255
P141 Fluorescence Spectroscopy Poster 141<br />
Characterization of P. abyssi NucS-PCNA complex <strong>for</strong>mation with<br />
branched DNA structures using fluorescence anisotropy, Fluorescence<br />
Resonance Energy Transfer and SAXS<br />
Alessio Ligabue 1 , Sergey P. Laptenok 1 , Christophe Creze 2 , Joelle Kuhn 1 , Marten H. Vos 1 ,<br />
Ursula Liebl 1 , Didier Flament 2 & Hannu Myllykallio 1<br />
1<br />
Laboratoire d'Optique et Biosciences INSERM U696 - CNRS UMR7645, Ecole Polytechnique,<br />
91128 Palaiseau (France)<br />
2<br />
Laboratoire de Microbiologie des Environnements Extrêmes IFREMER-Centre de Brest, CNRS<br />
UMR6197 29280 Plouzane (France)<br />
Pyrococcus abyssi NucS is a founding member of the novel endonuclease family that interacts with<br />
the replication clamp PCNA that coordinates the function of a large number of DNA replication and<br />
repair proteins [1] . In P. abyssi, a hyperthemophilic archaeon, NucS and PCNA <strong>for</strong>m a highly stable<br />
complex that can be isolated using gel filtration chromatography. Here we have investigated<br />
binding of the NucS-PCNA complex to branched DNA substrates using fluorescence anisotropy<br />
and fluorescence resonance energy transfer (FRET) techniques. Anisotropy experiments [2] , using<br />
several DNA probes carrying a terminal TAMRA label, demonstrated high specificity of NucS <strong>for</strong><br />
ssDNA. Moreover, our results reveal that PCNA is required <strong>for</strong> loading of NucS onto 5’ and 3’<br />
ssDNA flaps embedded in the duplex regions. We have not observed preferential association of<br />
NucS with 5’ or with 3’ flaps; this is a rare characteristic <strong>for</strong> enzymes acting on DNA. The<br />
interactions <strong>for</strong>med between the NucS-PCNA complex and dsDNA with a 5’flap were evaluated by<br />
measuring the dissociation constant as a function of salt concentration using anisotropy [3] . These<br />
results indicated the <strong>for</strong>mation of a higher order DNA complex that was stabilized by electrostatic<br />
[5 salt bridges (free binding energy of 20 kJ/mol at 150mM NaCl)] and non–electrostatic<br />
interactions (approximately 16 kJ/mol at 20°C). Biochemical experiments also suggest that PCNA<br />
keeps NucS in an inactive configuration until the complex encounters ssDNA extremities that are<br />
then rapidly cleaved, thus resulting into directed cleavage at the ss/dsDNA junction. Finally, FRET<br />
measurements [4] using double-labelled DNA flaps indicated that the complex <strong>for</strong>mation decreased<br />
the end-to-end DNA distance of the DNA substrates used, of roughly 20Å, thus indicating a marked<br />
DNA bending brought about by binding of the complex onto DNA. When combined with our small<br />
angle x-ray scattering (SAXS) data on the NucS-PCNA complex, revealing a solution structure of<br />
the complex, our measurements allow proposing a model how DNA is bound by the NucS-PCNA<br />
complex.<br />
This work was supported by ANR grants ARCREP and RETID(Y)NA.<br />
References: [1] B. Ren, et al., The EMBO Journal, 28 (2009) 2479. [2] V.J. LiCata and A.J. Wowor, Methods<br />
in Cell Biology, 84 (2008) 243. [3] T.M. Lohman and D.P Mascotti, Methods in Enzymology, 212 (1992) 400.<br />
[4] R. M. Clegg, et al., Proc. Natl. Acad. Sci. USA, 90 (1993) 2994.<br />
______________<br />
* Corresponding author: e-mail: hannu.myllykallio@polytechnique.edu<br />
256 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P142 Fluorescence Spectroscopy Poster 142<br />
The interplay between terbium/calcium binding and con<strong>for</strong>mational<br />
changes in self-splicing module of FrpC protein<br />
Petra Lišková 1 *, Radovan Fišer 1 & Ivo Konopásek 1<br />
1 Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague,<br />
Viničná 5, 128 44 Prague 2, Czech Republic<br />
Self-processing module (SPM) is a functional unit localised in several proteins produced among<br />
various bacterial species (Neisseria, Actinobacillus, Pseudomonas, etc.). While the function of<br />
some SPM-containing proteins is well-known, the significance of SPM <strong>for</strong> the protein<br />
function/activity remains unclear. In such a well described SPM in SPM-containing proteins, SPM is<br />
in general capable of self-cleavage in presence of calcium ions. FrpC protein, a SPM containing<br />
protein, is an iron-regulated product of bacterium Neisseria meningitidis. The intrinsic 177 AA long<br />
SPM of FrpC is a calcium-dependent autocatalytic domain that per<strong>for</strong>ms cleavage of a FrpC<br />
protein between Asp 414 and Pro 415 residues [1]. The tertiary structure of FrpC and also its<br />
self-cleaving function of SPM are fully dependent on calcium ions presence and binding. Inside the<br />
SPM, two putative calcium binding sites with homology to an EF-hand calcium binding motif were<br />
localised between 499-511 and 521-533 AA residues of FrpC [1]. While the reaction mechanism of<br />
SPM auto-cleavage is nearly about to be solved, the three-dimensional structure of FrpC and its<br />
SPM, their biological significance and relevance <strong>for</strong> pathogenesis are yet to be determined.<br />
Our work is aimed at characterizing of the metal ion binding sites of SPM together with the<br />
dynamics of the metal ion binding. At the same time, we also studied the structural changes of<br />
SPM induced by metal ion binding.<br />
For the description of the calcium-binding sites of SPM in FrpC we employed terbium, a<br />
luminescent analogue of calcium ions. Structural changes were observed using intrinsic<br />
fluorescence of tryptophans - using two single tryptophan mutants with point substitution of<br />
tryptophan to phenylalanine in positions 37 and 105 of SPM (W37F and W105F, respectively). In<br />
titration experiments with Tb 3+ , two dissociation constants were determined at 5 µM and 100 µM.<br />
The changes in tryptophan emission maxima reflecting con<strong>for</strong>mational changes of SPM were<br />
observed during Tb 3+ or Ca 2+ titration around 100 µM. We also described sequence of<br />
con<strong>for</strong>mational changes that SPM per<strong>for</strong>ms in particular Tb 3+ and/or Ca 2+ concentrations. Starting<br />
from unfolded unstructured protein, first Tb 3+ ion is bound to the protein. After some subtle<br />
con<strong>for</strong>mational change of SPM, the FRET between Trp37 and Tb 3+ is observed by the change in<br />
excitation spectra of Tb 3+ . While further increasing Tb 3+ concentration, folding of the protein<br />
proceeds while tryptophans are buried inside the structure. Since the protein is folded in suitable<br />
ion concentration, the cleavage begins.<br />
This work was supported by project of the Grant Agency of Charles University No. 354611 and by<br />
projects of the Ministry of Education, Youth, and Sports of the Czech Republic No. LC06034 and<br />
MSM 0021620858.<br />
References: [1] R. Osička, et al., J. Biol. Chem., 279 (2004) 24944–24956 .<br />
______________<br />
* Corresponding author: e-mail: fox.petra@gmail.com<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 257
P143 Fluorescence Spectroscopy Poster 143<br />
A new method <strong>for</strong> long-distance FRET analysis<br />
Vincenzo Manuel Marzullo 1,2 , Piotr Bojarski 3 , Leszek Kulak 4 , Katarzyna Walczewska-Szewc 3 ,<br />
Anna Synak 3 , Alberto Luini 2 & Sabato D’Auria 1*<br />
1<br />
Institute of Protein Biochemistry, CNR, Naples, Italy<br />
2<br />
Tigem, Naples, Italy<br />
3<br />
University of Gdansk, Institute of Experimental Physics, Molecular Spectroscopy Division, Wita<br />
Stwosza 57, 80-952 Gdańsk, Poland<br />
4<br />
Gdansk University of Technology, Faculty of Technical Physics and Applied Mathematics,<br />
Department of Theoretical Physics and Quantum In<strong>for</strong>matics, Narutowicza 11/12, 80-233<br />
Gdansk, Poland<br />
Förster resonance energy transfer [1] (FRET) has become a powerful tool to determine intramolecular<br />
distances and molecular con<strong>for</strong>mation variations of bio/molecules [2]. In fact, FRET may<br />
act as a spectroscopic ruler <strong>for</strong> measuring distances not exceeding 10 nm [3]. This property of<br />
FRET has been used extensively to study protein-protein interactions in mammalian cells.<br />
Cellular functioning is mediated by the activity of many protein complexes that constitute the<br />
cellular machines, which drive essentially all life processes including <strong>for</strong> example mRNA splicing,<br />
vesicle transport, cell migration or signal transduction. All these basic processes have in common<br />
that they involve multi-protein complexes with many components and tight spatio-temporal<br />
regulation of protein complexes. Un<strong>for</strong>tunately, many if not most complexes have sizes above 10<br />
nm. These have been analysed so far by biochemical methods such a yeast two-hybrid, tandem<br />
affinity purification, and mass spectrometry, as well as co-immuno-precipitation and PCA (proteinfragment<br />
complementation assays), but not in intact cells. There<strong>for</strong>e, our current understanding of<br />
these multicomponent systems is limited because of a lack of tools <strong>for</strong> characterizing their function<br />
in the cellular context where they per<strong>for</strong>m their functions [4].<br />
A new method <strong>for</strong> extending the utilizable range of FRET is proposed and tested by the Monte –<br />
Carlo technique. The obtained results indicate that the efficiency of FRET can be significantly<br />
enhanced at a given distance if the energy transfer takes place towards multiple acceptors closely<br />
located on a macromolecule instead of a single acceptor molecule as it is currently used in FRET<br />
analysis. On the other hand, reasonable FRET efficiency can be obtained at significantly longer<br />
distances than in the case of a single acceptor. Randomly distributed and parallel orientated<br />
acceptor transition moments with respect to the transition moment of the donor molecule are<br />
analyzed as two extreme cases. As expected, parallel orientation of donor and acceptors transition<br />
moments results in a more efficient excitation energy collection. This finding can be applied to<br />
reveal directly the assembly/de-assembly of large complexes of proteins inside the cell by<br />
fluorescence microscopy.<br />
This project is in the framework of the CNR Commessa” Diagnostica Avanzata” (SD) and NR 15<br />
0029/2009 (P.B).<br />
References: [1] Förster, Th. Ann.Physik 1948, 2, 55. [2] Lakowicz, J.R. Principles of fluorescence<br />
spectroscopy, 3rd ed.; Springer Verlag, 2006. [3] Stryer, L.; Haughland, R.P. Proc.Natl Acad Sci USA 1967,<br />
58, 719. [4] Shoemaker, B. A.; Panchenko, A. R. PLoS Comput. Biol. 2007, 3, 337–344<br />
______________<br />
* Corresponding author: e-mail: s.dauria@ibp.cnr.it<br />
258 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P144 Fluorescence Spectroscopy Poster 144<br />
Analysis of citrate in low-volume seminal fluid samples using a timegated<br />
measurement of europium luminescence [1]<br />
Robert Pal * , Andrew Beeby & David Parker<br />
Department of Chemistry, University of Durham, Durham, DH1 3LE, UK<br />
Low citrate concentration in prostatic and seminal fluid (SF, 30-50% prostatic origin) has been<br />
proposed as a possible indicator <strong>for</strong> prostate cancer (PCa). [2] Citrate levels in healthy SF levels<br />
have been previously identified to be 33(±8) mM. This value falls dramatically as PCa progresses<br />
due to a well-established metabolic change in prostatic epithelial cells, promoting measurements of<br />
seminal citrate levels as a clinical screening tool.<br />
The work presented aims to validate a rapid analytical method that enables the<br />
measurement of citrate in seminal fluid samples. [3] Alternative methods <strong>for</strong> the determination of<br />
citrate in this bio-fluid include NMR spectroscopy [4] and enzyme based assays based on citrate<br />
lyase; neither of these is suited to a high-throughput screening application.<br />
Each sample measured was obtained from men within 9 weeks of a vasectomy operation.<br />
Two age ranges were examined, between 40 to 43 and 50 to 53 years old, with nearly 100<br />
samples in each case. No patient clinical history was available <strong>for</strong> this anonymous study,<br />
simulating a random screening cohort. The concentration of citrate in 0.5 µL seminal fluid samples<br />
was assessed, using a luminescent europium (III) complex, involving the ratiometric analysis of two<br />
well-separated Eu(III) emission bands. Spectral data were obtained a tailor-made, time-gated<br />
spectrometer, and citrate concentrations were calculated aided by custom-made detection<br />
software. Citrate values were confirmed by independent measurements using a citrate lyase<br />
enzymatic assay and by 700 MHz 1 H-NMR analysis of seminal fluid samples.<br />
Citrate concentrations were not statistically different between age groups and averaged<br />
35.0(±14.6) mM <strong>for</strong> the 40-43 group, and 28.2(±12.7) mM <strong>for</strong> the 50-53 cohort; in each case a<br />
polymodal distribution was observed indicating the need <strong>for</strong> a larger clinical trial study.<br />
References: [1] R Pal, et al., JPBA, 2011, in press. [2] R. Clarke, et al., Cancers, 2(2010), 1125 and K.<br />
Singh, et al., Mol. Cancer, 15(2006), 5. [3] R Pal, et al., OBC, 7(2009), 1525. [4] J. Kavanagh, J. Reprod.<br />
Fert., 75(1985), 35 and N. Serekova, et al., Prostate, 68(2008), 620; and E. Kline, et al., J. Urol., 76(2006),<br />
2274.<br />
______________<br />
* Corresponding author: e-mail: robert.pal@dur.ac.uk<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 259
P145 Fluorescence Spectroscopy Poster 145<br />
Short-distance FRET applied to the polypeptide chain<br />
Ana Rei 1 , Indrajit Gohosh 1 , Roy D'Souza 1 , Amir Norouzy 1 , Maik H. Jacob 1* & Werner M. Nau 1<br />
1 School of Engineering and Science, Jacobs University Bremen, D-28759 Bremen, Germany<br />
To investigate and understand the structure and dynamics of the flexible polypeptide chain in the<br />
intrinsically disordered protein, or in the unfolded protein on the verge of folding into its globular<br />
native state, several techniques have been developed. All require to incorporate into the chain two<br />
optical probes at two selected positions. Among these techniques are PET (photo-induced electron<br />
transfer), TTET (triplet–triplet energy transfer) and single-molecule FRET (Förster resonance<br />
energy transfer). We introduced two methods based on DBO — sdFRET (short-distance FRET)<br />
and FCQ (fluorescence contact quenching) — that despite being limited to ensemble<br />
measurements yield in<strong>for</strong>mation not easily obtained from any other method: We demonstrate this<br />
on a widely studied model system, the Gly-Ser repeat chain, comparing the results of PET, TTET,<br />
CIFQ and sdFRET measurements.<br />
In the further development of short distance FRET, our focus is on quantifying the amount of<br />
FRET efficiency that is caused by donor-acceptor motions during the lifetime of the donor. With<br />
Förster distances in the 10Å domain, this amount is substantial and its proper treatment critical.<br />
We explore motion contributions to FRET efficiency in dependence of chain length, donor lifetime<br />
and viscosity. We delineate the efficiency contributions of the distance distribution, of the speed of<br />
diffusional motions and of the time available <strong>for</strong> diffusion to contribute to FRET, correlated to the<br />
donor lifetime. To analyze these contributions first on the level of FRET efficiency avoids pitfalls in<br />
the subsequent global analysis based on Steinberg`s differential equation.<br />
References: E. Haas et al, Biopolymers, 17 (1978) 11. H. Sahoo et al., J.Am. Chem. Soc. 129 (2007) 9762.<br />
J.R. Lakowicz et al. J. Phys. Chem., 370 (1991) 385.<br />
______________<br />
* Corresponding author: e-mail: m.jacob@jacobs-university.de<br />
260 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P146 Fluorescence Spectroscopy Poster 146<br />
Investigation of MADS box intrinsic fluorophores<br />
Barbora Řezáčová 1,2 *, Yves-Marie Coïc 3 , Christian Zentz 2 , Pierre-Yves Turpin 2<br />
& Josef Štěpánek 1<br />
1<br />
Institute of Physics, Charles University in Prague, Faculty of Mathematics and Physics, 5, Ke<br />
Karlovu, CZ-12116 Prague 2 (Czech Republic)<br />
2<br />
Laboratoire Acides nucléiques et biophotonique, FRE 3207, Université Pierre et Marie Curie, 2,<br />
place Jussieu, 75252 Paris Cedex 5 (France)<br />
3<br />
Unité de Chimie des Biomolécules, URA 2128 CNRS, Institut Pasteur, 28, Rue Du Dr. Roux,<br />
75724 Paris Cedex 15 (France)<br />
The MADS box family of transcription factors (over 200 members) plays a crucial role in the gene<br />
regulation of higher organisms. MADS box acronym is derived from initials of four of the originally<br />
identified members of the family: MCM1, AG, DEFA and SRF [1] . These transcription factors share<br />
a highly conserved DNA binding motif showing a wide sequence homology of 56 amino acids with<br />
many eukaryotic regulatory proteins.<br />
The serum response factor (SRF) plays a key role in the activation of genes that respond to<br />
mitogenes and in the regulation of muscle specific genes [2] . The transcriptional activity of SRF<br />
depends on its binding (as a symmetric dimer) to the so called CArG box element CC(A/T)6GG,<br />
one of the most extensively characterized DNA binding sequences recognized by MADS box<br />
proteins. Preceding studies on 20-mer oligonucleotides bearing the specific high affinity CArG box<br />
of the c-fos enhancer, disclosed an equilibrium between bend and linear con<strong>for</strong>mers [3] which is<br />
partly maintained even after the protein/DNA complex <strong>for</strong>mation [4] . Still not much is known about<br />
the recognition process. Thus the conserved MADS box motif may serve as a basic model <strong>for</strong><br />
study of common functional properties of DNA recognition within the whole protein family.<br />
We have investigated the structural and dynamic properties of the MADS box and its shorter<br />
segments by means of fluorescence and UV absorption spectroscopies. Acquired data have been<br />
treated by using an approach we have recently developed (based on factor analysis), which<br />
enabled us to detect even very weak spectral changes. We were also interested in the effects<br />
caused by variations of environmental conditions like pH.<br />
The MADS box contains three tyrosine residues serving as intrinsic fluorophores and no<br />
tryptophan. Fluorescence emission spectra of the MADS box segments provided in<strong>for</strong>mation about<br />
structural stability and protonation states of the tyrosines. It is though essential to elucidate and<br />
specify the effective contributions of fluorescence emission of these three tyrosines separately. In<br />
order to decompose the emission spectrum and to assign the fluorescence components to<br />
particular tyrosine residues of the MADS box, quenching experiments were per<strong>for</strong>med and<br />
analyzed (used quenchers Cs + , I - ).<br />
This work is supported by the Czech Science Foundation (project 202/09/0193), Grant Agency of<br />
Charles University (project 402111). B.R. gratefully acknowledges the French Government support<br />
<strong>for</strong> her stay in Laboratoire Acides nucléiques et biophotonique.<br />
References:<br />
[1]. P. Shore, A.D. Sharrocks, Eur. J. Biochem. 229 (1995) 1. [2] J. M. Miano, J. Mol. Cell. Cardiol. 35 (2003)<br />
577. [3] J. Stepanek et al., FEBS Journal 274 (2007) 2333. [4] J. Stepanek et al., BBRC 391 (2010) 203.<br />
______________<br />
* Corresponding author: e-mail: rezacova@karlov.mff.cuni.cz<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 261
P147 Fluorescence Spectroscopy Poster 147<br />
Medium viscosity effects on the fluorescence of betalains<br />
Ana Clara Beltran Rodrigues, Letícia Christina Pires Gonçalves & Erick Leite Bastos *<br />
Centro de Ciências Naturais e Humanas, Universiade Federal do ABC, Santo André, SP (Brazil)<br />
Betalains are water-soluble alkaloids classified as yellow betaxanthins or violet-red betacyanins,<br />
both originated from spontaneous non-stereoselective addition of amines or amino acids to<br />
betalamic acid. [1] Betaxanthins are the pigments responsible <strong>for</strong> visible fluorescence of flowers,<br />
e.g., yellow varieties of Mirabilis jalapa (four o'clock), [2] whereas betacyanins are natural phenolic<br />
non-fluorescent antioxidants. [3] In this work, we report the influence of the medium viscosity on the<br />
fluorescence intensity of betanin (Bn, betanidin 5-O-glucoside) and indicaxanthin (BtP, L-proline<br />
derived betaxanthin). The viscosity of some internal cell compartments of SK-OV-3 cells are<br />
around 260 cP; [4] there<strong>for</strong>e, the study of the effect of the medium viscosity on the fluorescence<br />
properties of vacuolar natural pigments, such as betalains, is relevant.<br />
Betanin was purified from red beetroot (Beta vulgaris, sup. vulgaris) juice and indicaxanthin was<br />
semisynthesized from betanin and L-proline. Both betalains were purified by RP-HPLC (purity<br />
99%+HPLC) and characterized by HRMS and NMR. Solutions of water and glycerol in different<br />
proportions (0, 15, 30, 45, 60, 75, and 90% glycerol) were prepared by weight and used as solvent<br />
in the acquisition of absorption and fluorescence spectra of Bn and BtP. The increase of the<br />
medium viscosity had no effect on the absorbance profile of Bn and BtP. However, although the<br />
fluorescence quantum yield (ΦFL) of BtP is at least two orders of magnitude higher than that of Bn,<br />
the area under the fluorescence curve, which is related to the ΦFL, increases with the increase of<br />
the medium viscosity <strong>for</strong> both Bn and BtP. This effect is higher <strong>for</strong> BtP than <strong>for</strong> Bn (Figure 1).<br />
These results are related to the internal charge transfer character of betalains, which depends on<br />
the presence of a 1,7-diazaheptamethine system.<br />
Figure 1 – Relative effect of medium viscosity on<br />
the area under fluorescence curve of Bn and BtP,<br />
[Bn] = 7,5 × 10 -6 mol L -1 , [BtP] = 9 × 10 -6 mol L -1 ,<br />
25 o C.<br />
This work was supported by grants from FAPESP, CNPq, CAPES and UFABC.<br />
References: [1] D. Strack, et al., Phytochem., 62 (2003) 247. [2] F. Gandia-Herrero, et. al., Planta, 222<br />
(2005) 586. [3] J. Kanner, et al., J. Agric. Food Chem. 49 (2001) 5178. [4] J. A. Levitt, et al., J. Phys. Chem.<br />
C 113 (2009) 11634.<br />
______________<br />
* Corresponding author: e-mail: erick.bastos@ufabc.edu.br<br />
262 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P148 Fluorescence Spectroscopy Poster 148<br />
Excited state lifetimes of riboflavine derivatives in solution<br />
Yvonne Schmitt 1 , Madina Mansurova 2 , Wolfgang Gärtner 2 & Markus Gerhards 1<br />
1<br />
Physikalische und Theoretische Chemie, Erwin-Schrödinger-Straße 52, D-67663 Kaiserslautern,<br />
Germany<br />
2<br />
MPI für Bioanorganische Chemie, Stiftstrasse 34–36, D-45470 Mülheim an der Ruhr<br />
Riboflavin plays an important role in the organism of human beings as well as in the<br />
photosynthesis of plants where it is part of photoreceptors. This diversity results from different<br />
possible oxidation states and a manifold of electronic states. The knowledge of these states is of<br />
importance to understand the processes which take place during the photosynthesis or other<br />
processes which are initiated via irradiation. In nature riboflavin is bounded via its ribityl chain.<br />
Thus it is important to analyse the derivatives as model systems in order to obtain in<strong>for</strong>mation of<br />
the subsiding biological processes.<br />
The derivatives chosen in this presentation are Tetraacetylriboflavin and 5-Deazariboflavin.<br />
These species are analyzed by UV-Vis-absorption and fluorescence spectroscopy as a function of<br />
different solvents. The effect on these spectra is discussed. Additionally the lifetimes of the<br />
electronically excited states are determined and result in 4,8 ns <strong>for</strong> 5-Deazariboflavin in water and<br />
5,5 ns <strong>for</strong> Tetraacetylriboflavin in acetonitrile.<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 263
P149 Fluorescence Spectroscopy Poster 149<br />
Fluorescence imaging of (phospho)lipase patterns using differential<br />
activity-based gel electrophoresis (DABGE)<br />
Maria Morak, Bojana Stojčić & Albin Hermetter<br />
Institute of Biochemistry, Graz University of Technology, Austria<br />
Patterns of active lipases and esterases from two different biological samples can be compared in<br />
one electrophoresis gel, using activity-based gel electrophoresis (DABGE) [1] . This approach is<br />
based on specific labeling of the active target enzymes with fluorescent suicide inhibitors followed<br />
by gel electrophoresis, fluorescence imaging and identification of the tagged proteins by MS/MS.<br />
For this purpose, a set of fluorescent suicide inhibitors emitting at different wavelengths was<br />
developed. These probes possess the same substrate analogous structures but carry different<br />
cyanine dyes (Cy2b, Cy3 and Cy5) as reporter fluorophores. Typically, two samples A and B are<br />
labeled with Cy3 and Cy5 probes, respectively. Cy2b is used as reference probe <strong>for</strong> labeling all<br />
enzymes in A and B. The labeled proteins are mixed, separated by 1-D or 2-D gel electrophoresis<br />
and imaged with a fluorescence laser scanner. Enzymes preferentially expressed in samples A or<br />
B show green Cy3 (A) or red Cy5 (B) fluorescence. Proteins equally abundant in A and B show<br />
both emissions. This method can be used <strong>for</strong> differential activity profiling of closely related<br />
proteomes differing with respect to genetic background or environment. Typical applications<br />
include measurements of changes in lipolytic proteomes due to effects of disease, genetic<br />
disorders as well as of nutrients or pharmacological drugs. Here we report on the differential<br />
analysis of (phospho)lipases in adipose tissues of mice deficient in enzymes catalyzing the<br />
degradation of glycerol(phospho)lipids. Deficiencies in these enzymes were associated with<br />
profound changes in the activity of several other lipolytic proteins. MS analysis of the affected<br />
proteins led to the identification of known (phospho)lipolytic enzymes as well as proteins with<br />
unknown functions. This example and further applications of the DABGE technique can be<br />
expected to provide entirely new in<strong>for</strong>mation on lipid enzymology in health and disease with high<br />
precision.<br />
This work was supported by grants from the Austrian Federal Ministry of Science and<br />
Research/FFG in the framework of the GEN-AU program (GOLD project).<br />
References: [1] M. Morak, et al., J. Lipid Res. 20 (2009) 1281-1292.<br />
______________<br />
* Corresponding author: e-mail: albin.hermetter@tugraz.at<br />
264 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P150 Fluorescence Spectroscopy Poster 150<br />
Con<strong>for</strong>mational transitions in the Mg 2+ -dependent 7S11 deoxyribozyme,<br />
a fluorescence study<br />
Elisa Turriani 1,2,* , Claudia Höbartner 2 & Thomas M. Jovin 2<br />
1 Scuola Normale Superiore di Pisa, Piazza dei Cavalieri 7, 56126 Pisa, Italy, and Dipartimento di<br />
Chimica e Chimica Industriale, via Risorgimento 35, 56126 Pisa, Italy<br />
2 Max Planck Institute <strong>for</strong> Biophysical Chemistry, Am Faβberg 11, 37077 Göttingen, Germany<br />
Since the discovery that DNA can act as a catalyst as well as a storage medium of genetic in<strong>for</strong>mation,<br />
many DNAs with catalytic activity have been synthesized in the laboratory. These DNA-enzymes or<br />
deoxyribozymes have high selectivity and can catalyze a wide range of reactions such as cleavage or<br />
ligation of RNA strands [1].<br />
The 7S11 deoxyribozyme under investigation in this project catalyzes the <strong>for</strong>mation of a 2'-5'<br />
branched-RNA. Such structures constitute the core of lariat RNAs, important intermediates in the<br />
splicing process.<br />
The reaction of 7S11 is Mg 2+ -dependent and proceeds via the nucleophilic attack of a 2’-OH<br />
group of a specific internal adenosine in one substrate RNA strand (L-RNA), on the 5'-triphosphate<br />
group of the second substrate RNA strand (R-RNA), leading to the release of inorganic phosphate [2].<br />
The active structure of the DNA:RNA complex is a three helix junction with 4 Watson-Crick basepairing<br />
regions between the DNA and its RNA substrates (Fig.1a) [3]. Although 7S11 has been<br />
extensively studied, the detailed reaction mechanism and kinetics, structural rearrangements, and Mg 2+<br />
binding sites are as yet unknown. The aim of this research has been to investigate these aspects using<br />
steady-state and time-resolved (T-jump, stopped-flow, lifetime) fluorescence spectroscopy.<br />
Two fluorescent probes were introduced into the RNA and DNA strands: 1) 2-aminopurine (AP),<br />
a fluorescent analog of adenosine, was incorporated into the sequence of the L-RNA substituting the<br />
branch site adenosine; and 2) small fluorogenic dye monobromobimane (mBBr) was conjugated to the<br />
γ position of the 5’-triphosphate on the R-RNA (Fig.1a). The incorporation of the dyes was tolerated by<br />
7S11, as was confirmed by kinetic measurements. AP and mBBr were also chosen because they are a<br />
good FRET pair, showing a convenient spectral overlap and almost no excitation crosstalk.<br />
The combined use of AP and mBBr allowed the elucidation of the mechanism of <strong>for</strong>mation of the<br />
paired regions in the DNA:RNA hybrid, including a readout of catalysis and product (pyrophoshate)<br />
release, manifested by an increase in intensity and depolarization of the mBBr. The role of Mg 2+ in the<br />
stabilization of the P4 paired region and in the structural rearrangements was also explored. Kinetic<br />
measurements (T-jump) confirmed that the presence of Mg 2+ is required <strong>for</strong> stabilizing the P4 paired<br />
region, the <strong>for</strong>mation of which is otherwise unfavorable. The FRET measurements demonstrated that<br />
Mg 2+ brings the branch site adenosine and the triphosphate closer together (Fig.1b).<br />
a) b)<br />
350 400 450<br />
λ(nm)<br />
500 550<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 265<br />
Fluorescence (a.u.)<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
2-aminopurine<br />
Donor<br />
mBBr<br />
Acceptor<br />
Figure 1: (a) 7S11 with its RNA substrates labeled with AP and mBBr, paired regions P1-P4 indicated. (b)<br />
Emission spectra of a sample containing AP and mBBr. Sensitized emission of mBBr and quenching of AP<br />
upon addition of Mg 2+ (solid, [Mg 2+ ] 0 mM; dashed, [Mg 2+ ] 40 mM)<br />
References: [1] S. K. Silverman, D. A. Baum, Methods Enzymol., 469 (2009) 95. [2] R. L. Coppins, S. K.<br />
Silverman, Nat. Struct. Mol. Biol., 11 (2004) 270. [3] R. L. Coppins, S. K. Silverman, J. Am. Chem. Soc., 127<br />
(2005) 2900.<br />
______________<br />
* Corresponding author: e-mail: elisa.turriani@sns.it
P151 Fluorescence Spectroscopy Poster 151<br />
The heart tissues differentiation by time resolved<br />
fluorescence spectroscopy<br />
Jonas Venius 1 , Edvardas Žurauskas 2 & Ričardas Rotomskis 1,3<br />
1<br />
Biomedical Physics Laboratory of Institute of Oncology, Vilnius University, Baublio 3B, LT- 08406,<br />
Vilnius, Lithuania<br />
2<br />
Medical Faculty of Vilnius University, Čiurlionio 21, LT-03100 Vilnius, Lithuania<br />
3<br />
Biophotonics Group of Laser Research Centre, Vilnius University, Saulėtekio 9, c.3, LT-10222,<br />
Vilnius, Lithuania<br />
Optical methods carry in<strong>for</strong>mation about intrinsic properties of the tissue and provide the unique<br />
possibility to study the objects none invasively. The fluorescence reflects the general composition of<br />
the tissue and depends mostly on the fluorophores present in the tissue. The fluorescence lifetime<br />
holds additional in<strong>for</strong>mation about the tissue fluorophores, and can be used <strong>for</strong> the tissue type<br />
differentiation.<br />
The heart conduction system (HCS) is a specific muscular tissue, where a heartbeat signal<br />
originates and initiates the depolarization of the ventricles. The muscular origin makes it complicated<br />
to distinguish HCS from the surrounding tissues and it could be damaged during the surgical<br />
intervention. There<strong>for</strong>e, there is an immense necessity <strong>for</strong> developing a method to visualize HCS<br />
during the operation time.<br />
The fluorescence spectrometer FLS920 (Edinburgh Instruments) was used <strong>for</strong> steady state<br />
and time resolved fluorescence measurements. The time resolved experiments on the human heart<br />
tissue specimens were per<strong>for</strong>med ex vivo by using 405 nm pulsed laser (pulse width 100ps). The<br />
HCS, myocardium (MC) and connective tissues (CT)) were preliminary marked by the pathologist<br />
and approved histologically after the spectral measurements.<br />
Two excitation wavelengths at 330 nm and 380 nm were used <strong>for</strong> the differentiation of the<br />
heart tissues on the basis of the steady state fluorescence changes in the spectral region of 400 nm<br />
– 550 nm. No specific fluorescence or fluorescence excitation band was observed <strong>for</strong> any type of the<br />
heart tissue. This suggests that there is no specific fluorophore in HCS, CT or MC. The observed<br />
differences in the fluorescence intensity could be explained by the uneven amount and different<br />
organization of structural proteins collagen and elastin.<br />
Time resolved fluorescence measurements were per<strong>for</strong>med in the 430 nm – 550 nm region<br />
with 5 nm step. The registered fluorescence decay could be approximated at least by two<br />
exponentials. This indicates that at least two fluorophores are responsible <strong>for</strong> the fluorescence in this<br />
region. τ1 could be attributed to collagen and τ2 could be attributed to elastin. The lifetimes (τ) and the<br />
fractional components of fluorescence intensity (F) registered at 460 nm are displayed in the table<br />
below.<br />
Exp. HCS CT MC<br />
No. F(%) τ(ns) F(%) τ(ns) F(%) τ(ns)<br />
1 41 2.1 53 2.4 46 2.2<br />
2 59 7.7 47 8.6 54 8.5<br />
τ1 is very similar <strong>for</strong> all three tissue types in the heart, whereas τ2 of HCS differs significantly from MC<br />
and CT. The fractional components also are different <strong>for</strong> muscular type tissue (HCS and MC) and<br />
CT. Muscular type tissues contain more elastin than collagen, there<strong>for</strong>e fractional component of<br />
elastin is higher. CT contain more collagen than elastin, there<strong>for</strong>e the fractional component of<br />
collagen is higher. Similar findings were also observed <strong>for</strong> fluorescence lifetimes registered at<br />
different wavelengths.<br />
The observed spectroscopic differences between HCS, CT and MC show a great possibility <strong>for</strong><br />
the visualization of human heart conduction system.<br />
This work was supported by the Lithuanian Science Council under grant No. LIG-25/2010.<br />
______________<br />
*Corresponding author: e-mail: jonas.venius@vuoi.lt<br />
266 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P152 Fluorescence Spectroscopy Poster 152<br />
Organization of ECF and ABC transporter modules during substrate<br />
translocation<br />
Joanna Ziomkowska 1* , Johanna Heuveling 2 , Daniela Weidlich 2 , Franziska Kirsch 3 , Thomas<br />
Eitinger 3 , Erwin Schneider 2 & Andreas Herrmann 1*<br />
1<br />
Institute of Biology/ Biophysics, Humboldt University of Berlin, Invalidenstraße 43, 10115 Berlin<br />
(Germany)<br />
2<br />
Institute of Biology/ Microbial Physiology, Humboldt University of Berlin, Chausseestr. 117, 10115<br />
Berlin (Germany)<br />
3<br />
Institute of Biology/ Microbiology, Humboldt University of Berlin, Chausseestr. 117, 10115 Berlin<br />
(Germany)<br />
ATP binding cassette (ABC) transporters can be found in all organism <strong>for</strong>m bacteria to man.<br />
Dysfunction of these transporters is often associated with diseases, like multi drug-resistance in<br />
cancer cells. Energy coupling factor (ECF) transporters are a novel defined class of permeases,<br />
found in archae and bacteria including many human pathogens. Both, ATP and ECF transporters,<br />
translocate substances across the cellular plasma membrane under the expense of ATP, consist of<br />
dimers of ATPase subunits known as the ATP binding cassette and of two transmembrane<br />
domains (TMDs). Whereas a soluble binding protein (BP) is required <strong>for</strong> transport in ABC<br />
importers, this module is lacking in the ECF permeases. Here, the substrate is interacting directly<br />
with one TMD (S-component) of the ECF transporter and oligomerisation with the energy-coupling<br />
module (=ABC subunit plus conserved TMD), enables high affinity substrate uptake with kinetics of<br />
a primary transport system. Strikingly, the S-component can function as a high-capacity low-affinity<br />
transporter with kinetic parameters of secondary transport in absence of the energy-coupling<br />
module[1]. The quaternary structure of the S-component accomplishing low-affinity transport is still<br />
under discussion. From in vitro studies of the thiamine transporter ThiT (L. lactis) a monomeric<br />
state of the transporter was proposed [2], whereas lifetime based Förster Resonance Energy<br />
Transfer (FRET) studies in living bacteria indicate oligomerisation of S-components <strong>for</strong> the biotin<br />
transporter BioMNY (R. capsulatus)[3]. To unravel whether oligomerised S-components are a<br />
general property of ECF systems or a special feature of the biotin transporters, the lifetime based<br />
FRET approach [Ref] is applied to S-components of other ECF transporters. Amplitude weighted<br />
fluorescent lifetimes of recombinant E. coli expressing mCerulean tagged S-components and<br />
recombinant E. coli co-expressing mCerulean and mYFP tagged S-components are compared with<br />
respect to a negative control. The histidine permease (HisQMP2) of S. typhimirium is a well<br />
characterised classic canonical ABC importer. For substrate translocation HisJ (BP with specificity<br />
<strong>for</strong> histidine) or LAO (BP with specificity <strong>for</strong> lysine/arginine/ornithine) are required. The BP interacts<br />
with the extracellular loops of the TMD and the binding constants <strong>for</strong> both proteins were found to<br />
be in a micro molar range [4]. To assess the con<strong>for</strong>mational dynamics of BP-TMD complexation<br />
during the catalytic cycle of substrate translocation, A FRET based binding assay is designed to<br />
investigate the dependency of this interaction on the catalytic state of the transporter. HisQMP2 is<br />
functionally reconstituted into large unilamellar vesicles containing rhodamine-PE (acceptor) and a<br />
monocysteine variant of the BP is chemically labelled with Alexa Fluor 488 (donor). By trapping the<br />
transporter in different steps of ATP hydrolysis, the amplitude weighted fluorescent donor lifetime is<br />
supposed to reveal the dynamics of the BP association during substrate translocation. For further<br />
specification of the binding at single molecule level, lateral diffusion of the AlexaFluor 633 labelled<br />
monocysteine HisQMP2 and AlexaFluor 488 BP in giant unilamellar vesicles will be determined by<br />
Fluorescence Cross Correlation Spectroscopy.<br />
References: [1] P. Hebbeln, et al., Proc Natl Acad Sci U S A, 104 (2007) 2909. [2] J. ter Beek, et al., J Biol<br />
Chem 286 (2011) 5471. [3] F. Finkenwirth, et al., Biochem J 431 (2010) 373. [4] G. F.-L. Ames, et al., J Biol<br />
Chem 24 (1996) 14264.<br />
______________<br />
* Corresponding author: e-mail: ziomkowj@hu-berlin.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 267
P153 Fluorescence Spectroscopy Poster 153<br />
The influence of polymer structure on the cationic polymer – DNA<br />
complexes revealed by time-resolved fluorescence studies<br />
Tiia-Maaria Ketola 1,* , Martina Hanzlíková 2 , Linda Cameron 2 , Arto Urtti 3 , Helge Lemmetyinen 1 ,<br />
Marjo Yliperttula 2 & Elina Vuorimaa 1<br />
1<br />
Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541,<br />
FI-33101 Tampere, Finland<br />
2<br />
Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, P. O. Box 56, FI-<br />
00014 University of Helsinki, Finland<br />
3<br />
Centre <strong>for</strong> Drug Research, Faculty of Pharmacy, P. O. Box 56, FI-00014 University of Helsinki,<br />
Finland<br />
Polyethyleneimine (PEI) is widely<br />
used cationic polymer that is able to<br />
complex DNA and mediate efficient<br />
DNA transfection in the cell<br />
culture [1] . Poly-L-lysine (PLL) can<br />
also pack and condense DNA into<br />
small nanoparticles (less than 100<br />
nm in diameter) but upon challenge<br />
by polyanionic glycosaminoglycans,<br />
like heparan sulfate, PLL-DNA<br />
complexes retain DNA in a tightly bound complex [2] . In cell studies, PEI has shown 100fold<br />
higher transfection activity than PLL [3] . We used a time-resolved fluorescence<br />
spectroscopic method to study the mechanism of PEI-DNA and PLL-DNA complex<br />
<strong>for</strong>mation and to investigate how mean molecular weight and branching of PEI affect the<br />
complexes. Analysis of fluorescence lifetimes and time-resolved spectra revealed that <strong>for</strong><br />
both linear and branched high-molecular weight PEI the complexation takes place in two<br />
steps, but the small-molecular-weight branched PEI complexed DNA at a single step [4] .<br />
According to the binding constants obtained from time-resolved spectroscopic<br />
measurements, the affinity of N/P complexation per nitrogen atom decreases in the order<br />
PLL > linear 22 kDa PEI > branched 0.7 kDa PEI > branched 25 kDa PEI. On the other<br />
hand according to in vitro transfection studies the transfection efficiency decreases in the<br />
order linear 22 kDa PEI > branched 25 kDa PEI > PLL > branched 0.7 kDa PEI. Thus, the<br />
binding constant alone does not give adequate measure <strong>for</strong> transfection efficiency. On the<br />
other hand, the two-step complexation equilibrium, which includes a loosely bound DNA-<br />
PEI intermediate complex, is seen with efficient gene delivery systems LPEI and BPEI.<br />
Small molecular weight PEI does not <strong>for</strong>m loose intermediate complex; it <strong>for</strong>ms complexes<br />
with DNA at high affinity per N-P pair, but the overall binding constant per small molecular<br />
weight PEI molecule is low compared to larger branched and linear PEI molecules. The<br />
high overall binding constant may provide adequate stability in the extracellular space,<br />
whereas the loosely bound intermediate complex state may facilitate DNA release in the<br />
cells, a prerequisite <strong>for</strong> DNA transcription.<br />
This work was supported by Academy of Finland.<br />
References: [1] O. Boussif, et al., Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 7297. [2] M. Ruponen, et al.,<br />
Biochim. Biophys. Acta,Biomembr. 1415 (1999) 331. [3] M. Männistö, M et al., J. Gene Med. 9 (2007) 479.<br />
[4] T.-M. Ketola, et al., J. Phys. Chem. B 115 (2011) 1895.<br />
______________<br />
* Corresponding author: e-mail: tiia.ketola@tut.fi<br />
268 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P154 Fluorescence Spectroscopy Poster 154<br />
Poly(β-amino ester) - DNA complexes: time-resolved fluorescence and<br />
cellular transfection studies<br />
Elina Vuorimaa-Laukkanen 1,* , Tiia-Maaria Ketola 1 , Jordan J. Green 2 , Martina Hanzlíková 3 ,<br />
Helge Lemmetyinen 1 , Robert Langer 4 , Daniel G. Anderson 4 , Arto Urtti 5 & Marjo Yliperttula 3<br />
1<br />
Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541,<br />
FI-33101 Tampere, Finland<br />
2<br />
Biomedical Engineering and the Institute <strong>for</strong> Nanobiotechnology, The Johns Hopkins University<br />
School of Medicine, 400 N Broadway, Smith Building 5017, Baltimore, MD 21231, USA<br />
3<br />
Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, P. O. Box 56, FI-<br />
00014 University of Helsinki, Finland<br />
4<br />
The David H. Koch Institute <strong>for</strong> Integrative Cancer Research, Massachusetts Institute of<br />
Technology, 77 Massachusetts Avenue , Cambridge, MA 02139, USA<br />
5<br />
Centre <strong>for</strong> Drug Research, Faculty of Pharmacy, P. O. Box 56, FI-00014 University of Helsinki,<br />
Finland<br />
A large number of different polymers have<br />
been developed and studied <strong>for</strong> application<br />
as DNA carriers <strong>for</strong> nonviral gene delivery.<br />
This study describes a method <strong>for</strong><br />
estimating the efficiency of nanoparticle<br />
<strong>for</strong>mation by time-resolved fluorescence<br />
measurements. The method was tested with<br />
a series of poly(β-amino esters), PBAEs,<br />
biodegradable polymers that condense DNA<br />
into small stable nanoparticles. From the<br />
large library of PBAEs [1] ten polymers with<br />
different transfection efficacies were chosen<br />
<strong>for</strong> this study. These polymers <strong>for</strong>m a<br />
homologous series with respect to the nature (all tertiary) and the density of the amine groups. We<br />
compared the DNA–complexation behaviour of the PBAEs in a wide amine to phosphate (N/P)<br />
range, from 1 to 100, to reveal the complexation efficiency and mechanism, and to determine the<br />
binding constants <strong>for</strong> the studied PBAEs. In addition, we investigated possible correlation between<br />
fluorescence parameters and transfection efficacy of PBAEs. The binding constants per amine<br />
were in the same order of magnitude <strong>for</strong> PBAEs and branched 25 kDa PEI [2] , but the overall<br />
binding constants per PBAE molecule were ~10 times smaller. This explains why the N/P ratios<br />
needed <strong>for</strong> the <strong>for</strong>mation of DNA:PBAE nanoparticles are ~10 times higher. Nanoparticle <strong>for</strong>mation<br />
efficiency is positively correlated with increased transfection efficacy <strong>for</strong> eight out of ten PBAE<br />
polymers. This demonstrates the usefulness of the described fluorescence method in DNA<br />
complexation studies within polymer libraries <strong>for</strong> the prediction of the transfection efficacy. This<br />
study helps elucidate how small changes to polymer structure make significant differences to gene<br />
delivery function.<br />
This work was supported by Academy of Finland.<br />
References: [1] J.J. Green, et al., Acc.Chem.Res. 41 (2008) 749. [2] T. Ketola, et al., J. Phys. Chem. B 115<br />
(2011) 1895.<br />
______________<br />
* Corresponding author: e-mail: elina.vuorimaa@tut.fi<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 269
P155 Fluorescence Spectroscopy Poster 155<br />
Modulation of raft-dependent immune functions by a monoclonal<br />
cholesterol-specific antibody: a biophysical study on the mechanism<br />
of action<br />
Andrea Balogh 1 , Emese Izsépi 1 , Anikó Molnár 1 , László Cervenák 2 , George Füst 2 , Zoltán<br />
Beck 3 , György Vámosi 4 , Gábor Mocsár 4 , Glória László 1 & János Matkó 1,*<br />
1<br />
Eötvös Loránd University, Insitute of Biology, Department of Immunology, H-1117 Budapest<br />
(Hungary)<br />
2<br />
Research Group of Immunogenomics, Hungarian Academy of Sciences at Semmelweis<br />
University, H-1089, Budapest (Hungary)<br />
3<br />
Institute of Medical Microbiology, University of Debrecen, H-4012, Debrecen (Hungary)<br />
4<br />
Cell Biology and Signaling Research Group of the Hungarian Academy of Sciences, Department<br />
of Biophysics and Cell Biology, Research Center <strong>for</strong> Molecular Biology, University of Debrecen, H-<br />
4012, Debrecen (Hungary)<br />
Monoclonal IgG anti-cholesterol antibodies (ACHA, clones AC1 and AC8), made in our laboratory,<br />
are unique tools to selectively label cholesterol-enriched plasma membrane microdomains, like<br />
lipid rafts [1] . The question is whether binding of the ACHA to the cell membrane has any important<br />
functional consequence in the immunological cell functions.<br />
We found that lipid raft-dependent immune functions can be modulated by ACHA. First, AC8<br />
antibody significantly inhibited infection and in vitro HIV production of human macrophages and T<br />
cells [2] . The new ACHAs, but not their Fab fragments can modulate the receptor/microdomain<br />
architecture at the surface of human macrophages and T cells, since they caused a remarkable<br />
lateral clustering of membrane rafts upon binding to both cell types and remodeled the interaction<br />
pattern of CXCR4 chemokine receptors (HIV-1 co-receptor) with both CD4 (virus receptor) and<br />
lipid rafts.<br />
Second, we have shown that AC8 augments antigen presentation in an APC-Th cell<br />
immunological synapse model. Formation of the immunological synapse between APCs and T<br />
cells is followed by enhanced calcium-mobilization in T cells when B cells, but not the Th cells are<br />
preincubated with ACHA.<br />
Thus, monoclonal IgG ACHAs, probably by promoting microlustering of membrane<br />
cholesterol, can be considered as potential modulators of several important immune functions<br />
dependent on lipid rafts, such as pathogen internalization or lymphocyte activation especially under<br />
conditions altering the epitope accessibility on their target cells (e.g. apoptosis, tumor, virus<br />
infection, etc.). Further studies on the mechanism of their immunomodulatory action, as well as,<br />
analysis of their sequence and structure are currently running in our laboratory.<br />
This work was supported by grants from Hungarian Ministry of Health (ETT grants 2003/102 to<br />
Z.B. and 2006/070 and 2006/065 to G.V.), the Hungarian National Science Fund<br />
(OTKA grants T49696 to J.M., F49164 to L.C., and T48745 and NK61412 to G.V., and OTKA CK<br />
grant 80935 to J.M.), the National Office of Research and Development (Pázmány Grant RET-<br />
06/2006 to J.M.)<br />
References: [1] A. Biro, et al., J lipid Res., 48 (2007) 19. [2] Z. Beck, et al., J Lipid Res 51 (2009) 286.<br />
______________<br />
* Corresponding author: e-mail: matko@elte.hu<br />
270 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P156 Fluorescence Spectroscopy Poster 156<br />
The action of chlorogenic acid on erythrocyte and model membranes<br />
Dorota Bonarska-Kujawa * , Hanna Pruchnik & Halina Kleszczyńska<br />
Department of Physics and Biophysics, Wroclaw University of Environmental and Life Sciences,<br />
C.K. Norwida 25/27, 50-375 Wroclaw, Poland<br />
Chlorogenic acid (CGA) is a compound that occurs in various parts of plant, e.g. in fruits,<br />
vegetables and herbs. It is present in large quantities in coffee beans, beans, potato<br />
tubers, berries, sunflower seeds, apple fruits and leaves. Many authors have reported on a<br />
protective effect of CGA on the human organism, ascribed to its very good antioxidant<br />
properties and also anticancer, fungicidal and bartericidal. However, the CGA action on<br />
biological system at the molecular and cell levels has not yet been elucidated. The studies<br />
presented aimed at determination of the effect of CGA on the biological and lipid<br />
membrane. In the fluorimetric method applied, various probes were used. In particular, the<br />
antioxidant activity of chlorogenic acid with respect to biological membranes was studied,<br />
its location within membranes and influence on temperature of the main phase transition of<br />
lipid membranes. Its antioxidant activity was tested with the DPH-PA probe. Changes in<br />
the hydrophilic region of membrane and changes in temperature of the main phase<br />
transition of lipid membranes were investigated using the laurdan and prodan probes,<br />
whereas changes in fluidity of the membrane hydrophobic region were assayed with the<br />
DPH and TMA-DPH markers.<br />
The results obtained confirm the very good antioxidant properties of CGA towards<br />
biological and model membranes. They also indicate that the compound incorporates<br />
mainly into the hydrophilic part of membrane, changing the packing order of the polar<br />
heads of lipids. No significant changes were recorded in membrane fluidity of the<br />
hydrophobic membrane region, <strong>for</strong> the fluorescence anisotropy did not change practically.<br />
One can thus infer that chlorogenic acid does not penetrate deep the hydrophobic area of<br />
the membrane. This is confirmed by the results on temperature of the main phase<br />
transition of lipids, which decreased slightly.<br />
Based on the results obtained, one can state that chlorogenic acid locates mainly in the outer<br />
hydrophilic part of membrane, does not penetrate deep into the hydrophobic region of the lipid<br />
bilayer, and hence does not effect the physical parameters and structure of the membranes<br />
studied. Thus it seems that the very good antioxidant properties of CGA with regard to biological<br />
membranes are due both to its free radical scavenging ability and the intensity of binding to the<br />
hydrophilic part of membrane.<br />
This work was sponsored by the Ministry of Science and Education, scientific project no. N N312<br />
422340 and N N304 173840 .<br />
______________<br />
* Corresponding author: e-mail: dorota.bonarska-kujawa@up.wroc.pl<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 271
P157 Fluorescence Spectroscopy Poster 157<br />
Fluorescence studies on new potential antitumoral 1,3-diarylurea<br />
derivatives of thieno[3,2-b]pyridines in solution and in nanoliposomes<br />
Elisabete M. S. Castanheira 1,* , Cátia N. C. Costa 1 , Ana Rita O. Rodrigues 1 , Daniela Peixoto 2<br />
& Maria-João R. P. Queiroz 2<br />
1 2<br />
Centre of Physics (CFUM) and Centre of Chemistry (CQ-UM), University of Minho, Campus de<br />
Gualtar, 4710-057 Braga (Portugal)<br />
Nanoliposomes are among technological delivery developments <strong>for</strong> chemotherapeutic drugs in the<br />
treatment of cancer. This technique can potentially overcome many common pharmacologic<br />
problems, such as those involving solubility, pharmacokinetics, in vivo stability and toxicity. [1,2] In<br />
this work, the fluorescence properties of new potential antitumoral 1,3-diarylureas in the thieno[3,2b]pyridine<br />
series, 1a-c, were studied in solution and in lipid membranes of different composition<br />
and charge, either cationic, neutral (zwitterionic) or anionic. Nanosized liposomes were prepared<br />
using egg yolk phosphatidylcholine (Egg-PC) dioctadecyldimethylammonium bromide (DODAB),<br />
dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG), with or<br />
without cholesterol (Ch). Compounds 1a-c present very reasonable fluorescence quantum yields in<br />
different solvents (0.10 ≤ ΦF ≤ 0.30) and exhibit a red shift in emission with increasing solvent<br />
polarity. However, they are not fluorescent in protic solvents (like alcohols and water).<br />
HN<br />
O<br />
H<br />
N<br />
N<br />
S<br />
1a R = H<br />
1b R = OCH 3<br />
1c R = CN<br />
NH 2<br />
R<br />
CO 2Me<br />
Figure 1. Normalised fluorescence spectra of compound 1a in<br />
lipid membranes of DPPC, Egg-PC and DPPC/Ch 7:3.<br />
All compounds exhibit reasonable fluorescence emission when incorporated in lipid membranes<br />
(Figure 1), indicating that they can be transported in the hydrophobic region of the lipid bilayer.<br />
Fluorescence anisotropy measurements of the compounds incorporated in nanoliposomes show<br />
notable anisotropy variations when fluidity increases above the melting transition temperature or by<br />
addition of cholesterol. These results may be important <strong>for</strong> future drug delivery applications of<br />
these potential antitumoral compounds using nanoliposomes as drug carriers.<br />
This work was supported by Fundação para a Ciência e a Tecnologia (Portugal), QREN and<br />
FEDER through CFUM, CQ/UM and Project PTDC/QUI/81238/2006 cofinanced by FCT and<br />
program FEDER/COMPETE (FCOMP-01-0124-FEDER-007467).<br />
References: [1] T. L. Andresen, et al., Prog. Lipid Res. 44 (2005) 68-97. [2] N.A. Ochekpe, et al., J. Pharm.<br />
Res. 8 (2009) 265-274; 275-287.<br />
______________<br />
* Corresponding author: e-mail: ecoutinho@fisica.uminho.pt<br />
272 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P158 Fluorescence Spectroscopy Poster 158<br />
Study of morphology, hydration and mobility of cationic<br />
lipophosphoramidates liposomes by fluorescent solvent relaxation<br />
Damien Loizeau 1 , Piotr Jurkiewicz 2 , Laure Deschamps 1 , Paul-Alain Jaffrès 1 , Martin Hof 2<br />
& Philippe Giamarchi 1,*<br />
1 Laboratoire CEMCA, CNRS UMR 6521, IFR 148 ScInBIoS, Université de Bretagne Occidentale,<br />
6 Avenue Le Gorgeu, 29238 Brest, France<br />
2 Institut of Physical Chemistry, Prague, Czech Republic<br />
We present a study on hydration and viscosity of liposome’s membranes of cationic phospholipids<br />
designed <strong>for</strong> DNA transfer and gene therapy. These original cationic phospholipids are<br />
characterised by the presence of two aliphatic chains placed on a phosphoramidate group which is<br />
also bonded to a cationic head via a linker. They are used to <strong>for</strong>mulate liposomes and then to<br />
condense DNA to obtain lipoplexes vectors [1, 2]. This work is a collaboration between the CNRS<br />
UMR 6521 laboratory (Brest, France) <strong>for</strong> the phospholipids synthesis and general fluorescence<br />
study; and Martin Hof laboratory (Prague, Czech Republic) <strong>for</strong> the advanced fluorescence solvent<br />
relaxation study [3, 4].<br />
We have followed the<br />
Stokes shift of the probe emission<br />
(Laurdan) in pure cationic<br />
liposomes. At 20°C the figure<br />
reveals gradual red shift of<br />
Laurdan spectra indicating a mix<br />
of the gel phase (Lβ) and liquid<br />
crystalline (Lα) phase. Generalized<br />
polarization study leads also to the<br />
same conclusion.<br />
To obtain additional<br />
in<strong>for</strong>mation we used the<br />
fluorescent solvent relaxation<br />
technique. By reconstruction of<br />
the time-resolved emission<br />
spectra we estimated the<br />
membrane hydration and the<br />
micro environment viscosity of the<br />
probe.<br />
We first worked on a mixture of our cationic phospholipids with DOTAP and DOPC as<br />
reference, and then on pure lipo-phosphoramidate liposomes. We found that the last ones are less<br />
hydrated and their microenvironment is less mobile than pure DOTAP or pure DOPC. Moreover, in<br />
mixtures, DOPC increases significantly the hydration and the fluidity of the membranes.<br />
References: [1] Fraix A., et al., Organic & Biomolecular Chemistry, 9 (2011) 2422. [2] Le Gall T., et al.,<br />
Journal of Medicinal Chemistry, 53 (2010) 1496. [3] J. Sykora, et al., Chemistry and Physics of Lipids, 135<br />
(2005) 213. [4] P. Jurkiewicz, et al., Langmuir, 22 (2006) 8741.<br />
______________<br />
* Corresponding author: e-mail: philippe.giamarchi@univ-brest.fr<br />
Fluorescence Intensity (au)<br />
1<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
DOTAP<br />
C18:1, As+<br />
C18:1, N+, N-Me<br />
400 450 500 550 600<br />
Emission Wavelength (nm)<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 273
P159 Fluorescence Spectroscopy Poster 159<br />
Characterization by steady-state and time resolved fluorescence<br />
spectroscopy of the lipid and protein organization in HBsAg particles<br />
Vanille J. Greiner 1 , Caroline Egelé 1 , Sule Oncul 1 , Frédéric Ronzon 2 , Catherine Manin 2 ,<br />
Andrey Klymchenko 1 1, *<br />
& Yves Mély<br />
1 Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg,<br />
Faculté de pharmacie, 74 route du Rhin, 67401 ILLKIRCH, France<br />
2 Sanofi pasteur, Marcy l’étoile, France<br />
Hepatitis B vaccines are based on recombinant hepatitis B surface antigen (HBsAg) particles<br />
adsorbed on aluminium hydroxide. HBsAg particles are 20 nm particles, composed of S surface<br />
viral proteins and host-derived lipids. Since the detailed structure of these particles is still missing,<br />
we characterized their lipid and protein organization by fluorescence techniques. Fluorescence<br />
correlation spectroscopy indicated that the particles are mainly monomeric, with about 70 copies of<br />
S proteins per particle. Furthermore, S proteins exhibit restricted movement, as expected from their<br />
tight association with lipids. The lipid organization of the particles was studied using viscositysensitive<br />
DPH-based probes and environment sensitive 3-hydroxyflavone probes, and compared<br />
to lipid vesicles and low density lipoproteins (LDLs), taken as models. Like LDLs, the HBsAg<br />
particles were found to be composed of an ordered rigid lipid interface and a more hydrophobic<br />
and fluid inner core, likely composed of triglycerides and free fatty acids. The lipid core of HBsAg<br />
particles was substantially more polar than the LDL one, probably due to its larger content in<br />
proteins and its lower content in sterols. Based on our data, we propose a structural model <strong>for</strong><br />
HBsAg particles where the S proteins deeply penetrate into the lipid core [1] . In a next step, we<br />
checked whether the adsorption on aluminium hydroxide does not affect the structure of the viral<br />
particles. Steady-state and time-resolved fluorescence data of the intrinsic fluorescence of the S<br />
proteins indicated that the adsorption/desorption of HBsAg particles on Al-gel did not modify the<br />
environment of the most emitting Trp residues, confirming that the con<strong>for</strong>mation of the S proteins<br />
remains intact. The lipid organization was also investigated, indicating that no significant<br />
modifications of the lipid core and the lipid membrane surface of HBsAg particles occurs during the<br />
adsorption/desorption process. In conclusion, this study demonstrates that adsorption of HBsAg<br />
particles on Al-gel does not affect the structure of the viral particles.<br />
This work was supported by a grant from Sanofi Pasteur.<br />
References: [1] V.J Greiner, et al., Biochimie 92 (2010) 994.<br />
______________<br />
* Corresponding author: e-mail: yves.mely@unistra.fr<br />
274 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P160 Fluorescence Spectroscopy Poster 160<br />
Spectroscopic studies of the novel, cationic gluconamide-type<br />
surfactants/DNA and lipid interactions<br />
Teresa Kral 1,2,* , Hanna Pruchnik 1 , Martin Hof 2 , Renata Skrzela 3 & Kazimiera A. Wilk 3<br />
1<br />
Wrocław University of Environmental and Life Sciences, C.K. Norwida 25, Wrocław (Poland)<br />
2<br />
J. Heyrovsky Institute of Physical Chemistry of the ASCR, v. v. i., Dolejškova 3, Prague (Czech<br />
Republic)<br />
3<br />
Wrocław University of Technology, Department of Chemistry, Wybrzeże Wyspiańskiego 27,<br />
Wrocław (Poland)<br />
Sugar-based surfactants (SBS) are considered to be interesting because they possess improved<br />
surface and per<strong>for</strong>mance properties, reduced environmental impact due to easy biodegradation,<br />
and have potential pharmaceutical and biomedical applications such a gene therapy.<br />
A major problem <strong>for</strong> the routine use of gene therapy is the efficient introduction of DNA into<br />
the cell nucleus. DNA condensing agents cause, by a variety of mechanisms, DNA molecules to<br />
collapse and aggregate into ordered, highly condensed states.<br />
In order to <strong>for</strong>m condensed states of DNA a number of intermolecular interactions need to be<br />
resolved and quantitatively described. Interaction of DNA and lipid molecules with sugar-based<br />
surfactants is a key issue in the design and construction of supramolecular assembly.<br />
We study the <strong>for</strong>mation of DNA–SBS complexes using the Fluorescence Correlation<br />
spectroscopy (FCS). FCS provides the unique capability of measuring the statistical distribution of<br />
particle size basing on single molecule detection. We have measured the interaction of<br />
fluorescently labeled DNA with various SBS (2-(alkyldimethylammonio)ethylgluconamide<br />
bromides) chain length (n = 10, 12, 14 and 16). The potency of DNA/SBS assembly <strong>for</strong>mation is<br />
enhanced both with increasing concentration of the surfactant (in relation to base pair of the DNA)<br />
and increasing chain length of the surfactant.<br />
We investigated lipid-SBS systems using steady-state fluorescence spectroscopy. As model<br />
systems were used small unilamellar DPPC-SBS liposomes. Presence of SBS has an impact on<br />
temperatures of the main and pre-transition of DPPC lipid phase and its fluidity. We can write that<br />
examined SBS narrowly decreasing the main phase transition temperatures (n = 10
P161 Fluorescence Spectroscopy Poster 161<br />
Effects of supplementation with L-proline or inositol on yeast<br />
membrane fluidity and ethanol tolerance<br />
Safri Ishmayana 1,2 , Ursula Kennedy 1 & Robert Learmonth 1*<br />
1<br />
Centre <strong>for</strong> Systems Biology, University of Southern Queensland, Toowoomba 4350 (Australia)<br />
2<br />
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran,<br />
Bandung (Indonesia)<br />
The decrease of fossil fuel availability has created a high demand <strong>for</strong> alternative fuels, including<br />
bioethanol produced by yeast fermentation of carbohydrate. Relatively low ethanol yields can be<br />
problematic, and approaches to increase efficiency have included genetic modification of yeast to<br />
improve metabolic flux, fermentation rate and ethanol tolerance. We investigated an alternative<br />
approach, aiming to evaluate novel yeasts <strong>for</strong> bioethanol production, and further to enhance their<br />
ethanol tolerance through modification of growth medium composition. We focused specifically on<br />
two important components which have been reported to positively affect yeast stress tolerance; the<br />
sugar inositol [1,2] and the amino acid L-proline [3] . Three yeast strains (a bakers’ yeast, a wine yeast<br />
and a sake yeast), known tolerate 17% ethanol, were studied under aerobic conditions. Cultures<br />
were supplemented with various concentrations of either inositol or L-proline, and sampled during<br />
respiro-fermentative growth (fermentation of glucose to ethanol) and respiratory growth on ethanol.<br />
Physiological parameters, viability, membrane fluidity (by laurdan GP) and tolerance to 18%<br />
ethanol were determined. Differences were noted in the growth, ethanol productivity and ethanol<br />
tolerance of the yeasts, with significant differences evident in laurdan GP in respiro-fermentative,<br />
but not respiratory phase cells. As expected, tolerance to ethanol was significantly higher in<br />
respiratory phase cells, also correlating with significantly higher laurdan GP which indicated lower<br />
membrane fluidity. However, on trialling L-proline levels from 0.1 to 3 g/L and inositol levels from<br />
0.002 to 0.2 g/L, no significant differences were found in laurdan GP or ethanol tolerance. Thus we<br />
could not confirm previous reports of positive effects of L-proline or inositol, due largely to high<br />
variability in the data from triplicate experiments. It is possible that the effects of these supplements<br />
may be strain dependant; the wine yeast tended towards lower membrane fluidity with 0.5 g/L<br />
proline, and in the sake yeast there was a trend towards improved ethanol tolerance with inositol<br />
levels above 0.005 g/L. Future experiments will investigate a wider variety of yeast strains and<br />
increase repetition to minimise data variability.<br />
The authors wish to acknowledge the support of a scholarship from the Ministry of National<br />
Education of the Republic of Indonesia to enable postgraduate study by Safri Ishmayana.<br />
References: [1] E. L. Krause et al., Indust. Biotechnol. 3 (2007) 260. [2] R. Ji et al., J. Agro-Environ. Sci. 27<br />
(2008) 2080. [3] H. Takagi et al., Appl. Env. Microbiol. 71 (2005) 8656.<br />
______________<br />
* Corresponding author: e-mail: Robert.Learmonth@usq.edu.au<br />
276 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P162 Fluorescence Spectroscopy Poster 162<br />
Study of the phase transition of mixtures of original cationic<br />
phospholipids by fluorescence anisotropy<br />
Sélim Mahfoudhi 1,2 , Damien Loizeau 1,2 , Laure Deschamps 1,2,* , Paul-Alain Jaffrès 1,2 , Tristan<br />
Montier 1,3 , Pierre Lehn 1,3 & Philippe Giamarchi 1,2<br />
1 1 a<br />
Université Européenne de Bretagne, Brest University, IFR ScInBioS<br />
2<br />
UMR CNRS 6521 CEMCA,<br />
3<br />
INSERM U613, 6 av. Le Gorgeu, 29285 Brest cedex, France<br />
Different types of cationic phospholipids have been synthesized in the laboratory with the aim of<br />
developing new DNA carriers <strong>for</strong> gene therapy [1, 2]. These original cationic phospholipids are<br />
characterised by the presence of two aliphatic chains placed on a phosphoramidate group which is<br />
also connected to a cationic head via a linker. Some precedent works have focussed on the<br />
physical and chemical properties of these vectors in order to establish correlations between their<br />
structure, some physico-chemical properties and biological activity [3, 4].<br />
In this work, the cationic lipids studied have all the same polar head, the same linker and<br />
number of unsaturations on the fatty chains. We studied the influence of mixing cationic lipids of<br />
various chain lengths (14 to 18 carbons) at different ratios on the membrane viscosity. The<br />
diphenyl-hexatriene (DPH) fluorescent probe was used to measure the anisotropy of the bilayer as<br />
a function of the temperature. The results obtained show an important evolution of the anisotropy<br />
(figure below).<br />
For each mixture, the<br />
evolution of the anisotropy as<br />
a function of the temperature<br />
has been modeled by an arctangent<br />
mathematical model.<br />
The minimum of the arctangent<br />
function allows to<br />
determine the transition<br />
temperature from the gel<br />
phase (Lβ) to the liquid<br />
crystalline phase(Lα).<br />
By the end, we can<br />
describe the evolution of the<br />
phase transition temperature<br />
versus the mixture ratio.<br />
These results allow to<br />
choose the phase transition<br />
temperature of the lipids<br />
Anisotropy (r)<br />
bilayer by choosing the lipids mixture ratio. In future evolutions, we will study the influence of phase<br />
transition temperature on the fusogenic properties of the liposomes by Fluorescent Resonant<br />
Energy Transfer (FRET).<br />
References: [1] A. Fraix, et al., Organic & Biomolecular Chemistry, 9 (2011) 2422. [2] T. Le Gall, et al.,<br />
Journal of Medicinal Chemistry, 53 (2010),1496. [3] L. Burel-Deschamps, et al., Journal of fluorescence, 18<br />
(2008) 835.[4] F. Lamarche, et al., Bioconjugate Chemistry, 18 (2007) 1575.<br />
______________<br />
*Corresponding author: e-mail: laure.deschamps@univ-brest.fr.<br />
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0.35<br />
0.3<br />
0.25<br />
0.2<br />
0.15<br />
0.1<br />
0.05<br />
0<br />
Model<br />
C18:0<br />
Slope<br />
0 10 20 30 40 50 60<br />
Temperature<br />
-0.002<br />
-0.004<br />
-0.006<br />
-0.008<br />
-0.01<br />
-0.012<br />
-0.014<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 277<br />
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P163 Fluorescence Spectroscopy Poster 163<br />
Dynamic organization of HIV co-receptors in the membrane of immune<br />
cells revealed by single quantum dot tracking<br />
Patrice Mascalchi 1 , Anne-Sophie Lamort 1 , Evert Haanapel 1 , Fabrice Dumas 1 , Bernard<br />
Lagane 2 , Fernando Arenzana-Seisdedos 2 , André Lopez 1 & Laurence Salomé 1,*<br />
1 Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse UPS/CNRS<br />
UMR5089, 205 route de Narbonne, BP 64182, F-31077 Toulouse (France)<br />
2 Unité de Pathogénie Virale, INSERM U819, Institut Pasteur, 75724 Paris Cedex 15 (France)<br />
Early steps of infection by a HIV viral particle involves several trimeric gp120 envelop proteins,<br />
each subunit binding sequentially to CD4, and then to CCR5 (or CXCR4) receptors at the immune<br />
cell surface. Thus, infection efficiency probably depends on the dynamic distribution of both CD4<br />
and CCR5 receptors.<br />
We previously showed by fluorescence based approaches that compartmentalization of<br />
these receptors in their basal state exists at the surface of a model cell line [1,2] . To validate these<br />
results and to determine the molecular mechanisms that control this lateral distribution, we pursue<br />
our study on natural targets of HIV: immune cells which express both CD4 and CCR5 native<br />
receptors. We implemented a single particle tracking method to investigate the behavior of<br />
individual receptors. It is based on the labelling of HIV receptors by means of specific antibodies<br />
labelled with Quantum Dots.<br />
By comparison with fluorescence recovery after photobleaching measurements, we first<br />
validated on a supported planar lipid bilayer that the fluorescent particles coupled to antibodies do<br />
not influence the diffusion of the tracked molecule. We then analyzed the diffusion of CD4<br />
receptors at basal state and observed 3 diffusion modes: random, confined or transitory confined<br />
diffusion. Addition of the envelop protein or other molecules disrupting the distribution of each<br />
receptor will allow us to determine which processes are involved in their dynamic membrane<br />
organization. These data will help to the design of new therapeutical strategies which aim at<br />
inhibiting the binding of viral particles to target cells.<br />
This work was supported by grants from ANRS and Sidaction.<br />
References: [1] G. Gaibelet, et al., J. Biol. Chem., 281-49 (2006) 37921. [2] A. Baker, et al., J. Biol. Chem.<br />
282-48 (2007) 35163.<br />
______________<br />
* Corresponding author: e-mail: laurence.salome@ipbs.fr<br />
278 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P164 Fluorescence Spectroscopy Poster 164<br />
Lipid raft detecting in membranes of live erythrocytes<br />
Ilya Mikhalyov 1* & Andrey Samsonov 2<br />
1<br />
Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul.<br />
Miklukho-Maklaya 16/10, Moscow 117997, Russia<br />
2<br />
University of Illinois Medical School, Department of Physiology and Biophysics, 835 S. Wolcott<br />
Ave., Chicago, IL 60612, USA<br />
The fluorescent probe N-(BODIPY ® -FL-propionyl)-neuraminosyl-GM1, bearing the fluorescent<br />
group in the polar head of ganglioside GM1 (BODIPY-GM1), was used to detect lipid rafts in living<br />
red blood cells (RBCs) membranes [1] . The probe was detected with fluorescence video<br />
microscopy and was found to be uni<strong>for</strong>mly distributed along plasma membrane at room<br />
temperature (23ºC). At 4ºC some probe clearly phase-separated to yield detectable bright spots<br />
that were smaller than spatial resolution. As measured by spectrofluorometry, in addition to a major<br />
fluorescence peak caused by emissions from monomers, the probe exhibited a red-shifted peak<br />
that is characteristic of a BODIPY fluorophore at high local concentrations, indicating that some<br />
probe had clustered. Red-shifted fluorescence was the greatest at 4ºC, intermediate at 23ºC, and<br />
the smallest at 37ºC (Fig. 1). The fluorescent GM1, bearing the residue of BODIPY-FL-C5 fatty acid<br />
in the hydrophobic part of the ganglioside molecule, was used as a control. Treating the RBCs with<br />
methyl-β-cyclodextrin to remove cholesterol eliminated the red-shifted peak. This strongly indicates<br />
that the presence of cholesterol was essential <strong>for</strong> phase separation of the probe. Fluorescence<br />
experiments indicate that rafts exist at 23ºC and at 37ºC, even though the membrane appears to<br />
be uni<strong>for</strong>m at the resolution of microscope. The distinct GM1 patches distributed over entire<br />
membrane of the erythrocytes were observed at both 23°C and at 37°C in RBCs stained with<br />
Alexa FL 647 cholera toxin subunit B conjugate (CTB-A647 ). Based on both fluorometry and<br />
fluorescence microscopy, some rafts clearly exist at 37ºC. So, our BODIPY-GM1 can be used as a<br />
marker of lipid rafts in cells.<br />
Fig. 1. Fluorescence emission spectra of BODIPY-GM1 and BODIPY ® FL C5-GM1 incorporated into RBCs.<br />
Spectra are shown <strong>for</strong> 4°C, 23ºC, and 37ºC, all normalized to the monomer peak at the 515 nm. A broad redshifted<br />
peak is more prominent <strong>for</strong> lower temperature <strong>for</strong> BODIPY- GM1 but not <strong>for</strong> BODIPY ® FL C5-GM1,<br />
labeled in the hydrophobic part of the ganglioside (the results are showed only <strong>for</strong> 4°C).<br />
References: [1] I. I. Mikhalyov and A. V. Samsonov, Biochim. Biophys. Acta, (2011), doi:<br />
10.1016/j/bbamem.2011.04.002.<br />
______________<br />
* Corresponding author: e-mail: Ilya.mikhalyov@gmail.com<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 279
P165 Fluorescence Spectroscopy Poster 165<br />
Domains II and III of Bacillus thuringiensis Cry1Ab toxin remain exposed<br />
to the solvent after Insertion of part of domain I into the membrane<br />
Liliana Pardo-Lopez*, Luis Enrique Zavala, Emiliano Cantón, Isabel Gómez, Mario Soberón<br />
& Alejandra Bravo<br />
Instituto de Biotecnología. Universidad Nacional Autónoma de México. Av. Universidad 2001 Col<br />
Chamilpa. Cuernavaca Morelos (México)<br />
Bacillus thuringiensis produces insecticidal proteins named Cry toxins, that are used commercially<br />
<strong>for</strong> the control of economical important insect pests. These are pore-<strong>for</strong>ming toxins that interact<br />
with different receptors in the insect gut, <strong>for</strong>ming pores in the apical membrane causing cell burst<br />
and insect death.<br />
Elucidation of the structure of the membrane-inserted toxin is important to fully understand its<br />
mechanism of action. One hypothesis proposed that the hairpin of α-helices 4-5 of domain I inserts<br />
into the phospholipid bilayer, whereas the rest of helices of domain I are spread on the membrane<br />
surface in an umbrella-like con<strong>for</strong>mation. However, a second hypothesis proposed that the three<br />
domains of the Cry toxin insert into the bilayer without major con<strong>for</strong>mational changes. In this work<br />
we constructed single Cys Cry1Ab mutants that remain active against Manduca sexta larvae and<br />
labeled them with different fluorescent probes that have different responses to solvent polarity.<br />
Different soluble quenchers as well as a membrane-bound quencher were used to compare the<br />
properties of the soluble and brush border membrane-inserted <strong>for</strong>ms of Cry1Ab toxin. The<br />
fluorescence and quenching analysis presented here, revealed that domains II and III of the toxin<br />
remain in the surface of the membrane and only a discrete region of domain I is inserted into the<br />
lipid bilayer, supporting the umbrella model of toxin insertion.<br />
This work was supported by grants from DGAPA/UNAM IN206209-3 and IN218409, CONACyT<br />
128883 and 81679-Q..<br />
______________<br />
* Corresponding author: e-mail: liliana@ibt.unam.mx<br />
280 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P166 Fluorescence Spectroscopy Poster 166<br />
Quantification of local water concentration in biomolecules using<br />
dual-fluorescence labels<br />
Vasyl G. Pivovarenko 1,2 , Oleksandr M. Zamotaiev 1 , Volodymyr V. Shvadchak 2 , Viktoriia Y.<br />
Postupalenko 2 , Andrey S. Klymchenko 2 & Yves Mély 2<br />
1<br />
Organic Chemistry Chair, Chemistry Faculty, Taras Shevchenko National University of Kyiv,<br />
01601 Kyiv (Ukraine)<br />
2<br />
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 du CNRS, Université de Strasbourg,<br />
Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, (France)<br />
Water plays a key role in the structure and function of biological systems, mainly as the medium <strong>for</strong><br />
biomolecular interactions between nucleic acids, proteins, carbohydrates, etc. However, the<br />
quantification of local water concentration [H2O]L at a given site of a biomolecular system using<br />
fluorescent probes has been never addressed so far. For most hydration-sensitive probes,<br />
quantification of [H2O]L requires discrimination of H-bonding interactions from effects of polarity<br />
(dipole-dipole interactions) and ions. Recently, a step towards this aim was made through the<br />
design of 3-hydroxychromone (3HC) probes [1,2].<br />
HO<br />
O<br />
O<br />
O<br />
N<br />
H O<br />
FC<br />
O<br />
O<br />
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HO<br />
O<br />
O<br />
O<br />
O<br />
H<br />
O<br />
HO<br />
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— MAF 12, Strasbourg, France, September 11-14, 2011 — 281<br />
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F6C F6A F4O<br />
In this work, we propose a method <strong>for</strong> measuring the local water content near protein backbones,<br />
based on the ratiometric response of 3HC fluorophores and the application of this method <strong>for</strong><br />
comparing peptide hydration levels in their free state and in peptide-DNA complexes. Using four<br />
labels of the 3HC family [1,2] displaying selective sensitivity to H-bond donors and poor response<br />
to other polar molecules, pH and ionic strength, an approach was elaborated <strong>for</strong> the estimation of<br />
local Hydrogen bond donor concentration in the probe surrounding both in solvent mixtures and in<br />
biomolecules. For each probe, the logarithm of the fluorescence intensity ratio of their two emissive<br />
<strong>for</strong>ms, Log(N*/T*), was found to be linearly dependent on the local concentration of H-bond donors<br />
and was thus used to estimate the water concentration [H2O]L in biomolecules. This method allows<br />
the determination of [H2O]L with a good precision at concentrations higher than 5 M (higher than<br />
10% by its partial volume). Next, the local concentration of water was determined <strong>for</strong> several<br />
labeled peptides in complex with ss-DNAs and ds-DNAs. The hydration of the probe surroundings<br />
was found to gradually decrease in the following order: free peptide >> peptide/short<br />
oligonucleotides >> peptide/ssDNAs > peptide/DNA hairpins > peptide/dsDNAs. In the last case, a<br />
very efficient screening of the probe from water was achieved through intercalation of the probe<br />
within the base pairs. The position of probe-to-linker connection was found to influence the probe<br />
response, especially in the case of complexes with ds-DNA. Thus, quantification of local water<br />
concentration in the studied biomolecules allows estimating fine features of the probe surrounding<br />
and helps revealing the nature of peptide complexes with different DNAs.<br />
This work was supported by grant from ARCUS program.<br />
References: [1] O.M. Zamotaiev et al., Bioconjugate Chem., 22 (2011) 101–107. [2] V.G. Pivovarenko et al.,<br />
Submitted.<br />
______________<br />
* Corresponding author: e-mail: pvg_org@ukr.net<br />
O<br />
H<br />
O<br />
O<br />
O<br />
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OH
P167 Fluorescence Spectroscopy Poster 167<br />
Biophysical state of plasma membrane and intrinsic efficiency of δopioid<br />
receptors; fluorescence spectroscopy studies of HEK293 cells<br />
expressing DOR-Giα1 fusion protein<br />
Lenka Roubalova 1,* , Pavel Ostasov 1 , Jan Sykora 2 , Jana Brejchova 1 , Martin Hof 2<br />
& Petr Svoboda 1<br />
1<br />
Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20<br />
Prague 4 (Czech Republic)<br />
2<br />
Institute of Physical Chemistry of Jaroslav Heyrovsky, Academy of Sciences of the Czech<br />
Republic, Dolejskova 3, 182 23 Prague 8 (Czech Republic)<br />
Delta-opioid receptor (DOR)-G protein coupling was analyzed in plasma membranes (PM) isolated<br />
from PTX-untreated and PTX-treated HEK293 cells stably expressing PTX-insensitive DOR-Gi1α<br />
(Cys 351 -Ile 351 ) fusion protein which were exposed to cholesterol-depleting agent, β-cyclodextrin (β-<br />
CDX). Results of receptor ligand binding and high-affinity [ 35 S]GTPγS binding assays indicated that<br />
cholesterol depletion did not alter the agonist binding site of DOR (Bmax and Kd) but the ability of<br />
DOR agonist DADLE to activate G proteins was markedly impaired: EC50 <strong>for</strong> DADLE-stimulated<br />
[ 35 S]GTPγS binding was shifted by one order to the right indicating the decrease in affinity of<br />
hormone response.<br />
With the aim to get in<strong>for</strong>mation about organization and dynamic state of PM, the intact, living<br />
cells were labeled by fluorescent derivative of cholesterol (NBD-cholesterol), exposed to β-CDX<br />
and the fluorescence signal of this probe was monitored by fluorescence life-time imaging (FLIM).<br />
The intensity of NBD-cholesterol fluorescence was decreased to half of the control level but the<br />
distribution of its life-time values along the cell surface was unchanged. Analysis of hydrophobic<br />
membrane interior by steady-state anisotropy of fluorescence of 1, 6-diphenyl-1´,3´,5´-hexatriene<br />
(rDPH) indicated that cholesterol depletion caused significant “fluidization” of the membrane. The<br />
time-resolved analysis of DPH fluorescence revealed that the average life-time of DPH was<br />
decreased indicating an increased polarity of membrane interior. Analysis of dynamic<br />
depolarization of DPH molecule according to the „wobble in cone“ model indicated that S-order<br />
parameter was decreased in parallel with increase of wobbling diffusion constant Dw. Thus, the<br />
decrease of the cell membrane cholesterol level was reflected in more chaotically organized<br />
aliphatic chains of fatty acids which provided more space <strong>for</strong> the movement of DPH dye. The polar<br />
head group region of the membrane /membrane-water interface was characterized by Laurdan GP.<br />
These measurements indicated an increased polarity of membrane surface area. When combined<br />
together, results of biophysical studies of HEK cell membrane indicated that decrease of<br />
cholesterol content was reflected in relatively small change of the cell membrane structure and<br />
dynamics; both hydrophobic membrane interior (represented by aliphatic chains of fatty acids) and<br />
membrane-water interface (region of phospholipids polar head-groups) were altered. Hydrophobic<br />
membrane interior became more chaotically organized /fluid and simultaneously more hydrated,<br />
i.e. more accessible to the surrounding polar/water environment.<br />
This work was supported by MSMT (LC 554 and LC 06063), GA AVCR IAA 500110606 and<br />
AV0Z50110509.<br />
______________<br />
* Corresponding author: e-mail: roubalova@biomed.cas.cz<br />
282 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P168 Fluorescence Spectroscopy Poster 168<br />
Interactions of α-synuclein with membranes<br />
Volodymyr V. Shvadchak 1* , Lisandro J. Falomir-Lockhart 1 , Dmytro A. Yushchenko 1,2<br />
& Thomas M. Jovin 1<br />
1<br />
Laboratory of Cellular Dynamics, Max Planck Institute <strong>for</strong> Biophysical Chemistry, am Faßberg 11,<br />
37077 Göttingen, Germany<br />
2<br />
Present address: The Molecular Biosensor and Imaging Center, Carnegie Mellon University,<br />
4400 Fifth Avenue, Pittsburgh 15213, PA, USA<br />
α-synuclein (AS) is the main constituent of pathological deposits in the midbrain of individuals<br />
affected by Parkinson’s disease. Evidence suggests that neurotoxicity may originate from the binding<br />
of oligomeric AS to cellular membranes, resulting in disruption and cell leakage. Defining the<br />
interactions of AS with membranes is thus essential <strong>for</strong> understanding its physiological and<br />
pathological functions.<br />
We per<strong>for</strong>med a systematic in vitro study of the effects of membrane charge, phase, curvature,<br />
defects and lipid unsaturation on AS binding using model vesicles and AS labeled with a new<br />
solvatochromic fluorescent probe MFE. This dye senses protein microenvironment via the ratio of<br />
two emission bands resulting from Excited <strong>State</strong> Intramolecular Proton Transfer (ESIPT). The<br />
emission spectrum of MFE strongly depends on the membrane properties, allowing clear<br />
discrimination of the protein bound to vesicles of different composition and corresponding affinities by<br />
competition experiment without per<strong>for</strong>ming titrations. The two-band ratiometric response of the MFE<br />
label also enables stopped-flow measurements of the kinetics of AS migration between membranes<br />
of different compositions [1], which are inaccessible by conventional methods. The interaction of AS<br />
with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes<br />
upon thermal transition to the Ld phase and transfers to vesicles with higher affinity, e.g. those that<br />
are negatively charged.<br />
Fluorescence, a.u.<br />
20 On Membrane<br />
15<br />
10<br />
5<br />
0<br />
Free<br />
α-Synuclein<br />
450 500 550 600 650<br />
S<br />
N<br />
O<br />
O<br />
Wavelength, nm<br />
Fluorescence, a.u.<br />
800<br />
600<br />
400<br />
200<br />
0<br />
Migration<br />
450 500 550 600 650<br />
Wavelength, nm<br />
AS bound to<br />
DPPC<br />
DOPG<br />
0.0 0.5 1.0 1.5 2.0<br />
Time, s<br />
By introducing the label at three different positions we were able to estimate the relative immersion of<br />
different AS domains into membrane, and how this feature changes depending on membrane<br />
composition and lipid-to-protein ratio.<br />
These findings provide insight into the relation between membrane physical properties and AS<br />
binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role<br />
of AS in the physiopathology of Parkinson’s disease as well as in its physiological repertoire.<br />
This work was supported by Marie Curie Actions postdoctoral fellowship to VVS and DAY, and by<br />
Alexander von Humboldt Foundation Georg Förster postdoctoral fellowship to LJFL.<br />
References: [1] V.V. Shvadchak, et al., J. Biol. Chem., 286 (2011) 13023. [2] V.V. Shvadchak, et al., in<br />
Lipids and Cellular Membranes in Amyloid Diseases, Wiley-VCH, 2011<br />
______________<br />
* Corresponding author: e-mail: vshvadc@gwdg.de<br />
O<br />
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O<br />
O<br />
OH<br />
N<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 283<br />
Fluorescence, a.u.<br />
5.5<br />
5.0<br />
4.5<br />
4.0<br />
3.5<br />
DPPC<br />
AS<br />
DOPG<br />
DPPC<br />
DOPG<br />
AS
P168 B Fluorescence Spectroscopy Poster 168 B<br />
Trans-membrane potential affects lateral microdomain organization of<br />
the yeast plasma membrane<br />
Jaroslav Vecer 1 , Petra Vesela 2 , Jan Malinsky 2 & Petr Herman 1*<br />
1<br />
Faculty of Mathematics and Physics, Institute of Physics, Charles University , Ke Karlovu 5,<br />
121 16 Prague, (Czech Republic)<br />
2<br />
Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videnska 1083,<br />
142 20 Prague, (Czech Republic)<br />
The cell membrane is recently understood as a laterally heterogeneous structure subdivided into<br />
specialized dynamic microdomains differing in size, structural arrangement and chemical<br />
composition, the lipid rafts. These structurally and chemically different membrane areas exhibit<br />
significantly different physical properties, host different proteins and modulate their activity.<br />
Formation and rearrangement of these plat<strong>for</strong>ms is involved in signal transduction, sensing,<br />
membrane transport, endocytosis, pathogenesis and other biological processes. Little is known<br />
about relation between membrane potential changes, plasma membrane structure and metabolic<br />
regulation. Membrane potential of ~100 mV corresponding to the trans-membrane electric field<br />
intensity of ~10 7 V/m was shown to affects membrane order of lipid vesicles [1] . Recently, a role of<br />
the membrane potential in the organization of the plasma membrane was discovered. The lateral<br />
distribution of specific proton symporters was demonstrated to be governed by trans-membrane<br />
potential [2] . In this work we explored redistribution of membrane domains in live yeast cells<br />
induced by depolarization of the plasma membrane. Redistribution of the microdomains was<br />
monitored by domain-selective trans-parinaric acid (trans-PA) that exhibits different fluorescence<br />
lifetime in ordered and unordered microenvironment [3] . In ordered membranes trans-PA displays<br />
~30 ns decay component that is missing in emission <strong>for</strong>m the unordered environment. We<br />
demonstrated that both the emission and anisotropy decays of trans-PA clearly report on a<br />
significant decrease of the ordered membrane fraction upon the cell membrane depolarization<br />
which corresponds to the ordered domain dissociation. As a reference, overall membrane order<br />
was monitored by fluorescence anisotropy of domain-nonspecific DPH.<br />
References: [1] P. Herman et al., J. Fluorescence 14 (2004), 79. [2]G. Grossmann at al., EMBO J 26, 1. [3]<br />
L.A. Sklar, et al. Biochemistry 16 (1977), 819.<br />
______________<br />
* Corresponding author: e-mail: herman@karlov.mff.cuni.cz<br />
284 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P169 Fluorescence Spectroscopy Poster 169<br />
Photo-induced modifications of model membranes<br />
Georges Weber 1,* , Maurício Baptista 2 , Adjaci F. Uchoa 2 , Rosângela Itri 3 , Carlos M. Marques 1 ,<br />
André P. Schröder 1 & Thierry Charitat 1<br />
1<br />
Institut Charles Sadron, UPR 22 CNRS, 23 rue du Loess, 67034 Strasbourg (France)<br />
2<br />
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo (Brazil)<br />
3<br />
Departamento de Física Aplicada, Instituto de Física, Universidade de São Paulo, São Paulo<br />
(Brazil)<br />
Photodynamic Therapy or PDT has been used to treat various solid tumors [1] . The method relies on<br />
the administration of a photosensitizer molecule that is suitable to induce the <strong>for</strong>mation of singlet<br />
oxygen ( 1 O2), a powerful oxidizing agent, able to induce necrosis or apoptosis in cancer cells [2] . At<br />
the molecular level, the well-known mechanism of 1 O2 induced peroxidation leads to a modification<br />
of the structural characteristics of the phospholipids including breaking of the lipid chain [3] ,<br />
<strong>for</strong>mation of ketones, aldehydes and carboxylic acids. In spite of extensive work on chemistry of<br />
phospholipids peroxidation, little is known of the deep repercussions that such molecular<br />
modifications have on the membrane cohesion and structure at the optical length scales in the 1-<br />
100 μm range, where the shape, adhesiveness and fluctuations of the membrane can be<br />
continuously monitored by optical techniques. We study the physical trans<strong>for</strong>mations induced on<br />
giant unilamellar vesicles (GUVs) by singlet oxygen species generated at the GUV surface by<br />
anchored photosensitive molecules. Lipid peroxidation results <strong>for</strong> our systems in the production of<br />
a significant surface excess [4] that we measure by micropipet aspiration. The correlation between<br />
fluorescence intensity of the photosensitizers and GUV area increase allows us to evaluate the<br />
fraction of oxidized lipids in the membrane. For different peroxidation levels, we measure the<br />
evolution of the different mechanical properties of the membrane by micropipet suction<br />
experiments.<br />
DOPC giant vesicle with 1% of photosensitizers that generate singlet oxygen. Left : Scheme<br />
representing the different steps occuring under irradiation. Right: (A) At time t = 0 s the vesicle is<br />
hold by a micropipet under a slightly sucking pressure. (B) Membrane area excess produced by<br />
lipid peroxidation is aspirated into the micropipette.<br />
This work was supported by grants from UDS and CNRS.<br />
References: [1] B. W. Henderson and T. J. Dougherty, Photochem. Photobiol., 55 (1992) 145. [2] D. P.<br />
Valenzeno, Photochem. Photobiol., 46 (1987) 147. [3] W. Caetano, et al., Langmuir, 23 (2007) 1307. [4] K.A<br />
Riske, et al., Biophys. J., 97 (2009) 1362.<br />
______________<br />
* Corresponding author: e-mail: georges.weber@etu.unistra.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 285
286 — MAF 12, Strasbourg, France, September 11-14, 2011 —
Fluorescent Proteins,<br />
Fluorescent Probes & Labels<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 287
288 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P170 Fluorescent Proteins, Fluorescent Probes & Labels Poster 170<br />
GFP—quantification by fluorescent lifetime measurements<br />
Dagmar Auerbach 1 , Benjamin Hötzer 2 & Gregor Jung 1*<br />
1<br />
Biophysical Chemistry, Saarland University, Campus B2.2, D-66123 Saarbrücken<br />
2<br />
Institut d'Electronique Fondamentale, Université Paris-Sud 11, Bātiment 220, F-91405 Orsay<br />
Cedex<br />
The green fluorescent protein (GFP) is of inestimable value <strong>for</strong> cell biology because of the<br />
possibility to attach it to other specific proteins. Particular problems have arisen with quantitative<br />
analysis of protein concentrations because of different extinction coefficients <strong>for</strong> GFP at 280 or 470<br />
nm 1 : the amount of protein calculated at 280 nm is higher than the calculation at 470 nm. Already<br />
15 years ago it was mentioned that at least 30% of the GFP molecules has failed to undergo the<br />
final dehydration reaction in the chromophore <strong>for</strong>mation 2 . Different applications are affected by this<br />
fact like FRET, ligand binding studies and metabolism analysis where uncompleted chromophores<br />
limit the dynamic range. Here, we present a method <strong>for</strong> the determination of the chromophore<br />
<strong>for</strong>mation efficiency (CFE) of GFP in a cuvette experiment. The quantification of the amount of the<br />
correctly folded proteins is carried out by lifetime measurements. A blue fluorescent dye with long<br />
lifetime acts as donor <strong>for</strong> FRET and is nonspecifically bound to GFP. A shortening of the<br />
fluorescence lifetime is interpreted as an energy transfer to the GFP with a completed<br />
chromophore. In contrast, an unaltered lifetime indicates a fail in the last step of the dehydration<br />
reaction or even earlier reactions. Analysis of the amplitude ratio of the fast and the unaltered<br />
lifetimes yields the CFE.<br />
Another approach <strong>for</strong> this issue of determination of the CFE is a titration experiment by Cu 2+ .<br />
By adding Cu 2+ to a GFP sample a concentration dependent shortening of the fluorescent lifetime<br />
of GFP is observed. On the basis of this experiment we even recently developed a copper sensor<br />
in the micro molar range with GFP in cuvettes, however a quantification of GFP can be per<strong>for</strong>med<br />
as well. Both approaches will be compared 3 .<br />
References: [1] Chalfie, M. Green Fluorescent Protein. Photochemistry and Photobiology 62, 651-<br />
656(1995). [2] Ormö, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science<br />
(New York, N.Y.) 273, 1392-5(1996). [3] Hötzer, B. et al. Determination of copper(II) ion concentration by<br />
lifetime measurements of green fluorescent protein. Journal of fluorescence accepted, (2011).<br />
______________<br />
*Corresponding author: e-mail: g.jung@mx.uni-saarland.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 289
P171 Fluorescent Proteins, Fluorescent Probes & Labels Poster 171<br />
A photoswitching chromoprotein <strong>for</strong> use in pcFRET applications<br />
Craig Don Paul 1 , Daouda AK Traore 1 , Csaba Kiss 2 , Alexander May 1 , Giuseppe Lucarelli 1 ,<br />
Rodney J Devenish 1 , Andrew Bradbury 2 , Matthew CJ Wilce 1 & Mark Prescott 1*<br />
1 Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash<br />
University, Clayton, Victoria, 3800, Australia<br />
2 Biosciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA<br />
Originally cloned from the jellyfish Aeqourea victoria some 17 year ago, the Green Fluorescent<br />
Protein (GFP) underpins the foundations of an indispensable molecular cell biological technology.<br />
In order to improve the properties of fluorescent proteins (FPs), considerable ef<strong>for</strong>t has been<br />
expended through engineering and isolation of new proteins [1] . Today bright photostable FPs<br />
covering the entire visible emission spectrum are now readily available, with many of these<br />
possessing additional useful and intriguing optical properties, such as photoswitching.<br />
Förster Resonance Energy Transfer (FRET) is a phenomenon that can be used to provide<br />
molecular proximity in<strong>for</strong>mation in living cell and tissues [2] . A range of FPs suitable <strong>for</strong> use as<br />
donor/acceptor pairs are currently available, and their careful selection can allow more than one<br />
combination to be monitored in the same experiment. However, in general and <strong>for</strong> most<br />
researchers, the rather broad excitation and emission spectra complicates analysis and restricts<br />
the number of probes emission that can be monitored to one at a time. The requirement to<br />
determine acceptor emissions to estimate FRET efficiency can be eliminated by measuring the<br />
fluorescent lifetime of the donor, but such techniques require expensive instrumentation not<br />
commonly available in most biomedical laboratories.<br />
Photoswitching acceptors such as some chemical dyes [3] and photoswitching FPs [4] have, in<br />
photochromic FRET (pcFRET) applications, been demonstrated as useful tools to determine FRET<br />
efficiency. These studies describe the use of acceptors that can be reversibly converted between a<br />
donor absorbing (‘on’) state and a donor non-absorbing (‘off’) state with the use of light. We report<br />
here on the properties and application of the first non-fluorescent reversibly photoswitchable<br />
protein useful <strong>for</strong> pcFRET applications.<br />
References: [1] R.Y. Tsien, Annu Rev Biochem., 67 (1998) 509. [2] E.A. Jares-Erijman and T.M. Jovin, Nat<br />
Biotechnol 21 (2003) 1387. [3] L. Giordano, et al., J Am Chem Soc. 124 (2002) 7481. [4] F.V. Subach, et al.,<br />
Chemistry & Biology 17 (2010) 745<br />
______________<br />
*<br />
Corresponding author: e-mail: mark.prescott@monash.edu<br />
290 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P172 Fluorescent Proteins, Fluorescent Probes & Labels Poster 172<br />
Photophysical properties of the Cyan Fluorescent Protein<br />
Asma Fredj 1 , Marie Erard 1 , Agathe Espagne 1 , Germain Vallverdu 1 , Isabelle Demachy 1 ,<br />
Jacqueline Ridard 1 , Bernard Levy 1 , Fabienne Mérola 1 & Hélène Pasquier 1,*<br />
1<br />
Laboratoire de Chimie Physique, UMR8000 CNRS, Université Paris Sud 11, Bat 349 Centre<br />
Universitaire, 91405 Orsay<br />
Highlighted during the 2008 Nobel Prize in chemistry, Green Fluorescent Protein from the jellyfish<br />
Aequoria victoria (GFPav), its many colored variants and its homologues from Anthozoa species, have<br />
become versatile tools, extensively used in pure and applied biological and biomedical research to<br />
monitor cellular events. They have allowed the understanding at the molecular, tissue and organism<br />
levels of a wide variety of biological processes. Many of these applications rely on the Förster-type<br />
resonant energy transfer (FRET) mechanism, where a non radiative dipole-dipole interaction leads to<br />
changes in the fluorescence properties of a donor fluorophore in the presence of a nearby acceptor<br />
molecule. Fluorescent Proteins (FPs) can also be used directly as local optical sensors, due to their<br />
intrinsic sensitivity to many physicochemical environmental parameters. In all these applications, a<br />
sound knowledge of FP’s photophysics and its dependence on the specific cellular and imaging<br />
conditions is necessary <strong>for</strong> a quantitative analysis of the fluorescence signals in living cells.<br />
We are focussed on the Cyan Fluorescent Protein (ECFP: GFPav- F64L, S65T, Y66W, N146T,<br />
M153T, V163A) which is one of the mostly used donor in the FRET and FLIM-FRET experiments<br />
despite several photophysical disvantages. We have undertaken a thorough characterization of the<br />
photophysical behaviors of this protein 1,2 . More precisely, we have compared the fluorescence decays<br />
of the purified ECFP and of a ECFP variant carrying a single mutation at the position 148, mutation that<br />
is characteristic of the brighter <strong>for</strong>m Cerulean. Both proteins display highly non-exponential<br />
fluorescence decays showing strong temperature and pH dependences. At neutral pHs, we have<br />
shown that this single mutation leads (ii) to a general increase in all fluorescence lifetimes and (ii) to the<br />
disappearance of a sub-population, estimated to more than 25 % of the total ECFP molecules,<br />
characterized by a quenched and red-shifted fluorescence. The fluorescence lifetime distributions of<br />
ECFP and its mutant remain otherwise very similar, indicating high structural and dynamic similarity of<br />
the two proteins in their major <strong>for</strong>m in agreement with theoretical approaches 3 . From thermodynamic<br />
analysis, it is concluded that the non-exponential fluorescence of ECFP cannot be ascribed to the slow<br />
con<strong>for</strong>mational change previously characterized by NMR studies 4 . The complex photophysics of ECFP<br />
involves dynamic processes, possibly taking place on the nanosecond time scale, that are not related<br />
to any of the currently described chemical and physical equilibria of the protein. Measurements in living<br />
cells on the relative photostability of ECFP and its main derivatives will be also presented.<br />
Dev(t)<br />
Counts (u.a)<br />
3<br />
-3<br />
10 5<br />
10 4<br />
10 3<br />
10 2<br />
10 1<br />
10 0<br />
10 -1<br />
0<br />
g(t) F(t)<br />
5<br />
10 15<br />
Time (ns)<br />
20<br />
25<br />
Amplitude (a.u.)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
4 6 0.1 2 4 6 1 2 4 6 10<br />
Lifetime (ns)<br />
ECFP fluorescence decay curve and lifetime distribution (T°=20°C, pH 7,4)<br />
References: [1] A. Villoing et al., Biochemistry 47 (47), 12483 (2008). [2] R. Grailhe, et al., ChemPhysChem<br />
7, 1442 (2006). [3] Vallverdu, G. et al., Proteins 78, 1040-54 (2010) [4] Seifert, M.H. et al., J. Am. Chem.<br />
Soc. 124, 7932-7942 (2002)<br />
______________<br />
* Corresponding author: e-mail: helene.pasquier@u-psud.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 291
P173 Fluorescent Proteins, Fluorescent Probes & Labels Poster 173<br />
AmCyan100 - new fluorescent protein <strong>for</strong> cell flow applications<br />
Oleg Guryev 1* , Marta Matvienko 2,3 , Barny Abrams 1 & Tim Dubrovsky 1<br />
1 BD Biosciences, Cell Analysis, 2350 Qume Drive, San Jose, CA 95131 (USA)<br />
2 UC Davis, One Shields Avenue, Davis, CA 95616 (USA)<br />
3 Current address: Life Technologies, 850 Lincoln Centre Drive, Foster City, CA 94404 (USA)<br />
We created a new variant of AmCyan protein, AmCyan100, specially designed <strong>for</strong> use with violet<br />
laser in multicolor flow cytometric applications. AmCyan (Ex max = 458 nm, Em max = 489 nm) is<br />
a fluorescent protein that belongs to the family of proteins isolated from coral reef organisms. This<br />
fluorescent protein is being used in combination with other dyes <strong>for</strong> the violet laser on the BD LSR<br />
and BD FACSAria cell flow cytometers. It can also be used as a reporter in fluorescent<br />
spectroscopy.<br />
As a violet laser dye, AmCyan is excited at 405-407 nm. Also, it is partially excited by the<br />
blue laser at 488 nm. In a multicolor flow cytometric analysis, when excited by the blue laser,<br />
AmCyan emits at 500 nm. There<strong>for</strong>e, its emission peak overlaps with that of FITC, which is also<br />
excited by the blue laser. This problem is also known as “AmCyan spillover into FITC channel”.<br />
Bright staining of cells with AmCyan can severely limit the resolution of the FITC channel. Our goal<br />
was to narrow the excitation peak of AmCyan and/or to shift this peak to the violet spectral region.<br />
The new AmCyan100 has an emission profile similar to AmCyan with an emission maximum at<br />
500 nm, but its excitation maximum at 395 nm is better fitted into violet laser than original AmCyan.<br />
The new protein has a Stokes shift of more than 100 nm, compared to 31 nm in its precursor. The<br />
AmCyan100 conjugates with antibodies are brighter than the AmCyan conjugates. We also<br />
showed that the AmCyan100 conjugates don’t have the significant fluorescence emission of the<br />
blue laser. Our data suggest that AmCyan100 conjugates do not have spillover into the FITC<br />
channel, and can replace existing AmCyan conjugates in multicolor flow cytometry without any<br />
changes in instrumental design.<br />
______________<br />
*Corresponding author: e-mail: oleg_guryev@bd.com<br />
292 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P174 Fluorescent Proteins, Fluorescent Probes & Labels Poster 174<br />
Pyrimidine derivatives as fluorescent sensors<br />
Sylvain Achelle 1,2,3 , Caroline Hadad 1,2 , Joaquin C. García-Martinez 2 , Julián Rodríguez-López 2<br />
& Nelly Plé 3<br />
1Institut<br />
de Chimie Organique Fine (IRCOF) UMR-CNRS 6014, INSA et Université de Rouen,<br />
BP08 76131 Mont-Saint-Aignan Cedex, France<br />
2<br />
Facultad de Química, Universidad de Castilla-La-Mancha, 13071 Ciudad Real, Spain<br />
3<br />
Sciences Chimiques de Rennes UMR CNRS 6226, IUT de Lannion, rue Edouard Branly BP<br />
30219, 22302 Lannion Cedex, France<br />
In the past two decades, π-conjugated organic compounds have attracted much attention due to their<br />
potential applications in light-emitting and nonlinear optical materials. Incorporation of heteroaryl<br />
moieties into π-extented conjugated systems is particularly useful to modify and to improve the<br />
luminescence properties. In π-conjugated materials, the pyrimidine ring, due to its aromaticity, its<br />
strong electron-withdrawing, its high dipolar interaction and its pH sensibility presents the requested<br />
characteristics to be incorporated in organic materials in view of luminescence sensing.<br />
Over the past five years, we have synthesized various pyrimidine derivatives with a π-conjugated<br />
scaffold.<br />
A first series of star-shaped and banana-shaped compounds (I and (II) has been developed with<br />
a pyrimidine central core and π-conjugated arms consisting of aromatics bearing electron-donor<br />
substituents (D). The position of the arms as well the nature of their substituents (D) were investigated<br />
leading to compounds with interesting light-emitting properties. Comparison of the optical properties of<br />
these oligomers with those having a benzene or a s-triazine ring as central unit has been achieved and<br />
highlighted that the pyrimidine compounds gave the best results.[1]<br />
Bis- and Tris(arylethynyl)pyrimidine oligomers have been synthesized. Comparison with bananashaped<br />
and star shaped pyrimidine core molecules without ethynyl linkers was carried out, highlighting<br />
that triple bounds generally enable a red shift of the absorption and emission spectra and upgrade<br />
fluorescence properties in terms of quantum yield.[2]<br />
The V-shaped 4,6-(arylvinyl)pyrimidines efficiently obtained by aldol condensation between 4,6dimethylpyrimidine<br />
and appropriate aromatic aldehydes have been studied. These compounds can be<br />
used as polarity and pH sensors: a strong emission solvatochromism is observed that is reflected by a<br />
large red shift in their fluorescence emission maxima on increasing the solvent polarity; the abilities of<br />
these molecules to function as colorimetric and luminescence pH sensor were demonstrated with<br />
dramatic color change and luminescence switching upon the introduction of acid. [3]<br />
Various 4-arylvinyl-2,6di(pyridine-2-yl)pyrimidines have been studied and could act as polarity<br />
and pH sensors as well as Zn(II) and Sn(II) sensors.[4]<br />
Figure 1: Some example or the<br />
chromophores studied.<br />
References: [1] S. Achelle, et al. Eur. J. Org. Chem. (2008) 3129-3140 [2] S. Achelle, et al Tetrahedron 64<br />
(2008) 2783-2791. [3] S. Achelle, et al. J. Org. Chem. 74(2009) 3711-3717. [4] a) C. Hadad, et al. J. Org.<br />
Chem. 76(2011) doi: 0.1021/jo200204u. b) C. Hadad et al. Inorg. Chem. Submitted<br />
______________<br />
* Corresponding author: e-mail: sylvain.achelle@univ-rennes1.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 293
P175 Fluorescent Proteins, Fluorescent Probes & Labels Poster 175<br />
New long-wave excitable, covalently coupled sensors <strong>for</strong> pH based on<br />
photoinduced electron transfer<br />
Daniel Aigner*, Sergey M Borisov & Ingo Klimant<br />
Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology,<br />
Stremayrgasse 9, 8010 Graz (Austria)<br />
pH is a key parameter in biotechnological, physiological and marine samples. Fluorescence<br />
constitutes a powerful tool <strong>for</strong> pH sensing since it allows on-line monitoring in a virtually contactless<br />
way.<br />
Several pH sensitive dyes are widely used in optical sensors, but most of them still are<br />
subject to individual limitations such as short-wave excitability, poor photostability or limited<br />
fluorescence brightness. However, the selection of chromophores <strong>for</strong> pH sensing can be<br />
dramatically extended by taking advantage of the photoinduced electron transfer (PET) process. It<br />
involves fluorescence quenching over the reduction of an excited chromophore by an organic<br />
amine [1] (figure 1).<br />
This contribution focuses on the application of two classes of indicators, perylene bisimides<br />
(PBIs) and rhodamines. Both chromophores are of high brightness and excitable at >550nm which<br />
is more long-wave than the majority of pH indicators of common use. Long-wave excitability is<br />
highly advantageous in biological samples. PBIs have already been presented by the authors as<br />
promising dyes <strong>for</strong> the application in pH sensors [2] . Here we aim on covalently coupled sensors as<br />
covalent linkage (e.g. to hydrogel or silica beads) is expected to minimize migration and<br />
aggregation processes. It also allows the employment of hydrophilic indicators without<br />
perturbations by dye leaching. This concept is thus also investigated with PET functional<br />
rhodamine dyes which show excellent pH sensitivities in aqueous solution. An ideal and versatile<br />
combination of indicator dye and immobilization matrix is crucial <strong>for</strong> optimal sensor per<strong>for</strong>mance<br />
over a variable sensitive range.<br />
H3C H3C CHROMOPHORE<br />
AMINO<br />
FUNCTION<br />
e- NO PET<br />
This work was supported by the Austrian Science Fund (FWF; Research Project No. P21192-N17).<br />
References:<br />
[1] R.A.Bissell, et al., Chem. Soc. Rev., 21 (1992) 187.<br />
[2] D.Aigner, et. al., Anal. Bioanal. Chem., in press.<br />
______________<br />
* Corresponding author: e-mail: daigner@tugraz.at<br />
294 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
PET<br />
e -<br />
CHROMOPHORE<br />
AMINO<br />
FUNCTION<br />
Strong Fluorescence Weak Fluorescence<br />
H3C H3C<br />
O<br />
N<br />
O<br />
R R<br />
R R<br />
C<br />
H 3<br />
O<br />
N<br />
O<br />
C<br />
H 3<br />
N +<br />
H<br />
O<br />
H +<br />
R = N<br />
OH<br />
N<br />
CH 3<br />
C<br />
H 3<br />
O<br />
O<br />
R<br />
R<br />
R<br />
R<br />
O<br />
N<br />
O<br />
CH 3<br />
CH 3<br />
CH3 CH3
P176 Fluorescent Proteins, Fluorescent Probes & Labels Poster 176<br />
Absorption and fluorescence spectroscopic study of Genistein<br />
isoflavone : towards a natural hormone replacement therapy<br />
Saadia Ait Lyazidi 1 , M. Haddad 1 , K. Benthami 1 , B. Bennetau 2 & S. Shinkaruk 3<br />
1<br />
Laboratoire de Spectrométrie, des Matériaux et Archéomatériaux , Unité de Recherche Associée<br />
au CNRST- URAC 11, Université Moulay Ismail, Faculté des Sciences, B.P 11201 Zitoune, 50 000<br />
Meknès, Morocco<br />
2<br />
Institut des Sciences Moléculaires (ISM), CNRS-UMR 5255, Université Bordeaux 1, 351 cours de<br />
la Libération, F-33405 Talence, France<br />
3<br />
Ecole Nationale d’Ingénieurs des Travaux Agricoles (ENITA) de Bordeaux 1, cours du Général de<br />
Gaulle -CS 40201, 33175 Gradignan, France<br />
Several recent works are focusing on natural Isoflavones because of their beneficial effects <strong>for</strong><br />
human health [1-3] namely in preventing hormono-dependant cancers, and also as potential<br />
hormone replacement therapy. This biological activity is found to be dependant on tissues, and<br />
isoflavone concentration in the blood. Genistein (called G) is the most studied isoflavone because<br />
of its high structural similarity with 17β-estradiol hormone. The aim of the present work is the<br />
photo-physics study of both solvent cage and solute concentration effects on the Genistein<br />
behaviour in its ground and excited states. In the ground state [4] , the Genistein is shown to exist in<br />
two different con<strong>for</strong>mations which are the 90° completely twisted geometry and the 50° less twisted<br />
one with respect to the angle between the benzopyrone and phenol rings. Specific interactions with<br />
a protic solvent cage as well as self-association process seem shifting the molecule from the<br />
perpendicular con<strong>for</strong>mation towards the less twisted one. The Genistein absorption spectrum<br />
shape is found to be concentration depending in non protic solvent, while it’s not showing any<br />
concentration effect in protic solvent. The study of the Genistein room temperature emission<br />
spectra, in parallel with the absorption ones, revealed a dual fluorescence depending on the<br />
solvent cage and solute concentration. The fluorescence spectrum does not show any<br />
fluorescence when the excitation is in the first nπ* absorption band. In contrast with this, the<br />
excitation within the second absorption π π*band leads to the apparition of two fluorescence bands<br />
located at 308 and 350 nm depending on the solvent and the solute concentration. The first<br />
emission is attributed to a S2* - S0 fluorescence originating probably from an ESPT as in the case<br />
of the 3-hydroxyflavone congener [5] . The second band, which depends on the excitation<br />
wavelength, behaves differently depending on the nature, protic or non protic, of the solvent cage.<br />
In the non protic solvent its relative intensity increases with the Genistein concentration, contrarily<br />
in the protic solvent the spectrum does not show any shape variation with concentration rising. So,<br />
in the first case the fluorescence band at 350 nm was attributed to an excimer (G…G)*originating<br />
from the conversion of a sandwich like excited dimer. In the second case this band was attributed<br />
to an exciplex (G…solvent)* fluorescence. The coincidence of the excimer and exciplex<br />
fluorescences implies a same configuration of the excited monomer (G*) isoflavone in both excited<br />
supermolecules. Hence this study revealed two aspects of the Genistein photophysics behaviour:<br />
i) S2* -S0 fluorescence, and ii) specific interactions with a protic solvent cage prevents completely<br />
self-association process in both ground and excited states.<br />
References: [1] S. Basak, et al., Mol. Cancer. Ther., 7 (2008) 3195. [2] S. Banerjee, et al., Cancer. Lett., 269<br />
(2008) 226. [3] Y. Zhang, et al., J. Steroid Biochem. & Mol. Biol. 94 (2005) 375. [4] K. Benthami, et al.,<br />
Spectrochim. Acta A 74 (2009) 385. [5] P. K. Sengupta, et al., Chem. Phys. Lett. 68 (1979) 382.<br />
______________<br />
* Corresponding author: e-mail: aitlyazidisaadia@yahoo.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 295
P177 Fluorescent Proteins, Fluorescent Probes & Labels Poster 177<br />
The novel terbium(III) chelate probe <strong>for</strong> hemoglobin determination using<br />
resonance energy transfer<br />
D. Aleksandrova, А. Yegorova * , I. Leonenko & V. Antonovich<br />
A.V. Bogatsky Physico-chemical Institute of the National Academy of Sciences of Ukraine, Odessa<br />
(Ukraine)<br />
Recently the long lifetime luminescence of lanthanide complexes has been widely used <strong>for</strong> the<br />
analysis of bioactive substances in solution [1] .<br />
A new, simple, sensitive luminescence method <strong>for</strong> the determination of hemoglobin has been<br />
developed and validated. The assay is based on the quenching of the luminescence of the terbium<br />
complexe with the ligand: 1-еthyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid-(4methyl-pyridin-2-yl)-amide<br />
(L) by hemoglobin (Hb).<br />
The key factor is the equimolar concentration of Tb 3+ and the ligand (1.0 µM). Ilum is<br />
maximum at pH 7.5 (Tris-HCl buffer). The excitation and emission maxima of the Tb-L complex in<br />
the presence of Hb are at 317 nm and 545 nm, respectively. Maximum Ilum was observed in water<br />
solution of this complex. The lifetime of the terbium ion in the Tb(III)-L complex (τ =734 мкс)<br />
decreases in the presence of a various concentrations of Hb (τ =324-165 мкс). Under optimum<br />
conditions, the quenched fluorescence intensity (Stern-Volmer calibration plot) is in proportion to<br />
the concentration of hemoglobin in the range of 0.6 - 36.0 µg/mL.<br />
Quenching mechanism of luminescence of complex by Hb is discussed. The fluorescence<br />
emission spectra of Tb(III)-L and the absorption spectra of Hb have a biggish overlap. Under these<br />
experimental conditions, we find that the maximum distance of the donor-acceptor is R0 = 47 Å. All<br />
of this supports the conclusion of non-radioactive energy transfer. It indicates that efficient energy<br />
transfer occurs from Tb(III)-L complex (donor) to Hb (acceptor).<br />
The long luminescence decay time of Tb-L makes the assay useful <strong>for</strong> time – resolved<br />
fluorescence measurements.<br />
RI<br />
1000<br />
800<br />
600<br />
400<br />
200<br />
0<br />
hemoglobin<br />
0 μg /mL<br />
60 μg /mL<br />
0<br />
0 10 20 30 40 50 60 70<br />
СHb , μg /mL<br />
500 550 600 650<br />
296 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
I 0 /I<br />
Wavelength, nm<br />
9<br />
8<br />
7<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
[20.05.2011 15:02 "/Graph1" (2455701)]<br />
Polynomial Regression <strong>for</strong> Data1_B:<br />
Y = A + B1*X + B2*X^2<br />
Parameter Value Error<br />
------------------------------------------------------------<br />
A 1,40613 0,1213<br />
B1 0,00918 0,01431<br />
B2 0,0017 2,44049E-4<br />
------------------------------------------------------------<br />
R-Square(COD) SD N P<br />
------------------------------------------------------------<br />
0,9866 0,24909 12
P178 Fluorescent Proteins, Fluorescent Probes & Labels Poster 178<br />
Spectroscopic investigations of fluorescent dyes and their chemical<br />
reactions<br />
Nicole Baltes*, Marcel Wirtz*, Gudrun Nürenberg & Gregor Jung*<br />
Prof. Dr. Gregor Jung, Saarland University, Biophysical Chemistry, Building B2.2, D-66123<br />
Saarbrücken (Germany)<br />
The visualization of a chemical reaction on the single-molecule level is our main scientific interest.<br />
We explored Bor-dipyrromethene-dyes (BODIPY-dyes) [1] with an external C=C-double-bond which<br />
are appropriate <strong>for</strong> single-molecule examinations. [2] They offer the ideal spectroscopic properties<br />
as the chromophoric unit can be easily expanded by extension of the BODIPY-core via the<br />
exocyclic double-bond. During the cleavage on an external C=C-double-bond of the dye the<br />
chromophore is shortened and resulting the fluorescent wavelength changes from orange to<br />
green. [3]<br />
Currently we are studying different reactions like catalytic or oxidative reactions with these<br />
dyes. The catalytic examination with Grubbs(I)-catalyst is already advanced, with characterization<br />
of many fluorescent products by mass spectrometry. It turns out that consecutive reactions are the<br />
main source <strong>for</strong> the product variety.<br />
The oxidative trans<strong>for</strong>mation is per<strong>for</strong>med with peroxides or metal-oxides. [4] In the oxidation<br />
with osmiumtetroxide and permanganate one green and one yellow fluorescent substance are<br />
generated which we characterized them by fluorescence spectroscopic analysis including FCS<br />
(Fluorescence Correlation Spectroscopy), TCSPC (Time Correlated <strong>Single</strong> Photon Counting), as<br />
well as by infrared spectroscopy and mass spectrometry.<br />
The observation of the kinetics and characterization of the various products and<br />
intermediates of a highly fluorescent compound helps to create a reactive system on which the<br />
chemical trans<strong>for</strong>mation of a single molecule can be studied in the near future.<br />
References:<br />
[1] A. Treibs, et al., Liebigs Ann., 718, (1968), 203.<br />
[2] Hinkeldey B., et al., ChemPhysChem, 9, (2008), 2019.<br />
[3] Jung G., et al., Ann.N.Y.Acad. of Sci., 1130, (2008), 131.<br />
[4] Schmitt A, et al., J.Phys.Org.Chem., 22, (2009), 1233.<br />
______________<br />
*Corresponding authors: e-mail: m.wirtz@mx.uni-saarland.de, n.bach@mx.uni-saarland.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 297
P179 Fluorescent Proteins, Fluorescent Probes & Labels Poster 179<br />
Photofluorescent systems based on chromones <strong>for</strong> three-dimensional<br />
archival optical memory<br />
Valery Barachevsky 1* , Olga Kobeleva 1 , Tatyana Valova 1 , Anton Ayt 1 , Igor Martynov 1 ,<br />
Konstantin Levchenko 2 , Vladimir Yarovenko 2 & Mikhail Krayushkin 2<br />
1<br />
Photochemistry Center of the Russian Academy of Sciences, 7a, bld.1. Novatorov Street, 119421,<br />
Moscow (Russia)<br />
2<br />
N.D.Zelinsky Institute of Organic Chemistry of thr Russian Academy of Sciences, 47, Leninsky<br />
Prospect, 119991, Moscow (Russia)<br />
Prospects <strong>for</strong> increasing the in<strong>for</strong>mation capacity of optical disks more than 1 Tbytes are related to<br />
the development of multilayer light-sensitive recording media <strong>for</strong> bitwise data recording and<br />
readout of optical in<strong>for</strong>mation. Light-sensitive organic recording media based on photochemical<br />
trans<strong>for</strong>mations of organic compounds have in principle a higher resolution than the currently used<br />
temperature-sensitive materials. Of particular interest is multilayer recording media with<br />
nondestructive fluorescent readout <strong>for</strong> 3D archival optical memory<br />
From the above reasoning it was carried out the synthesis and the spectral-kinetic study of<br />
photochemical trans<strong>for</strong>mations of chromones in solutions and polymer binders [1-4] . These organic<br />
compounds have no fluorescence in the initial <strong>for</strong>m A but <strong>for</strong>m the fluorescent photoproduct B<br />
under UV irradiation.<br />
R<br />
O O<br />
O<br />
A<br />
R 1<br />
O<br />
R 2<br />
298 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
hv<br />
O<br />
O<br />
B<br />
R 1<br />
O<br />
R 2<br />
R 1 R 2<br />
R, , = H, Alk, Ar, Het, FG<br />
Methods <strong>for</strong> obtaining chromones A were worked out. More than 200 new chromones synthesized<br />
of this type with the furyl group in position 2 including 3-acyl- derivatives of alkyl, phenyl, aryl, furyl,<br />
and thienyl heterocycles were studied on spectral absorptive and fluorescent properties.<br />
Dependencies between the chromone structure and positions of absorption bands of initial<br />
compounds and photoproduct as well as fluorescence bands of the photoproduct were established.<br />
The change of compound structures provides an effective control <strong>for</strong> position of chromone<br />
absorption bands in the spectral region 300-390 nm and, consequently, the choice of lasers with<br />
acceptable wave-length of radiation <strong>for</strong> writing optical in<strong>for</strong>mation. Besides, the influence of<br />
substituent nature on the efficiency of photochemical trans<strong>for</strong>mations and the quantum yield of<br />
photoproduct fluorescence in solutions and polymers was studied. Phenyl- and thienyl- derivatives<br />
of 2-furyl-chromones are characterized by the best fluorescence properties. It was found that the<br />
quantum yield of fluorescence may range up to 0.40 in solutions. The efficiency of fluorescence<br />
increased sharply in the polymer matrix, especially in poly(methyl methacrylate).<br />
Several chromones manifesting the best fluorescence properties in polymer binders were<br />
used <strong>for</strong> preparation of photofluorescent polymer recording media <strong>for</strong> functional investigations. The<br />
comparative spectral-kinetic study showed that a number of samples are characterized by<br />
properties acceptable <strong>for</strong> application as light-sensitive layers in multilayer optical disks of the<br />
archival type with nondestructive fluorescent readout of optical in<strong>for</strong>mation.<br />
This work was supported by the Russian Foundation of Basic Researches (the grant #10-03-<br />
00250-a).<br />
References: [1] V.A. Barachevsky, et al., ARKIVOC., IX (2009) 70. [2] M.M.Krayushkin, et al., ARKIVOC., IX<br />
(2009) 269. [3] M.M, Krayushkin, et al., New J. Chem, 33 (2009) 2267. [4] K.S.Levchenko, et al., Chem<br />
.Heterocycl. Comp.,N2(2011)198 (Rus.).<br />
______________<br />
* Corresponding author: e-mail: barva@photonics.ru<br />
O
P180 Fluorescent Proteins, Fluorescent Probes & Labels Poster 180<br />
A molecular-size thermometer<br />
Sara Bonacchi*, Daniele Cauzzi, Roberto Pattacini, Massimiliano Delferro, Marco Montalti,<br />
Luca Prodi, Nelsi Zaccheroni, Matteo Calvaresi & Francesco Zerbetto<br />
Dipartimento di Chimica “G. Ciamician”, Università di Bologna,via Selmi 2, 40126 Bologna, Italy<br />
Accurate measurement of temperature is gaining increasing importance due to the wide application<br />
range (electronic devices, biology, medical diagnostics). Nowadays, the technological frontier to<br />
cross is the measurement of local temperature in an extremely small volume. The thermometric<br />
response must be highly accurate, sensitive, stable, and presenting high spatial/temporal<br />
resolution [1-2]; in this context, one of the most intriguing challenge is to found suitable probes <strong>for</strong><br />
ultrasensitive spectroscopic techniques such as confocal fluorescence microscopy. We present an<br />
uncommon zwitterionic metallate cluster fulfilling these requirements.<br />
Figure 1<br />
Its photophysics, both in solid and in solution, is characterized by a temperature dependent<br />
emission and excited state lifetime that changes remarkably in a wide working range (fig.1). This<br />
allows <strong>for</strong> an unprecedented accuracy in temperature determination, and the lifetime dependence<br />
on temperature makes this compound a suitable probe <strong>for</strong> FLIM (fluorescence lifetime imaging<br />
microscopy) analysis; the emission lifetime, in fact, in contrast with its intensity, is insensitive to the<br />
concentration of the fluorophore, allowing temperature detection also in environments where an<br />
homogeneous distribution of dyes is hard to achieve, such as in biological matrices or complex<br />
devices.<br />
References: [1] J. Lee, N. A. Kotov, Nanotoday 2 (2007) 48. [2] S. Uchiyama, A. P. De Silva, et al. J. Chem.<br />
Ed. 83 (2006) 720.<br />
______________<br />
*Corresponding author: e-mail: sara.bonacchi3@unibo.it<br />
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P181 Fluorescent Proteins, Fluorescent Probes & Labels Poster 181<br />
Phosphorescent platinum(II) and palladium(II) complexes with donoracceptor<br />
Schiff bases – new red light-excitable indicators <strong>for</strong><br />
oxygen sensing<br />
Sergey M. Borisov 1 , * Robert Saf 2 , Roland Fischer 3 & Ingo Klimant 1<br />
1<br />
Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology,<br />
Stremayrgasse 9, 8010, Graz (Austria)<br />
2<br />
Institute of Chemistry and Technology of Materials, Graz University of Technology,<br />
Stremayrgasse 9, 8010, Graz (Austria)<br />
3<br />
Institute of Inorganic Chemistry, Graz University of Technology, Stremayrgasse 9, 8010, Graz<br />
(Austria)<br />
Phosphorescent metal complexes receive much attention due to their application in OLEDs,<br />
photovoltaic devices, optical sensors and as luminescent labels [1] . Red light and NIR-excitable<br />
complexes are particularly promising <strong>for</strong> sensing and labelling applications due to much lower levels<br />
of autofluorescence compared to the established UV-Vis dyes, lower light scattering and because of<br />
their potential <strong>for</strong> measurements in tissues and in living organisms (“smart tattoos”). Such dyes also<br />
find application in photodynamic therapy and in NIR OLEDs. Currently, red light-excitable<br />
phosphorescent dyes are limited to heavy metal complexes of porphyrins and their analogues [2] . In<br />
this contribution we present a novel type of NIR phosphorescent indicators which are based on Pt(II)<br />
and Pd(II) complexes with donor-acceptor Schiff bases. The indicators can be conveniently prepared<br />
in only 2 steps. The dyes possess efficient absorption in the red part of the spectrum (ε ∼ 120000<br />
l⋅mol -1 ⋅cm -1 , Fig. 1) and emit at RT in the NIR. Spectral properties can be tuned by varying the nature<br />
of the Schiff base. Pt(II) complexes, particularly, show fairly strong emission (QY up to 10%) with the<br />
decay time of 11 µs. The phosphorescence of the complexes is efficiently quenched by oxygen<br />
which is the basis <strong>for</strong> their application in optical oxygen sensors. The sensing materials are prepared<br />
by embedding the indicators into polystyrene. The photostability of the dyes and sensors was found<br />
to be lower than that of the benzoporphyrin complexes. Nevertheless, they may represent a<br />
promising alternative to the latter in oxygen sensing applications.<br />
Figure 1. Chemical structures of the Schiff base complexes and spectral properties of the Pt(II) dyes<br />
This work was supported by the Austrian Science Fund (FWF; Research Project No. P21192-N17).<br />
References: [1] D. B. Papkovsky, T. C. O’Riordan, J Fluoresc 15 (2005) 569. [2] A.V. Cheprakov, M.A.<br />
Filatov, J. Porphyrins Phthalocyanines 13 (2009) 291.<br />
______________<br />
* Corresponding author: e-mail: sergey.borisov@tugraz.at<br />
300 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P182 Fluorescent Proteins, Fluorescent Probes & Labels Poster 182<br />
Imaging intracellular viscosity of live cells by fluorescent<br />
molecular rotors<br />
Pei-Hua Chung a , James A. Levitt a , Marina K. Kuimova b , Gokhan Yahioglu b,c<br />
& Klaus Suhling a,*<br />
a Department of Physics, King’s College London, Strand, London WC2R 2LS, UK<br />
b Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AZ, UK<br />
c PhotoBiotics Ltd, 21 Wilson Street, London EC2M 2TD, UK<br />
Metabolism, chemical signalling and dynamic diffusion phenomena of cellular processes can be<br />
influenced by viscosity. A fluorescent molecular rotor, meso-substituted boron-dipyrromethene<br />
(BODIPY-C12) was used to monitor intracellular viscosity and the motion of organelles in HeLa cells<br />
via fluorescence lifetime imaging (FLIM), steady-state fluorescence anisotropy, and time-resolved<br />
fluorescence anisotropy measurements. The fluorescence lifetime of BODIPY-C12 is a function of<br />
viscosity, and the relationship between the fluorescence lifetime and the rotational correlation time<br />
of the dye in methanol-glycerol solutions follows the combination of the Förster Hoffmann equation<br />
and the Debye-Stokes-Einstein equation. [1,2] The steady-state anisotropy of the molecular rotors is<br />
a function of viscosity based on a modified Perrin equation, and can be used to obtain intracellular<br />
viscosity values. [3]<br />
Moreover, the BODIPY-C12 is insensitive to the surrounding polarity, and located in two<br />
different environments within the cells. Counterstaining experiments appear to indicate that the<br />
BODIPY-C12 is located in lipid droplets and the endoplasmic reticulum. Combining the<br />
measurement of the lifetime of fluorescent molecular rotors and the emission spectrum of Nile Red<br />
provides a method to obtain the viscosity and the polarity of the probes’ environment<br />
simultaneously.<br />
In summary, we have developed a practical and versatile approach to map the microviscosity<br />
in cells based on imaging fluorescent molecular rotors.<br />
References:<br />
[1] M. K. Kuimova et al., J. Am. Chem. Soc., 130 (2008) 6672<br />
[2] J. A Levitt et al., J. Phys. Chem. C, 113 (2009) 11634<br />
[3] J. A Levitt et al., Chem. Phys. Chem., 12 (2011) 662<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 301
P183 Fluorescent Proteins, Fluorescent Probes & Labels Poster 183<br />
Studies of apoptosis and lipid order of cell membranes using new<br />
fluorescent probes<br />
Zeinab Darwich, Oleksandr A. Kucherak, Youri Arntz, Pascal Didier, Andrey S. Klymchenko<br />
& Yves Mély *<br />
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg,<br />
Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch (France)<br />
Plasma membranes of mammalian cells exhibit a remarkable lipid asymmetry, where<br />
sphingomyelin is mainly present in the outer leaflet. Together with cholesterol, sphingomyelin<br />
<strong>for</strong>ms a highly packed ordered membrane phase, which is believed to play an important role in the<br />
function of membrane proteins. The aim of the present work was to study these phases in cell<br />
membranes and their changes in response to different lipid composition change (cholesterol<br />
extraction, apoptosis, cholesterol oxidation and hydrolysis of sphingomyelin). For this purpose, we<br />
used new probes developed in the laboratory, which are sensitive to environment and lipid phase<br />
and allow imaging of separate phase domains in giant unilamellar vesicles. Two families of dyes<br />
were studied, which stain selectively the outer leaflet of cell membranes. The first is based on 3hydroxyflavone<br />
dyes and exhibits a dual emission sensitive to the polarity and hydration of the<br />
environment. The second one is a Nile Red based dye, which shows a single emission band<br />
changing its position as a function of the environment properties. Our cellular studies showed that<br />
both families change their emission color in response to cholesterol extraction, cholesterol<br />
oxidation and hydrolysis of sphingomyelin, suggesting the transition from liquid ordered phase to<br />
liquid disordered phase. Moreover, these dyes are sensitive to apoptosis induced by different<br />
known agents and their response is similar to the effect of cholesterol extraction. Remarkably, the<br />
probes that are more sensitive to change of lipid order are more sensitive to apoptosis. Thus, the<br />
lipid order at the outer biomembrane leaflet decreases dramatically on apoptosis, probably<br />
because of the flip of sphingomyelin from the outer to the inner leaflet resulting in a loss of the<br />
liquid ordered phase. We observed a slow internalization of Nile red-based probes inside the cells.<br />
This internalization was blocked at 4 °C and after treatment with chlorpromazine and methyl-βcyclodextrin,<br />
which suggested that the probe internalizes via energy-dependent, clathrindependent,<br />
and raft-related endocytosis.<br />
This work was supported by CNRS, Region Alsace and Conectus.<br />
References: [1] Kucherak, O.A, et al., J.Am.Chem.Soc. , 132 (2010) 4907-16. [2] Oncul, S. , et al.,<br />
Biochim.Biophys.Acta., 1798 (2010) 1436-43. [3] Shynkar, V.V., et al. J.Am.Chem.Soc., 129 (2007) 2187-93.<br />
______________<br />
* Corresponding author: e-mail: yves.mely@unistra.fr<br />
302 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P184 Fluorescent Proteins, Fluorescent Probes & Labels Poster 184<br />
A novel PET probe <strong>for</strong> fluorescent detection and cellular imaging of<br />
hydrogen peroxide, and <strong>for</strong> oxidase-based enzymatic assays<br />
Axel Duerkop * , Dominik B. M. Groegel, Martin Link, Joachim Wegener & Otto S. Wolfbeis<br />
Institute of Analytical Chemistry, Chemo- and Biosensors, Faculty of Chemistry and Pharmacy,<br />
University of Regensburg, Universitaets Str. 31, 93040 Regensburg (Germany)<br />
Hydrogen peroxide (HP) belongs to the class of the so-called reactive oxygen species (ROS) that<br />
include e.g. singlet oxygen, hydroxyl radicals, superoxide anions or nitric oxide. If under oxidative<br />
stress, cells can be substantially damaged if the levels of those ROS exceed the tolerable<br />
physiological range. [1] HP also is vasoactive and plays key roles in inflammation and hypoxiareoxygenation<br />
of tissues. Despite its hazard to organisms, HP is ubiquitous as it is a by-product of<br />
many metabolic reactions and acts as a messenger in cellular signalling. [2] HP is also a widely<br />
used bleaching agent and its toxicity towards microorganisms is harnessed <strong>for</strong> cleaning and<br />
disinfection. Furthermore, it is a precursor <strong>for</strong> the production of explosives and employed as a<br />
rocket fuel. The state of the art in fluorescent probes <strong>for</strong> HP has been reviewed recently [3]<br />
including the use of lanthanide complexes. [4]<br />
We present a new probe (to which we refer as HP Green) <strong>for</strong> the detection and visualization<br />
of hydrogen peroxide (HP). It is based on the yellow (λmax = 456 nm) fluorophore 4-amino-1,8naphththalimide<br />
that was coupled to p-anisidine (a redox-active group) to <strong>for</strong>m a photoinduced<br />
electron transfer (PET) probe. The preparation is accomplished in four steps and with good yields.<br />
In absence of HP, the fluorescence of HP Green is strongly quenched due to a PET from the redox<br />
moiety (p-anisidine) to the naphthalimide luminophore. If p-anisidine is oxidized by HP, the PET is<br />
suppressed and the fluorescence intensity is strongly increased. The addition of 0.1 U/mL<br />
horseradish peroxidase (HRP) further accelerates the oxidation of HP Green. This is accompanied<br />
by an up to 11-fold increase of fluorescence intensity at 534 nm and a rising quantum yield (from<br />
0.32% to 4%). At acidic pH (≤ 6), PET is suppressed but the PET is active from pH 6-9. This<br />
renders HP Green suitable <strong>for</strong> experiments under physiological conditions (pH 7.4). The combined<br />
use of HRP and HP Green enables HP to be quantified from 100 nM to 5 µM and a limit of<br />
detection (LOD) as low as 64 nM (16 pmol/well) is achieved. This LOD is comparable to that of the<br />
standard Amplex Red/HRP assay.<br />
HP Green (10 mM) and HRP (0.1 U/mL) are also shown to enable the enzymatic assay of Dglucose<br />
or L-lactate, respectively, if glucose oxidase (1 U/mL) or lactate oxidase (1 U/mL) are<br />
employed to oxidize these substrates. Glucose can be determined from 2 to 30 µM with a LOD of<br />
0.64 µM (161 pmol/well) of D-glucose after 11 min incubation at 30°C. In case of the L-lactate<br />
assay, a dynamic range of 0.5 to 10 µM with a LOD of 164 nM (41 pmol per well) of L-lactate was<br />
achieved after 6 min incubation at 30°C. This LOD is more than 12-fold lower than that of standard<br />
NADH methods and 6-fold lower than the commercial biovision assay. The effect of oxygen ingress<br />
on the D-glucose and the L-lactate assay is compensated by the kinetic assay <strong>for</strong>mat and<br />
microplates do not require sealing. Hence, HP Green seems to be generally applicable <strong>for</strong> assays<br />
of substrates of oxidases.<br />
Furthermore, HP Green is an in-vitro probe <strong>for</strong> HP as proven by imaging experiments of NRK<br />
cells incubated with a 50 µM solution of HP Green <strong>for</strong> 30 min at 37 °C. HP Green is found to be<br />
easily internalized and mainly located around the nucleus. Its green fluorescence remains almost<br />
constant during observation under a fluorescence microscope <strong>for</strong> 80 min. The presence of 100 µM<br />
HP in the extracellular matrix causes a strong increase in brightness over time.<br />
This work was supported by a grant from the BMBF (TumorVision).<br />
References: [1] K. B. Beckman, B. N. Ames, Physiol. Rev., 78 (1998) 547. [2] M. Giorgio, et al., Nat. Rev.<br />
Mol. Cell. Biol., 8 (2007) 722. [3] M. Schäferling, et al., Microchim. Acta 174 (2011) 1. [4] O. S. Wolfbeis, A.<br />
Dürkop, et al., Angew. Chem. Int. Ed. 41 (2002), 4495<br />
______________<br />
* Corresponding author: e-mail: axel.duerkop@chemie.ur.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 303
P185 Fluorescent Proteins, Fluorescent Probes & Labels Poster 185<br />
Excited states of multichromophoric compounds based on<br />
2-aminoanthracene<br />
Miroslav Dvořák 1 , Numan Almonasy 2 , Martin Michl 1 & Miloš Nepraš 2<br />
1 Department of Physical Electronics, Faculty of Nuclear Sciences and Physical Engineering, Czech<br />
Technical University in Prague, V Holešovičkách 2, 180 00 Praha 8, Czech Republic<br />
2 Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of<br />
Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic<br />
The nature of excited states and their coupling in multichromophoric compounds is still not yet fully<br />
understood, though plenty of both experimental and theoretical studies have been carried out on<br />
this topic in recent years.<br />
In this contribution, we employed fluorescence anisotropy measurements and quantum<br />
chemical calculations to study excited states of multichromophoric compounds, which contain<br />
different number (1, 2 or 3) of 2-aminoanthracene chromophores. The 2-aminoanthracene (AA)<br />
chromophores are linked via sym-triazine ring (T) and the remaining positions are occupied by<br />
corresponding number of aniline groups (An). Hereafter, these compounds are referred to as<br />
AATAn2, AA2TAn and AA3T. The results, in combination with our previous study on analogous 1aminopyrene<br />
multichromophoric compounds, contribute to understanding of excited states<br />
dynamics of linked chromophores with broken D2h symmetry [1] .<br />
Fluorescence emission spectra are almost identical <strong>for</strong> all three compounds. The same holds<br />
<strong>for</strong> their absorption spectra except that the varying ratio between aminoanthracene and aniline<br />
groups is reflected in the corresponding band intensities. The most significant difference appears<br />
when fluorescence anisotropy excitation profiles are measured in frozen 2-methyltetrahydrofurane.<br />
The presence of additional aminoanthracene chromophores in AA2TAn and AA3T results into<br />
decrease of the fluorescence anisotropy value across the region 320-400 nm. This decrease is<br />
pronounced with the increasing number of the chromophores. But, at the very red edge of the<br />
spectra, the fluorescence anisotropy reaches almost the same value of about 0.3 <strong>for</strong> all three<br />
compounds.<br />
The quantum chemical calculations show that in AATAn2 there are two lowest excited states<br />
whose excitation energies fall into this region. These states closely resemble those of 2aminoanthracene<br />
and they seem to be responsible <strong>for</strong> the steady rise of the fluorescence<br />
anisotropy profile in AATAn2. Each additional chromophore gives rise to additional pair of<br />
analogous excited states. According to calculated electronic density redistributions upon excitation,<br />
these states are localized on individual chromophores what is in agreement with observed<br />
fluorescence and absorption spectra.<br />
Based on the abovementioned facts, we propose that the observed photophysical behavior<br />
of the studied compounds can be explained in terms of competition between excitation energy<br />
migration, which causes depolarization of fluorescence, and internal conversion into the emitting<br />
state.<br />
This work was supported by grants MSM 6840770022 and GAP208/10/0941.<br />
Anizotropy<br />
0.3<br />
0.2<br />
0.1<br />
0.0<br />
300 350 400<br />
Wavelength / nm<br />
Fig: Fluorescence anisotropy of AATAn2 (solid), AA2Tan<br />
(dashed) and AA3T (dotted) with absorption spectrum of<br />
AA3T (gray)<br />
References: [1] M. Dvořák, et. al., J Fluoresc (2010) DOI<br />
10.1007/s10895-010-0668-3<br />
304 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0.0<br />
Normalized absorbance
P186 Fluorescent Proteins, Fluorescent Probes & Labels Poster 186<br />
The homodimeric nucleic acid dyes 6-chloroYOYO-1 and 6-chloroTOTO-1<br />
John J. Naleway 1 , Ying Jiang 2 , Fiona K. Harlan 1 , Todor Deligeorgiev 3 , Nikolay Gadjev 3 ,<br />
Stefka Kaloyanova 3 , Nedyalko Lesev 3 , Aleksey Vasilev 3 , Iliana Timcheva 4 & Vera Maximova 5<br />
1 Marker Gene Technologies, Inc., 1850 Millrace Drive, Eugene, Oregon 97403, USA<br />
2 The University of Oregon, Eugene, Oregon 97403, USA<br />
3 University of Sofia, Faculty of Chemistry, 1164 Sofia, Bulgaria<br />
4 Bulgarian Academy of Sciences, Institute of Organic Chemistry, 1113 Sofia, Bulgaria<br />
5 Bulgarian Academy of Sciences, Institute of Molecular Biology, 1113 Sofia, Bulgaria<br />
Recently we have synthesized the homodimeric dye 6-chloro-YOYO-1 [1] and now we are<br />
proposing the homodimeric dye 6-chloro-TOTO-1 which are analogs of the commercial dyes<br />
YOYO-1 and TOTO-1. They differ from the commercial products, in that they have chloro<br />
substituent on the sixth position in the benzoxazole or benzothiazole moieties respectively and<br />
each dye has four trifluoroacetates as anions (Scheme 1). The specific anions of these dyes impart<br />
high water solubility of the products.<br />
Scheme 1: Molecular <strong>for</strong>mulae of 6-chloroYOYO-1 and 6-chloroTOTO-1<br />
The maximum absorptions of the studied dyes lie between 450 and 520 nm. They do not fluoresce<br />
in TE buffer but become strongly fluorescent after binding to dsDNA. The fluorescent maxima of<br />
the dye – DNA complexes are in the region 510 – 540 nm. The fluorescent quantum yield of 6chloroYOYO-1<br />
in the presence of dsDNA is 0.5 [1], while that of the newly synthesized 6chloroTOTO-1<br />
was determined to be 0.28 after binding to dsDNA.<br />
The staining properties of 6-chloro-TOTO-1 and 6-chloro-YOYO-1 dyes were evaluated using<br />
prestaining experiments in which BamH1 digested fragments of pCMV-Evoglow-PP1 were<br />
incubated with the respective dyes and analyzed using agarose gel electrophoresis. 6-Chloro-<br />
TOTO-1 and 6-chloro-YOYO-1 dyes exhibited at least a 2-fold increase in sensitivity over the<br />
unsubstituted TOTO-1 and YOYO-1 dyes. Staining of fixed human breast cancer cells (MDA-MB-<br />
231) also exhibited bright green staining of the chromatin, providing an alternate fluorescence<br />
detection of chromatin/DNA over DAPI. These new dyes have also found use <strong>for</strong> ultrasensitive<br />
detection of DNA in Loop Mediated Isothermal Amplification (LAMP) assays utilizing 4 primer<br />
sequences which recognize 6 distinct regions on target – bacteriophage DNA under isothermic<br />
conditions with a strand disrupting polymerase. They have also exhibited excellent staining of<br />
nuclear DNA in paraffin-embedded animal tissue sections.<br />
This work is funded in part by grant number NSF-IIP0923953 from the National Science<br />
Foundation-USA.<br />
Reference:[1] Furstenberg, A. et al; Ultrafast Excited-<strong>State</strong> Dynamics of DNA Fluorescent Intercalators: New<br />
Insight into the Fluorescence Enhancement Mechanism, J. Am. Chem. Soc.; 128(23); 7661-7669 (2006).<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 305
P187 Fluorescent Proteins, Fluorescent Probes & Labels Poster 187<br />
Fluorescent probes <strong>for</strong> studying the interactions of cryoprotective<br />
organic substances with biomembranes<br />
Tatyana S. Dyubko 1,2<br />
1<br />
SSI "Institute <strong>for</strong> <strong>Single</strong> Crystals", NAS Ukraine, 60, Lenin Ave., 61001 Kharkiv (Ukraine)<br />
2<br />
Institute <strong>for</strong> Problems of Cryobiology and Cryomedicine, NAS Ukraine, 23, Pereyaslavskaya Str.,<br />
61015 Kharkiv (Ukraine)<br />
Cryoprotectors (CPs) are the organic substances such as multinuclear alcohols, sugars, and<br />
polymers among others, which can be added into cell suspensions to help protect them against<br />
damage during freeze-thawing. The investigation of mechanisms of CP's is important and will help to<br />
develop methods <strong>for</strong> long-term storage of cells, tissues, and organs at low temperatures.<br />
Microenvironment-sensitive fluorescent probes (FPs) are well suited to study CP-initiated structural<br />
changes in natural and model membranes. We investigated a series of fluorescent probes DSM,<br />
3-DAB, K8-1350, and K8-1431 available from SETA BioMedicals (http://www.setabiomedicals.com)<br />
to study mechanism of the interaction of CPs with natural and artificial biomembranes and compared<br />
them to the flavonol-based probes DMAF and FME.<br />
For studying of CPs behavior in aqueous media (water, physiological solution, phosphate<br />
buffer, a variety of solvent systems applied <strong>for</strong> cells cryopreservation, etc.), FPs are suggested to<br />
have high quantum yield in aqueous cryoprotectant solutions, reasonable solvatochromic<br />
properties and sensitivity towards structural reorganizations of CPs molecular ensembles in<br />
aqueous solution. Fluorescent probes such as the flavonol based dye DMAF and the styryl dye<br />
DSM are known probes to estimate the cryoprotective behavior of CPs and their potential<br />
cytotoxicity on model systems without use of real biological objects. At the same time the use of<br />
fluorescent dyes to investigate effect of CPs in cell suspension is obstructed by the fact that they<br />
cannot mimic the effects which are caused by the CPs itself: e.g. changes in viscosity, polarity,<br />
dielectric permeability and other solvent parameters affect the characteristics of FPs as well as the<br />
interaction of FPs with biological species. An important requirement <strong>for</strong> FPs <strong>for</strong> studying of CPs is<br />
a low quantum yield when it is in free <strong>for</strong>m in cryoprotective solutions and a high quantum yield and<br />
preferably a large Stokes' shift when bound to biomembranes. A shift in the emission maxima<br />
when the FP is moved from cryoprotective medium into biomembrane is also desirable. Such a<br />
change e.g. is observed <strong>for</strong> the flavonol probe FME and the benzantrone-based probe 3-DAB. It is<br />
worth mentioning that due to the hydrophylic-hydrophobic characteristics, each CP has its own<br />
concentration range limiting the applicability of a specific FP. To select a FP <strong>for</strong> studying the CPs<br />
impact on various polar and non-polar areas of membranes it is helpful to measure the FP<br />
distribution coefficients (Kp) in an n-octanol — water system, which allows to estimate the FPs<br />
hydrophylic-hydrophobic properties. In particular, <strong>for</strong> the long-wavelength probes K8-1350 and K8-<br />
1431 the Kp is 1.78 and 31.68, respectively, which demonstrate that K8-1431 is higher hydrophobic<br />
probe that is favorable <strong>for</strong> studying of CPs interaction with non-polar area of lipid bilayer while K8-<br />
1350 can be used <strong>for</strong> studying the superficial area of membranes.<br />
CPs interact with the surface in biomembranes, substitute-bound water immobilized on the<br />
membrane surface and herewith affect the surface topography. There<strong>for</strong>e probes which are<br />
sensitive to bilayer hydration enabling the study of the CPs interaction with a membrane surface<br />
are of considerable interest. On the base of the fluorescent probes characteristics and the<br />
discussed criteria, we selected the most promising fluorescent probes <strong>for</strong> cryobiological research<br />
particularly <strong>for</strong> studying of CPs interaction with biomembranes. We found that the probes such as<br />
3-DAB, DSM, FME, and К8-1350 are very promising probes to study bilayer hydration and the<br />
interaction of CPs with membrane surfaces, in particular those containing various concentrations of<br />
cholesterol.<br />
______________<br />
* Corresponding author e-mail: tdyubko@mail.ru<br />
306 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P188 Fluorescent Proteins, Fluorescent Probes & Labels Poster 188<br />
Synthesis and spectral properties of modified nucleobases bearing<br />
multiparametric and environment sensitive 3-hydroxychromones<br />
Dmytro Dziuba 1 , Luliia Karpenko 1 , Benoit Michel 1 , Marie Spada<strong>for</strong>a 1 , Rachid Benhida 1 ,<br />
Victoria Y. Postupalenko 2 , Volodymyr V. Shvadchak 2 , Andrey S. Klymchenko 2 , Yves Mély 2<br />
& Alain Burger 1,*<br />
1 Laboratoire de Chimie des Molécules Bioactives et des Arômes, UMR 6001 CNRS, Institut de<br />
Chimie de Nice, Université de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France<br />
2 Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg,<br />
Faculté de Pharmacie, 74, Route du Rhin, 67401 Illkirch (France)<br />
2-Aryl-3-Hydroxychromones (3HCs) are oxygen-containing heterocyclic compounds which possess<br />
attractive properties to be used as fluorescent labels. Due to their specific electronic structure<br />
these dyes are able to tautomerize in the excited state by intramolecular proton transfer (ESIPT).<br />
This leads to the appearance of two well-defined bands in the fluorescent spectra, caused by<br />
emission of the normal and tautomer <strong>for</strong>ms. The position of the fluorescent maxima and the<br />
intensity ratio of these two bands strongly depend on the microenvironment of the fluorophore and<br />
this noteworthy property has stimulated the usage of 3HCs as reporting group in the construction<br />
of ratiometrical fluorescent sensors with wide field of applications [1].<br />
Being incorporated into DNA strand, 3HCs are suspected to become a powerful tool to elucidate<br />
the three-dimensional arrangement of DNA structure and to study the nucleic acid - ligand<br />
interactions.<br />
In this research our goal was to synthesize the fluorescent 3HC-containing oligonucleotides<br />
and to characterize their photophysical properties. Two ways have been envisioned to attain this<br />
goal. One of them is the covalent replacement of one of the natural bases by a 3-HC derivative [2]<br />
(Figure 1, strategy 1). Following this strategy a series of oligonucleotides containing 3HC as<br />
modified nucleobase was obtained and their structure and photophysical properties were explored<br />
using UV, circular dichroism and fluorescent spectroscopies. The ability of the developed<br />
fluorescent tool to detect, quantify and characterize the interactions between the NCp7 protein and<br />
the labelled DNA PBS sequence was explored.<br />
The second approach consists in the attachment of the fluorescent label to the natural<br />
nucleobase. According to this strategy the fluorescent deoxyuridine bearing 3-HC moiety was<br />
synthesised and the spectroscopic investigations of this later were made. The incorporation of the<br />
fluorescent nucleotide into the oligonucleotide sequences is the focus of our current research<br />
(Figure 1, strategy 2).<br />
HO<br />
OH<br />
O<br />
HO<br />
O<br />
S<br />
O<br />
Strategy 1<br />
O<br />
O<br />
Figure 1<br />
OH<br />
Strategy 2<br />
References: [1] A.P. Demchenko, FEBS Lett. 580 (2006) 2951. [2] M. Spada<strong>for</strong>a et al., Tetrahedron 65<br />
(2009) 7809.<br />
______________<br />
* Corresponding author: e-mail: burger@unice.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 307<br />
O<br />
O<br />
S<br />
HO<br />
OH<br />
OH<br />
O<br />
O<br />
N<br />
NH<br />
O
P189 Fluorescent Proteins, Fluorescent Probes & Labels Poster 189<br />
Profiling of functional group: an approach to develop a bio-sensitive<br />
quencher <strong>for</strong> the development of turn-on fluorescent probes<br />
Coraline Egloff 1 , Denis Weltin 2 & Alain Wagner 1,*<br />
1 Laboratoire des systèmes chimiques fonctionnels, UMR 7199 CNRS, Université de Strasbourg,<br />
Faculté de Pharmacie, 74, Route du Rhin, 67401 Illkirch (France)<br />
2 Phytodia, Pôle API, Boulevard Sébastien Brant, 67412 Illkirch (France)<br />
Fluorescent compounds are excellent tools to study biological phenomenon. In the literature,<br />
several turn-on fluorescent probes have been developed to detect biomolecules, metals and<br />
differences in pH in the cell. [1] These probes can be composed of a quencher and a fluorophore<br />
that are joined by a reactive linker and its cleavage switches on the fluorescence. [2] We decided to<br />
develop a compact probe composed of just a fluorophore and a bio-sensitive quencher.<br />
Using a screening strategy, we tested different chemical groups <strong>for</strong> their reactivity towards<br />
various biological compounds such as glutathione (GSH), cysteine, hydrogen peroxide (H2O2). One<br />
molecule containing an iminomalonitrile N-oxide function is reactive towards GSH and has a high<br />
absorption spectra. This molecule was derivatized into a carboxylic acid to be easily functionalized<br />
with a tetramethylrhodamine by peptide coupling to af<strong>for</strong>d the probe. Then, we assessed its<br />
potential as a turn-on fluorescent probe.<br />
Cells contain GSH at millimolar concentrations (0.5 mM-10 mM). [3] We tested our probe with<br />
different concentrations of GSH which led to an increase in fluorescence. A signal-to-noise ratio of<br />
11 in one hour was detected <strong>for</strong> 5 mM of GSH. This result is encouraging <strong>for</strong> future cellular assays.<br />
This screening has enabled the development of a new turn-on fluorescent probe sensitive to GSH<br />
composed of a quencher and a fluorophore. This probe could be used to determine the intracellular<br />
concentration of GSH in tumor cells.<br />
This work was supported by grant from region Alsace.<br />
References: [1] H. Kobayashi, et al., Chemical. Rev., 110 (2010) 2620. [2] I. Texier et al., Nuclear<br />
Instruments and Methods in Physics Res. A, 571 (2007) 165. [3] C. Boulègue et al., ChemBioChem, 8 (2007)<br />
591.<br />
______________<br />
* Corresponding author: e-mail: wagner@bioorga.u-strasbg.fr<br />
308 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P190 Fluorescent Proteins, Fluorescent Probes & Labels Poster 190<br />
Fluorescent sensor <strong>for</strong> Zn(II) based on coumarin Schiff base derivative<br />
Amel F. El Husseiny 1* , Elham S. Aazam 2 & Huda M. Al Amri 2<br />
1<br />
Department of Chemistry, Faculty of Science, Alexandria University, Egypt, P.O. Box 426,<br />
Ibrahimia, Alexandria 21321, Egypt<br />
2<br />
Department of Chemistry, Girls section, University of King Abdulaziz, Saudi Arabia<br />
Mononuclear Zn(II), Cd(II), Cu(II), Ni(II) and Pd(II) metal complexes of coumarin Schiff-base<br />
ligands: 4-methyl-7-(salicylidineamino) coumarin HL 1 , N 1 ,N 2 -bis [8-(1-ethylidene)-7-hydroxy-2Hchromen-2-one]<br />
ethane-1,2-diamine H2L 2 and 8-{(1E)-1-[(2-aminophenyl) iminio] ethyl}-2-oxo -2Hchromen-7-olate<br />
HL 3 .have been prepared and characterized.<br />
The fluorescence spectra were studied at room temperature in DMSO. The Schiff base<br />
ligands exhibited photoluminescence originating from intraligand (ð- ð*) transitions. Metal mediated<br />
enhancement is observed on complexation of the three organic ligands with Zn(II) and Cd(II),<br />
whereas metal mediated fluorescence quenching occurs in Cu(II), Ni(II) and Pd(II).<br />
The fluorescence spectra of the probe HL 1 showed a clear shift in emission wave length<br />
maxima upon Zn +2 binding, indicating its potential use as a Zn +2 sensor.<br />
______________<br />
*Corresponding author: e-mail: dr_amelfawzy@yahoo.com<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 309
P191 Fluorescent Proteins, Fluorescent Probes & Labels Poster 191<br />
Kinetic analysis of phagosomal production of reactive oxygen species<br />
Asma Tlili 1,2 , Sophie Dupré-Crochet 1,2 , Marie Erard 1,3,* & Oliver Nüße 1,2<br />
1 University of Paris-Sud, Orsay F-91405, France<br />
2 INSERM UMR-S757, Orsay F-91405, France<br />
3 CNRS UMR 8000, Orsay F-91405, France<br />
Phagocytes involved in the immune system produce large quantities of reactive oxygen species or<br />
(ROS) <strong>for</strong> pathogen killing, namely superoxide anion (O2 ●- ), hydrogen peroxide (H2O2), hydroxyl<br />
radical (HO ● ) or hypochlorous acid (HOCl). The initial production of O2 ●- in the phagosome of<br />
human neutrophils has been estimated at 2.5 mM.s -1 and the HOCl concentration could reach level<br />
as high as a few millimolar just after the phagosome closure.[1] However, the kinetics and<br />
amplitude of ROS production at the level of individual phagosomes are poorly understood. This is<br />
mainly due to the lack of appropriate methods <strong>for</strong> quantitative ROS detection with microscopic<br />
resolution (suitable size <strong>for</strong> phagocytosis, conventional excitation and emission wavelength or<br />
excitation power).<br />
We covalently attached the ROS-sensitive dye dichlorodihydrofluorescein (DCFH2) to yeast<br />
particles and investigated their fluorescence due to oxidation in vitro and in live phagocytes (figure<br />
A).[2] In vitro, the dye was oxidized by H2O2 plus horseradish peroxidase in its usual fluorescent<br />
oxidation product, DCF. Surprisingly HOCl also oxidized DCFH2 to a previously unrecognized<br />
oxidation product, called Xfluo, with red-shifted excitation and emission spectra and a<br />
characteristic difference in the shape of the excitation spectrum near 480 nm.. Millimolar HOCl<br />
bleached DCF as well as Xfluo. Inside phagosomes, DCFH2-labeled yeast were oxidized <strong>for</strong><br />
several minutes in a strictly NADPH oxidase-dependent manner as shown by video microscopy<br />
with no contribution of photooxidation (figure B&C). Inhibition of the NADPH oxidase rapidly<br />
stopped the fluorescence increase of the particles. Inhibition of myeloperoxidase, responsible <strong>for</strong><br />
the <strong>for</strong>mation of HOCl from H2O2, also modulated the yeast fluorescence.[3] DCFH2–yeast is thus a<br />
valuable and versatile tool to investigate the kinetics and amplitude of ROS production in individual<br />
phagosomes.[4,5]<br />
Figure: A Yeast (S cerevisiae) opsonized and covalently functionalised with DCFH2. B Sequence of<br />
snapshoot from a movie of internalization of DCFH2-yeast by neutrophile-like cells. C Mean fluorescence<br />
intensities of DCFH2-yeast variations in phagosome of wild type cells (square), cells deficient <strong>for</strong> the NADPH<br />
oxidase (KO, diamond) or outside the cell (empty symbol).<br />
References: [1] Winterbourn, C.C. et al J. Biol. Chem. ,281 (2006) 39860 [2] Wardman, P. Free Rad. Bio.<br />
Med. 43 (2007) 995 [3] Tlili A. et al Free Rad. Bio. Med. 50 (2011) 438 [4] Steinckwich, N.et al J Leuko. Biol.<br />
81 (2007) 1054 [5] Tlili A. et al submitted<br />
______________<br />
* Corresponding author: e-mail: marie.erard@u-psud.fr<br />
310 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P192 Fluorescent Proteins, Fluorescent Probes & Labels Poster 192<br />
Fluorescence properties of thioflavin-T and modeling its<br />
nonradiative processes<br />
Yuval Erez 1 , Dan Huppert 1 & Nadav Amdursky 2<br />
1<br />
Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv<br />
University, Israel<br />
2<br />
Department of Materials and Interfaces, Faculty of Chemistry, Weizmann Institute of Science,<br />
Rehovot, Israel<br />
Thioflavin-T (ThT) was first introduced in the 1960s as a fluorescent dye <strong>for</strong> the detection of<br />
amyloid fibrils. Since then, ThT has become the most effective fluorescence marker <strong>for</strong> the<br />
detection of amyloid fibrils, and an essential standard of measurement <strong>for</strong> any in vitro<br />
quantification, kinetics and inhibition measurements in the research field of amyloid-related<br />
diseases. The unique fluorescent photophysics of ThT is derived from its electronic<br />
characterization. The excited electronic state of ThT is composed from two states, a locally excited<br />
(LE) state and a twisted intramolecular charge-transfer (TICT). The rotation angle of a single C-C<br />
bond that connects the benzothiazole moiety to the dimethylanilino ring of ThT is crucial in the<br />
photophysics of ThT. The transition from the initially populated LE state to the TICT is induced by<br />
the rotation of this bond from 37° to 90°, respectively. The LE electronic state is radiative with an<br />
oscillator strength of ~1.1, whereas the TICT is a nonradiative electronic state with an oscillator<br />
strength of
P193 Fluorescent Proteins, Fluorescent Probes & Labels Poster 193<br />
Highly solvatochromic fluorophores based on 7-aryl-3-hydroxychromones<br />
Luciana Giordano 1,* , Dmytro A. Yushchenko 1,2 , Volodymyr Shvadchak 1 , Jonathan A.<br />
Fauerbach 3 , Elizabeth A. Jares-Erijman 3 & Thomas M. Jovin 1<br />
1<br />
Laboratory <strong>for</strong> Cellular Dynamics, Max Planck Institute <strong>for</strong> Biophysical Chemistry, Am Faßberg 11,<br />
Göttingen, D-37077, (Germany)<br />
2<br />
Present address: The Molecular Biosensor and Imaging Center, Carnegie Mellon University,<br />
4400 Fifth Avenue, Pittsburgh 15213, PA, (USA)<br />
3<br />
Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de<br />
Buenos Aires, 1428, Buenos Aires, (Argentina)<br />
Environment-sensitive fluorescent dyes are widely used as probes and labels in biology. [1]<br />
Particularly useful is a class of probes based on solvatochromism as the readout of biomolecular<br />
environment. [2] We have developed a family of new solvatochromic fluorophores based on 3hydroxychromone<br />
(3-HC) with an extended conjugation system. We synthesized 7-aryl-3hydroxychromones<br />
from the corresponding 7-bromo-3-hydroxychromones using the Suzuki<br />
coupling reaction. Substituents in position 2 were varied from hydrogen to diverse aryl groups.<br />
We reversed the direction of the dipole moment that is commonly ascribed to 3-hydroxyflavones<br />
and achieved improved solvatochromism. The spectroscopic characterization indicates that<br />
compared to Prodan, the new dyes exhibit up to 3-fold greater solvatochromism, as well as redshifted<br />
absorption and emission and substantial photostability due to significant prolongation of the<br />
conjugated system. The new probes display great sensitivity to membrane properties, with a >50<br />
nm difference of the emission band position depending on bilayer phase and charge. In 2-aryl-3hydroxychromones<br />
addition of aryl substituents in position 7 stabilize the highly polar excited state<br />
and decrease the rate of ESIPT (excited state intramolecular proton transfer) to carbonyl-4. The<br />
environment polarity can be sensed both by the emission band maxima shifts and the ratio of the<br />
normal and tautomeric state emission.<br />
We are functionalizing the probes <strong>for</strong> convenient covalent attachment to biomolecules.<br />
References: [1] S. A. Klymchenko, A. P. Demchenko, Methods in Enzymol, 450 (2008), 37. [2] A. P.<br />
Demchenko, et al., Biophys.J., 96 (2009), 3461.<br />
______________<br />
* Corresponding author: e-mail: luciana.giordano@mpibpc.mpg.de<br />
312 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P194 Fluorescent Proteins, Fluorescent Probes & Labels Poster 194<br />
Influence of Cu + and Cu 2+ cations on luminescent properties of new 3cyano-4-dicyanomethylene-5-oxo-4,5-dihydro-1H-pyrrol-2-olate<br />
anion<br />
Stanislav I. Gurskiy 1* , Viktor A. Tafeenko 1 & Andrey N. Baranov 1<br />
1<br />
Chemistry Department, Lomonosov Moscow <strong>State</strong> University, Leninskie Gory, 1, building 3, GSP-<br />
1, 119991, Moscow (Russia)<br />
In recent years, a lot of attention has been paid to the impact of molecular surrounding on<br />
luminescent properties (emission wavelength, quantum yield) of organic molecules. Intermolecular<br />
contacts such as π-π interactions, hydrogen bonds, coordination bonds with transition metal<br />
cations can strongly alter luminescence properties of fluorophore.<br />
Recently we synthesized new luminescent organic anion 3cyano-4-dicyanomethylene-5-oxo-4,5-dihydro-1H-pyrrol-2-olate<br />
(A) [1] .<br />
A is believed to be a convenient object to investigate effect of<br />
molecular surrounding on its luminescence on the basis of it's<br />
properties [2,3] :<br />
i) A can create π-π interactions with neighbor A, ionic, hydrogen and<br />
coordination chemical bonds with neighbor ions & molecules (A,<br />
alkaline metal cations, water molecules, d-block metal cations).<br />
ii) Strengthening of π-π interactions between neighbor A cause redshift<br />
of luminescence maximum wavelength. Hydrogen bonding<br />
between neighbor A cause blue-shift of luminescence maximum<br />
wavelength. Hydrogen bonding between A and water molecules cause red-shift of luminescence<br />
maximum wavelength.<br />
d-Block metal cations with incomplete d sub-shell usually quench luminescence upon<br />
complexation with fluorophore molecules, while completeness of d sub-shell nullifies this effect.<br />
However, <strong>for</strong> certain types of molecules luminescence enhancement may also be observed as a<br />
result of complexation with transition meta cations [4] .<br />
In present work we have investigated influence of metal cations with incomplete (Cu 2+ ) and<br />
complete (Cu + ) d sub-shell on luminescence of A. In crystalline state luminescence of A-Cu 2+ -<br />
based salts is totally quenched, while luminescence of A-Cu + -based salts is observed. However<br />
luminescence enhancement is observed upon addition of Cu 2+ to a solution of A in wateracetonitrile<br />
mixture. Mechanism of the luminescence enhancement effect is proposed on the basis<br />
of absorption spectroscopy and electrospray mass spectrometry studies. All salts in crystalline<br />
state were characterized by x-ray single crystal diffraction, luminescence spectroscopy and<br />
luminescence microscopy.<br />
References: [1] V.A. Tafeenko, et al., Acta Cryst., C59 (2003), m421. [2] V.A. Tafeenko, et al., Acta Cryst.,<br />
C61 (2005), m298. [3] V.A. Tafeenko, et al., Acta Cryst. C65 (2009), m52. [4] Z. Xu, et al., Chem. Commun.,<br />
46 (2010), 1679.<br />
______________<br />
* Corresponding author: e-mail: stanislav.gurskiy@gmail.com<br />
NC<br />
H<br />
N<br />
O O<br />
CN<br />
CN<br />
(A)<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 313
P195 Fluorescent Proteins, Fluorescent Probes & Labels Poster 195<br />
Synthesis, fluorescence and solvatochromism of novel 3aminoderivatives<br />
of benzanthrone<br />
Irena D. Ivanova 1,* , Natalja Orlova 1 & Elena M. Kirilova 2<br />
1 Faculty of Chemistry, University of Latvia, 48, Valdemara str., LV-1013, Riga (Latvia)<br />
2 Department of Chemistry, Daugavpils University, 13, Vienibas str., LV-5401, Daugavpils, (Latvia)<br />
Conjugated organic materials exhibit a variety of interesting optical properties. Derivatives of<br />
benzo[de]anthracene-7-one are known as effective luminophores [1] . They exhibit strong<br />
fluorescence, which accounts <strong>for</strong> their use in practice as active lasing media and fluorescent<br />
probes <strong>for</strong> investigation of biological objects. Earlier the series of benzanthrone amino derivatives<br />
were prepared [2,3] . The obtained results testify that the fluorescence of these amino derivatives is<br />
sensible to the changes on polarity of surrounding and fluorescence in the red region of spectrum<br />
contributes to the high analytical sensitivity of the method.<br />
The aim of the present investigation is to create novel fluorophores and study the effect of a<br />
molecular structure on its characteristics. A number of new derivatives of 3-aminobenzanthrone<br />
were synthesized. The influence of solvents with various polarities upon absorption and emission<br />
spectra was investigated. In summary, five new dyes were synthesized in good yields (80-87%) via<br />
the reduction of corresponding azomethine derivative by sodium borohydride in DMF solutions.<br />
O<br />
N R<br />
DMF, NaBH 4<br />
The structure of obtained compounds was confirmed by NMR and FT-IR spectroscopy and mass<br />
spectrometry. <strong>Single</strong>-crystal structures of obtained dyes were determined by X-ray diffraction<br />
studies. In addition, thermal stability of the synthesized chromophores has been undertaken using<br />
TG–DTA. The absorption and luminescent spectra of the novel compounds in several solvents of<br />
different polarity were investigated. The synthesized dyes absorb at 520-580 nm with high<br />
extinction coefficients, have relatively large Stokes’ shifts (about 100 nm), and emit at 650-720 nm<br />
showing both absorption and fluorescence solvatochromism similarly to studied earlier 3-amino<br />
derivatives of benzanthrone [2] . The results indicated that these dyes were strongly dependent on<br />
solvents and show generally bathochromic shifts as the polarity of solvents was increased. These<br />
characteristics of obtained dyes demonstrate their potential as biomedical probes <strong>for</strong> proteins,<br />
lipids and cells.<br />
References: [1] B.M. Krasovitskii, B.M. Bolotin, Organic luminescent materials. VCH Publishers, 1988. [2] E.<br />
M. Kirilova, et al., J.Fluoresc, 18 (2008) 645.[3] E. M. Kirilova, et al., (2009) . J.Luminesc, 129 (2009) 1827.<br />
______________<br />
* Corresponding author: e-mail: asaze@inbox.lv<br />
314 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
O<br />
H<br />
N<br />
R
P196 Fluorescent Proteins, Fluorescent Probes & Labels Poster 196<br />
Discovery of full-color tunable and predictable fluorescent core<br />
skeleton, 9-aryl-1,2-dihydropyrrolo[3,4-b]indolizin-3-one (Seoul-Fluor)<br />
Eunha Kim 1 , Minseob Koh 1 , Byung Joon Lim 1 & Seung Bum Park 1,2*<br />
1 2<br />
Department of Chemistry and Department of Biophysics and Chemical Biology, Seoul National<br />
University, Seoul 151-747, Korea<br />
A study of 9-aryl-1,2-dihydropyrrolo[3,4-b]indolizin-3-one, we name it Seoul-Fluor, were presented<br />
in this study.<br />
During our continuous ef<strong>for</strong>ts on building the small molecule library, based on DOS (Diversity<br />
Oriented Synthesis) strategy, we identified a novel fluorescent core skeleton, 1,2dihydropyrrolo[3,4-b]indolizin-3-one.<br />
Guiding with computational simulation, 24 fluorescent<br />
compounds library were constructed in combinatorial fashion, and which covers the full-color<br />
range. Further, bioapplication of the fluorescent system in the immunofluorescent was successfully<br />
demonstrated.<br />
After discovery of the 1,2-dihydropyrrolo[3,4-b]indolizin-3-one, we analyzed the fluorescent<br />
core skeleton more systematically and extensively. Finally we defined the 9-aryl-1,2dihydropyrrolo[3,4-b]indolizin-3-one<br />
fluorescent skeleton as Seoul-Fluor. Using a concise and<br />
practical one-pot synthetic procedure, a 44-member library of new fluorescent compounds was<br />
synthesized. The systematic perturbation of electronic densities on the specific positions of Seoul-<br />
Fluor, guided with the Hammett constant, allows tuning the emission wavelength in full-color range<br />
more systematically. Furthermore, on the basis of these observations and a computational<br />
analysis, we extracted a simple first-order correlation of emission wavelength with the theoretical<br />
calculation and accurately predicted the emission wavelength of Seoul-Fluor. There<strong>for</strong>e we clearly<br />
demonstrated that Seoul-Fluor could provide a powerful gateway <strong>for</strong> the generation of desired<br />
fluorescent probes without the need <strong>for</strong> tiresome synthesis and trial-and-error process.<br />
References: [1] E. Kim, et al., J. Am. Chem. Soc., 133 (2011) 6642–6649. [2] E. Kim, et al., J. Am. Chem.<br />
Soc. 130 (2008) 12206–12207<br />
______________<br />
* Corresponding author: e-mail: sbpark@snu.ac.kr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 315
P197 Fluorescent Proteins, Fluorescent Probes & Labels Poster 197<br />
Aminobenzanthrone derivatives as novel fluorescent probes <strong>for</strong><br />
membrane studies<br />
Valeriya M. Trusova 1 , Inta Kalnina 2 , Pavel Fedorov 1 , Elena Kirilova 2 , Georgiy Kirilov 2<br />
& Galyna P. Gorbenko 1<br />
1<br />
Department of Biological and Medical Physics, V.N. Karazin Kharkov National University, 61077<br />
Kharkov, Ukraine<br />
2<br />
Department of Chemistry and Geography, Faculty of Natural Sciences and Mathematics,<br />
Daugavpils University, 13 Vienibas, Daugavpils LV5401, Latvia<br />
e-mail: valtrusova@yahoo.com<br />
The unique photophysical properties of aminobenzanthrones have resulted in their extensive use<br />
as disperse dyes <strong>for</strong> textiles, polymers, daylight fluorescent pigments and laser dyes. [1] Despite the<br />
technological utilization of these compounds is continuously growing, their applicability as<br />
fluorescent probes in biological assays, so far remain scantily evaluated. Meanwhile, spectral<br />
characteristics of aminobenzanthrones satisfy all the requirements <strong>for</strong> an ideal fluorescent tracer.<br />
Bright fluorescence, high extinction coefficients, photo-, thermo- and chemical stability, and<br />
reduced background signal make benzanthrone dyes particularly attractive as bioimaging agents.<br />
Our previous studies revealed one prospective benzanthrone derivative sensitive to the<br />
con<strong>for</strong>mational and aggregation state of proteins. [2]<br />
In the present study we extended the evaluation of aminobenzanthrone per<strong>for</strong>mance in<br />
biological media and concentrated our ef<strong>for</strong>ts on evaluating the ability of these dyes to monitor the<br />
properties of lipid bilayers. The binding of five aminobenzanthrones referred to here as A4, A8,<br />
AM2, AM3 and AM4 (Fig. 1), to the lipid membranes composed of zwitterionic lipid<br />
phosphatidylcholine (PC) and its mixtures with sterol cholesterol (Chol) and anionic lipid cardiolipin<br />
(CL) was followed by significant increase in fluorescence intensity with small blue shift of emission<br />
maximum (~ 5nm). Quantitative description of the binding studies allowed us to determine the dye<br />
partition coefficients KP. The values of KP were found to fall in the range (0.36 – 6.2)×10 4 indicating<br />
that the examined dyes posses high lipid-associating ability. The enhancement of probe<br />
fluorescence in liposomal suspension was explained by two main factors: (i) the dye transfer to the<br />
lipid environment of reduced polarity, and (ii) immobilization of the probe molecules within the lipid<br />
bilayer resulting in strongly hindered fluorophore rotation. Interestingly, increasing CL content<br />
resulted in ambiguous changes in partition coefficients, suggesting that dye-membrane association<br />
is controlled mainly by hydrophobic interactions.<br />
O<br />
O<br />
H<br />
N<br />
A4<br />
N<br />
AM3<br />
O<br />
C<br />
H N(CH 3) 2<br />
316 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
O<br />
O<br />
N<br />
A8<br />
N CH 3<br />
N<br />
AM4<br />
C CH 3<br />
N(C 2H 5) 2<br />
O<br />
N<br />
AM2<br />
C CH 3<br />
N(CH 3) 2<br />
Fig. 1. Structures of<br />
aminobenzanthrones<br />
Analysis of red-edge excitation shift (REES) phenomenon allowed us to range the dyes according<br />
to the depth of their location. Within the framework of “dipstick” rule proposed by Chattopadhyay et<br />
al., magnitude of REES varies in direct correlation with the depth of probe penetration in the lipid<br />
bilayer – the less the REES, the deeper probe locates. [3] Examination of REES estimates in our<br />
systems showed that A8 exhibits the deepest penetration while the shallowest location<br />
corresponds to A4 and AM2.<br />
The results obtained strongly suggest that the employment of novel aminobenzanthrone<br />
derivatives may prove of particular usefulness in monitoring the physicochemical properties of lipid<br />
bilayers.<br />
References: [1] I. Grabchev, et al., Dyes and Pigments, 48 (2001) 143. [2] G.P. Gorbenko, et al., Chem.<br />
Phys. Lett. 495 (2010) 275. [3] A. Chattopadhyay, S. Mukherjee, J. Phys. Chem. B 103 (1999) 8180.
P198 Fluorescent Proteins, Fluorescent Probes & Labels Poster 198<br />
Application of amino benzanthrone based fluorescent probes <strong>for</strong><br />
studying of erythrocytes under ozonetherapy procedures<br />
Tatyana S. Dyubko 1,2 , Elena Kirilova 3* , Inta Kalnina 3 , Oksana A. Sokolyk 1 , Yuriy Kozin 4<br />
& Nikita V. Pereverzev 1<br />
1<br />
SSI "Institute <strong>for</strong> <strong>Single</strong> Crystals", NAS Ukraine, 60, Lenin Ave., 61001, Kharkov (Ukraine)<br />
2<br />
Institute <strong>for</strong> Problems of Cryobiology and Cryomedicine, NAS Ukraine, 23 Pereyaslavskaya Str.,<br />
61015, Kharkiv (Ukraine)<br />
3<br />
Daugavpils University, 13, Vienibas Street, Daugavpils, Latvia<br />
4<br />
Kharkov National Medical University, 4, Lenin Ave., 61058, Kharkov (Ukraine)<br />
In contrast to any other cells, erythrocytes contain intracellular haemoglobin that is capable to<br />
quench fluorescent probes. There<strong>for</strong>e the fluorescent probes <strong>for</strong> erythrocytes are suggested to<br />
have not only a pronounced affinity to cells but also an adequate fluorescence signal. We<br />
investigated applicability of amino benzantrones АВМ-1 (АВМ), АВМ-4, АВМ-6, АВМ-8<br />
synthesized in Daugavpils University as fluorescent probes <strong>for</strong> erythrocytes. These lipophilic<br />
probes are orange-red emitting N-substituted 3-amino derivatives of benzanthrones with various<br />
substituents such as morpholine, piperidine, piperazine residues. From previous investigations it<br />
was known that studied dyes are environment-sensitive fluorophores which display sensitivity to<br />
the polarity of the local environment. The concentration range was 10 –5 –10 –6 M. Emission spectra<br />
of the ABM probes changed after light exposure as shown on the Fig. 1. These probes have a<br />
pronounced affinity to erythrocyte membranes and emit in the visible spectral range. Fluorescence<br />
images obtained with АВМ-4 and АВМ-8 (Fig. 2) are brighter compared to those obtained with<br />
АВМ-1 and ABM-6.<br />
Fluorescence Intensity [a.u.]<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
ABM-4,<br />
6 days<br />
ABM-1<br />
Time zero<br />
ABM-1,<br />
6 days<br />
ABM-8 Time Zero<br />
ABM-6 Time zero<br />
ABM-6 6 days<br />
ABM-8 6 days<br />
ABM-4,<br />
Time zero<br />
500 600 700 800<br />
Wavelength [nm]<br />
Fig. 1. Photostability of ABM derivatives in<br />
water<br />
λex. 488 nm, λem 530 nm λex. 545 nm, λem 625<br />
nm<br />
Fig. 2. Human erythrocytes stained with АВМ-8<br />
Using the АВМ probes we studied impact of ozonotherapy on erythrocytes of patients with<br />
urogenital infections. The study was carried out using confocal fluorescent microscopy and flow<br />
cytofluorometry. Ozonation was found to decrease the content of erythrocyte hosts in the patient's<br />
blood and improve the cells morphological characteristics up to normal values. Theses effects can<br />
be more or less pronounced according to the kind of pathology and can be considered as the<br />
common biological and physiological ozonation principles acting by the "feedback" mechanism.<br />
______________<br />
* Corresponding author e-mail: elena.kirilova@inbox.lv<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 317
P199 Fluorescent Proteins, Fluorescent Probes & Labels Poster 199<br />
SeTau-647: The brightest label currently available <strong>for</strong> the Kr-ion laser line<br />
Oleksii P. Klochko 1 , Yuliia O. Klochko 1 , Yevgen A. Povrozin 1 , Ewald A. Terpetschnig 2<br />
& Leonid D. Patsenker 1,2<br />
1<br />
<strong>State</strong> <strong>Scientific</strong> <strong>Institution</strong> "Institute <strong>for</strong> <strong>Single</strong> Crystals", National Academy of Sciences of Ukraine,<br />
60, Lenin Ave., 61001 Kharkiv (Ukraine)<br />
2<br />
SETA BioMedicals, LLC, 2014 Silver Ct East, Urbana, IL 61801 (USA)<br />
Commercially available, long-wavelength dyes have certain shortcomings such as insufficient<br />
brightness, chemical and photochemical stability, which substantially limit their use in biomedical<br />
applications. We have developed and investigated a new, water soluble, extremely bright and<br />
stable near-infrared (NIR) fluorescent label SeTau-647. This dye contains a reactive NHS ester<br />
group <strong>for</strong> covalent attachment to biomolecules. Maleimides and azide versions are also available.<br />
The spectral characteristics of SeTau-647 and other commercially available labels like Alexa 647<br />
and ATTO 647N in phosphate buffer (pH 7.4) are listed in the Table below.<br />
Dye<br />
λmax<br />
(Abs)<br />
[nm]<br />
λmax<br />
(Em)<br />
[nm]<br />
ΦF<br />
[%]<br />
ε<br />
[M –1 cm –1 ]<br />
318 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
ΦF × ε /<br />
100%<br />
[M –1 cm –1 ]<br />
SeTau-647 649 695 61 200,000 122,000 3.1<br />
SeTau-647 — IgG conjugate (D/P = 1) 648 697 59 — — 3.4<br />
Alexa 647 649 668 32 237,000 75,840 1.0<br />
ATTO 647N 644 669 65 150,000 97,500 3.5<br />
Cy5 646 664 27 250,000 67,500 1.0<br />
ATTO 655 663 684 30 125,000 37,500 1.8<br />
SeTau-647 is a dye that combines a high quantum<br />
yield (ΦF) and extinction coefficient and the<br />
fluorescence lifetime (τ) is 3 times longer compared<br />
to Alexa 647. Importantly the quenching tendencies<br />
of this label even at higher degrees of labelling is<br />
low as can be seen from the plot of the brightness<br />
of SeTau-647 — antibody conjugates defined as<br />
ΦF multiplied by the extinction coefficient (ε) and the<br />
dye-to-protein ratio (D/P). SeTau-647 was found to<br />
exhibit also extreme chemical stability particular<br />
toward oxidizers such as hydrogen peroxide. It is<br />
also more photostable compared to ATTO-647N.<br />
The optimized properties of SeTau-647, commercially available from Seta BioMedicals make it a<br />
preferred fluorescent marker <strong>for</strong> applications in biological imaging, microarrays, clinical diagnostics<br />
and immunology.<br />
The work was supported by the STCU grant No.P313.<br />
______________<br />
* Corresponding author: e-mail: klochko@isc.kharkov.com<br />
Brightness [M -1 cm -1 ]<br />
400000<br />
300000<br />
200000<br />
100000<br />
SeTau-647<br />
Alexa 647<br />
τ<br />
[ns]<br />
Cy5<br />
0<br />
0 1 2 3 4 5<br />
Dye-to-Protein Ratio<br />
Brightness vs. D/P of K9-4149<br />
— IgG conjugates in phosphate buffer (pH 7.4)
P200 Fluorescent Proteins, Fluorescent Probes & Labels Poster 200<br />
Impact of the rotaxane-based protection on the properties of fluorescent<br />
squaraine dyes<br />
Yuliia O. Klochko 1 , Oleksii P. Klochko 1 , Inna Yermolenko 1 , Yevgen A. Povrozin 1 , Ewald A.<br />
Terpetschnig 2 & Leonid D. Patsenker 1,2<br />
1<br />
<strong>State</strong> <strong>Scientific</strong> <strong>Institution</strong> "Institute <strong>for</strong> <strong>Single</strong> Crystals", National Academy of Sciences of Ukraine,<br />
60, Lenin Ave., 61001 Kharkiv (Ukraine)<br />
2<br />
SETA BioMedicals, LLC, 2014 Silver Ct East, Urbana, IL 61801 (USA)<br />
Squaraine dyes and their derivatives are gaining significant interest in the field of supramolecular<br />
chemistry, both in host—guest recognition and self-assembly. The unique structure and properties<br />
of squaraine molecules has led to extensive research into their use as sensors, fluorescent probes<br />
and labels <strong>for</strong> biomedical research, dyes <strong>for</strong> optoelectronic applications such as xerographic<br />
devices, solar cells, optical recording media and other applications. Squaraine dyes contain<br />
electron-deficient squaric acid core making them prone to nucleophilic attack, which leads to<br />
decrease stability. As known, one of the ways to resolve this problem is to encapsulate the dye into<br />
a rotaxane structure.<br />
We have developed a series of hydrophobic rotaxane dyes and investigated their spectral<br />
properties and stability in PMMA thin films. Encapsulation of squaraine dyes in macrocycle to<br />
squaraine-rotaxanes was found to increase their photostability and thermal stability. Thus in PMMA<br />
thin films exposed to light with a 500 W halogen lamp at 50 ºC the unprotected squaraine Sq1<br />
photobleaches within 5 min while squaraine-rotaxane Sq1-R1 retains almost unchanged within<br />
tens hours.<br />
Normalized Absorption<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
Sq2-Rn2<br />
Sq2-Rn1 Sq1-Rn1<br />
0.2 Sq1<br />
Sq2<br />
0.0<br />
0 20 40 60 80 100 120 140<br />
Exposure Time (min)<br />
Decay of the normalized absorption of squaraines in<br />
PMMA thin films exposed to light with 500W lamp at<br />
50 ºC be<strong>for</strong>e (Sq1 and Sq2) and after (Sq1-Rn1,<br />
Sq2-Rn1 and Sq2-Rn2) encapsulation<br />
A<br />
Rn=<br />
O<br />
N<br />
NH O<br />
X<br />
O<br />
O<br />
NH<br />
NH<br />
X<br />
O<br />
NH<br />
2<br />
O HN<br />
N<br />
O<br />
N<br />
N<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 319<br />
HN<br />
HN<br />
HN<br />
O<br />
X<br />
O<br />
X<br />
O<br />
B<br />
Rn1: X=<br />
Sq1: A = B =<br />
Sq2: A = B =<br />
Sq3: A =<br />
Sq4: A =<br />
Sq5: A = B=<br />
N<br />
H<br />
N<br />
H<br />
Ph<br />
N<br />
B =<br />
N<br />
Rn2: X=<br />
N<br />
B = N<br />
Ph<br />
Ph<br />
Ph<br />
N((CH 2) 3COOH) 2<br />
In general, the encapsulation causes insufficient blue (Sq1, Sq5) or red (Sq2, Sq3, Sq4) shift in<br />
the absorption and emission maxima while the quantum yield and fluorescence lifetime may<br />
increase many times. Thus the fluorescence lifetime <strong>for</strong> Sq4 increases by factor of 3. Correlations<br />
of spectral, photophysical and photochemical properties of the squaraine-rotaxanes with their<br />
molecular structures are discussed.<br />
We expect that new materials can potentially find application in many fields, such as organic<br />
plastic solar cells or light-emitting diodes, can be a prototype <strong>for</strong> the development of new probes,<br />
labels, classification dyes, and standards <strong>for</strong> biomedical research.<br />
The work was supported by the STCU grant No.P313.<br />
______________<br />
* Corresponding author: e-mail: klochko@isc.kharkov.com<br />
COOBu
P201 Fluorescent Proteins, Fluorescent Probes & Labels Poster 201<br />
Functionalized phosphorescent Ir III - and Pt II -porphyrins – <strong>for</strong><br />
bioconjugation and polymer coupling<br />
Klaus Koren 1* , Sergey M. Borisov 1 , Ruslan Dmitriev 2 , Dmitri B. Papkovsky 2 & Ingo Klimant 1<br />
1 Institute of Analytical Chemistry and Food Chemistry, University of Technology, 8010 Graz (Austria)<br />
2 Laboratory of Biophysics and Bioanalysis, Department of Biochemistry, University College Cork (Ireland)<br />
Phosphorescent metalloporphyrins constantly attract scientific interest as oxygen indicators, emitters in<br />
OLEDs as well as labels <strong>for</strong> biomolecules. Although the potential application fields are enormous, the<br />
full potential of phosphorescent metalloporphyrins has not been exploited yet. A possible explanation is<br />
that tailor made porphyrin-dyes are either not available or the synthetic ef<strong>for</strong>t is too high.<br />
In this contribution we report two different systems enabling the functionalization of<br />
porphyrins with little synthetic ef<strong>for</strong>t. The first system is based on six coordinating Ir III -<br />
octaethylporphyrins (Ir-OEP), where the axial ligands can be changed and there<strong>for</strong>e alter the dye<br />
properties. The second system relies on the commercially available highly phosphorescent<br />
platinum(II)-meso-tetra(pentafluorophenyl)porphyrin (Pt-TFPP); within this system click chemistry<br />
can be used in order to introduce functional groups.<br />
Ir III -porphyrins are a new class of phosphorescent dyes [1]. In contrast to the established squareplanar<br />
Pt II and Pd II analogs iridium porphyrins have two additional axial ligands. A variety of groups<br />
can be introduced via straight<strong>for</strong>ward ligand exchange reactions. We synthesized symmetric and<br />
asymmetric Ir III -porphyrins with functional groups like carboxylic acids, norbornene units and<br />
lipophilic anchors. Besides introducing functional groups the ligands can also be used to modify the<br />
photophysical properties.<br />
Pt-TFPP can be functionalized by using click-chemistry based nucleophilic substitution of the<br />
labile para-fluorine atoms of the pentafluorophenyl groups [2]. With this method it is possible to<br />
introduce hydrophilic groups (e.g. acids, amines), monomers and even entire macromolecules like<br />
peptides or polymers.<br />
Low synthetic ef<strong>for</strong>t, high yields and a large variety of ligands or substituents make both<br />
systems attractive <strong>for</strong> applications in different fields. A few applications will be shown,<br />
demonstrating the wide scope of those methods and dyes.<br />
Financial support from the Austrian Science Fund (FWF; Research Project No. P21192-N17) is<br />
gratefully acknowledged.<br />
References: [1] K. Koren et al. Eur. J. Inorg. Chem. 10 (2011) 1531. [2] C. Becer, et al. Angew. Chem. Int.<br />
Ed. 48 (2009) 4900.<br />
______________<br />
* Corresponding author: e-mail: klaus.koren@tugraz.at<br />
320 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P202 Fluorescent Proteins, Fluorescent Probes & Labels Poster 202<br />
Trans<strong>for</strong>mation of chromones into fluorescent ones <strong>for</strong> archival optical<br />
memory<br />
M.M. Krayushkin 1 , V.N.Yarovenko 1 , M.A. Kalik 1 , K.S. Levchenko 2 , A. G. Devyatkov 2 , G. E.<br />
Adamov 2 , V. A. Barachevsky 3 , T.M. Valova 3 & O.I. Kobeleva 3<br />
1<br />
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47 Leninsky prosp.,<br />
119991 Moscow, Russian Federation<br />
2<br />
ОJSC CSRIТ «Technomash», Ivan Franco str.4, 121108 Moscow, Russian Federation<br />
3<br />
Photochemistry Center, Russian Academy of Sciences, 7a ul. Novatorov, 119421 Moscow,<br />
Russian Federation<br />
Considerable attention is given to the creation of novel photosensitive recording media <strong>for</strong> archival<br />
three-dimensional optical memory on multilayer discs with fluorescence in<strong>for</strong>mation reading. We<br />
proposed a method <strong>for</strong> the synthesis of earlier unknown derivatives of 3-acetyl-2(2’hetaryl)chromone<br />
(A) capable of undergoing irreversible changes under UV irradiation to <strong>for</strong>m<br />
photoluminescent products (B) that provide optical in<strong>for</strong>mation reading.<br />
R<br />
R<br />
1<br />
O<br />
O<br />
O<br />
O<br />
O<br />
R<br />
R 1<br />
X<br />
R 3<br />
R 1<br />
R 2<br />
R<br />
A<br />
O<br />
O<br />
R<br />
O<br />
S<br />
R 1<br />
O<br />
R 3<br />
4 5<br />
R 1 =<br />
R= H,<br />
S<br />
S<br />
O<br />
Pyr<br />
Cl<br />
O<br />
R<br />
R<br />
2<br />
R 2 =Hal, Alk,<br />
R 2<br />
O<br />
O<br />
R 1<br />
O<br />
O<br />
R<br />
S<br />
hv<br />
O<br />
O<br />
R 1<br />
X<br />
R 3<br />
R<br />
R<br />
t-BuOK<br />
R 2<br />
--OMe<br />
R<br />
SeO 2<br />
R<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 321<br />
B<br />
O<br />
O<br />
R 3 = Br, H,<br />
R<br />
O<br />
O<br />
R 1<br />
S<br />
O<br />
R 2<br />
R 3<br />
O<br />
O<br />
R 1<br />
6<br />
--OMe<br />
=OPh, -SAlk, -SPh, NAlk, -CH 2 -S-Ph, -CH 2 -OPh, -CH 2 -S-Alk, CH 2 -N-Alk<br />
The photochemical properties of choromones (6) were studied, including the spectra data <strong>for</strong> both<br />
individual substances and compounds incorporated into polymer matrices. The data obtained show<br />
that synthesized chromones are promising <strong>for</strong> use as luminescent media <strong>for</strong> superdense recording<br />
on a multilayer carrier.<br />
This work was supported by Russian Foundation <strong>for</strong> Basic Researches (Grant #10-03-00250-а)<br />
References: [1] V.A.Barachevsky, O.I. Kobeleva, T.M.Valova, A.O. Ait,A.A.Dunaev, A.M. Gorelik,M.M.<br />
Krayushkin, K.S. Levchenko, V.N. Yarovenko,V.V. Kiyko, E.P.Grebennikov; Optical Memory & Neural<br />
Networks (In<strong>for</strong>mation Optics), v.19, №2, 187-195, 2010.<br />
______________<br />
* Corresponding author: e-mail: mkray@ioc.ac.ru<br />
3<br />
O<br />
O<br />
O<br />
O<br />
X=O,S<br />
X<br />
R 3<br />
R 1<br />
X<br />
R 3<br />
R 2<br />
R 2
P203 Fluorescent Proteins, Fluorescent Probes & Labels Poster 203<br />
New environment-sensitive fluorescent dyes <strong>for</strong> biological applications<br />
Oleksandr Kucherak, Ludovic Richert, Youri Arntz, Pascal Didier, Yves Mely<br />
& Andrey Klymchenko<br />
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg,<br />
Faculté de Pharmacie, 74, Route du Rhin, 67401 Illkirch (France)<br />
Our aim was to develop advanced environment-sensitive fluorophores. In this respect, two classes<br />
of chromophores were developed. First one is based on fluorene bearing strong electron donor<br />
(dialkylamino) and acceptor (carbonyl) groups at 2,7-positions. These dyes can be considered as<br />
an extended conjugated analogue of Prodan, which is one of the best solvatochromic dyes [1] .<br />
These fluorene-based dyes showed red-shifted absorption (close to 400 nm), twice as large<br />
absorption coefficient (43,000 M-1×cm-1), and manifold larger two-photon absorption cross-section<br />
(ca 400 GM) compared to Prodan. In addition, studies in solvents revealed much stronger<br />
fluorescence solvatochromism of the new dyes, which is connected with their twice as large<br />
transition dipole moment (14.0 D). Similarly to Prodan, they exhibit high fluorescence quantum<br />
yields, while their photostability is largely improved. The second class of dyes is based on 3methoxychromone,<br />
characterized by strong electron donor and acceptor functionalities and<br />
different conjugation lengths [2] . These dyes are also characterized by significant absorption<br />
coefficient and two-photon absorption cross-section (up to 450 GM) and large fluorescence<br />
quantum yield. In comparison to the parent 3-hydroxychromone derivatives, they present<br />
dramatically improved photostability confirming that photodegradation of 3-hydroxychromones<br />
occurs from a product of ESIPT-reaction (tautomer <strong>for</strong>m). Importantly, all dyes showed remarkable<br />
fluorescence solvatochromism, which increased significantly with extension of their conjugation.<br />
For the 3-methoxychromone bearing 2-fluorenyl donor group the difference dipole moment,<br />
estimated from Lippert equation, reached 20D, which is among the largest reported value <strong>for</strong><br />
neutral dipolar fluorophores. In conclusion, two classes of new environment-sensitive dyes were<br />
synthesized and characterized. Their attractive fluorescence properties and high sensitivity to the<br />
environment make them attractive building blocks <strong>for</strong> advanced fluorescent probes and labels.<br />
This work was supported by grants from Conectus Alsace and ANR blanc.<br />
References: [1] O. Kucherak, et al., J. Phys. Chem. Lett., 1 (2010) 616. [2] O. Kucherak, et al.,submitted.<br />
______________<br />
* Corresponding author: e-mail: andrey.klymchenko@unistra.fr<br />
322 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P204 Fluorescent Proteins, Fluorescent Probes & Labels Poster 204<br />
ATP sensitive fluorescence turn-on probe based on a pyrenebipyridinium<br />
complex<br />
Thomas Lang & Michael Schäferling<br />
Institute of Analytical Chemistry, Chemo- and Biosensensors, University of Regensburg, 93053<br />
Regensburg (Germany)<br />
Nucleoside phosphates such as ATP or GTP play an important role in biological systems as carrier<br />
<strong>for</strong> chemical energy and as substrate in many enzymatic reactions. ATPase, <strong>for</strong> instance,<br />
harnesses the energy derived from the decomposition of ATP and controls several important<br />
processes in the human body, e.g. muscle contraction, ion transport and the cellular metabolism.<br />
Other important classes of enzymes are the phosphokinases which alter the reactivity and function<br />
of molecules (e.g. proteins) by phosphorylation, or adenylyl cyclases which <strong>for</strong>m the second<br />
messenger cAMP from ATP. Hence, significant in<strong>for</strong>mation on the regulation of these enzymes can<br />
be gained by monitoring ATP consumption. This is essential <strong>for</strong> the design of enzyme regulating<br />
drugs. Fluorescent ATP sensitive probes exhibit crucial advantages over established methods <strong>for</strong><br />
the determination of enzymatic activity using radioactively labelled [ 32 P]ATP or immunoassays. [1]<br />
We designed a new fluorogenic indicator system <strong>for</strong> the determination of ATP in neutral aqueous<br />
solution. It has been reported that derivatives of bipyridinium salts (viologens) carrying boronic acid<br />
groups can be used <strong>for</strong> the sensing of sugars. [2] The concept is based on the quenching of pyrene<br />
trisulfonic acids, e.g. 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) or 8-aminopyrene-1,3,6trisulfonic<br />
acid (APTS) by the <strong>for</strong>mation of adducts with viologen derivatives. We modified the<br />
viologen unit with phosphate-binding ammonium groups. The stacked ion pair disaggregates if<br />
ATP binds to the viologen entailing to a strong increase of the fluorescence of the pyrene moiety.<br />
We studied the response of this fluorescence turn on probe with several phosphates including<br />
ATP, ADP, AMP, cAMP, GTP, GDP, phosphate anions, and pyrophosphate at 37°C. The probe<br />
responds very fast and with sufficient selectivity between ATP and the other tested phosphate<br />
species. Thus, straight<strong>for</strong>ward assays can be developed <strong>for</strong> the real time monitoring of the activity<br />
of pharmaceutically relevant enzymes such as protein kinases, ATPases, or adenylyl cyclases, and<br />
<strong>for</strong> the screening of their regulators.<br />
APTS<br />
This work was supported by the Deutsche Forschungsgemeinschaf (DFG SCHA 1009/4-3).<br />
References: [1] C.M. Spangler et al., Anal. Biochem. 381 (2008) 86; [2] Z. Sharrett et al., Org. Biomol.<br />
Chem. 7 (2009) 1461.<br />
______________<br />
* Corresponding author: e-mail: michael.schaeferling@chemie.uni-regensburg.de<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 323
P205 Fluorescent Proteins, Fluorescent Probes & Labels Poster 205<br />
Discovery of new LD (Lipid Dropet) staining fluorescent compounds<br />
Eunha Kim 1 , Sanghee Lee 1 & Seung Bum Park 1,2*<br />
1 2<br />
Department of Chemistry and Department of Biophysics and Chemical Biology, Seoul National<br />
University, Seoul 151-747, Korea<br />
In this study, discovery of new LD (Lipid Droplet) staining fluorescent compounds was described. A<br />
screening of 9 different Seoul-Fluor compounds revealed the two different fluorescent compounds,<br />
sensitive and specific <strong>for</strong> LD. Through the solvatochromism study of Seoul-Fluor system, ICT<br />
(Intramolecular Charge Transfer) process in Seoul-Fluor system is crucial factor <strong>for</strong> the LD staining<br />
function. Compare to the traditional LD staining dyes, such as Nile Red, BODIPY-Fatty acid, and<br />
LipidTox green, discovered fluorescent compounds stained the LD without the phospholipid signal,<br />
washing step and fixation step. With the compounds, not only the LD in in vivo system, employing<br />
the C. elegans system, but also lipid metabolism in HeLa cells were clearly visualized. Moreover,<br />
autophagy related changes of LD pattern in HeLa cells, such as increase or decrease of LD<br />
<strong>for</strong>mation corresponding to the inhibition or induction of the autophagy state in the cells, were<br />
monitored in the living cells. Combining with the multiplexing flexibility, we envisioned that the<br />
discovered fluorescent compounds can function as a versatile tools to develop the image-based<br />
real time HCS assay system <strong>for</strong> the discovery of certain compounds regulating LD not only the lipid<br />
metabolism but also <strong>for</strong> lipid metabolism related signal cascade, such as autophagy.<br />
References: [1] E. Kim, et al., J. Am. Chem. Soc., 133 (2011) 6642–6649. [2] E. Kim, et al., J. Am. Chem.<br />
Soc. 130 (2008) 12206–12207. [3] E. Kim, et al., Chem. Commun. in press.<br />
______________<br />
* Corresponding author: e-mail: sbpark@snu.ac.kr<br />
324 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P206 Fluorescent Proteins, Fluorescent Probes & Labels Poster 206<br />
Turn-on fluorescent probe with activatable internal control<br />
Geoffray Leriche 1 , Ghyslain Budin 1 , Zeinab Darwich 2 , Yves Mély 2 , Andrey Klymchenko 2<br />
& Alain Wagner* ,1<br />
1 Laboratory of Functional Chemo-Systems, UMR 7199 CNRS, Université de Strasbourg, Faculté<br />
de Pharmacie, 74 Route du Rhin, 67401, Illkirch-Graffenstaden (France)<br />
2 Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg,<br />
Faculté de Pharmacie, 74, Route du Rhin, 67401 Illkirch-Graffenstaden (France)<br />
Fluorescence methods are widely used to study biological processes in living cells. Recently, turnon<br />
probes have been developed to measure cell enzymatic activity, pH alterations and to detect<br />
singlet oxygen, nitric oxide and highly reactive oxygen species (hROS). [1] This turn-on approach is<br />
particularly interesting since it increases the signal-to-noise ratio, which is a key <strong>for</strong> live cell<br />
imaging. Turn-on probes are not detectable be<strong>for</strong>e their activation and this can lead to artifacts.<br />
When there is a negative result (i.e. no or weak fluorescence increase), it is difficult to assess<br />
whether the probe could not enter the cell and localize in the appropriate compartment or the<br />
biological trigger was insufficient.<br />
To address these issues, we report the development of a turn-on fluorescent probe which<br />
can be activated both by biological stimuli and a bio-independent chemical reagent (see scheme).<br />
We assumed the azo-based quencher/dithionite reagent system would act as an internal control. In<br />
this system, the absorption of the azo-quencher could be efficiently altered by a reaction with<br />
sodium dithionite under mild and bio-compatible conditions. [2] Previously, we conducted an<br />
extensive reactivity study to determine the key structural features <strong>for</strong> the dithionite-triggered<br />
reductive cleavage of the azo group. [3] A highly reactive (4-hydroxy-2-methoxy-phenylazo) benzoic<br />
acid scaffold (HAZA) was selected as the quencher, which can be cleaved quickly under a low<br />
concentration of dithionite. Conjugated to a fluorescent dye, this quencher was used to investigate<br />
the dithionite triggered reduction. We found that addition of a reducing agent to the activatable<br />
probe resulted in an almost instantaneous increase of fluorescence in solution. Using the same<br />
probe, dithionite biocompatibility was also validated in living cells.<br />
Based on this fluorophore/quencher/reagent system, we developed a FRET-based probe to<br />
detect the protease activity. [4] A caspase-3 sensitive DEVD peptide was incorporated in a turn-on<br />
probe to image apoptotic cells. The concept of double cleavage was validated in solution with<br />
human recombinant caspase-3 and in Hela cells pre-treated with actinomycin D to induce<br />
apoptosis. We have demonstrated that our probe can distinguish between apoptotic and healthy<br />
cells and it also acts as an internal control to reveal its presence in healthy cells and apoptotic<br />
cells. This work can lead to the interesting development of fluorescent probes to quantify<br />
enzymatic activity in cells.<br />
References: [1] H.Kobayashi, et al., Chem. Rev. 110 (2010) 2620. [2] G. Budin, et al., ChemBioChem 11<br />
(2010) 2359. [3] G. Leriche, et al., Eur. J. Org. Chem. 2010 (2010) 4360. [4] G. Leriche, et al., ChemComm.<br />
(2011) submitted<br />
______________<br />
* Corresponding author: e-mail: wagner@bioorga.u-strasbg.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 325
P207 Fluorescent Proteins, Fluorescent Probes & Labels Poster 207<br />
Synthesis of fluorescent probes <strong>for</strong> the non-invasive imaging of<br />
cancer hallmarks<br />
Martha Mackay* & Mark Bradley<br />
School of chemistry, University of Edinburgh, King’s Building, West Mains Road, Edinburgh, EH9<br />
3JJ (United Kingdom)<br />
Caspases are intracellular endopeptidases that play an essential role in apoptosis, necrosis and<br />
inflammation. Until proteolytically activated by apoptotic stimuli, caspases remain as inactive<br />
zymogens. Uncontrolled proliferation of cells caused by non-activation of the Caspase Cascade is<br />
often deemed a ‘Hallmark of Cancer’. 1 Molecular imaging of Caspase 3 and 7 in vivo may reveal<br />
key pathological events and help the early diagnoses of diseases such as alzheimer’s and cancer 2 .<br />
Herein, we report internally quenched molecular reporters that will be used to detect activity<br />
of Caspase 3 & 7 via the use of fluorophores attached to a tribranched dendron through a Caspase<br />
specific peptide. 3,4 This allows internal self-quenching. Specific hydrolysis of the peptide by<br />
caspase 3 & 7 leads to rapid and efficient generation of intense fluorescent signal. These reporters<br />
could be used as tools <strong>for</strong> the early diagnosis and follow-up of diseases such as cancer.<br />
This work has been supported by funding from Cancer Research UK and the University of<br />
Edinburgh.<br />
References: [1] S.J. Riedl, et al., Nature Reviews 5, (2004) 897-906. [2] L.E. Edgington, et al., Nature<br />
Medicine 15, (2009) 967-974. [3] S. Lebreton, et al., Tetrahedron 59, (2003) 3945-3953. [4] J.M. Ellard, et<br />
al., Angewandte Chemie Communications 41, (2002) 3233-3236.<br />
______________<br />
Corresponding author: e-mail: m.mackay@sms.ed.ac.uk<br />
326 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P208 Fluorescent Proteins, Fluorescent Probes & Labels Poster 208<br />
Covalent labeling of His-tagged protein with a “turn-on” fluorescent<br />
probe utilizing a quencher-conjugated histidine peptide<br />
Atsushi Murata 1 , Satoshi Arai 2 & Shinji Takeoka 1*<br />
1<br />
Graduate School of Advanced Science and Engineering, Waseda University, TWIns, Japan<br />
2<br />
Consolidated Research Institute <strong>for</strong> Advanced Science and Medical Care, Waseda University,<br />
Japan<br />
Fluorescence imaging is a powerful tool to investigate functions of proteins under physiological<br />
conditions, because it is sensitive and has great temporal/spatial resolution. Labeling of the target<br />
protein with small chemical probes has the advantage of suppressing the movement obstruction of<br />
the target protein to minimum and being easy to be labeled. To improve the small chemical probes<br />
<strong>for</strong> the target protein labeling, stable labeling with covalent bond and fluorogenic property based on<br />
the recognition of the target protein are required. Here, we describe an approach of the target<br />
protein covalent labeling with a “turn-on” fluorecent probe. Because the fluorogenic probes have<br />
the advantage that a washing step to remove the probe that is not bound to the target protein is not<br />
necessary, this system is expected to simplify the labeling procedure. For a versatile fluorescent<br />
probe to label the target protein, we focused on hexahistidine-tag (His-tag). The His-tag has been<br />
widely used as an affinity tag <strong>for</strong> the purification of genetically engineered proteins by using affinity<br />
chromatography.<br />
As a fluorescent probe <strong>for</strong> covalent labeling of His-tagged protein, aryl azide which is one of<br />
the photo-cross linker conjugated nitrilotriacetic acid (NTA) derivartive was designed. NTA<br />
recognize imidazole ring of histidine by coordinaton bond. Photo-cross linker has the advantage<br />
that is stable in the physiological condition and controllable the reaction by UV irradiation.<br />
Tetramethylrhodamine and aryl azide conjugated NTA derivative (TMR-NTA-N3) was synthesized<br />
and the binding ability and fluorescence property of TMR-NTA-N3 in the presence of His-tag moiety<br />
were evaluated. First of all, the covalent labeling yield was evaluated by measurement of mass<br />
spectroscopy (MS) and fluorescence correlation spectroscopy (FCS). Mixture of the TMR-NTA-N3,<br />
NiCl2 and hexahistidine (His6) which is a model compound of His-tag was prepared. The shifting of<br />
molecular weight was monitored by measurement of MS be<strong>for</strong>e and after UV irradiation. After UV<br />
irradiation, ion of the covalent lebeling molecule was observed. Then, the covlent labeling was also<br />
evaluated using His-tagged ubiquitin by the diffusion time measurement of the TMR-NTA-N3. After<br />
UV irradiation to the complex of His-tagged ubiquitin and TMR-NTA-N3, the diffusion time was<br />
increased compared to the sample without UV irradiation in the presence of EDTA. This<br />
enhancement of the diffusion time also indicated that TMR-NTA-N3 bind to His-tagged protein<br />
covalently. The covalent labeling yield calculated by using the enhancement of diffusion time was<br />
approximately 33%. These results of MS and FCS measurement supported TMR-NTA-N3<br />
covalently bind to His-tag moiety. To evaluate the “turn-on” fluorescence property of TMR-NTA-N3,<br />
fluorescence intensity was measured utilizing quencher-conjugated histidine peptide (Dabcyl-<br />
His6) [1] . The fluorescence intensity of the TMR-NTA-N3 was decreased by the addition of Dabcyl-<br />
His6. Then, the fluorescence recovery efficiency was tested by adding either ubiquitin or Histagged<br />
ubiquitin to a solution of Dabcyl-His6 and TMR-NTA-N3. Although there was no significant<br />
enhancement in fluorescence after addition of an equimolar amount of ubiquitin, the fluorescence<br />
increased after the addition of His-tagged ubiquitin. There<strong>for</strong>e, this fluorescent probe was able to<br />
specifically and stably bind to His-tagged proteins.<br />
Reference: [1] A. Murata et al., Bioorg. Med. Chem. Lett., 20, 2285-2288 (2010).<br />
______________<br />
* Corresponding author: e-mail: takeoka@waseda.jp<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 327
P209 Fluorescent Proteins, Fluorescent Probes & Labels Poster 209<br />
Phosphorescence based intracellular oxygen sensing probes and assays<br />
Dmitri B. Papkovsky, Alexander V. Zhdanov, Ruslan I. Dmitriev & Andreas Fercher<br />
Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, Cork, Ireland<br />
It is known that depending on experimental conditions (e.g. levels of external pO2, cell density and<br />
metabolic activity, temperature, sample geometry, mass exchange conditions), local O2 gradients<br />
in respiring samples may vary greatly and thus contribute to the observed biological effects [1].<br />
Precise in situ control of cell oxygenation is there<strong>for</strong>e important, particularly when conducting<br />
complex metabolic studies and working under hypoxic conditions. Phosphorescence quenching<br />
technique allows non-invasive monitoring of O2 concentration, however its application to the<br />
monitoring of cellular O2 has been challenging, mainly due to non-optimal characteristics of the<br />
probes and measurement methodologies used.<br />
We present a family of new cell-permeable, self-loading O2-sensing probes which provide<br />
simple, reproducible, passive loading of different types of mammalian cells, high phosphorescent<br />
signals, low cytotoxicity and general convenience. The probes, which include i) Pt-coproporphyrin<br />
derivatives (small molecules), ii) their conjugates with cell-penetrating peptides, and iii) polymeric<br />
nanoparticle probes, allow accurate, real-time monitoring of cellular O2 levels (in μM), changes in<br />
metabolic status and responses to stimulation [2,3]. Multiple samples can be analysed by simple<br />
means using standard time-resolved fluorescent readers or live cell imaging systems.<br />
These intracellular O2 probes and sensing techniques have been extensively tested and<br />
validated with various cell lines and experimental conditions, and subsequently used in in a<br />
number of complex biological studies [3] and experiments under hypoxia [1] where they have<br />
demonstrated good analytical per<strong>for</strong>mance and high practical utility. The talk will cover rational<br />
probe design, characterisation and biological applications.<br />
This work was supported by the Science Foundation of Ireland (grant 07/IN.1/B1804) and by the<br />
European Commission (FP7 project CP-FP 214706-2).<br />
References: [1] Zhdanov AV et al., Integr. Biol. 2 (2010) 443. [2] Dmitriev RI et al, FEBS J. 277 (2010) 4651.<br />
[3] Zhdanov AV et al., Cell Mol Life Sci., 68 (2011) 903.<br />
______________<br />
* Corresponding author: e-mail: d.papkovsky@ucc.ie<br />
328 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P210 Fluorescent Proteins, Fluorescent Probes & Labels Poster 210<br />
Impact of hydrophilic groups on fluorescent properties of<br />
long-wavelength squaraine dyes<br />
Larysa I. Markova 1 , Sania U. Khabuseva 1 , Yevgen A. Povrozin, Ewald A. Terpetschnig 2<br />
& Leonid D. Patsenker 1,2 *<br />
1<br />
<strong>State</strong> <strong>Scientific</strong> <strong>Institution</strong> "Institute <strong>for</strong> <strong>Single</strong> Crystals", National Academy of Sciences of Ukraine,<br />
60, Lenin Ave., 61001 Kharkiv (Ukraine)<br />
2<br />
SETA BioMedicals, LLC, 2014 Silver Ct East, Urbana, IL 61801 (USA)<br />
Squaraine dyes, which are a subclass of cyanines,<br />
are used as the long-wavelength fluorescent labels.<br />
Aggregation of squaraines as well as other cyanines<br />
in aqueous solutions is known to be a negative factor<br />
decreasing fluorescence quantum yields and stability<br />
of dyes. This work investigates the quantum yields<br />
and photostability of squaraine dyes and their<br />
antibody (IgG) conjugates in terms of the number of<br />
sulfo-grops that are present in the dye: squaraines<br />
2Sq–5Sq with two to five sulfo groups were studied.<br />
The number be<strong>for</strong>e the abbreviation "Sq" indicates the<br />
number of sulfo groups in the molecule. The<br />
investigated dyes contain a single carboxy group (to avoid cross-linking) allowing covalent<br />
attachment to biomelecules. All measurements were done in phosphate buffer pH 7.4<br />
Squaraines 2Sq–5Sq absorb and emit in<br />
Quantum Yield [%]<br />
30<br />
20<br />
10<br />
4Sq<br />
2Sq 3Sq<br />
5Sq<br />
0<br />
0 1 2 3 4 5<br />
Dye to protein ratio<br />
Quantum yield vs. dye-to-protein ratio (D/P)<br />
of squarine dyes conjugated with IgG<br />
in phosphate buffer (pH 7.4)<br />
N N<br />
(CH2) 5 O R<br />
COOH<br />
3<br />
Sq<br />
2Sq R 1 , R 2 = Me, R 3 = Et<br />
3Sq R 1 , R 2 = Me, R 3 = (CH2) 4SO3H 4Sq R 1 , R 3 = (CH2) 4SO3H, R 2 = Me<br />
5Sq R 1 , R 2 , R 3 = (CH2) 4SO3H the long-wavelength spectral region. After<br />
binding to IgG the spectra are red-shifted by 4–<br />
8 nm. Squaraines have high extinction<br />
coefficients in the range of 284,000 –<br />
333,000 M –1 cm –1 . Their quantum yields (Q.Y.s)<br />
are low in water (4–9%) but these values<br />
increase by factor of 3–4.4 after binding to IgG<br />
(up to 15–27% at dye-to-protein ratio D/P = 1)<br />
or oligonucleotides. The increase of the number<br />
of sulfo groups causes the increase of the Q.Y.s<br />
in both the free (by factor of 2.3) and the IgGbound<br />
states (by factor of 1.5–1.8 at D/P = 1).<br />
However, <strong>for</strong> the conjugated dyes with four<br />
(4Sq) and five (5Sq) sulfo groups the Q.Y.s are<br />
of the same order. The fluorescence lifetimes<br />
<strong>for</strong> squaraines are in the range of 0.2–0.3 ns but<br />
noticeably increase up to 1.4–2.0 ns <strong>for</strong> the<br />
conjugates. The Q.Y.s and lifetimes decrease with the increase of the D/P but the decrease is<br />
more pronounced <strong>for</strong> the less hydrophilic dyes containing a lower number of sulfo groups. The<br />
photostability increases with the number of sulfo-groups.<br />
We can there<strong>for</strong>e conclude that the increase of the number of sulfo groups causing an<br />
increase of the hydrophilicity of squaraines is a promising way to improve their quantum yields and<br />
photostability. On the other hand, the increase in hydrophilicity reduces the sensitivity of the<br />
squaraine dyes toward the microenvironment, which makes them less useful as fluorescent<br />
probes.<br />
This work was supported by the Science and Technology Center in Ukraine, STCU project<br />
No. P313.<br />
______________<br />
* Corresponding author: e-mail: patsenker@isc.kharkov.com<br />
HO 3S<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 329<br />
R 1<br />
O<br />
R 2<br />
SO 3H
P211 Fluorescent Proteins, Fluorescent Probes & Labels Poster 211<br />
Comparison of the reaction rates of strain-mediated<br />
click-chemistry reagents<br />
Anatoliy Tatarets 1 , Yevgen A. Povrozin 1 , Leonid D. Patsenker 1,2 * & Ewald A. Terpetschnig 2<br />
1<br />
<strong>State</strong> <strong>Scientific</strong> <strong>Institution</strong> "Institute <strong>for</strong> <strong>Single</strong> Crystals", National Academy of Sciences of Ukraine,<br />
60, Lenin Ave., 61001 Kharkiv (Ukraine).<br />
2<br />
SETA BioMedicals, LLC, 2014 Silver Ct East, Urbana, IL 61801 (USA).<br />
The click chemistry reaction (CCR) has experienced a renaissance since its discovery by Huisgen<br />
several decades ago. The reaction shows several advantages: it proceeds quantitatively at RT in<br />
aqueous solution near neutral pH and is there<strong>for</strong>e well-suited <strong>for</strong> use in biomedical applications.<br />
Conventional CCRs require Copper(I) ions as a catalyst to facilitate the 1,3-dipolar cycloaddition<br />
reaction between the alkynyl and the azido group. Strain-mediated CCR that work without the use<br />
of a Cu-catalyst have been developed more recently in order to use the advantages of the CCR in<br />
cellular environments where the presence of Cu-ions is not desirable.<br />
SETA BioMedicals is developing and<br />
commercializing a series of new reagents related to<br />
CCRs. Some of these reagents containing the DBCO-<br />
moiety facilitate the strain-mediated reaction without<br />
the need of Cu-ions in aqueous solutions.<br />
The current study investigates the reaction rate<br />
of the reaction between a Seta-acceptor-labeled<br />
DBCO reagent and a Seta-dye-donor-labeled azide<br />
and aims to compare it to the only other commercially<br />
available strain-mediated product. A fluorescence<br />
energy transfer (FRET) based system is used <strong>for</strong><br />
these investigation. Reaction of the azide with the<br />
DBCO unit brins the donor and acceptor into close<br />
proximity thereby leading to FRET in the donoracceptor<br />
labeled triazole-system.<br />
The R0 <strong>for</strong> the FRET pair was calculated to be<br />
55 Å, which ensures complete energy transfer once<br />
the cyclo-addition reaction occurs. The ratio of the<br />
measured intensities at 520 and 670 nm plotted<br />
against the reaction time allows determining the<br />
temperature dependent reaction rates. Finally we also<br />
made an attempt to calculate the activation energy <strong>for</strong><br />
the two different strain-mediated cycloaddition<br />
reagents.<br />
This work was supported by the Science and Technology Center in Ukraine, STCU project<br />
No. P313.<br />
______________<br />
* Corresponding author: e-mail: patsenker@isc.kharkov.com<br />
Seta-650<br />
330 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
N<br />
O<br />
N 3<br />
O<br />
+<br />
FRET<br />
N<br />
Seta-650<br />
Seta-404<br />
N<br />
N<br />
N<br />
Seta-404<br />
Fig. Seta-dye labeled donor-acceptor pairs<br />
used in the FRET-based, comparative<br />
studies of the reaction rates of strainmediated<br />
CCRs
P212 Fluorescent Proteins, Fluorescent Probes & Labels Poster 212<br />
A new strategy towards fluorescent cyclotriveratrylenes<br />
L. Peyrard 1* , S. Chierici 2 , S. Pinet 1 , G. Jonusauskas 3 , P. Meyrand 4 & I. Gosse 1<br />
1<br />
Institut des Sciences Moléculaires, Groupe Nanosystèmes analytiques, Site ENSCBP, 16 Av. Pey<br />
Berland, 33607 Pessac, France<br />
2<br />
Université Joseph Fourier, Département de Chimie Moléculaire, BP 53 38041 Grenoble, France<br />
3<br />
Université de Bordeaux, Centre de Physique Moléculaire Optique et Hertzienne, 351 cours de la<br />
libération, 33400 Talence, France<br />
4<br />
Institut des Maladies Neurodégénératives, 351 cours de la libération, 33405 Talence, France<br />
The development of fluorescent probes <strong>for</strong> the detection of neuronal species in vivo, like<br />
acetylcholine, presents a growing interest <strong>for</strong> few years. In this context, we develop in our<br />
laboratory fluorescent probes having a cyclotriveratrylene (CTV) skeleton. 1<br />
CTV are bowl-shaped structures known to complex quaternary ammoniums like<br />
acetylcholine. 2,3 CTV can be fluorescent via photoinduced charge transfer (PCT) if conjugated<br />
withdrawing and donating groups are introduced onto the aromatic skeleton. However, up to now,<br />
none of the fluorescent CTV probes fulfills all the criteria required <strong>for</strong> an application in vivo<br />
(solubility in biological medium, high excitation wavelength, and selectivity <strong>for</strong> acetylcholine<br />
especially versus choline, its precursor and metabolite). In order to fulfill these requirements, more<br />
suitable withdrawing groups (like phosphonic acid or nitrile) can be introduced onto the aromatic<br />
skeleton of the CTV. These groups should improve the probe properties such as solubility in water<br />
and/or fluorescence ones (excitation wavelength, quantum yield. We also tried to extend the<br />
conjugation between the donating and the withdrawing groups to obtain a deeper hydrophobic<br />
cavity and thus a higher selectivity <strong>for</strong> acetylcholine. Furthermore, the extension of conjugation<br />
using organometallic coupling reactions should lead to more interesting absorption and<br />
fluorescence properties. In order to introduce various withdrawing groups, and so to get a small<br />
library of fluorescent CTVs, we have elaborated a new convergent strategy from a keyintermediate<br />
CTV, bearing iodine groups. 4<br />
W<br />
D<br />
W D W<br />
W = COCH3, PO(OH) 2, CN<br />
D = OMe, OH<br />
I<br />
D<br />
OMe<br />
Key intermediate<br />
I<br />
OMe<br />
I<br />
OMe<br />
First generation Second generation<br />
2 or 3 steps<br />
W'<br />
D<br />
D<br />
W' D W'<br />
W ' = conjugated groups + withdrawing groups<br />
Figure 1: Synthesis of two generations of CTVs from the key-intermediate<br />
In this communication, we will present first, the versatile synthesis of the key-intermediate CTV and<br />
the syntheses of the new fluorescent CTVs obtained. Then, we will concentrated on the<br />
spectroscopic properties and the detection results, like the determination of binding constants and<br />
competition studies in aqueous buffer.<br />
This work was supported by grants from Ministère de la Recherche, Bordeaux University.<br />
References: [1] M. Dumartin, et al., Organic and Biomolecular Chemistry 7, (2009) 2725-2728. [2] M.<br />
Hardie, Chemical Society Reviews 39, (2010) 516-527. [3] T. Brotin, J. Dutasta, Chemical Reviews 109,<br />
(2009) 88-130. [4] L. Peyrard, et al., Submitted<br />
______________<br />
* Corresponding author: lisa.peyrard@enscbp.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 331
P213 Fluorescent Proteins, Fluorescent Probes & Labels Poster 213<br />
Novel nonlinear octupolar merocyanine chromophores<br />
Yevgen Poronik 1 , Mireille Blanchard-Desce 2 & Daniel Gryko 1,*<br />
1<br />
Institute of Organic Chemistry Polish Academy of Science, Kasprzaka 44/52, 01-224 Warsaw<br />
(Poland)<br />
2<br />
Université de Rennes, Case 1003, Rennes Cedex 35042 (France)<br />
Design of novel chromophores possessing nonlinear optical (NLO) properties such as two-photon<br />
absorption (2PA) has been attracting considerable attention in recent years owing to the prospect<br />
of using them in a wide range of applications including two-photon excited fluorescence (TPEF)<br />
microscopy of biological objects, optical limiting, 3D microfabrication etc. [1]<br />
From the structural point of view, the majority of 2PA chromophores belong to the family of<br />
push-pull polyenes with electron donor and acceptor at the edges of the conjugated system. [1]<br />
Merocyanine dyes are similar to polyenes, have electrically neutral chromophore, but in contrast to<br />
the latter they are characterized by more efficient intramolecular charge transfer (ICT) between<br />
electron-donor and electron-withdrawing groups, [2, 3] which potentially results in higher two-photon<br />
cross-section at the shorter conjugated chain. Our research addresses to synthesis and<br />
investigation of novel octupolar conjugated merocyanine dyes as a combination of three dipolar<br />
chromophores in one system.<br />
Changing electron-donor properties of the end heterocyclic nuclei makes it possible to change the<br />
contribution of polyene and zwitterionic mesomeric <strong>for</strong>ms. This results in changing ICT and dipole<br />
moment within the dipolar arms of the octupolar chromophore, and hence allows to control 2PA. [4]<br />
Dyes synthesized are characterized by the intensive long-wavelength absorption bands within the<br />
range λmax = 560-660 nm (ε = 140000-220000) and short-wavelength bands at 418 – 530 nm.<br />
Merocyanine dyes of this series possess fluorescent properties and values of fluorescent quantum<br />
yield reaches 12%.<br />
According to our assumption above-mentioned dyes are characterized by the intensive<br />
nonlinear response, values of 2PA cross-section (σ) <strong>for</strong> such octupolar chromophores are in the<br />
range 1200-2000 GM.<br />
This work was supported by the Foundation <strong>for</strong> Polish Science (Grant TEAM 2009 4/3).<br />
References: [1] M. Pawlicki, et al., Andew. Chem. Int. Ed. 48 (2009) 3244. [2] A. Kulinich et al., Russ. Chem.<br />
Rev. 78 (2009) 141. [3] L. Padilha et al., J. Mater. Chem. 19 (2009) 7503. [4] D. Beljonne, et al., Adv. Funct.<br />
Mater. 12 (2002) 631.<br />
______________<br />
* Corresponding author: e-mail: daniel@icho.edu.pl<br />
332 — MAF 12, Strasbourg, France, September 11-14, 2011 —
P214 Fluorescent Proteins, Fluorescent Probes & Labels Poster 214<br />
Ratiometric fluorescent probes <strong>for</strong> sensing peptide interactions with<br />
nucleic acids and membranes<br />
Viktoriia Postupalenko 1,2 , Aleksandr Stryzhak 2 , Volodymyr Shvadchak 1 , Guy Duportail 1 ,<br />
Andrey Klymchenko 1 , Vasyl Pivovarenko 2 & Yves Mély 1,*<br />
1<br />
Laboratoire de Biophotonique et Pharmacologie, UMR-CNRS 7213, Université de Strasbourg,<br />
Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch-Cedex (France)<br />
2<br />
Chemistry Faculty, National Taras Shevchenko University of Kyiv, 62A, Volodymyrska st., 01033<br />
Kyiv (Ukraine)<br />
Fluorescence spectroscopy is one of the best tools <strong>for</strong> investigating protein interactions and<br />
con<strong>for</strong>mational changes. In this respect, environment-sensitive fluorescent probes, which monitor<br />
environment changes at a specifically labeled site, are of particular interest. We developed<br />
environment-sensitive fluorescent labels that sense peptide interactions with oligonucleotides and<br />
lipid membranes through changes in the intensity ratio of their two emission bands, originating from<br />
an excited-state intramolecular proton transfer., For investigating peptide-oligonucleotide<br />
interactions, a 3-hydroxychromone (3HC) dye which displays high sensitivity to protic solvents has<br />
been developed and coupled to the N-terminus of the HIV-1 nucleocapsid protein (NC), obtained<br />
using solid phase peptide synthesis. The fluorescence spectra of the labeled peptide was found to<br />
depend strongly on the nucleic acid sequence and strandedness. [1] Moreover, to further<br />
characterize the peptide-nucleic acid interaction and provide site-specific in<strong>for</strong>mation on the<br />
environmental changes induced by the interaction close to the labelling site, we synthesized a<br />
fluorescent amino acid analogue, which was inserted at selected positions of the NC peptide.<br />
Using different oligonucleotides whose structures in complex with NC have been solved, we found<br />
that the ratiometric response of the fluorescent amino acid correlates well with its proximity to the<br />
closest base in the complexes. There<strong>for</strong>e, this amino acid provides a new and universal approach<br />
<strong>for</strong> site-specific monitoring of peptide-nucleic acid proximity. [2] For investigation peptide-membrane<br />
interactions, we further developed a 3HC probe sensitive to polarity changes in apolar solvents. [3]<br />
To validate this probe, we synthesized melittin, magainin-2 and polylysine, which are known to<br />
interact with lipid membranes, and labeled their N-terminus with this probe. The observed intensity<br />
ratio of the probe was found to correlate well with the insertion depth of the N-terminal region of the<br />
peptides. Thus, the developed ratiometric fluorescent probes appear as attracting tools <strong>for</strong><br />
monitoring the interactions of the labeled peptides with different targets.<br />
This work was supported by ANR blanc, ANRS and ARCUS.<br />
References:<br />
[1] Shvadchak V.V., et al. Nucleic Acids Res. 37 (2009) e25.<br />
[2] Stryzhak A., Postupalenko V.Y., et al. (submitted).<br />
[3] Postupalenko V.Y., et al. Biochim. Biophys. Acta 1808 (2011) 424.<br />
______________<br />
* Corresponding author: e-mail: yves.mely@unistra.fr<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 333
P215 Fluorescent Proteins, Fluorescent Probes & Labels Poster 215<br />
Optical stimulation and imaging of cell activity with new<br />
optogenetic probes<br />
Matthias Prigge, Franziska Schneider, Satoshi P. Tsunoda & Peter Hegemann<br />
Experimentelle Biophysik, Humboldt-Universität zu Berlin, Institut für Biologie, Invalidenstraße 42,<br />
10115 Berlin<br />
Controlling cellular events with light has been a long standing goal in physiology. But only recently,<br />
since the discovery of channelrhodopsins (ChRs), a light driven cation channel from<br />
Chlamydomonas reinhardtii, such a technique became routinely applicable. Most commonly ChR2<br />
is specifically expressed in a subpopulation of neurons, which then can be stimulated with blue<br />
light. This optogenetic approach gains elegance due to its non-invasive character and its high<br />
temporal and spatial resolution [1]. Genetically-encoded optical reporters <strong>for</strong> measuring cellular<br />
properties such as membrane voltage or intracellular calcium concentration exhibit a spectral<br />
overlap with ChR2, hindering simultaneous application of an optical stimulator and a reporter in a<br />
single cell.<br />
To overcome this problem, we designed a new ChR chimera, named C1V1, featuring an 80<br />
nm red-shifted spectrum (λmax=540 nm) compared to that of ChR2 (470 nm) [2]. An ensuing<br />
widespread mutational analysis shifted the absorption spectra further to 550 nm. In addition C1V1<br />
showed 2 times larger photocurrent and better membrane targeting than those of ChR2 in HEK293<br />
cells and hippocampal neurons.<br />
We have transiently expressed C1V1 in HEK293 cell line permanently expressing the<br />
voltage-gated T-type calcium channel, CaV3.2, Activation of C1V1 with yellow light led to<br />
membrane voltage deflection and opening of CaV3.2 [3]. We first monitored the transient Ca2+<br />
influx by Fura-2, which is excited at 340/380 nm and shows a calcium dependent fluorescence at<br />
510 nm [3]. This influx could be abolished by mibefradil, a know CaV3.2 inhibitor.<br />
In a second approach we fused the genetically-encoded calcium sensor GCaMP3 in frame<br />
with C1V1 [4]. This construct was well expressed in HEK293 cells and showed calcium dependent<br />
fluorescence. Since GCaMP3 is excited at 440 nm and cross-activation with C1V1 should be<br />
avoided, only low intensity of light up to 0.5 mW/cm 2 was applied <strong>for</strong> calcium detection.<br />
Nevertheless, we were able to detect the light activated calcium influx through CaV3.2 by<br />
monitoring fluorescence of GCaMP3.<br />
In future, this approach will be transferred into neurons aiming <strong>for</strong> observation of single<br />
action potentials by transient increase in intracellular calcium.<br />
References:[1]K.Deisseroth, Nature Methods, 8 (2011) 26 [2]F.Zhang, M.Prigge et al.,Nature Neuroscience<br />
11 (2008) 631. [3] M.Prigge, A.Rösler et al., Channels 4 (2010) 241.[4] L.Tian, S.Hires et al.,Nature Methods<br />
6 (2009) 875<br />
334 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
A Shows spectral absorption of fura-2,<br />
GCaMP and C1V1. Both reporters still<br />
show some overlap with C1V1.<br />
At low excitation light intensities of the<br />
calcium reporter, C1V1 activation at<br />
560 nm causes a transient increase in<br />
calcium fluorescence signal.
P216 Fluorescent Proteins, Fluorescent Probes & Labels Poster 216<br />
Novel cationic near infrared fluorescent probes possessing ester,<br />
hydroxyl and amino functional groups in different combinations<br />
Bachu R. Raju 1 , A. Luísa S. Costa 2 , Paulo J. G. Coutinho 1 & M. Sameiro T. Gonçalves 2,*<br />
1 2<br />
Centro de Física and Centro de Química, Universidade do Minho, Campus de Gualtar, 4710-057<br />
Braga, Portugal<br />
Dyes that fluoresce above 600 nm are preferred in biosensing aplications, mainly when dealing<br />
with biological interferences in vivo measurement of metabolites such as glucose or lactate, given<br />
that tissue and serum fluorescence are minimal, and background scattering is limited to its lowest<br />
value in the long-wavelength region. As a result, fluorescence signal from the fluorophore can be<br />
determinate with greater accuracy, and consequently the long-wavelength region is superior to<br />
visible spectral regions <strong>for</strong> fluorescence-based diagnostic applications. [1]<br />
Benzo[a]phenoxaziniums are among the several dyes known to emit in the long-wavelength<br />
region and have been used as fluorescent probes <strong>for</strong> heavy metal ions, amino acids and other<br />
biomolecules. [2-4]<br />
Considering these facts and as a continuation of our research related to the design,<br />
photophysics and application of NIR fluorescence probes, the present work describes the<br />
synthesis of new mono- and bifunctionalised benzo[a]phenoxazinium chlorides possessing a<br />
combination of substituents having ester, hydroxyl, amino and methyl groups as terminals at the 5-<br />
and 9-amino positions of the heteroaromatic ring (Figure 1). The photophysical properties of all<br />
fluorescent compounds in homogeneous media as well as in DNA and in model biological<br />
membranes were measured and will be discussed.<br />
O<br />
O<br />
O<br />
O<br />
N +<br />
Cl -<br />
HO<br />
O<br />
N<br />
H<br />
HN<br />
Cl- N +<br />
O<br />
N<br />
HN<br />
R = H, NH 2, OH<br />
Figure 1. Structure of mono- and bifunctionalised benzo[a]phenoxazinium chlorides.<br />
Thanks are due to Fundação para a Ciência e Tecnologia (Portugal) <strong>for</strong> its financial support of a<br />
BPD to B. R. Raju (SFRH/BPD/62881/2009), Centro de Química and Centro de Física<br />
(Universidade do Minho). The NMR spectrometer Bruker Avance III 400 is part of the National<br />
NMR Network and was purchased in the framework of the National Program <strong>for</strong> <strong>Scientific</strong> Reequipment,<br />
contract REDE/1517/RMN/2005 with funds from POCI 2010 (FEDER) and FCT.<br />
References: [1] M. S. T. Gonçalves, Chem. Rev., 109 (2009) 190. [2] J. Jose, K. Burgess, Tetrahedron, 62<br />
(2006) 11021. [3] C. M. A. Alves et al., Tetrahedron Lett., 52 (2011) 112. [4] S. Naik, et al., Eur. J. Org.<br />
Chem. (2011), 2491.<br />
______________<br />
* Corresponding author: e-mail: msameiro@quimica.uminho.pt<br />
O<br />
O<br />
H<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 335<br />
N +<br />
Cl -<br />
R<br />
O<br />
N<br />
HN<br />
R
P217 Fluorescent Proteins, Fluorescent Probes & Labels Poster 217<br />
New fluorescence standards <strong>for</strong> the NIR<br />
Thomas Behnke, Martin Hübner, Christian Würth, Jutta Pauli, Katrin Hoffmann, Angelika<br />
Hoffmann & Ute Resch-Genger<br />
BAM Federal Institute <strong>for</strong> Material Research and Testing, D-12489 Berlin (Germany)<br />
Although functional chromophores absorbing and emitting above 650 nm are increasingly used in<br />
biology, molecular imaging, and clinical diagnostics as well as in optoelectronic devices, no<br />
dependable spectral fluorescence standards and quantum yield standards <strong>for</strong> the near-infrared<br />
(NIR) spectral region are available. [1] For example, in the case of the frequently used NIR quantum<br />
yield standard indocyanine green (ICG), the authors of one of the most fundamental and often<br />
cited publication explicitly state that the fluorescence quantum yield (Φf ) values provided present<br />
only trends due to questionable dye purity. [2] This hampers the reliability and comparability of<br />
fluorescence measurements in the NIR and the spectroscopic characterization of NIR dyes and<br />
functional chromophores concerning the determination of the fluorometric key parameter<br />
fluorescence quantum yield. [3] This calls <strong>for</strong> simple and internationally accepted fluorescence<br />
standards <strong>for</strong> NIR <strong>for</strong> instrument characterization and <strong>for</strong> fluorescence quantum yield standards <strong>for</strong><br />
this spectral region to improve the quality of data on relative Φf .<br />
F001 F002 F003 F004<br />
300 400 500 600 700 800 900 1000<br />
wavelength/ nm<br />
336 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
F005<br />
Figure1: Normalized corrected emission spectra of BAM Kit Spectral<br />
Fluorescence Standards supplemented by candidate NIR standards.<br />
This encouraged us to study a set of NIR dyes concerning their suitability as spectral fluorescence<br />
standards <strong>for</strong> the determination of the relative spectral responsivity of fluorescence measuring<br />
systems. This includes dye solutions as well as solid materials like transition metal ion-doped<br />
glasses. Criteria <strong>for</strong> the choice of these materials were broad and unstructured emission spectra,<br />
at least a moderate fluorescence quantum yield, and sufficient thermal and photochemical stability<br />
in solution. The corrected emission spectra of first candidate materials emitting > 700 nm are<br />
shown in Figure 1. These currently tested dyes enable the expansion of the BAM Kit Spectral<br />
Fluorescence Standards F001 to F005 [4] into the NIR wavelength region.<br />
In parallel, a first set of fluorescence quantum yield standards <strong>for</strong> the visible and NIR has<br />
been developed using a new custom-built integrating sphere setup <strong>for</strong> absolute measurements of<br />
fluorescence quantum yields.<br />
Financial Support by the MNPQ BMWi Grant 22/06 is gratefully acknowledged.<br />
References: [1] Kobayashi, H.; Ogawa, M.; Al<strong>for</strong>d, R.; Choyke, P. L.; Urano, Y. Chemical Reviews 2010, 110,<br />
2620-2640. [2] Benson, R. C.; Kues, H. A. Journal of Chemical Engineering Data 1977, 22, 379 - 383. [3] [1] U.<br />
Resch-Genger, et al., Reviews in fluorescence 2007, Springer Science+Business Media (2009) 1-33. [4] Pfeifer,<br />
D.; Hoffmann, K.; Hoffmann, A.; Monte, C.; Resch-Genger, U. Journal of Fluorescence 2006, 16, 581-587.<br />
______________<br />
* Corresponding author: e-mail: ute.resch@bam.de
P218 Fluorescent Proteins, Fluorescent Probes & Labels Poster 218<br />
Targeted luminescent NIR nanoprobes <strong>for</strong> in vivo imaging of tumor<br />
hypoxia<br />
Ute Resch-Genger 1 , Thomas Benke 1 , Joanna Napp 2 , C. Würth 1 , Lorenz Fischer 3 , Frauke<br />
Alves 2 & Michael Schäferling 3<br />
1<br />
BAM Federal Institute <strong>for</strong> Material Research and Testing, D-12489 Berlin (Germany)<br />
2<br />
Department of Hematology and Oncology, University Medical Center Göttingen, and Department<br />
of Molecular Biology of Neuronal Signals, Max-Planck-Institute <strong>for</strong> Experimental Medicine,<br />
Göttingen (Germany)<br />
3<br />
Institute of Analytical Chemistry, Chemo- and Biosensensors, University of Regensburg, D-93040<br />
Regensburg (Germany)<br />
Monitoring of pathophysiological changes in vivo is in high demand <strong>for</strong> medical research and<br />
requires robust, cost-effective, and efficient tools and strategies <strong>for</strong> the preferably simultaneous<br />
detection and analysis of multiple parameters in parallel. Near infrared (NIR)-emissive polystyrene<br />
nanobeads (size 100 nm) doped with an oxygen-sensitive phosphorescent Pd(II) porphyrin and an<br />
inert reference dye have been prepared to enable measurements of the hypoxic states of cells or<br />
tumorous tissue.<br />
The oxygen-sensitive probe and reference can be both excited with a 635 nm laser.<br />
Referenced dual wavelengths measurements of the oxygen partial pressure (pO2) are carried out<br />
by calculating the ratio of the NIR emissions of the probe (λem = 800 nm) and reference (λem = 675<br />
nm). Alternatively, fluorescence lifetime imaging can be applied to obtain intrinsically referenced<br />
measurements. We present the synthesis and spectroscopic characterization of these nanoprobes<br />
as well as their ratiometric response to oxygen. They were applied to assess oxygenation levels in<br />
vitro in cultured MHC macrophages and the hypoxic status was compared to the HIF1-α<br />
expression.<br />
Furthermore, the nanobeads were coated with PEG and functionalized with a monoclonal<br />
antibody (herceptin) targeted to the HER2/neu receptor which is overexpressed in different types of<br />
cancer. This paves the way <strong>for</strong> simultaneous cancer biomarker targeting and oxygen sensing and<br />
represents an example of a new generation of optical probes <strong>for</strong> oncology. [1] It can be<br />
demonstrated that the nanoprobes are applicable to in vivo imaging. Signals from probe and<br />
reference can be detected from a mouse tumor model by means of a commercially available<br />
animal imaging system.<br />
Fluorescence Intensity (a.u.)<br />
0,15<br />
0,10<br />
0,05<br />
Reference<br />
Oxygen<br />
0,00<br />
650 700 750 800 850 900 950<br />
Wavelength [nm]<br />
References: [1] Y.-E. Koo Lee, et al., Anal. Chem. 82 (2010) 8446.<br />
______________<br />
* Corresponding author: e-mail: ute.resch@bam.de<br />
Luminescence spectra of the<br />
referenced oxygen nanoprobe (exc:<br />
635 nm) in deaerated (solid line) and<br />
oxygen-saturated solution (dotted<br />
line).<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 337
P219 Fluorescent Proteins, Fluorescent Probes & Labels Poster 219<br />
Hydroxyacridizinium ions: a novel class of water-soluble photoacids<br />
Katy Schäfer 1 & Heiko Ihmels 1<br />
Universität Siegen, Organische Chemie II, Adolf-Reichwein-Str. 2, D-57068 Siegen, Deutschland<br />
Hydroxy-substituted aromatic compounds are of significant theoretical and practical interest<br />
because these compounds usually exhibit a significant enhancement of their acidity upon<br />
excitation. [1,2] For example, the well-investigated cyano-substituted naphthol derivatives serve as<br />
exemplary model compounds, because their excited-state acidity is significantly larger than the one<br />
of the corresponding unsubstituted naphthol. [3] Along these lines, we observed recently that the 8hydroxyacridizinium<br />
ion 1b OH also acts as a strong photoacid (Scheme 1), whose excited-state<br />
acidity originates from the strong donor-acceptor interplay between the oxyanion functionality and<br />
the acridizinium unit in the conjugate base 1b cB . Most notably, the water solubility of this photoacid<br />
enables the investigation of the acid-base equilibrium in aqueous media, which is somewhat limited<br />
with the more hydrophobic naphthol-type photoacids. [1,2]<br />
Photometric and fluorimetric titrations revealed a moderate ground-state acidity of 8hydroxyacridizinium<br />
(1b OH ) in water (pKa = 7.2) and a significantly increased acidity in the first<br />
excited singlet state (pKa* = –0.4). Moreover, the strong donor-acceptor interplay in 1b cB leads to a<br />
large Stokes shift (45872 cm –1 in water) and to a pronounced solvatochromic behavior. Thus, the<br />
emission maximum of the acridizinium 1b OH shifts from 468 nm in acetonitrile to 634 nm in ethanol<br />
(Figure 1).<br />
Scheme 1<br />
338 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
Figure 1. Normalized absorption spectra in water<br />
(1) and normalized emission spectra in<br />
acetonitrile (2), water (3) and ethanol (4) of 8hydroxyacridizinium<br />
ion (1b).<br />
To assess whether the photoacidity and the solvatochromism is a general feature of hydroxysubstituted<br />
acridizinium derivatives, the photochemical and photophysical properties of the<br />
hydroxyacridizinium isomers 1a–e were examined in detail and will be presented and discussed in<br />
this contribution.<br />
References: [1] N. Agmon, J. Phys. Chem. A 2005, 109, 13. [2] L.M.Tolbert, K.M. Solntsev, Acc. Chem.<br />
Res. 2002, 35, 19. [3] H. Ihmels, K. Schäfer, Photochem. Photobiol. Sci. 2009, 8, 309.
P220 Fluorescent Proteins, Fluorescent Probes & Labels Poster 220<br />
Luminescent properties of green emitting cyclometalated<br />
platinum(II) complexes<br />
Michael Schäferling 1 , Lorenz Fischer 1 , Cüneyt Karakus 2 & Elisabeth Holder 2<br />
1<br />
Insitute of Analytical Chemistry, Chemo- and Biosensensors, University of Regensburg, 93040<br />
Regensburg (Germany)<br />
2<br />
Functional Polymers Group and Institute of Polymer Technology, University of Wuppertal, 42097<br />
Wuppertal (Germany)<br />
The photo- and electroluminescent properties of platinum(II) complexes indicate their possible<br />
applications in chemosensors, [1] light emitting devices, [2] or photovoltaic cells. [3] Different<br />
mononuclear square planar platinum(II) complexes have been investigated in recent years.<br />
Foremost, Pt(II) porphyrins are employed as probes <strong>for</strong> oxygen sensing if they are incorporated in<br />
an appropriate polymer matrix. [4]<br />
We present the synthesis as well as the photophysical and electrochemical properties of a<br />
series of novel green to blue-green emitting cyclometalated heteroleptic Pt(acac) complexes.<br />
R<br />
R<br />
N<br />
O<br />
Pt<br />
O<br />
R = alkyl, X = halogen<br />
The luminescence intensities and lifetimes of these compounds were also studied with respect to<br />
their responses to oxygen and temperature. These cyclometalated Pt(II) complexes show generally<br />
only a very low sensitivity to oxygen and temperature. Particularly, the rather insensitivity to oxygen<br />
quenching is unusual <strong>for</strong> phosphorescent metal-ligand complexes. Accordingly, these complexes<br />
are promising candidates to be used as inert reference dyes in optical sensors. However, a<br />
complex with X = Br was found which displays a strong temperature quenching effect. The<br />
response to temperature was calibrated after incorporation in poly(vinylidene chloride-coacrylonitrile).<br />
This green-emitting temperature sensor represents an interesting alternative to the<br />
available mostly red emitting temperature-sensitive probes.<br />
References: [1] K. M.-C. Wong, et al., Inorg. Chem. 44 (2005) 1492. [2] Y. Y. Lin, et al., Chem. Eur. J. 9<br />
(2003) 1263. [3] J. E. McGarrah, et al., Inorg. Chem. 40 (2001) 4510. [4] J. Kavandi, et al., Rev. Sci. Instrum.<br />
61 (1990) 3340.<br />
______________<br />
* Corresponding author: e-mail: michael.schaeferling@chemie.uni-regensburg.de<br />
X<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 339
P221 Fluorescent Proteins, Fluorescent Probes & Labels Poster 221<br />
Long-wavelength benzodipyrrolenine based bis-cyanine and bis-styryl<br />
dyes<br />
Olga Semenova*, Oleksii Klochko, Iryna Fedyunyayeva, Saniya Khabuseva & Leonid Patsenker<br />
<strong>State</strong> <strong>Scientific</strong> <strong>Institution</strong> "Institute <strong>for</strong> <strong>Single</strong> Crystals", National Academy of Sciences of Ukraine,<br />
60, Lenin Ave., 61001 Kharkiv (Ukraine)<br />
Red and near-infrared absorbing and emitting cyanine and styryl dyes are widely used as<br />
fluorescent probes <strong>for</strong> biological research, biomedical assays, and fluorescence imaging<br />
applications. We describe the molecular structure and spectral properties of bis-cyanine dyes 1a–<br />
1e and bis-styryl dyes 2a–2c containing a benzodipyrrolenine moiety as the central bridge and<br />
compare them to the "monomeric" cyanines 3a–3e and styryls 4a–4c.<br />
Het<br />
N<br />
N<br />
Het<br />
2TsO<br />
1<br />
Het =<br />
S<br />
N<br />
(c),<br />
N<br />
(a),<br />
N<br />
(d),<br />
O<br />
N<br />
N<br />
340 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
N<br />
(b),<br />
3<br />
(e)<br />
Het<br />
I<br />
Ar<br />
2I<br />
Ar =<br />
N<br />
2<br />
N<br />
NMe 2<br />
NMe 2<br />
(a),<br />
(c)<br />
Ar<br />
N<br />
Ph<br />
N N<br />
4<br />
Ar<br />
I<br />
(b),<br />
Ph<br />
Conjugation of two "monomeric" chromophores 3a–3e and 4a–4c to a bis-chromophoric system 1a–<br />
1e and 2a–2c was found to cause a substantial red-shift (~100 nm) of the absorption and emission<br />
maxima, and and increase of the extinction coefficients and quantum yields. The dyes 3 and 4<br />
absorb and emit in between 490–700 nm while 1 and 2 in the red and near-IR spectral region (643–<br />
812 nm), showing negative solvatochromic (<strong>for</strong> all dyes 1–4) and insignificant solvatofluorochromic<br />
effects <strong>for</strong> "monomeric" cyanines 3, styryls 4 and bis-styryls 2. Bis-cyanines 1a–1e exhibit a more<br />
pronounced blue-shift of the emission maxima when chloro<strong>for</strong>m is replaced with methanol.<br />
Quantum chemical simulations using PM3 and<br />
PPP CI methods show that compared to bis-cyanines<br />
the fusion of two parent styryl chromophores to a bisstyryl<br />
system causes a pronounced con<strong>for</strong>mational<br />
rigidization: the molecule containing two conjugated<br />
styryl chromophores becomes less flexible and more<br />
planar, which affects the spectral properties of the dyes.<br />
Compared to biscyanines, the vibrational structures of<br />
the absorption and emission bands in bis-styryl dyes are<br />
much better defined (Fig.). Bis-cyanines 1a–1e exhibit<br />
higher extinction coefficients (ε 133,700 — 251,000 M –<br />
1 cm –1 ) and quantum yields (ΦF 3.8 – 28% in chloro<strong>for</strong>m)<br />
600 700<br />
Wavelength [nm]<br />
800<br />
than bis-styryls 2a–2c (ε 80,000 — 133,000 M –1 cm –1 , ΦF<br />
2.4 – 10%. Because of the higher polarity of the excited<br />
state of bis-styryls compared to bis-cyanines, ΦF differences between these dyes are even more<br />
pronounced in polar solvents. Both bis-cyanines and bis-styryls have relatively large Stokes' shift (430–<br />
900 cm –1 in chloro<strong>for</strong>m and 470–1540 cm –1 in methanol), but these values are lower compared to the<br />
"monomeric" dyes due to the more rigid structure and lower polarity of bis-dyes.<br />
The fluorescence intensity of bis-dyes 1a–1e and 2a–2c in aqueous solutions increases many times in<br />
presence of protein. We also noticed the extremely low cytotoxicity and photocytotoxicity of these dyes<br />
in cells. These characteristics demonstrate their potential as biomedical probes <strong>for</strong> proteins, lipids and<br />
cell staining.<br />
The work was supported by the National Academy of Sciences of Ukraine, project No.0110U000488.<br />
______________<br />
* Corresponding author: e-mail: patsenker@isc.kharkov.com<br />
Normalized Emission<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0.0<br />
3a<br />
4a<br />
1a<br />
2a
P222 Fluorescent Proteins, Fluorescent Probes & Labels Poster 222<br />
2- and 4-[2-(4-dimethylaminophenyl)-1-ethenyl]-1-alkylpyridinium tosylates<br />
(DSP) as the fluorescent probes <strong>for</strong> cells<br />
Oksana O. Sokolyk 1 , Tatyana S. Dyubko 1,2 , Yevgenia I. Smolyaninova 2 & Leonid D. Patsenker 1<br />
1<br />
SSI "Institute <strong>for</strong> <strong>Single</strong> Crystals", NAS Ukraine, 60, Lenin Ave., 61001 Kharkiv (Ukraine)<br />
2<br />
Institute <strong>for</strong> Problems of Cryobiology and Cryomedicine, NAS Ukraine, 23 Pereyaslavskaya Str.,<br />
61015 Kharkiv (Ukraine)<br />
The structural changes in cell membranes were evaluated with 2- (DSP-n-2) and 4-[2-(4dimethylamino<br />
phenyl)-1-ethenyl]-1-alkylpyridinium tosylates (DSP-n-4) as the fluorescent probes<br />
using spectro- photometry, fluorescence spectroscopy and confocal laser scanning microscopy<br />
methods. These dyes that differ in the length of their alkyl chain on the pyridinium ring (n = 1–13)<br />
and the position of the dimethylamino group are available from SETA BioMedicals<br />
(www.setabiomedicals.com). Red blood cells (RBC), mouse ovum and model artificial membranes,<br />
liposomes were used as model systems in our investigation.<br />
N(CH3) 2<br />
2<br />
4<br />
(H3C) 2N<br />
N C<br />
N CnH2n+1 OTs<br />
nH2n+1 OTs<br />
DSP-n-4<br />
DSP-n-2<br />
These dyes exhibit absorption maxima between 465–480 nm, extinction coefficients between<br />
24,600–50,500 M –1 cm –1 , emission wavelength in water between 585–610 nm and quantum yields<br />
between 1.9–3.2%. Importantly the ΦF's substantially increase upon binding to proteins, liposomes<br />
and cells. The most pronounced increase was obtained <strong>for</strong> DSP-13-4: 37-fold, ΦF = 26% in<br />
presence of HSA and 45-fold, ΦF = 38% with liposomes. Lengthening of the alkyl substituent,<br />
which resulted in an increase of the dyes hydrophobicity, was found to facilitate permeation of the<br />
dye molecules through cell membranes and thereby causing an increase of the cell brightness.<br />
Increased permeation through cell membranes and increased brightness was also observed upon<br />
shifting the dimethylamino group from position 2 (DSP-n-2) to position 4 (DSP-n-4). DSP-13-4 was<br />
found to yield the brightest images. The bright and high contrast fluorescence images that we<br />
obtained with these dyes can be used to determine the cell membrane heterogeneity. The brightest<br />
areas seem to correspond to the RBC "concaves" interacting with each other when RBC's move<br />
through capillaries. Furthermore, we used DSP probes to identify the structural changes in RBC's<br />
membranes caused by freezing-thawing. A pronounced increase in fluorescence of cells stained<br />
with DSP-13-4 was noted after freezing cells in presence of 5% DMSO (cryoprotector) at –196 ºС<br />
(Fig. a, b). The fluorescence increase was due to the increased permeability of cell membranes<br />
after freeze-thawing. Mouse ovum stained with a mixture of probes Square-670 (red fluorescent<br />
zona pellucida) and DSP-6-4 (green fluorescent cytoplasm) is shown on Fig. c. We can conclude<br />
that the DSP series of dyes, in particular DSP-13-4 are promising fluorescent probes to investigate<br />
cells using fluorescence spectroscopy and fluorescence microscopy.<br />
a b c<br />
Fig. RBC's stained with DSP-13-4 be<strong>for</strong>e (a) and after (b) freeze-thawing to –196 ºС in presence of DMSO<br />
and (c) mouse ovum stained with a mixture of probes Square-670 red fluorescent zona pellucida) and DSP-<br />
6-4 (green fluorescent cytoplasm).<br />
This study was supported by the National Academy of Sciences of Ukraine, project No.<br />
0110U000488.<br />
______________<br />
* Corresponding author: e-mail: ksenaksena@mail.ru<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 341
P223 Fluorescent Proteins, Fluorescent Probes & Labels Poster 223<br />
Charge separation and charge recombination excited states in<br />
pentacoordinated complex of zinc tetraphenylporphyrin and axially<br />
bounded isoquinoline N-oxide ligand<br />
K. Oberda 1 , I. Deperasińska 2 , Y. Nizhnik 3 , L. Jerzykiewicz 1 & A. Szemik-Hojniak 1*<br />
1 Faculty of Chemistry, University of Wroclaw, Joliot-Curie 14 st; 50-383 Wrocław, Poland<br />
2 Institute of Physics, Pol. Acad.of Sciences, Al.Lotników 32/46; 02-668 Warsaw, Poland<br />
3 Department of Molecular Biology, Biological and Organic Chemistry, Petrozavodsk <strong>State</strong><br />
University, pr. Lenina 33, 185910, Republic Karelia, Russia<br />
Metalloporphyrins, find many applications in different areas of science and technology. Their<br />
combination with Lewis bases, such as <strong>for</strong> example biologically activite heteroaromatic N-oxide<br />
ligands [1-2] results in a new type of hybrid materials with unique photophysical and electronic<br />
properties. The relaxation dynamics and assignment of excited states of these systems became a<br />
“hot” subject of recent photophysical studies devoted to these macrocycles [3]. Herein, we report<br />
photophysical properties of excited states of zinc tetraphenylporphyrin (ZnTPP) and isoquinoline Noxide<br />
(IQNO) complex (ZnTPP-IQNO) investigated by a combined solution-state study (steady<br />
state absorption, emission, time –resolved emission) as well as quantum-mechanical ab-initio and<br />
DFT calculations. A single-crystal X-ray studies show that the ZnTPP-IQNO complex is<br />
characteristic of a centrosymmetric triclinic unit cell, space group P-1 and that two molecules of<br />
adduct <strong>for</strong>m a supramolecular dimmer (Figure 1).<br />
342 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
Fig.1.Dimeric structure of solid ZnTPP-IQNO complex<br />
The DFT and TD DFT calculations revealed that different types of electronic transitions (LE ZnTPP,<br />
LEIQNO and CTcomplex) occur in this system and on excitation the photoinduced intramolecular<br />
electron transfer (PET) takes place both from the S2 (Soret band) and from the S1 state (Q band).<br />
The charge is shifted from the ZnTPP unit, as electron donor to the isoquinoline N-oxide ligand, as<br />
electron acceptor. An accurate analysis of the energy dissipation channels in the target system<br />
allowed us to distinguish the whole set of the CT states as those with the charge separation and<br />
the charge recombination character.<br />
References : [1] M.P.Bryn. et al J.Am.Chem.Soc. 115 (1993) 9480. [2] C.L.Hill, M.M.,Wiliamson,<br />
Inorg.Chem.32 (1985), 32 3024. (3) Y.Nizhnik et al.,. Cryst.Eng.Comm. 2009, 11, 2400.<br />
______________<br />
*Corresponding author: e-mail: anias@wchuwr.pl
P224 Fluorescent Proteins, Fluorescent Probes & Labels Poster 224<br />
The charge transfer nature of the emissive state of 3-aminoflavone<br />
Łukasz Wiśniewski 1 , Irena Deperasińska 2 , Bogusława Żurowska 1 & Anna Szemik-Hojniak 1*<br />
1 Faculty of Chemistry, University of Wroclaw, Joliot-Curie 14 st; 50-383 Wroclaw (Poland)<br />
2 Institute of Physics, Pol. Acad. of Sciences, Al.Lotników 32/46, 02-668 Warsaw (Poland)<br />
The flavones, are the sub group of flavonoids - known naturally occurring biologically active<br />
compounds, demonstrating, the antioxidant [1], cytostatic, antibacterial, anticancer [2] and other<br />
important pharmacological properties. In the excited state they show a complex photophysical<br />
properties including excited state intramolecular proton (ESIPT) or electron transfer processes.<br />
The <strong>for</strong>mer was found in many cases of natural and synthetic flavones, including 7- , 5- or-3hydroxyflavones<br />
[3]. In either of them the role of the solvent polarity is highlighted. In studied here<br />
3-aminoflavone (3-AF, Figure 1), the N-H…O hydrogen bonding geometry is unfavorable <strong>for</strong> the<br />
ESIPT to occur.<br />
energy [cm -1 ]<br />
30000<br />
25000<br />
20000<br />
15000<br />
10000<br />
5000<br />
0<br />
27231 cm -1<br />
FC<br />
S1 (4.58 D)<br />
μ(S 1)<br />
S0(3.04 D)<br />
μ(S 0)<br />
2211 cm -1<br />
S1(4.74 D)<br />
FC<br />
S0 (2.72 D)<br />
1933 cm -1<br />
Fig.1. Diagram of the energetics of 3-amino flavone in the gas phase.<br />
The DFT and TD DFT calculations show that the S1 Franck-Condon (S1 FC ) state has the CT<br />
character and in the S0 → S1 FC transition the transfer of charge occurs between the 62 π HOMO<br />
orbital of the amino group and 63 π LUMO orbital of the chromen ring. The CT nature of the<br />
emissive state of 3-AF and its dipolar changes (shown in diagram of Figure 1) result in the red<br />
shifted emission maxima observed with the solvent polarity increase (Stokes shift value in THF is<br />
6489 cm -1 ).<br />
Interesting experimental observation that the lifetimes are invariable with solvent polarity<br />
increase has indicated the constancy of the overall deactivation rate constant (k) [k = kf + ki] of 3-<br />
AF system. The TD DFT calculations with included PCM model of the solvent helped us to reveal<br />
the effectiveness of the nonradiative intersystem crossing process in strongly polar media as<br />
responsible <strong>for</strong> a constant (k) value.<br />
This work was supported by the following grants: Min.of Sci. and High Educ.(Poland) Nr N N204<br />
131338; ICMCM-Warsaw (G32-10) and WCSS-Wroclaw (G-61).<br />
References: [1] B.Hallivel. et al., J.Am.J.Clin.Nutr. 81 (2005) 268S., [2] B.Kośmider, R., Osiecka,<br />
Drug.Dev.Res. 63 (2004) 200; [3] P.K. Sengupta, M. Kasha, Chem.Phys.Lett. 68 (1979) 382.<br />
______________<br />
*Corresponding author: e-mail : anias@wchuwr.pl<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 343<br />
23087 cm -1
P225 Fluorescent Proteins, Fluorescent Probes & Labels Poster 225<br />
Controllable C=N isomerization <strong>for</strong> rational design of fluorescent sensors<br />
Jiasheng Wu, Ruilong Sheng, Weimin Liu & Pengfei Wang*<br />
Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of<br />
Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, P. R. China<br />
Fluorescent sensors are receiving considerable attention because of their high selectivity,<br />
sensitivity, and simplicity to guest species, together with their practical applications in biological<br />
fluorescence imaging, molecular catalysis, environmental analysis and detection, etc. 1 Exploration<br />
of new probing mechanisms between the recognition and signal units is of continuing interest <strong>for</strong><br />
design of new fluorescent sensors. 2 Conventional photophysical mechanisms, such as photoinduced<br />
electron transfer (PET), intramolecular charge transfer (ICT), metal-ligand charge transfer<br />
(MLCT), excited-state intramolecular proton transfer (ESIPT), fluorescence resonance energy<br />
transfer (FRET), and excimer/exciplex <strong>for</strong>mation, have been widely developed and used <strong>for</strong> design<br />
of new sensors. 3 Different from these conventional mechanisms, we have ever developed C=N<br />
isomerization as a new sensing mechanism based on an imine-derived sensor. 4 In this design,<br />
C=N isomerization is the predominant decay process in the excited states of imines with unbridged<br />
C=N structures so these imines are often non-fluorescent. In contrast, the fluorescence of their<br />
analogs containing covalently bridged C=N structures increases dramatically due to the<br />
suppression of C=N isomerization in the excited states. This can be also known from the fact that<br />
compounds with acyclic double bonds are usually non-fluorescent, while their analogues with cyclic<br />
double bonds are significantly fluorescent.<br />
In the course of our continuing exploration of the C=N isomerization mechanism, we have<br />
developed a coumarin-derived imine (ICZn) as a new fluorescence turn-on sensor <strong>for</strong> Zn 2+ ions<br />
based on restricting the rotation of C=N bonds via complexation with metal ions (Figure 1). ICZn<br />
was designed as a Zn 2+ -selective fluorescent sensor based on the consideration as follows, i) the<br />
methyl group as a minor control unit was incorporated into ICZn to tune its sensing selectivity <strong>for</strong><br />
Zn 2+ ; ii) a methyl-free analog M was prepared to study the selectivity <strong>for</strong> comparison; iii) the<br />
carbonyl in coumarin and the pyridyl hydrazine could <strong>for</strong>m a pseudocavity to increase its affinity to<br />
Zn 2+ , which will result in an obvious enhancement in fluorescence. As expected, ICZn and M give<br />
rise to significant fluorescence enhancements to Zn 2+ in aqueous solution due to the suppression<br />
of C=N isomerization. Moreover, ICZn displays much higher selectivity to Zn 2+ compared with M,<br />
indicating that the introduction of methyl to imine does play an important role to tune its sensing<br />
selectivity <strong>for</strong> C=N isomerization-based sensors. We believe that the controllable C=N<br />
isomerization as a general principle can be extended to other probing systems to improve the<br />
sensing selectivity, especially <strong>for</strong> specific recognition of Zn 2+ from Cd 2+ in the aqueous solution.<br />
Isomerization Isomerization inhibited<br />
Minor control unit to tune selectivity<br />
R<br />
N O O<br />
N<br />
H<br />
N<br />
ICZn: R=CH 3<br />
M: R=H<br />
N<br />
ZnCl 2<br />
Aqueous Solution<br />
N O O<br />
Non-fluorescencet Strongly fluorescent<br />
This work was supported by the youth foundation from Technical Institute of Physics and<br />
Chemistry, Chinese Academy of Sciences.<br />
References: [1] A. T. Wright, et al., Chem. Soc. Rev., 35 (2006) 14. [2] J. S. Wu, et al., Chem. Soc. Rev., 40<br />
(2011), 3483. [3] R. Martinez-Manez, Chem. Rev., 103 (2003) 4419. [4] J. S. Wu, et al., Org. Lett., 9 (2007)<br />
33.<br />
______________<br />
* Corresponding author: e-mail: wangpf@mail.ipc.ac.cn<br />
344 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
R<br />
Cl<br />
N<br />
Zn<br />
H<br />
N<br />
Cl<br />
N
P226 Fluorescent Proteins, Fluorescent Probes & Labels Poster 226<br />
New samarium (III) chelates as an efficient donors <strong>for</strong> fluorescence<br />
resonance energy transfer<br />
А. Yegorova 1* , D. Aleksandrova 1 , I. Leonenko 1 , D. Patsenker 2 & A. Tatarets 2<br />
1<br />
A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine,<br />
Odessa (Ukraine)<br />
2<br />
<strong>State</strong> <strong>Scientific</strong> <strong>Institution</strong> "Institute <strong>for</strong> <strong>Single</strong> Crystals", National Academy of Sciences of Ukraine,<br />
Kharkov (Ukraine)<br />
Förster resonance energy transfer (FRET) is a powerful tool in studying biomolecular interactions.<br />
Various ligands have been used to enhance the lanthanide fluorescence. Lanthanide chelates<br />
such as Eu or Tb complexes, used as donors offer several advantages over classical fluorescence<br />
probes in resonance energy transfer: large Stokes shifts and narrow emission lines arising from 4f–<br />
4f transitions. The lanthanide chelates together with organic dyes enable a straight<strong>for</strong>ward and<br />
sensitive assay technology in FRET applications.<br />
In this investigation, two energy transfer pairs, samarium(III) chelates (Sm-L1, Sm-L2) with<br />
thio-squaraine dye (K8-1642, Seta-632-mono-NHS, SETA BioMedicals) were studied. A strong<br />
emission of Sm(III) complexes with new ligands (1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3carboxylic<br />
acid-(4-methyl-pyridin-2-yl)-amide (L1) and 6-[(1-hydroxy-3-oxo-6,7-dihydro-3H,5Hpyrido[3,2,1-ij]quinoline-2-carbonyl)-amino]-hexanoic<br />
acid (L2)) could be seen as narrow bands in<br />
the wavelengths interval from 550 to 750 nm. Herein, we can see that the spectra overlap between<br />
the emission of Sm-L1 (L2) chelates and absorption of K8-1642 meets the prerequisite <strong>for</strong><br />
fluorescence energy transfer. Based on this, a novel efficient fluorescence energy transfer systems<br />
between Sm-L1 (L2) chelates as donors and K8-1642 as acceptor were developed. The donor<br />
emission (605 nm) intensity was decreased and the acceptor emission (654 nm) intensity was<br />
increased by the addition of K8-1642 (Figure).<br />
The key factor is the equimolar concentration of Sm 3+ and the ligand (0.1 mM). Ilum is<br />
maximum at pH 7.5 (HMTA-HCl buffer). The excitation and emission maxima of the Sm-L1 (L2)<br />
complexes are at 317 nm (300 nm) and 605 nm, respectively. Maximum Ilum was observed in water<br />
solution of these complexes. Spectral overlapping between the emission of the donor and the<br />
excitation of the acceptor was studied (Figure).<br />
Luminescence intensity, a.u..<br />
300<br />
250<br />
200<br />
150<br />
100<br />
1<br />
2<br />
1000<br />
800<br />
600<br />
400<br />
200<br />
1<br />
2<br />
0<br />
500 550 600 650 700 750 800<br />
Wavelength, nm<br />
50<br />
0<br />
550 600<br />
3<br />
650 700 750 800<br />
Wavelength, nm<br />
Luminescence intensity, a.u. .<br />
3<br />
The Forster radiuses and overlap integrals were calculated <strong>for</strong> both of the donors (Sm(III) chelates)<br />
with acceptor (K8-1642). The samarium chelates together with thio-squaraine dye enable a<br />
straight<strong>for</strong>ward and sensitive assay technology in FRET applications.<br />
______________<br />
* Corresponding author: e-mail: yegorova@interchem.com.ua<br />
Figure: The emission spectra of Sm-L1<br />
(1); Sm-L1-K8-1642 (2); К8-1642 (3). The<br />
inset corresponds to the excitation and<br />
emission spectra of the dye K8-1642<br />
(1,2), emission spectrum of samarium(III)<br />
chelate (3)<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 345
P227 Fluorescent Proteins, Fluorescent Probes & Labels Poster 227<br />
Two-color genetically targetable and switchable fluorescent probes<br />
Dmytro Yushchenko 1,2,* , Sujung Kim 1 , Ming Zhang 3 , Qi Yan 3 , Brigitte Schmidt 1 , Christopher<br />
Szent-Gyorgyi 1 , Alan Waggoner 1,2 & Marcel Bruchez 1,2,3<br />
1 Molecular Biosensor and Imaging Center, 2 Department of Chemistry, 3 Department of Biology,<br />
Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA)<br />
Genetically expressed fluorescent proteins (FPs) revolutionized the field of cell biology, especially<br />
live-cell studies. The limitations of FPs however are related to the photophysical properties of their<br />
fluorophores, mainly brightness and photostability, which are still are inferior to organic<br />
fluorophores. Biological tags technology is a good alternative to FPs since it allows application of<br />
synthetic dyes <strong>for</strong> fluorescence cell imaging. Fluorogen activating proteins (FAPs) are the tags that<br />
were recently developed in our group and may be easily fused to the protein of interest [1] . They<br />
bind specific nonrigid fluorophores (fluorogens) which have very low level of fluorescence in the<br />
absence of FAP; this binding leads to the activation of the fluorophore fluorescence by hundredsto-thousands<br />
fold. Because of a high brightness, specificity and an extremely low background<br />
FAPs were shown to be very useful in fluorescence microscopy of live cells including superresolution<br />
STED imaging [2,3] .<br />
In this work we present a methodology <strong>for</strong> development of two-color genetically targetable<br />
fluorescent dyes [4] . These are FRET based tandem dyes with Malachite Green (MG) as an<br />
acceptor which switch their fluorescence from one color to another upon binding to FAP. We show<br />
that tandem dyes based on cationic Tetramethylrhodamine (TMR) as a donor, namely MG-TMRi2,<br />
are suitable <strong>for</strong> intracellular labeling. They penetrate through the plasma membrane and are able<br />
to label different structures inside the live cells. In cells that do not express FAP MG-TMRi2<br />
predominantly stains mitochondria. In cells that do express FAP this dye labels structures<br />
expressing FAP in one color and mitochondria in other color. We also show that MG-TMRi2 may<br />
be used <strong>for</strong> ratiometric monitoring of mitochondrial membrane potential changes. Meanwhile MGbased<br />
tandem dyes with anionic Alexa fluorophores as the donors are shown to be suitable dyes<br />
<strong>for</strong> selective extracellular protein labeling and to be useful <strong>for</strong> 2-photon imaging of live cells.<br />
This work was supported by grants from the US National Institutes of Health (7U54RR022241 and<br />
1R01GM086237).<br />
References: [1] C. Szent-Gyorgyi, et al., Nat Biotechnol., 26 (2008) 235. [2] J.A. J. Fitzpatrick, et al.,<br />
Bioconjugate Chem., 20 (2009) 1843. [3] C. Szent-Gyorgyi, et al., J Am Chem Soc.,132 (2010) 11103. [4]<br />
D.A. Yushchenko, et al., manuscript in preparation.<br />
______________<br />
* Corresponding author: E-mail: dmytroyu@andrew.cmu.edu<br />
346 — MAF 12, Strasbourg, France, September 11-14, 2011 —
POSTER MAIN AUTHORS INDEX<br />
Red: Poster session 1 (Monday)<br />
Blue: Poster session 2 (Tuesday)<br />
Green: Poster session 3 (Wednesday)<br />
A<br />
ACHELLE Sylvain ______________ P174<br />
ACUÑA Ulises _________________ P122<br />
AGUILAR-CABALLOS Maria de la Paz P70<br />
AIGNER Daniel ________________ P175<br />
AIT LYAZIDI Saadia ____________ P176<br />
AJAJ Yathrib __________________ P21<br />
ALEKSANDROVA Daria _________ P177<br />
ALVEROGLU DURUCU Esra _____ P64<br />
ARAKI Tsutomu _______________ P37<br />
ARAMINAITÉ Rüta _____________ P104<br />
AUERBACH Dagmar ___________ P170<br />
B<br />
BALOGH Andrea ______________ P155<br />
BALTES Nicole ________________ P178<br />
BARACHEVSKIY Valery _________ P179<br />
BARANOWSKI Matthias _________ P123<br />
BARNEKOW Peter _____________ P71<br />
BENZ Christian ________________ P38<br />
BERBERAN-SANTOS Mario _____ P72<br />
BHATTACHARYA Bhaswati ______ P124<br />
BIRCH David __________________ P125<br />
BONARSKA-KUJAWA Dorota ____ P156<br />
BONACCHI Sara ______________ P180<br />
BORISOV Sergey ______________ P181<br />
BORISOVER Mikhail ___________ P112<br />
BOUDIER Christian ____________ P126<br />
BUJAK Lukasz ________________ P73<br />
C<br />
CALVET Amandine _____________ P127<br />
CANESI Eleonora V. ____________ P74<br />
CASTANHEIRA Elisabete M.S. ___ P157<br />
CHANG Young-Tae ____________ P1<br />
CHEN Jian-Hua _______________ P15<br />
CHUNG Pei-Hua _______________ P182<br />
CISCATO Luis Francisco ________ P113<br />
CISZAK Kamil _________________ P75<br />
CLAVIER Gilles ________________ P76<br />
COULTER Jonathan ____________ P128<br />
COUTINHO Paulo J.G. __________ P105<br />
CRISTEA Castelia _____________ P65<br />
CZECHOWSKI Nikodem ________ P77<br />
(Posters numbering)<br />
D<br />
DARWICH Zeinab _____________ P183<br />
DAS Dibyendu K. ______________ P22<br />
DAVIES Amy _________________ P78<br />
DE Dipanwita _________________ P117<br />
DELIGEORGIEV Todor ________ P79, P186<br />
DELON Antoine _______________ P16<br />
DEMCHENKO Alexander ________ P80<br />
DESCALZO Ana B. ____________ P81<br />
DEVAUGES Viviane ____________ P39<br />
DJIKANOVIC Daniela ___________ P82<br />
DOLEŽALOVÁ Iva _____________ P129<br />
DON PAUL Craig ______________ P171<br />
DOWD Peter Dylan ____________ P130<br />
DUERKOPAxel ________________ P184<br />
DVORAK Miroslav _____________ P185<br />
DYUBKO Tatyana _____________ P187<br />
DZIUBA Dmytro _______________ P188<br />
E<br />
EGLOFF Coraline ______________ P189<br />
EL HUSSEINY Amel ___________ P190<br />
ERARD Marie _________________ P191<br />
EREZ Yuval __________________ P192<br />
EROSTYAK Janos _____________ P2<br />
F<br />
FAVARD Cyril _________________ P17<br />
FELLER Karl-Heinz ____________ P11<br />
FEREIDOUNI Farzad ___________ P3, P4<br />
FERY-FORGUES Suzanne ______ P83<br />
FIDY Judit ____________________ P131<br />
FINKLER Björn ________________ P23<br />
FISER Radovan _______________ P132<br />
FOURATI Amine ______________ P67<br />
FREDJ Asma _________________ P172<br />
FRIAA Ouided ________________ P24<br />
G<br />
GAL Emese __________________ P133<br />
GEBA Maria __________________ P111<br />
GENOVESE Damiano __________ P84<br />
GHIGGINO Ken _______________ P68<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 347
GIAMARCHI Philippe ___________ P158<br />
GIORDANO Luciana ____________ P193<br />
GODET Julien _________________ P25<br />
GODOY-NAVAJAS Juan ________ P134<br />
GONÇALVES Leticia Christina ____ P135<br />
GORRIS Hans-Heiner ___________ P85<br />
GRAMATICA Andrea ___________ P40<br />
GREINER Vanille ______________ P159<br />
GRUSSMAYER Kristin __________ P41<br />
GUCHHAIT Biswajit ____________ P116<br />
GURSKIY Stanislaw ____________ P194<br />
GURYEV Oleg ________________ P173<br />
H<br />
HALASOVÁ Tereza ____________ P118<br />
HARZ Stephanie _______________ P66<br />
HIDAKA Chiharu _______________ P86<br />
HOFFMANN Katrin _____________ P103<br />
HOLMES-SMITH Sheila _________ P87<br />
HUNGERFORD Graham ________ P88<br />
HUYNH Ahn-Minh ______________ P23<br />
I<br />
ILIEN Brigitte __________________ P42<br />
IVANOV Denis ________________ P114<br />
IVANOVA Irena ________________ P195<br />
IZSEPI Emese ________________ P43<br />
J<br />
JOCKUSCH Rebecca ___________ P5<br />
JOHNSON Carey ______________ P26<br />
K<br />
KANG Nam Joo _______________ P135B<br />
KARABANOVAS Vitalijus ________ P106<br />
KEMPF Noemie _______________ P44<br />
KETOLA Tiia-Maaria ____________ P153<br />
von KETTELER Alexa ___________ P136<br />
KIM Eunha ___________________ P196<br />
KIRILOVA Elena ____________ P197, P198<br />
KIRSHNER Hagai ______________ P45, P46<br />
KISSANE Bridget ______________ P137<br />
KITAJIMA Ittetsu _______________ P89<br />
KLOCHKO Yuliia _______________ P200<br />
KLOCHKO Oleksii ______________ P199<br />
KOREN Klaus _________________ P201<br />
KRAL Teresa _________________ P160<br />
KRAJNIK Bartosz ______________ P90<br />
KRAYUSHKIN Mikhail __________ P202<br />
KRISHNAMOORTHY Guruswamy _ P138<br />
KUCHERAK Oleksandr __________ P203<br />
348 — MAF 12, Strasbourg, France, September 11-14, 2011 —<br />
L<br />
LAINE Romain ________________ P6<br />
LANG Thomas ________________ P204<br />
LEARMONTH Robert ___________ P161<br />
LEE Ji-Eun ___________________ P27<br />
LEE Sanghee _________________ P205<br />
LENHARDT Lea _______________ P139<br />
LERICHE Geoffray _____________ P206<br />
LI Boyan _____________________ P140<br />
LI Quiang ____________________ P7<br />
LIGABUE Alessio ______________ P141<br />
LISITSYNA Ekaterina ___________ P91<br />
LISKOVA Petra _______________ P142<br />
LYMPEROPOULOS Konstantinos _ P28<br />
M<br />
MACKAY Martha ______________ P207<br />
MACKENZIE Alexandra _________ P92<br />
MACMILLAN Alexander _________ P47<br />
MAHFOUDHI Selim ____________ P162<br />
MAILLOT Sacha _______________ P12<br />
MALVAL Jean-Pierre ___________ P93, P94<br />
MARQUER Catherine __________ P48<br />
MARZULLO Vincenzo M. ________ P143<br />
MASCALCHI Patrice ___________ P163<br />
MEALLET-RENAULT Rachel _____ P95<br />
MIKHALYOV Ilya ______________ P164<br />
MIYATA Tsuyoshi ______________ P8<br />
MIZUNO Takahiko _____________ P9<br />
MOJZISOVA Halina ____________ P49<br />
De MOURA Ana Paula __________ P96<br />
MRAVEC Filip ________________ P119<br />
MURATA Atsushi ______________ P208<br />
N<br />
NADIARNYKH Oleg ____________ P50<br />
NAGL Stefan _________________ P13<br />
NAKASHIMA Kenichi ___________ P120<br />
NORLIN Nils __________________ P51<br />
P<br />
PAL Robert ___________________ P144<br />
PANKAJAKSHAN Praveen ______ P52<br />
PAPKOVSKY Dmitri ____________ P209<br />
PARDO LOPEZ Liliana__________ P165<br />
PATSENKER Leonid _________ P210, P211<br />
PETER Sébastien _____________ P29<br />
PEYRARD Lisa _______________ P212<br />
PIATKOWSKI Tanja ____________ P63<br />
PIVOVARENKO Vasyl __________ P166<br />
POLLINGER Klaus _____________ P107<br />
PORONIK Ievgen ______________ P213<br />
POSTUPALENKO Viktoriia ______ P214<br />
PRIGGE Matthias ______________ P215
R<br />
RADU Beatrice M. ______________ P53<br />
RAJU Bachu Rama _____________ P216<br />
REI Ana ______________________ P145<br />
RESCH-GENGER Ute ______ P217, P218<br />
REZACOVA Barbora ___________ P146<br />
de ROCQUIGNY Hugues ________ P54<br />
RODRIGUES Ana Clara Beltran ___ P147<br />
ROSA Ieda L.V. _______________ P97<br />
ROUBALOVA Lenka ____________ P167<br />
ROWLEY Laura _______________ P55<br />
RYBINA Arina _________________ P30<br />
S<br />
SCHÄFER Katy ________________ P219<br />
SCHÄFERLING Michael _________ P220<br />
SCHEUL Teodora ______________ P56<br />
SCHLEIFENBAUM Frank ________ P57<br />
SCHMITT Yvonne ______________ P148<br />
SCHWARZER Roland __________ P58<br />
SEEFELD Anne _______________ P31<br />
SEMENOVA Olga ______________ P221<br />
SHANEHSAZ Maryam __________ P108<br />
SHVADCHAK Volodymyr ________ P168<br />
SIERRA-RODERO Marina _______ P14<br />
SKRIPKA Artiom _______________ P109<br />
SLIWA Michel _________________ P98<br />
SMITH Darren _________________ P59<br />
SOBOLEV Yaroslav ____________ P32<br />
SOKOLOWSKI Adam ___________ P33<br />
SOKOLYK Oksana _____________ P222<br />
SOLTYSINSKI Tomasz __________ P34<br />
SPIES Christian _______________ P20<br />
SPIELMANN Thiemo ___________ P60<br />
STOJCIC Bojana ______________ P149<br />
SUHLING Klaus _______________ P61<br />
SUZUKI Akihiro ________________ P99<br />
SZEMIK-HOJNIAK A. ___________ P223<br />
T<br />
TANNERT Sebastian ___________ P10<br />
TOKAR Valentyna _____________ P100<br />
TURRIANI Elisa _______________ P150<br />
V<br />
VALANCIUNAITE Jurga _________ P110<br />
VASILESCU Marilena __________ P115<br />
VECER Jaroslav _______________ P168B<br />
VENIUS Jonas ________________ P151<br />
VERMA Pramod Kumar _________ P121<br />
VODNIK Vesna _______________ P101<br />
VUORIMAA-LAUKKANEN Elina __ P154<br />
W<br />
WEBER Georges ______________ P169<br />
WIRTZ Marcel ________________ P178<br />
WISNIEWSKI Lukasz ___________ P224<br />
WU Jiasheng _________________ P225<br />
Y<br />
YAMAUCHI Hiroaki ____________ P19<br />
YEGOROVA Alla ______________ P226<br />
YILDIZ Alptekin _______________ P69<br />
YOO Hyejin __________________ P35<br />
YOSHIKAWA Kenichi ___________ P62<br />
YOSHIKAWA Yuko ____________ P36<br />
YUSHCHENKO Dmytro _________ P227<br />
Z<br />
ZHANG Yinan _________________ P102<br />
ZIOMKOWSKA Joanna _________ P152<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 349
350 — MAF 12, Strasbourg, France, September 11-14, 2011 —
12th Conference on Methods and Applications of Fluorescence<br />
Spectroscopy, Imaging and Probes<br />
Conference program and Book of abstracts<br />
Volume editors:<br />
Yves Mély<br />
Guy Duportail<br />
Strasbourg, August 2011<br />
Printed by:<br />
University of Strasbourg<br />
Imprimerie et Reprographie<br />
29 rue du Maréchal Juin<br />
F-67084 Strasbourg Cedex<br />
Copyright © 2011 by the 12th Conference on Methods and Applications of Fluorescence (MAF 12)<br />
Website:<br />
http://maf12.unistra.fr<br />
more about MAF history: http://www.maf-sip.com<br />
Contact address:<br />
Yves Mély<br />
Université de Strasbourg<br />
Faculté de Pharmacie<br />
UMR 7213<br />
Laboratoire de biophotonique et pharmacologie<br />
74 route du Rhin<br />
BP 60024<br />
F-67401 ILLKIRCH CEDEX<br />
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umr7213-secr@unistra.fr<br />
Programming of the general style and the composition of this book is by Ingrid Barthel,<br />
Marlyse Wernert and Guy Duportail, UMR 7213.<br />
Pictures: © Marlyse Wernert<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 351
Notes<br />
352 — MAF 12, Strasbourg, France, September 11-14, 2011 —
Notes<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 353
Notes<br />
354 — MAF 12, Strasbourg, France, September 11-14, 2011 —
Notes<br />
— MAF 12, Strasbourg, France, September 11-14, 2011 — 355
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