Book of abstracts - State Scientific Institution “Institute for Single ...

10 th Conference on 

Methods and Applications of Fluorescence 

Conference Programme 

and 

Book of Abstracts 

organized by: 

Institute for Analytical Chemistry, Chemo- and Biosensors 

University of Regensburg, Germany 

http://www-analytik.chemie.uni-regensburg.de/ 

Contact address: 

Otto Wolfbeis 

University of Regensburg 

Institute of Analytical Chemistry, Chemo- and Biosensors 

93040 Regensburg, Germany 

1

Volume Editors: 

Otto Wolfbeis 

Rudi Hutterer 

Matthias Stich 

Martin Link 

Regensburg, August 2007 

Printed by Digital Print Group O. Schimek GmbH 

Ludwig-Thoma-Str. 27, 93051 Regensburg 

© by the 10 th Conference on Methods and Applications of Fluorescence (MAF 2007) 

Conference Homepage: http://10.maf-sip.com 

more about MAF history: http://www.maf-sip.com 

2

Welcome to the Attendants of MAF 10 … 

It is a pleasure for me to welcome you at the 10 th Conference on Methods & Application of 

Fluorescence with its focus on spectroscopy, imaging and probes. The MAF series has become the 

largest conference in the area of fluorescence. It is highly interdisciplinary and covers areas that 

range from physics and chemistry to biology, medicine, pharmacy, and others. MAF conferences 

are typically attended by 350 people coming from all over the globe. 

The conference of the year 2007 is the 10 th in this series. It is fair to say that in the 20 years of its 

existence this conference has created numerous contacts and cooperation that have led to joint 

research, papers, proposals and projects, and it is hoped that it will stay like this. This year's 

conference is particularly voluminous by incorporating 45 lectures and more than 220 posters. It is 

with certain proud when presenting to you the abstracts of these lectures that represent a good 

fraction of the research that is going on in spectroscopy, imaging and probes. 

It is also fair to say that MAF conferences have a very specific flavor in that not only the scientific 

level is a very high one, but also that a number of social activities are accompanying the attendants 

for the duration of their stay. 

I would like to express my sincere thanks to all of those that have contributed to the organization 

of MAF including the Scientific Board (for making excellent suggestions for speakers), the 

speakers that have agreed to give lectures (it always takes more time than anticipated), the many 

authors of poster contributions (thank you for all the beautiful artwork and excellent science), and 

the Local Organizing Committee in Regensburg (it is both stress and fun!). 

Last, but certainly not least, my thanks go to our sponsors, supporters and exhibitors whose names 

are given on the following page. Without their support, the organization of an event like MAF 10 

would be impossible. 

I truly hope that this conference will be as rewarding as were the previous ones, and that it will 

become a pleasant experience for both attendants and accompanying persons. 

Regensburg, September 2007 

3

… and thanks to our Sponsors 

4

Scientific Programme Committee 

A. Ulises Acuña, CSIC, Madrid, Spain 

David J. S. Birch, Strathclyde University, Glasgow, Scotland, UK 

John Birmingham, Unilever Research, Merseyside, UK. 

Jean-Claude Brochon, Ecole Normale Superieure; Cachan, France 

A. P. Demchenko, MAM-TUBITAK, Gebze-Kocaeli; Turkey 

A. P. De Silva, Queen's University, Belfast; Northern Ireland, UK 

Jorg Enderlein, University of Tuebingen, Tuebingen, Germany 

Hans C. Gerritsen, Utrecht University, Utrecht, The Netherlands 

Enrico Gratton, University of Illinois, Urbana, USA 

Martin Hof, Acad. of Sciences; Prague; Czech Rep. 

Johan Hofkens, Kath. Universiteit Leuven; Heverlee, Belgium 

Totaro Imasaka, Kyushu University; Fukuoka, Japan 

David M. Jameson, University of Hawaii, Honolulu, USA 

Yun-Bao Jiang, Xiamen University, Xiamen, P. R. China 

Lennart Johansson, Umea University, Umea; Sweden 

Paavo K. J. Kinnunen, University of Helsinki, Helsinki; Finland 

Helge Lemmetyinen, University of Technology; Tampere, Finland 

Yves Mely, Université Louis Pasteur, Illkirch, France 

Janos Matko, Eötvös-Lorand University, Budapest; Hungary 

James N. Miller, Univ. of Technology, Loughborough; UK 

Ute Resch-Genger, Federal Institute of Materials Research (BAM), Berlin, Germany 

Wolfgang Rettig, Humboldt University, Berlin, Germany 

Claus Seidel, Heinrich-Heine University; Düsseldorf, Germany 

Bernard Valeur, Conservatoire National des Art et Metiers; Paris; France 

Antonie J. V. G. Visser, Wageningen Agricultural University; Wageningen; Netherlands 

Jerker Widengren, Royal Institute of Technology, Stockholm, Sweden 

Otto S. Wolfbeis (Chair), University of Regensburg, Germany 

Sergey M. Yarmoluk, Institute of Molecular Biology and Genetics, Kyiv, Ukraine 

Local Organizing Committee 

Otto S. Wolfbeis (Chairman) 

Doris Burger 

Axel Duerkop 

Rudi Hutterer 

Heike Mader 

Martin Link 

Michael Schaeferling 

Edeltraud Schmid 

Christian Spangler 

Matthias Stich 

5

Visit our Exhibitors: 

Amgen Res. GmbH 

Becker & Hickl GmbH 

Berthold Technologies 

Dyomics GmbH 

Edinburgh Instr. Ltd. 

Hamamatsu Photonics 

idQuantique 

ISS 

Jobin Yvon 

Leica 

L O T Oriel GmbH 

Nikon 

Olympus Austria 

PCO AG 

PicoQuant GmbH 

Raytest 

Sensovation AG 

Tecan 

Thermo Fisher Scientific 

TriPor Tech GmbH 

Varian 

http://www.amgen.com 

http://www.becker-hickl.com 

http://www.bertholdtech.com 

http://www.dyomics.com 

http://www.edinst.com/ 

http://www.hamamatsu.com/ 

http://www.idquantique.com/ 

http://www.iss.com 

http://www.jyhoriba.com 

http://www.leica-microsystems.com/ 

http://www.lot-oriel.com 

http://www.nikon.de 

http://www.olympus.at/ 

http://www.pco.de 

http://www.picoquant.com 

http://www.raytest.com 

http://www.sensovation.com/ 

http://www.tecan.de/ 

http://www.thermofisher.com/ 

http://www.triportech.de/ 

http://www.varianinc.com 

6

Table of Contents 

Welcome 3 

Sponsors 4 

Scientific Programme Committee 5 

Local Organizing Committee 5 

Exhibitors 6 

Table of Contents 7 

Scientific Programme 8 

Abstracts of Lectures 13 

Abstracts of Posters 

Part I: Fluorescence Spectroscopy 61 

Part II: Imaging and Microscopy 97 

Part II: Probes, Labels and Sensors 117 

Part IV: Fluorescence Correlation and Single Molecule Spectroscopy 173 

Part V: Upcoversion and 2-Photon Excitation 203 

Part VI: Nanomaterials 213 

Part VII: Other Materials 233 

Part VIII: Biophysics 247 

Part IX: Fluorescence in Biology, Medicine, Bioassays and Diagnostics 275 

Advertisements 311 

Author Index 

7

Scientific Programme 

Sunday, 9 Sep. 2007 

12:00 – 18:00 Exhibition Build-up 

15:00 – 18:00 Registration & Mounting of Posters (note that posters will be on display for the 

complete duration of the conference) 

17:00 – 17:15 Opening Ceremony 

17:15 – 18:00 Manfred Auer (Novartis; Vienna; AT): Single Bead, Single Compound, Single 

Molecule, Single Cell Fluorescence: Technologies for Drug Screening and 

Target Validation 

18:30 – 21:00 Welcome Reception (sponsored by Horiba – Jobin-Yvon) at Rosenhügel or 

Sheraton (depending on weather) 

Monday, 10 Sep. 2007 

Morning 

Advances in Fluorescence Spectroscopy and Fluorescent Materials 

08:30 – 09:00 Stefan W. HELL (Goettingen, DE): Breaking Abbe's Barrier: Diffraction- 

Unlimited Resolution in Far-Field Microscopy 

09:00 – 09:30 Maite COPPEY-MOISAN (Paris; FR): Picosecond Time-Resolved Imaging of 

FRET in Live Cells 

09:30 – 10:00 Michael S. STRANO (Urbana, US): Aspects and Applications of Single Walled 

Carbon Nanotube Photoluminescence 

10:00 – 10:30 Jicun REN (Shanghai; CN): Characterization of Water-Soluble Luminescent 

Quantum Dots by Single Molecule Methods 

10:30 – 11:00 Coffee Break 

11:00 – 11:30 Itamar WILLNER (Jerusalem; IL): Visualization of Biocatalytic Transformations 

and DNA-Bases Machines by FRET Processes Stimulated by Quantum Dots 

and Organic Dyes 

11:30 – 12:00 Seung B. PARK (Seoul, KR): Specific Targeting, Cell Sorting, and Bioimaging 

with Smart Magnetic Core-Silica Shell Nanomaterials 

12:00 – 12:30 Heinz LANGHALS (Munich, DE): Highly Stable Fluorescent Units, and their 

Applications in Functional Materials, Solar Energy Systems and Analysis 

8

12:30 – 13:30 Lunch Break 

13:30 – 14:30 Poster Session I and Coffee 

Afternoon Imaging – Microscopy – Arrays Micro- and Nanomaterials 

14:30 – 15:00 Alberto DIASPRO (Genova, IT): 

Confocal and Two-Photon 

Microscopy: Fundamentals, 

Applications and Advances 

15:00 – 15:30 Alberto BILENCA (Boston, US): 

A New Imaging Paradigm: 

Fluorescence Coherence 

Tomography 

15:30 – 16:00 Yong ZHANG (National University, 

Singapore): NIR-to-Visible 

Upconversion Fluorescent 

Nanoparticles for Cell and Animal 

Imaging 

16:00 – 16:30 Gerhard J. SCHUETZ (Linz; AT): 

Addressing Plasma Membrane 

Structure by Single Molecule 

Microscopy 

16:30 – 17:00 Jean-Louis REYMOND (Berne; CH): 

Substrate Arrays for Fluorescence- 

Based Enzyme Fingerprinting and 

High-Throughput Screening 

17:00 – 17:30 Margit BALAZS (Debrecen, HU): 

Array CGH and FISH Analyses 

Reveal New Genomic Alterations 

in Malignant Melanomas 

Zoe PIKRAMENOU (Birmingham, UK): 

Multi-colored Luminescent Lanthanide 

Complexes: from Nanoparticles to 

Biomolecule Recognition 

Joseph R. LAKOWICZ (Baltimore; MD): 

Plasmon-Controlled Fluorescence: 

A New Paradigm in Fluorescence 

Spectroscopy 

Mario BERBERAN-SANTOS (Lisbon; PT): 

The Fluorescence of Fullerenes: 

Singularities and Applications 

Weihong TAN (Gainesville, US): 

Bioconjugated Silica-Coated 

Nanoparticles: Characterization and 

Applications 

Suzanne FERY-FORGUES (Toulouse, FR): 

Nanoparticles of Organic Fluorescent 

Dyes: Self-Organization and Optical 

Properties 

Edin NUHIJI (Melbourne, AUS): 

Detection of Unlabelled Oligonucleotide 

Targets Using Whispering Gallery Modes 

in Single, Fluorescent Microspheres 

17:30 – 19:00 Evening Break and Session of the Permanent Steering Committee 

19:00 – 22:00 Dinner 

9

Tuesday, 11 Sep. 2007 

Morning 

Fluorescence Correlation and Single Molecule Spectroscopy 

08:30 – 09:00 Martin HOF (Prague, CZ): Principles and Applications of Fluorescence Lifetime 

Correlation Spectroscopy 

09:00 – 09:30 Nancy L. THOMPSON (Chapel Hill, US): Ligand-Receptor Interactions 

Measured by Total Internal Reflection FCS 

09:30 – 10:00 Gustav PERSSON (Stockholm, SW): Modulated Fluorescence Correlation 

Spectroscopy 

10:00 – 10:30 Joerg ENDERLEIN (Tuebingen; DE): Two-Focus Fluorescence Correlation 

Spectroscopy 

10:30 – 11:00 Coffee Break 

11:00 – 11:30 David P. MILLAR (La Jolla, US): Single-Molecule Studies of Biomolecular 

Folding and Assembly 

11:30 – 12:00 Klaus MUELLEN (Mainz, DE): Nano-emitters by Design 

12:00 – 12:30 Matthew J. LANG (Boston, Mass., US): Optical Force Fluorescence 

Measurements for Single Molecule Biophysics 

12:30 – 13:30 Lunch Break 

13:00 – 14:30 Poster Session II and Coffee 

Afternoon 

Fluorescent Probes, Sensors, and 

Labels 

Upconversion, 2-Photon Excitation, 

Delayed Fluorescence 

14:30 – 15:00 Guy DUPORTAIL (Strasbourg, FR): 

Revealing the Difference Between Gel 

and Liquid Ordered (Raft) Phases by 

Hydration-Sensitive Fluorescent Probes 

15:00 – 15:30 Hatsuo MAEDA (Kobe, JP): 

Highly Specific Fluorescent Probes for 

Reactive Oxygen Species 

15:30 – 16:00 Albin HERMETTER (Graz, AT): 

Novel Fluorescent Probes for Lipids 

and Lipases 

Pekka E. HÄNNINEN (Turku; FI): 

Two-photon Excitation Fluorescence 

Bioassays 

Petra SCHWILLE (Dresden; DE): 

Two-photon Fluorescence 

Correlation Spectroscopy in Cells 

and Developing Embryos 

Jean-Claude G. BUENZLI, (Lausanne, 

CH): Fluorescence Upconversion 

Using Lanthanide Compounds and 

Nanoparticles 

10

16:00 – 16:30 Gabor PATONAY (Atlanta, US): 

New Fluorophores for Wavelengths 

Beyond 900 nm 

16:30 – 17:00 Cristina LAGUNAS (Belfast, UK): 

Luminescent Au(I) Complexes: 

Implications for Sensors 

17:00 – 17:30 Christoph WEDER (Cleveland,US): 

Stimuli-Responsive Photoluminescent 

Polymer Blends 

Markus HAASE (Osnabrueck, DE): 

Upconversion Emission in Colloidal 

Solutions of Lanthanide-Doped 

Nanocrystals 

Tero SOUKKA (Turku, FI): 

Fluorescence Upconversion Based 

Enzymatic Assays 

Christoph J. FAHRNI (Atlanta, US): 

Rational Design of Metal-Ion 

Sensors for Two-Photon Microscopy 

18:00 – 19:00 Evening Break 

19:00 – 21:00 Reception by the Mayor of the city of Salzburg, followed by a Mozart Concert 

Wednesday, 12 Sep. 2007 

Morning 

Fluorescence in Bioassays, Biophysics, and Medicine 

08:30 – 09:00 Ilkka HEMMILÄ (Perkin-Elmer, FI): Recent Progress in Time-Resolved 

Methods for Diagnosis and Proteomics 

09:00 – 09:30 Catherine ROYER (Montpellier, FR): Nuclear Receptors as Studied by 

Fluorescence 

09:30 – 10:00 Markus SAUER (Bielefeld; DE): Multistep Energy Transfer Processes: 

Spectroscopy and Applications 

10:00 – 10:30 Anita JONES (Edinburgh, UK): Probing DNA Conformation and DNA-Enzyme 

Interaction by Time-Resolved Fluorescence 

10:30 – 11:00 Coffee Break; Dismantling of Posters 

11:00 – 11:30 Achim WAGENKNECHT (Regensburg, DE): Fluorescent DNA Base 

Modifications and Surrogates: Synthesis and Optical Properties 

11:30 – 12:00 Christian BLUM (Twente, NL): The Spectral Versatility of Fluorescent Proteins 

Revealed by Single Molecule Spectroscopy 

12:00 – 12:30 Closing Ceremony, Annoucement of MAF-11 

11

Lectures 

Abstracts 

13

Abstracts: Lectures 

LECT-1 

Single bead, single compound, single molecule, single cell fluorescence: 

Technologies for drug screening and target validation 

Martin Hintersteiner, Thierry Kimmerlin, Volker Uhl, Mario Schmied, Geraldine Garavel, 

Jan-Marcus Seifert, Christof Buehler, Nicole-Claudia Meisner, Manfred Auer 

Novartis Institutes for BioMedical Research, Discovery Technologies, Innovative Screening Technologies, 

A-1230 Vienna (Austria). E-mail: 

The modern drug discovery process is perceived as an increasingly cost-intensive, lengthy and complex 

multi-step process. [1] Despite the progress, the still unchanged classical concept of synthesizing and 

purifying several mgs of LMW compounds and building up of large solution or solid compound-archives 

for testing in HTS is associated with extensive storage, liquid handling and maintenance costs. All currently 

marketed drugs act on not more than 218 proven target proteins from only 6 major target classes. [2] The 

sequencing of the human genome, followed by several years of functional genomics and proteomics 

research, has so far failed to show the expected impact in increasing the number of successfully tackled 

drug targets. A good target protein needs to fulfill two requirements. Its up or down regulation must 

ameliorate or cure a disease and it must be drugable, i.e. susceptible to functional modulation by chemical 

or biological agents. To address the capacity-relevant processes of hit and lead finding, as well as the 

profiling of targets for their chemical drugability, we integrated all steps from chemical synthesis to 

deciphering the mechanistic mode of action of hit compounds in cells into a miniaturized chemical 

biophysics process. 

All major steps of this ICB process 

(single bead, single compound, 

single molecule, single cell 

technologies) are based on 

fluorescence spectroscopy and 

microscopy as detection methods. 

We therefore believe that the ICB 

methodology can be used to 

demonstrate the ultimate importance 

of fluorescence detection in lead 

discovery. 

D0 

Basic 

Research 

Chemistry 

single compounds 

on single beads 

“classical” HTS Process 

D0-1 Target D1 

D2a 

D2b 

HTS - High 

Identification Assay Throughput 

Validation Development Screening 

Hit/Lead 

Profiling 

Integrated Chemical Biophysics Process 

multiple targets 

but 1 single dye 

single 

molecule 

assays 

single cell 

microspectroscopy 

The exponential increase of the drug discovery costs since the 1980’s with the reduced numbers of new 

molecular entities brought to the market allows the conclusion that too much money and man power was 

invested into processes which were not sufficiently effective. ICB is the only fully integrated process 

described - from library design to cellular and, potentially, in-vivo confirmation. It is based on the 

systematic and early exclusion of artifacts connected with solid information on compound – target affinity 

(direct binding!). Its key feature is a linked tool set of high standard internal chemical, biological, and 

engineering technology developments, including single-molecule microspectroscopy, scanning and imaging 

as the key techniques to identify a low molecular weight compound (lmw) with high affinity to a target 

protein. This lmw compound is then used to mechanistically understand a biological problem in a 

quantitative way. The fast identification of stable foldamers opens up new avenues for target validation (by 

chemical inhibition), for new HTS strategies, and for tackling Protein Protein Interactions. 

References: [1] H. Federsel, Drug Discov. Today 11 (2006) 966-974. [2] P. Imming et al., Nat. Rev. Drug Discov. 5 

(2006) 821-834. 

15


LECT-2 

Breaking Abbe’s barrier: diffraction-unlimited resolution 

in far-field microscopy 

Stefan W. Hell 

Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, 

D-37077 Göttingen (Germany); e-mail: 

Ernst Abbe discovered in 1873 that the resolution of focusing (‘far-field’) optical microscopy is limited to 

d = λ ( 2nsinα 

) > 200 nm, with n sinα 

denoting the numerical aperture of the lens and λ the wavelength 

of light. While the diffraction barrier has prompted the invention of electron, scanning probe, and x-ray 

microscopy, in the life sciences 80% of all microscopy studies are still performed with lens-based 

(fluorescence) microscopy. The reason is that the 3D-imaging of the interior of (live) cells requires the use 

of focused visible light. Hence, besides being a fascinating physics endeavor, the development of a far-field 

light microscope with nanoscale resolution would facilitate observing the molecular processes of life. 

I will discuss novel physical concepts that radically break the diffraction barrier in focusing fluorescence 

microscopy. They share a common strategy: exploiting selected molecular transitions of the fluorescent 

marker to neutralize the limiting role of diffraction. More precisely, they establish a certain, signal-giving 

molecular state within subdiffraction dimensions in the sample [1]. 

The first viable concept of this kind was Stimulated Emission Depletion (STED) microscopy. In its simplest 

variant, STED microscopy uses a focused beam for fluorescence excitation, along with a red-shifted 

doughnut-shaped beam for subsequent quenching of fluorescent molecules by stimulated emission. Placing 

the doughnut-beam on top of its excitation counterpart in the focal plane confines the fluorescence near its 

central zero where stimulated emission is absent. The higher the doughnut intensity, the stronger is the 

confinement. In fact, the spot diameter follows d ≈ λ ( 2nsinα 

1 + I I s 

), with I denoting the intensity of 

the quenching (doughnut) beam and I s giving the value at which fluorescence is reduced to 1/e. Without 

the doughnut ( I = 0) we have Abbe’s equation, whereas for I I s → ∞ it follows that d → 0 , meaning that 

the fluorescence spot can be arbitrarily reduced in size. Translating this subdiffraction spot across the 

specimen delivers images with a subdiffraction resolution that can, in principle, be molecular! Thus, the 

resolution of a STED microscope is no longer limited by λ , but on the perfection of its implementation. We 

will demonstrate a resolution down to λ /45 ≈ 15-20 nm with nanoparticles and biological samples, i.e., 10- 

12 times below the diffraction barrier. 

The concept can be expanded by employing other molecular transitions that control or switch fluorescence 

emission, such as (i) shelving the fluorophore in a metastable triplet state, and (ii) photoswitching marker 

molecules between a fluorescent 'on' and 'off' state. Examples for the latter include photochromic organic 

compounds, and fluorescent proteins which undergo a photoinduced cis-trans isomerization or cyclization 

reaction. Due to their optical bistabilty/metastabilty, these molecules entail low values I 

s 

, meaning that the 

diffraction barrier can be broken at low I . A complementary approach is to switch the marker molecules 

individually and assemble the image molecule by molecule. By providing molecular markers with the 

appropriate transitions, synthetic organic chemistry and protein biotechnology plays a key role in 

overcoming the diffraction barrier. 

Finally, I will discuss more recent work of the group showing that the advent of far-field ‘nanoscopy’ has 

already solved fundamental problems in (neuro)biology, such as the fate of synaptic vesicle proteins after 

synaptic transmission. Besides, the emerging far-field ‘optical nanoscopy’ also has the potential to advance 

nanolithography, the colloidal sciences, and to help elucidate the self-assembly of nano-sized materials. 

References: [1] S.W. Hell, Far-field optical nanoscopy, Science 316 (2007) 1153. 

16


LECT-3 

Homo-FRET versus hetero-FRET to probe molecular interactions in living 

cells: fluorescence anisotropy and lifetime imaging microscopy 

Marc Tramier, Nicolas Audugé and Maïté Coppey-Moisan 

Institut Jacques Monod, F-75251 Paris (France). E-mail: 

Such progress has been made in fluorescence microscopy in both the methods and engineering of 

fluorescent probes that the biology of the cell can now be investigated at macromolecular levels in 

biological space and time. For example, it is possible to use FRET imaging to monitor protein-protein 

interactions [1] , biochemical reactions [2] and polymer organization [3] within living cells. The determination 

and the quantification of FRET are, however, difficult tasks to carry out under the microscope in living 

cells. Moreover, processes such as photoconversion, the occurrence of a “dark state”, photobleaching, or 

co-presence of other fluorescent species, can produce pitfalls in FRET determination [4] . Through 

standardized probes and biological examples, we will show how different methods for FRET imaging can 

bring reliable quantitative FRET determination in living cell. 

Fluorescence Lifetime Imaging Microscopy (FLIM) is the most reliable method for hetero-FRET 

measurement in living cell [5] . The time-correlated single photon counting (TCSPC) method provides the 

possibility to resolved multiexponential decay functions thanks to their high-time resolution. The single 

photon counting rate is however the limiting step in image acquisition. By combining multifocal 

multiphoton excitation and a fast-gated CCD camera we have created a novel confocal FLIM system 

(TRIM-FLIM), which provides fluorescence decay maps from the time-gated fluorescence intensity images 

at increasing intervals after excitation. We used this system to show that the nuclear map of the fraction of 

the acetylated EGFP-Histone H4 can be determined with high spatial resolution from the mean fluorescence 

lifetime images of EGFP-H4 in presence of mCherry-Bromo domain protein. 

Visualization of the fraction of acetylated EGFP- 

H4 (P FRET ) in live cells with the TRIM-FLIM. 

Two-photon EGFP-H4 images in the absence (A) 

and in the presence (D) of mCherry BD, and the 

corresponding mean fluorescence lifetime images 

(B, E) and the histogram of P FRET (C, F). 

We show that two-photon excitation steady-state fluorescence anisotropy imaging microscopy (TRIM- 

FAIM) is a powerful tool for the visualization of homo-dimerization of proteins in living cells and can be 

used for time-laspe homo-FRET. Hetero-FRET requires the use of two spectrally different chromophores. 

In contrast, homo-FRET can occur between like chromophores. This transfer does not change the 

fluorescence steady-state intensity nor the fluorescence lifetime. This homo-transfer can only be monitored 

by fluorescence anisotropy. Time-resolved fluorescence anisotropy decay and steady-state fluorescence 

anisotropy can be performed in microscopy[6]. 

References: [1] Y. Yan, G. Marriott, Curr. Opin. Chem. Biol. 7 (2003) 635-640. [2] Miyawaki A. Dev. Cell 4 (2003) 

295. [3] E. Delbarre et al. Hum. Mol. Genet. [4] G. Valentin et al. Nat. Methods 2 (2005) 801. [5] W. YU, W. 

Mantulin, E. Gratton, Emerging Tools for Single Cell Analysis (2000) New-York: Wiley. [6] M. Tramier et al. 

Methods Enzymol. 360 (2003) 580. 

17


LECT-4 

Optical modulation of single walled carbon nanotubes: 

fundamentals and biomedical applications 

Michael S. Strano 

Department of Chemical Engineering , Massachusetts Institute of Technology, 

Room 66-464, 77 Massachusetts Ave, Cambridge MA 02139 (USA) 

E-mail: 

Individually dispersed single walled carbon nanotubes (SWNT) are an excellent materials for optical 

sensors, as we have previously demonstrated 1-3 . Semiconducting SWNT fluoresce at near infrared (NIR) 

wavelengths 4,5 , and therefore offer great potential for use in biological environments and applications 

because of the low absorption of blood and tissue 4-6 , and the low autofluorescence of cells 7 in the NIR. 

SWNT are free of surface states 1,8 , and resistant to permanent photobleaching 9 . The optical transition 

energies (Eii) of SWNT are influenced by their local environment created by solvents and adsorbed 

molecules. Analysis of SWCNT photoluminescence (PL) energies in dielectric media is used to elucidate a 

semiempirical scaling relation for Eii shifts and nanotube structural properties. The SWCNT Kataura plot is 

corrected for a dielectric constant of unity and used to describe PL energy shifts in a broad range of media. 

We demonstrate direct detection of DNA hybridization on the SWNT surface using NIR fluorescence 

modulation of SWNT. Complementary DNA added to nanotubes with pre-adsorbed probe DNA cause a 

modulation in NIR photoluminescence. This system enables label-free nanoscale optical detection of 

hybridization. Further applications are currently being investigated. Complexes of DNA-encapsulated 

carbon nanotubes serve as sensitive markers for the activity of chemotherapeutic drugs which alkylate 

DNA. SWNT fluorescence is employed for optical transduction of drug binding events to the DNAnanotube 

complex. The complexes exhibit a concentration-dependent red-shift in emission energy of up to 

6 meV upon binding to alkylating agents such as nitrogen mustards and platinum compounds. 10,11 

Conformational changes of DNA are also detected by complexation with carbon nanotubes. The DNA- 

SWNT complex exhibits uptake into cellular vesicles where it functions as a real-time, non-cytotoxic, 

photobleaching-resistant sensor which remains functional in cells for up to 3 months. Fluorescence spectra 

of the complex within live cells exhibit changes upon contact with metal ions and chemotherapeutic agents. 

The nanotubes detect ion binding and alkylation in real time, functioning as a diagnostic for ion 

concentrations and chemotherapeutic drug uptake. 

We also report on near-infrared β-D-glucose sensors 1 that utilize a different mechanism: a photoluminescence 

modulation via charge transfer. Adsorbing glucose oxidase and ferricyanide ions to the 

surface of carbon nanotubes creates a flux-based β-D-glucose sensor. Reaction of glucose at the enzyme 

injects charge into the nanotube and modulates the fluorescence via two distinct mechanisms of signal 

transduction – fluorescence quenching and charge transfer. Detailed photo-physical experiments quantify 

these two effects for various (n,m) SWNT. The results demonstrate new opportunities for nanoparticle 

optical sensors that operate in strongly absorbing media of relevance to medicine or biology. 

References: [1]Barone, P. W. et al. Nature Mat. 4 (2005) 86; [2] Jeng, E. S. et al., 6 (2006) 371; [3] Heller, D. A. et. 

al., Science 311 (2006) 508; [4] O'Connell, M. J. et al., Science 297 (2002) 593; [5] Bachilo, S. M.; Strano, M. S.; 

Science 298 (2002) 2361; [6] Wray, S.; Cope, M., Biochim Biophys Acta 933 (1988) 184; [7] Weissleder, R. et al. 

Nature Med., 9 (2003) 123; [8] Saito, R. et al.; Physical Properties of Carbon Nanotubes; Imp. Coll. Press: London, 

1998. [9] Heller, D. et al. Adv. Mater. 17 (2005) 2793; [10] Delalande, O. et al., Biophys. J. 88 (2005) 4159; [11] 

Povirk, L. F.; Shuker, D. E.. Mutat Res. Rev. Genet. 318 (1994) 205. 

18


LECT-5 

Characterization of water-soluble luminescent quantum dots 

by single molecule methods 

Chaoqing Dong, Xiangyi Huang, Huifeng Qian, Hua He, Jicun Ren 

College of Chemistry and Chemical Engineering, Shanghai Jiaotong University, 

800 Dongchuan Road, Shanghai 200240 (P. R. China). E-mail: 

Quantum dots (QDs, also known as nanocrystals) are nanoscale inorganic particles composed of hundreds 

to thousands of atoms. Due to their quantum confinement of charge carriers in tiny spaces, QDs show some 

unique and fascinating optical properties, such as, sharp and symmetrical emission spectra, high quantum 

yield (QY), good chemical and photo-stability and size dependent emission wavelength tenability[1]. So 

far, QDs have been successfully used in biological systems, but some fundamental parameters and 

luminescence features are not clearly understood. We will present single molecule technologies for 

characterizing certain fundamental parameters of luminescent QDs synthesized in aqueous phase and will 

focus on the following aspects: 

1. We will present a method for characterization of molecular weight, molar extinction coefficient and 

bright fraction of QDs by combining fluorescence correlation spectroscopy (FCS) with ensemble molecular 

spectrometry. The principle is mainly based on the measurements of hydrodynamic diameters of QDs and 

the particle number of bright QDs in a small illuminated volume element using FCS technique [2]. 

Hydrodynamic diameters of a series of CdTe QDs were measured with FCS and the molecular weights 

were calculated assuming the measured hydrodynamic diameters as the diameters of QDs. The molar 

extinction coefficients of QDs at different excitonic absorption peak were calculated with the molecular 

weights. The bright fractions of QDs samples were characterized by measuring the concentration of the 

bright QDs and the total concentration of QDs. 

2. We will describe a new method for the measurement of the surface charge of QDs by combination of 

FCS with microchip electrophoresis. The principle is based on the measurement of the hydrodynamic radii 

and mobility of water soluble QDs in solution [3]. This technique has been successfully used to determine 

the surface charge of the different stabilizer modified CdTe QDs and study their transport properties in 

electric field. We found that the surface charge of QDs was remarkably associated with the type of 

stabilizers on QDs surface, buffer pH and other factors. 

3. We will show that total internal reflection fluorescence microscopy (TIRFM) can be used to visualize 

individual CdTe QDs by fluorescence emission spectroscopy. We find that individual CdTe QDs 

synthesized in mercaptopropionic acid (MPA) solution display non-blinking behavior [4]. Our experiments 

confirmed that MPA coating on CdTe QDs played key role for suppressing blinking of QDs. 

References: [1] X.Y. Huang, L. Li, H.F. Qian, et al., Angew. Chem. Int. Ed..45 (2006) 5140. [2] C.Q. Dong, 

H.F. Qian, et al., J. Phys. Chem. B, 110 (2006) 11069. [2] C.Q. Dong, H.F. Qian, et al., Small 2 (2006) 534. 

[4] H. He, H.F Qian, et al., Angew. Chem. Int. Ed. 45 (2006) 7588. 

19


LECT-6 

Visualization of biocatalytic transformations and DNA-based machines by 

FRET processes stimulated by quantum dots and organic dyes 

Itamar Willner 

The Hebrew University of Jerusalem, Institute of Chemistry, Jerusalem 91904 (Israel) 

E-mail: 

Quantum dots (QDs) are employed as optical labels for probing biocatalytic transformations. This will be 

exemplified by the analysis of telomerase activity and tyrosinase activity in different types of cancer cells. 

The telomerization of a primer nucleic acid linked to CdSe QDs with the incorporation of the Texas-Red 

dye into the telomer units allows the analysis of telomerase by a dynamic fluorescence resonance energy 

transfer (FRET) process. [1] Tyrosinase is analyzed by the biocatalytic transformation of L-DOPA-capped 

CdSe QDs to the respective dopaquinone units that quench the fluorescence of the QDs. [2] A further 

method that applies QDs for following biocatalytic transformations will involve dye-modified CdSe QDs 

for the fluorescent detection of NADH as a versatile system for the analysis of NAD + -dependent enzymes. [3] 

The incorporation of these QDs into cells enables us to monitor metabolic intracellular pathways. 

Recent activities of our laboratory include the development of isothermal DNA-based machines for the 

analysis of DNA, proteins, and low molecular weight substrates. [4] The use of FRET processes to follow 

the operation of DNA machines will be discussed. This will be exemplified with an aptamer-based machine 

for the fluorescent detection of cocaine, and the use of a protein/DNA machine for the fluorescent analysis 

of the Tay-Sachs genetic disorder mutant. The synthesis of programmed protein nanowires by DNA 

machines, and the fluorescent imaging of the nanowires will also be presented. 

References: [1] F. Patolsky et al., J. Am. Chem. Soc. 125 (2003) 13918. [2] R. Gill et al., J. Am. Chem. Soc. 128 

(2006) 15376. [3] R. Freeman et al., unpublished results. [4] (a) Y. Weizmann et al., Angew. Chem. Int. Ed. 45 (2006) 

2238. (b) Y. Weizmann et al., Angew. Chem. Int. Ed. 45 (2006) 7384. (c) B. Shlyahovsky et al., J. Am. Chem. Soc. 

(2007) in press. 

20


LECT-7 

Specific targeting, cell sorting and bio-imaging 

with smart magnetic core-silica shell nanomaterials 

Taejong Yoon, 1 Kyeongnam Yu, 2 Junsung Kim, 2 Eunha Kim, 1 

Myung-Haing Cho, 2 Jin-Kyu Leek*, 1 Seung Bum Park, 1* 

1 

Department of Chemistry, Seoul National University, Seoul 151-747 (Korea); 

2 

College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, 

Seoul 151-742 (Korea). E-mail: 

Magnetic nanoparticles (MNPs) have been used in numerous areas.[1] Rapidly developing applications 

include magnetic resonance imaging (MRI), targeted drug delivery, rapid biological separation, biosensing, 

and therapy.[2] J.–K. Lee et al. reported [3] on the synthesis of an organic dye-incorporated silicacoated 

core-shell MNP[MNP@SiO 2 (OD)] (where OD stands for an organic dye) that has controllable shell 

thickness and is taken up by various cells. These MNP@SiO 2 (OD) particles have a magnetic motor effect. 

We have modified the surface of MNP@SiO 2 (OD) with poly(ethylene glycol) (PEG) and amine. The 

purpose of these modifications was to increase a biocompatibility of MNPs by PEG, and also to increase 

their applicability by introducing an amine moiety which can function as a linker between MNPs and a 

maleimide moiety which can be used as a bioconjugation linker.[4] Subsequently, antibodies were 

immobilized on these nanomaterials. Specific targeting was observed, and we could confirm that the 

"smart" magnetic core-silica shell nanoparticle can be applied to antigen-antibody specific targeting in 

biomedical systems. 

Interestingly, the MNP@SiO 2 (OD) particles with 

antibody also exhibited a magnetic motor effect 

which has been observed in previous experiments. 

Furthermore, we have applied our smart 

nanoparticles to the field of bio-imaging. We 

envision that such nanomaterials can be used in a 

number of biomedical applications in nanobiotechnology 

such as targeting, bio-imaging, cell 

sorting, drug delivery, and therapy systems. 

References: [1] Zeng, H. et al. Nature 420 (2002) 395. [2] Willner, I., Katz, E. Angew. Chem. Int. Ed. 42 (2003) 

4576. [3] Yoon, T.-J. et al. Small 2 (2006) 209. [4] Saul, J.M. et al. J. Controlled Release 92 (2003) 49; Gao, X. 

et al., Nature Biotechnol. 22 (2004) 969. 

21


LECT-8 

Highly stable tailor-made fluorescent units and their applications 

to functional materials, solar energy systems, and analysis 

Heinz Langhals 

LMU University of Munich, Department of Chemistry and Biochemistry, D-81377 Munich (Germany). 

E-mail: 

The perylene bisimides 1 exhibit unique properties such as high photo stability and fluorescence quantum 

yields close to 100%; these are good prerequisites for applications where strong irradiation has to be 

handled such as in solar collectors or in dye lasers. Intense and stable fluorescence signals can be obtained 

with the chromophore of 1 in analytics. Thus, fluorescent labels with anchor groups were developed for 

many biologically important structures such as naturally occurring amines. The incorporation of 1 into 

liposomes and their linkage to antibodies allowed single-antibody-tracing with a simple fluorescence 

microscope; see the figure. 

O 

O 

R 

N 

N 

R 

O 

1 

O 

Single-antibody-tracing of fluorescent immuno 

liposomes doped with 1 (up) by means of a fluorescence 

microscope. Detection of chirality with bichromophoric 

derivatives of 1 (bottom). 

The wavelength of absorption, fluorescence and the Stokes’ shift of derivatives of 1 can be controlled by 

substituents at the aromatic core as general properties such as the solubility by the groups R attached to the 

nitrogen atoms. Thus, the attachment of anchor groups to R in 1 resulted, for example, in reagents for 

recognizing amines and aldehydes, respectively, and the incorporation of such structures into membranes 

allowed a continuous detection of aldehydes. 

An even larger manifold of possibilities is given be the interaction of two ore more chromophores within 

the same molecule because of their exciton interactions. Tailor-made functional dyes could be established 

on the basis of such interaction. Thus, for example, chirality could be visualized with a special arrangement 

of chromophores.in one molecule, the Stokes’ shifts of fluorescent dyes could be increased by means of 

exciton interaction, and systems for an efficient collection of light could be constructed. Finally, devices for 

molecular electronics were established where the direction of energy transport was determined by 

molecular geometry. 

References: [1] H. Langhals et al. Eur. J. Org. Chem.. (2007) in press. [2] H. Langhals, O. Krotz, Angew. Chem. 

Int. Ed. Engl. 2006, 45, 4444. [3] H. Langhals et al., Chem. Eur. J. 2006, 12, 4642. [4] H. Langhals, K. Fuchs, Coll. 

Czech. Chem. Commun. 2006, 71, 625. [5} H. Langhals, H. Jaschke, Chem. Eur. J. 2006, 12, 2815. [6] H. Langhals 

et al., Eur. J. Org. Chem. 2005, 4313. [7] Review: H. Langhals, Helv. Chim. Acta. 2005, 88, 1309-1343. 

22


LECT-9 

Confocal and two-photon microscopy: from 3D to 7D 

Alberto Diaspro 

LAMBS-IFOM, MicroScoBIO Res. Center, Dept. of Physics, University of Genoa, I-16146 Genova (Italy). 

E-mail: , URL: www.lambs.it 

There are plenty of new tools in optical microscopy consolidating the bridge that connects a variety of 

disciplines from biology to engineering, from medicine to physics, from computer science to biophysics. 

Optical microscopy is rapidly moving to nanoscopy [1] exploiting the running multiphoton revolution [2] that 

brought a dramatic and wide-reaching change in optical microscopy methods [3] . As well, it is still unique in 

allowing to explore the 3D (three-dimensional) space occupied by biological systems - from 

macromolecules to cells, from tissues to organs - while temporal changes occur within a temporal scale 

from microseconds to several hours and days. Imaging 3D structure has been aided by the introduction of 

computational and confocal optical methods [4] . 

However, two- and multi-photon excited fluorescence microscopy (2PE/MPE) is probably the most 

important advance in optical microscopy since the introduction of confocal imaging in the eighties, and 

related non-linear optical methods. The advantages of 2PE over confocal and wide-field 3D imaging are 

still being evaluated [4, 5] . In order to collect 3D data, a substantial volume of the specimen has to absorb 

light, with an inevitable concomitant photobleaching and phototoxicity, which may be particularly severe 

when ultra-violet-excited fluorochromes are used. In principle, 2PE is superior to conventional single 

photon scanned imaging (e.g. confocal), in that absorption can be limited to a very small volume at the 

focus of the objective lens at any one time. It can be the most efficient way of collecting 3D information in 

the sense of using the lowest time-integrated dose of radiation to the volume of interest. Besides this 

efficiency, 2PE has other advantages in imaging. It uses longer wavelengths (often infra-red), which are 

not only less scattered by the specimen but also may fail to excite background autofluorescence. This is 

particularly important in the imaging of single molecules [3] . Although the introduction of 2PE appeared 

incremental in the sense of providing similar optical sections to those obtained with confocal optics, and 

using similar scanning apparatus, it was also revolutionary, in that it represented a novel application of 

quantum physics. 

2PE has stimulated the application of other non-linear optical processes. It’s worth outlining currently 

developing approaches within a 7D observation framework: from 3D to time until spectral information (5D) 

plus lifetime (6D) and high-order harmonics (7D) [3] . FRAP and FRET methods are complementing the 

multidimensional approaches. As well, one of the most recent applications of 2PE is given by the 

photoactivation in confined volumes of phoactivatable proteins [6] . This really defines a new window in 4D 

(x, y, z, t). In fact, the coupling with new fluorescent molecules, including photoactivatable and 

photoswitchable ones, makes the “microscopical machine” an enormously powerful tool in scientific 

research. 

References: [1] S. W. Hell et al., Science 316 (2007) 1153. [2] W. Denk et al., Science 248 (1990) 73. [3] A. Diaspro 

et al., Quart.Rev.Biophys. 38 (2005) 1. [4] Diaspro A. (ed) Confocal and Two-Photon Microscopy: Foundations, 

Applications, and Advances; Wiley-Liss (2001). [5] P.J. Verveer et al., Nat Methods 4 (2007) 311. [6] M. Schneider 

et al., Biophys. J. 89 (2005) 1346. 

23


LECT-10 

A new imaging paradigm: fluorescence coherence tomography 

Alberto Bilenca, Brett E. Bouma and Guillermo J. Tearney 

Harvard Medical School and Wellman Center for Photomedicine, Massachusetts General Hospital, 

50 Blossom Street (BAR 7), Boston, MA 02114 (USA). E-mail: 

Large area, cross-sectional fluorescence imaging is of great interest in the life sciences as it has the capacity 

to view the ‘big picture’ of biology and offers the potential for studying molecular expressions in an 

organismal context. Different optical sectioning mechanisms have been suggested, including spatial 

filtering, nonlinear excitation, and perpendicular illumination-observation volumes 1 . We report on a new, 

intriguing approach dubbed ‘Fluorescence Coherence Tomography’ (FCT) that is based on the 

manipulation of low coherence fluorescence fields to obtain a deep look into specimens. FCT employs 

concepts of optical coherence gating and produces tomograms of fluorescently labeled structures over a 

wide field (mm scale) and a large depth range with high axial resolution (a few microns) using low NA 

objectives. The axial and transverse resolutions in FCT are decoupled. The axial resolution is determined 

by the coherence length of the fluorophore, the transversal resolution is governed solely by the optics. 

Two realizations of FCT are possible 2 : A spectral-domain (SD) setup and a time-domain (TD) arrangement. 

In SD-FCT, the entire sample depth is excited, and fluorescence self-interference, imaging spectrometry 

and Fourier signal processing are employed to localize fluorophores across the illumination sheet without 

axial scanning. In TD-FCT, the entire sample is excited with a wide-field light. Then, fluorescence selfinterference, 

axial scanning and demodulation processing are used to detect the position of fluorophores 

across the whole sample. In both FCT implementations, fluorescence self-interference is accomplished by a 

4π-like interferometer 3 . TD-FCT requires axial scanning; yet, it provides full-field imaging capabilities. 

SD-FCT does not require axial scanning; however, 3D imaging involves scanning of the light-sheet along 

the transverse direction. 

We have measured the narrow axial intensity PSF’s of an SD-FCT system employing an NA of 0.06. The 

FWHM extents from 3.29-3.45 µm were obtained at different depths (Fig. (a)) and were comparable to the 

theoretical value of 3.2 μm. We then demonstrated the ability of SD-FCT to image a fluorescent duallayered 

phantom over a transversal field greater than 1 mm and a depth range greater than 100 µm without 

scanning (Fig. (b)). These results are a precursor to eventual whole-organism fluorescence coherence 

imaging as simulated in Fig (c). Lastly, we have developed an extension of FCT based on the phase of the 

FCT signal, to allow for nm localization of fluoro-phores along the optical axis. A localization precision of 

19 nm was achieved (Fig. (d)), thereby indicating the potential of this technology to image individual 

molecules with nm accuracy. 

Fluorescence coherence 

tomography. (a) Axial 

intensity psf of FCT. (b) 

Localization phase-sensitivity 

of FCT. (c) Measured FCT 

tomogram of a layered 

phantom. (d) Simulated FCT 

tomogram of Drosophila 

nervous system (bar = 50 µm). 

Acknowledgements: A. B. acknowledges the support of the European Commission under the Marie Curie Fellowship. 

Special thanks to Drs. Laurel A. Raftery and Jing Cao from the CBRC at MGH for providing the original Drosophila 

image. 

References: [1] P. J. Keller et al., Curr Opin Cell Biol. 18 (2006) 117. [2] A. Bilenca et al., Opt. Express 14 (2006) 

7134. [3] S. Hell & E. H. K. Stelzer, J. Opt. Soc. Am. A 9 (1992) 2159. 

24


LECT-11 

NIR-to-visible upconversion fluorescent nanoparticles 

for cell and animal imaging 

Yong Zhang, 1 Zhengquan Li, 2 Dev Kumar Chatterjee, 1 Rufaihah Binte Abdul Jalil 1 

1 Division of Bioengineering, National University of Singapore, 7 Engineering Drive 1, 

Singapore 117574 (Singapore). E-mail: 

2 Singapore - MIT Alliance, National University of Singapore, Singapore 117576 (Singapore) 

Monodisperse infrared-to-visible upconversion nanoparticles have been developed in our lab, which have a 

wide range of biological and clinical applications. Optical window for in vivo imaging of cells and tissues 

are in the wavelength range of 700–1100 nm. The upconversion fluorescent nanoparticles are excited using 

NIR laser at a wavelength of 980nm which falls in the optical window. The upconversion nanoparticles 

have the following advantages: high light penetration depth in tissues, no photodamage to living organisms, 

weak auto-fluorescence from cells or tissues, low background light and high sensitivity for detection. 

Furthermore, only UV-visible detectors are required for detection which are usually equipped with normal 

fluorescence microscopes. Infrared detector (which er more expensive, insensitive, and less stable) are not 

required. 

The pictures on the right side show TEM images of 

NaYF 4 : Yb, Er/Tm nanocrystals and photographs of the 

upconversion fluorescence from the nanocrystals. The 

upconversion nanoparticles are also used for imaging of 

live cells and animals. 

An efficient and user-friendly method has been developed 

for synthesis of uniform β-phase NaYF 4 nanocrystals with 

strong upconversion fluorescence, by consuming fluorine 

reagents completely before the growth and ripening of the 

nanocrystals. NaYF 4 nanoplates, nanospheres and 

nanoellipses are produced and all these nanocrystals 

showed strong upconversion fluorescence. The 

fluorescence from the nanoplates can be observed by eye 

even when the power density of the laser is reduced to 

about 1 W cm -2 . 

Furthermore, the inorganic upconversion nanoparticles are chemically and photochemically stable (not 

photo-bleaching), and biocompatible (much less toxic than quantum dots). The nanoparticles are also well 

dispersed in some common organic solvents, and more importantly, in water. The surface of the 

nanoparticles can be functionalized so biomolecules can be attached to the nanoparticles. These 

upconversion nanoparticles are used for long term continuous imaging of live cells and animals for which 

most of downconversion fluorescent materials can not be used. 

References: [1] Z. Q. Li, Y. Zhang, Angew. Chem. Int. Ed. Engl. 45 (2006) 7732. [2] F. Wang, D. Chatterjee, et. al. 

Nanotechnology 17 (2006) 5786. [3] J. C. Boyer, F. Vetrone, et. al J. Am. Chem. Soc. 128 (2006) 7444. 

25


LECT-12 

Single molecule microscopy in vitro and in living cells 

Gerhard J. Schütz 

Johannes Kepler University Linz, Biophysics Institute, Altenbergerstr. 69, A-4040 Linz (Austria). 

E-mail: 

Current research throughout the natural sciences aims at the exploration of the Nanocosm, the collectivity 

of structures with dimensions between 1 and 100 nm. The cellular plasma membrane represents one of the 

most complex matrices heterogeneously organized on this length scale. We apply single molecule 

fluorescence microscopy to study the organization of the plasma membrane below the diffraction-limit of 

light microscopy by employing the high precision for localizing biomolecules of ~15 nm. Minimum 

invasive labeling via fluorescent Fab fragments is sufficient to image the lateral diffusion of individual 

protein molecules on a sub-millisecond time scale. We applied this technology to study the motion of single 

glycosylphosphatidylinositol- (GPI-) anchored proteins in the plasma membrane of living cells. In contrast 

to results obtained by tracking gold-labeled membrane proteins, the single molecule fluorescence data 

reveal free Brownian motion of the proteins down to length scales of ~70 nm, indicating no constitutive 

confinement zones [1, 2]. 

While single molecule tracking offers a strategy to measure protein interactions via their effect on mobility, 

the brightness contains information on the association of proteins to larger complexes. Based on brightness 

analysis, we developed a technique to detect molecular cluster formation in the cellular plasma membrane 

of living cells [3]. With this methodology, individual aggregates can be selectively imaged, and the load of 

each cluster can be determined. We applied this technique to investigate the association of a fluorescent 

lipid analogue in living Jurkat T cells. Aggregates containing up to 4 probe lipids were observed to diffuse 

freely as stable platforms in the plasma membrane, shedding new light on the current debate concerning the 

existence of “lipid rafts”. 

The development of ultra-sensitive detection schemes also has a strong impact on bioanalysis, as the 

sensitivity of biochemical assays could be dramatically increased. Whenever the available amount of 

sample is the limiting factor for unambiguous diagnosis e.g. in medical diagnostics, bioanalytics with single 

molecule sensitivity can be expected to become even an enabling technology. To specifically address this 

aspect, we developed a device for single molecule imaging on large surface areas such as biochips [4]. We 

applied this technology for RNA expression profiling down to the single molecule level [5]. The 

applicability of the system to PCR amplification-independent gene expression profiling of minute samples 

was demonstrated by complex hybridization of cDNA derived from the equivalent of only 10 4 cells; the 

results are in good agreement with data obtained in ensemble studies on large samples. 

References: [1] S. Wieser et al., Biophys J 92 (2007) 3719. [2] K. Drbal et al., Int. Immunol. 19 (2007) 675. [3] M. 

Moertelmaier et al., Appl Phys Lett 87 (2005) 263903. [4] J. Hesse et al., Anal Chem 76 (2004) 5960. [5] J. Hesse 

et al., Genome Res 16 (2006) 1041. 

26


LECT-13 

Substrate arrays for fluorescence-based enzyme fingerprinting and 

high-throughput screening 

Jean-Louis Reymond 

Department of Chemistry and Biochemistry, University of Berne, CH-3012 Berne (Switzerland). 

E-mail: 

High-throughput enzyme activity assays are indispensable tools in enzyme engineering for biotechnology, 

drug discovery, and medical screening. In these applications simple and robust methods with high 

information content are preferred, in particular by using enzyme specific yet stable reference substrate 

which are either fluorogenic or can be detected indirectly [1]. 

We have developed a series of highly specific fluorogenic substrates and product sensors for generic 

detection of various enzyme classes based on indirect mechanism for fluorescence release [2]. These 

systems are particularly robust against non-specific signals, and can be used in whole cell assays. When 

used in arrays, these substrate open a new window on enzyme activity by delivering enzyme specific 

activity fingerprint, which can distinguish between related enzymes, for example point mutations and cyclic 

permutations of the same enzyme. 

Fingerprinting setups in the form of cocktails and microarrays are possible [3]. In the case of lipases and 

esterases, novel enzyme from the metagenome produced very different fingerprints from reference enzymes 

[4]. Analysis of the fingerprinting data by principal components facilitates vizualization of the 

multidimensional datasets. Recent enzyme fingerprinting assays using combinatorial libraries of more than 

50'000 substrates will also be discussed. 

Lipase fingerprinting 

microarrays [3]. The 

substrates with various 

acyl chain length are 

printed as single and 

binary mixture substrates. 

The lipase 

cleaves the ester and 

unmasks a periodate 

sensitive 1,2-diol which 

can be tagged with the 

carbonyl reactive 

hydrazine. 

R 

O 

HO 

O 

C2-C12 esters 

1. Lipase HO 2. NaIO 4 

HO 

3. Rhodamine 

sulfohydrazide (1) 

TAG 

O 

S 

HN 

H 

O 

N 

References: [1] a) Enzyme Assays: High-throughput Screening, Genetic Selection and Fingerprinting, Ed. J.-L. 

Reymond. Wiley-VCH, Weinheim, Germany, 2006. [2] Recent example: R. Sicart et al., Biotechnol. J. 2 (2007) 221. 

[3] J. Grognux, J.-L. Reymond, Mol. Biosys.2 (2006), 492. [4] C. Elend et al., Appl. Environ. Microbiol. 72 (2006) 

3637. 

27


LECT-14 

Array CGH and fluorescence in-situ hybridization analyses reveal 

new genomic alterations in malignant melanomas 

Margit Balázs, 1 Viktória Lázár, 1 Zsuzsa Rákosy, 1 Laura Vízkeleti, 1 Szilvia Ecsedi, 1 

Ágnes Bégány, 2 Gabriella Emri, 2 Róza Ádány 1 

1 Department of Preventive Medicine, School of Public Health, 2 Department of Dermatology, 

Faculty of Medicine, University of Debrecen, Medical and Health Science Centre, Debrecen (Hungary). 

E-mail: 

Fluorescence in-situ hybridization (FISH) technology became a powerful tool not only in basic science but 

also in clinical genetics[1]. Application of FISH made it possible to analyze chromosome copy number and 

structural alterations not only on metaphase chromosomes but also in interphase cells. It has the capability 

to simultaneously visualize different DNA targets in multiple, distinct colors. One of the most recent 

development of FISH is array based comparative genomic hybridization (aCGH). Compared to 

chromosomal CGH, which resolution is limited to 10-20 Mb, array based CGH permits highly accurate 

mapping of chromosome copy number alterations throughout the entire genome[2,3]. CGH is a helpful 

starting point to search for candidate oncogenes and tumor suppressor genes affected by amplifications and 

deletion in the tumor genomes. 

Cutaneous malignant melanoma is known to be one of the most resistant cancers to therapies. It exhibits a 

large degree of molecular heterogeneity. Our aim was to search for genomic alterations in the melanoma 

genome, to identify gene amplifications and deletions that are associated with the aggressive behavior of 

the disease. The array-platform (HumArray 3.1 UCSF) contained 2464 FISH verified BAC clones, with an 

average spacing between clones of 1.4 Mb[4]. We also aimed to detect chromosome copy number 

alterations at a single cell level by using FISH. 

The Figure shows the clustered amplification of 

chromosome 11 in a malignant melanoma sample at 

the 11q13 region as detected by array CGH (A). 

Using FISH high level amplifications were detected 

on the Cyclin D1 gene (B). Melanoma cell nuclei are 

labeled with the blue fluorescent DAPI, amplified 

CCDN1 Gene appear as red color (SpectrumRed), 

whereas the green fluorescent spots represent copy 

numbers of chromosome 11. Amplification of the 

CCDN1 oncogene was associated with the 

amplification of the surrounding genes, including 

fibroblast growth factor 3 (FGF3), FKBP16, and 

FTHL6. 

Log2 Mean Ave RawRatioA 

B 

2,5 

chromosome 11 

2 

1,5 

1 

0,5 

0 

-0,5 

-1 

-1,5 

0 50000 100000 150000 

Among the genetic changes described in primary melanoma, high level amplifications were seen on the 

7q22.1 (CUTL1) 7q31.2 (TES), 7q32.1-32.2 (NRF1), 8q21 (RUNX1T1) 8q24 (MTSS1, TRIB1), 11q13 

(CCND1), 15q21.3 (RAB11A, ADAM10, MADM), 15q26.3 (ISG20) 20q12-q13 (PTPRT, PREX1. 

Amplification of the genes was detected by interphase FISH. Additional studies are in progress to further 

characterize and refine aCGH alterations. (supported by NKFP1-00003/2005 and OTKA-T 048750). 

References: [1] D. Pinkel et al. Proc Natl Acad Sci USA. 85 (1988) 9138; [2] J. B. Geigl et al. Nat Protoc. 1 (2006) 

1172; [3] D. Pinkel & D. G. Albertson. Annu Rev Genomics Hum Genet. 6 (2005) 331; [4] A. M. Snijders et al., 

Methods Mol Biol. 256 (2004) 39. 

28


LECT-15 

Multicoloured luminescent lanthanide complexes: 

From nanoparticles to biomolecule recognition 

S. P. Hammond, D. J. Lewis, P. B. Glover, M. Solomons and Z. Pikramenou* 

School of Chemistry, The University of Birmingham, Edgbaston, Birmingham, B15 2TT (UK). 

E-mail: 

We are interested in ligand design for the assembly of luminescent lanthanide complexes with recognition 

features that allow probing of interactions in sensing schemes, biomolecules or nanoscale systems. [1-3] In 

one approach we use ligands based on bis-amides of diethylene triamine pentaacetic acid (bis-DTPA). 

Upon complexation to the lanthanide ion they form a rigid hairpin structure with pendant arms that can be 

modified to target specific recognition sites. 

Red-emissive, water-soluble nanoscale labels 

have been prepared based on europium 

complexes. [4-5] Bis-DTPA ligands with thiol 

arms have been used. The binding of the 

Eu(III) complexes to platinum or gold 

nanoparticles (NPs) has been demonstrated 

by different techniques. Results suggest that 

the system can be optimised to allow 

minimum quenching of the luminescence by 

the surface of NPs. 

O 

H 

N 

C 

O 

N O 

O 

O 

N Eu 

O O 

N O 

C 

N 

H 

SH 

SH 

Au NP 

The thiol active bis-DTPA system sensitizes visible and near infra-red emission from many lanthanide ions. 

Its versatile nature has allowed the formation of bi-colour emissive lanthanide compounds. [5] 

To address DNA recognition using the lanthanide metal as a reporter luminescent probe we have 

incorporated platinum terpyridyl units as arms in the lanthanide bis-DTPA complex. Luminescent Ln-Pt 2 

metallohairpin complexes have been developed and their intercalative recognition with DNA has been 

demonstrated with linear dichroism spectroscopy. [5-6] The heterotrimetallic complexes were formed in onestep 

reaction, by assembly of a derivative of a DTPA bisamide, a platinum terpyridine unit and the 

lanthanide salt. The metallohairpin complexes bear a neutral lanthanide moiety and two positively charged 

platinum containing intercalating units. The Nd(III) and Eu(III) analogues are luminescent in the near infra– 

red and the visible respectively. 

References: [1] P.B. Glover et al., Chem. Eur. J. (2007) in press. [2] M. M. Castaño-Briones et al., Chem. Comm. 

(2004) 2832. [3] A. P. Bassett et al. Inorg. Chem. 44 2005 6410. [4] D. J. Lewis et al., Chem. Commun. (2006) 1433. 

[5] unpubl. results [6] P. B. Glover et al., J. Am. Chem. Soc. 125 (2003) 9918. 

29


LECT-16 

Plasmon-controlled fluorescence: 

a new paradigm in fluorescence spectroscopy 

Joseph R. Lakowicz 

University of Maryland School of Medicine, Center for Fluoresc. Spectrosc., Dept. Biochem. Mol. Biol., 

725 W Lombard St, Baltimore, MD 21201 (USA); E-mail: 

Since the beginning of fluorescence spectroscopy the observed emission propagates in free space and is 

detected in the far-field. By free-space we mean a transparent dielectric medium. Under these conditions, 

the radiative decay rate of a fluorophore remains essentially constant and is determined by the transition 

probability or extinction coefficient. Aside from the classical experiments of Drexhage in 1974, there have 

been few attempts to modify the free-space properties of fluorophores. We have developed methods to 

modify the intrinsic properties of fluorophores by modification of the electrodynamic properties or photonic 

mode density (PMD) near the fluorophore. 

Figure 1 (top right) shows the interactions of a fluorophore with a nearby 

metallic colloid. Several effects occur. The rate of excitation can be 

increased because the electric fields of the incident light are collected and 

concentrated by the metal. Our finite-difference time-domain (FDTD) were 

used to calculate the near-field around an excited fluorophore in the 

absence (middle) and presence (bottom) of a silver colloid. The spatial 

distribution of the radiation is changed dramatically by a nearby silver 

colloid (bottom). These effects occur because of near-field interactions 

between the excited fluorophore and the colloid, which induces a charge 

distribution in the colloid. We refer to the fluoropore-metal complex as a 

plasmofluor. 

The opportunities for using fluorophore-metal complexes can be shown by 

specific examples. Figure 2 shows single molecule images of Cy5-DNA 

alone and when single Cy5-DNA molecules are bound to single silver 

particles. The brightness depends strongly on particle size, with the highest 

signal coming from the 50 nm particle. 

We have examined the effects of silver colloids on FRET from Cy5 to 

Cy5.5 on a DNA oligomer. The length of the oligo is designed so that the 

amount of FRET in the absence of metal is about 10%. The FRET 

efficiency increased about 4-fold for the D-A pair on a 15 nm colloid. The 

FRET efficiency increases with colloid size (Fig. 2). The extent of FRET 

increases with the near-field intensity around the colloids of each size (not 

shown). Figure 2 shows the effects of silver particle size on single 

molecules of Cy5-DNA. Note the difference in the color scales. 

In summary, the use of fluorescence with metallic nanostructures provides 

an opportunity to exert control over the excited state fluorophores and 

direct their emission. This control will result in a new generation of devices 

for fluorescence detection. 

Fig. 1: ↑; Fig. 2: ↓ 

30


LECT-17 

The fluorescence of fullerenes: singularities and applications 

Mário N. Berberan-Santos 

Centro de Química-Física Molecular, Instituto Superior Técnico, P-1049-001 Lisbon (Portugal). 

E-mail: 

The most common fullerenes, C 60 and C 70 , are spheroidal structures containing a relatively large number of 

atoms, and can be viewed either as large carbon molecules or as tiny carbon nanoparticles. Their 

photophysical and photochemical properties result from the many delocalized pi electrons present and also 

from the high symmetry and curvature of the structures. 

The photophysics of fullerenes, and in particular of C 60 and derivatives, has been the subject of 

considerable investigation in the last 15 years. Much attention was paid to the triplet state. In fact, the 

fluorescence quantum yield of these compounds is usually quite low (ca. 5×10 -4 ), owing to a very efficient 

intersystem crossing. Nevertheless, interesting results were obtained from the study of the usually weak 

fluorescence of C 60 , C 70 , and derivatives. A number of peculiar features were disclosed, including: 

unpolarized or weakly polarized fluorescence; multiple emitting states; anomalous heavy-atom quenching; 

and exceptionally strong thermally activated delayed fluorescence. 

The graph shows a picture of films of C 70 in a polymer under 

UV light. In the absence of molecular oxygen, and owing to 

thermally activated delayed fluorescence, the red 

fluorescence is easily perceived by the naked eye. Upon 

heating, the fluorescence intensity further increases. 

A general view of the fluorescence properties of fullerenes will be presented, with an emphasis on the 

external heavy-atom effect and on the delayed fluorescence, and including recent applications in 

temperature and molecular oxygen sensing. 

References: [1] M.N. Berberan-Santos, J.M.M. Garcia, J. Am. Chem. Soc. 118 (1996) 9391. [2] M. Rae et al., J. 

Chem. Phys. 119 (2003) 2223. [3] M. Rae et al., J. Phys. Chem. B 110 (2006) 12809. [4] C. Baleizão et al., Chem. 

Eur. J. 13 (2007) 3643. [5] S. Nagl et al., Angew. Chem. Int. Ed. 46 (2007) 2317. [6] C. Baleizão, M.N. Berberan- 

Santos, J. Chem. Phys, in press. 

31


LECT-18 

Nanoparticles for bioanalysis and molecular imaging 

Weihong Tan 

Department of Chemistry and Shands Cancer Center, Center for Research at the Interface of Bio/nano; 

University of Florida, Gainesville, FL 32601 (USA). E-mail: 

Bionanotechnology is an evolving field which covers a vast and diverse array of nanomaterials and devices 

derived from engineering, biology, physics and chemistry. There has been great interest in applying 

nanomaterials for biotechnology and biomedical studies. In this talk, we will report our recent work on 

bioconjugated nanoparticles for bioanalysis, trace amount biomolecule collection and molecular imaging. 

We have prepared silica nanoparticles which are doped with a variety of luminescent, magnetic and metallic 

materials. The size of the nanoparticle can be controlled as small as 2 nm. Bioconjugation of these 

nanoparticles adds unique features for their efficient biotechnological and bioanalytical applications, which 

ideally links biologically significant molecules with nanomaterials. 

We have used these bioconjugated nanoparticles for a variety of applications: luminescent nanoparticles for 

rapid single bacterium monitoring and for cell imaging; luminescent nanoparticles for ultrasensitive 

DNA/mRNA analysis; magnetic nanomaterials for trace gene collection. Specifically, we will discuss 

fluorescent nanoparticles (NPs) with multiple emission signatures by a single wavelength excitation for 

multiplex bioanalysis and molecular imaging. We prepared silica NPs encapsulated with three organic dyes 

using a modified Stober synthesis method. By varying the doping ratio of the three tandem dyes, FRETmediated 

emission signatures can be tuned to have the NPs exhibit multiple colors under one single 

wavelength excitation. These NPs, shown in Figure 1, are intensely fluorescent, highly photostable, uniform 

in size, and biocompatible. The acceptor emission of the FRET NPs has generated a large Stokes shift, 

which implicates broad applications in biological labeling and imaging. 

Molecular recognition moieties, such as biotin, can be covalently attached to the nanoparticle surface to 

allow for specific binding to target molecules. These multicolor FRET NPs can be used as barcoding tags 

for multiplexed signaling. By using these NPs, one can envision a dynamic, multicolor, colocalization 

methodology to follow proteins, nucleic acids, molecular machines, and assemblies within living systems. 

We will discuss these topics as well as our most recent results in bionanotechnology. 

The Figures show FRET nanoparticles with different doping dye combinations under single wavelength 

illumination. Right: Schematic representation of triple FRET dye-doped silica nanoparticles. 

References: 1. L. Wang, W. Tan, Nano Lett. 6 (2006) 84; 2. X. Zhao et al., PNAS 101 (2004) 15027; 3. L. Wang, 

et al., Bioconj. Chem. 18 (2007) 297. 4. L. Wang et al., Nanomed. 1 (2006) 413. 

32


LECT-19 

Detection of unlabelled oligonucleotide targets using 

whispering gallery modes in single, fluorescent microspheres 

Edin Nuhiji, Paul Mulvaney 

School of Chemistry & Bio21 Institute Level 2 North, 30 Flemington Road, University of Melbourne 

Parkville, VIC, 3010 (Australia). E-mail: 

Sequence specific nucleic acid detection has become an important goal in an array of biotechnology and 

biomedical disciplines as well as in forensic analysis. However a disadvantage of most current nucleic acid 

detection systems is the necessity to label the analyte. [1, 2] Labelling the target molecule can be costly, 

laborious and may interfere with the binding affinity of the target molecule. In this work we demonstrate 

the development and characterization of an innovative, highly-sensitive, label-free oligonucleotide specific 

biosensor. [3] 

Whispering gallery modes (WGM) have emerged as a powerful signal transduction mechanism that could 

be utilized in ultra-sensitive biosensors. [4] In dielectric microspheres WGM are produced when incident 

radiation is internally trapped as standing waves, resulting in a scattering spectrum composed of numerous, 

sharp peaks. Light is introduced into the microsphere by coupling through an optic fibre or more simply by 

attaching a fluorescent molecule to a microspheres surface and illuminating with a UV lamp. The 

fluorescent microspheres within this work comprise a silica microsphere functionalised with a fluorophore 

and a dense monolayer of 5’ tethered, single-stranded oligonucleotides. The adsorption of the unlabelled 

complementary (cDNA) probe then causes nanometre shifts in the emission spectrum of the microsphere. 

Intensity/ a.u. 

9000 

8000 

7000 

6000 

5000 

4000 

550 560 570 580 590 600 610 620 630 640 

λ/ nm 

Pre-Hybridized 

Post-Hybridized 

WGM spectra from a single microsphere 

hybridization assay using 7.5μm 

oligonucleotide (70 bases) specific microspheres. 

WGM emission spectra of the same 

tetramethylrhodamine (TMR) modified 

microsphere were acquired pre and post cDNA 

probe treatment using an optical microscope 

coupled to a CCD detector. The peak 

wavelengths exhibit red-shifts (in brackets) at 

575 (1.1nm), 585 (1.5nm), 595 (1.1nm) and 

605nm (1.1nm) following a 90s exposure of the 

target microspheres to the cDNA probe. 

These highly sensitive deviations in the emission signal can be registered using an optical microscope 

coupled to a CCD detector. The assays are run using a gridded array plate system that enables the relocation 

and scrutiny of a single particle. This capability alleviates the need for statistical data analysis and laborious 

data processing of samples which is associated with current micro-array and microfluidic DNA based 

systems. The assay is capable of detecting sub-picomolar levels of unlabelled-oligonucleotide targets and 

delineating between control reagent and non-specific oligomeric sequences. The spectral shifts can be used 

to monitor both the hybridisation kinetics and the denaturation of duplex DNA at elevated temperatures. 

Oligonucleotides with more than 30 bases are most readily detected, while a complete assay takes only a 

few minutes. This inexpensive and highly sensitive nucleic acid-specific microsphere assay system provides 

an alternative bio-molecular recognition format with tunable specificity (proteins, anti-bodies) and easy 

implementation in high-throughput screening and point of care systems. 

References: [1] M. Y. Han et al., Nature Biotechnology 19 (2001) 631; [2] B. J. Battersby et al., Chem. Comm. 2002, 

1435; [3] E. Nuhiji and P. Mulvaney, Small, 2007, in press. [4] F. Vollmer et al., Biophys. J. 84 (2003 295A. 

33


LECT-20 

Principles and applications of fluorescence lifetime correlation spectroscopy 

Aleš Benda, Jana Humpolíčková, Jan Sýkora, Martin Hof 

Dept. of Biophys. Chem., J. Heyrovský Institute of Phys. Chem., Acad. of Science of the Czech Republic, 

Dolejškova 3, CZ-18223 Praha 8 (Czech Republic). E-mail: 

Fluorescence correlation spectroscopy (FCS) analyses fluorescence intensity fluctuations of labeled 

molecules in a “cuvette” determined by diffraction limited focus of the laser beam. It allows for 

determination of diffusion coefficients and absolute concentration of the fluorophores. Additionally, also 

spectral information of the labeled molecules can be considered, which enables to distinguish between 

different fluorophores and their interaction can be revealed. This concept is known for more than ten years 

as dual-color cross-correlation spectroscopy and requires two laser lines with well overlapping foci and two 

detectors. 

In 2002 fluorescence lifetime correlation spectroscopy (FLCS) has been suggested by J. Enderlein. [1] 

Instead of different spectral properties, fluorescence lifetime is used for signal separation. It employs only 

one laser, one detector and additionally a compact TCSPC card for data acquisition. [1,2] FLCS can be 

straightforwardly used to suppress noise or afterpulsing [1] or to analyze mixtures of two dyes with different 

lifetimes. [2] Apart from that, however, advanced applications seem to be enormously interesting. 

In our lab we have recently developed several new applications of FLCS: 

a) Distinguishing between signals from individual leaflets of supported phospholipids bilayers 

(SPB’s) with the help of surface mediated lifetime tuning, [3,4] 

b) Simultaneous monitoring of 2-D and 3-D diffusion of lipid molecules using single dye labeling, [3] 

c) Characterizing DNA (plasmids) condensation dynamics, and 

d) Determining the dynamics of protein-lipid (SPB’s) interactions. 

We would like to acknowledge the financial support of the Ministry of Education of the Czech Republic 

(grant No. LC06063). 

References: [1] P. Kapusta et al., J. Fluoresc., 17 (2007) 43. [2] A. Benda et al., Rev. Sci. Instrum. 76 (2005) 33106. 

[3] A. Benda et al., Langmuir 22 (2006) 9580. [4] M. Przybylo et al., Langmuir 22 (2006) 9096. 

34


LECT-21 

Ligand-receptor interactions measured by total internal reflection 

fluorescence correlation spectroscopy 

Nancy L. Thompson 

Department of Chemistry, Campus Box 3290, University of North Carolina at Chapel Hill, 

Chapel Hill, NC 27599-3290 (USA). E-mail: 

The combination of total internal reflection illumination with fluorescence correlation spectroscopy (TIR- 

FCS) allows one to examine in quantitative detail a variety of biophysical properties related to the motions 

and interactions of fluorescent molecules near the interface of a transparent planar surface and an adjacent 

solution. Several experimental and theoretical aspects of this combination will be discussed. 

TIR-FCS has allowed characterization of local diffusion coefficients and concentrations of fluorescently 

labeled antibodies in solution but very close to substrate-supported phospholipid bilayers. TIR-FCS has 

also been used to examine the interaction kinetics of fluorescently labeled mouse IgG specifically and 

reversibly associating with the mouse receptor FcγRII, which was purified and reconstituted into substratesupported 

planar membranes. The use of quantum dot blinking for measuring submicroscopic distances will 

also be described. 

35


LECT-22 

Modulated fluorescence correlation spectroscopy 

Gustav Persson, Per Thyberg and Jerker Widengren 

Royal Institute of Technology, Department of Applied Physics, Experimental Biomolecular Physics, 

S-106 91 Stockholm (Sweden). E-mail: 

We introduce and have experimentally verified a method to retrieve the full correlation curves from 

fluorescence correlation spectroscopy (FCS) measurements with modulated excitation and arbitrarily low 

fraction of active excitation. 

Many of the best fluorescent markers, synthetic organic dyes and fluorescent proteins exhibit flickering due 

to e.g. triplet formation, trans-cis-isomerization, electron transfer or protonation. Whereas each of these 

properties may be exploited to probe the environment, they sometimes complicate the measurements or data 

analysis by obscuring some other process of interest falling within the same time range. It has been shown 

previously, with other methods, that the amount of triplet formation may be controlled by modulating the 

excitation with pulse widths and periods in the range of the transition times of the involved states [1] . This 

should also be true for other photo-induced processes, i.e. all of the processes mentioned above, except 

protonation. Suppressing the triplet is also useful because it is a way of decreasing the photobleaching. 

However, modulating the excitation in FCS measurements normally destroys correlation information and 

induces ringing in the correlation curve, making it hard to interpret for any time range. 

We will show, for the case of the dye rhodamine 6G (Rh6G) in water, that modulated excitation can be 

applied to FCS experiments to suppress the triplet build-up more efficiently than by reducing excitation 

power with continuous wave (CW) excitation. Further, we demonstrate the usefulness of the method by 

measurements that were done on fluorescein at different pH, where suppression of the triplet significantly 

facilitates the analysis of the protonation kinetics which generate fluorescence blinking in the same time 

range as that of the the triplet state kinetics. 

We conclude that the method of combining the advantages of modulated excitation with the power of FCS 

will most likely prove very beneficial for many future studies. Full correlation curves can be retrieved from 

FCS measurements with excitation modulated in any time regime. 

Reference: [1] T. Sandén et al., Anal. Chem. 79 (2007), in press. 

36


LECT-23 

Two-focus fluorescence correlation spectroscopy 

Anastasia Loman, 1,3 Thomas Dertinger, 1,2 Iris von der Hocht, 1 

Ingo Gregor, 1 Jörg Enderlein 1,3 

1 Institute for Neuroscience and Biophysics 1, Forschungszentrum Jülich, D-52425 Jülich (Germany). 

2 Department of Chemistry & Biochemistry, Univ. of California, Los Angeles (USA). 

3 Institute of Phys. and Theoret. Chem., Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 

D-72076 Tübingen (Germany). E-mail: 

Thermally induced translational diffusion is one of the fundamental properties exhibited by molecules 

within a solution. Via the Stokes-Einstein relation it is directly coupled with the hydrodynamic radius of the 

molecules [1]. Any change in that radius will change the associated diffusion coefficient of the molecules. 

Such changes occur to most biomolecules – in particular proteins, RNA and DNA – when interacting with 

their environment (e.g. binding of ions or other biomolecules) or performing biologically important 

functions (e.g. enzymatic catalysis) or reacting to changes in environmental parameters such as pH, 

temperature, or chemical composition (e.g. protein unfolding). Therefore, the ability to precisely measure 

diffusion coefficients has a large range of potential applications, for monitoring e.g. conformational 

changes in proteins upon ion binding or unfolding. However, many biologically relevant conformational 

changes are connected with rather small changes in hydrodynamic radius on the order of Ångstrøms (see 

e.g. [2]). To monitor these small changes, it is necessary to measure the diffusion coefficient with an 

accuracy of better than a few percent. 

An elegant technique capable of measuring diffusion coefficients of fluorescent molecules at nanomolar 

concentrations is Fluorescence Correlation Spectroscopy (FCS) which was originally introduced by Elson, 

Magde and Webb in the early seventies [3]. In its original form it was invented for measuring diffusion, 

concentration, and chemical/biochemical interactions/reactions of fluorescent or fluorescently labelled 

molecules at nanomolar concentrations in solution. However, standard FCS is prone to a wide array of 

optical and photophysical artefacts which make precise quantitative and absolute measurements of e.g. 

diffusion coefficients rather difficult [4]. The main problem of standard FCS is the absence of a reliable 

extrinsic length scale in the measurements, which is, however, necessary for obtaining absolute values of 

the diffusion coefficient. 

Here, we report on our recently developed new technique of 2-focus fluorescence-correlation spectroscopy 

[5], allowing for measuring the hydrodynamic radius of molecules at pico- and nanomolar concentrations 

with sub-Angstrom precision. In 2fFCS, the problem of an extrinsic length scale is solved by generating 

two excitation foci with well defined distance from each other. Several applications of 2fFCS are presented, 

for example monitoring conformational changes of proteins upon ion binding, or monitoring protein 

unfolding curves upon chemical and thermal denaturation. 

References: [1] A. Einstein Investigations on the Theory of the Brownian Movement, Dover, New York, 1985. [2]. A. 

M. Weljie et al., Protein Science 12 (2003) 228; [3]. D. Magde et al. Phys. Rev. Lett. 29 (1972) 705. [4] J. Enderlein 

et al. ChemPhysChem. 6 (2005) 2324. [5] T. Dertinger et al. ChemPhysChem. 8 (2007) 433. 

37


LECT-24 

Single-molecule studies of biomolecular folding and assembly 

Stephanie Pond, Joshua Gill, David Millar 

Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037 (USA). 

E-mail: 

Single-molecule fluorescence methods provide powerful new tools to investigate the folding, assembly and 

dynamic behavior of biological macromolecules and complexes. Single-molecule measurements typically 

reveal discrete sub-populations that are hidden in conventional ensemble experiments. Moreover, kinetic 

information can be obtained under conditions of thermodynamic equilibrium without the need to 

synchronize a population of molecules. These capabilities can be exploited in dissecting complex assembly 

processes and in monitoring dynamic conformational changes that underpin the biological function of a 

variety of macromolecular machines. 

We have used single-molecule fluorescence methods to study the assembly of large ribonucleoprotein 

(RNP) complexes that are formed during replication of the HIV-1 virus. The Rev protein of HIV-1 

promotes the nuclear export of unspliced and partially spliced viral mRNAs encoding the structural proteins 

required for virion assembly. Rev binds to a highly structured portion of the viral mRNA, the Rev 

Responsive Element (RRE), where it forms an oligomeric RNP complex. The details of this assembly 

process are not fully understood and the role of other proteins present within the infected host cell is not 

known. We have developed a single-molecule TIRF imaging technique to visualize the assembly of 

fluorescently-labeled Rev on individual RRE molecules immobilized on a quartz surface. Using this 

approach, we have been able to monitor discrete steps in the assembly pathway and to quantify each of the 

microscopic rate constants. The RNPs are highly dynamic and show discrete assembly intermediates 

consistent with both sequential monomer binding and direct binding of preformed Rev oligomers to the 

RRE. Similar experiments have been performed in the presence of the nuclear export receptor CRM-1, 

which is known to interact with the Rev-RRE complex. The CRM-1 protein does not influence the kinetics 

of Rev binding to the RRE, but it retards dissociation of each of the RNA-protein complexes formed during 

the assembly process. 

We have also used single-molecule fluorescence methods to monitor dynamic conformational changes of a 

single DNA polymerase molecule as it interacts with both DNA and nucleotide substrates. Our studies 

utilized the Klenow fragment of E. coli Pol I (KF) and oligonucleotide primer/templates as a model 

polymerase-DNA system. Donor and acceptor dyes were attached to different domains of KF and/or to the 

DNA primer/template in order to monitor conformational changes by means of fluorescence resonance 

energy transfer (FRET). The DNA primer/templates were immobilized on a quartz surface and the KF 

molecules were present in the surrounding solution. A two-color TIRF imaging system was used to acquire 

FRET time trajectories following the binding of a single KF molecule to an immobilized primer/template. 

This system has been used to monitor conformational changes of KF during DNA binding and also during 

the subsequent binding of an incoming nucleotide substrate (either correct or incorrect). These studies 

reveal protein conformational changes that occur during the recognition of a correct incoming nucleotide 

and thereby provide insights into the molecular basis for DNA polymerase fidelity. 

Supported by grants GM44060 and GM66669 from the U.S. National Institutes of Health. 

38


LECT-25 

Nano-emitters by design 

Andreas Herrmann and Klaus Müllen 

Max-Planck-Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz (Germany). 

E-mail: 

Although organic dyes are among the oldest objects of organic synthesis, their importance as colorants and 

the fascination with color in general remain undiminished. [1] The field of dye chemistry has evolved to 

include not only synthetic methodology, but physical and materials sciences, requiring a commensurate, but 

continuously fruitful evolution. Recent developments in, both, fundamental science and technology have 

defined even more urgent needs for e.g. 

– control of absorption and emission wavelength of dyes including the NIR-range, 

– high fluorescence quantum yield and high light fastness, 

– processability including the creation of supramolecular order. 

In addition, chemical functionalization [2] must be possible for controlled attachment to e.g. conjugated 

polymers [3,4,5] (energy transfer), semiconductor surfaces (electron transfer), or biopolymers (tagging). We 

introduce a new family of dyes derived from commercial perylene-tetracarboxdiimide. The members of this 

perylene dye series open new avenues for the fabrication of electronic and optoelectronic [6] devices, 

biolabelling, [7] polymer-morphology studies [8] and laser writing. Of particular importance is their active 

roles in single molecule spectroscopy [9] for which dendritic multichromophores [10-12] and 

organic/inorganic [13] or biosynthetic hybrids [14] are rewarding objects of study. 

References: [1] A. C. Grimsdale, K. Müllen, Angew. Chem.- Intl. Ed. 44 (2005) 5592. [2] F. Nolde et al., Chemistry - 

Eur. J., 11 (2005) 3959. [3] B. Muls et al., ChemPhysChem 6 (2005) 2286. [4] T. D. M. Bellet al., Chem. Comm. 

(2005) 4973. [5] E. Fron et al., J. Am. Chem Soc. 129 (2007) 610. [6] R. Metivieret al., Phys. Rev. Lett. 98 (2007) 

47802. [7] M. Zhang et al., J. Am. Chem. Soc., 2007, in press. [8] T. Weil et al., Biomacromol. 6 (2005) 68. [9] H. 

Uji-i et al., Polymer 47 (2006) 2511. [10] G. Hinze et al., J. Phys. Chem. A 109 (2005) 6725. [11] R. E. Bauer et al., 

in: Functional Molecular Nanostructures, vol. 245 (2005) p. 253ff (Springer, 1. ed.). [12] F. C. De Schryver et al., 

Acc. Chem. Res.38 (2005) 514. [13] M. Cotlet et al., J. Am. Chem. Soc.127 (2005) 9760. [14] Y. J. Jung et al., 

Macromol. Chem. Phys. 206 (2005) 2027. [15] A. Margineanu et al., J. Phys. Chem. B 108 (2004) 12242. 

39


LECT-26 

Optical force fluorescence measurements for single molecule biophysics 

Matthew J. Lang 

Massachusetts Institute of Technology, Departments of Biological Engineering and Mechanical 

Engineering, Cambridge, MA 02139 (USA). E-mail:< mjlang@mit.edu> 

The ability to combine optical trapping and single molecule fluorescence measurements in the most 

common coincident arrangement enables measurements that mechanically probe a structure with force 

while simultaneously watching structures using fluorescence. In single molecule rupture experiments, this 

method can report the precise location of a break [1]. In conformational change measurements, this method 

can be used to monitor conformation as a function of load. The method has been limited due to fluorophore 

photobleaching in the intense flux of trapping photons requiring non-coincident application of the method 

in many cases. 

We report a technical advance where we demonstrate a FRET measurement in a coincident geometry with a 

model system consisting of a classic DNA hairpin opening mechanical transition assay controlled with 

force applied across the hairpin using the optical trap, Tarsa et al [2]. A FRET pair placed at the base of the 

hairpin, consisting of a donor Cy3 fluorophore and an Alexa acceptor molecule, reports the conformational 

state of the hairpin as open or closed. We demonstrate reversible mechanical control over the state of the 

hairpin while simultaneously watching the hairpin conformation through both mechanical displacement of 

the trapped bead and FRET reporting, identifying the precise location of the transition. 

Our solution to the photobleaching problem to alternate the application of trapping and fluorescence lasers, 

outlined in a paper by Brau et al. [3], makes this measurement possible. Work is currently underway to 

compare the behavior of a range of fluorophores in response to both continuous and alternating application 

of the trap. 

References: [1] M. J. Lang et al., Nature Methods 1 (2004) 133. [2] P. B. Tarsa et al., Angew. Chem., Intl. Ed. 46 

(2007) 1999. [3] R. R. Brau et al., Biophy. J. (2006) 1069. 

40


LECT-27 

Revealing the difference between gel and liquid ordered (raft) phases 

by a hydration-sensitive fluorescent probe 

Guy Duportail, Gora M’Baye, Yves Mély and Andrey S. Klymchenko 

Photophysique des Interactions Biomoléculaires, UMR 7175 du CNRS, Faculté de Pharmacie, 

Université Louis Pasteur, 67401 Illkirch (France). E-mail: 

So far, the existing fluorescence probe techniques cannot distinguish between gel phase and liquid ordered 

(raft) phases since their physicochemical properties appear quite similar. In the present study, we used a 

recently developed 3-hydroxyflavone fluorescent probe (F2N8), which exhibits high sensitivity to 

hydration of lipid bilayers [1]. Experiments performed at 20 °C in large unilamellar vesicles composed of 

sphingomyelin or DPPC with different concentrations of cholesterol reveall the strong dehydration of the 

bilayer above a critical concentration of cholesterol which results in raft formation. 

For the same samples, the anisotropy of TMA-DPH remains roughly constant, indicating no changes in the 

viscosity upon transition from the gel phase to the liquid ordered phase. Further transition of the liquid 

ordered phase to the liquid crystalline phase by an increase in the temperature results in a dramatic increase 

in the bilayer hydration, while transition from gel to liquid crystalline phase does not affect hydration 

significantly. Opposite tendencies are observed by measuring the anisotropy of TMA-DPH since the gelliquid 

crystalline transition changes the anisotropy to a much larger extent than the liquid ordered-liquid 

crystalline phase transition does. 

Thus, while viscosities of the gel and liquid ordered phases are similar, the liquid ordered phase is much 

less hydrated. This is in line with the high density packing of the ordered phase due to strong interaction of 

lipids with cholesterol, which results in a decrease in the void space available for water molecules and thus 

in dehydration of the bilayer. 

Fluorescence spectra of probe F2N8 in 

large unilamellar vesicles composed of 

sphingomyelin (black line) and 

sphingomyelin with 35 mol% of 

cholesterol (red dotted lines), at 20 °C. 

Fluorescence Intensity 

1.2 

0.9 

0.6 

0.3 

0.0 

450 500 550 600 650 700 

Wavelength, nm 

Sphingomyelin (gel) 

Sphingomyelin (raft) 

Reference: [1] A. S. Klymchenko et al., Biochim. Biophys. Acta 1665 (2004) 6-19. 

41


LECT-28 

Highly specific fluorescent probes for reactive oxygen species 

Hatsuo Maeda 

Hyogo University of Health Sciences, School of Pharmacy, Kobe 650-8530 (Japan) 

E-mail: 

We have proposed a novel strategy for designing fluorescent probes based on protection-deprotection 

chemistry involving fluoresceins (1) and their benzenesulfonyl (BES) derivatives (2) (eq. 1 in Chart 1). 

Compound 2 exhibits almost no fluorescence, and hence will work as a specific fluorescent probe toward a 

target molecule when the BES group is deprotected selectively by reaction with the molecule. This strategy 

has been successfully applied in the design of novel florescent probes toward H 2 O 2 , [1] O –• 2 , [2,3] thiols, [4] and 

selenols. [5] Herein we describe the performance of our probes useful for measurements of extra- and 

intracellularly generated H 2 O 2 and O –• 2 . 

BESH 2 O 2 (Chart 1) works as a useful probe for H 2 O 2 with a high specificity over HO•, tBuOOH, 1 O 2 , NO•, 

and ONOO – . The measurements of intracellularly generated H 2 O 2 in human Jurkat T cells as well as 

Chlamydomonas reinharadtii, a freshwater green alga, were successfully achieved with its acetyl derivative 

BESH 2 O 2 -Ac (Chart 1). As for O –• 2 , BESSo (Chart 1) functions as a sensitive probe, and exhibits high 

specificity over GSH as well as ROS such as H 2 O 2 , NaOCl, tBuOOH, 1 O 2 , NO•, and ONOO – . 

–• 

The release of O 2 from neutrophils after stimulation with phorbol myristate acetate was sensitively 

followed by the microtiter plate assay with BESSo. Its acetoxymethyl derivative BESSo-AM (Chart 1) is 

useful for the measurement of intracellular O –• 2 . By fluorescence microscopy with this probe, O –• 2 generated 

in human Jurkat T cells stimulated with butyric acid was clearly visualized. 

References: [1] H. Maeda et al., Angew. Chem. Int. Ed., 43 (2004) 2389. [2] H. Maeda et al., J. Am. Chem. Soc. 127 

(2005) 68. [3] H. Maeda et al., Chem. Eur. J. 13 (2007) 1946. [4] H. Maeda et al., Angew. Chem. Int. Ed. 44 (2005) 

2922. [5] H. Maeda et al., Angew. Chem. Int. Ed. 45 (2006) 1810. 

42


LECT-29 

Novel fluorescent probes for lipids and lipases 

Albin Hermetter 

Graz University of Technology, Institute of Biochemistry, A-8010 Graz (Austria). 

E-mail: 

Glycerolipids are components of cell membranes, intracellular lipid droplets and extracellular lipoproteins. 

Modifications of these biomolecules, e.g. due to hydrolytic degradation or oxidation, modulate their 

multiple functions as supramolecular building blocks, second messengers and metabolites. The focus of this 

presentation will be on novel fluorescent lipid analogs for monitoring the processes of lipid modification, 

the properties of the modified lipids and the lipid-specific enzymes involved. 

For high-throughput screening for lipases and phospholipases, we have developed fluorogenic substrate 

analogs containing a fluorophore and a quencher fatty acid. [1] Under the influence of the lipolytic enzymes, 

the labeled fatty acids are released from the glycerol backbone. This leads to a time-dependent increase in 

fluorescence intensity reflecting the progress of lipolysis. 

For discovery and identification of the lipolytic enzymes, we have established a library of substrate 

analogues including fluorescent phosphonate inhibitors. [2] These probes react specifically, 

stoechiometrically, and covalently with the active sites of hydrolases. After protein separation by 2-D gel 

electrophoresis, the fluorescent enzyme-lipid complexes can be detected by laser scanning followed by 

identification using HPLC-MS/MS. The probes the identification of the lipolytic proteomes of mouse 

adipose tissue, liver, liver subfractions, and other samples of animal or microbial origin. The probes also 

can be used for enzyme screening in protein chip technology. [3] Active lipases can be identified using a 

fluorescent inhibitor, whereas substrate selectivity can be analyzed by screening an array of unlabeled 

inhibitors with a fluorescently labeled enzyme. 

For monitoring free radical-mediated oxidation of lipids, we established continuous fluorescence assays 

based on diphenylhexatriene-labeled lipid analogs. [4] These probes show the same oxidation susceptibility 

as compared to natural poly-unsaturated fatty acids. Lipid oxidation and its inhibition by antioxidants can 

be determined from the time-dependent decrease in fluorescence in biological samples such as serum, 

membranes, lipoproteins and food samples. 

Fluorescent lipid analogs were developed to determine the uptake, localization and biological targets of 

phospholipid oxidation products in cultured cells. [5] In a fluorescence microscopy study, we identified the 

subcellular localizations of these compounds after costaining with organelle-specific fluorophores. In order 

to identify the primary molecular targets of oxidized phospholipids containing amino reactive groups, the 

proteins of the labeled cells were separated by 2-D gel electrophoresis followed by imaging of the 

fluorescent lipid-protein complexes. MS/ MS analysis revealed the identity of the labeled proteins which 

may be considered potential initiation sites of oxidized lipid signalling. 

In summary, we have established a fluorescence-based platform for the screening of glycero(phospho)lipid 

modification and the identification of the involved proteins. This task has been achieved by combining the 

techniques of fluorescence spectroscopy, lipid chemistry and protein analysis. The strategies described here 

are not restricted to lipid-associated proteins. They can of course be extended to other examples of 

functional proteomics, provided useful molecular models for the interactions of the proteins with their small 

substrates or ligands are available. 

References: [1] R. Birner-Grünberger et al., in: Enzyme Assays and Enzyme Profiling, J.-L. Reymond (ed), Wiley- 

VCH, Weinheim, Germany, 2006, p. 241ff. [2] R. Birner-Gruenberger et al., Mol. Cell. Proteomics 4 (2005) 1710. 

[3] H. Schmidinger et al. ChemBiochem 7 (2006) 527. [4] G.O. Fruhwirth et al., Anal. Bioanal. Chem. 384 (2006) 

703. [5] A. Moumtzi et al., J. Lipid Res. 48 (2007) 565. 

43


LECT-30 

New fluorophores for wavelengths beyond 900 nm 

Gabor Patonay, Lucjan Strekowski, Maged Henary and Jun-Kim Seok 

Department of Chemistry, Georgia State University, Atlanta, GA 30303 (USA) 

E-mail: 

Near-Infrared (NIR) absorbing chromophores have been used extensively in analytical and bioanalytical 

chemistry, in areas such as determination of properties of biomolecules including DNA sequencing, 

immunoassays, capillary electrophoresis (CE) separations, etc. The major analytical advantage of these 

dyes is the low background interference afforded by the NIR spectral region and their high molar 

absorptivities. In addition, NIR chromphores that do not possess chirality can exhibit induced circular 

dichroism (CD) upon binding to biomolecules. Most of the NIR dye applications utilize dyes that absorb in 

the 680-800 nm range. Advanced dye synthesis has allowed the design of highly stable long wavelength 

(900 nm and beyond) NIR chromophores. These dyes open up new analytical applications and they are 

promising fluorophores for bioanalytical use by moving detection further out in the NIR where fewer 

naturally occurring fluorophores contributing to the background. 

The extension of a polymethine chain of a cyanine by one vinyl (-CH=CH-) group results in a 

bathochromic shift of about 100 nm. Unfortunately, starting with nonamethine cyanines there is a 

substantial decrease in stability. On the other hand, a largely neglected fact is that the extended conjugation 

within each of the terminal heterocyclic subunits of a cyanine also contributes significantly to the desired 

bathochromic shift. Trimethylene-bridged heptamethines substituted with polybenzo-fused heterocyclic 

subunits have been synthesized in our labs. The large heteroaromatic subunits and the trimethylene bridge 

at the heptamethine chain give rise to low rates of internal conversion and cis/trans photoisomerization due 

to a reduced number of vibrational degrees of freedom. This, in turn, results in an increased quantum yield 

of fluorescence and a longer lifetime of fluorescence and increased stability. 

HO 

N 

I 

O 

Cl 

NaO 

N 

O 

The NIR dyes that typically absorb in the 900-1200 nm range can 

be used like their shorter wavelength counterparts, dependent on 

their functional moieties. For example, to be used as a covalent 

label, an NHS-ester or –SCN moiety could be introduced, or pH 

sensitivity can be achieved by replacing the central –Cl by –OH. 

Due to the lower excitation energy, these probes typically exhibit 

larger wavelength changes upon interacting with the analyte. This 

presentation will discuss different synthetic approaches to 

obtaining these fluorophores. 

Several analytical applications of these fluorophores will be presented; these vary from simple probe 

applications to detect pH changes to detection of the presence of metal ions for covalent and non-covalent 

labeling applications. Due to the hydrophobic nature of NIR chromophores non-covalent labeling may be a 

viable alternative. Typical dye structures that exhibit large binding constants to biomolecules will be 

compared in order to characterize non-covalent applications. In addition several other examples will be 

presented to illustrate the utility of NIR dyes in other applications. 

References: [1] S. A. Hilderbrand et al., Bioconjugate Chem. 16 (2005) 1275-1281. [2] N. Narayanan et al., 

J. Org. Chem. 62 (1997) 9387. [3] J.C. Mason et al., Heterocycl. Commun. 3 (1997) 409-411. [4] G. Patonay 

et al. Molecules 9 (2004) 40-49. 

44


LECT-31 

Luminescent Au(I) complexes: implications for sensors 

M. Cristina Lagunas 

School of Chemistry and Chemical Engineering, Queen’s University Belfast, Stranmillis Rd., 

Belfast BT9 5AG (UK). E-mail: 

The emissive properties of many d 10 -metal complexes are often influenced by the presence of metal⋅⋅⋅metal 

or metallophilic interactions, which are particularly strong in the case of Au(I). This phenomenon can be 

exploited for the development of luminescent sensors or ion probes, 1 i.e., by favouring or restricting 

metallophilic contacts as a response to an analyte, the emission of Au(I) compounds can be switched ‘on’ or 

‘off’. We have shown that the use of diphosphines with various bite angles and flexibilities allows some 

control over the Au⋅⋅⋅Au distance in dinuclear Au(I) complexes, 2 and that these interactions can be strong 

enough to also exist in solution. 3 

In order to evaluate their 

potential as sensors, we have 

explored the changes in the 

optical properties of the 

complexes when exposed to 

other d 10 metals and/or 

solvents. 4 One example is 

shown on the right, where 

Au⋅⋅⋅Au interactions are 

favoured by addition of Cu(I), 

thus trigering a luminescent 

response. 

N 

C 

C 

Au 

Ph 

Ph 

P 

O 

Ph 

P 

Ph 

Au 

C 

C 

N 

Cu + 

r.t. 

Ph 

Ph 

P 

O 

Au 

P 

Au 

Ph 

Ph 

77 K 

r.t. 

N 

Cu 

N 

Ph 

Ph 

P 

Au 

N 

Cu 

N 

O 

Au 

P 

Ph 

Ph 

2+ 

References: [1] V. V.-W. Yam et al., Angew. Chem. Int. Ed. 37 (1998) 2857; V. V.-W. Yam et al., Dalton Trans. 

(2003) 1830; C.-K. Li et al., Inorg. Chem. 43 (2004) 7421. [2] A. Pintado-Alba et al., Dalton Trans. (2004) 3459. 

[3] H de la Riva et al., Chem. Commun. (2005) 4970. [4] H. de la Riva et al. Inorg. Chem. 45 (2006) 1418. 

45


LECT-32 

Stimuli-responsive photoluminescent polymer blends 

Jill Kunzelman, Brent R. Crenshaw, Christoph Weder 

Case Western Reserve University, Department of Macromolecular Science and Engineering, Cleveland, 

OH 44106-7202 (USA). E-mail: 

An overview of a new technology platform for the design of chromogenic polymer materials with “selfassessing” 

capabilities will be presented. Cyano-substituted oligo(p-phenylene vinylene)s (cyano-OPVs) 

are members of a family of photoluminescent (PL) dyes that exhibit strong tendencies toward excimer 

formation and charge-transfer complexes. As a result the emission and/or absorption color of the dye 

molecules can strongly depend on the extent of their aggregation. 

This effect is used for the design of molecular sensors that are easily integrated into a polymer of interest 

and allow one to monitor mechanical deformation, exposure above a threshold temperature, or exposure to 

moisture. Small amounts of the sensor molecules are blended with conventional host polymers. The phase 

behavior and nano-scale structure of the resulting blends or nanocomposites is responsive to external 

stimuli such as temperature, mechanical deformation, or moisture which can cause a pronounced, easy-todetect 

variation of the fluorescence and/or absorption color of the sensor molecules. 

For example, phase-separated systems with nanoscale dye aggregates can be produced by quenching meltprocessed 

blends of semicrystalline polymers and cyano-OPVs. Mechanical deformation of these blends 

leads to a pronounced change of the materials PL and/or absorption characteristics. The inverse mechanism, 

i.e. kinetically trapping molecular mixtures of cyano-OPVs and amorphous host materials in a 

thermodynamically unstable glassy state, which spontaneously phase separates when the material is heated 

above its glass transition. 

A similar mechanism can be applied to hygroscopic polyamides where exposing an initially quenched blend 

to water would plasticize the polyamide, and trigger the self-assembly of sensor molecules. The sensing 

approach bears significant potential for exploitation in safety and security applications, for example lowcost 

materials with built-in indicators that provide an early failure warning, evidence for tampering, or 

exposure to inappropriate temperatures or moisture. 

Pictures of stimuli-responsive cyano-OPV/polymer blends 

exposed to (a) mechanical deformation, (b) temperatures 

above the polymer’s T g , and (c) water. Blends undergo PL 

color changes due to a difference in the extent of 

aggregation of the dye molecules upon exposure to stimuli. 

The samples are shown under illumination with UV light of 

a wavelength of 365 nm. 

References: [1] M. Kinami et al., Chem. Mater. 18 (2006) 946. [2] B. R. Crenshaw, C. Weder, Macromolecules 39 

(2006) 9581. [3] B. R. Crenshaw et al., Macromol. Chem. Phys. 208 (2007) 572. [4] B. R. Crenshaw et al., 

Macromolecules 40 (2007) 2400. [5] J. Kunzelman et al., Macromol. Rapid Commun. 2006, 27, 1981-1987. 

46


LECT-33 

Two-photon excitation fluorescence bioassays 

Pekka E. Hänninen, Marko Tirri, Janne O. Koskinen, Teppo Stenholm, Rina Wahlroos 

and Juhani T. Soini 

University of Turku, Institute of Biomedicine, Laboratory of Biophysics, 

Tykistökatu 6, FIN-20520 Turku (Finland). E-mail: 

As two-photon excitation (TPE) was first introduced to biosciences in 1990 by Denk, Strickler and Webb in 

their well cited article[1], expectations were high. The technique could essentially replace confocal 

microscopy, at least when using UV dyes or when specimen are dense. Also, the manufacturers reacted 

quick, and commercial instruments rapidly appeared on the market. The commercialization very quickly 

spread the technique around the world. Although successful in microscopy, the number of TPE applications 

on other fields of biosciences have remained low – mainly due to lack of suitable instrumentation. 

Two-photon imaging requires an ultra-fast pico- or femtosecond pulsed laser for reasonable image 

acquisition times. Such a device is usually not very small in size nor has a low price tag making it difficult 

to construct a low-cost laboratory device. When this recording speed requirement is removed, much simpler 

lasers may be opted. With these low-cost lasers other positive features of two-photon excitation may be put 

to use, features that have little or no meaning in imaging applications: TPE instrument can be built to be 

simple and robust, and the technique has practically no instrumental background making TPE an excellent 

technique for measurements requiring high sensitivity. 

A laboratory plate-reader instrument was 

developed based on a pulsed microchip laser 

operating at 1.064 µm [2]. The basic idea of the 

instrument is, similarly to a common flow 

cytometer, record particle (cell, carrier microparticle 

etc.) triggered fluorescence signals as the 

particles of interest enter the focal volume. The 

instrument includes detection channels for 

fluorescence and microparticles, an optical scanner 

and xy-plate scanner. 

This TPX- Plate Reader can be utilized in fluorescence based immunoassays[3], cellular assays as[4] well 

as in different types of research assays[3]. Recent efforts have concentrated on the development of a 

method for detection of important infectious pathogens[4,6]. The lecture will focus on the steps of 

developing the TPX-technology and will also present the latest results on TPX-bioassays. 

References: [1] Denk et al., Science 248 (1990) 73; [2] Hänninen et al., Nature Biotechnol. 18 (2000) 548; 

[3] Waris et al., Diabet Med. 22 (2005) 1123; [4] Stenholm et al.; manuscript submitted (2007). [5] Vaarno et al., 

Nucleic Acids Res. 32 (2004) e108. [6] Koskinen et al., JCM, manuscript in revision (2007). 

47


LECT-34 

Fluorescence correlation spectroscopy in cells and delevoping embryos 

Petra Schwille 

TU Dresden, Biophysics/BIOTEC, Tatzberg 47-51, D-01307 Dresden (Germany). 

E-mail: 

Cell and developmental biology are immensely complex and rapidly growing fields that are particularly in 

need of quantitative methods to determine their key processes. With all the data known about protein 

interactions and interaction networks from biochemical analysis, there still remains the important task of in 

situ proteomics, i.e. determining the thermodynamic and kinetic parameters of certain reactions in the 

cellular environment. Further, to understand how cells polarize and develop into organisms, we need 

quantitative methods to determine concentration gradients and diffusion coefficients of key factors such as 

morphogens. 

Fluorescence Correlation Spectroscopy (FCS) is a powerful means for the study of concentrations, 

translocation processes, molecular association or enzymatic turnovers. It is fair to state that this technique 

raises strong hopes for the possibility of in situ proteomics, but also for a more quantitative access to 

developmental processes. We have applied FCS to a variety of cell-associated phenomena, among them 

protein-protein binding, enzymatic reactions, endocytosis, and gene delivery. To study processes on cell 

membranes, and to elucidate the delicate interplay between membrane proteins and the surrounding lipids, 

we devised cell-like model membrane systems mimicking the formation of membrane domains whose 

cellular counterparts are potentially active as recruitment platforms for signalling proteins. 

We have established one- and two-photon scanning FCS for processes on membranes which are too slow 

for standard FCS observation with a fixed beam. Performing circular scanning FCS on developing embryos 

of C.elegans, we show how the motion of labelled proteins is non-uniformly distributed in the cortex during 

cell polarization. Additionally, scanning FCS overcomes the problems of photobleaching and low statistical 

accuracy commonly encountered in FCS with fixed measurement volume, when applied to slowly moving 

molecules. By using two-photon excitation one additionally benefits from the possibility of long 

measurement times without disturbing the embryo development. 

48


LECT-35 

Europium bimetallic helicates as luminescent stains for in vitro imaging 

of cancerous cells 

Jean-Claude G. Bünzli, Anne-Sophie Chauvin, Caroline C. B. Vandevyver, 

Bo Song, and Steve Comby 

École Polytechnique Fédérale de Lausanne (EPFL), Laboratory of Lanthanide Supramolecular Chemistry, 

BCH 1402, CH-1015 Lausanne (Switzerland); E-mail: 

Lanthanide ions have stirred a new incentive in the development of luminescent molecular probes because 

of their peculiar properties including easily recognizable line-like emission spectra, long excited-state 

lifetimes allowing time-resolved measurements, and large Stokes shifts upon ligand excitation. [1],[2] They 

are now commonly used as luminescent responsive probes in immunoassays and for the quantitative 

determination of analytes present in biological fluids. Building on these applications, the next step is to 

integrate them into luminescent stains for bio-imaging purposes. [3] To date however, only a few labels are 

available, presumably in view of the numerous requirements to be fulfilled by lanthanide probes for timeresolved 

applications in vitro and in vivo. The latter include featuring groups suitable for their conjugation 

to bio-specific probes, thermodynamic stability, kinetic inertness, and large sensitization of the metalcentered 

luminescence. In recent years, we have been tailoring homo- and hetero- bimetallic helicates with 

the final purpose of designing bi-functional lanthanide probes. [4],[5] 

In this presentation, we report the properties of triple-stranded 

R 

R = 

R' 

N 

OH 

O 

N 

N 

O 

O 

L 

O 

[Eu 2 (L) 3 ] bimetallic helicates in in vitro conditions, their 

interaction with the human cervical adenocarcinoma cell line 

HeLa [6] and MCF-7 and we assess their ability for the imaging of 

these cells. The molecular design of the ligands relies on a 

ditopic hexadentate receptor. To enhance the solubility of the 

neutral helicates, polyoxyethylene arms are grafted in the para 

position of the pyridine ring and will lend themselves to 

numerous future derivatizations for biological coupling and 

targeting. 

The helicates are shown to be robust under physiological 

conditions, even in presence of excesses of edta, dtpa, citrate, 

ascorbate, or zinc. Their photophysical properties are very good, with quantum yields up to 18 % and 

lifetimes up to 2.4 ms. In addition the ligand sensitizes the luminescence of other lanthanide ions such as 

Sm, Tb and Yb. 

Staining of Hela cells with the Eu helicate. The 

cells were incubated in the presence of different 

concentrations of the complex in RPMI-1640 for 

6 hrs at 37°C. The images were taken using a 

Zeiss LSM 500 META confocal microscope 

(Objective: Plan-Apochromat, 63/1.30 oil ; Eu III 

luminescence excited at 405 nm and detected 

after filtration with a LP 505 filter). Scale bar: 17 

µM. 

N 

O 

N 

N 

OH 

R' 

O R'' 

R 

250 μM 125 μM 50 μM 

References: [1] J.-C. G. Bünzli, Acc. Chem. Res. 2006, 39, 53. [2] J.-C. G. Bünzli, C. Piguet, Chem. Soc. Rev. 2005, 

34, 1048. [3] S. Pandya, J. H. Yu, D. Parker, Dalton Trans. 2006, 2757. [4] M. Elhabiri et al. J. Am. Chem. Soc. 1999, 

121, 10747. [5] T. B. Jensen et al. Inorg. Chem. 2006, 45, 7806. [6] C.D.B. Vandevyver et al., Chem. Commun. 2007, 

1716. 

49


LECT-36 

Upconversion emission in colloidal solutions of lanthanide-doped nanocrystals 

Helmut Schäfer, Pavel Ptacek, Anja Hischemöller, Claudia Walter, 

Karsten Kömpe, Markus Haase 

University of Osnabrueck, Institute of Chemistry, D-49076 Osnabrück (Germany) 

E-mail: 

Colloidal solutions and re-dispersible powders of NaYF 4 : Yb, Er and NaGdF 4 : Yb, Er nanocrystals have 

been prepared in high-boiling coordinating solvents by different methods. All procedures yield gram 

amounts of highly crystalline nanoparticles which display upconversion emission in colloidal solution. The 

efficiency of the upconversion emission strongly depends on the size, the crystal phase and surface 

properties of the nanocrystals. A procedure providing water-soluble particles will be presented which has 

been used to apply the upconversion nanocrystals as luminescent biolabels. In addition, the growth process 

of the nanocrystals in colloidal solution and the optical properties of europium-doped samples will be 

discussed. 

50


LECT-37 

Upconversion-based enzyme assay using quenched substrate 

Tero Soukka, Marja-Leena Järvenpää, Terhi Rantanen, Katri Kuningas, Timo Lövgren 

University of Turku, Department of Biotechnology, FIN-210014 Turku (Finland). 

E-mail: 

Internally quenched dual-labeled fluorescent peptide and oligonucleotide substrates are frequently used for 

measurement of activity of specific proteases or nucleases and for screening their potential inhibitors. 

Sample autofluorescence, which commonly limits the applicability of conventional fluorophores, can be 

excluded by using upconverting phosphor (UCP) reporters emitting green or red anti-Stokes 

photoluminescence upon excitation at near-infrared [1] . UCPs can be used as donors in fluorescence 

resonance energy transfer (FRET) to conventional acceptor fluorophores [2] enabling measurements also in 

strongly colored samples such as whole blood [3] . The relatively large dimensions of UCPs yet limit their 

applicability in conventionally designed fluorogenic assays, as the UCP emission cannot be entirely 

quenched, resulting in background fluorescence and a compromised limit of detection. An advanced assay 

design based on UCP in combination with a dual-labeled fluorescent substrate, whereon the cleavage of the 

substrate results in an increase in the sensitized acceptor emission, is provided to eliminate the problem. 

Upconversion-based fluorogenic model assay was constructed using quenched fluorescent 10-base 

oligonucleotide substrate containing a biotin moiety and AlexaFluor 680 (AF680) at 5’-end and BlueBerry 

Quencher 650 at 3’-end. The proportion of substrate cleaved by endonuclease was observed by measuring 

the non-quenched FRET-sensitized emission of AF680 (acceptor) after capturing the biotinylated AF680- 

labeled oligonucleotide fragments by streptavidin-coated UCP (NaYF 4 : Er 3+ , Yb 3+ ; approx. 340 nm in 

diameter; donor). The UCP was excited at 980 nm and the sensitized AF680 emission was measured 

simultaneously at 730 nm using an anti-Stokes plate fluorometer [1] . The upconversion-based assay was 

performed in two stages to avoid the reduced enzyme activity with UCP bound substrate; the enzyme 

reactions were first incubated without UCP. 

Maximal signal-to-background ratio up to 20 was achieved for completely cleaved 2 nmol/L substrate 

compared to intact substrate. Dynamic range of the assay for the endonuclease concentration was over one 

decade and limit of detection of the cleaved substrate less than 100 pmol/L measured both in buffer and in 

presence of 20% whole blood. The presented assay design is advantageous for fluorogenic assays based on 

particulate photoluminescent donor, as also the background emission originating from the radiative energy 

transfer (reabsorption of the donor emission by an acceptor) was eliminated. 

The assay design and upconversion FRET described can facilitate enzyme activity assays using quenched 

fluorescent substrate even in whole blood, where conventional fluorescence based assays provide limited 

success due to autofluorescence and sample absorption. The sensitized emission of the acceptor is measured 

free of directly excited acceptor emission and scattered excitation light. In addition, as an alternative to 

biotin-streptavidin interaction, the capture of the acceptor-labeled substrate can be based on recognition of 

terminal oligonucleotide or peptide sequence. 

References: [1] T. Soukka et al., J. Fluoresc. 15 (2005) 513-528. [2] K. Kuningas et al., Anal. Chem. 77 (2005) 

7348-7355. [3] K. Kuningas et al., Clin. Chem. 53 (2007) 145-146. 

51


LECT-38 

Rational design of fluorescent Zn(II) sensors for two-photon 

excitation microscopy 

Christoph J. Fahrni, S. Sumalekshmy, Yonggang Wu, Maged M. Henary, 

PaDreyia Lawson, Jean-Luc Brédas, Nisan Siegel, Joseph Perry 

Georgia Institute of Technology, School of Chemistry and Biochemistry, Petit Institute of 

Bioengineering and Bioscience, 901 Atlantic Drive, Atlanta, GA 30332-0400 (USA) 

E-mail: 

Two-photon laser scanning microscopy (TPM) is increasingly utilized in biological research; however, 

traditional fluorophores used in non-linear optical microscopy are not optimized for efficient two-photon 

excitation. Their brightness is typically compromised due to a small two-photon absorption (TPA) cross 

section. To design a Zn(II)-selective fluorescent probe optimized for TPM imaging, we explored the utility 

of an oxazole donor-acceptor system in which the metal binding moiety was attached to the electron 

accepting site. Saturation of the probe with Zn(II) under simulated physiological conditions resulted in a 

red-shifted emission maximum accompanied by a significantly increased TPA cross section. Two-photon 

imaging experiments demonstrated that the probe was readily taken up by live cells, thus giving rise to 

bright intracellular staining. The modular fluorophore architecture readily allowed for tuning of the Zn(II) 

binding affinity from the micromolar to nanomolar concentration range. The overall design approach 

should be readily adaptable for the development of other cation-selective fluorescent sensors with improved 

non-linear optical sensitivity for two-photon imaging microscopy. 

52


LECT-39 

Recent progress in time-resolved methods for diagnostics and functional assays 

Ilkka Hemmilä, Ville Laitala, Janne Ketola, Jari Peuralahti, Lassi Jaakkola, 

Veli-Matti Mukkala 

University of Turku, Department of Biochemistry, and PerkinElmer LAS, Wallac Oy, 

P.O. Box 10, FIN 20101 (Finland). E-mail: 

Time-resolved fluorometry has become a standard way to improve assay sensitivity and robustness through 

temporal discrimination of autofluorescence background. Temporal resolution in emission detection also 

creates an efficient way to measure homogeneous assay technologies as well as multiplexing. Lanthanide 

chelates probes with long-decay luminescence emission have proven successful in diagnostic and research 

applications. Wide variety of chelate based labels have been developed for that purpose, based both as 

single soluble complexes or as complexes embedded into nanobeads. 

The aim of the present work is to further develop lanthanide chelate labels and assay technologies by 

improving signal strength in direct binding assays, such as immunoassays or immunocytometry, or by 

improving their energy donating properties in respect to spectral features, temporal behavior, avoiding cross 

talk and improving their biocompatibility and stability. 

We used aza crown as a flexible backbone to study the effects of substituents on the critical features using 

pyridine or furane derivatives as the light harvesting antenna moieties. The symmetrical derivatives produce 

optimal emission profile allowing high spectral resolution, and substituents on the aromatic part had a 

substantial effect on excitation maximum, intensity, and FRET. 

The novel chelates are evaluated in various cell signaling assays including protein-protein interactions, 

receptor activation, and kinase cascade. Exploitation of different lanthanides, and their excited energy 

levels allow, not only sensitive analytical assay, but also multiplexing, providing versatile tools for 

bioanalytical assays. 

53


LECT-40 

Nuclear receptor interactions studied by fluorescence approaches 

Catherine A. Royer 

INSERM U554, Centre De Biochimie Structurale, F-34090 Montpellier (France) 

E-mail: 

Nuclear receptors constitute a large family of eukaryotic transcriptional regulators that modulate gene 

expression in response to the binding of specific hydrophobic ligands. One large subfamily is represented 

by the hormone receptors such as Estrogen receptor, androgen receptor and glucocorticoid receptor. 

Another subfamily is the retinoïX receptor family whose members form heterodimers with the RXR 

receptor and modulate transcription upon binding fatty acids, retinoic acids and bile acids. 

The activity of both subfamilies has been implicated not only in homeostasis, development and growth, but 

also in a number of human pathologies such as certain cancers (breast, prostate, uterine, leukaemia) and in 

metabolic diseases such as type 2 diabetes and obesity. Their action is mediated by multi-functional coregulator 

proteins that act on chromatin structure and recruit the transcription machinery. We have sought 

to understand the molecular basis for the ligand dependent specificity of interactions within this family 

using fluorescence based techniques both n vitro and in live cells. 

54


LECT-41 

Design and control of unidirectional multistep energy transfer through 

individual molecular photonic wires 

Mike Heilemann, Robert Kasper, Philip Tinnefeld, Markus Sauer 

Applied Laser Physics and Laser Spectroscopy, Bielefeld University, Universitäts-Str. 25, 

D-33615 Bielefeld (Germany). E-mail: 

1. Nanometer scale optical architectures are of great interest as photonic and electronic devices with 

potential applications in dense optical circuits, optical data storage and materials chemistry. While classical 

optical waveguides rely on propagating modes in the far field, nanometer-sized molecular photonic devices 

guide light via near-field interactions of molecules in close proximity. That is, molecular photonic wires 

transfer light via electronic excitation transfer (EET). On the level of nanometer-sized molecular devices 

the transport of excitation energy is advantageous because it circumvents the connection problem present in 

electric wires, i.e. the bottleneck that occurs when trying to connect molecular with macroscopic devices. In 

the case of a molecular photonic wire excited state energy is induced into an input unit by means of light, 

transported through transmission elements, and finally emitted at another wavelength and location by an 

output unit or the energy is used for an electron transfer reaction, i.e. the conversion of excited state energy 

into an electric charge with the possibility for subsequent chemical reactions. 

2. We present our efforts to synthesize and study DNA-based molecular photonic wires that carry 

several chromophores arranged in an energetic downhill cascade and exploit fluorescence resonance energy 

transfer (FRET) as transport mechanism. As increasing heterogeneity is coming along with the complexity 

of the supramolecular devices, we adopt single-molecule fluorescence spectroscopy (SMFS) to dissect the 

intricate relationship between structure and function as well as to evaluate different sources of 

heterogeneity. The developed strategy enables detailed measurements of energy transfer between up to five 

individual chromophores along a distance of ~14 nm with an overall efficiency of up to 90%. 

Immobilization under conditions also relevant for biomolecular single-molecule studies provides the basis 

for minimization of heterogeneity and points towards new approaches for controlling conformational 

flexibility of complex multichromophoric systems. 

References: [1] M. Heilemann et al., J. Am. Chem. Soc. 126 (2004) 6514; [2] P. Tinnefeld, M. Sauer, Angew. Chem. 

Int. Ed. 44 (2005) 2642. [3] P. Tinnefeld et al., ChemPhysChem 6 (2005) 217; [4] M. Heilemann et al., J. Am. Chem. 

Soc. 128 (2006) 16864. 

55


LECT-42 

Probing DNA conformation and DNA-enzyme interaction by 

time-resolved fluorescence 

Anita C. Jones, Robert K. Neely, Eleanor Y. M. Bonnist and David T. F. Dryden 

School of Chemistry and Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre 

(COSMIC), Univ. of Edinburgh, Edinburgh EH9 3JJ, UK. E-mail: 

The dynamic behaviour of the DNA bases plays an important role in processes that are critical to the 

maintenance and function of the duplex, including electron transport and many fundamental DNA-enzyme 

interactions. The conformational properties of DNA can be probed using the fluorescent adenine analogue, 

2-aminopurine (2AP). 2AP forms Watson-Crick base pairs with thymine and, therefore, does not disrupt the 

DNA double helical structure. The absorption maximum of 2AP (~305 nm) is red-shifted relative to the 

natural bases, allowing selective excitation, and its fluorescence properties are sensitive to the local 

molecular environment. 

We have used time-resolved fluorescence measurements of 2AP-labelled DNA, in solution, single crystals 

and frozen matrices at 77K, to investigate the influence of base dynamics on the populations and properties 

of the conformational states of the duplex. Measurements on rigid duplexes at 77K [1] reveal that the 

predominant conformation of the duplex in solution at room temperature can be attained only through 

thermal motion of the bases, it is not a minimum energy structure of the duplex. This conformation does 

not, therefore, correspond to the duplex geometry that we perceive from low temperature crystal structures. 

The DNA duplex undergoes conformational change in response to interaction with agents such as enzymes 

and drugs. A particularly remarkable example of localised conformational distortion is the phenomenon of 

nucleotide flipping, induced by DNA methyltransferase enzymes. This involves 180 o rotation of the target 

nucleotide around the phosphate backbone, out of the DNA helix and into the reactive site of the enzyme. 

Time-resolved fluorescence measurements 2AP-labelled DNA duplexes complexed with methyltransferase 

enzymes, in single crystals and in solution, 

have allowed us to explore in detail the 

nature of the interaction between enzyme 

and duplex and the conformational 

properties of the nucleotide-flipped 

complex. [2,3] The picture on the right 

shows the crystal structure of a 

methyltransferase enzyme bound to a 

synthetic DNA duplex in which 2AP is at 

the target site. The 2AP nucleotide, shown 

in yellow, is flipped out of the DNA 

duplex into the catalytic cleft of the 

enzyme. The fluorescence decaycurve of 

the flipped-out 2AP in this crystalline 

complex is shown in yellow, in 

comparison with the decay of unflipped, 

intrahelical 2AP (red). 

References: [1] R.K. Neely, A.C. Jones, A.C., J. Am. Chem. Soc. 128 (2006) 15952. [2] R.K. Neely et al., Nucleic 

Acids Res. 33 (2005) 6953. [3] T. Lenz et al., J. Am. Chem. Soc. 129 (2007) 6240. 

56


LECT-43 

Fluorescent DNA base modifications and surrogates: 

Synthesis and optical properties 

Hans-Achim Wagenknecht 

University of Regensburg, Institute of Organic Chemistry, D-93040 Regensburg (Germany). 

E-mail: 

Fluorescent probes that are sensitive to the local environment within DNA duplexes represent important 

tools for DNA hybridization and for the detection of physiologically important DNA base mismatches or 

lesions. As a consequence, there is an continuously increasing demand for fluorescent DNA modifications 

having a clear and specific range of spectral characteristics. Ways to create such DNA assays include either 

the replacement of DNA bases by chromophores or the attachment of fluorophores to common DNA bases. 

We have synthesized new photochemical DNA assays in order to investigate the mechanism of 

photoinduced charge transfer processes in DNA. A variety of fluorescent DNA base modifications or 

surrogates have been applied as charge donors. They can photoinitiate electron transfer through the DNA 

which results in a characteristic modulation or quenching of the emission. We have shown that fluorescence 

quenching via photoinduced charge transfer between ethidiums in a fluorescent artificial DNA base and 7- 

deazaguanine is significantly different in the presence of a single intervening single base mismatch. 

With respect to potential applications in DNA analysis, one goal of our research efforts is to enhance and 

modulate the fluorescence properties by the incorporation of several adjacent fluorophores into DNA. Most 

importantly, the sequence-selective base-pairing properties of such modified oligonucleotides should be 

maintained in order to apply them as probes in molecular diagnostics with DNA. One suitable and 

important way to fulfil these requirements is to attach chromophores covalently to natural DNA bases. 

Recently, we functionalized DNA duplexes with up to five adjacent pyrene (Py-U) moieties. A helical and 

regularly structured -stack can only be formed if more than three chromophores are synthetically 

incorporated into the oligonucleotide. The DNA systems with five adjacent Py-U units showed a 

remarkably strong fluorescence enhancement that is sensitive to DNA base mismatches and thermal 

denaturation of the duplex. 

H 

N 

O 

O 

O 

N 

O O 

N 

O H 

Et 

Pe 

+ 

N 

O 

N 

O 

NH 2 

NH 2 

O 

O 

O 

In 

O 

H 

N 

N O 

O O 

O 

Py-dU 

O 

O 

N 

H 

O 

O 

To 

+ 

N 

S 

N CH 3 

References: Reviews: [1] H.-A. Wagenknecht (Ed.), Charge Transfer in DNA, Wiley-VCH, Weinheim, 2005. 

[2] H.-A. Wagenknecht, Nat. Prod. Rep. 23 (2006) 973. [3] H.-A. Wagenknecht, Angew. Chem. Int. Ed. 45 (2006) 

5583. [4] H.-A. Wagenknecht, Curr. Org. Chem. 8 (2004) 251. [5] H.-A. Wagenknecht, Angew. Chem. Int. Ed. 42 

(2003) 3204. [6] H.-A. Wagenknecht, Angew. Chem. Int. Ed. 42 (2003) 2454. Research papers: [3] L. Valis et al., 

Proc. Natl. Acad. Sci.103 (2006) 10192. [4] C. Wagner, H.-A. Wagenknecht, Org. Lett. 8 (2004) 4191. 

[5] C. Wanninger, H.-A. Wagenknecht, Synlett 2006, 2051. [6] J. Barbaric, H.-A. Wagenknecht, Org. Biomol. Chem. 

4 (2006) 2088. [6] L. Valis et al., Bioorg. Med. Chem. Lett. 16 (2006) 3184. [7] E. Mayer-Enthart, H.-A. 

Wagenknecht, Angew. Chem. Int. Ed. 45 (2006) 3372. [8] L. Valis et al., Org. Biomol. Chem 3 (2005) 36. 

57


LECT-44 

Insights into the spectral versatility of fluorescent proteins 

by single molecule spectroscopy 

Christian Blum 1 , Alfred Meixner 2 , Vinod Subramaniam 1 

1 Biophysical Engineering Group, University of Twente, 7500 AE Enschede (The Netherlands) 

2 Institut für Physikalische und Theoretische Chemie, University of Tübingen, D-72076 Tübingen 

(Germany). E-mail: 

The palette of genetically-encodable fluorescent proteins for in vivo cellular labelling is constantly 

growing, both by mutagenesis of known proteins and by discovery of new fluorescent proteins in different 

species. By now a range of proteins emitting from the blue to the far red is at hand for applications in cell, 

molecular and developmental biology. Despite the widespread use of fluorescent proteins as reporters and 

sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense 

research. Understanding the photophysics of these reporters is essential for interpretation of the processes 

illuminated by the fluorescent proteins as well as for the development of biosensors based on fluorescent 

proteins. 

We used spectrally resolved single molecule spectroscopy to analyse aspects of fluorescent protein 

photophysics that are not accessible by conventional ensemble spectroscopy. We were able to identify and 

characterize different sub-ensembles and spectral forms of a range of fluorescent proteins. We could also 

follow transitions between different spectral forms on a single molecule level and draw conclusions on the 

underlying molecular origins of the various species [1]. 

The nanoenvironment the chromomophore of a fluorescent protein is defined by the sequence of the protein 

that encapsulates the chromophore. By changing the sequence of the protein the nanoenvironment of the 

chromophore can be modified. Hence, fluorescent proteins are excellent systems to analyze the interaction 

between chromophore and its nanoenvironment which is the basis of the use of single molecules as local 

nanoprobes. We find that for the fluorescent proteins studied the induced chemical variations in the 

chromophore vicinity does not play a dominant role in determining the width of the distribution of the 

single molecule emission maximum positions which is strictly correlated with the flexibility of the 

chromophore nanoenvironment [2]. 

The emission maximum positions of the 

predominant forms from the DsRed 

group of proteins were assembled into 

histograms. The width of the 

distribution is clearly characteristic for 

each variant and is correlated with the 

conformational flexibility of the 

chromophore nanoenvironment in the 

different variants. 

Further we analyzed the emission spectra from single fluorescent protein tetramers to analyze the coupling 

of different chromophores within one tetramer by fluorescene resonance energy transfer. Our results 

indicate that in the majority of the tetramers the different chromophores are indeed effectively coupled. 

However, we find that in a fraction of the tetramers which is characteristic for each analyzed variant, the 

different chromophores are not effectively coupled. For these tetramers we propose an interruption of the 

energy transfer chain within the multichromophoric system by proteins lacking a chromophore. 

References: [1] C. Blum et al., Biophys. J. 87 (2004) 4172. [2] C. Blum et al., J. Am. Chem. Soc. 128 (2006) 8664. 

58


LECT-45 

Characterization of water-soluble luminescent quantum dots by 

single molecule methods 

Chaoqing Dong, Xiangyi Huang, Huifeng Qian, Hua He, Jicun Ren 

College of Chemistry and Chemical Engineering, Shanghai Jiaotong University, 800 Dongchuan Road, 

Shanghai 200240, P. R. China. E-mail: jicunren@sjtu.edu.cn 

Quantum dots (QDs, also known as nanocrystals) are nanoscale inorganic particles composed of hundreds 

to thousands of atoms. Due to their quantum confinement of charge carriers in tiny spaces, QDs show some 

unique and fascinating optical properties, such as, sharp and symmetrical emission spectra, high quantum 

yield (QY), good chemical and photo-stability and size dependent emission wavelength tenability[1]. So 

far, QDs have been successfully used in biological systems, but some fundamental parameters and 

luminescence features are not clearly understood. In the talk, we presented some single molecule 

technologies for characterizing certain fundamental parameters of luminescent QDs synthesized in aqueous 

phase, and our work mainly includes the following aspects: 1. We presented a method for characterization 

of molecular weight, molar extinction coefficient and bright fraction of QDs by combining fluorescence 

correlation spectroscopy (FCS) with ensemble molecular spectrometry. The principle is mainly based on 

the measurements of hydrodynamic diameters of QDs and the particle number of bright QDs in a small 

illuminated volume element using FCS technique [2]. Hydrodynamic diameters of a series of CdTe QDs 

were measured with FCS and the molecular weights were calculated assuming the measured hydrodynamic 

diameters as the diameters of QDs. The molar extinction coefficients of QDs at different excitonic 

absorption peak were calculated with the molecular weights. The bright fractions of QDs samples were 

characterized by measuring the concentration of the bright QDs and the total concentration of QDs. 2. We 

developed a new method for the measurement of the surface charge of QDs by combination of FCS with 

microchip electrophoresis. The principle is based on the measurement of the hydrodynamic radii and 

mobility of water soluble QDs in solution [3]. This technique has been successfully used to determine the 

surface charge of the different stabilizer modified CdTe QDs and study their transport properties in electric 

field. We found that the surface charge of QDs was remarkably associated with the type of stabilizers on 

QDs surface, buffer pH and other factors. 3. We used a total internal reflection fluorescence microscopy 

(TIRFM) setup to visualize individual CdTe QDs, and investigated their fluorescence emission behavior. 

We found that individual CdTe QDs synthesized in mercaptopropionic acid (MPA) solution presented nonblinking 

behavior [4]. Our experiments confirmed that MPA coating on CdTe QDs played key role for 

suppressing blinking of QDs. 

References: [1] X.Y. Huang, L. Li, H.F. Qian, et al., Angew. Chem. Int. Ed., 45 (2006) 5140. [2] C.Q. Dong, H.F. 

Qian, et al., J. Phys. Chem. B, 110 (2006) 11069. [2] C.Q. Dong, H.F. Qian, et al., Small, 2 (2006) 534. [4] H. He, 

H.F Qian, et al., Angew. Chem. Int. Ed. 45 (2006) 7588. 

59

Posters 

Part I 

Fluorescence 

Spectroscopy 

61

Abstracts Poster – Part I: Fluorescence Spectroscopy 

FLUO-1 

Fluorescence behaviour of cyclodextrin inclusion complexes: 

a versatile playground in graduate education 

Rudi Hutterer 

University of Regensburg, Institute of Analytical Chemistry, Chemo- and Biosensors, 

D-93040 Regensburg (Germany). E-mail: rudolf.hutterer@chemie.uni-r.de 

Fluorescence spectroscopy plays an essential role not only at the frontiers of analytical, bioanalytical and 

biochemical sciences but also in the curriculum of graduate education for all students of chemistry, biology, 

pharmacy and so on. On the other hand, supramolecular chemistry and the study of inclusion complexes is 

an important topic. In this contribution we show how these concepts put together can be used as a lab 

period in a (bio)analytical or screening lab for advanced students. 

Cyclodextrins (CD) are a well known class of host molecules known to form inclusion complexes with a lot 

of different compounds. They consist of 6 (α-), 7 (β-) or 8 (γ-) molecules of α-D-glucose linked by α-1,4- 

glycosidic bonds forming a cavity of different size and relatively hydrophobic character, respectively. 

Reversibly bound guest molecules often show pronounced changes in properties, for example in emission 

wavelengths, quantum yields or acidity constants (pK S -values). The fluorescence behaviour of several 

fluorophores can be used to detect their ability to form inclusion complexes as a function of pH and cavity 

size and to evaluate their relative association constants using a modified Benesi-Hildebrand plot [1]. 

1 1 1 

= + 

I − I k⋅K ⋅Q k⋅ 

Q 

[ F ][ CD ] [ F ] 

0 app 0 0 0 

Q is the quantum yield, k as a constant depending on the apparatus used, I is the measured fluorescence 

intensity and I 0 the intensity in absence of the host molecule. F 0 and CD 0 are the total concentrations of 

guest and host molecule, respectively. Some fluorophores do not show any major chance in fluorescence 

intensity or emission maximum, like e.g. 9-anthracenecarboxylic acid. This can be either due to the fact that 

its emission is already quite strong in buffer, thus yielding no further increase after incorporation into the 

cyclodextrin cavity or due to a very small binding constant, i.e. hardly any formation of an inclusion 

complex. 

Other dyes, like p- or o-aminobenzoic acid show a dramatic (but pH dependent) increase of fluorescence 

and a blue shift in their emission maximum in presence of cyclodextrin, with a preference for α-CD of the 

para-isomer and for β-CD of the ortho-isomer giving insight to their different steric requirements. 

The study of week acids like 1- and 2-naphthol leads to interesting observations regarding the deprotonation 

equilibria in the excited versus ground state. While at pH values of 1.5 and 4 only the protonated 

species is expected to absorb (pK S ≈ 9.3), only the red-shifted emission from the deprotonated form is seen 

for 1-naphthol due to the huge drop of the acidity constant in the excited state. This behaviour changes, 

however, in presence of β-CD. The additional short wavelength band of the protonated species shows that 

the deprotonation is influenced by forming the inclusion complex with β-CD. These observation prepare the 

field for a discussion of both kinetic and thermodynamic effects: on the one hand the pK S * -value may 

change due to formation of the inclusion complex. On the other hand deprotonation may slow down in the 

restricted and more hydrophobic inside of the cavity, allowing fluorescence emission to take place also 

from the non-deprotonated state. 

In summary these experiments allow both qualitative screening for preferences of different fluorophores to 

form inclusion complexes considering steric and charge effects, the quantitative determination of 

association constants and an insight in excited state acid-base behaviour and its dependence on inclusion 

complex formation. 

Reference: [1] G.C. Catena et al. Anal. Chem. 61 (1989) 905. 

63


FLUO-2 

Frequency domain fluorescence lifetime study of crude petroleum oils 

Peter Owens 1 , Alan Ryder 1,2 , and Nigel Blamey 1 

1 

Nanoscale Biophotonics Laboratory, Department of Chemistry, National University of Ireland, Galway. 

E-mail: peter.owens@nuigalway.ie 

2 

National Centre for Biomedical Engineering and Science, National University of Ireland, Galway, 

Ireland. 

Petroleum oils are complex mixtures of aliphatic, aromatic, and high molecular weight organic compounds 

and due to this heterogeneity, the chemical analysis of petroleum oils is complex and time consuming. 

Fluorescence techniques have been used for many years as a fast and non-destructive tool in the analysis of 

crude oils [1]. Fluorescence lifetimes are potentially more useful for characterising crude oils as the 

measurements are relatively insensitive to intensity fluctuations, sample turbidity, and sample morphology. 

Previous studies using Time Correlated Single Photon Counting (TCSPC) methods have shown that one 

can correlate various aspects of oils composition with changes in lifetime [2,3]. However, these studies 

also showed that accurate quantitative measurements were not possible. Another complicating factor with 

TCSPC measurements was the difficulty in data analysis when fitting multi-exponential decays. 

An upright, confocal Fluorescence Lifetime Imaging Microscope (Alba system, ISS, Champaign, Illinois) 

was used to measure fluorescence lifetimes. The excitation source was a 405 nm violet diode modulated 

using a frequency synthesiser (1 to 300 MHz range) in conjunction with an RF amplifier. The detector gain 

was also modulated and the phase shift and demodulation ratios of the fluorescence emission were 

measured and used to calculate fluorescence lifetimes. In this work, we calculated the fluorescence lifetimes 

for 32 bulk crude oils for a series of wavelength ranges (426-477 nm, 465-500 nm, 480-520 nm, 510-560 

nm, 542-582 nm, 573-613 nm, and 600-650 nm). This covers most of the steady state emission spectrum for 

most crude oils. The 32 oils tested have a wide range of oil maturities (from 12 to 50 API gravity) and are 

sourced from diverse geographical locations and rock types. 

Fluorescence lifetimes are generally shorter for heavy (less mature) oils and longer for lighter (more 

mature) oils. Lifetimes also tend to increase with increasing wavelength band-pass, with lifetimes being 

shorter in the 426-477 nm bandpass than the 600-650 nm bandpass. The effect of the fitting model 

(discrete, Gaussian or Lorentzian distributions) on the values of average lifetimes obtained was compared to 

average lifetimes calculated from TCSPC data. The lifetime data was then correlated with various 

compositional measurements and with density (API gravity). We discuss the merits of using frequency 

domain lifetime data to characterise crude oil composition. 

References: [1] Analysis of crude petroleum oils using fluorescence spectroscopy. A.G. Ryder, Reviews in 

Fluorescence, Annual volumes 2005, 169-198, (2005). [2] Time-resolved fluorescence spectroscopic study of crude 

petroleum oils: influence of chemical composition. A.G. Ryder. Applied Spectroscopy, 58(5), 613-623, (2004). 

[3] Time-resolved fluorescence microspectroscopy for characterizing crude oils in bulk and hydrocarbon bearing fluid 

inclusions. A.G. Ryder, M.A. Przyjalgowski, M. Feely, B. Szczupak, and T.J Glynn, Applied Spectroscopy, 58(9), 

1106-1115, (2004). 

64


FLUO-3 

Picosecond dynamics of acrylodan in ethanol and dimethylformamide solution 

János Erostyák a , Andrea Buzády a , Ida Z. Kozma b,c , Jürgen Kuhl b , János Hebling a 

a Department of Experimental Physics, University of Pécs, Ifjúság u. 6., H-7624 Pécs (Hungary). 

E-mail: erostyak@fizika.ttk.pte.hu 

b MPI für Festkörperforschung, Stuttgart (Germany). 

c Menlo Systems GmbH, D-82152 Martinsried, Munich, (Germany). 

Excited state relaxation dynamics of a protein labeling dye acrylodan (AC) [1] in solution has been studied 

earlier by femtosecond transient absorption spectroscopy (fs TRABS) [2]. This fs study revealed the fastest 

components in the excited state processes. Already in this time scale a significant difference was seen 

between ethanol and dimethylformamide (DMF) solutions of AC. 

Picosecond fluorescence spectroscopy (ps FS) was applied to see the temporal evolution of AC’s emission 

on a longer time scale. The samples were excited by the frequency doubled output of a Spectra-Physics 

Tsunami Ti:Sapphire-laser, and the emitted light was detected by a streak camera. 

In the earlier measurements excited state solvation dynamics was characterized by multi-exponential 

behavior in both solvents. In DMF solution two time constants, 1.5 ps and 7.8 ps were assigned to solvation 

relaxation around excited AC, in ethanol solution solvation relaxation component of 3.8 ps was determined 

[2]. 

The ps time-emission matrices of ethanol and DMF solution show significant differences. In the ps time 

scale an additional effect, not present in DMF, is observed in ethanol solution. It is assigned to 

isomerization of AC in the excited state. 

The Figure shows a time-emission matrix of AC in ethanol. After the initial excited state relaxations 

(resolved in [2], but not visible in the Figure), the emission maximum is ~520 nm. Later, between 10-80 ps, 

this maximum moves towards 580 nm. Finally, 

after ~130 ps it is stabilized at 490 nm, which 200 

otherwise is the maximum in the steady-state 

emission spectrum of AC. On the basis of the 

Acrylodan in ethanol 

data of earlier fs TRABS and the present ps 150 

fluorescence measurements, detailed schemes 

of energy levels and relaxation processes are 

now proposed in case of both solutions. In 

100 

ethanol solution of AC, the time dependence 

of transient spectra is interpreted in terms of 

fast solvent relaxation followed by excited 

50 

state isomerization of the dye. It is worth 

mention, that in the ethanol solution of AC, in 

the steady-state emission spectrum, only one 

0 

wide spectral band can be seen, thus the 

underlying excited state processes remain 

450 500 550 600 650 700 

entirely hidden from the observers eye. 

Emission wavelength (nm) 

Time (ps) 

References: [1] F. G. Prendergast et al., J. Biol. Chem. 258 (12) (1983) 7541. [2] A. Buzády et al., J. Phys. Chem. B 

2003, 107, 1208. 

65


FLUO-4 

Excited state electron transfer in systems containing a coumarin derivative 

and different electron donors 

Cristina Tablet, a Rositca Nikolova, b Sorana Ionescu a 

a 

University of Bucharest, Department of Physical Chemistry, Bd. Regina Elisabeta 4-12 Bucharest 

(Romania). E-mail: sorana@gw-chimie.math.unibuc.ro 

b University of Sofia, Department of Organic Chemstry, J. Bourchier Av. 1, Sofia 1126 (Bulgaria). 

Coumarin derivatives are intensively studied due to their wide range of applications, from biological 

activity to laser dyes. [1,2] Upon light absorption, they are submitted to electron transfer in solution in the 

presence of aromatic or aliphatic amines used as electron donors. [3] The present paper aimed at studying the 

processes that take place in the presence of different electron donors of a previously newly synthesised 

phosphorus containing coumarin derivative, namely 7-diethylamino-3-diethylphosphonocoumarin (CumP). 

This is the first step in a more complex study regarding electron transfer in binary systems containing an 

aromatic and a N or S containing compound and the conditions necessary for excited state complex 

formation. Firstly we performed steady-state fluorescence measurements of this compound in solvents of 

different polarity in order to check the solvent effect on the emission spectrum and the nature of the first 

excited state, as other 7-diethylaminocoumarin derivatives were proved to have charge transfer excited 

states of the type twisted intramolecular charge transfer (TICT) and by consequence to present dual 

fluorescence. [4] This is due to the presence of the diethylamino fragment with both donor character and a 

free degree of rotation along a single bond. Only one band was observed in the emission spectrum of the 

studied compound and a charge transfer character for the first excited state. The fluorescence quantum 

yields were measured. The second step was to measure the fluorescence spectrum in the presence of 

different electron donors, such as aliphatic or aromatic amines, phenoxathine or thianthrene. The data were 

rationalised according to the Stern-Volmer equation for calculating the quenching constants and to Marcus 

theory in order to check the electron transfer nature of the processes that take place in solution upon 

irradiation. The results for the quenching of the fluorescence of CumP by diphenylamine (DFA) are 

presented in the graph below. 

2.0 

Stern-Volmer plot of the fluorescence 

quenching of CumP by diphenylamine in 

acetonitrile. The fluorescence intensity of 

CumP was measured at different 

diphenylamine concentrations. The 

dependence is linear even at high 

quencher concentration and so the nature 

of quenching is purely dynamic. 

I 0 

/I 

1.8 

1.6 

1.4 

1.2 

1.0 

0.00 0.02 0.04 0.06 0.08 0.10 0.12 

c DFA 

(M) 

Quantum chemical calculations were performed for the ground and excited states in order to explain the 

experimental data and to gain an insight on the photophysical phenomena. The geometry optimisation for 

the planar and twisted conformations confirmed the lack of a TICT excited state for this molecule. 

References: [1] F. Gao, Dyes Pigments 52 (2002) 223. [2] G. Jones II, J.A.C. Jimenez, J. Photochem. Photobiol. B: 

Biol. 65 (2001) 5. [3] C. Tablet, M. Hillebrand, J Photochem. Photobiol A: Chemistry 189 (2007) 73. [4] T. Lopez 

Arbeloa, F. Lopez Arbeloa, M. J. Tapia I. Lopez Arbeloa, J. Phys. Chem. 97 (1993) 4704. 

66


FLUO-5 

Matrix isolation spectroscopy applied to phosphorescent organo-transitionmetal 

materials for OLEDs 

Hartmut Yersin 

Institut für Physikalische Chemie, Universität Regensburg, D-93040 Regensburg, Germany 

E-mail: hartmut.yersin@chemie.uni-r.de 

The outstanding importance of triplet emitters for OLED applications is well established. [1,2] Thus, a deeper 

understanding of the photophysics of these organo-transition-metal emitters is required and indeed came 

into the focus of spectroscopists. In this contribution, it is introduced to the photophysics of the triplet state. 

It usually splits into three substates each of which exhibits its specific emission behavior. Investigations at 

low temperature, high magnetic fields, and by use of high-resolution spectroscopy allow to determine 

properties of the individual triplet substates, such as zero-field splittings (ZFS), substate decay times, spinlattice 

relaxation times, vibronic coupling activities (Franck-Condon/Herzberg-Teller), singlet-triplet 

splitting, intersystem crossing time, etc.. 

The results of these investigations provide an insight into the vibronic origin of the spectral band width 

(colour purity), the ambient temperature emission decay time, and the ZFS. In particular, the ZFS displays 

directly the importance of the MLCT (metal-to-ligand-charge transfer) character in the emitting triplet 

state. [2] Interestingly, the triplets of the most successful emitters for OLEDs, such as Ir(ppy) 3 , Ir(btp) 2 (acac), 

Ir(pic) 2 (acac), Pt(Me 4 -salen), etc., exhibit significant MLCT character. A corresponding systematic will be 

presented. 

References: [1] H. Yersin (ed.) Highly efficient OLEDs with phosphorescent materials, Wiley 2007; [2] H. Yersin; 

Top. Curr. Chem. 241 (2004) 1 

67


FLUO-6 

On the cleavage process of N-trifluoromethylsulfonyloxy-1,8-naphthalimide 

as a photoacid generator 

Jean-Pierre Malval, Fabrice Morlet-Savary, Xavier Allonas, Jean-Pierre Fouassier 

Department of Photochemistry, UMR CNRS 7525, Université de Haute Alsase, 

3 rue Alfred Werner. 68093 Mulhouse, France 

Shota Suzuki, Shigeru Takahara, Tsuguo Yamaoka 

Department of Information and Image Science, Faculty of Engineering, Chiba Univer.siht 1-33 Yayoi-cho, 

Inage-ku, Chiba 263-8522, Japan 

Photoacid generators (PAG) are of primary importance in microlithography, particularly for applications in 

the microelectronics industry[1]. Even new efficient molecular structures are currently under development, 

primary photoconversion mechanisms of some PAG remain uncleared. Iminosulfonates and imidosulfonates 

appear as attractive PAG as a N-O bond is easily cleaved by light irradiation. N-trifluoromethylsulfonyloxy-1,8-naphthalimide 

(NIOTf), is also a well known PAG, however its 

photocleavage process is source of controversy[2, 3]. 

A comprehensive mechanism of photoacid generation from N-trifluoromethylsulfonyloxy-1,8-naphthalimide 

is reported. Several convergent results from stationary fluoresence measurements, picosecond 

transient spectroscopy, infrared analysis and DFT calculations strongly supported an homolytic N-O 

photocleavage followed by an unusual internal cyclic rearrangement of 1,8 naphthalimide core. 

0.25 

O 

O 

N 

O 

S 

CF 3 

Abs. 

O 

O 

NIOTf 

0.00 

200 250 300 350 400 450 500 550 600 650 

Fluo. (a.u.) 

425 450 475 

0 

200 250 300 350 400 450 500 550 600 650 

wavelength (nm) 

References: [1] H.Ito, C.G.Willson, Polymers in Electronics, in, T. Davidson (Ed.), ACS Symp. Ser., Washington 

DC, 1984. [2] F. Ortica, J.C. Scaiano, G. Pohlers, J.F. Cameron, A. Zampini, Chem. Mater. 12 (2000) 414-420. 

[3] M. Saotome, S. Takano, A. Tokushima, S. Ito, S. Nakashima, Y. Nagasawa, T. Okada, H. Miyasaka, Photochem. 

Photobiol. Sci. 4 (2005) 83-88. 

68


FLUO-7 

Ultrafast spectroscopy of combustion products in a propane flame 

A. Bruno 1 , F. Ossler 2 , C. de Lisio 1 , P. Minutolo 3 , A. D’Alessio 4 , N. Spinelli 1 

1 CRS “Coherentia” - CNR-INFM and Dip. Scienze Fisiche, Università Federico II, Naples, Italy; 

2 Combustion Physics Department, Lund University, Lund, Sweden 

3 Istituto di Ricerche sulla Combustione, CNR, Naples, Italy 

4 CNISM and Dip. Ingegneria Chimica, Università Federico II, Naples, Italy 

Several toxicological and epidemiological studies have demonstrated that ultrafine particulate with organic 

functionalities can deeply penetrate into pulmonary alveoli, circulatory system and can also deposit into the 

brain [1]. 

In the last few years there has been an increasing interest in charcterizing the optical properties of 

nanoparticles produced in combustion systems. Carbonaceous atmospheric aerosols represent 

approximately 50% of the total particulate matter and a non negligible part of it comes from combustion 

processes. Understanding the formation mechanism of particulate matter due to incomplete combustion is 

fundamental to control the atmospheric impact of combustion systems. The most unclear point of this 

mechanism is the soot inception, and, consequently, there is an increasing interest in developing and 

studying innovative techniques for direct analysis of the incipient nanoparticles of organic matter with 

typical sizes of 2-3 nm besides soot particles. 

The mean size of NOC particles was determined for the first time by in situ measurements of light 

scattering/absorption in premixed flames [2]. Afterwards, some other ex-situ sizing techniques have been 

used to analyse NOC sampled from flames and from exhausts of engines [3]. Nevertheless, some 

difficulties arise in developing diagnostics in the nanometric size range, and, as a consequence, there is a 

great demand of new diagnostics capable to detect nanometric pollutants. 

Laser-induced fluorescence has proven to be a very sensitive technique for measurements of small 

polyatomic species in combustion, also at high temperatures. For larger molecules, the rates of internal 

energy redistribution and conversion into non-radiative states strongly increase with temperature causing 

considerable quenching and spectral shifts and broadening.Therefore there is the need to use laser 

techniques based on short-pulse (in femtosecond to picosecond scale) to resolve the dynamics of larger 

molecular species and to characterize their size and chemical properties [4]. 

In this experiment, the combustion products are spectroscopically analyzed directly in a propane Bunsentype 

diffusion flame at atmospheric pressure. The flame is probed by the second harmonic of a femtosecond 

laser. The time resolved fluorescence signals are analyzed as a function of wavelength in order to obtain 

complementary information on the nature of chromophores and their sizes. Using the temporal decay of the 

fluorescence anisotropy ratio, the volume of polyatomic species and nanoparticles can then be measured 

and, simultaneously, one can explore how the spectral properties of the fluorescence depend on the size of 

nanoparticles. This technique will open up new opportunities to measure the size of very small 

nanoparticles down to the size of a nanometer or below. 

References: [1] Oberdoster G. et al., Journal of Toxicology and Environmental Health – part A 65 (2002) 1531. 

[2] D’Alessio et al., J. Aerosol Sci., 29 (1998) 397. [3] L.A. Sgro et al. / Chemosphere 51 (2003) 1079 [4] A. Bruno 

et al., Optics Express 13 (2005) 5393. 

69


FLUO-8 

Different conformations of free based dinuclear phthalocyanins 

Christian Litwinski, Eugeny A. Ermilov, Sebastian Tannert, Beate Röder 

Humboldt-University of Berlin, Institute of Physics, Newtonstr. 15, 12489 Berlin (Germany) 

Sergey Makarov, Olga Suvorova 

G.A. Razuvaev Institute of Organometallic Chemistry of Russian Academy of Sciences, 

Nizhny Novgorod (Russia) 

Dieter Wöhrle 

Institute of Organic and Macromolecular Chemistry, University of Bremen, P.O. Box 330440, 28334 

Bremen (Germany) 

Ines Corral, Leticia Gonzalez 

Institut für Chemie und Biochemie, Physikalische und Theoretische Chemie, Freie Universität Berlin, 

Takustrasse 3, Berlin 14195 (Germany) 

Phthalocyanines (Pcs) - the most popular porphyrin analogues - have a wide range of applications as 

molecular photoconductors, optical limiters, catalysts for photodegradation of pollutants, sensitizers for 

photovoltaic devices and photodynamic therapy. One way of modifying the electronic properties of Pcs is 

the synthesis of conjugated oligomers. The consequently increasing of the conjugated π-electron system is 

of substantial interest for the design of new functional materials. [1,2,3] 

In the present study the photophysical properties of 

a metal-free binuclear Pc solved in toluene are 

investigated. The Pc units are connected through a 

common annulated benzene ring. Using the 

combination of different steady-state and timeresolved 

optical methods it was clearly shown for 

the first time, that three species with different 

photophysical properties exist in the chemically 

pure compound. This observation derives from 

different conformations of the dimer as was pointed 

out by quantum-mechanical ab initio calculations. 

These conformations differ in the respective 

hydrogen atoms orientation in the center of the tetrapyrrole ring of each Pc unit in the annulated dimer. In 

the first conformation (fluorescence maximum λ fl = 840 nm) both pairs of hydrogen atoms lie parallel to the 

connection line of two Pc units (see figure). Second conformation (λ fl = 858 nm) has two pairs with 

perpendicular orientation and the third species (λ fl = 874 nm) has one perpendicular and one parallel pair of 

hydrogen atoms. The fluorescence lifetime of the last two isomers is approximately two times longer 

compared to the first one. 

References: [1] S. Makarov et al., Chem. Eur. J. 2006, 12, 1468–1474. [2] S. Makarov et al., Eur. J. Inorg. Chem. 

2007, 546-552. [3] A. Tsuda, A. Osuka, Science 2001, 293, 79-82. 

RO 

RO 

R = 

H 3 C 

H 3 C 

RO 

N 

N 

NH 

RO 

N 

N 

OR 

HN 

N 

N 

OR 

4 

RO 

N 

N 

NH 

RO 

N 

N 

OR 

HN 

N 

N 

OR 

OR 

OR 

70


FLUO-9 

Comparative investigation in different soybean cultivars via 

fluorescence spectroscopy 

Anderson R. L. Caires 1,* , Gian P. G. Freschi 1 , Luis H. C. Andrade 2 , Sandro M. Lima 2 , 

Maria R. O. Teixeira 3 

1 Grupo de Óptica Aplicada, UFGD, Dourados, MS, Brazil. 

2 Grupo de Espectroscopia Óptica e Fototérmica, UEMS, Dourados, MS, Brazil. 

3 Centro de Pesquisa Agropecuária do Oeste, Embrapa, Dourados, MS, Brazil. 

*E-mail: andercaires@ufgd.edu.br 

The development of the new tool for soybean cultivars identification is extremely necessary to make 

improvements in the soybean production. Soybean cultivars have been characterized mainly by 

morphological and biochemical traits. However, these methods have not been efficient to characterize the 

large number of cultivars eligible to receive protection under the Brazilian Cultivar Protection Act [1] . In this 

context, the fluorescence spectroscopy can be applied to identify different soybean varieties. This method 

is a powerful tool to analyze molecular and atomic behavior in different kinds of materials. 

In this work we have analyzed the fluorescence response of two varieties of soybean, BRS181 and 

BRS244RR, that are cultivated in the south region of Brazil. The excitation wavelength was 355 nm and the 

fluorescence spectra were collected in the range between 400 nm and 800 nm. The RF-1501 (Shimadzu) 

Spectrophotometer was utilized to realize the measurements. 

The fluorescence emission of both soybean varieties showed the same behavior with a maximum at Bluegreen 

region (460-530 nm) and in the chlorophyll a fluorescence region (720 nm). However, different peak 

intensities was observed in a comparative analyze of the fluorescent signals: the BRS181 sample exhibits 

the peak intensity in the blue-green region higher and in the chlorophyll a fluorescence region smaller than 

BRS244RR sample. The observed difference in the fluorescence response results from a stronger reabsorption 

of blue-green fluorescence in the BRS244RR samples caused by photosynthetic pigments 

(carotinoids and chlorophylls). Our results show the potentiality of the fluorescence spectroscopy to 

investigate different soybean cultivars. The method can be used as a descriptor tool for soybean cultivars 

identification. 

Reference: [1] R. H. G. Priolli et al., Genetics and Molecular Biology. 25 (2002) 185. 

71


FLUO-10 

Quantitative time-resolved FRET spectroscopy reveals structural changes 

in the calcium sensor YC 3.60 

J.W. Borst, S.P. Laptenok, A.H. Westphal, N.V. Visser, J. Aker, A. van Hoek, 

A.J.W.G. Visser 

Microspectroscopy Centre, Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA 

Wageningen, The Netherlands. E-mail: Ton.Visser@wur.nl 

Förster Resonance Energy Transfer (FRET) is a widely used method for monitoring interactions between 

or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence 

lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. 

However, these lifetimes may originate from interacting and non-interacting molecules, which hampers 

quantitative interpretation of FRET data. Here, we describe a method for the detection of FRET by 

monitoring the rise time of the fluorescence intensity of acceptor upon donor excitation, thereby 

measuring only those molecules undergoing FRET. Time-dependent fluorescence anisotropy measured in 

parallel with fluorescence intensity provides additional structural information about the relative 

orientation between donor and acceptor chromophores. As a model system, the calcium sensor protein 

Yellow Cameleon 3.60 (YC3.60) was chosen. YC3.60 changes its structure upon calcium binding thereby 

increasing the FRET efficiency. A structural model was designed of the two fluorescent proteins moieties 

and the calmodulin-M13 complex of YC3.60 (-/+ Ca 2+ ) from the obtained distances and orientational 

angles. In the closed, calcium bound conformation the distance between the chromophores was set at 2.3 

nm and a relative angle between the transition dipole moments of 77° was used for construction. For the 

open conformation, in the absence of calcium, values of 4.6 nm and 66° were used. 

Fig. 1. Structural model of YC3.60 in the closed (A) and open (B) conformation. Calcium ions are shown 

as green balls in the closed conformation. The left, cyan barrel (ECFP) is connected at the N-terminus of 

the red calmodulin part (A, top; B, middle). The C-terminus of the M13 helix (gold) finally connects to 

the right, yellow barrel (Venus). 

In figure 1 a structure of this FRET sensor could be modelled taking distances and orientation angles 

between the FRET moieties in YC3.60 into account (see Fig. 1.). This spectroscopic approach can be 

used as an alternative, rapid method for modelling changes in sensor conformation when ligands are 

bound. 

72


FLUO-11 

Eliminating the effects of the instrument detection optics and 

reaction kinetics on fluorescence and luminescence spectra 

Reija-Riitta Harinen, Jorma Lampinen, Hanna Granö-Fabritius 

Thermo Fisher Scientific, P.O.Box 100, FI-01621 Vantaa (Finland). 

E-mail: reija-riitta.harinen@thermofisher.com 

Monochromator based spectral scanning instruments can be used to measure the spectra of labels used in 

fluorometric and luminometric assays. This spectral information can be used to optimize the assay 

conditions and to select the optimal filters. In this paper we describe the factors which cause the measured 

technical spectra to differ from the true chemical spectra of fluorometric and luminometric labels. 

The wavelength dependent efficiency of the instrument has an effect on the peak area of the fluorometric 

emission spectrum. The efficiency is affected by the photomultiplier tube detector and optical parts of the 

monochromator grating system. The technical spectra does not precisely represent the true chemical spectra 

of the label. The differencies in the technical and true chemical spectra are dependent on the wavelength. If 

the emission peak of the label is on the wavelength area where the instrument’s performance changes 

rapidly, the peak wavelengths of the technical and chemical spectra can differ remarkably. 

Luminescence emission is normally not as stable as fluorescence emission, so the total signal intensity can 

change during the spectral scanning. Therefore the kinetics of the luminescence reaction can also affect the 

shape of the spectrum. The faster the luminescence signal changes and the longer the measurement time in 

spectral scanning is, the more it shifts the spectrum. 

The effects of the instrument detection optics and the luminescence reaction kinetics should be eliminated 

to get the correct spectra. Thermo Scientific Varioskan Flash is a spectral scanning multitechnology 

microplate reader, which has a unique spectral correction feature. This feature can be used to correct the 

shift of the technical spectra caused by the effects of the instrument’s detection optics. The correction 

feature is valuable in assay optimization when small changes in wavelengths can affect the assay 

performance considerably. This paper also shows how the shift caused by the effect of the kinetics can be 

mathematically corrected to get the true luminescence spectra of the label. 

73


FLUO-12 

Water effect on the spectral behavior of 2-(benzimidazol-2-yl)- 

3-hydroxychromone in alcohols 

Denis Svechkarev, Lubov Lukatskaya, Andrey Doroshenko 

V. N. Karazin Kharkov National University, Institute for Chemistry, 

61077 Kharkov (Ukraine). E-mail: aod@univer.kharkov.ua 

Derivatives of 3-hydroxychromone have been objects of extensive interest and research for over decades. 

Particularly, their dual-band fluorescence due to the ongoing process of the excited state intramolecular 

proton transfer (ESIPT) made them prospective for sensitive fluorescent ratiometric probes design [1] . Thus, 

the question of systematic studies of spectral and fluorescent behavior of these compounds in different 

media, as well as influence of intermolecular H-bonding and other solvent effects, became important. In this 

domain, derivatives of 3-hydroxychromone showing remarkable solvatochromogenic properties were 

shown to be capable of use as probes for different parameters of their environment on the molecular level [2- 

4] . 

236.5 

212.8 

189.2 

165.5 

141.9 

118.2 

94.60 

70.95 

47.30 

23.65 

Normal-to-tautomer fluorescence ratio 

0.000 

25 

400 

24 

417 

23 

435 

22 

455 

21 

476 

20 

500 

19 

526 

18 

556 

17 

588 

16 

625 

15 

667 

0 20 40 60 80 100 

Concentration of water, % (v/v) 

In the present communication we are demonstrating influence of the concentration of water on the spectral 

and fluorescence behavior of 2-(benzimidazol-2-yl)-3-hydroxychromone. While the concentration of water 

in ethyl alcohol increases, a long wavelength band at 425 nm appears with increasing intensity. In the 

fluorescence spectra, the normal emission band intensity increases with corresponding decreasing of the 

phototautomer band. This could be caused by the formation of intermolecular H-bonds with water 

molecules acting as proton accepting species, which affects the ESIPT process. A near-linear dependence of 

the normal-to-tautomer intensity ratio on the water concentration allows to consider this compound as 

prospective fluorescence probe for water contents monitoring in organic solvents and probably in biological 

systems as well. 

References: [1] D. McMorrow, M. Kasha, J. Amer. Chem. Soc. 105 (1983) 5132. [2] W. Liu, Y. Wang et al., 

Anal. Chim. Acta 383 (1999) 299. [3] A. Roshal, A. Grigorovich et al., Photochem. Photobiol. A 127 (1999), 89. 

[4] V. Shynkar, A. Klymchenko et al., J. Phys. Chem. A 108 (2004) 8151. 

74


FLUO-13 

Multicomponental fluorimetric determination of aluminium, gallium 

and indium 

Šimon Vojta, Luděk Jančář and Lumír Sommer 

Institute of Environmental Protection, Faculty of Chemistry, Brno University of Technology, 

Purkyňova 118, 612 00 Brno (Czech Republic), e-mail: vojta@fch.vutbr.cz 

For the fast characteristics of mixtures of Aluminium, Gallium and Indium the fluorimetric analysis with a 

multivariant calibration in overdetermined systems can be used. Especially the Multiple Linear Regression 

(MLR), the Principal Components Regression of the measured variable against the real analyte 

concentrations (PCR) and the regression of latent variables of measured quantities against latent variables 

of concentration values of analytes with full projection to latent structures - Partial Least Squares (PLS) [1, 2] , 

are convenient. The prediction error of the enquired analyte concentrations depends on the character and the 

overlapping of the particular spectra of the components, the number and selection of wavelengths, the 

number of the calibration solutions and the design of used statistical plan of the calibration set. The Kalman 

filtering [3] is suitable for the interpretation of very similar spectra of the particular components as well as 

the derivation of spectra enabling the better distinguishing of signals of the particular components [4] . 

The PLS, Kalman filtering and MLR were tested and compared in this paper for the fluorimetry of Al, Ga 

and In complexes with 8-Hydroxyquinoline-5-sulphonic acid under various evaluation conditions. 

References: [1] H. Martens, T. Naes, in: Multivariate Calibration Wiley, Chichester 1989. [2] P. Geladi, 

B. R. Kowalski, Anal. Chim. Acta. 185 (1986) 1. [3] S. D. Brown, Anal. Chim. Acta. 181 (1986) 1. 

[4] R. Kostrhounová-Štěpánková, L. Jančář, L. Sommer, Chem. Listy 97, (2003), 269. 

75


FLUO-14 

Determination of Dy(III) at the presence of Tb(III) with use of 

time resolved luminescence 

M.P. Tsvirko*, А.V. Кiriiak, S.B. Meshkova 

*Institute of Chemistry and Environmental Protection, Jan Dlugosz University, 42-200 Częstochowa, Armii 

Krajowej Av. 13/15 (Poland). 

*National Scientific-Research Centre of Ozonesphere Monitoring of Belorussia State University, 220067, 

Minsk, Kurchatova Street, 7 (Belarus); E-mail: m.tsvirko@ajd.czest.pl 

A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, 

65080 Odessa, Lustdorfskaya Doroga 86 (Ukraine); E-mail: s_meshkova@ukr.net 

The decrease of both detection limit and sensibility of lanthanide (Ln) determination was observed in series 

of cases at the use of spectrofluorometry with the isolation of long-lifetime component (200-1000 mks) of 

luminescence signal. 

Analytical possibilities for the isolation of short-lifetime luminescence component of weak luminescence 

Ln (Dy, Sm) at the background of long-lifetime intense luminescence (Tb, Eu), especially in pairs of 

neighbor elements Sm – Eu and Tb – Dy, were not studied. 

We have used complexes of Tb(III) and Dy(III) with pyrazole-5-carbonic acids, which relative quantum 

yields and luminescence life time were established. Complexes with 3-(6-benzo-dioxanil)-pyrazole-5- 

carbonic acid (BOPA) are featured by the highest value of ϕ and τ. On the base of research of luminescence 

spectra with time resolution of Tb(III) and Dy(III) complexes with BOPA the principal possibility to 

determine Dy(III) in presence of Tb(III) through the isolation of short -lifetime component of dysprosium 

has been established in spite of practically complete recovering of analytical bands Dy (575 nm) and Tb 

(585 nm) (Fig.). 

Fig. Schematic showing of isolation of shortlifetime 

component of Dy(III) luminescence 

(λ=575 nm) – 1 in presence of Tb(III) (λ=585 

nm) – 2. C Tb, Dy =1·10 -5 M; C BOPA =1·10 -4 M). 

Dy(III) in luminescence materials – scandium-borates doped by terbium and dysprosium (Table) was 

determined with the help of proposed method. 

Table. Results of determination of Dy(III) in luminescence materials (n=5, Р= 0.95) 

Sample Content Dy, % Found Dy, % S r 

ScBO 3 Tb 1.5 % Dy 5.0 % 5.00 4.94±0.08 0.013 

ScBO 3 Tb 1.5 % Dy 1.0 % 1.00 1.02±0.06 0.047 

ScBO 3 Tb 1.5 % Dy 0.5 % 0.50 0.46±0.03 0.054 

As follow from table data it is good coincidence between content and bound quantity of Dy, %. 

76


FLUO-15 

Theory of solvatochromic shift and broadening of the phosphorescence 

spectrum of molecular oxygen in solutions 

Vladimir S. Pavlovich 

NASB, Institute of Molecular and Atomic Physics, Luminescence,220072 Minsk (Belarus). 

E-mail: pavlovich@imaph.bas-net.by 

In presented work the experimental data from Wessels and Rodgers [1] on solvent effect on the peak 

1 

3 − 

position of the 0-0 band ν max of the a Δ 

g 

→X 

Σ phosphorescence of molecular oxygen are described 

g 

through a new relation for solvatochromic shift due to dispersion and induction interactions, [2] obtained 

with help Onsager’s cavity model. Thus 

2 

Δα 

eg 

3E00I 

s 

ν 

max 

= 7882.4 

+ Δν 

rep 

+ Δν 

Σ 

− P( 

n, 

ε, 

I ) 

3 

s 

, P( n, 

ε, 

I 

s 

) = Pn 

+ k 

BT 

P 

2 2 

ε 

, 

aO 

I 

s 

− E00 

where Δν rep and Δν Σ denote the effect of repulsive and multipole interactions, Δα eg is the change of 

1 

3 − 

polarizability under a Δ 

g 

→X 

Σ transition, a 

g 

O is Onsager radius, E 00 =7882.4 cm -1 (energy gap of solute 

2 

n −1 

in the gas phase taken from Herzberg), I s is the ionization potential of solvent molecule, P n 

= , n 

2 

+ 2 

ε −1 

P 

ε 

= with n and ε being the reflective index and the dielectric constant of solvent. 

2ε + 1 

As shown by Figure the used solvents are divisible into 

three principle groups with virtually alike linear 

correlation between ν max and the P(n,ε,I s ) with slope 

−0.073 ±0.004. Say the first group includes n-alkanes, n- 

alcohols (but methanol), benzene and its halogen 

derivatives, acetone, tetrahydrofuran, C 2 Cl 4 , toluene and 

benzonitrile (20 solvents). The second group includes 

methanol, CCl 4 , CHCl 3 , dioxane and CS 2 . The one 

exception to all solvents is represented by water and 

acetonitrile (stars). After detail analysis we came to the 

main conclusion that the red shift results in the dipoledipole, 

dispersion and induction, interactions as well as 

from the interactions of multipoles, among which 

quadrupole-quadrupole interactions play a dominant role. 

The repulsion makes the blue shift of 0-0 band, which is a more or less alike for all solvents that early 

pointed by Schmidt. [3] It has obtained with a O =1.37 Å that the polarizability in a 1 Δ g state is 0.19 ±0.03 Å 3 

above than that in X 3 Σ g state. There is a need to point that our result for Δα eg =0.19 Å 3 do not support that 

of −0.08 Å 3 early obtained by quantum mechanical calculations from Ogilby et al. [4] 

Broadening of 0-0 band caused by fluctuations of O 2 -solvent interactions due to intermolecular and 

intramolecular vibrations in solvent environment is studied too. A fairly good agreement between the 

theoretical results and the experimental half-width data [1] is observed within the groups of n-alkanes, n- 

alcohols, and halogen derivatives of benzene individually. 

This work is supported by grant F06-177 from the Belarusian RFFR. 

References: [1] J. M. Wessels, M. A. J. Rodgers, J. Phys. Chem. 99 (1995) 17586. [2] V. S. Pavlovich, J. Appl. 

Spectrosc. 74 (2007) in press. [3] R. Schmidt, J. Phys. Chem. 100 (1996) 8049. [4] T. D. Poulsen, P. R. Ogilby, 

K. V. Mikkelsen, J. Phys. Chem. 102 (1998) 8970. 

77


FLUO-16 

Thermally activated delayed fluorescence as a cycling process between 

excited singlet and triplet states. Application to the fullerenes 

Carlos Baleizão, Mário N. Berberan-Santos 

Centro de Química-Física Molecular, Instituto Superior Técnico, P-1049-001 Lisboa (Portugal). 

E-mail: carlos.baleizao@ist.utl.pt 

In efficient thermally activated delayed fluorescence (TADF) [1] the excited chromophore alternates 

randomly between the singlet and triplet manifolds a large number of times before emission occurs. In this 

communication we obtain an expression for the average number of cycles n in terms of photophysical 

parameters and show that n + 1 is the intensification factor of the prompt fluorescence intensity, owing to 

the occurrence of TADF. [2] The maximum possible intensification factor is 1/(1-Φ T ), where Φ T is the 

quantum yield of triplet formation. A new method of data analysis for the determination of the quantum 

yield of triplet formation, combining steady-state and time-resolved data in a single plot, is also presented. [2] 

Application of the theoretical results to the TADF of [70]fullerenes, [3,4] whose average number of excited 

state cycles can exceed 100, shows a general good agreement between different methods of fluorescence 

analysis, and allows the determination of several photophysical parameters. 

100 

80 

Computed average number of S 1 →T 1 →S 1 

cycles as a function of temperature for 

C 70 in polystyrene. 

n 

60 

40 

20 

0 

-50 0 50 100 150 200 

T (ºC) 

The additional study of the temperature dependence of the phosphorescence intensity allows to take into 

account the temperature dependence of the T 1 →S 0 intersystem crossing and thus to refine the above 

calculations. 

Acknowledgements: This work was supported by FCT (Portugal) and POCI 2010 (POCI/QUI/58535/2004). 

C. Baleizão is grateful for a postdoctoral fellowship from FCT (SFRH/BPD/28438/2006). 

References: [1] B. Valeur, Molecular Fluorescence, Wiley-VCH, Weinheim, 2002. [2] C. Baleizão, M.N. Berberan- 

Santos, J. Chem. Phys., submitted. [3] M.N. Berberan-Santos, J.M.M. Garcia, J. Am. Chem. Soc. 118 (1996) 9391. 

[4] C. Baleizão et al., Chem. Eur. J. (2007), in press. 

78


FLUO-17 

Enhancement luminescence of the heteronuclear f-p-complexes with 

diethylenetriaminepentaacetic acid 

Sergii Smola 1 , Natalya Rusakova 1 , Elena Martsinko 2 , Inna Seifullina 2 , Eugeny Ermilov 3 , 

Yuriy Korovin 1 

1 Department of Chemistry of Lanthanides, A.V.Bogatsky Physico-Chemical Institute, 

65080 Odessa (Ukraine). E-mail: lanthachem@te.net.ua 

2 Department of Chemistry, I.I. Mechnikov Odessa National University, 65026 Odessa (Ukraine). 

3 Humboldt - University of Berlin, Physics Department, D-12489, Berlin (Germany). 

Recently lanthanide complexes with diethylenetriamine-N,N,N´,N´´,N´´-pentaacetic acid (DTPA) and its 

derivatives have attracted attention, first of all, as potential contrast agents. But there are only few reports 

dedicated to the investigation of the spectroscopic properties (in particular, 4f-luminescence) in complexes 

with DTPA [1]. Is it possible to increase 4f-luminescent characteristics without of functionalization of 

DTPA by aromatic substituents-sensitizers? We tried to apply non-traditional objects for this purpose, 

namely heteronuclear f-p-complexes based on the DTPA. For this moment there is insufficient information 

about the photophysical properties of the heteronuclear f-p-complexes [2] and practically no data about 

lanthanide luminescence in them. 

Therefore we reported the results to gain data on the spectral-luminescent properties of the lanthanidegermanium 

complexes with DTPA (Ln = Sm, Eu, Tb and Dy). All data analysis obtained with the help of 

different physico-chemical methods allows assuming that f-p-complexes are three nuclear ones (the ratio 

Ln: ligand : Ge is equal 1:2:2). Coordinated polyhedron of germanium is the same as in complex acid 

[Ge(OH)(H 2 DTPA)]⋅H 2 O. On the basis of obtained data and taking into account the coordination figures, 

oxidation degrees characterized typically for investigated metals (as well as isostructural of synthesized 

complexes), their structure schemes can be proposed as it is given in figure. Coordination polyhedron of 

lanthanide is the “distorted octahedron” forming for account of tridentate coordination of two complex 

anions [Ge(OH)DTPA] 2- and [Ge(OH)HDTPA] - with the closing of four glycine metal cycles. 

In considered complexes the 4f-luminescence of three-charged ions (europium, terbium, samarium and 

dysprosium) with corresponding maxima in visible range is realized at UV-excitation [3]. It is noteworthy 

that it is the first observation of 4f-luminescence in water solutions of heteronuclear f-p-complexes. The 

luminescence intensity of heteronuclear samarium, terbium or dysprosium complexes at various lengths of 

excitation waves was higher (up to 1.8, 2.1 and 2.5 times, respectively) than in the mono-complexes. At the 

same time, the luminescence of heteronuclear europium complex was higher (up to 1.7 times) as compared 

to Eu-DTPA complex only in the excitation region of 310-330 nm. Nontrivial approaches to the increasing 

of the 4f-luminescence (for Ln 3+ ions) in these complexes are described and discussed. 

Our recent data indicate that heteronuclear Ln-Bi complexes with DTPA posses unusual luminescent 

properties also, which will be discussed as well. 

References: [1] M.A. Abubaker et al., Anal. Lett. 26 (1993) 1681. [2] V. Stavila et al., Inorg. Chem. Commun . 7 

(2004) 634. [3] N. Rusakova et al., J. Fluorescence. 17 (2007), in press. 

79


FLUO-18 

Charge-transfer states in pyrene-triazine compounds 

Martin Michl, a 

Numan Almonasy, b Prokop Hapala, a Vlastimil Fidler, a and Miloš Nepraš b 

a 

Dept. of Physical Electronics, Faculty of Nuclear Sciences & Physical Engineering, Czech Technical 

University, V Holešovičkách 2, 180 00 Prague 8, Czech Republic. E-mail: michlm@troja.fjfi.cvut.cz 

b 

Dept. of Technology of Organic Compounds, Faculty of Chemical Technology, University of Pardubice, 

Studentská 95, 532 10 Pardubice, Czech Republic. 

In this contribution, we compare the properties of CT states which are important for photophysics of 

compounds where pyrene is connected to triazine ring either directly or via an amino-group. In previous 

studies, a large difference in photophysical properties of N-substituted 1- and 2-aminopyrenes has been 

found [1,2]. Briefly put, the substitution of amino-group into 2-position of pyrene represents less severe 

perturbation of electronic structure of pyrene both from the molecular symmetry point of view and also 

because this position corresponds to nodal plane of pyrenes frontier orbitals. Besides other properties, the 

N-triazinylated 1-aminopyrene derivatives show, in contrast to analogous 2-aminopyrene derivatives, 

strong dependence of fluorescence quantum yields on the solvent polarity. This effect is even more 

pronounced when a chlorine atom is attached to the triazine ring and 2-N-(1-aminopyrenyl)-4,6-dichloro- 

1,3,5-triazine does not even fluoresce at all. This behaviour can be explained in terms of CT states, which 

mediate the non-radiative de-excitation of the molecule. On the other hand, 2-(1-pyrenyl)-4,6-dichloro- 

1,3,5-triazine (where the triazinyl ring is directly attached to pyrene) exhibits a very large solvatochromic 

shift of fluorescence band, indicating CT character of the emitting state. (See the picture of electron density 

redistribution in the molecule upon excitation to S 1 state according to ZINDO-CI calculation.) Moreover, 

the fluorescence quantum yield of this 

compound is relatively high (~ 0.8) and does 

not decrease significantly with solvent polarity. 

The aforementioned properties would make 

this compound very attractive as a polarity 

probe, e.g. for studying biomembranes by the 

solvent relaxation method [4]. However, this 

compound is susceptible to substitution of the 

chlorine atoms, and their presence seems to be 

also here substantial for the formation of the 

CT state. Even the replacement of one of the 

atoms by such as methoxy-group results in 

complete loss of the pronounced 

solvatochromic properties. 

References: [1] P. Kapusta et al., Fluorescence Microscopy and Fluorescent Probes 3 (1999 ) 145. [2] P. Šoustek 

et al., Dyes and Pigm. submitted. [3] P. Kapusta et al., Fluorescence Microscopy and Fluorescent Probes 2 (1998) 

133. [4] J. Sýkora et al., Langmuir 18 (2002) 571. 

80


FLUO-19 

Decay kinetics in fluorescence resonance energy transfer (FRET) sensors 

for metal ions 

Jens-Uwe Sutter, Olaf J. Rolinski and David J.S. Birch 

University of Strathclyde, Department of Physics, John Anderson Building, 107 Rottenrow, 

Glasgow G4 0NG, UK. E-mail: o.j.rolinski@strath.ac.uk 

Fluorescence lifetime sensing techniques recover the parameters of the assumed model decay functions by 

fitting them to the experimental data, usually in terms of the χ 2 goodness of fit criteria. 

For example, when FRET is used as a sensing mechanism, the Förster model of the decay function (for a 

random distribution of acceptors) or a monoexponential decay with shortened lifetime (for the donoracceptor 

pairs at a fixed distance), are used as model decays, and the acceptor concentration or donoracceptor 

separation can be determined, respectively. 

However, in many real FRET sensing applications, the experimental decays do not follow precisely the 

model curves, as the actual kinetics is more complex, frequently due to the original (unquenched) decay of 

the donor being not monoexponential, the actual donor-acceptor distribution being different to the one 

assumed in model kinetics, or FRET not being the only mechanism of quenching. 

In this poster, we report our attempts to develop more adequate models of FRET kinetics observed in some 

real systems [1] designed for detection of metal ions used as acceptors. In our approach, a model-free 

deconvolution based on maximum entropy method [2] is applied first to determine the lifetime distributions 

g(τ) rather than the fluorescence decay functions I(t), where 

∞ 

() ( τ ) 

0 

t 

− 

τ 

I t = ∫ g e dτ 

The recovered lifetime distributions can be then compared with the analytical g(τ) functions obtained for 

alternative models of excited-state kinetics. 

The results obtained for a number of the donor/metal ion pairs will be presented and usefulness of using 

changes in donor g(τ) as an indication of metal ions will be discussed. 

References: [1] D.J.S.Birch, O.J.Rolinski, Res.Chem.Intermed. 27, 4-5(2001)425. [2] J.C.Brochon, in: Methods in 

Enzymology, vol.240 (1994) Chapter 13, 262. 

81


FLUO-20 

Steady-state fluorescent polarization techniques in investigation on 

interactions of porphyrins with polynucleotides 

Victor Zozulya, Olga Ryazanova, Igor Voloshin 

B. Verkin Institute for Low Temperature Physics and Engineering, NAS of Ukraine, Department of 

Molecular Biophysics, 47 Lenin ave., 61103, Kharkov (Ukraine). E-mail: zozulya@ilt.kharkov.ua 

The interactions of tetracationic porphyrin compound, meso-tetrakis(N-methyl-4-pyridyl)porphine 

(TMPyP4), and its modified tricationic derivative bearing a peripherical carboxymethyl chain (Fig.1) with 

synthetic single- and double-stranded polynucleotides of various base compositions, as well as with fourstranded 

poly(G) and a G-quadruplexes of telomeric oligonucleotides were investigated by the methods of 

steady-state polarized fluorescence spectroscopy. The binding of the porphyrins to the polynucleotides were 

studied over a wide range of molar polymer-to-dye ratios (P/D) in aqueous buffered solutions, pH 7, at low 

and physiological ionic conditions, measuring the intensity and polarization degree of porphyrins emission 

upon titration experiments. 

The fluorescence technique was effective in recognition of complex formations between porphyrins and 

polynucleotides investigated. The three types of strong binding mechanisms were revealed, namely 

intercalation, embedding of porphyrins into polynucleotide groove and external cooperative stacking of 

their chromophores on polyanionic backbones. For these types of complex formation the changes in 

porphyrin fluorescence spectra, polarization degree and quantum yield are different. The porphyrin 

stacking-association is observed at low P/D values. It is characterised by strong fluorescence quenching. In 

pure form it was modeled by the dye binding with polyanionic matrix of polyphosphate. At the same time, 

the different types of assemblies were revealed for two porphyrins investigated: H-aggregates for TMPyP4 

and J-aggregates for the tricationic derivative. However, in some cases the outside stacking-association of 

porphyrins is not realized, for instance, upon binding to double-stranded poly(A)·poly(U). 

CH 3 

N 

Fig.1. Molecular structures of TMPуP4 

tricationic derivative. 

NH N 

O 

N O (CH 2 ) 4 C 

N HN 

OCH 3 

CH 3 

CH 3 

N 

This work is partially supported by Science and Technology Center in Ukraine (Project #3172). We thank Dr. 

I. Dubey for tricationic porphyrin synthesis. 

82


FLUO-21 

Sub-picosecond transient signal spectroscopy of prodan in 

dimethylformamide solution 

János Erostyák a , Pasi Myllyperkiö b , Andrea Buzády a , Jouko Korppi-Tommola b 

a Department of Experimental Physics, University of Pécs, Ifjúság u. 6., H-7624 Pécs (Hungary). 

E-mail: erostyak@fizika.ttk.pte.hu 

b Nanoscience Center/Department of Chemistry, University of Jyväskylä, P.O.Box 35, 

FIN-40014 Jyväskylä (Finland). 

Fluorescent probes are widely used in studying ultrafast unfolding and hydration dynamics of proteins. The 

knowledge of their electronic states and of the details of their ultrafast relaxations is a key to their 

successful application in biophysical and biological studies. 

6-propionyl-2-dimethylaminonaphthalene (Prodan) is a frequently used probe attached to human serum 

albumin (HSA) and other proteins [1]. With HSA it occupies the warfarin binding site (hydrophobic and 

electrostatic mode of binding) in domain II. It’s steady-state fluorescence spectrum depends very 

sensitively on the solvent polarity. Prodan’s fluorescence has already been studied in details with timeresolved 

techniques, e.g. sub-nanosecond solvation dynamics of Prodan was described in ionic solvents [2]. 

In more conventional solvents, the details of femtosecond dynamics of Prodan has not yet been measured. 

In this paper, the temporal evolution 

of earliest part of Prodan’s excited 

state absorption in DMF is reported. 

The experimental setup consisted of 

one-box fs laser (LIBRA, 

COHERENT) producing ~1mJ pulses 

of ~80 fs duration at 1 kHz. LIBRA 

was used to pump two NOPAs (noncollinear 

optical parametric amplifier) 

that can be independently tuned in the 

wavelength range ~450-750 nm. The 

pulses can be compressed down to ~25 

fs. 

Relative transient signal 

1,2 

1,0 

0,8 

0,6 

0,4 

0,2 

0,0 

λ probe 

= 590 nm 

λ probe 

= 620 nm 

λ probe 

= 650 nm 

λ probe 

= 680 nm 

The Figure shows representative -0,5 0,0 0,5 1,0 1,5 2,0 2,5 

pump-probe decays at several 

wavelengths of the S 1 →S 2 transition 

Pump-probe delay (ps) 

of Prodan. Calculation of the first spectral moment of this band is presented on the fs-ps time scale. A 

comparison to similar parameters of the fluorescent dye Acrylodan is also given. 

References: [1] J. K. A. Kamal et al., PNAS, 101(37) (2004) 13400. [2] P. K. Mandal et al., CURRENT SCIENCE, 

90(3) (2006) 301. 

83


FLUO-22 

Self-aggregation of 2 I ,3 I -O-(o-xylylene) α- and β-cyclodextrin derivatives in 

aqueous solution: Fluorescence and molecular modelling 

M. José González Álvarez a , Patricia Balbuena b , Carmen Ortiz Mellet b , José M. García 

Fernández c , Francisco Mendicuti a 

a 

Dpto. Química Física, Universidad de Alcalá, 28871 Alcalá de Henares, Spain. 

E-mail: francisco.mendicuti@uah.es 

b 

Dpto. Química Orgánica, Fac. de Química, Universidad de Sevilla, E-41012, Sevilla, Spain. 

c 

Instituto de Investigaciones Químicas, CSIC, E-41092 Sevilla, Spain. 

Cyclodextrins (CDs) are cyclic donut-shaped oligosaccharides which are widely used as host moieties in 

supramolecular chemistry. [1,2] Chromophore groups can be attached to CDs to obtain hosts which are useful 

for sensors based on the guest-induced response of such modified CDs. [3] 

MeO OMe 

We have recently engaged in a project aimed at the synthesis and 

O 

OMe 

OMe 

O 

characterization of several 2 I ,3 I -O-(o-xylylene) permethylated CDs. 

MeO 

O 

O 

MeO 

O 

OMe Dynamic 1 H NMR spectra recorded in D 2 O allowed us to infer the 

OMe 

MeO 

O 

O existence of temperature-dependent conformational equilibria 

OMe OMe OMe 

between arrangements where the secondary entrance into the cavity is 

O 

O 

O OMe 

O 

either hindered (capped) or not (open) by the xylylene group. 

O OMe 

O O 

Comparison of NMR spectra obtained by changing the temperature 

OMe OMe 

n = 1, 2 and/or CD concentration also indicated the existence of aggregation 

phenomena associated to that conformational equilibrium. An 

20 

18 

16 

14 

12 

10 

8 

6 

4 

aggregation number n = 2 was determined from dilution experiments, 

supporting the presence of dimeric structures. Fluorescence, 

Molecular Mechanics (MM) and Molecular Dynamics (MD) were 

employed to study the dimerization processes for 2 I ,3 I -O-(oxylylene)-per-O-Me-α- 

and -β-CDs. Emission spectra upon excitation 

of the xylylene moiety (260 nm) for modified α- and βCDs showed a 

single band at ∼288 nm. Decay profiles, fitted to three-exponential 

decay functions, also supported the existence of temperaturedependent 

2CDCD 2 equilibria. Thus, in addition to a short lifetime 

scattering component, two other components ascribed to the monomer 

and dimer species, respectively, were identified. The dimer/monomer 

ratio increased with concentration and decreased with temperature, 

0 2 4 6 which is in agreement with an enthalpy-driven dimerization process. 

[CD] /mM 

vs. [CD] at 5 ºC (); 25ºC 

() and 45ºC() for modified α- 

(open symbols) and βCD (filled). 

The dimerization equilibrium constants (K) were obtained from nonlinear 

regression analysis of the plots of average lifetimes, 

against [CD] in the 5-45ºC temperature range. A standard van’t Hoff 

analysis for K allowed us to obtain ΔH 0 and ΔS 0 associated to dimer 

formation. MM and MD calculations in the presence of water were 

also employed to study the conformational behaviour of isolated CDs, as well as the dimerization processes. 

/ns 

Acknowledgements: This work was supported by the Spanish MEC (projects CTQ2006-15515C02-01/BQU, CTQ2004- 

05854/BQU and CTQ200504710/BQU), the Junta de Andalucía (P06-FQM-01601) and the Comunidad de Madrid (S- 

055/MAT/0227). 

References: [1] J. Szejtli, T. Osa (Eds.), Comprehensive Supramolecular Chemistry, Pergamon Press, Oxford, 1996, 

Vol. 3. [2] V.T. D’Souza, K.B. Lipkowitz (Eds.), Chem. Rev. 98 (1998) 1741. [3] S. R. McAlpine, M.A. García et al., 

J. Am. Chem. Soc. 120 (1998) 4269. 

84


FLUO-23 

Frequency domain spectrofluorometry enhaced with pulsed LEDs 

Petr Herman and Jaroslav Vecer 

Charles University, Faculty of Mathematics and Physics, Inst. of Physics, 121 16 Praha (Czech Republic). 

E-mail: herman@karlov.mff.cuni.cz 

We present a simple way for extension of the time resolution of standard frequency-domain (FD) 

fluorometer by use of pulsed LEDs as an excitation source. Frequency-domain or phase-modulation 

measurements are widely used for time-resolved spectroscopy of biopolymers, fluorescence sensing, and 

lifetime imaging. Maximal temporal resolution of this methodology requires the excitation light to be 

modulated up to the highest possible frequencies with high modulation depth. Commercial phase 

fluorometers equipped with classical PMT and a cw light source with a Pockels cell modulator can typically 

work up to 200 MHz. Directly modulated LEDs allow for extension of this range up to 250-300 MHz [1, 2]. 

Higher modulation frequencies can be reached by utilization of a harmonic content of a pulse-train typically 

generated by expensive and complicated mode-locked laser systems. 

We used harmonic content of subnanosecond pulsed LEDs for generation of modulated excitation light. By 

simple replacement of the light source we immediately tripled the frequency range of the FD fluorometer 

equipped with an ordinary PMT. The frequency range increased from 200 MHz up to 600-700 MHz. The 

high-frequency cutoff was caused mainly by a frequency response of the PMT. Besides the increased time 

resolution, this approach allowed for elimination of a part of an expensive hardware (a synthesizer with an 

RF power amplifier) normally required for FD measurements. Examples of fluorescence and anisotropy 

decays acquired on a standard phase fluorometer equipped with the pulsed LED light source are presented. 

Our data demonstrate that pulsed LEDs can serve as an inexpensive alternative to pulsed laser sources for 

frequency domain fluorescence spectroscopy. 

References: [1] H. Szmacinski, Q. Chang , Appl. Spectroscopy 54 (2000), 106. [2] P. Herman et al., J. Microscopy. , 

203 (2001), 176. 

. 

85


FLUO-24 

Intramolecular dynamics of donor → acceptor energy transfer 

Miroslav Dvořák 1 , Philipp Wagener 2 , Vlastimil Fidler 1 , and Jörg Schroeder 2 

1 Department of Physical Electronics, Czech Technical University in Prague, 180 00 Praha 8, 

Czech Republic. E-mail: dvorakm@km1.fjfi.cvut.cz 

2 Institute of Physical Chemistry, University of Göttingen, D-37077 Göttingen, Germany 

The photophysics of rigidly linked multichromophoric molecular systems has attracted considerable interest 

spurred by the aim to perform an elemental (opto)electronic function (e.g. switching) by a single-molecule 

electronic device. 

With this motivation in mind we have studied bi- and tri-chromophoric molecules consisting of aminopyrene 

derivatives as donors and amino-benzanthrone derivatives as acceptors rigidly linked by a triazine 

ring [1]. We showed that (i) donor and acceptor moieties are practically decoupled in the electronic ground 

state and (ii) excitation of the donor part causes ultra-fast electronic energy transfer (EET) – probably of the 

through bond type – to the acceptor part [2]. The next logical step is to understand the role of the third 

substituent at the triazine ring and its possible influence on the photo-induced EET process. In the context 

of these investigations we report here on new results of corresponding fluorescence and ps/fs transient 

absorption studies that help to characterize additional aspects of EET dynamics in these systems. In 

particular, we compare the behaviour of differently substituted 2-(3-benzanthronylamino)-4-(1- 

pyrenylamino)-6-X-1,3,5-triazine molecules, (APyTXABa), where X is chlorine, aniline or amino-pyrene. 

In these molecules, the amino-pyrene acts as a donor, the amino-benzanthrone as an acceptor, and the 

triazine ring serves as a spacer. In this report, we discuss the differences in time evolution of emission and 

absorption of the acceptor fluorescing state following excitation into the absorption band of either the donor 

or the acceptor part. 

APyTCABa APyTAnABa APyTAPyABa 

Chemical structures of the compounds compared in this contribution. 

For comparison, we also studied the corresponding model donor and acceptor molecules which closely 

mimic the photophysical properties of the respective sub-units in the bichromophore [2, 3]. 

Based mainly on fluorescence kinetics and ps/fs transient absorption spectra, possible transient states are 

discussed that could participate in the electronic excitation energy transfer from donor to acceptor. In 

particular, the role of the donor/spacer localised CT states [3] will be considered. 

References: [1] M. Nepraš et al., Dyes and Pigments 35, 31-44 (1997), [2] V. Fidler et al., Z. Phys. Chem. 216, 

589-603 (2002), [3] Michl et al., Charge-transfer states in pyrene-triazine compounds, MAF 2007. 

86


FLUO-25 

Fluorescence behaviour and complexation with cyclodextrins of R and 

S-[1,1’-binaphthalene]-2,2’-diols 

Raquel de Francisco, Gema Marcelo, Francisco Mendicuti 



Cyclodextrins (CD) are cyclic oligosaccharides capable of forming complexes with a great variety of low 

and high molecular weight guest molecules. The complexation ability is very selective and depends on the 

size and polarity of the guest molecule relative to the inner CD cavity. When guests contain chromophore 

groups, fluorescence is very helpful to get information on the thermodynamics of complexation. 

Recognition of chiral guest compounds in solution by modified CDs attached to stationary phases for 

liquid chromatography is one of the current research topics. [1,2] 

fluorescence (a.u.) fluorescence (a.u.) 

HO 

OH 

HO 

OH 

R-BINOL 

340 360 380 400 420 440 460 480 500 

λ /nm 

[βCD] 

340 360 380 400 420 440 460 480 500 

λ /nm 

R-BINOL-αCD 

22.6mM 

[αCD] 

R-BINOL-βCD 

12.6mM 

Fluorescence emission spectra for R-BOH 

in the absence and in the presence of α- 

and βCD at different concentrations (25ºC) 

0 

0 

S-BINOL 

Steady-state and time-resolved fluorescence techniques were 

used on isolated R and S-1,1’-binaphthalene-2,2’-diol (R- and 

S-BINOL) and in the presence of α- and βCD to obtain 

stoichiometries, binding constants and enthalpy and entropy 

changes accompanying the formation of complexes in aqueous 

medium. Molecular Modelling contributes to clarifying the 

structure of the complexes as well as to extending the 

knowledge on the interactions involved in such processes. [3] 

Emission spectra either for free S- and R-BINOL or in the 

presence of CDs showed two bands placed at ∼355 nm and 

∼380 nm. Fluorescence intensity and average lifetimes depend 

on the guest and [CD] types, increasing with [CD]. Complexes 

have 1:1 stoichiometries and the association constants at 

different temperatures which were obtained from these 

changes, are relatively low. ΔH 0 < 0 and ΔS 0 < 0 were obtained 

for all systems by using van’t Hoff plots. Attractive van der 

Waals host-guest interactions are characterized by ΔH 0 < 0. For 

guests that penetrate only partially into the cavity ΔS 0 < 0 are 

expected. Fluorescence anisotropies for guest/CD solutions 

increase with [CD] due to the increase in the fraction of the 

complexed form. Quenching experiments give information 

about the accessibility of the guest to the quencher in the 

complex and its location. Both BINOLS hardly penetrate into 

the CD cavities, although they do slightly more so into the 

βCD cavity than into the αCD one. Molecular Mechanics 

showed that van der Waals interactions are the most important 

contribution to the complex formation and inferred that the 

guests hardly penetrate into de CD cavities. 

Acknowledgements: This research was supported by Comunidad de Madrid (CAM project: S-055/MAT/0227) and by 

the Spanish Ministerio de Educación y Ciencia (project: CTQ2005-04710/BQU). G. Marcelo acknowledges a FPU 

fellowship from the Spanish government. 


Vol. 3. [2] A. Harada, Acc.Chem. Res. 34 (2001) 456. [3] A. Di Marino et al., J. Incl. Phenom. Macroc. Chem. 56 

(2006) 225. 

87


FLUO-26 

Fluorescence and molecular mechanics of the inclusion complexes of 

dimethyl 2,3-naphthalene dicarboxylate with 2-hydroxypropyl cyclodextrins 

in aqueous media 

Ruben Usero, M. José González Álvarez, Francisco Mendicuti 



Cyclodextrins (CD) are toroid-shaped host molecules with a capacity to form non-covalent binding guesthost 

inclusion complexes with low molecular weight compounds and polymers. [1,2] The inner surface of 

the cavity is relatively hydrophobic because it is linked by glycosidic oxygen bridges. In contrast the 

exterior surface is hydrophilic due to the presence of hydroxyl groups at both entrances. Thus, depending 

on the CD type and the extent of the penetration, both microviscosity and the polarity of the medium 

surrounding the guest molecule can be substantially modified. These changes influence the fluorescence 

characteristics of the aromatic guest. 

R 

1.00 α-HPCD 

β-HCD 

0.95 

γ-HPCD 

0.90 

0.85 

0.80 

0.75 

0.70 

0.65 

0.00 0.01 0.02 0.03 0.04 

[HPCD] /mM 

Variation of R with [HPCD] 

at 25ºC 

Fluorescence and Molecular Modelling techniques were 

employed to study the inclusion complexes of dimethyl 2,3- 

naphtalenedicarboxylate (23DMN) with α-, β- and γ-2- 

hydroxypropyl cyclodextrins (CDs). Emission spectra of 23DMN 

show two bands whose intensity ratio R is very sensitive to the 

medium polarity. [3,4] From the variation of R with [CD] and 

temperature, the stoichiometry, the formation constants, and the 

changes of enthalpy and entropy upon inclusion of the complexes 

formed were obtained. Results showed identical stoichiometry 

(1/1) for the three complexes with α-, β- and γCDs. The estimated 

formation constants at 25ºC were ∼160 M -1 , ∼1100 M -1 , and ∼90 

M -1 , respectively. ΔH 0 and ΔS 0 were obtained from linear van’t 

Hoff plots. R at [CD]→∞ allows us to estimate the effective 

dielectric constant of the medium surrounding the guests when 

complexed. The later values and the fluorescence anisotropy, 

quenching with (CH 3 CO) 2 and average lifetime measurements 

can also give additional information about the guest location and 

the geometry of the complexes. Molecular Mechanics 

calculations were also employed to study the formation of 

complexes of 23DMN with α-, β- and γ-HPCDs. This study was 

mainly performed in the presence of water as a solvent. The 

driving forces for the inclusion processes, in agreement with the 

thermodynamic parameters, were dominated by non-bonded van 

der Waals host:guest interactions. 

Acknowledgements: This research was supported by Comunidad de Madrid (CAM project: S-055/MAT/0227) and 

by the Spanish Ministerio de Educcación y Ciencia (project: CTQ2005-04710/BQU). 


Vol. 3. [2] A. Harada, Acc.Chem. Res. 34 (2001) 456. [3] A. Di Marino et al., J. Incl. Phenom. Macroc. Chem. 56 

(2006) 225. [4] C. Alvariza et al., Spectrochimica Acta Part A (2007) in press. 

88


FLUO-27 

Photoinduced switching in fluorophores as a mean for ultrahigh 

resolution microscopy 

Andriy Chmyrov*, Stefan W. Hell**, Jutta Arden-Jacob***, Alexander Zilles***, 

Karl-Heinz Drexhage***, Jörg Reichwein ****, Jerker Widengren* 

* Royal Institute of Technology, Department of Applied Physics, Stockholm (Sweden); 

** MPI Biophysical Chemistry, Department of NanoBiophotonics, Göttingen (Germany); 

*** University of Siegen, Department of Physical Chemistry, Siegen (Germany); 

**** ATTO-Tec GmbH, Siegen (Germany) 

In the EU project SPOTLITE we aim at establishing molecular resolution with focused visible light. The 

diffraction resolution limit inherent to an imaging system is to be broken by application of a reversible 

saturable optical transition, induced by a spatial intensity distribution featuring a local minimum. The 

intended approach is similar to STED (stimulated emission depletion), developed by Stefan Hell and 

coworkers. STED has been successfully implemented; however, its range of application is limited by the 

high intensities needed to induce stimulated emission. 

We present an investigation of switching properties of fluorophores to and from different, more long-lived 

transient states, for which reversible saturable optical transition can be achieved at considerably lower 

intensities. First, we investigated the triplet state properties of several fluorophores, with respect to their 

possible use for reversible photo-switching and resolution enhancement. At present, we believe to have 

identified at least one candidate dye having a considerable triplet quantum yield, and yet a relatively limited 

quantum yield of photobleaching. 

As another possible photoswitching mechanism, we also investigated trans-cis isomerisation of specially 

designed carboxycyanine dyes. This mechanism possibly benefits from a higher photostability, since triplet 

states are not involved. 

References: [1] S.W. Hell, Nat. Biotechnol. 21 (2003) 1347. [2] S.W. Hell et al., Curr. Opin. Neurobiol. 14 (2004) 

599. [3] J. Widengren et al., J. Phys. Chem. 99 (1995) 13368. [4] Widengren J., Schwille P., J Phys Chem A 104 

(2000) 6416. 

89


FLUO-28 

Fluorescence studies on functionalized polypropylene supports using an 

amino-sensitive fluorogenic pyrylium label 

Katrin Hoffmann, Ute Resch-Genger, Renate Mix, Joerg. F. Friedrich 

Federal Institute for Material Research and Testing, D-12489 Berlin (Germany). 

E-mail: katrin.hoffmann@bam.de 

Tailor-made polymer surfaces are of ever increasing importance in the area of material science and bio 

analysis. Plasma-chemical functionalization of polymer surfaces with e.g OH-, NH 2 -, or CHO-groups 

enables to control e.g. the biocompatibility, the hydrophilicity as well as adsorption and wetting properties 

of materials and provides the basis for the attachment of bio- and sensor molecules for different 

(bio)analytical and biomedical applications. [1] Crucial are here not only strategies towards a chemically 

defined surface modificafition, but also simple and robust analytical tools for the reliable characterization of 

these materials with respect to the type and density of the reactive functional groups at the surface. The 

application of extremely sensitive fluorescence labelling techniques is well established for this purpose in 

the areas of biological, biomedical as well as polymer chemistry. The fluorometric analysis of surface 

species, however, is complicated by different factors [2] , amongst others by non-specific adsorption of 

(unreacted) fluorescent probes. To overcome these limitations, sophisticated fluorescent reporters such as 

the recently introduced pyrylium dye Py-1, [3] are desired, that reveal strong changes in energy and intensity 

in absorption and emission upon covalent attachment to functional groups. Even though pyrylium dyes 

have been successfully exploited for the detection of amino functionalities in proteins, [3] no attempts have 

been yet reported to adapt this strategy to the analysis of polymer surfaces. 

This encouraged us to investigate the potential of Py-1 to label and to monitor amino groups at complex 

plasma-chemically modified polymer surfaces. The straightforward strategy towards the sensitive 

fluorometric surface analysis is based on the transformation of the pyrylium dye Py-1 into its pyridinium 

analogue. Intriguingly, also for these polymer materials, strong binding-induced hypsochromic shifts and an 

increased fluorescence quantum yield have been observed which enable to spectroscopically distinguish 

between covalently linked and unreacted free dyes, i.e., non-specifically adsorbed molecules and labels 

diffused into the polymer. [4] 

Fig. 1. Intensity profile of a plasma-chemically 

amino-modified polymer support (thickness ca. 

100 µm) after exposure to Py-1 recorded by 

Confocal Laser Scanning Microscopy (excitation 

wavelength 543 nm). 

The intense emission of Py-1-labeled solid supports can be excited between 470 and 530 nm matching 

several laser lines commonly used for fluorescence-based bio-analytical techniques thereby elegantly 

circumventing the simultaneous excitation of unbound dye and decreasing undesired background 

emission.The results of fluorescence spectroscopic and microscopic studies represent a first step towards an 

improved direct fluorometric characterization of surface functionalities at plasma-chemically modified solid 

polypropylene supports revealing a complex surface chemistry and a modification-induced porosity. 

References: [1] C. Oehr, Nucl. Instr. and Meth. in Phys. Res., B 208 (2003) 40-47. [2] A. Holländer, Surf. Interf. 

Anal., 36 (2004) 1023-1026. [3] B. K. Hoefelschweiger, A. Duerkop, O. S. Wolfbeis, Anal. Biochem. 344 (2005) 

122-129. [4] K. Hoffmann, U. Resch-Genger, R. Mix, J.F. Friedrich, Langmuir (2006), in press. 

90


FLUO-29 

Application of the maximum entropy method in time-resolved 

FRET measurements 

Alexander A. Maskevich, Vitali I. Stsiapura, Sergey V. Hoh 

Yanka Kupala Grodno State University, 230023 Grodno (Belarus). E-mail: amaskevich@grsu.by 

Forster resonance energy transfer (FRET) is widely used in fluorescence spectroscopy. As a result of 

energy transfer both intensity and kinetics of fluorescence for donor and acceptor molecules change 

significantly. Therefore measurements of fluorescence decay can provide important information about 

distance between donor and acceptor and their mutual orientation. However, decay law functions for donor 

and acceptor molecules have complicated character and their reconstruction without a priori assumptions 

represents ill-posed mathematical problem. 

The case, when donor and acceptor are connected by flexible covalent linkage, is considered. Donoracceptor 

distance for the ensemble of molecules can be characterized by ρ ( r) 

distribution and fluorescence 

decay law for donor is following 

∞ 

⎡ 

6 

t t ⎛ R ⎤ 

0 ⎞ 

FDA() t = F0 ∫ρ( r) 

exp ⎢− 

− ⎜ ⎟ ⎥dr 

, 

⎢⎣ 

τ τ ⎝ r ⎠ 

0 

D D ⎥⎦ 

where τ D – decay lifetime of donor in the absence of acceptor, R 0 – Forster radius. 

ρ r distribution without a priori 

Maximum Entropy Method (MEM) was used to determine ( ) 

assumptions. According to MEM such a function ρ(r) must be selected among possible distributions, that 

maximize functional ψ = S − μ( χ 2 −1) 

, where χ 2 – Pearson parameter, S – entropy function [1], and μ - 

regularization parameter. 

Figure: Distance distribution function 

ρ(r) between tryptophanyl residue in 

HSA and covalently bound PLP. Solid 

line – native protein, dashed line – 

denatured by 6M urine. PLP/HSA ratio 

= 1.4. 

Capability of the developed method to reconstruct ρ ( r) 

was tested in model calculations, where ρ ( r) 

function was represented by mono- and bimodal Gaussian distributions. It was shown that recovered 

parameters of peaks (position, width) did not differ from the true ones by more than 2-5%. Application 

of the developed method to study protein structure and dynamics was demonstrated for human serum 

albumin (HSA) labeled by pyridoxal-5’-phosphate (PLP). Tryptophanyl residue of HSA plays the role 

of the energy donor and covalently bound PLP – of the acceptor. Reconstructed distribution ρ ( r) 

indicates (Figure) that two types of binding centers for PLP on the protein exists. Protein denaturation 

ρ r distribution, which testifies the 

in the presence of 6М urine leads to significant change of the ( ) 

ρ(r) 

suitability of the developed method to monitor changes in spatial structure of macromolecules. 

This work was supported by the grants Х06Р-115 and F06-351 of Belarus Foundation for Fundamental Research. 

Reference: [1] J.-C. Brochon // Methods Enzymol. 240 (1994) 262. 

0,5 1,0 2,0 

r/R 0 

91


FLUO-30 

Steady state fluorescence study on the cyclodextrin inclusion complexes of some 

cardiovascular drugs 

Laura Soare, Iulia Matei, Cristina Tablet, Mihaela Hillebrand 

University of Bucharest, Department. of Physical Chemistry, 

Bd. Regina Elisabeta, 4-12, Bucharest, Romania. E-mail: mihh@gw-chimie.math.unibuc.ro 

The photophysical properties of three cardiovascular drugs, atenolol, indapamide and simvastatin in organic 

solvents and aqueous media are reported and discussed. The experimental data, reflecting the nature of the 

first excited singlet state are rationalized in terms of solvent dependent semiempirical calculations. Since 

the guest-cyclodextrin interaction can be used as a model for a following study on the drug-protein 

interaction, the host-guest complexes of the mentioned drugs with the native α-,β-,γ- cyclodextrin and the 

modified 2-hydroxypropyl-β- and 2-hydroxypropyl -γ-cyclodextrin were investigated by means of steadystate 

fluorescence spectroscopy. 

The fluorescence spectra of atenolol in 

the presence of cyclodextrins (Spectra 

1-10 in figure, in order of increasing 

the concentration of the β- 

cyclodextrin) reveal a complex process 

characterized by the quenching of the 

main band of atenolol and the 

appearance of a new band at a longer 

wavelength. In the case of the other 

drugs, the complexation process is 

evidenced only by the quenching of 

the fluorescence band. The association 

constants and the stoichiometry of the 

complexes were estimated by nonlinear 

regression analysis. Depending 

on the drugs we have found complexes 

with 1:1, 1:2 and mixtures of 1:1 and 

1:2 stoichiometries. The optimized 

geometry of the complexes and the 

I (u.a.) 

500 

400 

300 

200 

100 

0 

300 350 400 450 500 

interaction energies were estimated by in vacuo and water –dependent molecular mechanics (MM) 

calculations. The calculations allow for the estimation of the relative electrostatic and van der Waals 

contributions to the interaction energy. 

1 

10 

10 

1 

λ(nm) 

92


FLUO-31 

Synthesis and photophysical properties of [60]fullerene–naphthalimide diads 

Marek Klučiar, a Carlos Baleizão, a Uwe Pischel b and Mário N. Berberan-Santos a 

a Centro de Química-Física Molecular, Instituto Superior Técnico, 1049-001 Lisboa, Portugal 

b Instituto de Tecnología Química, UPV/CSIC, 46022 Valencia, Spain 

E-mail: marek.kluciar@mail.ist.utl.pt 

In the past 15 years extensive efforts have been devoted to the development of molecular donor-acceptor 

assemblies as fascinating candidates for the design of molecular electronic devices and artificial systems for 

light energy harvesting. [1] The setup of efficient molecular assemblies requires an intelligent photophysical 

engineering of the underlying excited state processes like photoinduced electron transfer or electronic 

energy transfer. In this respect, fullerenes constitute preferential building blocks, owing to their 

multifaceted redox properties and rich photochemistry. [2] 

Generally, the fullerene is playing the role of an electron and/or energy acceptor. As counterpart a large 

variety of electron- and energy donor moieties has been used in search for improved electron- and energy 

transfer in donor-acceptor assemblies. [3] For instance, the use aromatic dicarboximides (perylenediimide) as 

antenna unit has been recently devised for the design of fullerene diads with improved light absorption 

properties. [4] 

O 

O 

O 

R 1 R 

O O 2 

N 

O 

R 3 

Upon intelligent design of the donor antenna it is further possible to influence its photophysical and redox 

characteristics, which might result in altered efficiencies of electron and energy transfer to the linked 

fullerene. The properties of 1,8-naphthalimide derivatives can be conveniently fine-tuned by 

straightforward and cost-effective synthetic procedures. This motivated us to synthesize (via a modified 

Bingel reaction) and photophysically investigate (absorption spectroscopy, steady-state and time-resolved 

fluorescence spectroscopy) of a series of novel [60] fullerene–1,8-naphthalimide diads. 

Acknowledgements: This work was supported by FCT (Portugal) and POCI 2010 (POCI/QUI/58535/2004). 

M. Kluciar was supported by a doctoral fellowship from FCT (SFRH/BD/18699/2004). C. Baleizão is grateful for a 

postdoctoral fellowship from FCT (SFRH/BPD/28438/2006). U. Pischel thanks the Spanish Ministry of Education 

and Science, Madrid, for a Ramón y Cajal grant. 

References: [1] D. M. Guldi, M. Prato, Acc. Chem. Res. 33 (2000) 695. [2] D. M. Guldi, Chem. Soc. Rev. 31 (2002) 

22. [3] S. Nascimento et al., J. Fluoresc. 16 (2006) 245. [4] Y. Shibano et al., Org. Lett. 8 (2006) 4425. 

93


FLUO-32 

Anisotropy of fluorescence as a tool for studying photoisomerization dynamics 

Alexander P. Blokhin a , Maxim F. Gelin b 

a 

Institute of Molecular & Atomic Physics, National Academy of Sciences of Belarus, 220072 Minsk, 

(Belarus). E-mail: lsfm@imaph.bas-net.by 

b Department of Chemistry and Biochemistry, University of Maryland, College Park, 20742 Maryland 

(USA). E-mail: mhelin@ umd.edu. 

Photoisomerization results in changes of molecular structures, and also can induce alternations of directions 

of transition dipole moments in molecular frames. Clearly, these phenomena manifest themselves in 

significant changes of the time development of the anisotropy of fluorescence of the ensemble of 

photoproducts. As a consequence, monitoring of the anisotropy decay allows one to obtain unique 

information about photoisomerization dynamics, both for isolated [1] and solvated [2] molecules. 

The subject of our work is to develop a simple, convenient for practical applications but general enough 

theory of the anisotropy decay in the course of photoisomerization of polyatomic molecules, both under 

collisionless conditions and in a solution. The basic idea is embodied in the assumption that a characteristic 

time, during which changes of molecular structure occur, is substantially less than a characteristic time of 

molecular reorientation. This allows us to describe mapping of the parent angular momentum into that of 

product by introducing the pertinent conditional probabilities and to use the formalism of orientational 

correlation functions [3-4]. 

We derive general expressions for the anisotropy time evolution in case of asymmetric top parent and 

product molecules. As the input parameters, the theory contains the standard quantities describing parent, 

product, and (if necessary) transition state structures, viz. the main moments of inertia, directions of 

transition dipole moments in molecular frames, and lifetimes in excited states. 

The explicit calculations are performed of both the steady-state anisotropy and its time evolution in cases, 

when characteristic times of photoprocesses are much less or grater than that of molecular reorientation. 

The value of steady-state anisotropy is calculated for symmetric top molecules for various mutual 

orientations of reactants and products, relationships between their moments of inertia and directions of 

transition dipole moments in molecular frames. Anisotropy is also analyzed for an ensemble of 

photoisomers, when the reactant and product molecule are planar asymmetric tops. Our analysis shows for 

which molecules one should expect the most prominent changes in the anisotropy decay. It is argued that 

the detection of the polarized response allows one to estimate the characteristic timescale of the 

photoreaction and to determine intramolecular orientation of absorption and emission dipole moments. 

References: [1] J.S. Baskin et all., J. Chem. Phys.100 (1996) 11920. [2] G. Haran et all., J. Phys. Chem. A., 103 

(1999) 2202. [3] A.P. Blokhin et all, J. Chem. Phys. 110 (1999) 978. [4] A.P. Blokhin and M.F. Gelin, Phys. Chem. 

Chem. Phys. 4 (2002) 3356. 

94


FLUO-33 

Photoreactions of polycyclic aromatic hydrocarbon vapors with oxygen 

Galina Zalesskaya, Andrey Kuchinsky, Olga Galay 

Institute of Molecular and Atomic Physics of NAS of Belarus, 70 Procpect, Nezavisimosty, 220072 Minsk, 

(Belarus), E-mail: zalesskaya@imaph.bas-net.by 

Polycyclic aromatic hydrocarbons (PANs) entering the atmosphere as results of vehicle exhausts and 

industrial emissions play an important role in atmospheric photochemistry and belong to the most 

dangerous environmental pollutants. The fate of PAHs in the atmosphere depends to a large degree on the 

photochemical reactions with oxygen. To date, due to methodical difficulties, there are a few works devoted 

to the study of the oxygen quenching of electronically excited PANs in the vapor phase. In this study, the 

oxygen quenching of singlet and triplet states of PAN vapors is investigated for a set of PANs (anthracene 

derivatives, pyrene, chrysene, phenanthrene, fluorantene, carbazole) differing by the positions of the 

electronic levels and by the oxidation potentials. The method used was oxygen quenching of PAN 

fluorescence and delayed fluorescence. 

The oxygen quenching rate constants of the singlet and triplet states are determined and the most promising 

approaches to modeling oxygen-induced photoreactions are considered. In contrast to the generally 

accepted opinion that the quenching in the gas phase is always controlled by collisions, the quenching 

efficiencies of the singlet state S 1 in the PAHs studied are found to be both comparable with the gas-kinetic 

efficiencies and two order of magnitude lower, while the quenching efficiencies of the triplet state T 1 are 

found to vary in the range from 2.3·10 -4 to 4.0·10 -2 . The dependences of the rate constants on the free 

energy of the electron transfer, free energy of triplet-triplet energy transfer, vibration energy excess of 

interacting molecules are analyzed for the photoreactions of PANs with oxygen. It is established that the 

quenching rate constants of singlet and triplet states as well as the fraction of both quenched states and 

forming singlet oxygen are shown to vary in wide range and depend on the ionization potentials and the 

exothermisity in the electron transfer process. Experimental dependences of the quenching rate constants 

for singlet and triplet states on the free energy of the full electron transfer are described by the Marcus 

equation but are not consistent with predicted ones for the full electron transfer from PAN to oxygen. 

Conclusion is made that the process of electron transfer participates in oxygen quenching of the excited 

states PANs but only partial charge separation exists in the encounter complex. The results obtained are 

essential to understand the mechanisms governing the PAN residence time in the atmosphere and the singlet 

oxygen formation by PAN interaction. 

95

Part II 

Imaging 

and Microscopy 

97

Abstracts Poster – Part II: Imaging and Microscopy 

IMMI-1 

Read-out of dual sensors by means of a digital color camera 

Matthias I. Stich, Sergey M. Borisov, Michael Schäferling, Otto S. Wolfbeis 


D-39040 Regensburg (Germany), e-mail: matthias.stich@chemie.uni-regensburg.de 

In the bigger part of imaging applications, cooled black-and-white CCD-cameras are used to monitor the 

emission intensity of the indicators. When two or more analytes are considered, the signal of interest has to 

be separated from the others. Usually, this is accomplished by using emission filters or, in case of lifetime 

imaging, by evaluating the different luminescence decay times of the indicators. A novel approach for 

signal separation is presented here. It takes advantage of the fundamental setup of color CCD and CMOS 

cameras, making readout of different colors accessible. The principle of color cameras relies on the 

incorporation of three different types of pixels (CCD) or layers (CMOS), all sensitive towards another 

wavelength range. The idea was to utilize this technique for signal separation. The information about the 

color is saved on different areas on the sensor chip and is transported in different channels. In other words, 

due to the spatial distribution of three different types of pixels, it is possible to monitor the intensity of three 

different colors with one single image. 

For the proof of principle, a dual sensor was designed that exhibits emissions in the blue and in the red area, 

respectively. HPTS (8-Hydroxypyrene-1,3,6-trisulfonate) as a CO 2 -indicator [1] (which has blue emission 

for the deprotonated form) and Eu(tta) 3 (pat) as a temperature indicator [2] (with red emission) were applied. 

Both dyes can be excited at 405 nm and their emissions do not overlap. The two probes, HPTS (in 

ethylcellulose) and Eu(tta) 3 (pat) (in poly(vinyl methyl ketone)) were cast on a solid support, containing one 

dual sensor area and two single component sensors as reference. The response of the sensor was calibrated 

at seven temperatures and six carbon dioxide concentrations. For image acquisition, the sensor foil was 

illuminated continuously and the emitted light was recorded with a digital camera. The images obtained 

were divided in their RGB channels, resulting in additional three black-and-white images, containing the 

intensity information of the three color channels, respectively. The response of the sensor system agrees 

very well with theory and reference measurements. 

(a) 

(b) 

Relative Absorption/Emission 

1.0 

0.8 

0.6 

0.4 

0.2 

Emission CO 2 

-Indicator 

O O 

HO S 

ONa 

NaO 

ONa 

S S 

O O O O 

Emission T-Indicator 

N 

N N 

N N N 

N 

N 

Eu 

O O 

S 

F 

F F 

3 

450 500 550 600 

wavelength [nm] 

Fig. 1.: (a) Chemical structure and emission spectra of the indicators applied. (b) Sensitivity of 

HPTS to CO 2 in RGB color and in the two channels concerned 

References: [1] S.M. Borisov et al., Adv. Mater. 18 (2006) 1511; [2] C. Yang et al., Angew. Chem. Int. Ed. 43 

(2004) 5010. 

99


IMMI-2 

Confocal fluorescence lifetime imaging (FLIM): A tool for analysis of structure 

changes of protein adsorbed onto solid surfaces 

Denisio Togashi 1,2 and Alan G. Ryder 1,2 

1) Nanoscale Biophotonics Laboratory, Department of Chemistry, National University of Ireland, Galway. 2) 

National Centre for Biomedical Engineering Science, National University of Ireland, Galway. 

In biomaterials research, the interaction of surface coatings with proteins is of fundamental importance. It 

is important to be able to determine the quantity and rate of deposited protein on surfaces and also observe 

the effect that the material surface has on the structure of adsorbed proteins. It has been suggested that the 

structural changes of adsorbed proteins can affect biological response, causing unwanted effects such as 

inflammation, thrombosis, and/or biofilm growth [1]. The majority of analytical techniques only quantify 

the amount of the protein adsorbed on surfaces. Here we introduce a new strategy to observe 

conformational changes of adsorbed proteins by applying confocal FLIM microscopy. Using fluorescent 

labelled proteins in combination with confocal FLIM should enable the observation of changes in protein 

structure during adsorption onto surfaces. In this work we present FLIM data from the adsorption of 

labelled Bovine Serum Albumin (BSA). BSA is covalently linked to fluorescein molecules (BSA-FITC), 

and also double labelled with tetramethylrhodamine (BSA-FITC-TMR). Both proteins are allowed to 

adsorb onto solid glass (hydrophilic) and trimethylsilalized glass (hydrophobic) substrates. The average 

lifetimes of a thin layer of adsorbed BSA-FITC and BSA-FITC-TMR formed by the contact between the 

protein bulk solution and the substrate surface were measured by multifrequency modulation and phase 

shift. Different average lifetimes were obtained and correlated to different structures and geometrical 

disposition of protein on the surfaces. 

Acknowledgements: This work was supported by Science Foundation Ireland under Grant number (02/IN.1/M231), 

and by an equipment grant by Ireland’s Health Research Board (Grant EQ/2004/29). 

References: [1] “Molecular basis of biomaterial-mediated foreign body reactions”. W.J. Hu, J.W. Eaton, 

T.P. Ugarova, L. Tang. Blood 98 (2001), 1231-1238. 

100


IMMI-3 

Imaging of gas-concentration fields in the aqueous boundary layer 

Achim Falkenroth, Alexandra Herzog, Bernd Jähne 

University of Heidelberg, Institute of Environmental Physics (IUP), Gas Exchange and Waves, 

and Interdisciplinary Centre of Scientific Computing (IWR), Digital Image Processing 

D-69120 Heidelberg (Germany). E-mail: bernd.jaehne@iwr.uni-heidelberg.de 

Laser-Induced Fluorescence (LIF) techniques are applied to visualize gas exchange across the air–water 

interface in wind–wave facilities. The difficulty in studying inter-phase exchange processes is due to the 

small thickness (30 µm to 1 mm) of the mass boundary layers on both sides of a free air–water interface 

which is undulated by wind waves. Therefore, none of the traditional fixed sampling methods is applicable. 

Two techniques were investigated. Dissolved oxygen is made visible by the phosphorescence of an organic 

ruthenium complex (tris(4,7-diphenyl-1,10-phenanthroline disulfonic acid) ruthenate(II), 

Ru(dpp ds) 3 ). [1] This dye is very soluble in water, shows no surface activity, and has – due to its long 

lifetime – a high quenching constant of K = 11300 L/mol. Therefore it is much more sensitive to oxygen 

than other dyes such as pyrene butyric acid (PBA) [2] which were used in previous studies. Concentration 

fields of acid or alkaline volatile species such as CO 2 , HCl, or diethyl amine can be made visible by the 

fluorescent pH indicator 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS). [3] Fluorescence of both dyes can 

be stimulated simultaneously by a 473 nm DPSS laser. 

Pseudo-color image of concentration 

fields in a laser-light sheet. High gas 

concentrations (red) are observed in the 

boundary layer where oxygen penetrates 

the water surface to reach the degassed 

bulk. Turbulence structures below the 

surface are resolved with a 640x480 

pixel camera at 185 Hz and reveal the 

mechanisms of gas transport across the 

diffusion boundary layer with a pixel 

resolution of 25 µm/pixel. The mirror 

image at the top is due to total reflection 

at the surface. 

The novel visualization technique significantly increased the poor signal-to-noise ratio inherent to 

previously published LIF techniques. The significant temperature dependence of the phosphorescence of 

the Ru-complex is not a problem because the water temperatures are kept constant during the experiments. 

Digital image processing methods were developed [4] to analyze the image sequences. After the feature 

extraction of the surface position and image registration, the boundary-layer thickness z* was extracted 

from the concentration profiles. This value depends on the gas flux through the air–water interface. From 

this, the transfer velocity can be computed and compared to reference measurements. 

The form of the concentration profile in the water was compared to the theoretical descriptions to decide 

about the suitable description of the turbulence structures near the phase boundary. 

The project is funded by the German Research Society DFG, Graduiertenkolleg 1114: http://www.grk1114.de 

References: [1] F. N. Castellano, J. R. Lakowicz, Photochemistry and Photobiology 67:2 (1998) 179. [2] Herlina, 

G. H.Jirka, Exp. Fluids 37 (2004) 341. [3] O. S. Wolfbeis et. al., Fresenius Z. Anal. Chem. 314 (1983) 119. 

[4] A. Falkenroth, PhD-Thesis, University of Heidelberg (2007). 

101


IMMI-4 

Single-molecule detection of allophycocyanin (APC) entrapped in a silica sol-gel 

glass under physiological conditions 

Alexander M. Macmillan 1 , Jan Karolin 1 , Colin D. McGuinness 2 , Dalibor Pánek 1 , 

John C. Pickup 2 and David J. S. Birch 1 

1 Centre for Molecular Nanometrology, Department of Physics, John Anderson Building, 

University of Strathclyde, 107 Rottenrow, Glasgow G4 0NG, UK. E-mail: djs.birch@strath.ac.uk 

2 Department of Chemical Pathology, Guy’s, King’s, and St Thomas’s Hospitals School of Medicine, 

Guy’s Hospital, London SE1 9RT, UK 

Allophycocyanin (APC) is a highly fluorescent protein (quantum yield = 0.68), that belongs to the 

phycobiliprotein family found in the light-harvesting system in blue-green algae. Because of its large molar 

extinction coefficient (ε 650 = 7 x 10 5 M -1 cm -1 ); emission around 660 nm where cellular autofluorescence is 

low, and because it can be excited using standard diodes and HeNe lasers, it has found widespread use in 

both immunoassay and sensor applications [1]. 

Here we demonstrate how APC molecules can be spatially localized within nanometer sized silica cavities 

filled with water and thus be studied down to single molecule level under near physiological conditions. We 

show that the entrapment is critically dependent on the removal of methanol released by tetramethyl 

orthosilicate (TMOS) during the formation of the inorganic silica matrix [2], as well as on the pre-aging of 

the sol allowing particles to form and grow before addition of the biomolecule. We report on time-resolved 

photophysics observed in both the chromophoric phycocyanobilin groups when exited at 634 nm as well as 

on amino acid emission observed from the polypeptide backbone when excited using recently developed 

pulsed UV light emitting diodes[3,4]. 

Figure showing the emission spectra of APC in trimeric and monomeric form when encapsulated in a silica 

sol-gel pore. 

References: [1] L. J. McCartney et al. Anal. Biochem. 292 (2001) 216. [2] J.Karolin et al. Meas. Sci & Techn 13 

(2002) 21. [3] C. D. McGuinness et al. Meas. Sci. & Techn. 15 (2004) 11. [4] C. D. McGuinness et al. Appl. Phys. 

Lett. 89 (2006) 977. 

102


IMMI-5 

Development of a novel microscope platform for multiparameter 

fluorescence imaging 

Patricia R. Richardson 1,2 , Aongus McArthy 3 , Steven W. Magennis 2 , Jochen Arlt 2 , 

Gerald S. Buller 3 and Anita C. Jones 1,2 

1 School of Chemistry and 2 Collaborative Optical Spectroscopy, Micromanipulation and Imaging 

Centre (COSMIC), The University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK. 

3 School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK. 

E-mail: p.r.richardson@ed.ac.uk 

The research microscope has had the same physical design for the last 200 years, in spite of a fundamental 

shift in recent decades to the use of fluorescence intensity as the predominant contrast method in biological 

microscopy. This outmoded platform inhibits the use of the most recent advances in laser sources, optics 

and detectors, and optical micromanipulation. For live cell imaging, conditions are dynamic with limited 

time available to acquire data. The quasi-simultaneous measurement of multiple parameters is, therefore, 

highly desirable, as is the ability to combine such measurements with optical manipulation or stimulation. 

The scope for such multiplexing is very limited on conventional microscopes. 

We report the development of a radically new platform for fluorescence imaging (below) that makes multiparameter 

measurement straightforward and is easily reconfigured for the implementation of new sources, 

detectors and imaging techniques. The use of this system to carry out fluorescence lifetime imaging 

microscopy (FLIM) on optically trapped particles and cells will be demonstrated. 

103


IMMI-6 

Use of FLIM with novel fluorescent probes for mapping temperature with 

sub-degree resolution in microfluidic systems 

Emmelyn M. Graham 1,2 , Kaoru Iwai, 3 Seiichi Uchiyama, 4 A. Prasanna de Silva 5 , 

David A. Mendels 6 , Steven W. Magennis 2 and Anita C. Jones 1 

1 School of Chemistry and 2 Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre, 

University of Edinburgh, Edinburgh EH9 3JJ, UK. 

3 Department of Chemistry, Faculty of Science, Nara Women's University, Kitauoya-Nishimachi, 

Nara 630-8506, Japan. 4 Graduate School of Pharmaceutical Sciences, The University of Tokyo, 3-1, 

7-Chome, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 

5 School of Chemistry and Chemical Engineering, Queen's University, Belfast BT9 5AG, UK. 6 National 

Physical Laboratory, Hampton Road, Teddington, TW11 0LW, UK. E-mail: a.c.jones@ed.ac.uk 

The precise control and measurement of temperature in microfluidic lab-on-a-chip devices is essential for 

many chemical and biochemical applications, such as polymerase chain reaction (PCR) for the 

amplification of DNA. Previously, we have demonstrated the efficacy of fluorescence lifetime imaging 

microscopy (FLIM) as a tool for quantitative spatial mapping of mixing in microfluidic systems. 1 We now 

report the use of FLIM, in combination with novel fluorescent probes, for quantitative mapping of 

temperature within aqueous microfluidic devices. The large variation with temperature of the fluorescence 

lifetime of these fluorophores enables temperature resolution of 0.1 o C to be achieved, in combination with 

high spatial resolution. 

The fluorescence lifetime map on the left 

shows the temperature distribution within a 

spherical, moulded polymer, microfluidic 

chamber. The high quality of the image 

obtained, despite the imperfect optical quality 

of the chamber material, illustrates the 

immunity of FLIM to effects such as 

inhomogeneity of the optical path, scattering, 

variation in the fluorophore concentration and 

photobleaching, that cause distortion and 

compromise quantitation in intensity-based 

imaging techniques. 

50 _m 

The use of the quantitative data obtained from FLIM to validate computational models of diffusion and heat 

transfer on the microscale will be illustrated, with a view to the development of a priori methods for the 

design of microfluidic devices. 

Reference: [1] S.W. Magennis, E.M. Graham and A.C. Jones, Angew. Chem. Int. Ed.44 (2005) 6512. 

104


IMMI-7 

Label free detection of single proteins using deep-UV based laser 

fluorescence lifetime imaging microscopy 

Qiang Li, Eugene Riaplov, Stefan Seeger 

University of Zürich, Institute of Physical Chemistry, Winterthurerstrasse 190, CH-8057 Zürich, 

Switzerland. E-mail: sseeger@pci.unizh.ch 

A large number of biological species have intrinsic fluorescence excited in the UV region of 260-280 nm, 

UV laser excitation is an attractive alternative to tag these compounds with fluorescence labels excited at 

visible region. In this contribution we present a deep UV fluorescence lifetime imaging microscopy system 

based on a mode-locked diode-pumped picosecond deep UV laser. The described setup is well-suited for 

biological applications for ultrasensitive detection of intrinsic fluorescence. 

(1) Label-free detection of single protein molecules. 

We investigated the bursts of autofluorescence photons 

from tryptophan residues in β-Galactosidase molecules 

from Escherichia coli (Ecβ Gal) and fluorescence 

correlation spectroscopy of Ecβ Gal. The results 

demonstrate that deep UV laser-based fluorescence 

lifetime microscopy is useful for identification of 

biological macromolecules at the single molecule level 

using intrinsic fluorescence. 

(2) Label-free detection of antibody/antigen and 

protein/drugs interactions. 

A label free method for detection of Ecβ Gal/anti- Ecβ 

Gal interactions and protein/drugs interactions have 

been demonstrated by means of steady-state and timeresolved 

fluorescence spectroscopy. The interaction 

can be monitored by fluorescence lifetime changes 

between free components in the interaction system and 

corresponding complex. Energy transfer between 

tryptophan and bound drug in protein-drugs complexes 

has been observed. 

(3) One-dimension miniaturized polyacrylamine gel 

5 

0 

electrophoresis with native fluorescence detection. 

0 1 2 3 

The mixture of three biological compounds (β- 

Time (s) 

Galactosidase from Escherichia coli, apo-Transferrin 

4 5 

and bovine serum albumin) have been separated using miniaturized gel electrophoresis and a staining free 

detection limit below 80 pg per band has been achieved. 

References: [1] Q. Li, et al., J. Phys. Chem. B, 108 (2004) 8324. [2] Q. Li, S. Seeger, Anal. Chem., 78 (2006) 2732. 

[3] Q. Li. S. Seeger, submitted. [4] E. Riaplov, et al.,submitted. 

Counts 

30 

20 

10 

0 

20 

10 

15 0 

10 

15 0 5 

10 

15 0 5 

10 

15 0 5 

10 

15 0 5 

10 

(a) 

(b) 

(d) 

(e) 

(f) 

(c) 

(g) 

105


IMMI-8 

Live tissue multiphoton autofluorescence spectral imaging 

Jonathan Palero 1 , Henriëtte S. de Bruijn 2 , Angélique van der Ploeg van den Heuvel 2 , 

Henricus J.C.M. Sterenborg 2 and Hans C. Gerritsen 1 

1 Molecular BioPhysics, Utrecht University, 3584 CC Utrecht, Netherlands 

2 Department of Radiotherapy, University Medical Center Rotterdam-Erasmus MC 3008 AE Rotterdam, 

Netherlands. E-mail: j.palero@phys.uu.nl 

In recent years, studies in nonlinear microscopy based on tissue autofluorescence and second harmonic 

generation has steadily increased [1, 2]. Nonlinear microscopy is a general term for any microscopy 

technique based on nonlinear optics which include multiphoton microscopy (MPM), second- and thirdharmonic 

(generation) imaging (SHG, THG), and coherent anti-Stokes Raman scattering (CARS) 

microscopy. Because MPM allows the excitation of UV transitions using longer wavelengths, it has the 

ability to image deep into optically thick specimens, while restricting photobleaching and phototoxicity. 

Second harmonic generation (SHG) is another promising contrast mechanism for microscopy on superficial 

tissues. For instance, collagen fibers are the predominant structural component of superficial tissues and 

exhibit a strong second harmonic signal. Biochemical information from tissues can be obtained through 

autofluorescence spectroscopy. Tissue contains several endogenous fluorophores including NAD(P)H, 

FAD, keratin, elastin, collagen. 

A 

In this study, three-dimensional multiphoton autofluroescence 

spectral imaging microscopy was used for living mouse tissue 

imaging. A simple method of converting the spectral image data 

into RGB images enabled us to distinguish different structures 

within the skin tissue specimens. Our results showed 

morphological and emission spectral differences between 

excised tissue section, thick excised tissue and in vivo tissue 

samples of mouse skin. Results on collagen excitation at 

different wavelengths suggested the origin of the narrowband 

emission to be collagen Raman peaks. 

We also show results on depth-resolved tissue autofluroescence 

spectroscopy and 3D tissue microvolume autofluroescence 

spectroscopy which aid the identification of tissue components 

and possible endogenous fluorophores. 

Time-lapse multiphoton autofluorescence spectral imaging 

enabled us to track metabolic changes in keratinocytes and basal 

cells during acute administration of anaesthesia and tissue 

exposure to anoxia, respectively. 

Overall, multiphoton autofluorescence spectral imaging provided 

a wealth of information not easily obtainable with present 

conventional multiphoton imaging systems. 

References: [1] J. A. Palero, et al., Opt. Express 14, (2006), 4395- 

4402. [2] J. A. Palero, et al., Biophys. J. 93, (2007). 

B 

depth-integrated intensity [a.u.] 

10µm 

SHG 

X20 

Raman 

thin excised tissue 

thick excised tissue 

in vivo tissue 

Autofluorescence 

350 400 450 500 550 600 

emission wavelength [nm] 

(A) Spectral image of a living 

mouse tissue at a focal depth of 10 

µm. (B) Spectra of skin tissues 

showing SHG, Raman and 

autofluorescence. 

106


IMMI-9 

A solid-state time-gated luminescence microscope with UV LED excitation 

and EMCCD detection 

Russell Connally and James Piper 

Center for Laser Applications, Macquarie University, Australia 

E-mail: rconnall@ics.mq.edu.au 

Autofluorescent samples were spiked with europium beads and excited with pulsed UV from a LED source 

fitted to an Olympus BX51 microscope. Following the excitation pulse, a Hamamatsu electron multiplying 

CCD camera was gated on to yield a 15-fold increase in signal-to-noise ratio within a single 33 ms capture 

cycle. 

Time-gated luminescence (TGL) techniques exploit the large difference in lifetime that exists between most 

autofluorophores and lanthanide chelates. We previously reported the design of a UV LED excited TGL 

microscope for use with europium fluorophores[1]. Conventionally, a gated image-intensified CCD IICCD) 

camera is used to acquire images for TGL microscopy. EM-CCD cameras are the solid-state equivalent of 

IICCD cameras and afford a 3-fold improvement in quantum 

sensitivity. The BX51 microscope was fitted with a Hamamatsu 

C9100-02 electron multiplying CCD camera (EMCCD) and a UV 

LED mounted in the filter cube. Water samples containing 

phycoerythrin rich algae were spiked with 1 µm europium polymer 

microspheres with a luminescence lifetime of 700 µs. The LED was 

pulsed on for 500 µs and the camera triggered following LED 

extinction. Signal-to-noise ratio (SNR) enhancement was determined 

as the ratio of the brightest signal region versus brightest background 

for both TGL modes and prompt fluorescence mode. The 14 bit images 

were down-sampled to 8-bit using the software supplied with the 

camera. In the Figure, the bright pair of FluoSpheres embedded within 

the algal matrix had an average S PROMPT 8-bit value of 227 whereas the 

brightest region of auto-fluorescence was 255. The same regions were 

sampled in TGL mode, S TGL was 243 and background (B TGL ) was 18, 

corresponding to a 15-fold enhancement. In an earlier design TGL 

microscope, we employed a gated IICCD (DiCAM-PRO) to capture 

TGL images. This camera required up to 255 excitation / integration 

cycles before a useful image could be obtained. The EMCCD camera 

used here had a maximum gain of 2000; lower compared to the figure 

of 10,000 for the IICCD. Enhanced SNR was achieved within a single 

capture cycle using only a moderate EM camera gain of 400. Luminescent emission (λ= 620 nm) from the 

FluoSpheres was strong and use of the solid-state TGL microscope for practical biological applications 

would require efficient labelling of the target. Our previous reports on the preparation of monoclonal 

immuno-fluorophores against Giardia lamblia cysts showed that efficient, high brightness TGL labels can 

readily be prepared [2]. Importantly, TGL techniques provide a means to discriminate TGL-signal in 

strongly autofluorescent environments for the detection of rare pathogens that would otherwise be 

impossible to detect. 

References: [1] Connally, R., E, D. Jin, and J. Piper, High Intensity Solid-state UV Source for Time-Gated 

Luminescence Microscopy. Cytometry: Part A, 2006. 69A: p. 1020-1027. [2] Connally, R., E, D. Veal, A, and 

J. Piper, Time-resolved fluorescence microscopy using an improved europium chelate BHHST for the in-situ detection 

of Cryptosporidium and Giardia. Microscopy Research and Technique, 2004. 64: p. 312-322. 

107


IMMI-10 

Fluorescence lifetime imaging nanoscopy (FLIN) 

Klaus Kemnitz a , Marco Vitali a , Werner Zuschratter b 

a 

EuroPhoton GmbH., Berlin, Germany. E-mail: klauskemnitz@aol.com; 

b 

Leibniz-Institut for Neurobiology, Magdeburg, Germany 

FLIN, as recently introduced in SingleMotorFLIN 1 , provides groundbreaking tools for the study of single 

molecules (SM) and single molecular motors (SMM), as well as a broad array of phenomena in NanoWorld. 

Classical limitations in SM/SMM studies, such as resolution, short observation times, and photo-dynamic 

reactions are overcome by minimal-invasive picosecond FLIN. FLIN is the extension of the extremely 

successful fluorescence lifetime imaging microscopy (FLIM) into the nano-domain, with down to 5 nm 

space-resolution. FLIN results from the combination of nanoscopy (such as multi-colour, wide-field, pointspread- 

function (PSF) modelling microscopy) with novel ultrasensitive, non-scanning imaging detectors, 

based on time- and space-correlated single photon counting (TSCSPC) that allows ultra-low excitation 

levels. This results, for example, in long-period (>1 hour), minimal-invasive observation of living cells and 

SM/SMM, without any cell damage or irreversible bleaching. Minimal-invasive FLIN with global PSFmodelling 

allows observation of point-source movement at 1-nm accuracy and distance determination at the 

5-nm level, while simultaneously acquiring multi-exponential pico/nanosecond fluorescence dynamics. 

FLIN opens a wide avenue of novel applications, such as SMM-tracking, FRET-verification, dualpolarisation 

tracking, and super-background-free 2-photon TIRF-FLIN. SingleMotorFLIN will examine the 

behaviour of SMM and their dependence on energy-input. Enhanced basic understanding of biological and 

artificial machines/motors will lead to advanced models and proceed one day to artificial systems, 

revolutionising the interface of biological and non-biological worlds. Since biological SMM are involved in 

many human disorders, the novel FLIN method will help to show how these motors operate and how they 

break down in disease. 

Reference: [1] Supported by: EC-projects NMP4-CT-2004-013880 and MRTN-CT-2005-019481. 

108


IMMI-11 

Inkjet printing of near infrared fluorescent patterns 

Stefan Köstler, Martin Reischl, Martina Schaffenberger, Andreas Rudorfer, Volker Ribitsch 

Joanneum Research ForschungsgesmbH, Steyrergasse 17, A-8010 Graz (Austria) 

E-mail: stefan.koestler@joanneum.at 

University of Graz, Institute of Chemistry, Physical Chemistry Division A-8010 Graz (Austria). 

E-mail: volker.ribitsch@uni-graz.at 

Inkjet printing is a method attracting much interest in materials science for the deposition and patterning of 

functional molecules, polymers, and particles. It has been used for the fabrication of organic electronic 

devices, (bio)sensor arrays, photonic components, nanoparticle assemblies,… . [1] 

A broad range of near infrared (NIR) dyes having absorption maxima in the region about (700-900 nm) is 

known, some of them also showing fluorescence in the near infrared spectral region. Among them are 

several phthalocyanines, metal complexes, polymethines, azo-dyes an some others .[2] In this study several 

cyanine dyes were used as fluorophores. Exploiting the absorption window of biological tissue in the near 

infrared, this class of dyes is often used for in-vivo imaging applications. [3] 

These cyanine dyes tend to form J-aggregates in solution, thus changing their photophysical properties like 

absorption spectra and quantum yield. This undesired J-aggregate formation can be controlled by the 

preparation of supramolecular complexes. [4] 

In this work the formulation of well jettable aqueous and solvent based inks containing complexes of 

polyelectrolytes and near infrared emitting fluorophores is described. These inks were characterized 

regarding their photophysical and colloid chemical properties. 

1,0 

0,9 

absorbance [a.u.] 

0,8 

0,7 

0,6 

0,5 

0,4 

0,3 

Absorption spectra of different 

complexes consisting of indocyanine 

dye – and different polyelectrolytes 

in aqueous media. 

These were evaluated and 

characterised for inkjet printing of 

NIR fluorescent structures. 

0,2 

0,1 

0,0 

300 400 500 600 700 800 900 

! [nm] 

A scientific drop on demand (DOD) inkjet printing system was used for the formation of NIR fluorescent 

patterns on different types of paper substrates. Jetting parameters were adjusted to allow high resolution 

printing of aqueous and solvent based inks. 

Imaging of the produced patterns was accomplished using a setup consisting of a LED or laser diode as 

excitation light sources. A CCD camera and an optical bandpass filter were used for NIR-fluorescence 

detection. 

References: [1] P. Calvert, Chem. Mater. 13 (2001) 3299. [2] J. Fabian, et al., Chem. Rev.. 92 (1998) 1197. 

[3] A. Zaheer et al., Nature Biotech. 19 (2001), 1148. [4] T. V. S. Rao, Tetrahedron 54 (1998) 10627. 

109


IMMI-12 

Two channel near and far field fluorescence microscopy 

Dorinel Verdes, Michael Rabe, Christian Gojak, Stefan Seeger 

University of Zürich, Institute of Physical Chemistry CH-8057 Zürich (Switzerland). 

E-mail: d.verdes@pci.unizh.ch 

We report a new two channel fluorescence microscopy technique for surface-generated fluorescence. The 

realized fluorescence microscope allows high resolution imaging of aqueous samples. The core element of 

the instrument is a parabolic mirror objective that is used to collect the fluorescence at large surface angles 

above the critical angle of the water/glass interface. An aspheric lens, incorporated into the solid parabolic 

element, is used for diffraction limited laser focusing and for collecting fluorescence at low angles with 

respect to the optical axis. By separated collection of the fluorescence emitted into supercritical and 

subcritical angles, two detection volumes strongly differing in their axial resolution are generated at the 

water/glass interface. The collection of supercritical angle fluorescence (SAF) (by the parabolic mirror) 

results in a strict surface confinement of the detection volume whereas collecting below the critical angle 

(by the aspheric lens) allows gathering the fluorescence emitted several microns deep inside the aqueous 

sample. Consequently, the signals from surface-bound and unbound diffusing fluorescent molecules can be 

obtained simultaneously. [1] 

Unlike in TIRF geometry, the parabolic mirror objective easily achieves a diffraction limited excitation 

volume at water/glass interface. The excellent surface selectivity is obtained on the basis of the dipole 

emission profile near a dielectric interface. [2] Its angular distribution is a superposition of traveling and 

evanescent waves, which both can be detected in the far field using the parabolic mirror objective. 

Scheme of the parabolic mirror objective. 

The outer diameter limits the minimum 

angle collection to 62°, whereas an 

opaque aperture acts for angle collection 

above 75°. The aspheric lens is embedded 

into the parabolic mirror and performs as 

an objective to focuss the light at the 

surface of the sample. Subsequently, the 

emited fluorescence up to 24° is collected 

by the asperic lens in a confocal 

geometry. 

The dashed line illustrate the emission 

profile of the dipole near the dielectric 

interface. 

We detected single fluorescent molecules adsorbed non-specifically on a glass coverslip using the SAF 

geometry. Further, adsorption measurements of fluorescently labeled proteins were performed to reveal 

their adsorption mechanism on a hydrophilic glass surface. We show for instance that the two channel 

microscope can be applied to investigate the protein layer structures on dielectric interfaces with single 

molecule sensitivity. Consequently, we have acquired cell images with both channels simultaneously 

showing major differences between parts of the cell laying in close proximity of the interface and the cell as 

a whole. 

An excellent signal-to-background ratio at moderate illumination intensity, diffraction limited resolution, 

radical reduction of the detection volume along the optical axis, easy handling and stability, make the two 

channel fluorescence microscope a powerful technique for surface fluorescence measurements down to the 

single molecule level. 

References: [1] D. Verdes, et al. J. Biomed. Optics, in press. [2] T. Ruckstuhl, S. Seeger, Opt Lett 29 (2004) 569; 

T. Ruckstuhl, D. Verdes, Optics Express 12(8) (2004) 4246. 

110


IMMI-13 

Fluorescence imaging techniques for curing monitoring of hybrid thermosets 

Maria González, Ozlem Tari, B. Serrano, Juan Carlos Cabanelas and Juan Baselga 

University Carlos III de Madrid, Departament of Materials Science and Engineering and Chemical 

Engineering, Av de la Universidad 30, 28911 Leganés (Spain). E-mail: jbaselga@ing.uc3m.es 

Epoxy thermosets are commonly used in adhesives, coatings and polymer matrix composites[1]. It has been 

recently proposed [2] the use of polysiloxane multifunctional hardeners to increase toughness and thermal 

resistance and to decrease water up-take of epoxy systems. Siloxane compounds and epoxy resins are 

mutually insoluble and the use of reactive polysiloxanes, where the polysiloxane is chemically bonded to 

the epoxy component partially solves the problem. The reactive mixture, which is initially heterogeneous, 

becomes a transparent and partially homogeneous solid during the curing proccess [3]. 

Morphology evolution during curing of an hydrogenated derivative of diglicidylether of bisphenol A 

(HDGEBA) with a synthetic labelled polysiloxane poly(3-aminopropylmethylsiloxane) (PAMS) was 

followed in-situ at 40ºC by confocal fluorescence microscopy (LSCM). This technique makes possible 

measuring interphase thickness and compositional gradients, as well as monitoring the reactive 

compatibilization proccess. As it is shown in the Figure, initial mixture shows a dispersion of quasispherical 

fluorescent PAMS-rich domains in an epoxy-rich matrix, and as curing proceeds the size of the 

domains increases and composition gradients decrease leading to a more homogeneous material. 

PAMS of different molecular weights consisting in chains and cycles were labeled with four different 

molecular probes: dansyl, rhodamine, naphtalimide and nitrobenzofurazan. Influence of the molecular 

weight and chains/cycles ratio in the curing proccess and morphology was studied and, since the labels used 

have different molar volume, influence of the rigidity of the medium on the fluorescent response was 

characterized in the different epoxy/PAMS systems. 

LSCM images of 

HDGEBA/PAMS 

system taken at 

different times during 

curing at 40ºC. 

PAMS was labeled 

with rhodamine B 

sulphonyl chloride. 

Bright zones correspond 

to PAMS-rich 

domains and dark 

zones to epoxy-rich 

regions. 

t=8min t=21min t=33min 

References: [1] J.P. Pascault et al.: Thermosetting Polymers, Ed. Marcel Dekker Inc. (2002). [2] J.C. Cabanelas et al., 

Macromol. Rapid Commun. 22 (2001) 694. [3] J.C. Cabanelas et al, Polymer 46 (2005) 6633. 

111


IMMI-14 

A programmable light engine for quantitative TIRF microscopy of 

single quantum dots 

Marcel van ‘t Hoff, Vincent de Sars, Martin Oheim 

INSERM U603, Paris, F-75006 France; Université Paris Descartes, Laboratory of Neurophysiology & 

New Microscopies, Paris, F-75006 France ; CNRS UMR8154, Paris, F-75006 France. 

E-mail: marcel.vanthoff@univ-paris5.fr 

A spatially and temporally programmable light engine based on two crossed acousto-optical deflectors can 

create any intensity profile in the back focal plane (BFP) of a high-numerical aperture objective used for 

combined total internal fluorescence (TIRF) and epifluorescence imaging (Stout & Axelrod 1989). 

Fluorescence images taken with the spot in the four cardinal positions illustrate how single-spot 

illumination suffers from scattering and interference, resulting in a spatially non-uniform evanescent-field 

illumination (Schapper et al. 2003). In contrast, rapid circular spinning the spot averages over these 

perturbations (Mattheyses et al. 2006), while spatially redistributing and hence diluting the scattered light, 

resulting in a perfectly homogenously lit field-of-view. Spiral scans are shown to produce a homogenous 

evanescent field with variable penetration depth, whereas a raster or Lissajous scan produces whole-field 

epi-illumination with no extra light-source required. We demonstrate quantitative evanescent-field imaging 

of cortical mouse astrocytes tagged with functionalized semiconductor nanocrystals. 

112


IMMI-15 

FLIM with TIRF and confocal microscopy 

Ria Oosterveld-Hut 

Lambert Instruments, Leutingewolde, The Netherlands 

E-mail: ria@lambert-instruments.com ; www.lambert-instruments.com ; +31-50-5018461 

FLIM (Fluorescence Lifetime Imaging Microscopy) is a technique to map the spatial distribution of 

lifetimes within microscopic images and it allows measurements in living cells as well as in fixed materials. 

The fluorescence lifetime is the exponential decay in emission after the excitation of a fluorescent material 

has been stopped. It is independent of bleaching and intensity variations in the sample. Some phenomena do 

affect fluorescence lifetimes, therefor the applications of FLIM are various: ion imaging, oxygen imaging, 

FRET (Fluorescence Resonance Energy Transfer) microscopy, etc. When two fluorescent molecules are in 

very close proximity, the energy of the one fluorescent (donor) molecule is transferred in a nonradiative 

process to the other fluorescent (acceptor) molecule. So in case of FRET, the lifetime of the donor molecule 

decreases and this change can be measured quantitatively by FLIM. Lambert Instruments has developed a 

dedicated system (LIFA) that allows image acquisition and generation of lifetime images within one 

second. The nanosecond lifetime information can be extracted pixel-by-pixel, see figure 1A. 

The LIFA can be attached to any fluorescence widefield microscope and is compatible to several 

techniques, like Total Internal Reflection Fluorescence (white-TIRF as well as laser-TIRF) and multi-beam 

confocal microscopy (by spinning disk). TIRF microscopy is an ideal method for studying fluorescently 

labelled proteins located e.g. in the plasmamembrane, up to 100nm from the coverslip, see figure 1B. The 

multi-beam confocal microscopy is a technique used to increase micrograph contrast, but then at any focus 

plane in the cell, see figure 1C. It is also used to reconstruct three-dimensional images by eliminating outof-focus 

light in specimens that are thicker than the focal plane. The confocal image is obtained by the 

Nipkow disk with a spiral pattern of pinholes arranged to scan the specimen with an array of light beams. 

A B C 

Figure 1. A, FLIM image with lifetime in pseudo colours and intensity in grey scale. The cells show a 

decreased lifetime (FRET) at the centrosomes. B, FLIM-laser-TIRF image. These cells show vesicles 

with GFP-Rab6 that originate from the Golgi complex (courtesy of Optical Imaging Centre, Erasmus MC, 

NL). C, Confocal FLIM image prepared with LIFA with the CSU22 spinning disk. These are erythrocytic 

cells with membrane proteins fused to Cerulean or Citrine (courtesy of INSERM and Plateforme 

d'Imagerie Dynamique, Paris, France). 

113


IMMI-16 

Bothersome photochemistry in autofluorescent proteins and its impact to 

fluorescence lifetime imaging microscopy 

Gregor Jung 

Saarland University, Biophysical Chemistry, D-66041 Saarbrücken (Germany). 

E-mail: g.jung@mx.uni-saarland.de 

Autofluorescent proteins, in which the fluorescent moiety is formed out of several amino acids of the 

protein sequence, became versatile tools in the life sciences. In the beginning, labelling of gene products 

was the main application; purposeful photochemical transformations are gaining interest for tracking 

protein diffusion or high resolution microscopy. Accidental occurring photochemical reactions, however, 

are detrimental to the brightness of autofluorescent proteins in microscopy: light-driven isomerization, 

which is analyzed by Fluorescence correlation spectroscopy (FCS), diminishes the fraction of fluorescent 

species, and photoconversion reduces the fluorescence lifetime τ Fl [1, 2]. The examples show that the 

fluorescence properties of the chromophore are sensitive to the rigidity inside the protein barrel [2, 3]. 

Recently, it was shown that ubiquitous photoconversion obscures the determination of τ Fl in Fluorescence 

Lifetime Imaging Microscopy [4].While the photoconverted proteins with an anionic chromophore form, 

R pc - , still exhibit green fluorescence, the quantum yields as well as its photodynamics are altered compared 

to the chromophore in the native protein, R eq - . The pronounced intensity dependence of τ fl and a kinetic 

description of the photochemical processes in a confocal volume are used to determine the corresponding 

quantum yields (see figure). 

The extreme susceptibilty of τ Fl to light exposure allows for establishing a radiation dosimeter for cellular 

applications. The requirements of such a device with regard to photophysical parameters of autofluorescent 

proteins are discussed. 

References: [1] G. Jung, A. Zumbusch, Microsc. Res. Techn. 69 (2006) 175. [2] G. Jung et al., Biophys. J. 88 (2005) 

1932. [3] S.Veettil, G.Jung, in preparation. [4] G. Jung et al., submitted. 

114


IMMI-17 

Time-resolved “Deep UV” confocal fluorescence microscopy 

Trevor A. Smith, Peter Wichta and Craig N. Lincoln 

Ultrafast & Microspectroscopy Laboratory and ARC Centre of Excellence for Coherent X-Ray Science, 

School of Chemistry, The University of Melbourne, Victoria 3010, (Australia). 

E-mail: trevoras@unimelb.edu.au 

Fluorescence imaging of many samples, in particular biological systems, often requires the use of 

exogenous fluorescent tags or probes, and subsequent one- or multi-photon excitation. The addition of 

foreign fluorescent probes, including dyes, quantum dots or fluorescent proteins, to the sample of interest is 

often time consuming, and may induce often unknown and undesirable changes in the local environment or 

molecular conformation of the system under investigation. Many samples exhibit intrinsic fluorescence 

arising from proteins, coenzymes and other components of the cellular materials, and this 

“autofluorescence” may be exploited in certain circumstances for fluorescence imaging. 

Current commercially available microscopes have low efficiency in the wavelength regions below 400 nm, 

where the amino acid residue tryptophan (Trp), responsible for most native fluorescence, absorbs (280 nm) 

and fluoresces (340-360 nm). We have developed a microscope that uses deep ultraviolet excitation 

wavelengths derived from the harmonic generation of ultrashort laser pulses for the direct excitation of the 

amino acids, in particular Trp. We also utilise the short pulsed nature of the excitation source to include 

time-resolved fluorescence imaging (or fluorescence lifetime imaging (FLIM)) capabilities. This overcomes 

some of the issues associated with intensity-based measurements and provides additional information 

regarding the local environment of the amino acids, and an added mode of discrimination of the desired 

emission over other forms of intrinsic fluorescence and scattered light. A number of different time-resolved 

fluorescence imaging techniques have been assessed for this application and these will be discussed. 

Despite the advantages, and perhaps due to the disadvantages (e.g. the low efficiency combined with the 

poor photostability of fluorophores such as Trp), there are surprisingly few reports published concerning 

confocal fluorescence imaging in this region.[1] 

Applying the above techniques may have the potential to provide new insights into the function of 

membrane bound proteins, in particular, those associated with the asexual reproduction of the human 

malaria parasite P. falciparum within red blood cells. The system is also of potential use in the study of a 

range of botanical samples. 

References: [1] Q. Li and S. Seeger, Anal. Chem. 78, 2732 (2006). 

115

116

Part III 

Probes, Labels 

and Sensors 

117

118

Abstracts Poster – Part III: Probes, Labels and Sensors 

PRLS-1 

Sensitized luminescence in trivalent lanthanide complexes Eu(III)/ quinaldinic 

acid and Eu(III)/1,4-dihydro-oxo-quinoline-3-carboxylic acid 

Ababacar Sadikhe Ndao a , Andrea Buzády b , János Erostyák b , István Hornyák b 

a 

Department of Physics, Cheikh Anta Diop University, Dakar-Fann (Senegal). 

E-mail: asndao@yahoo.com 

b 

Department of Experimental Physics, University of Pécs, Ifjúság u. 6., H-7624 Pécs (Hungary). 

E-mail: buzady@fizika.ttk.pte.hu 

Spectroscopic properties of trivalent lanthanide ions have continuous interest, especially when they are 

chelated with appropriate organic ligands. The lanthanide chelates have been widely used for many 

applications such as laser materials or luminescent labels in clinical chemistry and molecular biology [1]. 

The specific physical and chemical properties of the lanthanide which make them useful in the studies of 

biological systems [2] are the consequence of their electronic structure. On the basis of energy level and 

quantum yields consideration, the europium ion can be considered as one of the best lanthanide ion for 

sensitized luminescence. In this paper, the luminescence properties of the complexes Eu/QA and 

Eu/DOQCA both in powder form and in water solution are reported. 

The steady-state luminescence measurements (emission and excitation spectra) were made with a Jobin– 

Yvon Fluorolog Tau3 spectrofluorometer at room temperature. The time-resolved spectra and the 

luminescence decays of the Eu(III) in the complexes were obtained using a laser pulsed fluorometer, where 

the samples were excited by a N 2 laser (337.1 nm, 1ns). The data were measured by a SRS SR250 boxcar 

averager and processeed by a PC. 

Detailed analysis of decay curves and spectra were done. Luminescence decays are mostly one exponentials 

except in case of 5 D 1 → 7 F j transitions in solution, where two- or three exponential fits are adequate. This 

reflects that the 

5 D 1 level has a 

transitional position in the energy 

transfer chain. 

Eu/QA complex in water solution. ! ex 

= 320 nm 

In the spectra, the 

5 D i → 

7 F j 

transitions are identified and their 

relative intensities are given for both 

compouds in powder form and in 

water solution. The most extreme 

spectrum is shown on the Figure, 

displaying that in case of Eu/QA in 

Intensity 

8x10 4 

7x10 4 

6x10 4 

5x10 4 

4x10 4 

water solution the 

5 D 0 → 

7 F 1 

transition gives far the most intense 

spectral band, which is quite rare 

among the Eu(III) complexes. 

A further analysis is given by 

deconvolving the spectral bands in 

the emission spectra. One can 

3x10 4 

2x10 4 

1x10 4 

0 

identify the subbands originating 

from the splitting of the europium 

levels by the ligand field. 

The scheme of energy transfer pathways is also given. 

520 540 560 580 600 620 

Emission wavelength (nm) 

References: [1] P. R. Selvin, Ann. Rev. Biophys. Biomol. Struct. 31 (2002) 275. [2] O. S. Wolfbeis et al., U. S. Pat. 

No. 7067275. (2006). 

 

D1 - 

7 F0 

5 

D1 - 

7 F1 

5 

D1 - 

7 F2 

5 

D0 - 

7 F0 

5 

D0 - 

7 F1 

5 

D0 - 

7 F2 

5 

119


PRLS-2 

Lifetime-based sensing film for carbon dioxide using resonance energy transfer 

Merima Cajlakovic, Alessandro Bizzarri, Christian Konrad, Volker Ribitsch 

Institute of Chemical Process Development and Control, JOANNEUM RESEARCH, 

A-8010 Graz (Austria). E-mail: merima.cajlakovic@joanneum.at 

The sensing scheme adopted for the development of the CO 2 sensor is the resonance energy transfer (RET) 

from a long lifetime inert donor to a pH sensitive acceptor. The use of luminescent ruthenium complexes as 

donors offers a way to design resonance energy transfer-based sensors with decay times in the µs range. [1] 

Several pH indicators as suitable acceptors have been firstly spectroscopic investigated. Donor emission 

and acceptor absorption overlapping integral indicated that thymol blue, bromothymol blue, bromophenol 

blue, Sudan III and Texas red are most appropriate candidates for used sensing principle (Figure 1). 

The sensing film consisted of a Ru complex as a donor dye, pH indicator as an acceptor and a quaternary 

ammonium hydroxide as a phase transfer agent embedded in hydrophobic matrix such as sol-gel or ethyl 

cellulose. Fluorescence lifetime as a pCO 2 -dependent parameter was selected as the sensing parameter, 

measured in frequency domain using phase modulation fluorometry. 

Since the Ru complexes are known as quenchers of molecular oxygen, the effect of molecular oxygen was 

investigated in detail. It was found out that sol-gel based film using Tris(2,2’-bipyridyl) ruthenium complex 

showed almost negligible oxygen cross-sensitivity. The sensors based on this dye as donor were further 

characterised in terms of sensitivity, response time, temperature and stability. Another possibility to 

overcome oxygen cross-sensitivity is by using Eu-chelate complex as an inert long-lifetime donor dye 

molecule or by compensating the cross sensitivity by the simultaneous measurement of O 2 , in which case 

another O 2 -dependent luminescent dye was used. 

Future activities are devoted to development on fiber optical sensor for medical applications. 

Spectral overlap -1 [M cm -1 nm 4 ] * 1E14 

1800 

1600 

1400 

1200 

1000 

800 

600 

400 

20 

15 

10 

5 

0 

Bromophenyl Blue 

Bromothymolblue 

Cresol Purple 

Naphtofluorescein 

Phenol Red 

Sudan III 

Texas Red 

Tymol Blue 

Ru(bpy) 

Ru(dpp) 

Ru(PAN) 

Ru(phen) 

Ru(bpy) 

Ru(dpp) 

Ru(PAN) 

Ru(phen) 

Ru(bpy) 

Ru(dpp) 

Ru(PAN) 

Ru(phen) 

Ru(bpy) 

Ru(dpp) 

Ru(PAN) 

Ru(phen) 

Ru(bpy) 

Ru(dpp) 

Ru(PAN) 

Ru(phen) 

Ru(bpy) 

Ru(dpp) 

Ru(PAN) 

Ru(phen) 

Ru(bpy) 

Ru(dpp) 

Ru(PAN) 

Ru(phen) 

Ru(bpy) 

Ru(dpp) 

Ru(PAN) 

Ru(phen) 

Figure 1: Spectral overlapping between donors and acceptors 

Reference: J. R. Lakowicz; Principle of Fluorescence Spectroscopy, Plenum Press, New York, 1983, p. 257 

120


PRLS-3 

Thiosquarylium dyes as highly-photostable biomedical markers 

Yelena Obukhova 1 , Anatoliy Tatarets 1 , Olga Kolosova 1 , Yevgeniy Povrozin 1 , 

Iryna Fedyunyayeva 1 , Ewald Terpetschnig 2 , Leonid Patsenker 1,2 

1 State Scientific Institution "Institute for Single Crystals", National Academy of Sciences of Ukraine, 

60 Lenin Ave., UA-61001 Kharkov (Ukraine). E-mail: patsenker@isc.kharkov.com 

2 SETA BioMedicals, LLC, Urbana, IL, USA. E-mail: ewaldte@setabiomedicals.com 

Next to brightness (extinction coefficient and fluorescence quantum yield) the photostability of dyes plays 

an important role for their use in biomedical applications. We measured the photostability of a series of 

recently developed thio-squarylium dyes 1 and 2 and their BSA conjugates and compared these data with 

those for oxo-squaraines 3 and open-chain cyanines such as Cy5 and Alexa Fluor 647. The relative photostability 

was determined via measurement of the relative change in absorption and fluorescence intensity 

upon exposure to light from a halogen lamp but also by using a fluorescent microscope equipped with a 

Cy5 filter set. While the conventional cyanines and oxo-squarylium dyes such as 3 photobleach upon 

exposure to light, the absorbance and emission intensity of the thio-squaraines 1 and 2 were found to 

increase. This effect can be attributed to a photo-induced hydrolysis of the thio-squarylium C–S group 

whereby the thionated dyes 1 and 2 are transformed into oxo-squarylium dyes 3. Because the photodecomposition 

of 3 is much slower than the hydrolysis rate of 1 and 2, and because 3 has a higher extinction 

coefficient and higher quantum yield than 1 and 2, the absorption and fluorescence intensity increases 

until 1 and 2 is totally transformed to 3. During this process only a small blue-shift of the absorption and 

emission (no more than 16 nm) is observed. Neither squaraine 1 nor 2 hydrolyze in the absence of light. 

Increase of fluorescence intensity of protein bound thio-squaraines upon light exposure was found to be 

even more pronounced than that for the free dyes. 

X 

R 1 Y 

Y R 1 

H 2 O, h! 

R 2 R 2 

Y 

R 1 R 2 R 2 

O 

Y 

R 1 

N 

N 

N 

S 

R 1 = H, SO 3 H 

O 

1 X = O; 2 X = S R 2 = Me, (CH 2 ) 5 COOH, (CH 2 ) 4 SO 3 H 

3 

N 

The figure shows the relative photostability of dyes 

1 and 3 (R 1 =SO 3 H, R 2 =(CH 2 ) 5 COOH) determined 

via measurement of the relative change in 

fluorescence intensity upon exposure to light from a 

halogen lamp (150 W). 

Due to their favorable photophysical properties 

(long wavelength absorption and emission, high 

extinction coefficients and fluorescence quantum 

yields) and high photostability as compared to Cy5 

and Alexa Fluor 647, thio-squarylium dyes 1 and 2 

show potential for use in biomedical assays and 

biological imaging to investigate the structure and 

function of cells. 

Normalized Fluorescence, 0 I/I 

1.6 

1.4 

1.2 

1.0 

0.8 

0.6 

0.4 

0 20 40 60 80 100 

Light Exposure Time, min 

1 

3 

Alexa Fluor 647 

Cy5 

The work was supported by the STCU grants No. 3804 and P313. 

121


PRLS-4 

New red and near-infrared squarylium probes for biomedical applications 

Olga Kolosova, Anatoliy Tatarets, Sania Khabuseva, Yuliya Kudryavtseva, 

Leonid Patsenker 


60 Lenin Ave., UA-61001 Kharkov (Ukraine). E-mail: tatarets@isc.kharkov.com 

Squarylium dyes, a subclass of cyanines, absorb and emit light in red and near-IR spectral region. They are 

used as fluorescent probes and labels for biomedical research but also as the dyes for optoelectronic 

applications such as xerographic devices, solar cells, optical recording media and other applications. 

We synthesized a series of new symmetrical and unsymmetrical hydrophobic squarylium dyes (1–9), 

investigated their spectral properties and used them for biological staining of cells. 

O 

X 

N 

N 

N 

O 

O 

6 

1- 5 

O 

1: X = S, R = H; 

2: X = O, R = H 

3: X = CH=CH, R = H 

N 

4: X = C(CH 3 ) 2 , R = NO 

O 

2 

5: X = C(CH 3 ) 2 , R = N(CH 3 ) 7 

2 

R 

O 

O 

N 

N 

R 

N 

O 

O 

8, 9 

N 

8: R = NO 2 

9: R = N(CH 3 ) 2 

R 

The nature of the terminal heterocyclic moiety has strong influence on the absorption and emission 

properties of these dyes. They have long-wavelength absorption and emission maxima in chloroform 

between 606 and 714 nm and high extinction coefficients up to 300,000 M –1 ⋅cm –1 . All dyes except 7 

possess high quantum yields (Q.Y. up to 47 %) in chloroform while the quinolinium moiety in 7 

dramatically reduces the fluorescence yield (Q.Y. 0.3%). The absorption and emission maxima are shifted 

in the order: diphenyloxazole < benzoxazole < indolenine < benzothiazole < 5-nitro-indolenine < 5- 

dimethylamino-indolenine. Absorption and emission spectra in methanol are blue-shifted by 10–25 nm 

compared to chloroform and the quantum yields are decreased. All these dyes except dimethylaminosquaraines 

5 and 9 show negative fluorosolvatochromism. The Stockes' shifts (Δν) for these dyes are 250– 

500 cm –1 in chloroform and 250–600 cm –1 in methanol. Compound 7 with a quinoline moiety has Stockes' 

shifts of 660 cm –1 in chloroform and 860 cm –1 in methanol. Both the low Q.Y. and high Stockes' shifts for 

this compound are due to its asymmetry but also the nature of the heterocyclic end-groups. 

The figure presents absorption and emission 

1.0 

spectra of dimethylamino-squaraine 5 in 

methanol (– – –) and chloroform (–––). An 0.8 

introduction of dimethylamino group causes 

essential increase in Stokes' shifts (1040 cm –1 in 0.6 

chloroform and 1980 cm –1 in methanol). In 

addition, dyes 5 and 9 exhibit positive fluorosolvatochromism. 

0.4 

0.2 

Nitro-squaraines 4 and 8 were found to have 

extremely high photostability compared to other 0.0 

squarylium dyes. 500 600 700 800 


Normalised absorption, a.u. 

Normalised fluorescence, a.u. 

The fluorescence intensity of these dyes increases upon binding to biological materials. These dyes are in 

particular useful as fluorescent stains for biological imaging, which was demonstrated by staining dog 

spermatozoa, human fibroblasts and Saccharomyces Cerevisiae yeast cells. 

122


PRLS-5 

Water-soluble, pH-sensitive fluorescent labels based on squaraine dyes 

Olga Kolosova 1 , Anatoliy Tatarets 1 , Yelena Obukhova 1 , Yevgeniy Povrozin 1 , 

Vadim Sidorov 1 , Larisa Markova 1 , Ewald Terpetschnig 2 , Leonid Patsenker 1,2 


60 Lenin Ave., UA-61001 Kharkov (Ukraine). E-mail: kolosova@isc.kharkov.com 


In recent years there has been an increased interest in the use of red and near-IR labels for intracellular and 

biomedical studies. Most of the existing pH-sensitive fluorescent dyes that are used in these applications are 

known to emit between 350 and 550 nm and they also do not contain any functionality for biolabelling. We 

investigated a series of new squaraine-based pH-sensitive fluorescent dyes available from SETA 

Biomedicals (www.setabiomedicals.com) as free carboxylic acids, amine-reactive N-hydroxysuccinimidyl 

esters and thiol-reactive maleimides. Their absorption and emission spectra, extinction coefficients, 

quantum yields, fluorescence lifetimes and polarization were measured in aqueous media, free in solution 

and after binding to BSA and IgG. Protonated forms of the free dyes absorb between 634 – 693 nm with 

extinction coefficients (ε) 87,000–188,000 M –1 cm –1 and fluoresce between 646 – 714 nm. The excitation of 

these labels with a 635 or 670-nm diode laser results in improved signal-to-noise ratios due to reduced 

background from the biological sample. The additional short-wavelength absorption band (ε 12,000–30,000 

M –1 cm –1 ) in the spectra of these dyes allows excitation with a 380-nm, 405-nm or 436-nm diode laser. 

Importantly the quantum yields are independent of the excitation wavelength. These new dyes exhibit 

adequate quantum yields in aqueous media and when covalently bound to protein. 

Absorption and emission spectra of 

K8-1405 at pH 5.3 and 9.0 

Absorbance 

pH = 5.3 

pH = 9.0 

400 500 600 700 800 


Fluorescence 

Characteristics of pH sensitive labels 

Label pKa pH 

Range 

λ (Ab), 

nm 

λ (Fl), 

nm 

K8-1405 7.17 5.2–9.0 653/535 671/663 

K8-1675 8.65 7.8–9.5 662/543 679 

K8-1365 8.86 6.5–11.0 672/537 694 

K8-1765 9.37 7.3–11.1 641/514 668 

K8-1375 9.56 8.8–11.5 693/557 714 

K8-1775 9.92 8.2–11.6 662/539 684 

K8-1665 10.29 8.4–11.8 640/519 656 

K8-1610 10.65 9.4–12.8 634/520 646 

In basic environment the long-wavelength absorption band of these dyes decreases and a new absorption 

band at 520–560 nm appears whereby the fluorescence of almost all the investigated dyes is totally 

quenched. An exception is K8-1405, where both forms, the protonated as well as the deprotonated, are 

fluorescent. The Stocke's shift of deprotonated form of K8-1405 is extremely large — more than 3,600 cm – 

1 , which is one of the largest Stokes’shifts that has been observed in a cyanine-based dye. 

The pKa values of the dyes are in the range between 7.17 and 10.65. The pKa values of the IgG and BSAconjugates 

are similar as those for the free dyes. These pH-probes are easily coupled to antibodies and other 

proteins using standard procedures. Applications are in biological, pharmaceutical and biomedical research, 

clinical diagnostics, and high-throughput screening for the investigation of biological cells, membranes, and 

the role of intracellular pH in diverse physiological and pathological processes. 


123


PRLS-6 

Polymerisable phosphorescent transition metal complexes 

Nadja Noormofidi, Andreas Pein, Astrid C. Knall, Christian Slugovc* 

Institute for Chemistry and Technology of Organic Materials (ICTOS), Graz University of Technology, 

Stremayrgasse 16, A-8010 Graz, E-mail: noormofidi@tugraz.at 

Transition metal complexes bearing polypyridine ligands have been intensively investigated in recent years 

due to their outstanding photophysical properties and promising applications in photoactive devices. [1-3] Of 

particular interest in this context are homo- and heteroleptic complexes comprising bidentate 

imidazophenanthroline type ligands as well as tridentate terpyridine and bis(pyridyl)triazine derivatives. 

In this contribution the synthesis and characterisation of ruthenium and europium complexes bearing 

norbornene functionalised imidazophenanthroline, terpyridine and bis(pyridyl)triazine derivatives is 

presented. Thereby we place special emphasis on the absorption and emission characteristics of the free 

ligands and the corresponding metal complexes. For example, by replacement of one terpyridine ligand in a 

homoleptic complex by a bis(pyridine)triazine derivative, prolongation of excited-state lifetimes is 

expected. 

N 

N 

O O O 

11 

O O O 

11 

N 

N 

N 

N 

N 

O O O 

11 

HN 

N 

N 

N 

F 3 C 

N 

O 

N 

O 

N 

S 

N 

R 

N 

N 

N 

N 

N 

N 

N 

R = H 

R = (CH 2 ) 6 CH=CH 2 

R = (CH 2 ) 11 OH 

Ligand tool box for transition metals like ruthenium and rare-earth metals e.g. europium with focus on 

imidazophenanthroline-, bis(pyridyl)trianzine- and terpyridines. 

Another main focus of our research deals with spectral changes upon protonation/deprotonation. 

Luminescence of imidazophenanthroline vanished in acidic ambience while luminescence intensity of 

imidazophenanthroline ruthenium complexes with the general structure [Ru II (bipy) 2 phen] 2+ increased upon 

protonation. 

Apart from the above mentioned photophysical properties the incorporation of the complexes into polymers 

using ring opening metathesis polymerisation (ROMP) will be discussed. 

Financial support by the Austrian Science Fund FWF (project number P17410-B10) in the framework of the Austrian 

Nano Initiative (Research Project Cluster 0700 - Integrated Organic Sensor and Optoelectronics Technologies – 

Research Project 0701) is gratefully acknowledged. 

References: [1] P. Sun, J. Duan, J. Lih, C. Cheng, Adv. Funct. Mater. (2003), 13, 639. [2] E. C. Constable, Chem. 

Soc. Rev. (2007), 36, 246. [3] E. A. Medlycott, G. S. Hanan, F. Loiseau, S. Campagna, Chem. Eur. J. (2007), 13, 

2837. 

124


PRLS-7 

Optical humidity sensor materials based on europium 

Astrid C. Knall 1 , Andreas Pein 1 , Nadja Noormofidi 1 , Martin Tscherner 1 , Christian Konrad 2 , 

Volker Ribitsch 2,3 , Georg Uray 3 , Franz Stelzer 1 and Christian Slugovc 1* 

1) Graz University of Technology, Institute for Chemistry and Technology of Organic Materials, 

Stremayrgasse 16/I, A-8010 Graz, Austria. E-mail: a.knall@TUGraz.at 

2) Joanneum Research, Institute of Chemical Process Development and Control, Steyrergasse 25, 

A-8010 Graz, Austria 

3) Institute of Chemistry, Karl-Franzens University Graz, Heinrichstraße 28, A-8010 Graz, Austria. 

Humidity control is an important issue in various applications ranging from health monitoring devices to 

trace humidity determination in high-purity gases for semiconductor applications. 

The quenching of the luminescence of europium by water is a well known and has been utilized by several 

research groups to determine the number of water molecules coordinated to the europium in the first 

coordination sphere 1 . Moreover, it has been shown that the luminescence lifetime of Eu(III)chloride 

nanoparticles decreases with increasing amounts of water, both in the gaseous 2 and liquid 3 state. 

Herein, this effect is used for an optical sensor for the determination of relative humidity based on 

europium luminescence lifetime, which is, to the best of our knowledge, a completely new concept. To 

overcome the small molar absorption coefficient of Eu(III), we used antenna dyes to achieve higher 

quantum yields and make the quench effect acessible to phase-modulation fluorometry. A set of several 

europium complexes was benchmarked also using different polymer matrix materials in terms of 

sensitivity, response-time and dynamic range. From best performing combinations sensor spots were 

prepared in a flow-through cell equipped with a tailored emission lifetime-based instrument allowing for 

phase sensitive lifetime measurement of water vapour. 

Acknowledgements: Financial support by a fellowship from the fForte Wissenschafterinnenkolleg FreChe Materie 

and by the Austrian Science Fund (FWF) in the framework of the Austrian Nano Initiative (Research Project Cluster 

0700 - Integrated Organic Sensor and Optoelectronics Technologies – Research Projects 0701 and 0703), by the 

FWF under contract no. P 19387 is kindly acknowledged. The authors would also like to express their thanks to the 

FFG and the Consortium (EUREKA-project SENSIC) and Isovolta AG. 

References: [1] W. D. Horrocks, D. R. Sudnick, J. Am. Chem. Soc. 101 (1979) 334. [2] A.A. Petushkov et al J. 

Lumin. 116 (2006) 127. [3] S. Lis, G. R. Choppin, Ana.l Chem. 63 (1991) 2542. 

125


PRLS-8 

Nanoparticles of organic fluorescent dyes: self-organization and 

optical properties 

Jean-François Lamère, Marion Mille, Mouhammad Abyan, Suzanne Fery-Forgues * 

Laboratoire des Interactions Moléculaires Réactivité Chimique et Photochimique, UMR CNRS 5623, 

Université Paul Sabatier, F-31062 Toulouse cedex 9, France. *E-mail : sff@chimie.ups-tlse.fr 

Fluorescent organic nanostructured materials are of increasing interest for applications in the fields of 

bioanalysis, photocatalysis, photonics and OLEDs. [1] However, their development is still a challenge for 

chemists. Their preparation is difficult to control, and their optical properties are difficult to predict, 

because they depend on both the chemical structure of the constituting molecules, and on the numerous 

intermolecular associations that take place in the solid state. 

A simple method based on a solvent-exchange process is currently used in our group to prepare 

nanoparticles from various fluorescent dyes. For instance, using this method, 4-octylamino-7- 

nitrobenzoxadiazole leads to nanocrystals. These crystals are some tens of micrometers long, but their 

thickness does not exceed 80 nm. Their physical characteristics can be tuned by the presence of various 

macromolecules (polymers, dendrimers, and calf thymus DNA) placed as additives in the reprecipitation 

medium. [2-4] Homogeneous populations of microcrystals with well defined shape and size are then obtained 

(Fig. 1a and b). Interestingly, nanocrystals of different habit display different fluorescence behaviour, 

although they are all made of the same dye. 

When the method is applied to coumarin derivatives, the latter assemble spontaneously to give 

microcrystals, hollow nanofibers, or solid nanofibers, depending on the substituent they bear (Fig. 1c). In 

this case, in spite of the different structures obtained, the fluorescence properties of the nanoparticles are 

rather similar. 

These two examples show the complexity of this research area. The interest and limitations of the 

preparation method used, the optical properties and possible applications of the fluorescent nanostructures 

will be discussed. 

a b b 

c 

1 µm 

20 µm 10 µm 

10 µm 

Fig. 1. Fluorescence microscopy image of microcrystals of 4-octylamino-NBD grown in the presence of 

poly(acrylic acid) sodium salt (a) and calf thymus DNA (b). Observation by transmission electronic 

microscopy of nanofibers obtained with a coumarin derivative (c). 

References: [1] H. Masuhara et al., Single Organic Nanoparticles, Springer-Verlag, Berlin, 2003. [2] F. Bertorelle 

et al., J. Am. Chem. Soc. 125 (2003) 6244. [3] M. Abyan et al., Langmuir 21 (2005) 6030. [4] L. Birla et al., 

Langmuir 22 (2006) 6256. 

126


PRLS-9 

GFP Mutant as Biosensor for Ion Concentration 

Silke Altmeier, Benjamin Hötzer, Gregor Jung 

Saarland University, Biophysical Chemistry, Building B2.2, D-66123 Saarbruecken (Germany), 

E-mail: s.altmeier@mx.uni-saarland.de 

Since its discovery Green Fluorescent Proteins (GFPs) have been used as biological marker in a variety of 

applications. Apart from employing them as fusion tag or reporter gene GFPs are also applied as 

biosensors. GFP variants have been used for ratiometric measurement of Ca 2+ concentration via calmodulin 

and fluorescence resonance energy transfer (FRET). [1] Halides and some other anions cause fluorescence 

quenching due to protonation of the chromophore upon anion binding in variants of Yellow Fluorescent 

Protein (YFP). [2] The pH dependant fluorescent behaviour of wild-type GFP and some mutants is adopted in 

ratiometric or ecliptic pHluorins. [2] Depending on histidine residues, wild-type GFP and certain variants 

show strong affinity for Cu 2+ and less affinity for other metal cations. [3] Binding of the cations results in 

fluorescence quenching that is assayed using a fluorescence plate reader. [3] 

We are analysing fluorescence quenchings via a microscopic setup. In a first example we show that higher 

concentrations of Cu 2+ cause a diminishment of the lifetime of our GFP variant while higher concentrations 

of Ni 2+ exhibit no influence. 

The fluorescence lifetime of a variant of Green Fluorescent Protein changes upon the addition of Cu 2+ . 

References: [1] R. Y. Tsien, Annu. Rev. Biochem. 67 (1998) 509-544. [2] M. Zimmer, Chem. Rev. 102 (2002) 

759-781. [3] T. A. Richmond et al., Biochem. Biophys. Res. Commun. 268 (2000) 462-465. 

127


PRLS-10 

Luminescent probes for nucleoside phosphates, and their application to the 

determination of enzyme activity 

Corinna M. Spangler, Christian Spangler, Michael Schäferling 


D-93040 Regensburg (Germany). E-mail: michael.schaeferling@chemie.uni-regensburg.de 

A large number of enzymatically catalyzed reactions are related to the conversion of ATP by (a) kinases in 

phosphorylation reactions, (b) adenylyl cyclases in the formation of cAMP, and (c) by ATPase in the 

decomposition of ATP. The effect of a series of adenosines (ATP, ADP, AMP, cAMP) and guanosines 

(GTP, GDP) and of pyrophosphate (PP) and phosphate (P) on the luminescence of the europium 

tetracycline (EuTC) complex has already been studied. The highly different quenching effects of the 

phosphonucleosides on the luminescence of EuTC found its application in the determination of creatine 

kinase activity as a model for non-membrane-bound kinases. [1] Compared to other methods, the advantage 

of using a fluorescent probe for phosphonucleoside determination is obvious. This approach is affordable, 

straight-forward, versatile, and the application of radio-labelled substrates or rather complicated 

immunoassays becomes redundant. Currently, we attempt to transfer the method to an adenylyl cyclase 

model system using the highly active enzyme edema factor (EF) from bacillus anthracis. EF is a calciumand 

calmodulin-dependent adenylyl cyclase that raises cellular cAMP levels in the presence of the cofactor 

Mg 2+ by consumption of ATP. [2] In view of certain drawbacks in the EuTC-cyclase assay (like nonspecificity 

and interferences by temperature effects, pH, ionic strength and various quenching agents) we 

are looking for alternative luminescent probes. 

Therefore, we develop improved probes for selective determination of ATP or PP. Poly-3-(1-propanoxy-3- 

triethylammonium)-4-methyl-thiophene bromide (PT-1) was reported to form strong complexes with 

oligonucleotides. Upon binding of an oligonucleotide, the polythiophene turned from yellow to red. [3] We 

tested the response of PT-1 to adenosine phosphoric esters. Fluorescence measurements were less 

promising due to similar quenching properties of ATP, ADP and PP. On the other hand, two new 

absorption bands arise at 538 nm and 585 nm exclusively in case of ATP addition. It can be assumed, that a 

planar conformation is induced in the polythiophene backbone and a supramolecular assembly is formed 

due to interactions with ATP, resulting in a change of the absorption spectrum. This effect can be utilized to 

establish a colorimetric assay for ATP consumption. 

A variety of Tb complexes also was tested for its response to adenosine phosphates. Most of these 

complexes show a dependence on phosphate esters but lack specificity. The Tb-norfloxacin complex is the 

most promising probe. [4] ATP and ADP increase the luminescence of Tb-norfloxacin, whereas PP and P act 

as quenchers. cAMP only has negligible effect on the fluorescence emission. Calibration plots recorded for 

different mole fractions of ATP, cAMP and PP as well as ATP, ADP and P showed a good linear response. 

The adenylyl cyclase assay is used now to study the kinetic response to the conversion of ATP to cAMP 

and PP. Finally, we validate the applicability of PT-1 and Tb-norfloxacin as probes for the determination of 

enzymatic activity and for the screening of enzyme inhibitors by means of the adenylyl cyclase EF model 

system. 

References: [1] M. Schäferling, O.S. Wolfbeis, Chem. – Eur. J. 13 (2007), in press. [2] F.J. Maldonado-Arocho et al., 

Mol. Microbiol. 61 (2006) 324. [3] H.-A. Ho et al., Angew. Chem. Int. Ed. 41 (2002) 1548. [4] Y. Miao et al., J. 

Lumin. 116 (2006) 67. 

128


PRLS-11 

Sensitive terbium(III) probes for luminescent determination of alkaline 

phosphatase and codeine phosphate 

Axel Duerkop 1 , D. Aleksandrova 2 , Y. Scripinets 2 , А.Yegorova 1,2 , E.Vityukova 2 

1 


D-93040 Regensburg (Germany); E-mail: axel.duerkop@chemie.uni-regensburg.de; 

2 National Academy of Sciences of Ukraine, A.V. Bogatsky Physico-Chemical Institute, 

UA-65080 Odessa (Ukraine) 

The main role of aPase in human organism is the transport of calcium as well as of phosphate. Elevated 

levels of aPase in blood serum or plasma can indicate primary and secondary liver cancer or bone tumors. 

Several luminescent methods for aPase detection have been presented, among them FIA assay, [1] ELISA, [2] 

and solid state room temperature phosphorescence. [3] The release of phosphate due to aPase action can be 

used for the design of aPase assays employing new probes for phosphate. [4] 

We present new assays for the determination of the activity of alkaline phosphatase (aPase) and for the 

determination of codeine phosphate (CP). The assays are based on the luminescence quenching of terbium 

complexes with the ligands 4–hydroxy-1-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid (5-ethyl- 

[1,3,4]-thiadiazol-2-yl)-amide (L 1 ) and 1-ethyl-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid 

(4-trifluoromethyl-phenyl)-amide (L 2 ) by phosphate ions. 

Phenylphosphate acts as substrate for aPase which releases phenol and phosphate. Phosphate induces 

luminescence quenching of TbL 1 at 545 nm (λ ex = 320 nm). Equimolar concentrations of Tb 3+ and ligand L 1 

(1.0 µmolL -1 ) at pH 8.0 (10 mmolL -1 Tris-HCl buffer) are required. The Stern-Volmer calibration plot is 

linear from 0.1-70 mU mL -1 of alkaline phosphatase (LOD = 0,05 mU mL -1 = 

4 ng mL -1 = 40 pmolL -1 ). The determination of aPase in synthetic samples yielded good recoveries 

(between 2.34 % and 4.92 %). The effect of organic solvents, various surfactants and donor-active additives 

on the luminescence intensity has been investigated. 

Luminescence quenching of the 

Tb 3+ -L 1 complex in the absence of 

aPase (top spectrum) and in the 

presence of incresing concentrations 

of aPase (bottom spctra); Conditions: 

c Tb = c L1 = 1.0 µM ; TRIS buffer pH 

8.0; λ ex = 320 nm) 

luminescence intensity 

10 

8 

6 

4 

2 

0 

c aPase 

/ mU mL -1 

0.0 

0.1 

0.3 

1.0 

3.0 

5.0 

7.0 

10.0 

30.0 

50.0 

70.0 

480 510 540 570 600 

wavelength / nm 

The method for the determination of codeine phosphate uses the luminescence quenching of TbL 2 upon 

complexation of phosphate released from codeine phosphate. The excitation and emission maxima are at 

320 nm and 545 nm, respectively. The Stern-Volmer calibration plot is linear within the concentration 

range 0.3-20 µg mL -1 of codeine phosphate (LOD = 120 ng mL -1 ). This method has been used to determine 

the amount of active ingredient of codeine phosphate in solution per tablet in “Pyatirchatka IC” and 

“Codterpin IC” tablets with standard additions. 

References: [1]. M. Masoom, P. J. Worsfold, Anal. Chim. Acta 179 (1986) 217. [2] L. C. V. Allen et al., Science 68 

(2000) 231. [3] J.-M. Liu et al., Anal. Biochem. 357 (2006) 173. [4] A. Duerkop et al., Anal. Chim. Acta 555, (2006) 

292. 

129


PRLS-12 

A novel europium (III) complex for luminescent determination of 

oxeladin citrat 

А.Yegorova, D. Aleksandrova, Y. Scripinets, E.Vityukova 

A.V. Bogatsky Physico-chemical Institute of National Academy of Sciences of Ukraine, 86, 

Lustdorfskaya doroga, Odessa 65080, Ukraine 

The opportunity of analytical use of the luminescence sensitization of Ln ions and its either decrease or 

increase effects by some inorganic and organic ions has been found out. Uses of these effects are 

perspective for the determination some drugs, which are not Ln luminescence sensitizers. 

We present a luminescent europium probe for determination of oxeladin citrate. The assay is based on the 

sensibilized of the luminescence of the europium complex with the ligand - 9-fluoro-7-hydroxy-3-methyl-5- 

oxo-N-[2-(1-piperazinyl)ethyl]-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinoline-6-carboxamide (L) by citrate 

ions. 

OH 

O 

F 

N 

H 

N 

NH . HCl 

N 

O 

v 

CH 3 

oxeladin citrate 

(L) 

The key factor is the concentration of Eu 3+ (100 µM) and the ligand (50 µM). I lum is maximum at pH 8,0 

(Tris-HCl buffer). The excitation and emission maxima of the complexes are at 320 and 616 nm, 

respectively. Optimum concentration and acid-basic conditions of complex Eu:L=1:1 formation has been 

established. A ratio of the energy of triplet state of the ligand (22200cm - 1 ) and the first emitting level of Eu 

(III) ion (17300 cm -1 ) as well as high extinction coefficients of L determine the high intensity of a 4fluminescence 

(I lum ) of complex. 

It has been established, that introduction of second ligand - oxeladin citrate in Eu – L system results to the 

increasing luminescence intensity of europium (III) ions. Formation of ternary complex of Eu-L-citrate ions 

was proved by luminescent method. The ratio metal: L: second ligand is 1 : 1 : 1 for investigated complex. 

Influence of organic solvents, amount and nature of surfactants, donor-active additives on I lum of ternary 

complex has been investigated. Maximum I lum has been observed in water solution of this complex. 

The method for the high-sensitive luminescent determination of oxeladin citrate concentration has been 

developed by using dissociation of oxeladin citrate (on oxeladin and citrate ions) and complex Eu:L. The 

calibration plot is linear from 2,5-35 µg/ml of oxeladin citrate (LOD is 1,0 µg /ml). This method has been 

used to assay of the active ingredient - oxeladin citrate of dosage forms (for example, capsuls “Paxeladine”- 

40mg, sirop “Paxeladine”- 2 mg/ml). 

The long luminescence decay time of Eu-L makes the assay useful for time – resolved fluorescence 

measurements. 

130


PRLS-13 

3-[2-(boronophenyl)benzoxazol-5-yl]alanine derivatives as 

monosaccharide sensors 

Daria Jażdżewska, Katarzyna Guzow, Wiesław Wiczk 

University of Gdańsk, Faculty of Chemistry, Sobieskiego 18, 80-952 Gdańsk (Poland). 

E-mail: kasiag@chem.univ.gda.pl 

Phenylboronic acid has been widely utilized for the design of chemosensors in the detection of saccharides 

over the past decade because of a reversible and fast equilibrium interaction of boronic acid group with 

monosaccharide. It can be incorporated in many different systems giving large possibilities for the 

development of analytical devices for the recognition and detection of the sugars which could find 

important applications, especially for diabetics. [1] Because of that we synthesized 3-[2- 

(boronophenyl)benzoxazol-5-yl]alanine methyl ester derivatives (Fig. 1) according to published procedures 

[2] and studied their interaction with glucose and fructose by means of absorption and fluorescence 

spectroscopy. 

Y = 

CH 3 

O O 

O 

Y 

H 2 N CH C 

N 

H 2 

1 

B 

OH 

OH 

HO 

2 

B 

OH 

Figure 1. Structures of the compounds synthesized. 

The acid-base titration of the compounds studied with and without the sugar revealed that the presence of 

D-fructose lowers pK a for about 2 units in contrast to D-glucose which influence is very small. The studies 

of D-glucose and D-fructose affinity to the compounds synthesized were performed in phosphate buffer 

(pH=7.5). The presence of the monosaccharide in the solution of the benzoxazolylalanine derivative in each 

case results in the small increase of the absorbance. Also, the higher concentration of the sugar in the 

solution, the higher fluorescence intensity of the fluorophore, except for compound 2 and D-glucose for 

which the fluorescence quenching was observed. In all cases the small blue shift of the emission spectrum 

in the presence of the sugar was observed indicating that the studied compounds are probably the internal 

charge transfer chemosensors. [1] The compounds studied showed preference for D-fructose over D-glucose 

as indicating much higher values of the apparent binding constants for this sugar. Those kind of the results 

are characteristic for the monoboronic acids. [3] Moreover, the compound 1 had higher affinity to diols 

studied than compound 2 probably as a result of its lower pK a according to the literature. [4] 

Acknowledgements: This work was financially supported by the Polish Ministry of Science and Higher Education 

under grants KBN 1005/T09/2003/24 and DS 8351-4-032-7. 

References: [1] Topics in Fluorescence Spectroscopy, Vol. 11: Glucose Sensing, Ch. D. Geddes, J. R. Lakowicz 

(eds.), Springer Science+Business Media, Inc., New York, 2006. [2] K. Guzow et al., J. Photochem. Photobiol. 

A:Chem. 175 (2005) 57. [3] G. Springsteen, B. Wang, Tetrahedron 58 (2002) 5291. [4] J. Yan et al., Tetrahedron 60 

(2004) 11205. 

131


PRLS-14 

Sensing of ATP anion in aqueous solutions and mitochondria by a fluorescent 

3-hydroxyflavone probe 

Olga B. Vadzyuk, a Dmytro A. Yushchenko, b,c Sergiy O. Kosterin, a Guy Duportail, c Yves 

Mély, c and Vasyl G. Pivovarenko b 

a 

O.V. Palladin Institute of Biochemistry, Kyiv 01030 (Ukraine). E-mail: olga_vadzyuk@hotmail.com 

b 

Department of Chemistry, Kyiv National Taras Shevchenko University, 01033 Kyiv (Ukraine) 

c 

Photophysique des Interactions Biomoléculaires, UMR 7175-LC1 du CNRS, Institut Gilbert Laustriat, 

Faculté de Pharmacie, Université Louis Pasteur, 67401 Illkirch (France) 

Despite the key role of adenosine-5’-triphosphate (ATP), no accurate method of determination of its local 

concentration in living cells is actually available. The determination of local ATP concentration and its 

changes with time in live cells is a complex problem not only due to the restricted space and/or time scale 

limitations, but also due to the selectivity required in the determination of this peculiar anion. In this respect 

multi-channel 3-hydroxyflavone fluorescent probes might be effective tools for resolving the problem. 

We demonstrate the formation of complexes between the tetra-anion ATP and 3-hydroxy-4’-(dimethylamino)flavone 

(FME). Two kinds of complexes are evidenced. The higher affinity 1:1 complex corresponds 

to a stacked configuration between the aromatic moieties of the two molecules and leads to a strong 

hypochromicity of the absorption spectrum of the dye. The lower affinity (ATP) 2 -FME complex results in a 

strong increase of the fluorescence intensity (~ 20-fold), mainly due to the appearance of the anionic form 

of FME, as shown by the important red-shift (60 nm) of both excitation and emission spectra. The collected 

data indicates that this anionic form results from the 

deprotonation induced by the phosphate groups of the 

second ATP molecule [1,2]. In the presence of 250 mM 

sucrose, the interaction with the second ATP molecule 

appears to weaken the spectral effect, which 

nevertheless remains appreciable. This red shift of 

excitation spectrum together with a strong enhancement 

of fluorescence intensity due to the formation of the 2:1 

complex should enable the quantitative evaluation of the 

ATP concentration in both physiological conditions and 

physiological range. A first set of experiments appears 

promising to monitor the succinate-induced production 

of endogeneous ATP in mitochondria. 

Fluorescence Intensity 

15 

a 

10 

5 

0 

[ATP], mM: 

0 

0.019 

0.058 

0.135 

0.287 

0.587 

1.13 

2.16 

4.09 

350 400 450 500 550 

Wavelength (nm) 

b 

O 

H 

O 

O 

FME 

CH 

N 3 

CH 3 

Fluorescence excitation spectra (a) of FME probe in 15 mM TRIS buffer pH 7.4. Emission wavelength is 

555 nm. (b) Structure of the FME and (ATP) 2 -FME complex obtained by quantum chemical simulation. 

Carbon and phosphorus atoms are in dark grey, oxygen and nitrogen atoms are in light grey, hydrogen 

(shown only on right drawings) in white. 

References: [1] V.G. Pivovarenko, O.B. Vadzyuk, S.O. Kosterin. J. Fluorescence 16 (2006) 9-15. [2] V.V. Shynkar, 

A.S. Klymchenko, Y. Mély, G. Duportail, V.G. Pivovarenko. J. Phys. Chem. B 108 (2004) 18750-18755. 

132


PRLS-15 

www.fluorophores.org – Database of Fluorescent Dyes 

H. Wiltsche, T. Mayr 

Institute of Analytical Chemistry, Workgroup Chemo- and Biosensors, Stremayrg. 16/III, 

8010 Graz, Austria, E-mail: torsten.mayr@TUGraz.at 

In the everyday life of a scientist, who is working with fluorescence, the search of a dye with special 

demands can often be a challenging task. Browsing dye catalogues or searching in achieves owned by 

oneself is time consuming and bothering. To make the life of scientist easier we recently launched 

Fluorophores.org, a www database for luminescent dyes. The objective of fluorophores.org is to provide a 

free, accurately, interactive and comprehensive catalogue of fluorescent dyes, their properties and 

applications. Data is collected by individual submission via a web form and the upload of fluorescence 

spectra data and structures. All records are reviewed after their submission to provide high quality data. 

Submission is open for profit as well as for non-profit organisations. The database can be searched for 

various items including wavelength maxima, applications, name, substance classes, CAS-number etc. A 

browse option for quick search is also implemented. This is in contrast to existing databases [1-4] which 

lack of interactivity and search functions. Fluorphores.org uses open-source software including MySQL- 

Database, PHP-scripts and GraPHPite for plotting the spectral data. All data can be viewed by a generic 

web browser without the use of propietary plug-ins or Java-applets. 

The highlight features of Fluorophores.org include: a) plot of absorption and fluorescence spectra; 

b) listing of applications; c) listing of the optical properties, e.g. extinction coefficients, decay time, 

quantum yield; e) listing of availability (commercial/ academic source); f) listing of References, g) export 

of data in various data formats (PDF; CSV); h) spectral data of several records can be displayed 

simultaneously in the Multiple Spectra Viewer. This will be extend by the possibility to show filter and 

light source spectra. Currently the database containes over 500 spectra of the most popular fluorescent 

substances including organic dyes, metal ligand complexes, quantum dots and fluorescent proteins. 

Figure: Screenshot of the displayed data for one record. 

References: [1] G. McNamara, C. Boswell – PupSpecta - http://www.mcb.arizona.edu/ipc/fret/default.htm, [2] Du, H., 

R.-C. A. Fuh, J. Li, L. A. Corkan et al., Photochem. Photobiol. 68 (1998) 141 [3] Invitrogen - 

http://probes.invitrogen.com/resources/spectraviewer/ [4] BDBiosciences - http://www.bdbiosciences.com/spectra 

133


PRLS-16 

Phenanthrene ring-fused boron-dipyrromethenes as new highly emitting 

red visible/near-infrared dyes 

Ana B. Descalzo, 1 Hai-Jun Xu, 2 Zhen Shen, 2 Knut Rurack 1 

1 Div. I.5, Bioanalytics. Federal Institute for Materials Research and Testing (BAM). 12489 Berlin 

(Germany); 2 Coordination Chemistry Institute, State Key Laboratory of Coordination Chemistry, 

Nanjing University, 210093 Nanjing (China). E-mail: ana.descalzo@bam.de 

There is still a strong need for the identification of new, more effective dyes that absorb and emit in the red 

visible / near-infrared (NIR) region of the spectrum. The search for this kind of fluorophores is of interest in 

many different fields of chemistry, ranging from optical sensors and imaging applications to materials 

chemistry related issues, such as molecular switches and devices, lasing media, or electrooptical 

applications [1]. Particularly advantageous for biological and sensing applications is the spectral region 

between 650 and 900 nm—the so-called “biological window”—as absorption and fluorescence of the 

sample matrix and light scattering is significantly reduced at such long wavelengths. 

Key requirements for fluorescent dyes to be suitably employable as fluorescent sensors and labels are 

efficiency, in terms of a high extinction coefficient and a high fluorescence quantum yield, and versatility 

with respect to the dyes synthesis as well as to its functionalization. However, design and synthesis of NIR 

dyes is more demanding with respect to chromophores absorbing in the visible region, as problems due to 

aggregation, photobleaching, and low fluorescence quantum yields are more often encountered. 

A promising starting point for the construction of highly emissive 

dyes is the boron-dipyrromethene (BDP) chromophore. BDP dyes 

are highly rigidized, polymethine-like fluorescent dyes that have 

found widespread application as laser dyes, as well as in biological 

research. They possess many advantageous photonic properties, 

such as high extinction coefficients, high fluorescence quantum 

yields and good photostability. Recent efforts have been focused 

on tuning the fluorescence emission to the NIR region by chemical 

modification of the BDP core, for example, by attaching strongly 

electron-donating groups, by rigidifying the structure or by 

extending the conjugation of the system [2]. 

1 

N 

B 

N 

F F 

Here we present a new family of BDP dyes with an extended chromophoric system achieved by fusing 

phenanthrene rings to the BDP core. This strategy seems to be promising as, for example, fluorescence of 

derivative 1 can be found at > 640 nm with a very intense and narrow emission band (fluorescence quantum 

yield of even 0.95). We will discuss here the spectroscopical properties of the phenanthrene-BDP 

derivatives and evaluate the influence of solvent polarity or pH on the absorption and emission bands. 

References: [1] R. Raghavachari (ed.), in: Near-Infrared Applications in Biotechnology, Practical Spectroscopy Series 

Vol. 25, Marcel Dekker, Inc. 2001. [2] H. Kim et al., Chem. Commun. (1999) 1889; Z. Shen et al., Chem. Eur. J. 10 

(2004) 4853; W. Zhao, E. M. Carreira, Angew. Chem. Int. Ed. 44 (2005) 1677. 

134


PRLS-17 

8-Hydroxyquinoline based turn-on fluorescent chemosensors for 

heavy transition metal ions 

Han Zhang, Qiang-Li Wang, Li-Feng Han, Yun-Bao Jiang* 

Department of Chemistry, College of Chemistry and Chemical Engineering, and the MOE Key Laboratory 

of Analytical Sciences, Xiamen University, Xiamen 361005 (China). 

E-mail: ybjiang@xmu.edu.cn 

8-Hydroxyquinilone (8-HQ) is the second important ligand after EDTA, showing good affinity towards a 

variety of metal ions. It is nonfluorescent before metal chelating because of an efficnet intramolecular 

proton transfer from the 8-OH to quinolino N atom, and becomes fluorescent upon metal binding as this 

proton transfer channel is blocked. 8-HQ has therefore been a candidate for constructing turn-on 

fluorescent chemosensors for metal ions. A drawback of parent 8-HQ as a chemosensor for metal ions is 

the low selectivity in its fluorescent resposne. In order to enhance the selectivity, structural modifications 

were made to the parant 8-HQ nucleus, but most to the aromatic ring. As the ether derivatives of 8-HQ are 

strongly fluorescent, effort in deriving 8-OH group has not been that much as at the aromatic ring. We have 

been interested in developing 8-OH derived molecules as turn-on fluorescent chemosensors for metal, in 

particular heavy transition metal ions. To this end we have tried to derive the 8-OH group in that the 

resultant molecules are nonfluorescent. Below show two series of 8-HQ derivatives with 8-OH being 

esterized or etherized. The derivatives were found nonfluorescent and turned to be highly fluorescent upon 

metal binding. In particular, it was found that they showed highly selective enhancements in fluorescence 

emission in the presence of heavy transition metal ions such as Hg 2+ and Cu 2+ , known for their efficient 

fluorescence quenching character. 

Chemical structures of 8-HQ based turnon 

fluorescent chemosensors with 8-OH 

group being derived. 8-HQ benzoates (1) 

and 8-HQ ethers (2a and 2b). 

O 

O 

N 

O 

O 

N 

N 

O 

O 

O 

N 

1 2 a 2b 

We showed that with 1 an efficient radiationless channel existed because of the nπ* character of the lowest 

excited state, which changed to be of ππ* character upon binding to metals such as Hg 2+ , resulting in over 

1000-fold fluorescence enhancement. With 2 a sequential singlet-singlet intramolecular energy transfer to 

the ketone moiety, intersystem crossing in the ketone, and triplet-triplet energy transfer from ketone back to 

the quinoline moeity was identified that led to fluorescence quenching. In the metal complex, however, the 

initial singlet-singlet eneygy transfer was stopped as the singlet energy of the complex is lower than that of 

the ketone. In this case fluorescence enhancement of 10 2 orders of magnitude was observed and 2b showed 

a high selectivity in its fluorescence reposnse toward Zn 2+ . The selective fluorescence enhancement of the 

newly developed 8-HQ based chemosensors suggested that they could be promising candidates as in vivo 

fluorescence bio-imagining reagents for metal ions of biological inetrests. 

References: [1] H. Zhang, et al., Org. Lett. 7 (2005) 4217. [2] H. Zhang, et al., Tetrahedron Lett. submitted (2007). 

135


PRLS-18 

Flow-injection chemiluminescent determination of aspartic acid using 

tris (2,2'-bipyridyl) ruthenium (II)-Ce (IV) System 

S M Wabaidur 1 , Hee Kyung Lee 2 , Sang Hak Lee 1 , Seikh Mafiz Alam 1 , Chi Wan Jeon 3 

1 

Kyungpook National University, Department of Chemistry, Daegu 702-701, Republic of Korea 

E-mail: tarabai22@yahoo.com.sg 

2 

Department of Nanoscience and Technology, Kyungpook National University, Daegu, 702-701, 

Republic of Korea 

3 

Korea Institute of Geoscience & Mineral Resources, Daejon, 305-350, Republic of Korea 

Aspartic acid, also known as aspartate, the name of its anion, is one of the 20 natural proteinogenic amino 

acids which are the building blocks of proteins. Aspartic acid is a major excitatory amino acids in the 

nervous system. It is of paramount importance in the metabolism during construction of other amino acids 

and biochemicals in the citric acid cycle. It causes much of the damage that occurs after a stroke and is also 

probably the chief neuron-killing villain in neurodegenerative diseases [1] . Many methods have been 

reported for the determinatioin of aspartic acid such as electrochemical [2] , chemiluminescence (CL) [3] and 

laser induced fluorescence (LIF) detectors [4] .In this work, a rapid and sensitive chemiluminescence (CL) 

method using flow-injection (FI) was developed for the determination of aspartic acid. The method is based 

on the CL reaction of aspartic acid with Ce (IV) and tris (2,2'-bipyridyl) ruthenium (II), Ru (bipy) 3 2+ . After 

optimization of the different experimental parameters, a calibration graph was obtained over a 

concentration range of 0.13 µg/ml - 13.3 µg/ml with minimum detectability of 0.075 µg/ml (S/N = 3). The 

correlation coefficient was 0.9910 (n = 7) with a relative standard deviation (%R.S.D.) of 1.51%. 

References: [1] C. Holden, Science 300 (2003) 1866. [2] Z. H. He et al. Anal. Biochem. 313 (2003) 34. 

[3] X. J. Huang et al. Anal. Chim. Acta 414 (2000) 1. [4] D. M. Pinto et al. Anal. Chem. 69 (1997) 3015. 

136


PRLS-19 

Native and genetically functionalized S-layer proteins for binding receptor 

molecules, fluorophores, and quantum dots into perfectly ordered arrays in 

optical sensor systems 

Birgit Kainz 1 , Helga Lichtblau 1 , Dominik Rünzler 2 , Dietmar Pum 1 , Nicola Ilk 1 , Sylvia 

Scheicher 3 , Stefan Köstler 3 , Uwe B. Sleytr 1 

1 Center for NanoBiotechnology, University of Natural Resources and Applied Life Science, 

Vienna, Austria 

2 Max F. Perutz Laboratories, Department of Chemistry, University of Vienna, Campus Vienna Biocenter, 

Vienna, Austria 

3 Karl Franzens University Graz, Institute of Physical Chemistry, Graz, Austria 

The main component of modular biosensor systems is most often a matrix which allows a well defined 

binding of molecules at the nano-scale. In particular, the development of a sensor system based on 

optochemical principles can be achieved, for example, by immobilizing a resonance energy transfer (RET) 

system with molecular precision. 

This presentation is focussing on the reassembly of native and genetically functionalized S-layer proteins 

on solid supports and, in particular, on their usage as matrices for the templated assembly of receptor 

molecules, fluorophores, and quantum dots into highly ordered superlattices. [1] Two-dimensional bacterial 

surface layer proteins (S-layer proteins), isolated from prokaryotic organisms (bacteria and archaea), have 

the intrinsic tendency to self-assembly into two-dimensional arrays in suspension, at solid supports (e.g. 

silicon wafers), at the air-water interface, at floating lipid monolayers and at vesicles (liposomes and 

nanocapsules). The incorporation of single or multifunctional domains in S-layer lattices by genetic 

engineering opened a new horizon for the tuning of their structural and functional features. Proof-ofprinciple 

was shown for genetically engineered S-layer streptavidin fusion protein which was capable to 

bind biotinylated ferritin molecules (12nm diameter) into ordered arrays. [2, 3] Based on this work a broad 

range of S-layer fusion proteins with different functionalities, such as GFP (green fluorescent protein) or 

metal binding peptides, has already been developed. This concept is of a more general nature and is 

currently used in the development of new optical biosensors, affinity matrices, diagnostics, biocompatible 

surfaces, microcarriers, and biological templating, or specific biomineralisation strategies on surfaces. 

References: [1] M. Sára et al., In: Kumar, C., Biological and Pharmaceutical Nanomaterials, Wiley-VCH, 2006, 

p. 219 ff. [2] D. Moll et al., PNAS 99 (2002) 14646. [3] C. Huber et al., Small 2 (2006) 142. 

137


PRLS-20 

Spectroscopic study of benzanthrone 3-N-derivatives as new highly fluorescent 

probes for biomolecules 

E. M. Kirilova 1 , I. Meirovics 2 

1 Chemistry Department, Daugavpils University, 13 Vienibas str., LV5401 Daugavpils, Latvia; 

E-mail: elen@dau.lv 

2 

Riga Technical University, Riga, Latvia 

Today many techniques use fluorescent dyes for the labelling of biological objects. New practical uses call 

for the synthesis of new fluorescent probes with improved properties. 

Recently, we have synthesized a number of 3-aminobenzanthrone N-derivatives by nucleophylic 

substitution of bromo atom in 3-bromobenzathrone with corresponding secondary amines, obtaining highly 

fluorescent compounds [1]. Synthesized dyes are the analogues of cell membrane hydrophobic probe – 

3-methoxybenzanthrone, but new derivatives are long-wavelength light-emitting fluorescent dyes and have 

lower citotoxicity. 

The spectral behaviour of the obtained dyes was investigated. We have studied the spectral properties of 3- 

aminobenzanthrone N-derivatives – absorption and fluorescence spectra in various solvents and binding 

with liposomes and human blood albumin as well as binding with human peripheral blood lymphocytes. 

For obtained dyes large Stokes shift values (about 100 nm) are observed, excitation maxima of the dyes are 

located near 500 nm and emission maxima near 650 nm. In addition these compounds are showed strong 

fluorescent solvatochromism. 

It was found that many of synthesized compounds are quite sensitive to the surrounding environments and 

are potential fluorescent probes for screening structural and functional alterations of cell membranes and for 

estimation of the immune state [2, 3] 

References: [1] E.M. Kirilova, I. Meirovics, S.V. Belyakov, Chemistry of Heterocyclic Compounds, 7 (2002) 896. 

[2] I.Kalnina, T.Zvagule, R.Bruvere, I. Meirovics, J. Fluoresc., 15 (2005) 105. [3] I.Kalnina, R.Bruvere, T.Zvagule, 

N.Gabrusheva, A.Volrate, G.Feldmane, L.Klimkane, E.Kirilova, I.Meirovics, Proceedings of the Latvian Academy of 

Science, 60 (2006) 113. 

138


PRLS-21 

Novel europium(III) chelates as viable probes for optical sensing and 

imaging of temperature 

Sergey M. Borisov, Ingo Klimant 

University of Technology of Graz, Institute of Analytical Chemistry and Radiochemistry, 

Stremayrgasse 16, 8010 Graz (Austria). E-mail: sergey.borisov@tugraz.at 

Knowing temperature is of the highest importance in a broad variety of fields and applications. Besides, it 

is essential for optical sensing of many important analytes (such as oxygen, CO 2 , etc.) since quenching is 

always temperature dependent. Europium(III) chelates are very promising for temperature sensing and 

imaging since they emit in the narrow optical window, possess highly temperature-dependent luminescence 

and long luminescence decay times in order of several hundred microseconds. [1, 2] Common drawbacks 

include excitation in the UV region and (usually) low to moderate brightnesses (BS). We have investigated 

the two possibilities of increasing sensitization wavelength of the Eu(III) luminescence: (a) by making use 

of the long-absorbing β-diketones and (b) by using antenna chromophores excitable in visible region. For 

the second group of the dyes the absorption and emission maxima can be extended well beyond 400 nm. 

Thus, very efficient excitation (ε > 60.000 M −1·cm −1 ) by bright LEDs with peak wavelength of 425, 435 and 

450 nm becomes possible for the first time. Moreover, high emission quantum yields (∼ 0.4 at r.t.) allow for 

high BS. The limits of sensitization wavelength were also investigated. Excitation in the charge-transfer 

band located at ∼ 440 nm does not result in detectable luminescence from Eu 3+ ion. 

The probes are shown to be very promising for temperature 

sensing and imaging when dissolved in a 

polymer film, or, alternatively, when immobilized into 

nano- and microbeads. Dyed poly(styrene-co-vinylpyrrolidone) 

nanobeads can be used for sensing 

purposes in aqueous media. When incorporated in 

microbeads of gas-blocking polymers, the probes 

exhibit virtually no cross-sensitivity to oxygen. On the 

other side, such matrixes as reversed phase silica allow 

for the highest temperature coefficient. The particles of 

both kinds can be very promising as components of 

optical dually sensing materials used in such widespread 

formats as planar sensor foils, microsensors or pressuresensitive 

paints. 

Luminescence Intensity, a.u. 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

Excitation 

Emission 

! = 300 - 400 µs 

300350400450500550600650700750 


References: [1] G. Khalil et al., Rev. Sci. Instrum. 75 (2004) 192. [2] S. M. Borisov, O. S. Wolfbeis, Anal. Chem. 78 

(2006) 5094. 

139


PRLS-22 

Combinatorial approaches for the synthesis of novel fluorescent library based 

on 1,2-dihydropyrrolo[3,4-β] indolizin-3-one 

Eunha Kim, Jihoon Ryu, Seung Bum Park* 

Department of Chemistry, Seoul National University, Seoul 151-747 (Korea). 

E-mail : sbpark@snu.ac.kr 

Due to high sensitivity and ease of handling, fluorescence material has been used extensively as a research 

tool in biological science, clinical diagnosis, and drug discovery. In addition to their application in 

biomedical research, Fluorescent materials became hotter research area because of their industrial 

application as organic light emitting dyes (OLEDs). Despite these high demands, the discovery of novel 

fluorescence core skeletons has been quite limited. To fill this gap, we initiated the development of novel 

core skeletons with tunable fluorescence property using combinatorial approach, which can serve as an 

efficient method for systematizing the synthesis of various molecules in parallel. 

In our strategy, we developed novel 

fluorescent core skeleton with fused 

tricyclic compounds through 

domino reactions. The reaction step, 

Tsuge cyclization followed by 

aromatization, provides a room to 

introduce various appendices, which 

control tunable fluorescence 

properties. 

We have synthesized a novel 

fluorescence library in parallel with 

various emission wavelength, 

simply by changing a reaction 

component using solid phase 

chemistry. This library was 

designed to have a high potential for 

their application to various fields by 

changing their functional handles 

Normalized PL Intensity 

500 

400 

300 

200 

100 

0 

380 430 480 530 580 630 680 

Wavelength 

using efficient chemical transformations, which do not affect a fluorescence property. We demonstrated the 

successful application of this fluorescence compounds as a bioprobe in biomedical research. 

(nm) 

B2 

C2 

A1 

C1 

A7 

B4 

C4 

C7 

E3 

E4 

E7 

E5 

E6 

References: [1] Soper, S. A et al. Anal. Chem. 70 (1998) 477-494. [2] Chen, C. H. Chem. Mater. 16(2004) 4389. [3] 

Nathaniel, S. Finney. Current Opinion in Chemical Biology 10(2006) 238-245. [4] Tsuge, O. Bull. Chem. Soc. Jpn. 

59(1986) 3631. 

140


PRLS-23 

A fiber optic fluorescent 1-(2-pyridylazo)-2-naphthol pH-sensor in 

aqueous solution 

Sang Hak Lee 1 , Jong Ha Choi 2 , Hye Young Chung 1 , Wook Hyun Kim 1 , Yeoun Suk Suh 1 

1 Kyungpook National University, Department of Chemistry, Daegu 702-701, Republic of Korea. 

E-mail: shlee@knu.ac.kr 

2 Department of Chemistry, Andong National University, Andong, 760-749, Republic of Korea. 

Fluorescent pH-sensors are analytical tools widely used in chemistry, biology, medicine and the 

environment protection. Some new developments in this area are related to the synthesis and application of 

fluorescent organic compounds with spectral characteristics highly sensitive to the different environmental 

changes. A fiber optic pH sensor has been fabricated using 1-(2-pyridylazo)-2-naphthol entrapped in an 

ammonia catalyzed silica sol-gel film coated on glass substrate by dip-coating. The sensor was fixed on the 

end of an optical fiber. The sensor showed pH sensitivity when dipped into liquids at different pHs. Linear 

and reproducible responses were obtained in standard buffer solutions in the pH range 7.2 - 8.4, which 

encompasses the clinically-relevant range. The effects of interferences on the determination of pH were also 

investigated. The sensors were successfully applied to the determination of pH in different commercial 

ionic drinks. 

141


PRLS-24 

Optical sensor for norfloxacin based on emission of KMnO 4 -Na 2 SO 3 -Tb 3+ 

System 

Sang Hak Lee 1 , Jun Hee Kwak 1 , Chi Wan Jeon 2 , Yeoun Suk Suh 1 

1 Kyungpook National University, Department of Chemistry, Daegu 702-701, Republic of Korea. 

E-mail: shlee@knu.ac.kr 

2 Korea Institute of Geoscience & Mineral Resources, Daejon, 305-350, Republic of Korea. 

Norfloxacin (NX) [1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline -carboxylic acid] is a 

synthetic fluoroquinolone derivative, which has demonstrated broad-spectrum activity against many 

pathogenic gram-negative and gram-positive bacteria and is highly effective in the treatment of a wide 

variety of infectious diseases. [1,2] Norfloxacin has been determined by polarography, adsorptive stripping 

voltammetry and high-performance liquid chromatography(HPLC). HPLC methods generally require 

tedious procedures and higher analytical costs. Terbium ions show unique fluorescent properties when 

complexed with organic ligands. The strong ion emission of these complexes originates from an intrachelate 

energy transfer from the triplet state of the ligand to the excited energy levels of the lanthanide ion. 

Methods for the selective and sensitive determination of several organic compounds, which serve as energy 

donors to lanthanides, have been developed. In this study, permanganate was immobilized on the resin and 

the resin in permanganate was packed into the flow–cell. The immobilized reagent was retained in the flowcell 

in a configuration perpendicular to the optical fiber bundle. The effects of pH, concentration of Tb(III) 

ion, KMnO 4 and Na 2 SO 4 solutions and flow rate of the norfloxacin solution on the chemiluminescence 

intensity were studied to find the optimum experimental conditions to determine norfloxacin. The emission 

intensity increased linearly with increasing norfloxacin concentration from 1.0×10 -8 to 1.0×10 -8 M and the 

detection limit (3σ) was 8.7×10 -9 . The applicability of the present method was demonstrated by 

determination of norfloxacin in pharmaceutical formulations. The influence of several usually interferences 

on the determination of norfloxacin has been investigated. 

References: [1] J.L. Vazquez et al., Int. J. Pharmaceut. 171(1998) 75. [2] P.G. Gigosos et al., J. Chromatogr. B 

871(2000) 31. 

142


PRLS-25 

New stable and efficient dyes for fluorescence labeling 

Jutta Arden-Jacob, Monika Hamers-Schneider, Norbert U. Kemnitzer, Alexander Zilles, 

Karl H. Drexhage 

University of Siegen, Department of Chemistry, D-57068 Siegen (Germany); 

ATTO-TEC GmbH, Am Eichenhang 50, D-57076 Siegen (Germany). 

E-mail: norbert.kemnitzer@gmx.de 

Very important properties of fluorescent labels for biomolecules are, among others, high fluorescence 

quantum yield and good stability. Not only photochemical stability under prolonged irradiation is required, 

also stability towards aggressive chemicals may be crucial. Both fluorescence efficiency and photochemical 

stability depend on temperature, solvent etc. Since most biomolecules of interest are investigated in 

aqueous environment, good dye properties in water are essential. 

The chromophore of the frequently used cyanine dyes, e.g. Cy5 TM , contains a flexible chain of methine 

groups. Therefore such dyes usually exist as an equilibrium mixture of several cis-trans isomers with 

varying optical properties. Moreover the relative amounts of the various isomers will change, when the 

equilibrium shifts due to coupling and/or adsorption to target biomolecules. Furthermore open chains of 

conjugated double bonds are prone to attack by aggressive chemicals like ozone. 

In contrast to open-chain cyanines, polynuclear systems are rigid and do not form mixtures of isomers. 

Therefore they have higher fluorescence efficiency and show better resistance towards chemical attack. We 

report on new polynuclear dyes belonging to the oxazine and carbopyronin class. The carbopyronin dye 

ATTO 647N, with absorption and 

fluorescence spectra almost identical to 

Cy5, has in aqueous solution a 

fluorescence quantum yield of 65 %, 

more than twice the quantum yield of 

Cy5. The new dye is also much more 

stable than Cy5 when irradiated with 

the light of a tungsten-halogen lamp. 

Similarly oxazine label ATTO 655 

shows excellent photo-chemical 

stability. Actually, oxazines appear to 

be the most stable fluorescent dyes 

known in the red region of the 

spectrum. 

a 

n 

c 

e 

a 

b 

s 

o 

rb 

ATTO 655 

Cy5 TM 

0 10 20 30 40 50 60 

time of irradiation, min 

Furthermore carbopyronins and oxazines show excellent resistance towards ozone, at least two orders of 

magnitude better than Cy5. This is very important in microarray applications, where the dye molecules, 

located at the surface, are directly exposed to ozone present in ambient air. Therefore the fluorescence from 

microarrays with new ATTO 655 is of unprecedented stability, results are highly reproducible. 

143


PRLS-26 

Standardization of fluorescence techniques: 

Where do we stand and what do we need? 

Ute Resch-Genger, K. Hoffmann, A. Hoffmann 

Federal Institute for Material Research and Testing, D-12489 Berlin (Germany). 

E-mail: ute.resch@bam.de 

The use of fluorescence techniques is been ever increasing in the life and material sciences with new 

instrumentation and promising techniques quickly evolving. The comparability of luminescence data across 

instruments is, however, hampered by instrument-specific contributions to measured signals, [1,2] that are 

not only wavelength- and polarization-dependent, but, due to aging of instrument components, also timedependent. 

To rule out instrumentation as a major source of variability and to improve the comparability of 

fluorescence data, reliable, yet simple chemical and physical standards in combination with tested protocols 

for instrument characterization and performance validation are required, thereby also meeting the increasing 

desire for quantification from measurements of fluorescence intensities. [1-4] This will eventually provides 

the basis for the application of fluorescence techniques in strongly regulated areas like e.g. medical 

diagnostics. 

Here, easy-to-operate liquid and solid fluorescence standards developed by BAM are presented, that enable 

the determination and control of a broad variety of fluorescence parameters such as the spectral responsivity 

of the emission channel, the wavelength accuracy, the spectral resolution, and the homogeneity of 

illumination of different types of fluorescence instruments like e.g. spectrofluorometers and confocal laser 

scanning fluorescence microscopes. [3,5] In addition, they can be applied as day-to-day intensity standards 

thereby providing a measure for the control the long-term performance of fluorescence instruments and a 

tool for the consideration of instrument drift. The standards, that will be available in different fomates, are 

designed for use under routine measurement conditions and enable the linkage of fluorescence 

measurements to radiometric units. With the use of the recently certified liquid spectral fluorescence or 

emission standards BAM-F001 to BAM-F005 covering the spectral region from 300 to 770 nm, that have 

been tested by all the National Metrology Instiutes active in the fluorescence area, a comparability of 

fluorescence measurements better than 5 % can be established. 

References: [1] U. Resch-Genger, D. Pfeifer et al. J. Fluoresc. 15 (2005) 325; [2] J. Hollandt, R. D. Taubert et al. 

J. Fluoresc. 15 (2005) 311. [3] U. Resch-Genger, K. Hoffmann et al. J. Fluoresc. 15 (2005) 347. [4] L. Wang, 

A. K. Gaigalas et al. Biophotonics Int. (2005) 42. [5] K. Hoffmann, U.Resch-Genger et al. manuscript in preparation. 

144


PRLS-27 

Symmetric benzothiazole and benzoselenazole squaraine dyes as 

fluorescent probes for proteins detection 

Vladyslava B. Kovalska, 1 Kateryna D. Volkova, 1 Artur Bento, 2 Lucinda V. Reis, 2 Paulo F. 

Santos 2 , Paulo Almeida, 3 and Sergiy M. Yarmoluk 1 

1 Institute of Molecular Biology and Genetics, NASci of Ukraine,03143 Kyiv, Ukraine. 

E-mail: sergiy@yarmoluk.org.ua 

2 Dep. Chemistry, Universidade de Trás-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal. 

3 Dep. Chemistry, Universidade da Beira Interior, 6201-001 Covilhã, Portugal. 

Fluorescence detection of proteins at long-wavelength excitation is widely used for biomedical applications 

due to such benefits of near-infrared-based methods as possibility to use non-expensive diode lasers as 

excitation sources and decreased autofluorescence from biomolecules beyond 600 nm. [1] Due to their 

physical-chemical properties such as light absorption in the visible and near-infrared (NIR) regions and 

sharp and intense fluorescence squaraine dyes are suitable for those purposes. 

A series of symmetrical squaraine dyes based on benzothiazole and benzoselenazole heterocycles was 

studied as fluorescent probes for the specific detection of various proteins (Figure). Spectral-luminescent 

properties of the squaraines were measured in the presence of bovine serum albumin (BSA), human serum 

albumin (HSA), avidin from hen egg white (AVI), lysozyme, insulin and carbonic anhydrase, as well as in 

the presence of a BSA/SDS mixture. The influence of the dye molecules structures on selectivity towards 

certain protein was studied. 

X 

O 

X 

X 

O 

X 

X 

O 

X 

N 

R1 N R1 

I 

CF 3 

SO 4 

- 

N + 

N + 

R1 

N 

N 

R1 

N + 

R1 

O 

N 

R1 

Figure. Structures of studied squaraine dyes 

X=S, Se R=C 2 

H 5 

, C 6 

H 13 

For the studied squaraines in unbound state and in protein presence excitation and emission maxima were 

placed correspondingly in the range 640- 700 nm and 650-720 nm. Generally it should be mentioned that 

emission intensity of benzothiazole squaraines both when unbound and in the presence of proteins was 

higher than the corresponding values of their benzoselenazole analogues. All dyes demonstrated low 

intrinsic emission intensity, while for the dyes with N-ethyl pendent groups in the heteroaromatic nuclei this 

value is in a few times higher than for their analogues with N-hexyl groups. 

It was shown that the length of the N-alkyl pendent group and the nature of the substituents in the squaric 

ring significantly influence on the binding specificity of the dyes. Studied unsubstituted squaraines (O - ) 

demonstrated sensitivity to BSA and gave considerable fluorescent response (several hundred times 

emission enhancement) on the presence of this protein. Unsubstituted dyes containing N-hexyl tails increase 

their emission in the thousands times in the presence of BSA/SDS micelles and thus could be interesting as 

probes for non-specific protein detection or as membrane probes. 

All dyes containing N-ethyl pendent groups demonstrated significant sensitivity to HSA; emission intensity 

of these dyes in HSA presence exceeded corresponding values for dye-BSA complexes. Due to the bright 

emission of the formed dye-protein complexes unsubstituted squaraines with short N-alkyl tails could be 

proposed as dyes for specific HSA detection. 

As a rule, dyes with N-methylamino or N,N-diethylamino substituents into squaric ring are less bright in 

protein presence than the corresponding unsubstituted dyes. It was shown that dyes containing alkylamino 

groups in the central squaric ring interact with AVI giving fluorescent enhancement in 10-15 times. In the 

presence of lysozyme, insuline and carbonic anhydrase the studied dyes slightly change their spectralluminescent 

properties. 

Reference: [1] E. Terpetschnig et al., Anal. Biochem. 217 (1994) 197. 

145


PRLS-28 

Development of near-infrared fluorescent probes for in vivo bioimaging 

Hirotatsu Kojima, Kazuki Kiyose, Sakiko Aizawa, Tetsuo Nagano 

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, 113-0033; CREST, JST 

(Japan). E-mail: kojima@mol.f.u-tokyo.ac.jp 

The number of reports on new techniques in molecular imaging have been recently increasing because of 

their usefulness in biological, medical, and clinical research. Fluorescence imaging methods are generally 

superior in terms of sensitivity, selectivity and ease of use. Cyanine dyes have been employed as 

fluorescent labels in fluorescence imaging studies of biological mechanisms. In particular, tricarbocyanines 

have the advantage that light at their emission and absorption maxima in the near-infrared (NIR) region 

around 650-900 nm is relatively poorly absorbed by biomolecules, and so can penetrate deeply into tissues. 

There is also less autofluorescence in this region. In addition to cyanine dyes for straightforward 

fluorescence labeling, we successfully developed cyanine dyes whose fluorescence intensity changes upon 

specific reaction with nitric oxide, which is an important signaling molecule involved in the regulation of a 

wide range of physiological and pathophysiological mechanisms, and many disorders. [1] The mechanism of 

fluorescence modulation, however, involves photoinduced electron transfer, and consequently imaging with 

these dyes is influenced by the dye concentration, cellular environment (hydrophobicity etc.), and 

photobleaching. To overcome these limitations, ratiometric fluorescent sensors are preferred. 

We synthesized a series of amine-substituted tricarbocyanines in order to examine the correlation between 

the electron-donating ability of the amine and the fluorescence peak wavelength. We found that changing 

the electron-donating ability of the amine substituent altered the absorption and emission wavelengths. 

Then, we synthesized dipicolylcyanine (DIPCY), consisting of tricarbocyanine as a fluorophore and 

dipicolylethylenediamine as a heavy metal chelator, and investigated its response to various heavy metal 

ions. Upon addition of zinc ion, a red shift of the absorbance maximum was observed. Namely, DIPCY 

can work as a ratiometric fluorescent sensor for zinc ion (Zn 2+ ) in the NIR region. [2] For over a century, 

Zn 2+ has been known as an essential trace element, acting as a structural component of proteins or in the 

catalytic site of enzymes. In general, Zn 2+ is tightly associated with proteins and peptides. However, recent 

advances in cell biology have revealed a fraction of Zn 2+ that is free or chelatable in some organs. 

Therefore, this NIR probe for Zn 2+ should be useful in such research. 

Moreover, we have recently developed several pH probes based on the amine-substituted tricarbocyanine 

fluorophore. We could measure pH with these fluorescent probes by a ratiometric monitoring method. We 

believe that this fluorescence modulation of amine-substituted tricarbocyanines should be also applicable to 

dual-wavelength measurement of other biomolecules or enzyme activities. 

References: [1] E. Sasaki et al. J. Am. Chem. Soc. 127 (2005) 3684. [2] K. Kiyose et al. J. Am. Chem. Soc. 128 

(2006) 6548. 

146


PRLS-29 

New fluorescent amino acid with boradiazaindacene derivative (BODIPY) 

as a substituent – synthesis and photophysical properties 

Kinga Kornowska, Katarzyna Guzow, Wiesław Wiczk 

University of Gdańsk, Faculty of Chemistry, Sobieskiego 18, 80-952 Gdańsk (Poland). 

E-mail: ww@chem.univ.gda.pl 

The well-known nowadays fluorophore 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) and its 

many derivatives, because of their favourable photophysical properties, are widely used in many different 

areas. [1] Because of that we synthesized new unnatural amino acid possessing BODIPY fluorophore as a 

substituent (N-Boc-3-[2-(4-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacenyl)phenyl)benzoxazol-5-yl)alanine 

methyl ester (1), Fig. 1) applying literature procedures. [2,3] Because of structural 

similarity of benzoxazol-5-ylalanine to tryptophan this compound may be applied to analysis of protein 

conformational change through its position-specific incorporation. [4] The obtained compound consists of 

two fluorophores – benzoxazole and BODIPY moieties. Thus, also the parent compound 4,4-difluoro- 

1,3,5,7-tetramethyl-8-(4-methyl)phenyl-4-bora-3a,4a-diaza-s-indacene (2, Fig. 1) was synthesized to 

determine the influence of the benzoxazolylalanine moiety on BODIPY fluorophore properties. 

H 3 C 

H 3 C 

O 

CH 3 

O 

O 

N 

H 

CH 

CH 3 

O 

C 

H 2 

CH 

H 3 C 

3 

CH 

H 3 C 

3 

O 

N F 

N F 

B 

B 

F 

F 

N 

N 

N 

H 3 C 

H 

CH 3 C 

3 

CH 3 

1 2 

Figure 1. Structures of the compounds synthesized 

The spectral and photophysical properties of the synthesized compounds in methanol, acetonitrile and 

cyclohexane were studied by means of absorption and steady-state and time-resolved fluorescence 

spectroscopy. Both compounds studied have a narrow absorption band with maximum at about 500 nm, 

however, amino acid derivative (1) has additional broad absorption band with maximum at about 300 nm as 

a result of benzoxazole ring absorption. Also, the benzoxazolylalanine derivative of BODIPY (1) has higher 

values of molar absorption coefficients (up to 72 000 dm 3 mol -1 cm -1 ) which increase with solvent polarity 

and ability to form hydrogen bonds in contrast to the parent compound (2) for which the opposite is true. 

The influence of the solvent polarity on the position of the absorption band is stronger in the case of 

compound 2 for which hypsochromic shift is observed with its increase. The opposite dependence is 

observed for the other compound (1). The similar dependences for each compound are observed for their 

emission spectra. However, the emission band of 1 (maximum at about 520 nm) is batochromically shifted 

in comparison to the spectrum of 2 (maximum at about 510 nm) but for both compounds quite strong 

overlap of absorption and emission spectra is observed. The fluorescence quantum yields of the compound 

2 are about twice times higher than those of 1. Moreover, their values increase with solvent polarity in 

contrast to those of compound 1. Also, the fluorescence intensity decays are different – compound 1 has 

biexponential whereas compound 2 monoexponential. 

Acknowledgements: This work was financially supported by the Polish Ministry of Science and Higher Education 

under grant DS 8351-4-032-7. 

References: [1] R. P. Haughland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc. 

1996. [2] K. Guzow et al., J. Photochem. Photobiol. A:Chem. 175 (2005) 57. [3] M. Baruah et al., J. Org.Chem. 70 

(2005) 4152. [4] D. Kajihara et al., Nat. Methods 3 (2006) 923. 

147


PRLS-30 

Fluorescent sensing effected by conformational mobility 

Krisztina Nagy a , Szabolcs Béni b , Péter Kele a , Zoltán Szakács a , Béla Noszál b and 

András Kotschy a 

a Institute of Chemistry, Eötvös Loránd University, Pázmány Péter sétány 1/A, H-1117 Budapest, Hungary 

b HAS Research Group for Drugs of Abuse, Department of Pharmaceutical Chemistry, 

Semmelweis University, Hőgyes Endre utca 9, H-1092 Budapest, Hungary 

E-mail: kelep12@hotmail.com 

The role of steric perturbation and conformational constraints in photoinduced electron transfer sensors 

have rarely been investigated so far. In our recent report we have shown that conformational mobility of the 

donor site’s surroundings has a profound effect on its signalling potential [1]. Following this lead we 

directed our efforts at understanding signal generation in common sensor types to track down the effects of 

conformational dynamics on their signal generation process. 

Four 18-crown-6 based sensors were selected for the present study: 1 contains an azacrown host unit and an 

attached coumarin fluorophore, while 2a-c have a 1,10-diazacrown core with either two coumarin units (2a) 

or pendant coumaryl and benzyl (2b) or tert-butoxycarbonyl-methyl groups (2c). According to the classical 

working hypothesis the electron donating nitrogen atoms of the aza-crown moieties quench the 

luminescence of the attached coumarins as long as they are not “distracted” by any secondary interaction. 

On complexation, hydrogen bonding or protonation the redox potential of the donor nitrogen is increased, 

weakening its donating capabilities that leads to an increase in the fluorescence intensity. 

N 

R 

R = 

MeO 

O 

O 

O 

O 

O 

O 

O 

O 

N N 

R 

O O 

1 2 

O 

2a R ' = R 

2b R ' = Bn 

2c R ' = -CO 2 t Bu 

R ' 

R '' -NH 3 ClO 4 

3 

3a R" = n Bu 

3b R" = Cy 

3c R" = i Pr 

3d R" = t Bu 

3e R" = CH 2 t Bu 

To examine the equilibrium and stability aspects of the complexation between selected guests and sensors 

we conducted a series of 1 H NMR spectroscopic experiments. A set of coordination experiments were 

conducted using fluorescence measurements with hosts 1 and 2 in the presence of different organic 

ammonium salts (3a-e). These ammonium salts as guests were selected on the basis of their similar binding 

modes but various steric demand. The obtained results support our formerly established theory that direct 

coordination and conformational dynamics both contribute to signal generation in PET sensory systems. 

Reference: [1] Kele, P. et al. Angew. Chem. Intl. Ed., 45 (2006) 2565. 

148


PRLS-31 

Dicyanomethylene squarylium dyes as the red and near-infrared fluorescent 

probes for proteins and cells 

Anatoliy Tatarets 1 , Leonid Patsenker 1,2 , Sania Khabuseva 1 , Ewald Terpetschnig 2 


60 Lenin Ave., UA-61001 Kharkov (Ukraine). E-mail: tatarets@isc.kharkov.com 


We synthesized a series of symmetrical and unsymmetrical dicyanomethylene squarylium dyes 1–10 and 

investigated their spectral properties free in solutions and after interaction with Bovine Serum Albumine 

(BSA) and cells such as human fibroblasts and Saccharomyces Cerevisiae yeast. 

NC CN NC CN 

R 3 

X 1 

X 2 

NC 

CN 

O 

R 3 N 

N 

N 

R 1 O R 2 O 

1 R 1 = R 2 = Me, R 3 = H; 

2 R 1 = R 2 = (CH 2 ) 2 OH, R 3 6 X 1 = CMe 2 , X 2 = O; 

= H; 

3 R 1 = Me, R 2 = (CH 2 ) 2 OH, R 3 7 X 1 = CMe 2 , X 2 = S; 

= H; 

4 R 1 = Me, R 2 = (CH 2 ) 5 COOH, R 3 8 X 1 = O, X 2 = S; 

= H; 

5 R 1 = R 2 = Me, R 3 9 X 1 = S, X 2 = S; 

= NO 2 

N 

N 

O 

10 

N 

Depending on the nature of the terminal heterocyclic moiety these dyes absorb and emit in a wide spectral 

range. They have long-wavelength absorption and emission maxima in chloroform between 647 and 

757 nm, extinction coefficients between 104,000 and 208,000 M –1 ⋅cm –1 and quantum yields as high as 80%. 

The terminal heterocyclic moieties cause a red-shift of the absorption and emission maxima in the order: 

diphenyloxazole < benzoxazole < indolenine < benzothiazole < 5-nitro-indolenine while alkylsubstitution at 

the heterocyclic nitrogen atom has only a minor influence on the spectral properties. All dicyanomethylene 

squaraine dyes exhibit additional absorption bands in the 378–396 nm range with extinction coefficients of 

about 29,000 – 44,000 M –1 ⋅cm –1 . Dye 5 absorbs not only in the red and UV but also in blue spectral region 

(468 nm) with extinction of 32,000 M –1 ⋅cm –1 . This makes dicyanomethylene squarylium dyes also suitable 

for use with the blue (380, 405 and 470-nm) diode lasers excitation. Absorption and emission spectra in 

methanol are blue-shifted by 10–30 nm compared to chloroform and the quantum yield are somewhat 

lower. 

These dyes form non-fluorescent aggregates in aqueous media. As a result the long-wavelength absorption 

band becomes broader and a new band appears. The fluorescence intensity of dyes 1–4 and 6 substantially 

increases in presence of BSA with quantum yields as high as 95%. These dyes were found to readily stain 

cells of different nature (figure below). 

The figure on the right shows a human fibroblast cell stained 

with squarylium dye 4. Dyes 1–4 and 6 are perfectly suited for 

in-situ biological imaging and fluorescence-based 

quantification of proteins. 


149


PRLS-32 

A polyoxometalate luminescence probe applied to the study of protein 

adsorption to fluorescent polymers for implant and tissue construct purposes 

Graham Hungerford 1,2 , Mark Green 1 and Klaus Suhling 1 

Johan Benesch 3,4 , João F Mano 3,4 and Rui R Reis 3,4 

1 Physics Department, King’s College London, Strand, London WC2R 2LS, UK 

2 Departamento de Física, Universidade do Minho, 4710-057 Braga, Portugal. 

3 3B´s Research Group - Biomaterials, Biodegradables and Biomimetics, Department of Polymer 

Engineering, University of Minho, 4710-057 Braga, Portugal 

4 IBB - Institute for Biotechnology and Bioengineering, PT Government Associated Laboratory, Braga, 

Portugal. e-mail: graham@fisica.uminho.pt 

The study of protein adsorption to biodegradable polymers is of high importance as changes in protein 

conformation upon interaction with these materials, when utilised as tissue constructs or implants, can illicit 

an adverse reaction. Fluorescence is a very sensitive technique by which to monitor changes in protein 

conformation and interaction with the local environment. This can take advantage of the intrinsic 

fluorescence of certain amino acids, eg. tryptophan or by making use of an extrinsic probe. The former 

avoids perturbation of the protein by addition of the probe, while the latter can be tailored to elucidate 

specific information and is necessary when monitoring intrinsic fluorescence is precluded. Some polymers 

of choice for tissue construct and implant applications, such as poly-caprolactone (PCL) and starchethylene 

vinyl alcohol (SEVA-C), exhibit fluorescence in the wavelength region where intrinsic protein 

fluorescence is observed. Selection of an extrinsic probe is therefore advantageous, but as proteins adsorb 

as a thin layer to the construct surface, even a small amount of background fluorescence can prove to be 

significant in relation to the probe originated fluorescence. Thus even choosing a wavelength away from the 

peak emission of the polymer may not be sufficient to clearly view the fluorescence emanating from the 

protein and has led us to previously employ a variety of fluorescence techniques [1] making use of the 

commonly used probe, fluorescein isothiocyanate [2], by resolving the probe and background emission. 

In this work we present the use of a new europium containing polyoxometalate [3] for use as a protein 

label. The luminescence intensity of this compound was found to increase in the presence of increasing 

quantities of serum albumin, and a change in the time-resolved behaviour was also observed. Contrast 

between labelled protein and the fluorescent polymers was achieved by time gating the luminescence signal 

to discriminate against that originating form the polymers employed, (eg PCL and SEVA-C). As well as 

making use of both steady state and time-resolved techniques imaging was performed to ascertain the 

degree of coverage on the polymer substrate. 

References: [1] J. Benesch et al., J.Coll. Int. Sci.(2007) in press. [2] G. Hungerford et al., Photochem. Photobiol. Sci. 

6 (2007) 152. [3] M. Green et al., J. Am. Chem. Soc. 127 (2005) 12812. 

150


PRLS-33 

Interaction of xanthene dyes with bovine serum albumin 

Negmat Nizomov 1 , Eldar Kurtaliev 1 , Zafar Ismailov 1 , Shavkat Nizamov 1 , Gairat 

Khodjayev 2 , Yelena Obukhova 3 , Leonid Patsenker 3 

1 Samarkand State University, University Blvd. 15, 140104 Samarkand (Uzbekistan). 

E-mail: nnizamov@yandex.ru 

2 Samarkand Agricultural Institute, M.Ulugbek St. 77, 140103 Samarkand (Uzbekistan). 

E-mail: gayrat_kh@mail.ru 


60 Lenin Ave., UA-61001 Kharkov (Ukraine). 

E-mail: kolosova@isc.kharkov.com 

Xanthene dyes are widely used as fluorescent markers for biomedical applications [1] . Therefore, 

investigation of interaction of these dyes with proteins and other biological molecules is not only of basic 

science interest but also has considerable practical importance. We synthesized a series of xanthene-based 

rhodamines (R-160, R-164, R-193, R-195) and pyronins (R-291, R-292) and examined their spectral 

properties in aqueous solutions in free state and in presence of Bovine Serum Albumin (BSA). These dyes 

contain the same chromophore system with delocalized unit positive charge but different substituents in the 

xanthene moiety. 

N 

O 

NEt 2 

N 

O 

N 

Et 2 N O NEt 2 

R-160 

COO 

R-164 

COOH 

CI 

R-193 

N 

COO 

N 

O 

N 

Et 2 N O NEt 2 

N 

O 

N 

HSO 4 

R-195 

N 

COOH 

CI 

R-291 

R-292 

HSO 4 

Primarily we investigated the absorption and emission spectra of the dyes vs. concentration and found these 

spectral data to be constant in the concentration range between 10 –5 and 10 –6 M. This evidences that the dye 

molecules exist in the above solutions in the non-aggregated (monomeric) form. The extinction coefficients 

(ε), oscillator strengths (f e ), fluorescence quantum yields (B), excited state lifetimes (τ), and frequency of 

pure electronic transition (ν 0-0 ) of the monomeric forms were determined and analyzed. Furthermore, 

interaction of the dyes with BSA was investigated. Binding parameters such as the binding constant (K) and 

the number of binding sites (N) were calculated as described in [2] . The data obtained evidence that the noncovalent 

attachment of the dyes such as R-193 and R-291 is due to electrostatic interaction of the xanthene 

oxygen atom to protein molecule. In contrast, in the julolidinium dyes R-164, R-195 and R-292 the oxygen 

atom is sterically hindered and, as a result, the binding constants of these dyes are in two orders of 

magnitude lower than that for R-193 and R-291. The binding constant of the unsymmetrical monojulolidinium 

dye R-160 is in between the values for symmetrical diethylamino (R-193 and R-291) and dijulolidinium 

dyes R-164, R-195 and R-292. Thus just the sterical availability of the xanthene oxygen atom 

causes more pronounced effect on the interaction of the investigated dyes with BSA while the influence of 

carboxy-aryl group was found to be much lower. 

References: [1] M. Sauer et al., in Near-Infrared Dyes for High Tehnology Applications, O. S. Wolfbeis et al. (eds.), 

Kluwer, London, 1998, p.57-87. [2] E. K. Baulie, J. P. Raynaud, Eur. J. Biochem. 13 (1970) 293. 

151


PRLS-34 

Can Ru(II) polypyridyl dyes measure pH directly using the 

luminescence lifetime? 

Laura Tormo, Nelia Bustamante, Guillermo Orellana* 

Laboratory of Applied Photochemistry, Department of Organic Chemistry, Faculty of Chemistry, 

Complutense University of Madrid, E-28040 Madrid (Spain). E-mail: orellana@quim.ucm.es 

Rapid and continuous monitoring of pH is required in practically all kinds of areas including chemical, 

biomedical and environmental sciences [1]. Although the pH electrode is probably irreplaceable in most 

situations, optical sensors are an excellent option due to their simplicity, small size and robustness for a 

wide variety of applications. In particular, optodes based on luminescence lifetime measurements present 

some decisive advantages over intensity-based devices since the effect of lamp and detector fluctuation/drift 

and indicator leaching/bleaching are avoided. However, instrumentation for nanosecond emission lifetime 

determinations is expensive and many fluorescent indicator dyes do not display a change in their excited 

state decay kinetics with pH. 

Ruthenium(II) polypyridyl complexes have carried fiber-optic oxygen sensing on to commercial 

applications [2] due to their intense absorption in the visible region, large Stokes shift, high emission 

quantum yields, excellent photochemical and thermal stability and almost diffusion-controlled oxygen 

quenching, together with μs excited state lifetimes. Similarly, it would be desirable to design pH indicator 

dyes from the family of Ru(II) polyazaheterocyclic complexes in order to benefit from the advantages and 

opto-electronic instrumentation already developed for O 2 monitors [2]. A proper design of the pH 

indicator/solid support couple must ensure little or zero cross-sensitivity to such ubiquitous gas. 

Chemical estructure of 

the pH-sensitive Ru(II) 

coordination complexes 

used in this 

study. The acidic 

hydrogen atom is the 

only one depicted in 

the Figure (in cyan). 

With these features in mind we set out to prepare two novel luminescent Ru(II) complexes for pH 

optosensing using lifetime based measurements, namely Ru(bds) 2 (F 15 ap) 2– and Ru(dpps) 2 (pyim) 2– (see 

Figure; bds = 2,2'-bipyridine-4,4'-disulfonate, dpps = diphenyl-1,10-phenanthroline-4,7-disulfonate, F 15 ap = 

1,10-phenanthroline-5-perfluorooctanamide, pyim = 2-(2-pyridyl)imidazole). We have investigated the 

changes in the emission properties of these complexes at different pH values in several buffer solutions 

using absorption spectroscopy, steady-state and time-resolved luminescence techniques. Taking into 

account the results obtained, we are now able to assess the suitability of Ru(II) indicator dyes for pH 

measurements based on whether they show an excited state acid-base equilibrium or a (deceiving) 

irreversible proton transfer reaction. 

Acknowledgements: This work has been funded by the Community of Madrid (grant no. S-0505/AMB/00535), the 

European Regional Development Fund, the European Social Fund and Interlab IEC, Madrid. 

References: [1] G. K. McMillan, R. A. Cameron, Advanced pH Measurement and Control (3rd ed.), ISA, Boca 

Raton, Florida, 2004. [2] G. Orellana, D. García-Fresnadillo in: Optical Sensors: Industrial, Environmental and 

Diagnostic Applications, R. Narayanaswamy, O. S. Wolfbeis (Editors), Springer Series on Chemical Sensors and 

Biosensors Vol. 1, Springer, Berlin-Heidelberg, 2004, p. 309. 

152


PRLS-35 

K7-1045 as a new fluorescence probe for pharmaceutical research, 

clinical diagnostics and biological imaging 

Inna Yermolenko, Oksana Sokolik, Tatyana Dyubko, Sania Khabuseva, Leonid Patsenker 

State Scientific Institution "Institute for Single Crystals", National Academy of Sciences of Ukraine, 

60 Lenin Ave., UA-61001 Kharkov (Ukraine). 

E-mail: patsenker@isc.kharkov.com 

Our group has extensive experience with albumin-specific fluorescent probes such as K35 for pharmaceutical 

and biomedical applications [1]. K35 absorbs at 448 nm, has a fluorescent quantum yield (Q.Y.) of 

0.3% in water and 12% in presence of albumin (HSA). Here we describe the new probe K7-1045 with 

improved spectral and photophysical properties. 

The new probe has absorption maximum at 456 nm and is excitable with a 405, 436 or 470-nm diode laser. 

This probe has a very low quantum yield of 0.2% in water. The Q.Y. increases substantially upon noncovalent 

binding to HSA (50%). Thus the fluorescence intensity is 250-times increased in presence of HSA. 

Absorption and emission maxima of the К7-1045/HSA complex are at 430 nm and 527 nm, respectively. 

Like K35 the new probe binds to albumin at two different binding sites. The dye/HSA binding constant 

(K c ) was found to be 3.4×10 5 М –1 and the number of binding sites (N) was 1.8. Importantly, 

autofluorescence of HSA is substantially quenched in presence of К7-1045. The HSA molecule is known to 

have two types of binding sites (Type I and Type II). Drugs such as warfarin and phenylbutazone bind to 

Type I while propranolol binds to Type II sites. К7-1045 competes with these drugs for the both binding 

sites. 

Furthermore, we investigated efficiency of the substitution of К7-1045 on the HSA binding sites with 

cryoprotectants such as glycerol, 1,2-propanediol, ethylene glycol, DMSO, and DMF. This probe is 

sensitive to interaction of HSA with the cryoprotectants of different hydrophobic/hydrophilic nature, which 

allows using this probe to study molecular mechanisms of cryoprotection. 

К7-1045 is also useful as very bright fluorophore for biological 

imaging. As an example, the figure shows that this dye 

can be used to obtain fluorescent images of Saccharomyces 

Cerevisiae yeast cells. Cytoplasmatic proteins and organelles 

of these cells stained with К7-1045 exhibit bright yellowishgreen 

fluorescence. 

К7-1045 is an albumin-sensitive fluorescent probe which promises useful applicability for drug screening, 

biological imaging and investigation of interaction of albumin with cryoprotectants and other small organic 

molecules. 

Reference: [1] Serum albumin in clinical medicine, Book 2, Yu. А. Grysunov, G. Е. Dobretsov (eds.), Geotar, 

Moscow, 1998 (In Russian). 

153


PRLS-36 

Fluorescent probes and labels for biomedical applications 

Leonid Patsenker 1,2 , Olga Kolosova 1 , Anatoliy Tatarets 1 , Iryna Fedyunyayeva 1 , 

Yevgeniy Povrozin 1 , Inna Yermolenko 1 , Yuliya Kudryavtseva 1 and Ewald Terpetschnig 2 



2 SETA BioMedicals, LLC, Urbana, IL, USA. E-mail: ewaldte@setabiomedicals.com, 

http://www.setabiomedicals.com 

We have developed extremely bright and sensitive fluorescence materials for use in biological and 

pharmaceutical research, clinical diagnostics, and high-throughput screening (HTS): Reactive Red and 

Near-infrared (NIR) Fluorescent Labels of the Square and SETA series for covalent attachment to 

biomolecules such as proteins, amino-acids, peptides, oligonucleotides, DNA, RNA, lipids and drugs; 

Fluorescent Probes for proteins, lipids and cells; Fluorescence Lifetime (FLT) Probes and Labels of 

SeTau series for FLT and fluorescence polarization based applications; Dark quenchers of the SQ series 

for Fluorescence Resonance Energy Transfer (FRET) applications; Classification Dyes for single or 

multiple encoding of microspheres. 

These dyes, probes and labels have several advantages as compared to other commercially available probes 

and labels: Square and SETA dyes absorb and emit in the 500–900 nm spectral range. Unlike dyes of the 

Cy and Alexa Fluor series, these red and NIR emitting markers can be excited not only with the red, 635- 

nm and 670-nm diode lasers but also with the blue, 380-nm, 405-nm and 436-nm lasers or light emitting 

diodes (LEDs). These dyes have high extinction coefficients (up to 265,000 M –1 cm –1 ) and protein 

conjugates of these labels are extremely bright (quantum yields up to 70%). The environment- sensitive 

lifetimes dyes have lifetimes in the range of 500 ps to 3 ns, while conventional polymethines such as Cy5 

and Alexa Fluor 647 exhibit almost no or much smaller changes in lifetime after binding to protein. Some 

of the probes exhibit high affinity for proteins, biomembranes and lipoproteins and can be used to detect 

and quantitate these analytes. A series of pH-sensitive markers for pH 5.5–12.0 was also developed. 

The figure on the right shows the image of a 

human fibroblast cells stained with the red 

cyanine dye K8-1500 (left) and a newly 

developed potential-sensitive dye K5-1000 

(right). These new dyes exhibit higher 

photostability as compared to Cy or Alexa 

Fluor dyes, which is especially important for 

high-throughput screening and biological 

imaging applications. 

SeTau tracers show fluorescence in the blue and green spectral region and have FLTs up to 40 ns in water. 

SeTau dyes are perfectly suited for use in homogeneous fluorescence polarization assay of high molecularweight 

antigens and substantial polarization increases are observed upon binding of the high molecularweight 

tracers to the antibody. 

The newly developed reactive Dark Quenchers of the SQ series that absorb in the 500–800 nm spectral 

range have several times higher extinction coefficients than Black Hole Quenchers; they do not exhibit 

any residual fluorescence and are perfectly suited for covalent labelling of proteins, peptides and oligonucleotides 

for use in FRET and real-time PCR based applications. 


154


PRLS-37 

Determination of DNA via fluorescence and resonance light scattering using 

new ruthenium derived luminescent probes 

Doris M. Burger, Otto S. Wolfbeis 

University of Regensburg,Institute of Ananlytical Chemistry, Chemo- and Biosensors 

D-93040 Regensburg (Germany). E-Mail: Doris.Burger@chemie.uni-regensburg.de 

The detection and determination of DNA is important for a multitude of biological applications. These 

range from standard molecular biological to diagnostic techniques. Intercalator dyes represent one of the 

most popular tools since these intercalate between the base pairs or move themselves in the major or minor 

groove of the DNA. Ethidium bromide, the so called Hoechst dyes, and the fluorophores of the TOTO and 

YOYO family have been commercially most successful. Besides these organic dyes, fluorescent transition 

metal complexes have become more and more interesting as intercalators in the recent years. 

Many efforts have been made to study the influence of ligands of ruthenium complexes on the binding 

mode to DNA. We demonstrated that various ruthenium complexes are capable of binding to DNA by an 

intercalative mode. New ruthenium(II) complexes were synthesised based on the binding motif 

for the mononuclear ruthenium complex and 

X-(CH 2 ) 2 –N + (R) 3 

X–NH-CS-NH-(CH 2 ) n -N + (R) 2 -(CH 2 ) m -N + (R) 2 -(CH 2 ) n - NH-CS-NH-X 

for the dinuclear complexes, where X is [Ru(bpy) 2 (phen)] 2+ , R is methyl, n is 2 or 3 and m is 3, 4 or 6 (see 

Fig. 1). 

The probes display an increase in fluorescence intensity upon addition of DNA. Absorption titrations, 

lifetime measurements were performed and melting curves established. Fluorescence titration experiments 

with DNA, allows the determination of affinity constants according to the model of McGhee and Hippel [1] . 

Binding constants in the range of 10 6 M were calculated. Furthermore, it was found that DNA can enhance 

the resonance light scattering (RLS) signal of the probes. RLS measurements were performed by scanning 

excitation and emission synchronously at ∆λ = 0. Under optimum conditions, the RLS signal is enhanced 

by a factor of 20 following the addition of DNA to the solution of the probes. DNA can be determined in 

concentrations as low as 2.4 ng/mL. 

+6 

N 

N 

N 

Ru 

N 

N 

Ru 

N 

(PF 6 ) 6 

N 

N 

N 

NH 

C 

S 

NH 

(CH 2 )n N (CH 2 )m N (CH 2 )n NH NH 

N 

C 

S 

N 

N 

Fig. 1. General structure of the dinuclear complexes 

Reference: [1] J. D. McGhee, P. H. von Hippel, J. Mol .Biol. 86 (1974), 469-489. 

155


PRLS-38 

A “piggyback” fluorescent protein marker 

Martin Link, Otto S. Wolfbeis 


D-93040 Regensburg (Germany). E-mail: martin.link@chemie.uni-r.de 

We envisioned that the use of a label carrying more than one fluorophore may increase the brightness (Bs; 

defined as the product of ε and quantum yield) of a tagged protein [1]. This would have the advantage of 

blocking less reactive sites of a protein and thus not compromising its function and charge (which is 

critical in case of electrophoresis). 

We chose tris-(3-aminopropyl)-amine (TAPA) as the starting molecule since it possesses three amino 

groups to which fluorescent molecules may be attached. Dansylchloride turned out to be the label of choice 

for synthesizing the triply substituted TAPA derivatives shown in Fig. 1. TAPA was first triply labeled with 

dansyl chloride, and a carboxy group was introduced thereafter which can be activated (via its NHS ester) 

to give an amino-reactive label. The absorption maximum (λ max ) of the activated dye is located at a 

wavelength of 343 nm with a molar absorption of 9700 L/(mol*cm) in ethyl alcohol. For protein labeling, 

the NHS-ester is added to a solution of bovine serum albumin (BSA) and bicarbonate buffer solution (pH 

9). After stirring for 18 h in the dark at room temperature, the dye-protein conjugate (Fig. 1) is purified by 

size-exclusion chromatography. The normalised excitation and emission spectra of the BSA-conjugate are 

shown in Fig. 1. The excitation peak is at 347 nm, and the emission at 497 nm (in phosphate buffer). The 

large Stokes' shift facilitates the separation of excitation and emission. 

If the dye-to-protein ratio (DPR) remains the same, the fluorescent signal increases with the utilisation of 

piggyback labels in comparison to classical probes with only one fluorophore. Consequently, the DPR can 

be minimized by the application of a multifluorophore marker and the probability of denaturation (as a 

result of labeling functional amino groups) is strongly reduced. 

NMe 2 

excitation and emission 

1,0 

0,8 

0,6 

0,4 

0,2 

0,0 

347 n m 

150 nm 

exc. 

497 n m 

em. 

300 400 500 600 

wavelength in nm 

Me 2 N 

O 2 

S 

H 

N 

O 

N 

Br 

SO 2 

NH 

N 

H 

O 2 

S 

NMe 2 

HN 

BSA 

Fig.1. Chemical structure of the triply labeled fluorophore attached to BSA (right), and its excitation and 

emission spectra (left). We refer to these labels as piggybacks because the label carries three fluorophores 

on its backbone. 

Reference: [1] V.V. Martin et al., Tetrahedr. Lett. 40 (1999), 223-226 

156


PRLS-39 

Novel quinolinium and isoquinolinium platforms for fluorescent probes, 

sensors and labels 

Wolter F. Jager , Otto van den Berg and Stephen J. Picken 

Nano Organic Chemistry & NanoStructured Materials, Delft University of Technology, Julianalaan 136, 

2628 BL Delft, (The Netherlands). E-mail: W.F.Jager@tudelft.nl 

Luminescent chromophores are used as fluorescent platforms for constructing sensors, probes and labels. 

The basic requirements for a suitable fluorescent platform are a high inherent quantum yield, good thermal 

and photochemical stability and facile functionalization at multiple positions. The different functionalities 

that may be attached are signaling subunits for detecting (different) analytes, and groups that enable 

attachment to a solid substrate. High fluorescence lifetimes are also desirable since increasing the lifetime 

τ F will increase the sensitivity towards changes taking place in the vicinity of a chromophore. For 

constructing fluorescent labels, a high and specific reactivity towards well-defined functional groups is an 

additional requirement. Here we report on the synthesis, properties and applications of 7-fluoro-1-methylquinolinium 

iodide (1), 5,7-difluoro-1-methyl-quinolinium iodide (2), and 6-fluoro-1-methylisoquinolinium 

iodide (3). These novel fluorescent platforms are used as as fluorescent labels, and for 

constructing fluorescent probes and sensors. 

X 

R 

F 

R 1 

I - N + 

N + F 

I - 

N 

I - 

R 1 

N 

N+ NHR 1 R 2 

1: X=H 3 

R 2 

1b: R=H 

R 2 

3b 

2: X=F 2b: R=F 

2c: R=NR 1 R 2 

N+ 

I - 

Scheme 1. Fluorescent labels 1-3 and the probes and sensors 1b-3c derived from them. 

Compounds 1-3 are highly reactive molecules that specifically react with soft nucleophiles like amines and 

thiols in water by a nucleophilic aromatic substitution reaction. This reaction, illustrated in Scheme 1, 

enables the synthesis of a wide variety of fluorescent probes, sensors and monomers. Alternatively 1-3 can 

be used as labels for functionalizing (bio)macromolecules that contain amines or thiols. The reactivity of 

these molecules will discussed, along twith the photophysical properties of the resulting probes and sensors, 

and examples of functionalized polymers will be given. 

Derivatives of 1-3, like 1-methyl-7-dimethylamino quinolinium tetrafluoroborate, [1] have been used as 

color-shifting mobility sensitive probes for detecting glass transition temperatures in amorphous and semicrystalline 

polymers. [2] In addition, physical ageing [3] and the detection of phase transitions in various media 

were reported using these probes. For this application the excellent thermal and photochemical stability, 

(for which the tetrafluoroborate anion is responsible to a large extend) along with a high fluorescence 

quantum yield in polymer films, were important. It should be noted that the molecules employed are nonfunctionalized, 

i.e. only having alkyl substituents, and that the inherent Charge Transfer properties of these 

molecules explains the observed sensitivity towards medium mobility. 

Fluorescent pH sensors were synthesized by reacting 1, 2 and 3 with various piperazines and other 

diamines. The resulting compounds were fluorescent only, when the external (piperazine) amine was 

protonated. Non-protonated sensors were quenched presumably by a PET mechanism. [4] Using a modular 

approach, we have synthesized fluorescent off-on sensors with pK A values ranging from 2.5 to 10 in this 

manner. 

References: [1] O. van den Berg, O, W.F. Jager et all, J. Org. Chem. 71 (2006) 2666. [2] O. van den Berg, O, 

W.F. Jager et all, Macromol. Symp. 230 (2005) 11. [3] O. van den Berg, O, W.F. Jager et all,. Macromolecules 39, 

(2006) 224. [4] A.P, da Silva, H. Q. N Gunaratne et all,. Chem. Rev. 97 (1997) 1515. 

157


PRLS-40 

Fluorescence modulation through conformational dynamics: development of a 

new class of PET sensors 

Péter Kele a , Krisztina Nagy a , Szabolcs Béni b , Zoltán Szakács a , Béla Noszál b and 

András Kotschy a 

a Institute of Chemistry, Eötvös Loránd University, Pázmány Péter sétány 1/A, H-1117 Budapest, Hungary 

b HAS Research Group for Drugs of Abuse, Department of Pharmaceutical Chemistry, 

Semmelweis University, Hőgyes Endre utca 9, H-1092 Budapest, Hungary 

E-mail: kotschy@chem.elte.hu 

The majority of present day fluorescent sensors, consisting of a receptor and a fluorophore unit, exploit the 

so called photoinduced electron transfer (PET) phenomenon [1]. Guest binding to the receptor module 

results in a coordinative interactions with the host donor site, whose redox potential is altered causing the 

change of the fluorescent signal. In certain cases, however, the observation of an enhanced fluorescent 

signal could not be explained satisfactorily [2] by the formation of secondary interactions. 

O 

O 

O 

n = 1, 2 

O 

n O 

n O 

N R O 

O 

O 

O 

N 

R 

R = 

MeO 

O 

O 

1 2 

Recently we have shown that, parallel to secondary interactions, changes in the conformational mobility 

around the donor site do also have a profound effect on its fluorescence [3]. Based on this principle we 

designed and synthesized a series of new sensors (1,2) harvesting the effects of conformational changes on 

signal generation, and obtained fluorescence enhancements comparable to or even greater than conventional 

PET sensors. Binding studies on 1 and 2 with different ammonium salts revealed that these new systems are 

more sensitive to steric factors, and less sensitive to the acidity of the guest, offering a new type of 

selectivity in sensing. Parallel to the fluorescence measurements NMR studies were also conducted to 

understand the recognition and signal generation process in more detail. 

References: [1] Callan, J. F. et al. Tetrahedron 61 (2005), 8551. [2] Gawley, R. E. et al. J. Am. Chem. Soc 124 

(2002) 13449. [3] Kele, P. et al. Angew. Chem. Intl. Ed., 45 (2006) 2565. 

158


PRLS-41 

Hoechst33258 and pyranine as a fluorescent probes for quantitative 

assessment of myoglobin 

B. M. Murari 1 , S. Anand 1 , N. K. Gohil 1 , N. K. Chaudhury 2 

1 Centre for Biomedical Engineering, Indian Institute of Technology Delhi, India. 

2 Institute of Nuclear Medicine and Allied Sciences, Delhi, India. 

E-mail: bhaskarmurari@hotmail.com 

Assessment of myoglobin (Mb) is of clinical importance. Mb is the smallest protein cardiac marker, 

diffuses rapidly throughout the vascular system and provides the earliest indication of acute myocardial 

infarction (AMI) or heart attack. 

We have studied the interaction of Hoechst33258 

(H258) and Pyranine (HPTS) with Mb. It is 

observed that fluorescence of H258 (503nm) and 

HPTS (513nm) quench linearly with increasing 

concentrations of Mb. The quenching of emission 

intensity is attributed to collisional quenching. 

Anisotropy and lifetime values of H258 and 

HPTS with Mb remained unaltered suggesting no 

complex formation. Therefore, the observed 

decrease in emission intensity of probes H258 

and PY is attributed to the presence of other 

strongly absorbing moiety in Mb, the heme 

porphyrin which has absorption band at 410nm 

with high extinction coefficient at 410nm. The 

role of heme porphyrin and interaction of these 

two probes with other proteins were further 

established by studies on hemoglobin (four heme 

porphyrin) and serum proteins fibrinogen and 

bovine serum albumin (BSA) which is analogous to human serum albumin (without heme porphyrin). H258 

with BSA showed increase in its emission intensity with blue shift along with altered anisotropy and 

lifetime which suggested H258-BSA complex formation. HPTS fluorescence properties remained unaltered 

with BSA. Both these probes did not interact with fibrinogen. Both these probes showed quenching with 

Mb concentration upto 0.2µM (500ng/mL). Thus, H258 and HPTS have potential for development of 

fluorescence based sensing system for Mb. HPTS have better prospects as it did not interact with BSA. A 

simple, rapid sensing of Mb based on fluorescence could facilitate fast processing with short turnaround 

time for better management of AMI. 

Reference: Hanbury, C.M., Miller, W.G. and Harris, P.B. (1997). Clin. Chem; 43:11, 2128-2136. 

11000 

10000 

Intensity 

9000 

8000 

7000 

6000 

5000 

4000 

3000 

2000 

1000 

0.5µ! 

15µ! 

513nm 

0 

400 450 500 550 600 650 


Fluorescence quenching 

of HPTS in presence 

of myoglobin (5-15µ!) 

159


PRLS-42 

Functionalised Ru(II) bipyridyl probes for dicarboxylic anion and 

aminoacid detection 

Emanuela Berni, Sandra Pinet, Nešo Sojic, Isabelle Gosse 

University of Bordeaux I, I.S.M. UMR 5255, Groupe Nanosystèmes Analitiques 

ENSCPB, 16 Avenue Pey Berland 33607 Pessac (France). E-mail: berni@enscpb.fr 

Anions are well known to play several fundamental roles in a wide range of chemical, biological and 

environmental processes. For this reason, optical and electrochemical sensing of anionic species in aqueous 

and nonaqueous media is an area of great interest in current research. However, anion binding has generally 

proved to be challenging because of their lower charge to radius ratio, pH sensitivity and range of 

geometries. Switterionic aminoacids recognition is still more challenging due to immense biological 

importance of these molecules. This is to be linked to the great interest in designing methods for the 

detection of neurotransmitters in intra- and extra-cellular medium. Fluorescent molecular sensors are 

especially convenient since they allow real time and real space monitoring of the activity of the desired 

analyte, that should be exploited for imaging. [1] Among potential switterionic aminoacids, L-Glutamate is 

a target of choice: it performs essential functions inside the Central Nervous System (CNS) and an 

alteration of its capacity in sending stimuli between neurons induces great damages of the cerebral structure 

and consequently neurological disorders. [2] 

In the perspective of developing fluorescent sensors for glutamate, we synthesized a 

N 

N 

N 

2+ 

Ru 

N 

N 

N 

O 

H 

N 

O 

N 

H 

n = 1 L1 

n = 3 L2 

n = 5 L3 

H 2 N 

N 

H 

( ) n 

( ) n 

H 

N 

4 X - 

H 2 N 

NH 2 

NH 2 

new series of 

guanidinium functionalised Ru(II) bipyridyl 

receptors L1-L3, with arms of different length. 

Indeed, the tris(2,2’-bipyridyl)ruthenium(II) 

([Ru(bpy) 3 ] 2+ ) system has been extensively 

investigated due to its chemical stability, redox 

properties, excited-state reactivity, and 

luminescent emission. For exemple, Beer and 

coworkers have incorporated this moiety into 

acyclic, macrocyclic and calix[4]arene 

structural frameworks to obtain efficient sensors 

that shown to coordinate via hydrogen bonds to 

anions such as Cl - , H 2 PO - 4 , HSO - 4 .[3] 

In the case of our Ru(II) complexes L1-L3, 

anion addition induces an increase in luminescent intensity with a concomitant hypsokromic (blue) shift in 

the emission λ max . Binding properties of these complexes have been investigated towards various anions. 

Their guanidinium moieties allow to sense dicarboxilic acid but also biologically important anions such as 

glutamate. The arm length of the bipyridyl receptor is shown to influence the rigidity of the complexes 

during anion recognition and thus the fluorescence reponse. 

Moreover, Ru(II) receptors present the advantage to be fluorescent sensors with electrochemical properties. 

We are especially interested in the possibility to study anion recognition by electrochemiluminescence 

(ECL), that is converting electrical energy into radiative energy, or in other words, converting electrical 

stimuli in image. Electrochemiluminescent detection of metal ion has been already described [5] and we 

aim to extend its application to anionic substrates. 

References: [1] A. W. Czarnik, Chem. Biol., 2, (1995), 423. [2] E. J. Fletcher, D. Loge, In An Introduction to 

Neurotransmitters in Health and Disease; P. Riederer, N. Kopp, J. Pearson, Eds; Oxford University Press: New York, 

1990; Chapter 7. [3] a) P. D. Beer , E. J. Hayes, Coord. Chem. Rev.. 240, (2003) 167. [4] M. M. Richter, Chem. Rev., 

104, (2004), 3003. 

160


PRLS-43 

Novel porphyrin-phthalocyanine triads: Light-harvesting and 

charge separation in one unit 

Eugeny A. Ermilov, a Sebastian Tannert, a Michael T. M. Choi, b Dennis K. P. Ng, b and 

Beate Röder a 

a Institut für Physik, Photobiophysik, Humboldt-Universität zu Berlin, Newtonstr. 15, D-12489 Berlin 

(Germany). E-mail: ermilov@physik.hu-berlin.de 

b Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong (China). 

E-mail: dkpn@cuhk.edu.hk 

Porphyrin-phthalocyanine heteromers are an attractive 

class of light-harvesters and charge separation systems 

exhibiting an easy route of synthesis and high chemical 

stability. In the present work we report the results of 

photophysical investigations of two novel non-sandwichtype 

porphyrin – silicon(IV) phthalocyanine heterotriads, 

where two porphyrins (H 2 TPP or ZnTPP) are linked to the 

central silicon atom of the phthalocyanine moiety. The 

steady-state absorption spectra of the triads (H 2 Tr and 

ZnTr) are well described as superposition of the monomer 

spectra, since strong coupling between chromophores is 

prevented by the axial to peripherical linking of 

phthalocyanine (Pc) and porphyrin (P) moieties via 

oxygen. That type of ligation also hinders planar stacking 

of the π-systems. 

It has been found that the photophysical properties of the 

triads in polar (dimethylformamide) and nonpolar (toluene) 

solvents are strongly affected by two different types of interaction between the P and Pc parts, namely 

excitation energy transfer (EET) and photoinduced charge transfer. The first one results in appearance of 

the Pc fluorescence when the P-part is initially excited and for free-base triad plays the dominant role in fast 

depopulation of the first excited singlet state of the P moiety. Whereas EET supersedes the electron transfer 

(ET) for H 2 Tr in both solvents, both transfer channels become comparable for ZnTr solved in toluene, and 

the probability of ET is approximately 3 times higher for this triad in polar DMF. 

If the first excited singlet state of the Pc-part is populated (directly or via EET), it undergoes fast 

depopulation by hole transfer (HT) to the charge-separated (CS) state. In polar DMF the CS state is the 

lowest excited state and the charge recombination occurs directly to the ground state. Using transient 

absorption spectroscopy the lifetime of the CS state was estimated to 30 ps and 20 ps for H 2 Tr and ZnTr, 

respectively. In nonpolar toluene the energy gap between the first excited singlet state of the Pc-part and the 

CS state is very small, and back HT occurs in both triads resulting in appearance of “delayed fluorescence” 

of the Pc-part with a decay time similar to the lifetime of the CS state (190 ps and 280 ps for H 2 Tr and 

ZnTr, respectively). Since the CS state of ZnTr solved in toluene has a lower energy than that one of H 2 Tr, 

the probability of BHT for ZnTr is lower, too. This was clearly proved by decay associated fluorescence 

spectra measurements. 

N 

N M 

N 

N 

N 

N 

N 

N 

S i 

N 

N 

N 

O 

O 

N 

N 

N 

M 

N 

N 

M = H 2 , Zn 

161


PRLS-44 

Optochemical oxygen sensing using Pt(II)-porphyrin dye immobilised on 

S-layer matrices 

Sylvia Scheicher 1 , Stefan Köstler 1,3 , Birgit Kainz 2 , Christian Konrad 3 , Michael Suppan 3 , 

Alessandro Bizzari 3 , Dietmar Pum 2 and Volker Ribitsch 1,3 

1) Karl Franzens University Graz, Institute of Physical Chemistry, A-8010 (Austria). 

2) University of Natural Resources and Applied Life Sciences, Centre of Nanobiotechnology, 

A-1180 Wien (Austria) 

3) Joanneum Research, Institute of Chemical Process Development and Control, A-8010 Graz (Austria) 

Crystalline bacterial surface layers (S-layers) appeared to be perfect matrices for immobilizing functional 

molecules in a highly ordered structure. S-layers are monomolecular arrays of a single type of protein and 

exhibit different types of lattice symmetry depending on the protein structure. The capability of S-layer 

proteins to reassemble in suspension, on solid surfaces and liquid films makes them an ideal substrate for 

immobilising (macro) molecules. [1-2] The protein SbpA, used in this study, forms an S-layer lattice of 

square symmetry with a center-to-center spacing of 13.1 nm. Each morphological unit is composed of four 

identical subunits. 

Pt(II) complexes of porphyrins show strong phosphorescence at room temperature with decay times in the 

range of several tens of µs. The excited triplet states of these dyes can be quenched by molecular oxygen. 

This leads to a marked decrease of luminescence lifetime and intensity and can be exploited for optical 

oxygen sensing. [3-4] 

S-layer protein SbpA was recrystallised onto glass substrates. The free carboxylic groups of the protein 

were used to covalently bind the sensitive dye via the formation of active ester intermediates. 5,10,15,20- 

Tetrakis-(4-aminophenyl)-porphyrin-Pt-(II) was subsequently immobilized to the protein layer. 

The sensor slides were mounted in a flowthrough-cell, 

and used for dissolved oxygen 

sensing in water. A LED with an emission 

maximum at 405 nm was used as excitation light 

source. Signal detection was accomplished using 

a photomultiplier tube and LockIn Amplifier. 

Modulation of the excitation light and analysis of 

the resulting phase shift allowed for lifetime 

based oxygen sensing. Analysis of luminescence 

intensity and phase signals showed clear and 

reversible dependence on oxygen concentration. 

References: [1] U.B. Sleytr, et al., Ang Chem Int Ed 38 (1999) 103. [2] T.J. Beveridge, Curr Op Struct Bio 4 (1994) 

204. [3] D.B. Papkovsky, T.C. O´Riordan, Journal of Fluorescence, 15 (2005) 569. [4] D.B. Papkovsky et al., Anal. 

Chem. 67 (1995) 4112. 

162


PRLS-45 

Characterization of new near infrared dyes for molecular imaging 

Jutta Pauli 1 , Tibor Vag 2 , Romy Haag 2 , Werner A. Kaiser 2 , Ingrid Hilger 2 , and Ute Resch- 

Genger 1 

1 Federal Institute for Material Research and Testing, D-12489 Berlin (Germany). 

E-mail: jutta.pauli@bam.de 

2 

Friedrich-Schiller-University Jena, Institute for Diagnostic and Interventional Radiology, 

D-07747 Jena (Germany). E-mail: tabor.vag@med.uni-jena.de 

The sensitivity of near-infrared fluorescence (NIRF) imaging depends to a strong extent on the 

spectroscopic properties of the chosen fluorescent reporters. Suitable dyes are characterized by e.g. a high 

molar absorption coefficient at the excitation wavelength and a high fluorescence quantum yield under 

application-relevant conditions. Aiming at the introduction of new fluorescent tools for medical diagnostics, 

we spectroscopically studied the NIR hemicyanine dyes DY-676, DY-681, DY-731, DY-751, and DY-776 

in phosphate buffered saline solution (PBS) and in a solution of bovine serum albumin (BSA) in PBS 

modelling body fluid and compared their absorption and fluorescence properties to that of indocyanine 

green (ICG), the only clinically approved fluorescent dye until now. 

The absorption and fluorescence properties of the DY dyes and ICG are controlled by dye hydrophilicity, 

dye aggregation, dye-protein interactions, and the energy gap rule. The fluorescence quantum yields of all 

the hemicyanine dyes in PBS and in PBS/BSA are always higher than the φ f values of ICG rendering the 

DY dyes attractive diagnostic reagents. In all cases, the fluorescence quantum yields of the dyes in 

PBS/BSA exceed those in PBS suggesting specific dye-albumine interactions. [1,2] This is supported by 

corresponding spectral shifts in absorption. These shifts, that can be most likely used as an indicator of dye 

hydrophility, point e.g. to an increased hydrophilicity of DY-676, DY-681, DY-731, and DY-751 as 

compared to ICG. The maximum fluorescence quantum yields in PBS/BSA were found for DY-681 and in 

PBS for DY-681, DY-731, and DY-751. The reduced values of φ f resulting for DY-676 and DY-776 in PBS 

are caused by aggregation of the dye molecules as also indicated by the broadening of the absorption 

spectra. 

References: [1] P. Czerney, et al., Biol. Chem. 382 (2001)495. [2] T. Vag , et al., submitted to Invest. Radiology. 

163


PRLS-46 

Sensing molecular oxygen in live mammalian cells by 

phosphorescence quenching 

Dmitri B. Papkovsky, Tomas C. O’Riordan, Alexander V. Zhdanov 

Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, Cork, Ireland; 

E-mail: d.papkovsky@ucc.ie 

Molecular oxygen is an informative marker of cell metabolism and cellular responses to various stimuli. 

Analysis of cellular oxygen and oxygen consumption rates provides important information on the status of 

the cell, particularly its mitochondrial function [1]. Quenched-phosphorescence oxygen sensing allows 

probing of oxygen consumption in complex biological samples in a minimally invasive manner and on a 

micro-scale [2]. However, application of this methodology to the analysis of oxygen distribution in 

individual mammalian cells and to monitoring of physiological responses of live mammalian cells still 

remains very challenging. 

We have developed several new methodologies for sensing intracellular oxygen and alterations in oxygen 

consumption in populations of live mammalian cells and in individual cells [2,3]. A family of 

supramolecular oxygen-sensitive probes based on phosphorescent metallopoprhyrin dyes was specially 

designed and optimised for intracellular use, thus enabling a range of new bioassays and high-utility 

applications performed in relatively simple and robust measurement formats. These formats include: 

• High-throughput analysis of intracellular oxygen in populations of cells using MitoXpress® oxygen 

probe and lifetime-based oxygen sensing on a time-resolved fluorescent plate reader; 

• High-content analysis of intracellular oxygen in individual cells using highly-photostable NIR oxygen 

probes and live cell fluorescent microscopy. 

Corresponding probe chemistries, detection principles and measurement set-ups will be presented in the 

talk and illustrated with particular experiments performed with different cells. 

References: [1] Will Y. et al., Nature Protocols, 2006, 1(6): 2563-2572; [2] O’Riordan T.C. et al., Am. J. Cell 

Physiol., 2007 [Epub ahead of print]. PMID: 17170232; [3] O’Donovan C., et al., Lab-on-Chip, 2006, 6: 1438-1444; 

164


PRLS-47 

Sensing intracellular oxygen in neuronal cells using phosphorescent oxygen 

probe and time-resolved fluorescence plate reader detection 

Alexander V. Zhdanov, Tomas C. O’Riordan, Dmitri B. Papkovsky 

Biochemistry Department, University College Cork, Cork, Ireland 

High energy costs of neurotransmitter (NT) release by neurons need to be quickly compensated to prevent 

damage and regenerate the cells for subsequent stimulation. Refilling of ATP stocks is mainly achieved by 

the mitochondrial machinery, through enhanced oxidative phosphorylation and increased consumption of 

oxygen as the key metabolite. 

To allow studying of these processes and their mechanisms, we have developed a simple method for realtime 

monitoring of intracellular oxygen in live neural cells, under resting conditions and upon stimulation. 

The method relies on dynamic quenching by O 2 of a supramolecular oxygen probe based on phosphorescent 

Pt-porphyrin dye [1] , which is loaded into the cells and then monitored by time-resolved fluorescence. The 

method includes the following main steps: i) growing the cells in collagen coated 96-well plates; ii) loading 

the cells with MitoXpress® probe using transfection reagent; iii) washing the cells and monitoring them on 

the TR-F reader Victor2 at 37°C, measuring periodically probe signal at two different delay times; iv) 

adding effector compounds to the cells during the measurements. Profiles of phosphorescence lifetime of 

intracellular probe thus obtained are indicative of oxygen levels in resting cells, and their changes upon 

stimulation. 

The method was applied to investigate alterations in oxygen consumption by differentiated neuronal cell 

line PC12 ( d PC12) in response to different compounds known to induce NT release. The electron transport 

chain uncoupler FCCP caused a negative spike in intracellular oxygen indicating an increase in respiration, 

with specific ‘bell-shape’ profile reaching maximum at ~20 min. Conversely, ETC inhibitors Rotenone 

(complex I) and Antymycin A (complex III) caused a small reduction in probe lifetime, due to the blockage 

of respiration bringing intracellular oxygen levels to that of bulk solution. Ryanodine and CMC (ryanodine 

receptor agonists mediating Ca 2+ release from the sarcoplasmic reticulum) showed only a slight increase of 

O 2 consumption, whereas 100mM K + (i.e. above plasma membrane depolarisation threshold) induced rapid 

and large transient increase in respiration. The naïve PC12 cells, also prone to NT synthesis and release, 

demonstrated response to 100mM K + with significantly lower amplitude then d PC12. 

Taken together, the results demonstrate that the new method provides sensitive, specific, minimally 

invasive monitoring of changes in intracellular oxygen in populations of resting and stimulated mammalian 

cells, including the very gentle and sensitive neuronal cells. 

Figure: 

Typical responses of d PC12 cells to 

100mM K + , 4 µM FCCP and 1µM 

Rotenone in comparison with the 

resting cells (no drug), presented in 

lifetime scale. 

Lifetime [us] 

40 

39 

38 

37 

36 

35 

34 

33 

32 

Drug application 

100mM K+ 

4uM FCCP 

1uM Rotenone 

NO drug 

31 

30 

10 20 30 40 50 60 70 

Time [min] 

Reference: [1] Y. Will et al., Nature Protocols, 1 (2006): 2563–2572. 

165


PRLS-48 

Photophysical characteristics of a new reactive fluorescent dye and 

some of its bioconjugates 

Alexander A. Karasyov 1 , Ulrich Schmeisser 1 , Otto S. Wolfbeis 2 

1 

Active Motif Chromeon GmbH, Von-Heyden-Str.12, D-93105 Tegernheim (Germany) 

2 


D-93040 Regensburg (Germany). E-mail: karasyov@activemotif.com 

Synthetic fluorescent dyes play a key role in the fluorescent visualization of biological objects. Fluorescent 

confocal microscopy, flow cytometry, gel-electrophoresis, and staining of membranes, proteins as well as 

DNA usually are performed with fluorescent dyes of different structures and functionality. A considerable 

number of dyes have been synthesized in the past 20 years [1-3] . 

Nevertheless, dyes like fluorescein and its reactive form (fluorescein isothiocyanate; FITC) are in use for 

many years. FITC has an absorption maximum at ~494 nm if conjugated to proteis, and thus can be excited 

with the argon ion laser line at 488 nm. Its molar absorption and quantum yield are acceptable, as is its 

solubility in water. Nevertheless, two drawbacks essentially restrict its applicability as a label, namely low 

photostability and its strongly pH-dependent fluorescence intensity and lifetime. 

We introduce a new reactive dye for protein labelling which is much more photostable than FITC, has a 

comparable fluorescence quantum yield both in buffer solutions and in the form of its conjugates to 

antibodies or avidin/streptavidin. Its fluorescence intensity is virtually independent of pH in the range from 

6.5 – 9.0. Antibody conjugates of Chromeo 488 have almost identical absorptions as the conjugates of 

Alexa Fluor 488 comjugates but their emission is longwave shifted by 5 – 10 nm. This allows the use of 

these conjugates along with Alexa 488 filter sets in fluorescence microscopy. 

Fig. 1. pH dependence of the fluorescence 

intensities of labels Alexa Fluor 488, 

FITC, and Chromeo 488. 

0,8 

0,6 

0,4 

Fluorescence Intensity1,0 

AF 

C 

F 

Alexa Fluor 488 (AF) 

FITC (F) 

Chromeo 488 (C) 

0,2 

5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 

pH 

Chromeo 488 displays good solubility in water and fairly constant fluorescence intensity in the 

physiological pH range. It therefore represents a useful label for a wide range of applications in biological 

research. 

References: [1] A.S. Waggoner, Curr. Opin. Chem. Biol. 10 (2006) 62. [2] C. Sun et al., J. Chromatogr. B. 803 

(2004) 173; [3] B. Wetzl et al., J. Chromatogr. B 793 (2003) 83. 

166


PRLS-49 

Novel fluorescent dyes for selective fibrillar α-synuclein detection 

Kateryna D. Volkova a , Vladyslava B. Kovalska a , Anatoliy O. Balanda a , Mykhaylo Yu. 

Losytskyy a , Rolf J. Vermeij b , Vinod Subramaniam b and Sergiy M. Yarmoluk a 

a Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 

150 Zabolotnogo St., 03143 Kyiv (Ukraine). E-mail: sergiy@yarmoluk.org.ua 

b MESA+ Institute for nanotechnology, University of Twente (the Netherlands) 

Parkinson's disease and other related disorders are characterized by the accumulation of fibrillar aggregates 

of α-synuclein (ASN) inside brain cells [1]. For today there exist only several dyes that are used extensively 

to detect amyloid inclusions, therefore designing of new dyes for this approach has substantial importance 

for basic research [2]. 

With the aim of searching novel fluorescent probes for selective fibrillar α-synuclein detection the spectralluminescent 

properties of the series of benzothiazole monomethine cyanines were studied in unbound state 

and in the presence of native and fibrillar ASN using Thioflavin T as a reference dye. 

3 

R 

Cl 

S 

+ 

N 

Thioflavin T 

N 

X 

S 

+ 

N 

R 

S 

N 

1 2 

Figure. Structures of Thioflavin T and studied cyanine dyes 

R 

L-43 R 1 = R 2 = Me; 

R 3 = H; X = SO 4 

L-414 R 1 = R 2 = Et; 

R 3 = NH 2 

; X = Cl 

T-284 R 1 = Me; R 2 = Et; 

R 3 = NEt 2 ; X = Cl 

For the monomethine cyanines in protein-containing solutions excitation and emission maxima were placed 

correspondingly in the range 400-444 nm and 474-573 nm. Firstly in was shown that asymmetrical 

monomethines demonstrated strong fluorescence responses on fibrillar α-synuclein presence, while in 

monomeric ASN presence weakly increase their fluorescence. 

Dye 

In free form in buffer 

In native ASN 

presence 

In fibrillar ASN presence 

λ ex , nm λ em , nm I 0 , a.u. λ em , nm I N , a.u. I F , a.u. I F /I 0 I F /I N 

L-43 400 481 17 481 12.4 28.3 1.66 2.3 

T-414 437 520 3.2 516 3.3 11.3 3.6 3.4 

T-284 443 580 2.9 573 2.2 21 7.34 9.5 

Thio-T 442 478 1.8 478 2.1 6.1 3.4 2.9 

λ ех (λ еm ) – maximum wavelength of fluorescence excitation (fluorescence emission) spectrum; 

I 0 (I N , I F ) – emission intensity of free dye in buffer (in native BLG presence, in fibrillar ASN presence). 

Analysis of structure-function dependences enable us to make a supposition that amino- or diethylaminosubstituents 

in the 6 position of the dye benzothiazole heterocycle could increase its fluorescent response on 

the aggregated α-synuclein. Such fluorescence intensity enhancement in fibrillar ASN presence could be 

explained with incorporation into the dye molecule of amino group, which is known to enhance 

dye/amyloid fibril complex stability. 

For the most efficient 6-diethilaminosubstituted dye T-284 the constant of binding (Kb = 1,78 µM) to the 

ASN fibrils was estimated. 

These studies present new class of amyloid specific fluorescent dyes for application in selective fluorescent 

detection of aggregated α-synuclein. 

References: [1] M.R.H. Krebs et al., J. Struct. Biol. 149 (2005) 30. [2] J.F. Kelly, Curr. Opin. Struct. Biol. 6 (1996) 11. 

167


PRLS-50 

4-Oxo-4,6,7,8-tetrahydropyrrolo[1,2-a]thieno[2,3-d]pyrimidinestyrylcyanines - 

novel fluorescent dyes for TPE detection of RNA 

Analoiy O. Balanda, Vladyslava B. Kovalska, Mykhaylo Yu. Losytskyy, Kateryna D. 

Volkova, Valentyna P. Tokar 1 , Vadym M. Prokopets 1 and Sergiy M. Yarmoluk 

Institute of Molecular Biology and Genetics, NASci of Ukraine, 03143 Kyiv, Ukraine. 


1 Physic Department of Kyiv Taras Shevchenko National University (Ukraine) 

Recently interest in the design and characterization of fluorescent compounds with potentially high twophoton 

absorption (TPA) cross-section has been increased because of their demonstrated application in a 

number of multidisciplinary areas, particularly in the rapidly developing fields of multiphoton fluorescence 

imaging. Previously series of benzothiazole styrylcyanines were synthesized and described as DNA 

sensitive fluorescent probes with high TPA cross-section values. [1] 

As continuation of these studies we firstly synthesized a series of fluorescent dyes based on 4-oxo-4,6,7,8- 

tetrahydropyrrolo[1,2-a]thieno[2,3-d]pyrimidine heterocycle (see Figure).These dyes were obtained using 

modified method of styrylcyanines synthesis. [2] 

Spectral-luminescent properties of novel dyes were evaluated in presence of nucleic acids. It was shown 

that dyes with aliphatic substituents in 2 and 3 positions of the heterocycle demonstrated RNA-binding 

preference. For the studied dyes in RNA-containing solutions excitation and emission maxima were placed 

correspondingly in the range 542-547 nm and 587-593 nm. For the developed dyes in RNA presence, 

fluorescence spectra after two-photon excitation (TPE) by 1064 nm radiation of YAG:Nd 3+ 15 ns pulsed 

laser were obtained. 

Fluorescence intensity, a.u. 

1.0 

0.8 

0.6 

0.4 

0.2 

Stp-3 in RNA presence, TPE 

Rhodamine 6G in ethanol, TPE 

Stp-3 in RNA presence, SPE 

0.0 

500 550 600 650 700 


decreased in 13 times 

Among studies styrylcyanines Stp-3 dye demonstrates the highest emission intensity increase (up to two 

orders) in RNA presence. Fluorescence spectra of styryl Stp-3 in RNA presence upon SPE (single photon 

excitation) at 532 nm and TPE, compared with this of Rhodamine 6G upon TPE are presented in Figure. 

The studied styrylcyanines in RNA complexes demonstrate medium values of TPA cross-section values, 

which are up to 0.7×10 -50 cm 4 s. 

Thus described dyes could be proposed as fluorescent probes for RNA detection upon TPE. 

This work was supported by the Science and Technology Center in Ukraine (STCU) grant #U3104k 

References: [1] V.P. Tokar et. al., J. Fluoresc. 16 (2006) 783. [2] A.O. Balanda et. al., Dyes Pigments (in press). 

R2 

R1 

S 

S 

I 

I 

O 

N 

O 

N 

+ 

R3 

N 

N 

R1,R2 = Alk, Ar; R3 = Alk. 

+ 

Stp-3 

N 

N 

168


PRLS-51 

Highly selective turn-on fluorescent chemodosimeters for Cu(II) in 

aqueous solutions 

Ai-Fang Li, Yi-Bin Ruan and Yun-Bao Jiang* 

Department of Chemistry, College of Chemistry and Chemical Engineering, and the MOE Key Laboratory 

of Analytical Sciences, Xiamen University, Xiamen 361005 (China). 

E-mail: ybjiang@xmu.edu.cn 

An ideal chemosensor shall exhibit a unique selectivity for a specific analyte. Chemodosimeters that probe 

analytes via highly selective chemical reactions induced by the analytes fulfill this requirement. Transition 

metal ion Cu 2+ plays an important biological as it participates in a variety of fundamental physiological 

processes in organisms and in enzyme-catalyzed reactions. Only few fluorescent chemodosimeters for Cu 2+ 

were exploited with enhanced fluorescence signal, which were based on Cu 2+ promoted hydrolysis and 

rearrangement reactions. N-Acylhydrazones have been widely employed in inorganic, organic, and 

analytical chemistry, mainly in terms of metal ligands. We previously found that the charge transfer dual 

fluorescent N-(p-dimethylaminobenzoyl)hydrazone showed in CH 3 CN a highly selective fluorescent 

response for Cu 2+ , despite similar absorption spectral variations being also observed with other metal ions 

such as Ni 2+ , Cu 2+ , Zn 2+ , Cd 2+ , Hg 2+ , and Pb 2+ . This suggested that N-acylhydroazone in this case 

functioned not only as a ligand. We thus extended our investigations by removing the excited-state charge 

transfer reaction channel and designed a variety of N-benzoylhydrazones. Highly selective fluorescent 

response for Cu 2+ was again observed in both CH 3 CN and CH 3 CN-H 2 O solutions, with unexpected 

structured emission, see for example the emission spectra of 1 in the presence of Cu 2+ in CH 3 CN-H 2 O. 

Detailed experiments established that Cu 2+ promoted the oxidative cyclization of the originally 

nonfluorescent N-acylhydrazones into highly fluorescent 1,3,4-oxadazoles (Figure 1). N-acylhydrazones 

were therefore shown to be a new kind of turn-on fluorescent chemodosimeters for Cu 2+ . 

CH 3 CH 2 O 

O 

N 

H 

N 

1 

O 

4 

Fluorescence intensity, a.u. 

3 

2 

1 

0 

Cu(ClO 4 ) 2 /CH 3 CN 

[ Cu 2+ ], µmol L !1 

CH 3 CH 2 O 

350 400 450 500 0 40 80 120 160 

Wavelength, nm [Cu 2+ ], µmol L !1 

Figure 1. Fluorescence spectra of 1 (10 µM) in a mixture of CH 3 CN and Tris-HCl aqueous buffer solution 

(20/80, v/v) in the presence of increasing concentration of Cu(ClO 4 ) 2 . Excitation wavelength was 283 nm. 

Under optimal conditions, 1 was found applicable for highly selective and sensitive determination of Cu 2+ 

in aqueous solutions over 1.0×10 -6 - 1.6×10 -4 M with a detection limit of 0.30µM (Figure 1). Other metal 

ions such as Co 2+ , Ni 2+ , Zn 2+ , Cd 2+ , Hg 2+ , Mg 2+ , Ca 2+ , and Ba 2+ were checked to show no interference on the 

fluorescent sensing of Cu 2+ . 

References: [1] R. Krämer, Angew. Chem., Int. Ed., 37 (1998) 772-773. [2] Z. C. Wen, et al., Chem. Commun., 

(2006) 106-108. 

160 

140 

120 

3.0 

2.0 

1.0 

0.0 

80 

70 

60 

50 

40 

30 

20 

10 

0 

I / I 0 

N 

O 

N 

O 

169


PRLS-52 

Metal ion-quenched fluorogenic oligonucleotide probes 

Roland Krämer 

University of Heidelberg, Institute of Inorganic Chemistry, 

D-69120 Heidelberg (Germany). E-mail: roland.kraemer@urz.uni-heidelberg.de 

Oligonucleotide probes that fluoresce upon hybridization (“molecular beacons”) have been first described 

in 1996 by Tyagi and Kramer and provide a powerful tool for diagnostic assays related to nucleic acid 

sequence detection. They posses a self-complementary stem-loop structure and are terminally modified 

with a fluorophore and organic quencher molecule, respectively. We have recently introduced labile 

transition metal complexes of polypyridyl ligands as a new type of highly effective, intramolecular 

fluorescence quenchers in oligonucleotide probes. [1] These probes enabled for the first time the direct 

monitoring of the formation and dissociation of single metal complexes using single-molecule fluorescence 

spectroscopy. [2] 

Schematic representation 

of a metal ion (M 

= Cu 2+ , Zn 2+ ) quenched 

oligonucleotide probe 

which fluoresces upon 

hybridization with a 

complementary target 

DNA sequence. The 

strength of metalfluorophore 

interaction 

is tunable by incorporation 

of chelating 

groups into the fluorophore. 

metal ion-quenched 

oligonucleotide probe 

Metal ions do not only have a dramatic effect on fluorescence of the probes but also on their biological 

acivity. We have monitored by flow cytometry and confocal laser scanning microscopy that cellular uptake 

and control of gene expression by a usually membrane impermeable PNA probe (PNA = peptide nucleic 

acid, a biostable DNA analog with a polyamide backbone) is triggered by Zn 2+ ions. [3] This might open 

new perspectives to the selective systemic delivery of oligonucleotide probes or drugs, since zinc 

distribution is highly cell-type and disease specific. Our ongoing studies are directed toward a better 

understanding of the relationship between extra/intracellular zinc binding and biological activity of the 

probe. The possibility of tuning the interaction strength of fluorophore and metal ion-quencher by attaching 

chelators to the fluorophore is advantageous for the design of “stem-free” probes, the control of metal ion 

affinity and the minimization of background fluorescence. 

References: [1] J. Brunner, R. Krämer, J. Am. Chem. Soc. 126 (2004) 13626-13627. [2] A. Kiel et al., Angew. Chem. 

Int. Ed. 46 (2007) in press (publ. online 2 Apr 2007). [3] A. Fuessl et al. J. Am. Chem. Soc. 128 (2006) 5986-5987. 

170


PRLS-53 

Selective, in vivo protein labeling with extracellularly adminstered, luminescent 

lanthanide complexes 

Nivriti Gahlaut, Harsha Rajapakse, Lawrence W. Miller 

University of Illinois at Chicago, Department of Chemistry, 845 W. Taylor St., MC111 

Chicago, IL 60607 (USA). E-mail: lwm2006@uic.edu 

The large Stokes shifts (>150 nm), long luminescent lifetimes (up to ca. msec) and high photostability of 

organic chromophore-sensitized, coordination complexes of lanthanide ions (particularly terbium and 

europium) allow for time-resolved spectroscopic or microscopic detection with high signal-to-background 

ratio. Thus, lanthanide complexes have been extensively developed as luminescent probes for in vitro 

bioassays and, to a much more limited extent, as cellular imaging agents. [1] Versatile cellular imaging 

probes must diffuse readily into cells from culture medium, partition only to the desired sub-cellular 

compartment, organelle or protein target of interest, and be easily detected using fluorescence microscopy. 

Individual proteins can be selectively labeled with cell-permeable luminescent probes using one of a 

number of ligand-receptor protein labeling schemes. [2] One such labeling scheme leverages the strong (K D 

= ~10 nM), orthogonal non-covalent interaction between the antifolate trimethoprim (TMP) and Esherichia 

coli dihydrofolate reductase (eDHFR). [3,4] We have prepared a series of TMP-linked lanthanide complexes, 

and have shown microscopically that these molecules diffuse into Chinese Hamster Ovary (CHO) cells and 

bind selectively to overexpressed fusions of recombinant eDHFR. 

TMP and carbostyril 124 

(cs124) were linked to 

diethylene triamine pentaacetic 

O 

acid (DTPA), triethylene 

N 

tetraamine hexaacetic acid 

H 

(TTHA), and 1,4,7,10-tetra- 

H 

azacyclododecane- N, N', N’, 

N 

N’’’ tetraacetic acid (DOTA) 

(see figure). When complexed 

O 

with trivalent terbium, the 

probes display characteristic 

lanthanide luminescence and 

have overall charges of 0, +1, 

O 

NH 

and –1, respectively. Incubation 

of CHO cells that overexpress 

nucleus-localized eDHFR with 

10-100 µM complex in cell 

O 

growth medium allowed for 

epi-fluorescence imaging of 

nucleus luminescence 

(excitation 340-360 nm, emission 405 nm LP). 

N 

H 

cs124 

O 

NCH 2 COO - n 

O 

O 

O 

N H 

COO - N 

O 

N 

N 

N 

HN 

COO - 

n = 3; cs124-DTPA-TMP 

n = 4; cs124-TTHA-TMP 

H 3 CO 

O 

OCH 3 

cs124-DOTA-TMP 

NH 2 N 

N 

NH 2 

TMP 

H O 

3 CO 

O 

O 

N O 

H 

OCH 3 

NH 2 N 

N 

NH 2 

Our results are the first example of microscopic detection of a recombinant fusion protein labeled with an 

extracellularly administered lanthanide complex in living cells. We used conventional fluorescence 

microscopy to detect Tb complex-labeled eDHFR in living mammalian cells. We anticipate that timeresolved 

microscopy will allow for detection of lanthanide-labeled proteins with extremely high signal-tobackground 

ratio. These studies will allow us to chemically optimize lanthanide complex structure to 

enhance cell permeability and target specificity, enabling, for example, the use of lanthanide complex 

protein labels as long-lifetime luminescent donors in resonant energy transfer studies of protein-protein 

interactions. 

References: [1] S. Pandya, et. al., Dalton Trans. (2006) 2757. [2] L.W. Miller, V.W. Cornish Curr. Opinion Chem. 

Biol. 9 (2005) 56. [3] L.W. Miller, et. al., Nat. Methods 2 (2005), 255. [4] N.T. Calloway, et. al., ChemBioChem 8 

(2007), 767. 

171


PRLS-54 

Labeling lipids with non-polar fluorescent markers 

A.Ulises Acuña, 1 Valentín Hornillos, 1 Javier Delgado, 1 LauraTormo, 1 Francisco Amat- 

Guerri 2 

1 

Department of Biophysics, Institute of Physical Chemistry, C.S.I.C., 28006-Madrid (Spain), 

2 

Department of Organic Synthesis, Institute of Organic Chemistry, C.S.I.C., 28006-Madrid (Spain) 

E-mail: roculises@iqfr.csic.es 

Lipid molecules intervene in a myriad of signaling and regulating cellular processes. In addition, nonnatural 

lipids have been developed with useful pharmacological properties, as e.g. antineoplastic, 

antiparasite or immuno–regulatory activity. Important details of these complex lipid functions may be 

obtained by the current highly sensitive methods of fluorescence (micro) spectroscopy, with increased 

space and time resolution. The crucial issue in this application is, of course, the lack of fully competent 

fluorescent lipid analogs. Lipids are relatively small molecules, with extended linear conformation and 

well-defined amphipathic properties; as a result of that the incorporation of bulky fluorescent groups with 

different electron density distribution and conformation often fails to produce a bioactive analog. 

We are presenting here a strategy of incorporation of non-polar fluorescent markers to lipids, by which the 

biological properties of the parent molecule may be preserved. Since the number and spatial location of the 

ionizable groups of the polar head are frequently essential for the process of lipid recognition, the original 

hydrophilic part of the molecule is conserved. Instead, non-polar fluorescent tags were developed to be 

inserted into the lipid alkyl chains, trying to reproduce as much as possible the original extended 

conformation and chemical properties. Phospholipids labeled with fluorescent phenylpolyene [1] (PTE), 

phenylpolyenyne [1,2] (PTRI), diphenylpolyene (DPH) and borondipyrromethene (BODIPY) groups obtained 

in this way are presented. These emitting groups are characterized by different spectroscopic properties and 

photochemical stability, which determine the type of fluorescence technique most appropriate for their 

application. 

X 

PTE 

X 

R 2 

N B 

N 

F 

F 

R 1 

PTRI 

DPH 

BODIPY 

Acknowledgments. Work supported by the Spanish MEyC (Project BQU2003/4413), MSyC (Project PI061125) and 

CSIC Grant 200680F0171. 

References: [1] Saugar, J.M. et al. (2007), submitted. [2] Quesada, E. et al., Eur. J. Org. Chem. (2007) 2285-2295. 

172

Part IV 

Fluorescence Correlation 

and Single Molecule 

Spectroscopy 

173

174

Abstracts Poster – Part IV: Fluorescence Correlation and Single Molecule Spectroscopy 

FCSM-1 

CMOS: a promising technology for future single photon detection applications 

A.Rochas, A.Pauchard, L.Monat, O.Guinnard, L. Widmer, and G.Ribordy 

id Quantique SA, Chemin de la Marbrerie 3, 1227 Carouge/Geneva, Switzerland 

E-mail : leonard.widmer@idquantique.com 

In the past several years, the detection of single photons at visible wavelengths has received a growing 

interest from academic researchers and industrial companies in various fields. During a long period of time, 

the PMTs (PhotoMultiplier Tubes) optimized for single photon detection has been the unique commercial 

solution. In the early 1990s, a product based on a silicon APD (Avalanche PhotoDiode) combined with an 

active quenching electronic circuit has been successfully introduced on the market. This product provided a 

very high detection probability which peaks in the red at more than 60%. However, it suffers from a poor 

timing resolution and the technology is not scalable in arrays. Scientists have optimized the timing 

resolution using avalanche photodiodes exhibiting thinner depletion regions. The processes are claimed 

CMOS (Complementary Metal Oxide Semiconductor) compatible [1][2], which means that the photodiode 

can be fabricated on a CMOS production line. However, the co-integration of electronic circuits on the 

same chip has not yet been achieved: it would required a huge effort comparable to the effort developed by 

a silicon foundry to propose a new process to customers. 

In this paper, we use a well-established CMOS process as a starting point [3] to produce single photon 

detectors. The process is qualified for automotive applications, which is a guarantee of high material quality 

and long-term stability. In the Geiger mode, the electric field in the depletion region is deliberately high, 

peaking at several hundreds of kV/cm, providing a sufficient internal gain to operate without any external 

electronic amplification circuit. The cost to pay for such a high gain is the requirement of a quenching 

circuit to lower the bias voltage close to the breakdown level and stop the avalanche. The id100 module 

family and id101 OEM family are fully integrated optical microsystem, which combines an avalanche 

photodiode and the electronic circuit for the quenching and recharging of the diode. 

In biology, fluorescence lifetime measurements, fluorescence correlation spectroscopy, total internal 

reflection fluorescence, fluorescence energy transfer and time-correlated single photon counting for single 

molecule detection would benefit from a high count rate, best-in-class timing resolution, small IRF shift at 

high count rates. 

id100 module family id101 OEM family 

References: [1] J. C. Jackson, J. Donnelly, B. O'Neill, A-M. Kelleher, G. Healy, A. P. Morrison and A. Mathewson, 

Integrated Bulk/SOI APD Sensor: Bulk Substrate Inspection with Geiger-Mode Avalanche Photodiodes, Electronics 

Letters, vol. 39, no. 9, pp.735-736, May 2003. [2] S. Cova, M. Ghioni, A. Lotito, I. Rech, and F. Zappa, Evolution 

and prospects for single-photon avalanche diodes and quenching circuits, Journal of Modern Optics, vol. 51, pp.1267- 

1288, 2004. [3] A. Rochas, M. Gani, B. Furrer, G. Ribordy, P.A. Besse, N. Gisin, and R.S. Popovic, Single photon 

detector fabricated in a complementary metal-oxide-semiconductor high-voltage technology, Review of Scientific 

Instruments, vol.74, n°7, pp.3263-3270, 2003. 

175


FCSM-2 

Probing metal complexes on the single molecule level 

Alexander Kiel 1 , Janos Kovacs 2 , Andrij Mokhir 2 , Roland Krämer 2 , Dirk-Peter Herten 1 

1 University of Heidelberg, Institute of Physical Chemistry, Im Neuenheimer Feld 253, 

D-69120 Heidelberg (Germany). E-mail: dirk-peter.herten@urz.uni-hd.de 

2 University of Heidelberg, Institute of Inorganic Chemistry, Im Neuenheimer Feld 270, 

D-69120 Heidelberg (Germany). 

Single molecule spectroscopy found widespread application in studying structures and dynamics of 

complexe biological systems, like enzymes or protein/protein interaction. Only few experiments in purely 

chemical systems, like polymers or catalytic reactions on crystal surfaces have been published up to date [1- 

3]. The focus on biological applications is due to the superior ability of single molecule methods to directly 

observe dynamic processes even in thermodynamic equlibrium and to resolve the static and dynamic 

heterogeneities of complex molecular systems. On the other hand some chemical systems like catalytically 

active metal-organic compounds are not yet fully understood as the direct observation of certain 

intermediate states remains hidden in the observation of the ensemble characteristics. An essential 

prerequisite for application of single molecule fluorescence spectroscopy (SMFS) to study any molecular 

dynamics, like association, dissociation or conformational changes, is the connection of the molecular event 

to a change in the characteristics of the fluorescence emission of an associated fluorescent label. To this end 

we are currently working on the development of fluorescent probes that able to indicate changes in 

transition-metal complexes by altering fluorescence intensity, lifetime or emission spectrum of covalently 

attached fluorescent dyes [4]. As a first step towards the investigation of transition-metal complexes by 

SMFS we started with the development of fluorscent ligand systems that are able to probe the presence of 

certain metal ions, like Cu 2+ or Ni 2+ . The probes are based on the 

rigid scaffold of a short double stranded DNA fragment (see 

figure) that is labeled with the fluorescent dye TMR on the 3’- 

end of oligonucleotide B and the bidentate ligand 2,2’- 

bipyridene-4,4’-dicarboxyacid on the 5’-end of oligonucleotide 

A. Fluorescence quenching by various metal ions was 

characterized using different spectroscopic methods, e.g. Stern- 

Volmer plots and fluorescence lifetime measurements, proving 

that TMR is specifically quenched by formation of transitionmetal 

complexes of Cu 2+ or Ni 2+ with the attached ligand. For 

SMFS studies we are using a confocal setup with a small 

observation volume in the order of ~10 -15 l (femtoliter). As the 

average residence time of an oligonucleotide in the observation 

volume is limited by diffusion to only a few miliseconds we 

attached a biotin to the 5’-end of oligonucleotide B for 

immobilization of the probe on strepavidin coated surfaces. This 

allows us to determine the positions of individual probes by 

microscopic imaging and to subsequently record the timeresolved 

fluorescence emission of a single probe. The 

measurements show stochastic fluctuations in the fluorescence emission between a high fluorescent (on) 

and a low fluorescent state (off) that are associated with individual binding events of the metal ion to the 

ligand. Statistical analysis of the duration of the on/off-states was used to determine the 

association/dissociation kinetics of the respective copper(II)-complex in thermodynamic equilibrium. With 

these experiments we demonstrated for the first time the applicability of methods from SMFS to molecular 

dynamics of metal complexes in homogenous solution. 

We gratefully acknowledge the financial support by the Deutsche Forschungsgemeinschaft (DFG, SFB 623). 

References: [1] D.A. Vanden Bout et al. Science 1997, 277, 1074-1077. [2] J.L. Young, et al. Chem. Phys. Chem. 

2005, 6, 2404-2409. [3] H. Uji-I et al. Polymer 2006, 47, 2511-2518. [4] A.Kiel et al. Angew. Chem. Intl. Ed. 2007, 

46, 3363-3366. 

176


FCSM-3 

Photokinetics of BODIPY dyes with a high triplet population 

Babette Hinkeldey, Alexander Schmitt, Gregor Jung 

University of Saarbrücken, Biophysical Chemistry, D-66123 Saarbrücken (Germany). 

E-mail: b.hinkeldey@mx.uni-saarland.de 

Fluorescence Correlation Spectroscopy (FCS) has become a well established method to investigate the 

photokinetics of fluorescent dyes. By applying this technique, different photophysical parameters, such as 

rate constants for intersystemcrossing or singlet/triplet state populations are extracted. 

In order to obtain a correlation between photophysical properties and chemical structure, different BODIPY 

(bordipyrromethene) dyes are synthezised and characterized by UV-Vis and fluorescence spectroscopy, 

FLIM and FCS. 

In our contribution we present a BODIPY dye with a high triplet population but no visible photobleaching, 

represented by a constant diffusion time at increasing excitation intensity. 

177


FCSM-4 

Monitoring protein stability in fluorescence correlation spectroscopy 

Dianwen Zhang, Aufried Lenferink, Ine Segers-Nolten, Vinod Subramaniam, Cees Otto 

MESA+ Institute for Nanotechnology, Biophysical Engineering Group, University of Twente, 

(the Netherlands). E-mail: d.w.zhang@tnw.utwente.nl 

Proteins are generally not stable any longer when they are highly diluted to very low concentration (e.g. 

nM), and thus the performance of fluorescence spectroscopy on single protein molecule may be strongly 

affected. However, Circular dichroism spectroscopy and calorimetry, et. al. common techniques for the 

study of protein stability, cannot work at such a low concentration protein solution. We developed the 

fluorescence correlation spectroscopy technology based on a confocal laser scanning confocal microscope 

to achieve the real-time and fast diffusion parameter determination of fluorescence or fluorescence labelled 

molecule in a time of the order of second. This technology has been directly used to monitor the protein 

dynamics in aqueous solution at similar single molecule level for the study of kinetic stability and 

biological function stability of fluorescence proteins in aqueous solution. This work manifests that high 

diluted proteins (~nM concentration) in aqueous solution can be excellently stable in the presence of 

detergent in room temperature. The present results provide valuable new insights into the contribution of 

forces governing protein stability. 

178


FCSM-5 

Zero-mode waveguides: a powerful tool for single-molecule optical studies 

Aurélien Crut 1 , Daniel A. Koster 1 , Zhuangxiong Huang 1 , Jue Lin 2 , Elizabeth H. Blackburn 2 , 

and Nynke H. Dekker 1 

1 

Delft University of Technology, Kavli Institute of Nanoscience, Lorentzweg 1, 2628 CJ Delft, 

The Netherlands. 

2 

University of California, San Francisco, Biochemistry and Biophysics, Box 2200, San Francisco, 

CA 94143-2200, USA. Email: huang@mb.tudelft.nl 

Fluorescence approaches for studying the dynamics of single molecules are based on the detection of 

individual, fluorescently-labelled molecules in an “observation volume”, often produced by a diffractionlimited 

laser spot. In such a case the observation volume has a typical size of ~0.1-1 femtoliter, which limits 

the maximal working concentrations of fluorescent molecules to the nanomolar range. However, many 

biologically relevant processes, such as the incorporation of nucleotides by polymerases, require at 

micromolar concentrations. Therefore, their proper study via single-molecule methods requires a 1000-fold 

decrease of the observation volume size. 

Zero-mode waveguides (ZMW) provide an elegant solution to 

this problem [1]. The principle of ZMWs is based on the 

creation of a small hole in a metal cladding on a microscope 

coverslip. Such a metal-clad waveguide exhibits a cut-off 

wavelength above which no propagating mode can exist inside 

the waveguide. Illuminating ZMWs with light of a wavelength 

larger than the cut-off wavelength results in an evanescent 

field, i.e. light intensity decays exponentially along the length 

of waveguide. In this way, the observation volume can be 

reduced by about three orders of magnitude, down to the 

zeptoliter (10 -21 L) range. We have been able to fabricate 

ZMWs as small as ~100nm in diameter (figure at right). In 

addition, numerical simulations of the optical properties of 

ZMW have been carried out to understand their optical 

properties, aiming to optimize the geometric design of the 

ZMW. 

For biological applications, an efficient surface coating in ZMWs is required to prevent non-specific 

adsorption of biomolecules on the waveguide surfaces. We have succeeded in reproducibly coating our 

ZMWs with PEG, which we will use for single-molecule studies of the kinetics of DNA polymerization by 

polymerase or telomerase. We will demonstrate our ability to detect polymerase activity with surfacetethered 

DNA templates in PEG-coated ZMWs. 

Reference: [1] M. J. Levene, et al., Science 299 (2003): 682. 

179


FCSM-6 

An ensemble and single molecule evaluation of functionalisable and water 

soluble substituted naphthalene diimides (SANDIs) as novel fluorescent labels 

Toby D. M. Bell, 1 Chintan Jani, 2 Steven J. Langford 2 and Kenneth P. Ghiggino 1 

1 School of Chemistry and Bio21 Institute, The University of Melbourne, Parkville, Victoria 3010 

(Australia). E-mail: tbell@unimelb.edu.au 

2 School of Chemistry, Monash University, Clayton, Victoria 3800, Australia. 

There is an ongoing need for new and improved fluorophores for labelling applications in the rapidly 

expanding field of single molecule (SM) detection and imaging, with water soluble labels being particularly 

sought after for use in biological and biochemical systems. The key requirements for such fluorophores are 

absorption and emission in the visible spectrum (or NIR), brightness (high quantum yield of fluorescence) 

and photo-stability (low quantum yield of photo-bleaching). Other desirable properties include sensitivity 

of the emission to the local environment and ease of functionalisation for ready incorporation into the target 

system. We report results from an ensemble and single molecule spectroscopic evaluation of four new 

substituted alkylamino naphthalene diimide (SANDI) compounds, two of which are water soluble. 

Structures of the disubstituted 

SANDI compounds 

studied in this 

work. Mono-substituted 

versions were also studied 

in which one alkyamino 

side group on the 

naphthalene core is 

replaced by a hydrogen 

atom. 

The compounds meet the requirements listed above well. The allyl versions are highly photo-stable and 

emit strongly (fluorescence quantum yields > 0.5) in the visible spectrum.[1] The water soluble systems 

show high QYs (> 0.7) in a number of solvents and QY > 0.1 in water. Furthermore, the emission of these 

compounds is sensitive to the nature and number of substituents attached to the aromatic core, and to the 

surrounding environment. For example, in toluene as solvent, the mono-substituted allyl SANDI compound 

emits at 510 nm, whereas the di-substituted allyl system emits at 630 nm. The compounds also display 

relatively long fluorescence decay times in the range of ~5 – 15 ns. 

Single molecules of the di-allyl SANDI embedded in poly(methyl methacrylate) films show very low yields 

of photobleaching and very few fluorescence intermittencies or “blinks”. These properties make these 

systems ideal candidates for use at the SM level, for example, as FRET labels. The Förster critical transfer 

distance for resonance energy transfer between the two allyl SANDIs was determined to be 41 Å, ideal for 

FRET studies in the 2-8 nm range. It is proposed that rotation of the substituent(s) attached directly at the 

NDI core is important in determining the emission characteristics of these SANDI molecules. 

Reference: [1] T. D. M. Bell, et al., Proc. SPIE, 6444 (2007) 644404/1. 

180


FCSM-7 

Single molecule fluorescence studies of the interaction of transcriptional 

regulators of metabolic genes with their operator DNAs and modulator 

molecules 

Silvia Zorrilla a , Denis Chaix b , Emmanuel Margeat b , Carlos Alfonso c , Alvaro Ortega b , 

Pilar Lillo a , German Rivas c , Nathalie Declerck b and Catherine A. Royer b 

a Instituto de Química Física Rocasolano. CSIC. Madrid. b Centre de Biochimie Structurale. CNRS, 

INSERM, UNIV MONTPELLIER-1. c Centro de Investigaciones Biológicas. E-mail: silvia@iqfr.csic.es 

Glycolysis is one of the most important metabolic pathways in Bacillus subtilis. A key point of control of 

this crucial route is the oxidation of glyceraldehyde 3-phosphate into 1,3-diphosphoglycerate, a reaction 

catalyzed by the enzyme Gap A in the glycolytic direction and by Gap B in the gluconeogenic one [1]. The 

expression of these two enzymes is regulated at the transcriptional level by two repressors, CggR acting on 

gapA [2] and CcpN acting on gapB [3]. Additionally, CcpN is a repressor of pckA, an enzyme necessary for 

efficient gluconeogenesis from Krebs cycle intermediates. Our research seeks to determinate the physical 

parameters underlying the interaction of these newly identified repressors with their target nucleic acid 

sequences and modulator molecules, using mainly state of the art single molecule fluorescence 

spectroscopy approaches in combination with other biophysical methods. The research accomplished so far 

has allowed to propose a model for the interaction of CggR repressor with its operator DNA, and it has 

outlined the presence of two different affinity binding sites for FBP on CggR repressor, one related with an 

inductor role and another one having and unknown function[4]. Steady-state fluorescence anisotropy 

binding titrations of the tetramethylrhodamine labeled repressor in conjunction with isothermal titration 

calorimetry experiments have confirmed the presence of this high affinity site (Kd 6µM). By means of twophoton 

fluorescence correlation spectroscopy experiments we have shown that FBP interferes with CggR 

oligomerization; although two color, two photon fluorescence cross-correlation spectroscopy measurements 

conducted show that FBP binding does not indeed destroy CggR dimer. Complementary experiments lead 

to the conclusion that dimeric CggR changes its conformational dynamics and is further stabilized upon 

FBP binding. On the other hand, the interaction of CcpN with its two target DNAs has been investigated by 

two-photon fluorescence cross-correlation spectroscopy, using fluorescein labeled CcpN and Atto 647N 

labeled DNAs, in order to determine the affinity of the interactions. The stoichiometry of binding of the 

repressor to target DNAs containing the full or half of the operator binding site is currently being 

investigated by analytical ultracentrifugation, in order to be able to propose a thermodynamic model for the 

interaction. The methodological approaches included in this study have rarely been used to address 

transcriptional regulation issues and therefore, they contribute to the development of methods that could be 

of general applicability to other systems. Furthermore, these biophysical studies are contributing to the 

understanding of the function of recently identified transcriptional regulators with high level of homology 

in different gram positive bacteria, some of which are pathogenic. 

References: [1] Fillinger, S., Boschi-Muller, et al. (2000) J Biol Chem 275(19), 14031-14037. [2] Doan, T., and 

Aymerich, S. (2003) Mol Microbiol 47(6), 1709-1721. [3] Servant, P., Le Coq, D. et al. (2005) Mol Microbiol 55(5), 

1435-1451. [4] Zorrilla, S., Doan, T.et al. (2007) Biophys. J., biophysj.106.095109. 

181


FCSM-8 

Application of a genetic algorithm for the optimised analyses of time-correlated 

single-photon counting measurements 

Oleg Opanasyuk, Denys Marushchak, Lennart B.-Å. Johansson 

Umeå University, Department of Chemistry; Biophysical Chemistry, S-90187 Umeå, Sweden 

E-mail: oleg.opanasyuk@chem.umu.se 

A new method, in which a genetic algorithm (GA) was combined with Brownian dynamics and Monte 

Carlo simulations, has been developed for the analyses fluorescence depolarisation data, which are 

collected by the time-correlated single photon counting (TCSPC) technique. The fact there is a gradual need 

to use more elaborate methods in the analysis of TCSPC data is one important motivation for using GA 

optimisation. 

General questions in the analysis of TCSPC data concern the development of realistic physical models, and 

the use of powerful methods for accurately determining the relevant physical parameters. The parameters 

are considered to be linearly independent and they are varied until the best fit to the data is achieved. 

Several strategies exist for the optimisation process. The Levenberg-Marquardt algorithm is a widely used 

gradient method which, unfortunately, has proven to be overly sensitive to local minima. Hence, by using 

different initial guesses of the parameters, one may find several local solutions. In the search for the global 

minimum of χ 2 (cf. Fig. 1) an obvious but very time-consuming method would be to scan the whole 

parameter space when applying a grid. In contrast, the more sophisticated method employing the GA is not 

sensitive to the local minima and it is therefore suitable for analysing ill-behaved parameter spaces(1). 

Recently the GA was implemented in fluorescence spectroscopy (2) and applied for exploring the structure 

of non-covalent protein polymers (3). 

1,4 

! 2 

1,2 

1,0 

0 

50 

# DC 

, ° 

100 

150 

0 

50 

150 

100 

" DC 

, ° 

Figure 1. The typical layout of a GA analysis 

of fluorescence depolarisation data that were 

obtained from TCSPC experiments. The 

system studied was a non-covalent protein 

polymer. The 3-D plot displays the 

statistical χ 2 -parameter as a function of two 

parameters (α DC , β DC ), which describe the 

orientation of the fluorophore group with 

respect to the main symmetry axis of the 

polymeric structure. The α DC , β DC represents 

two out of six structural parameters 

determined. 

In the present work a GA was applied for analysis of energy migration within pairs of photophysically 

identical fluorescent groups separated at a fixed distance. The energy migration is described by the 

extended Förster theory(4). 

References: (1) P Charbonneau: Genetic Algorithms in Astronomy and Astrophysics. Astrophys. J. Suppl. Ser. 101 

(1995) 309-34. (2) JJ Fisz, M Buczkowski, MP Budzinski, P Kolenderski: Genetic algorithms optimization approach 

supported by the first-order derivative and Newton-Raphson methods: Application to fluorescence spectroscopy. 

Chem. Phys. Letters 407 (2005) 8-12. (3) D Marushchak, S Grenklo, T Johansson, R Karlsson, LB-Å Johansson: 

Fluorescence Depolarisation Studies of Filamentous Actin Analysed with a Genetic Algorithm. Biophys. J. Submitted 

(2007). (4) P Håkansson, M Isaksson, P-O Westlund, LB-Å Johansson: Extended Förster Theory for Determining 

Intraprotein Distances.1. The k2-Dynamics and Fluorophore Reorientation. J. Phys. Chem. B 108 (2004) 17243-50. 

182


FCSM-9 

Diffusion of myelin specific proteins in OLN-93 investigated by raster-scanning 

image correlation spectroscopy (RICS) 

Ellen Gielen a , Ben De Clercq b,c , Nick Smisdom b , Martin vandeVen b , Rik Gijsbers d , Zeger 

Debyser d , Yves Engelborghs a , Marcel Ameloot c 

a Laboratory for Biomolecular Dynamics, Catholic University Leuven, Belgium. 

b Laboratory for Cell Physiology, Biomedical Research Institute, Hasselt University, Belgium. 

c Eindhoven University of Technology, The Netherlands. 

d Molecular Virology and Gen Therapy, Catholic University Leuven, Belgium. 

E-mail: marcel.ameloot@uhasselt.be 

The plasma membrane of various mammalian cell types is heterogeneous in structure and may contain 

microdomains, which can impose constraints on the lateral diffusion of its constituents. These membrane 

inhomogeneities comprise the so-called lipid rafts, [1] built mainly of cholesterol and saturated lipids, and 

"corrals" made up by the membrane-associated actin cytoskeleton (fences) and by rows of transmembrane 

proteins anchored to it (pickets). [1] 

Oligodendrocytes (OLs) are the myelin-producing cells of the central nervous system. Evidence for lipid 

rafts in the OL membrane is almost exclusively based on detergent methods. [2] However, as application of a 

detergent can alter the membrane phase behaviour, [3] it is important to investigate membrane heterogeneities 

in living cells. This can be accomplished by using microfluorimetric methods for monitoring the diffusion 

of molecules in the plane of the membrane. [4] Recently we have been able to demonstrate using Z-scan 

fluorescence correlation spectroscopy (FCS) that the lipid probe DiD exhibits hindered diffusive motion in 

the plasma membrane of the OLN-93 oligodendroglial cell line. [5] In the current work we investigate the 

diffusion behavior of the Myelin Oligodendrocyte Glycoprotein (MOG). A stable OLN-93 cell line 

expressing MOG-eGFP (with eGFP linked to the intracellular C-terminus of MOG) was generated by 

means of lentiviral vector technology. 

The diffusion of MOG-eGFP in OLN-93 appears to be too slow to be monitored by conventional FCS due 

to photobleaching. Therefore, we used the recently developed RICS (raster-scanning image correlation 

spectroscopy) technique. [6] In RICS, a temporal stack of images is taken with a laser-scanning confocal 

microscope. The spatial correlation of this series of images yields information about the molecular 

dynamics on different timescales determined by the motion of the scanning laser beam, and the average 

density of the labelled protein. Data are obtained at room temperature with a Zeiss LSM 510 META onephoton 

confocal microscope with a 40x oil/NA 1.3 objective. Control measurements on FITC-dextrans and 

fluorescent beads as well as simulations are performed to validate the method and the home-made software 

for data analysis. RICS-analysis of the MOG-eGFP data yields diffusion coefficients of the order of 0.1 

µm 2 /s. The average number of MOG-eGFP molecules is a few thousand per µm 2 . As information about the 

mobile fraction of the molecules is difficult to obtain by RICS, complementary FRAP (fluorescence 

recovery after photobleaching) measurements have been performed. Values for the diffusion coefficients 

obtained via FRAP corroborate well with those obtained from RICS measurements. It appears that most of 

the MOG-eGFP proteins are mobile. 

We thank Prof. C. Richter-Landsberg (Oldenburg University, Germany) for the OLN-93 oligodendroglial cells, Dr. W. 

Baron and Prof. D. Hoekstra (University of Groningen, The Netherlands) for adapting the OLN-93 MOG-eGFP cell 

line, Prof. E. Gratton and Dr. M. Digman (University of Irvine, USA) for their help with RICS data analysis. This 

work has been supported by the Research Council of the UHasselt, transnational University of Limburg, the 

K.U.Leuven (GOA/2006/02) and the IWT (Flanders). 

References: [1] Kusumi, A., et al., Traffic. 5 (2004) 213. [2] Gielen, E., et al., Glia 54 (2006) 499. [3] Heerklotz, H., 

Biophys. J. 83 (2002) 2693. [4] Marguet, D., et al., EMBO J. 25 (2006) 3446. [5] Humpolíčková, J., et al., Biophys. J. 

91 (2006) L23. [6] Digman, M., et al., Biophys. J. 89 (2005) 1317. 

183


FCSM-10 

FCS data analysis by quantified maximum entropy method 

Jean-Claude Brochon*, Elvire Guiot*, Eric Deprez*, Stephen F. Gull + 

*LBPA, Laboratoire de Biotechnologie et Pharmacologie génétique Appliquée, C.N.R.S. UMR8113, 

Ecole Normale Supérieure de Cachan, 61 av. du Président Wilson, F94235 Cachan (France). 

E-mail: brochon@lbpa.ens-cachan.fr 

+ Cavendish Laboratory, Cambridge (U.K.) E-mail: gull@maxent.co.uk 

Fluorescence correlation spectroscopy is used to measure the lateral diffusion behaviour of macromolecules 

such as protein, nucleic acids and their state of assembly or association. In analysing the fluorescence 

intensity autocorrelation function, the maximum entropy method yields distribution of diffusion times [1, 2] 

as well as contribution of additional photophysical phenomenon to the fluorescence fluctuations. 

The quantified version of maximum entropy method, QMEM [3], yields additional inferences from the final 

distribution (posterior knowledge) such as confidence intervals, given an estimate of the uncertainty in the 

result, functional of the image with associated error bars. In a more general approach a set of posterior 

images near the optimum value can be generated showing the rather stable moieties in the image in contrast 

to large fluctuations in the other parts. So, we focus FCS analysis on quantifying the recovered parameters: 

position, amplitude and width of peaks in the distribution and their associated variances. 

Practically all the probabilities derived in the classic maximum entropy analysis are conditional on the 

choice of model, noise amplitudes or, and the definition of the kinetic functional [3]. 

A key point in FCS is the determination of the signal-to-noise ratio. Several ways of calculation of the 

standard deviations are compared in running analysis of mocked data: -1) averaging a great number of 

successive measurements of an autocorrelation curve, -2) empirical computation [4], -3) a fully analytical 

approach [5]. In addition, a simplest way supported by QMEM, if the level of noise in the data is not 

precisely determined, is to rescale automatically the standard deviations σ by a variable coefficient and 

therefore to maximize the evidence accordingly during iterations to the optimum solution. The iterations are 

not ended when χ2 does not change within an arbitrarily percentage but in QMEM analysis, an automatic 

stopping criterion is ending iterations along the maximum entropy "trajectory" leading to the best posterior 

probability distribution. 

Data samples from enzymatically labelled spumavirus integrase at increasing concentration were analysed 

in order to monitor the monomer-dimer transition. The recovered lateral time diffusion distributions are 

compared to the corresponding rotational correlation times distributions obtained by quantified maximum 

entropy method of analysis of time-resolved fluorescence anisotropy data. 

References: [1] P. Sengupta et al., Biophys. J. 84 (2003) 1977. [2] K. Modos et al., Eur. Biophys. J. 33(2004) 59. 

[3] J. Skilling in « Maximum Entropy in Action » (B. Buck and V.A. Macaulay, eds.) Oxford Press (Clarendon) 1991. 

[4] Starchev et al., J. Coll. Inter. 233 (2001) 50. [5] S. Saffarian, E. L. Elson Biophys. J. 84 (2003) 2030. 

184


FCSM-11 

Fluorescence correlation spectroscopy as a tool to analyse Na+/H+ exchangers 

in the red blood cell membrane 

Seena Koyadan Veettil, Gregor Jung, Aravind Pasula, Ingolf Bernhardt 

Universität des Saarlandes, Biophysikalische Chemie, D-66123, Saarbrücken, Germany. 

E-mail: s.veettil@mx.uni-saarland.de 

Fluorescence Correlation Spectroscopy (FCS) in combination with Fluorescence Microscopy has been used 

for measuring intermolecular diffusion in living cells. FCS uses the time averaging fluctuation analysis of 

small molecular ensembles with maximum sensitivity of statistical confidence. 

We report on a successful application to the analysis of 

single fluorescently labeled Na + /H + exchangers in the 

Red Blood Cell membrane. A Confocal laser scanning 

microscopy, specially designed for single molecule 

study is used for the experiment. Excitation source is a 

continuous argon ion laser at 488nm and a fiber laser at 

546 nm. 

A schematic diagram of Confocal set up is shown in the 

figure. 

A two component fitting model is used for extracting 

diffusion constant from the autocorrelation curve as 

follows: 

( 

G( 

) = 1 + 

& 

$ 

fi 

$ 

1 

N $ 

1 + 

% 

( 

( d 

1 

# 

! 

! + 

! 

" 

( 1 ' fi) 

N 

& 

$ 

$ 

1 

$ 

1 + 

% 

( 

( d 

2 

# 

! 

! 

! 

" 

Na + /H + exchanger is important in the regulation of intracellular pH, cell volume and initiation of cell cycle 

events by growth factors. Our aim is to investigate the molecular dynamics and fluorescent fluctuations of 

Na + /H + exchangers in the Red Blood Cell membrane. For the above purpose we choose experimental and 

control model as Bodipy Fl amiloride and octadecyl rhodamine B chloride labeled Red Blood Cells 

respectively. Using these models we are able to elucidate the diffusion constant(1.92×10 -10 cm -2 s) for 

Bodipy labeled Na + /H + exchanger in the Red Blood Cell membrane. 

References: [1] Y. Takahashi et al., Optical Rev. 6 (2003) 596. [2] P.Schwille et al.,Cytometry 36 (1999) 176. 

[3] J. Orlowski, S. Grinstein., J Biol Chem.272:36 (1997) 22373. 

185


FCSM-12 

RNA expression profiling at the single molecule level 

Jaroslaw Jacak 1 , Jan Hesse 1 , Maria Kasper 2 , Gerhard Regl 2 , Thomas Eichberger 2 , 

Fritz Aberger 2 , Max Sonnleitner 3 , Robert Schlapak 3 , Stefan Howorka 3 , Leila Muresan 4 , 

Annemarie Frischauf 2 , and Gerhard J. Schütz 1 

1 Biophysics Institute, Johannes Kepler University Linz, Altenberger Str.69, 4040 Linz, Austria; 

2 Division of Genomics, Department of Molecular Biology, University of Salzburg, Austria; 

3 Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, Scharitzerstr.6-8, 

A-4020 Linz, Austria; 

4 Department of Knowledge-based Mathematical Systems, Johannes Kepler University Linz, 

Altenberger Str.69, 4040 Linz, Austria 

We present a microarray analysis platform, which enables detection of hybridized DNA sequences at the 

level of single molecules. Fluorescence detection is performed on an ultra-sensitive biochip reader. 

Oligonucleotide microarrays were printed on custom-made aldehyde-functionalized glass coverslips 

(UAR). The platform was evaluated by hybridizing fluorescent 60mer oligonucleotide to its complementary 

sequence covalently immobilized on the biochip surface. The Dynamic Range, dependent on the unspecific 

binding of sequences on non-complementary spots, reaches 4.7 orders of magnitude. Furthermore we 

analyzed mRNA expression of HaCat cells by hybridization of reverse transcribed cDNA out of 200ng total 

RNA. 

Such wide range in detection sensitivity needs reliable methods for exact data quantification. At low 

concentration the signal of each spot was quantified by counting the molecules; additionally the brightness 

of individual molecules was estimated by fitting a 2-dimensional Gaussian function. For high 

concentrations, the number of molecules per spot was inferred from the total signal per spot. 

Good correlation with experiments on commercial microarrays using hundredfold higher sample amounts 

indicates the feasibility of this approach, which avoids application of error prone amplification methods. 

References: 1) Jaroslaw Jacak, Jan Hesse, Maria Kasper, Fritz Aberger, Annemarie Frischauf, Stefan Howorka, and 

Gerhard J.Schütz - Proc.SPIE, 5699(2005), 442-449. 2) Hesse, J.; Sonnleitner, M.; Sonnleitner, A.; Freudenthaler, G.; 

Jacak, J.; Hoglinger, O.; Schindler, H.; Schutz, G. J. - Analytical Chemistry, 76 (2004), 5960-5964. 3) J.Hesse, 

J.Jacak, M.Kasper, G.Regel, T. Eichberger, M.Wikelmayr, F. Aberger, M. Sonnleitner, R. Schlapak, S. Hovorka, 

L. Muresan, Anna-Maria Frischauf, Gerhard J. Schütz – Genomic Research., 2006, 16, 1041-45. 

186


FCSM-13 

Single-molecule detection of allophycocyanin (APC) entrapped in a silica 

sol-gel glass under physiological conditions 

Alexander M. Macmillan 1 , Jan Karolin 1 , Colin D. McGuinness 2 , Dalibor Pánek 1 , John C. 

Pickup 2 and David J. S. Birch 1 

1 Centre for Molecular Nanometrology, Department of Physics, John Anderson Building, 

University of Strathclyde, 107 Rottenrow, Glasgow G4 0NG, UK. E-mail: djs.birch@strath.ac.uk 

2 Department of Chemical Pathology, Guy’s, King’s, and St Thomas’s Hospitals School of Medicine, 

Guy’s Hospital, London SE1 9RT, UK 

Allophycocyanin (APC) is a highly fluorescent protein (quantum yield = 0.68), that belongs to the 

phycobiliprotein family found in the light-harvesting system in blue-green algae. Because of its large molar 

extinction coefficient (ε 650 = 7 x 10 5 M -1 cm -1 ); emission around 660 nm where cellular autofluorescence is 

low, and because it can be excited using standard diodes and HeNe lasers, it has found widespread use in 

both immunoassay and sensor applications [1]. 

Here we demonstrate how APC molecules can be spatially localized within nanometer sized silica cavities 

filled with water and thus be studied down to single molecule level under near physiological conditions. We 

show that the entrapment is critically dependent on the removal of methanol released by tetramethyl 

orthosilicate (TMOS) during the formation of the inorganic silica matrix [2], as well as on the pre-aging of 

the sol allowing particles to form and grow before addition of the biomolecule. We report on time-resolved 

photophysics observed in both the chromophoric phycocyanobilin groups when exited at 634 nm as well as 

on amino acid emission observed from the polypeptide backbone when excited using recently developed 

pulsed UV light emitting diodes[3,4]. 

Figure showing the emission spectra of APC in trimeric and monomeric form when encapsulated in a silica 

sol-gel pore. 

References: [1] L. J. McCartney et al. Anal. Biochem. 292 (2001) 216. [2] J.Karolin et al. Meas. Sci & Techn 13 

(2002) 21. [3] C. D. McGuinness et al. Meas. Sci. & Techn. 15 (2004) 11. [4] C. D. McGuinness et al. Appl. Phys. 

Lett. 89 (2006) 977. 

187


FCSM-14 

Two photon FLIM and FCS investigation of the intracellular oligomerization of 

HIV-1 Vpr protein 

Joëlle Fritz, Pascal Didier, Emmanuel Schaub, Hugues de Rocquigny and Yves Mély 

Institut Gilbert Laustriat, CNRS UMR 7175, Faculté de Pharmacie, Université Louis Pasteur 

67401 Illkirch (France). E-mail: yves.mely@pharma.u-strasbg.fr 

Viral Protein R (Vpr), from HIV type I virus, is a 96 amino acid protein critically involved in several 

cellular processes during the viral cycle. Indeed, the Vpr protein facilitates the entry of the HIV preintegration 

complex through the nuclear pore, induces G2 cell cycle arrest and cell apoptosis, increases 

transcription from the long terminal repeat and enhances viral replication [1]. Structure/activity relationship 

studies revealed that the N-terminal part of Vpr is involved in virion incorporation, nuclear localization and 

formation of ion channels. On the other hand, the C-terminal part is involved in the G2 cell cycle arrest, 

apoptosis and interaction with HIV-1 nucleocapsid protein and nucleic acids. The structure of Vpr solved 

by NMR in organic solvents [2, 3] is characterized by three well-defined alpha-helices surrounded by 

flexible N and C-terminal domains. Moreover, Vpr likely forms a dimer through the formation of a Leu 

zipper. Nevertheless, the structure and the oligomeric state of Vpr in the cellular context are still unknown. 

In this context, our aim was to investigate the Vpr oligomerization in a cellular context by using GFP and 

mCherry fusion proteins. These fluorescent proteins were used respectively, as a donor and acceptor in 

resonant energy transfer experiments so that by measuring the fluorescence lifetime of the donor (with and 

without acceptor), it is possible to obtain information on the interacting proteins. Time-resolved imaging 

was performed with a home-made two-photon laser scanning microscope. From energy transfer 

measurements, Vpr-Vpr interaction is shown in HeLa cells mainly at the nuclear envelop level but also in 

the cytoplasm and nucleus. The energy transfer was found to depend on the position of the fluorescent 

protein on the N or C terminus in Vpr. Deletion or substitution of amino acids putatively involved in the 

Vpr tridimensional folding elicits a large decrease in energy transfer while mutation of other residues does 

not hamper Vpr oligomerization. In addition, two photon fluorescence correlation spectroscopy (FCS) 

indicated that oligomers were heterogenous and composed of two to eight monomers. 

References: [1] A. Deniaud, C. Brenner, G. Kremer, Mitochondrion 4 (2004) 223. [2] N. Morellet et al., J. Mol. Biol., 

327 (2003) 215. [3] S. Bourguibot et al., Biochem. J. 387 (2005), 333. 

188


FCSM-15 

Elucidation of conformational changes in protein C3 during activation of the 

complement system using single molecule fluorescence 

Aike Stortelder 1,2 , Lucio Gomes 2 , Dave J. van den Heuvel 1 , Bert J. C. Janssen 2 , Piet Gros 2 , 

Hans C. Gerritsen 1 

1 Department of Molecular Biophysics, Debye Institute and 2 Department of Crystal and Structural 

Chemistry, Bijvoet Centre for Biomolecular Research, Faculty of Sciences, Utrecht University, 

P.O. Box 80000, 3508 TA Utrecht, The Netherlands. E-mail: a.stortelder@phys.uu.nl 

The complement system is a key part in the innate immune system. The complement protein C3 binds to 

pathogens to select them for elimination. Activation of this so-called alternative pathway involves 

activation of C3 by C3 convertase, leading to the active form C3b. During activation a large conformational 

change takes place and a disulfide bridge is broken. The resulting exposed sulfur group will covalently bind 

to the pathogen surface. Subsequently, a series of reactions involving binding of cofactors and 

fragmentation of C3b leads to elimination of the pathogen cell. 

In recent years, many crystal structures of the various complexes playing a role in complement have 

become available [1,2] and have led to a deeper understanding of the alternative pathway immune reaction. 

However, since the crystal structures only represent static conformations, knowledge on the dynamics of 

system becomes of great interest. Single molecule fluorescence is then a useful method to explore these 

dynamics. 

The first goal of the research is to perform single molecule FRET experiments on activated C3 (C3b) to 

assess its orientation with respect to the surface to which it binds, and to what degree it is free to move 

when bound. A second goal is to monitor the conformational changes in the structure of C3 during 

activation. Also, the kinetics and efficiency of binding of activated C3 to a surface may be studied, as well 

as dynamic and conformational properties of cofactors. 

First step in the proposed model 

for the conformational pathway of 

C3: activation and binding to 

pathogen surface 

References: [1] B.J.C. Janssen et al., Nature 437 (2005) 505. [2] B.J.C. Janssen et al., Nature 444 (2006) 213. 

189


FCSM-16 

Proton transfer along lipid membranes 

Tor Sandén*, Magnus Brändén*, Peter Brzezinski**, Jerker Widengren* 

* Royal Institute of Technology, Department of Applied Physics, Experimental Biomolecular Physics, 

Albanova University Center, Stockholm (Sweden). E-mail: tsanden@kth.se 

** Stockholm University, Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural 

Sciences, Stockholm (Sweden). 

For cells, pumped protons are believed to be largely restricted in their equilibration with the bulk solution, 

such that a direct coupling between the proton pumps and the proton consumers (e.g. ATP:ases) can exist 

along their membrane surfaces. However, although of fundamental interest, and extensively investigated, 

the mechanisms for this slow equilibration are still strongly debated. We have addressed this issue using 

fluorescence correlation spectroscopy. As a model system, we used liposomes with various compositions, 

in which only one of the lipid head groups was covalently labeled with a pH sensitive dye. The influence of 

charge and buffering capacity of the membrane on the protonation kinetics of the attached dye could be 

followed, at dye concentrations low enough not to disturb the proton exchange, and at steady-state 

protonation conditions. We show that the lipid head groups collectively act as a proton-collecting antenna, 

dramatically accelerating proton uptake from water to a membrane-anchored proton acceptor. Furthermore, 

the results show that proton transfer along the surface can be significantly faster than that between the lipid 

head groups and the surrounding water phase. Thus, ion translocation across membranes and between the 

different membrane protein components is a complex interplay between the proteins and the membrane 

itself, where the membrane acts as a proton-conducting link between membranespanning proton 

transporters. 

Reference: [1] Magnus Brändén et al., Proc. Nat. Acad. Sci. 103 (2006) 19766-19770. 

190


FCSM-17 

Study of transglutaminase mediated C-terminal TAMRA-labeled spumavirus 

integrase by fluorescence correlation spectroscopy and resonance energy 

transfer 

Olivier Delelis, Elvire Guiot, Kevin Carayon, Patrick Tauc, Jean-François Mouscadet, 

Jean-Claude Brochon & Eric Deprez 

Ecole Normale Supérieure de Cachan. CNRS UMR 8113. Laboratoire de Biotechnologies et 

pharmacologie génétique appliquée. 61 Av. Président Wilson, 94235 Cachan cedex (France). 

E-mail: deprez@lbpa.ens-cachan.fr 

We have successfully applied the specific labelling of a retroviral integrase (IN) with TAMRA by guinea 

pig transglutaminase (TGase). In contrast to chemical labelling, the TGase-mediated C-terminal 

fluorophore labelling occurs specifically on a glutamine residue of a peptide substrate (PKPQQFM), which 

was fused to IN at the C-terminal extremity. In such a case, TGase can catalyze acyl transfer reaction 

between the γ-carboyamide group of Gln and cadaverine-TAMRA molecule. This led to a specifically 

labelled IN at a unique position by a TAMRA probe. TAMRA-labeled IN was then particularly suitable for 

fluorescence correlation spectroscopy and FRET analysis concerning studies of self-association properties 

of IN as well as IN-DNA interactions. Spumavirus integrase was found to be more soluble than HIV-1 

integrase. Time-resolved fluorescence anisotropy as well as gel-chromatography revealed a monomer-dimer 

equilibrium for spumavirus unlabeled IN at micromolar concentrations whereas in this concentration range, 

only aggregates were detected for HIV-1 IN. Using labelled protein, FCS confirms the monomer-dimer for 

spumavirus IN. FCS measurements were performed under two-photon excitation on a home-built system 

using an inverted microscope. In parallel, using TAMRA-labeled IN and fluorescein-labeled DNA substrate 

of increasing sizes (from 21- to 300-mer), resonance energy transfer study of the IN-DNA substrate 

interaction was performed to get deeper insight into the positioning of IN onto DNA substrates as a 

function of DNA size. DNA substrates were fluorescein-labeled at the 5’-extremity of double-stranded 

DNA, either localized at the processed extremity or on the opposite extremity. We found that the maximum 

FRET efficiency was strongly dependent on the DNA length: for a given protein concentration, the FRET 

efficiency was higher for short DNAs. This result is opposite to the one expected from apparent affinity 

which decreases as the DNA size decreases; it suggests that the positioning of IN onto DNA is the main 

factor modulating the FRET efficiency and is compatible with polymerisation of IN onto long DNA 

substrates. Interestingly, for a given DNA size, the FRET efficiency was found systematically higher when 

the fluorescein donor was attached on the processed side of DNA for 45-, 100- and 300-mer DNA. No such 

difference was observed for the 21-mer DNA substrate. This result indicates a significant preference of IN 

binding on the processed end. This is consistent with the absence of bias for the short 21-mer DNA for 

which a differential FRET efficiency between the two extremities is not expected to occur as the overall 

size of IN is comparable with the DNA length. For the first time, using a more soluble IN, we reveal a 

specific DNA-binding of integrase. 

191


FCSM-18 

Extended Förster theory of partial donor-donor energy migration 

Nils Norlin, Per-Olof Westlund, Lennart B.-Å Johansson 

Umeå University, Department of Chemistry; Biophysical Chemistry, S-901 87 Umeå, Sweden 

E-mail: Nils.Norlin@chem.umu.se 

An Extended Förster Theory (EFT) is described for partial donor-donor energy migration between two 

chemically identical but photophysically non-identical fluorophores denoted D A and D B (cf. Fig 1) 

* * 

D A 

D B 

1/τ A 

ω ΑΒ = ω ΒΑ = ω 

1/τ B 

Figure 1. Both donor groups D A and D B are 

excited with an equal probability and the 

rates of energy transfer are equal (= ω) The 

D A and D B groups are, however, assumed to 

exhibit fluorescence lifetimes (τ A , τ B ), which 

are significantly different 

D A D B 

The D A and D B groups, which might undergo reorienting motions on the same timescale as the electronic 

energy migration, can be described by a Stochastic Master equation (SME) derived from the Stochastic 

Liouville equation. The solution to the SME give the excitation probabilities {χ A (t), χ B (t)}. Using these 

solutions one can derive expression for the observed fluorescence decay {S(t)}, as well as the fluorescence 

anisotropy {r(t)}. One obtains that 

1 p 

s 

p 

s 

S( t) 

= < ! 

A 

( t) 

+ ! 

B 

( t) 

+ ! 

B 

( t) 

+ ! 

A 

( t) 

> 

(1) 

2 

r(0) 

< " 

r( 

t) 

= 

AA 

( t) 

! 

p 

A 

( t) 

+ " 

< ! 

p 

A 

AB 

s 

( t) 

! 

s 

( t) 

+ ! 

B 

B 

( t) 

+ " 

( t) 

+ ! 

BB 

p 

B 

( t) 

! 

p 

B 

( t) 

+ " 

s 

( t) 

+ ! ( t) 

> 

A 

BA 

s 

( t) 

! 

A 

( t) 

> 

(2) 

In the Eqs. 1 and 2 the brackets () represent the average of a stochastic equation, and the superscripts 

s and p denote the secondary and primary excited donor, respectively. Moreover, ! is a shorthand 

notation for the orientational correlation function: 

[ ˆ 0 ˆ ] 

" ( t ) = P µ ( ) ! µ ( t ) N,M = A or B 

MN 2 M N 

The EFT is applied to the analyses of PDDEM data obtained from time-correlated single photon 

experiments. Synthetic data have been generated that mimics true experiments for known values on the 

lifetimes, molecular orientations, migration and reorientation rates. These data were then re-analysed by 

using EFT theory combined with a previously described simulation-deconvolution method [1,2] 

References: [1] P Håkansson, M Isaksson, P-O Westlund, LB-Å Johansson: Extended Förster Theory for 

Determining Intraprotein Distances: 1. The κ 2 - Dynamics and Fluorophore Reorientation. J Phys. Chem. B 108 (2004) 

17243-50. [2] M. Isaksson, P Hägglöf, P Håkansson, T Ny, LB-Å Johansson: Extended Förster Theory for 

Determining Intraprotein Distances: 2. An Accurate analysis of Fluorescence Depolarisation Experiments. Phys. 

Chem. Chem. Phys. (2007), Accepted. 

MN 

192


FCSM-19 

2D polarisation single molecule spectroscopy of multichromophoric systems 

Oleg Mirzov, Ralph Hania, Hongzhen Lin, Daniel Thomsson, Ivan Scheblykin 

Lund University, Department of Chemical Physics, SE-22100 Lund (Sweden). 

E-mail: oleg.mirzov@chemphys.lu.se 

We present an enhancement of single-molecule spectroscopy (SMS) with a “2-dimensional” (excitationemission) 

polarisation feature. The technique consists in measuring single-molecule fluorescence light 

polarisation as a function of the excitation light polarisation. The emission polarisation is measured by 

rotating a linearly polarising analyser in front of a detector. The excitation polarisation is linear and is 

continuously rotated in the course of the measurement. Due to these two polarisation rotations the result of 

the measurement is a 2D-plot whose axes represent the polarisation angles and the colour code represents 

fluorescence intensity (Fig.1). This technique incorporates all the information provided by previously 

reported polarisation SMS approaches (where only emission or only excitation polarisation was addressed 

— “1D”), but goes much further than that. Apart from some information on the conformation of a single 

chain, the obtained 2D-plots provide more information on the intramolecular energy transfer efficiency: the 

average “rotation” angle of an exciton transition dipole moment in the course of energy transfer is one of 

the extracted parameters. 

The technique was tested on single chains of the π-conjugated polymer poly[2-methoxy-5-(2´ethylhexyloxy)-1,4-phenylene 

vinylene] (MEH-PPV). We performed a series of measurements on SMS 

samples of MEH-PPV prepared using different solvents and polymer matrices. It was found possible to 

simulate the obtained 2D plots very well with a simple formal model representing the chain as a set of three 

dipoles with energy transfer between them. Using this model, we were able to characterise energy transfer 

efficiency numerically and present statistics of model parameters for different sample preparation recipes. 

This technique has a potential for application to any other multichromophoric systems with energy transfer 

where the latter needs to be investigated as a function of conformation. 

Fig.1. Examples of polarisation 2D plots for the cases of poor and good “rotational” energy transfer. 

193


FCSM-20 

Single molecule detection in concentrated solutions of fluorescently 

labelled nucleotides 

Martin Gaplovsky, Rita Kröschel, Stefan Seeger 

Physikalisch-Chemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich 

(Switzerland) E-mail: sseeger@pci.unizh.ch 

Detection of trace compounds in complex mixtures are often performed using fluorescence methods. The 

goal is to provide fluorescence sensing methods which are able to detect single molecules selectively in 

presence of a high concentration of labelled marker molecules [1] . However, the size of diffraction-limited 

fluorescence detection volume allows only a low concentration of labelled molecules in bulk. A 

straightforward way, to confine the detection volume to a water–glass interface is parabolic based total 

internal reflection (TIRF) for fluorescence detection [2] . A detection volume of a few attoliters can be 

obtained using the paraboloid mirror objective together with confocal optics since only molecules with a 

surface distance below 100 nm contribute to the fluorescence signal. However, single molecule detection at 

surfaces is very sensitive towards the unspecific adsorption of fluorescent species increasing the 

background signal due to molecules not relevant for the analysis. This, because it is difficult to differentiate 

between adsorbed labelled molecules and their desired interaction with the sensing molecule. To overcome 

these complications not only detection volume has to be kept as small as possible, the surface properties 

must prevent unspecific adsorption of labelled nucleotides [3] . 

Fluorescence image of the Cy5-labelled 

primers hybridised with DNA-strands 

covalently attached to the polyacrylic acid 

(PAC)/polyethylene imine (PEI) 

functionalised surface in an aqueous 

solution pH 7.5 of 10 -7 M Cy5-dUTP. 

10 µ m 

300 350 400 450 500 

Counts 

We demonstrate that consecutively adsorbed PEI/PAC multilayer surface coating has the ability to repel 

Cy5-labelled nucleotides and suppress their unspecific binding to the surface. DNA molecules hybridised 

with Cy5 labelled primer were anchored to the PEI/PAC surface through the reaction of C6 linked amino 

group with carboxy group of polyacrylic acid. The ability of the scanning paraboloid TIRF microscope 

setup to detect single molecules in presence of concentrated solution of fluorescently labelled nucleotides as 

high as 10 -7 M is shown for the first time. 

References: [1] V. Cornish Peter, T. Ha, ACS Chem. Biol. 2 (2007) 53. [2] T. Ruckstuhl, S. Seeger, Opt Lett 29 

(2004) 569. [3] A. Krieg, T. Ruckstuhl, S. Seeger, Anal. Biochem. 349 (2006) 181. 

194


FCSM-21 

Quantitative fluorescence correlation spectroscopy 

B. Krämer, S. Rüttinger, F. Koberling, B. Ewers, V. Buschmann, U. Ortmann, M. Patting, 

M. Wahl and R. Erdmann 

PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany 

Fluorescence Correlation Spectroscopy (FCS) is used to determine concentrations and diffusion constants 

in the pico- to nano-Molar region with broad applications in Biology and Chemistry. However, the method 

is correlated to a broad range of measurement parameters and other factors as background contributions 

which make quantitative results very often difficult to obtain. In addition quantitative results rely on the size 

of the confocal volume which has to be determined experimentally. The confocal volume is difficult to 

measure in situ and is sensitive to saturation of the dye molecules, optical aberrations and variations of the 

index of refraction as observed in biological specimen. 

In the first part we will present three methods for the determination of the confocal parameters for standard 

FCS and compare the results: Starting with a dilution series of a sample has the advantage to be applicable 

to any dye with known concentration. The second method based on a known diffusion coefficient applies 

FCS curve fitting without having the need to determine the sample concentration exactly. The third method 

measures directly the confocal volume via raster scanning of a sub-resolution fluorescent bead with high 

precision. The effective confocal volume could be determined with all methods with an uncertainty of 10% 

[1]. 

To increase the precision even further we apply dual focus FCS (2fFCS) [2], a technique which relies on the 

pre-determined distance of two laser foci acting as a ruler for diffusion time determination. The two foci 

can be realized and individually addressed by using perpendicular pulsed interleaved excitation (PIE) [3] in 

combination with a Nomarski prism. In this case there is no necessity to have prior information about the 

size and shape of the confocal volume. We show the implementation of this technique into the 

MicroTime 200 [3] confocal microscope, first results and applications. 

The pulsed excitation allows in addition to discriminate dyes as well as artifacts not only by their spectral 

properties but also via the fluorescence lifetime. Fluorescence lifetime correlation spectroscopy (FLCS) [4] 

enables therefore not only for a very efficient detector afterpulsing removal but also for the possibility to 

carry out cross-correlation measurements (FCCS) between different dyes without the need to overlap two 

excitation volumes spatially. 

References: [1] S. Rüttinger, R. Macdonald, B. Kraemer, F. Koberling, M. Roos, E. Hildt, Journal of Biomedical 

Optics Vol.11, 2, (2006), 024012 [2] T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, 

J. Enderlein, ChemPhysChem, Vol. 8, 3, (2007) 

[3] http://www.picoquant.com/products/microtime200/microtime200.htm 

[4] M. Böhmer, M. Wahl, H.-J. Rahn, R. Erdmann, J. Enderlein, “Time-resolved fluorescence correlation 

spectroscopy“, Chemical Physics Letters Vol. 353, (2002), 439–445 

195


FCSM-22 

Translational and rotational motions of albumin sensed by a non-covalent 

associated porphyrin: A fluorescence correlation spectroscopy and time 

resolved anisotropy study 

Suzana M. Andrade a , Silvia M.B. Costa a , Jan Willem Borst b , Arie van Hoek b , 

Antonie J.W.G.Visser b 

a Centro de Química Estrutural, Complexo 1, Instituto Superior Técnico, 1049-001 Lisboa (Portugal) 

E-mail: sandrade@mail.ist.utl.pt; b Wageningen University, MicroSpectroscopy Centre, NL-6703 HA 

Wageningen (Netherlands) 

In recent years, porphyrins and related compounds have been explored as potential therapeutic drugs with 

use in areas of cancer detection and as photosensitizers in PDT. Biological effects of porphyrins largely 

depend on their physicochemical properties which in turn lead to important changes in their photophysical 

behavior. In particular, aggregation and axial ligation induce alterations on the porphyrin absorption 

spectra, fluorescence quantum yield, fluorescence lifetime and triplet state lifetime. The aggregation 

properties of the anionic water-soluble porphyrin, meso-tetrakis (p-sulfonatophenyl) porphyrin sodium salt 

– TSPP, have been extensively studied. Under suitable conditions of pH and ionic strength this molecule 

forms highly ordered molecular J and H aggregates. These aggregates can be promoted by interaction with 

serum albumins (HSA and BSA) at much lower porphyrin concentrations [1, 2]. TSPP monomer bound to 

these albumins is detected at high protein-to-porphyrin molar ratios (≥ 10). In order to further characterize 

the porphyrin-albumin interactions we have applied fluorescence correlation spectroscopy (FCS). The 

technique is a highly sensitive tool to measure concentration and diffusion coefficients from which we may 

determine binding/dissociation equilibria in the nanomolar range [3]. At low protein-to-porphyrin ratios 

(


FCSM-23 

A rhodamine 19 conjugated amino acid as a probe in fluorescence correlation 

spectroscopy (FCS) with human serum albumin (HSA) 

José A. B. Ferreira a) , Sílvia M. B. Costa a) , Pedro M. B. Vila a) , Carlos A. M. Afonso b) , 

Juan A. Organero c) , Abderrazzak Douhal c) 

a) Universidade Técnica de Lisboa, Instituto Superior Técnico, Centro de Química Estrutural, Av. Rovisco 

Pais, Complexo Interdisciplinar 1049-001 Lisboa (Portugal) b) Universidade Técnica de Lisboa, Instituto 

Superior Técnico, Centro de Química-Física Molecular, Av. Rovisco Pais, Complexo Interdisciplinar 1049- 

001 Lisboa (Portugal) c) Universidad de Castilla-La Mancha, Facultad de Ciencias del Medio Ambiente, 

Departamento de Quimica Fisica, Campus Tecnologico de Toledo, Avenida Carlos III, S.N., 45071 Toledo 

(Spain). E-mail: bracons@mail.ist.utl.pt 

Human serum albumin (HSA) is the most abundant protein in blood plasma. It has important binding and 

transport properties for many substances acting as specific ligands. The two HSA major binding sites are 

known as Site I and Site II. Site I provides conditions for hydrophobic binding whereas Site II that is 

cationic allows that binding associations based on stronger electrostatic interactions occur [1]. We have 

studied the association of rhodamine 19 4-(N-benzyloxycarbonyl-l-phenylalaninyloxymethyl)- 1- 

phenylmethyl ester iodide [2] - the probe molecule - to HSA in phosphate buffer at pH 7 using steady state 

electronic absorption, steady state and time resolved emission spectroscopy. It is found that rhodamine 19 

conjugated amino acid molecule displays a higher ability to associate with HSA relatively to that of 

rhodamine 6G, showing quenching effects reflected by nonexponential fluorescence decay kinetics. 

Fluorescence correlation spectroscopy (FCS) studies provided autocorrelations analyzed considering pure 

diffusion [3]. Respectively, diffusion coefficients obtained for rhodamine 6G, rhodamine 19 conjugated 

amino acid and rhodamine 19 conjugated amino acid in the presence of HSA were: D=28x10 1 µm 2 /s, 

D=15x10 1 µm 2 /s and D=5x10 1 µm 2 /s, compatible with the cationic π-system bound to a long hydrophobic 

group interacting with the protein environment. 

Normalized fluorescence autocorrelation 

functions, G(τ) of: (A) rhodamine 6G in water; 

(B) rhodamine 19 conjugated amino acid in 

water and (C) rhodamine 19 conjugated amino 

acid in the presence of HSA. Lines result from 

least-squares fit to the autocorrelated 

fluorescence fluctuations. Rhodamine 

chromophores were excited with a diode laser 

(638 nm, 40 MHz) in a PicoQuant MT-200 

system [4] 30 µm above glass/solution interface 

using an oil-immersion objective (100x/1.30 NA) 

in a confocal setup with a bandpass filter 

(695AF55) a 75 µm pinhole and a SPAD 

(IRF FWHM =0.6 ns, Perkin Elmer). 

G ( ! ) * 

1 

0.1 

0.01 

A B C 

0.01 0.1 1 10 

! / ms 

The observation that a conjugated amino acid rhodamine can be used to follow protein diffusion with 

excitation at the red end side of S 1 ←S 0 can be important for in situ diagnosis and phototherapies. 

Acknowledgements: POCI/QUI/57387/2004 (FCT, P); SAN-04-000-00 (JCCM, S). SFRH/BPD/24724/2005 (FCT, P). 

References: [1] A. Douhal et al., Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 18807. [2] C. A. M. Afonso et al., 

Synthesis 17 (2003) 2647. [3] J. Widengren et al., J. Phys. Chem. 99 (1995) 13368. [4] D. M. Togashi et al., 

Biophys. Chem. 119 (2006) 121. 

197


FCSM-24 

(Un)confined diffusion of CD59 in the plasma membrane of living cells 

determined by high-resolution single molecule microscopy 

Stefan Wieser 1 , Manuel Moertelmaier 1 , Elke Fürtbauer 2 , Hannes Stockinger 2 , 

Gerhard J. Schütz 1a 

1 Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr.69, A-4040 Linz, Austria 

2 Department of Molecular Immunology, Center of Biomolecular Medicine and Pharmacology, 

Medical University of Vienna, Lazarettgasse 19, A-1090 Vienna, Austria 

a E-mail: gerhard.schuetz@jku.at 

There has been emerging interest whether plasma membrane constituents are moving according to free 

Brownian motion or hop diffusion. In the latter model, lipids, lipid-anchored proteins and transmembrane 

proteins would be transiently confined to periodic corrals in the cell membrane, which are structured by the 

underlying membrane skeleton. The model of hop diffusion was further used as basis for unraveling 

properties of lipid microdomains. As this fundamentally important hypothesis for cell biology is based 

exclusively on results provided by one experimental strategy – high resolution single particle tracking –, we 

attempted in this study to confirm or amend it using a complementary technique. 

We developed a novel strategy which employs single molecule fluorescence microscopy to detect 

confinements to free diffusion of CD59 – a GPI-anchored protein – in the plasma membrane of living T24 

(ECV) cells. With this method, minimum invasive labeling via fluorescent Fab fragments was sufficient to 

measure the lateral motion of individual protein molecules on a millisecond time scale, yielding a positional 

accuracy down to 22 nm. The results rule out strong confinement to 120nm corrals in these cells as 

proposed by the single particle tracking approach (Murase, K., T. Fujiwara, Y. Umemura, K. Suzuki, R. 

Iino, H. Yamashita, M. Saito, H. Murakoshi, K. Ritchie, andA. Kusumi. 2004. Ultrafine Membrane 

Compartments for Molecular Diffusion as Revealed by Single Molecule Techniques. Biophys J 86(6):4075- 

4093). 

198


FCSM-25 

Single molecule fluorescence microscopy as a tool for biometrology 

Steffen Rüttinger * , Alex E. Knight 

National Physical Laboratory, Hampton Road, Teddington, Middlesex, TW11 0LW, UK 

* Physikalisch-Technische Bundesanstalt, Abbestrasse 2 – 12, 10587 Berlin, Germany 

Metrology increasingly seeks to define units and underpin measurements in terms of the numbers or 

properties of fundamental entities. Examples include the use of single trapped ions in atomic clocks; the use 

of single electrons to underpin electrical standards; attempts to redefine the kilogram in terms of the 

Avogadro constant; and the proposal to redefine the candela by counting photons. We are seeking to extend 

this approach to the life sciences by using single molecule fluorescence microscopy to investigate the 

quantity and behaviour of individual biological molecules. 

Single molecule measurements have many advantages and have been used to investigate many biological 

systems, such as molecular motors and enzymes. Single molecule approaches provide information that is 

lost in the “averaging” process intrinsic to conventional ensemble measurement techniques. This averaging 

can affect both the “static” characteristics of the molecules – such as conformation – and the “dynamic” 

behaviours involved in the activity of the protein molecules, such as enzyme catalysis and receptor-ligand 

interactions. Fluorescence imaging offers a versatile route to single molecule detection. 

To this end we have built a system that can be used to make these measurements of biological molecules 

and also to investigate the underlying measurement issues. Our system is based on the Total Internal 

Reflection Fluorescence Microscopy (TIRFM) technique [1, 2] . Here, an evanescent wave is used to excite 

fluorescence in a thin layer near a surface – such as a microscope slide – thereby achieving a high signal to 

noise ratio. We selected the prism-based TIRF approach, as this has been shown to have a higher signal to 

noise ratio than objective-based TIRF [3] and permits control of the angle of incidence. To achieve the 

widest possible application, our system has three laser sources at 488 nm, 532 nm, and 635 nm to cover the 

most commonly used fluorescent dyes. Imaging is through an Andor Electron Multiplication CCD camera. 

The entire system is under 

computer control, permitting 

automated exchange of filters, 

control of lasers and shutters, stage 

motion, focussing, and adjustment 

of angle of incidence. 

We are currently investigating 

applications to genomics and 

receptor-ligand interactions. We 

hope in the future to expand the 

range of timescales that we can 

access by adding a Total Internal 

Reflection - Fluorescence Correlation 

Spectroscopy (TIR-FCS) 

capability to the instrument [4] in the 

near future. 

References: [1] D. Axelrod, Methods In Cell Biology. 30 (1989): p. 245-270. [2] A. Knight, G. Mashanov, et al., 

European Biophysics Journal. 35 (2005): p. 89. [3] P. B. Conibear and C. R. Bagshaw, J Microsc. 200 Pt 3 (2000): p. 

218-29. [4] A. M. Lieto, R. C. Cush, et al., Biophysical Journal. 85 (2003): p. 3294-3302. 

199


FCSM-26 

Low-temperature spectral dynamics of single TDI molecules in 

alkane matrixes 

Sebastian Mackowski, Stephan Wörmke, Christoph Bräuchle 

Ludwig-Maximilian-University Munich, Department of Chemistry and Biochemistry and Center for 

Nanoscience, D-81377 Munich (Germany). E-mail: sebastian.mackowski@cup.uni-muenchen.de 

Single molecules embedded in host matrixes are perfect local probes of structural changes of their 

immediate environment. [1] The experiment commonly relies on monitoring frequency changes of the zerophonon-line 

(ZPL) excitation as a function of time. Since at cryogenic temperatures the linewidth of the 

ZPL could be lifetime-limited, small frequency changes can be detected. The limitation of this approach is 

however relatively narrow region of available frequency changes given by the scanning range of the laser. 

Recently proposed approach, which is based on using broadband laser excitation tuned into the vibronic 

band of the molecule and monitoring spectrally dispersed fluorescence has been shown to overcome this 

issue. [2] Using this method the frequency changes larger than 100 wavenumbers could be easily measured. 

We apply this approach to study the influence of the host matrix on the low-temperature spectral dynamics 

of single terrylenediimide (TDI) molecules embedded in hexane, heptane, pentadecane, and hexadecane. 

All the host materials are known to form Shpol’skii matrixes at low temperatures. For every matrix, 

fluorescence trajectories of several tens of single molecules were measured over 10 minutes each with the 

excitation power kept always the same. The acquisition time for a single spectrum was 1 sec. 

TDI in pentadecane 

T=1.5K 

TDI in heptane 

T=1.5K 

Fluorescence traces of TDI 

molecules embedded in pentadecane 

(left) and heptane (right) 

taken at 1.5K. The wavelength 

range is in both cases the same. 

In the case of long-chain alkanes (pentadecane and hexadecane), the fluorescence of single TDI molecules 

has been found to be quite stable, showing occasional spectral jumps of moderate frequency (


FCSM-27 

Single molecule spectroscopy of peridinin-chlorophyll-protein complex 

reconstituted with chlorophyll mixtures 

Stephan Wörmke, Tatas Brotosudarmo, Sebastian Mackowski, Hugo Scheer * , 

Christoph Bräuchle 

Ludwig-Maximilian-University Munich, Department of Chemistry and Biochemistry, D-81377 Munich 

(Germany). *Department of Biology. E-mail: stephan.woermke@cup.uni-muenchen.de 

A monomer of water soluble peridinin - chlorophyll a - protein (PCP) light - harvesting complex from 

dinoflagellate Amphidinium carterae consists eight peridinins (Per) and two chlorophyll (Chl) a molecules 

arranged into two clusters. [1] Recently it has been shown that the N-domain of the PCP apoprotein can be 

reconstituted with various Chl molecules characterized with different spectral properties. [2,3] The successful 

reconstitution opens a way to study energy transfer processes as well as chlorophyll-chlorophyll and 

chlorophyll-protein interactions in this relatively simple light-harvesting antenna. 

In this work we study reconstituted PCP complexes using single molecule spectroscopy at room 

temperature with the laser wavelength of 532 nm, which corresponds to Per absorption. The Chl excited 

states are therefore populated via very efficient sub-picosecond energy transfer. [4] This approach reduces the 

impact of inter-Chl Förster energy transfer and - thanks to very weak coupling between the two Chls - 

allows to simultaneously monitor the fluorescence of both Chl molecules comprising the complex. Indeed, 

the fluorescence trajectories detected for reconstituted complexes feature two intensity steps, each 

attributable to single chlorophyll's fluorescence. 

(Left) Fluorescence trace of Chl a 

emission showing spectral jump 

660 

at about 12 s. (Right) 

Fluorescence spectra of single 

630 

PCP complexes reconstituted 

with Chl a, Chl b, and both Chl a 

600 

and Chl b. 1 5 9 13 17 21 25 

Time [seconds] 

720 

Wavelength [nm] 

690 

70,00 

68,00 

66,00 

64,00 

62,00 

60,00 

58,00 

56,00 

54,00 

52,00 

50,00 

48,00 

46,00 

44,00 

42,00 

40,00 

Fluorescence intensity 

T=300 K 

! EX 

=532 nm 

Chl b only 

Chl a/Chl b 

Chl a only 

620 640 660 680 700 

Wavelength [nm] 

As a result of structural changes of the protein nanoenvironment the fluorescence of single PCP complexes 

fluctuates with time (left panel in the figure). The dynamics of the system is demonstrated via excitation 

power dependence of the fluorescence linewidth, where we observe monotonic increase of an average 

linewidth with the excitation power. This effect is accompanied by substantial broadening of the 

distribution of maximum emission wavelength for increasing excitation power. 

In the case of single PCP complexes reconstituted with Chl a and Chl b (energy difference of 400 cm -1 ), the 

fluorescence features two distinct lines each attributed to the respective Chl emission (right panel in the 

figure). The analysis of the experimental data together with Monte Carlo simulations allows us to describe 

energy transfer between two spectrally different Chls in this system. The energy transfer is found to occur 

not only from Chl b to Chl a, but also in less energetically preferential direction, from Chl a to Chl b. 

The financial support by Deutsche Forschungsgemeinschaft through SFB 533 (TP A6, B7) and by the Alexander von 

Humboldt Foundation is gratefully acknowledged. 

References: [1] E. Hofmann et al., Science, 272 (1996) 1788. [2] T. Polìvka et al., Photosyn. Res. 86 (2005) 217. [3] 

T.H.P. Brotosudarmo et al., FEBS Lett. 580 (2006) 5257. [4] T. Polivka, V Sundström, Chem. Rev. 104 (2004) 2021. 

201


FCSM-28 

Assessing the anomalous diffusion and nano-scale viscoelasticity of intracellular 

fluids by fluorescence correlations spectroscopy 

Matthias Weiss 

German Cancer Research Center, Cellular Biophysics Group (B085), D-69120 Heidelberg (Germany), 

E-mail: m.weiss@dkfz.de 

Diffusion is the basic means of intracellular transport, e.g. for membrane-bound and soluble proteins. 

However, due to macromolecular crowding and oligomerization processes, the random walk may be 

obstructed and a change in the diffusion characteristics towards anomalous diffusion (subdiffusion) is 

anticipated. Using fluorescence correlation spectroscopy (FCS) in combination with computer simulations, 

we were able to determine and quantify the subdiffusive motion of transmembrane proteins in the 

endoplasmic reticulum and the Golgi apparatus [1] and the strongly anomalous diffusion of fluorescently 

tagged, inert tracer particles (dextrans and nanometer-sized gold beads) in the cytoplasm and the nucleus of 

living cells [2,3]. While the observed anomalous diffusion of membrane proteins is most likely a signature 

of a dynamic oligomerization process that is needed for maintaining the secretory pathway [4], the 

subdiffusion in the cytoplasm and the nucleus is a consequence of the highly crowded state of the respective 

biofluids. In the latter case, quantifying the anomalous diffusion allows one to determine the viscoelastic 

properties (i.e., the shear modulus) of the fluids on the nanoscale in vivo. Indeed, all tested cell lines showed 

a strong viscoelastic characteristics for the cytoplasm and the nucleoplasm, with almost equal viscous and 

elastic moduli over a wide frequency range. 

To explore how changes of the crowded state alter the associated viscoelastic behavior, we have stressed 

cells osmotically. Under osmotic stress, the diffusion was seen to become less anomalous and the elastic 

modulus decreased with respect to the viscous modulus. On a heuristic level, the experimental data are well 

described by the Zimm-model for polymer solutions with varying solvent conditions, which indicates that 

the protein/DNA entanglement in the crowded cytoplasm and nucleus, respectively, may undergo a partial 

collapse when water is extracted due to osmotic stress. This reasoning is supported by in vitro data, e.g. for 

frog egg extracts. 

Based on the observation, that anomalous diffusion is indeed a fairly common phenomenon, we finally 

discuss how generic cellular processes (e.g. pattern formation) are altered in the presence of subdiffusion. 

References: [1] M. Weiss et al., Biophys. J. 84 (2003) 4043. [2] M. Weiss et al., Biophys. J. 87 (2004) 3518. 

[3] G. Guigas et al., Biophys. J. 93 (2007). [4] M. Elsner et al., submitted (2007). 

202

Part V 

Upconversion and 

2-Photon Excitation 

203

204

Abstracts Poster – Part V: Upconversion and 2-Photon Excitation 

UC2P-1 

Development of a broad-band femtosecond time-resolved fluorescence set-up 

in the UV: application to biological and chemical systems 

Olivier Bräm, Andrea Cannizzo, Oskouei Ahmad Ajdarzadeh, Frank van Mourik, Andreas 

Tortschanoff and Majed Chergui 

Laboratoire de Spectroscopie Ultrarapide, Ecole Polytechnique Fédérale de Lausanne, 

Ch-1015 Lausanne-Dorigny (Switzerland). E-mail: olivier.braem@epfl.ch 

Time-resolved luminescence spectroscopy is beyond all doubts a powerful tool to investigate dynamical 

behavior of many physical systems, in particular in the condensed phase. The advancement of pulsed laser 

technology to the femtosecond time scale brought a new approach of time-resolved spectroscopy, making 

use of ultrashort pulsewidths of these lasers. In the case of fluorescence up conversion based setups the non 

liner phenomenon of sum frequency generation is used to mimic a sub picosecond shutter. [1] It permits 

broad-band detection of time-resolved emissions with femtosecond resolution allowing ultrafast dynamics 

studies of different molecular systems, in particular proteins, and different phenomena such as solvation 

relaxation and cooling processes. In this respect, the present work is framed in a wider one aimed to probe 

the earliest dynamics of a wide variety of wild-type proteins, by means of ultrafast broad-band emission 

detection of optically active amino acids, as tryptophan, tyrosine and phenylalanine, [2] which all absorb in 

the UV range. Though up-conversion-based set-ups have been successfully implemented in the visible 

range, both for single wavelength and broad-band detection, only monochromatic detection measurements 

have been carried out in the UV. Here, we present for the first time a broad-band ultrafast fluorescence upconversion 

set-up in this spectral range, with a detection range of 300-550 nm range, a state-of-the-art time 

resolution of 330 fs, and a tunable excitation from 250 to 300 nm. We will present results of time-resolved 

fluorescence emission study of some UV dyes in order to characterize the setup. As a first application we 

report a preliminary investigation of tryptophan in water. 

References: [1] J. Shah, IEEE Journal of Quantum Electronics, 24 (1998) 276. [2] S. Schenkl et al., Science 309 

(2005) 917. 

205


UC2P-2 

Development of luminescence probes for bio-active systems based upon 

multi photon excited functionalised lanthanide complexes 

Lars-Olof Pålsson, Andrew Beeby, Robert Pal, Benjamin S.L. Murray and David Parker 

Department of Chemistry, Durham University, South Road, DH1 3LE Durham, (United Kingdom). 

E-mail: lars-olof.palsson@durham.ac.uk 

Functionalised lanthanide (Ln III ) complexes are emerging as powerful and versatile photo luminescence 

(PL) probes for biological systems including living cells 1,2 . The emission of the probes is based upon sharp 

and well defined bands of the f-f transitions of the Ln III ions.. The Eu III emission spectral profile is therefore 

an excellent probe and complexes can be engineered to report local environment factors such as pH, pM 

and pX. 1,2,3 . As the transitions of the 4f electrons are Laporte forbidden, the emission lifetime is orders of 

magnitude more long-lived than the organic fluorescence (ms compared to ns). This is utilised in 

microscopy as the emission can be monitored using time gated detection techniques thus discriminating 

against the autofluorescence of the bio-assay. Electronic excitation is achieved through an intra molecular 

electronic energy transfer process from a chromophore-ligand complex. The development of emissive 

probes has been focused on the nature of the complex which has to be engineered both with respect to the 

desired opto electronic properties of the Ln III ion and the ability to permeate cells and localise selectively to 

different cell organelles. To date, several complexes have been defined that localise successfully with the 

lysosomes, nucleus or the ribosomes 4,5 . 

While these complexes are readily excited 

through one photon excitation process of the 

chromophore, we can also show that the 

complexes can be excited through a two 

photon excitation process. We demonstrate the 

use of these functionalised Ln III complexes for 

bio assays using multiphoton excitation. 

This technique is extremely advantageous as 

the near IR radiation is less harmful to 

biological system as compared to UV 

excitation, with the avoidance of for instance 

photo oxidation and local heating. Using the 

long-lived Ln III emission will also allow for 

time gated detection with the possibility to 

discriminate against scattering and 

autofluorescence background signals. This 

will also filter out the ligand fluorescence. 

Due to the inherent need for a strong focus in 

multiphoton excitation spectroscopy these 

probes are well suited to be used in 

microscopy applications monitoring small 

sample volumes (10 -19 

specimen level. 

m 3 ) on a single 

Intensity [a.u.] 

1000 

800 

600 

400 

200 

MeO 

O 

O 

S 

N 

0 

350 400 450 500 550 600 650 700 

wavelength [nm] 

The figure above shows the Eu thiaxanthone complex 6 

excited at 758 nm. The blue line spectrum is the 

complex dissolved H 2 O, while red line spectrum is with 

HCO 3¯ added. Addition of HCO – 

3 leads to a large 

increase in the hypersensitive ΔJ = 2 transition at 616 

nm as bound water is displaced. A logarithmic plot of 

the integrated PL – excitation power relation yields a 

linear relation with a slope of 2.15 consistent with a two 

photon excitation process. 

Ph 

H 

N 

O 

HN 

N 

N O 

Eu 

N 

O N H 

NH 

OH 2 

Ph 

3+ 3Cl - 

References: [1] D. Parker Chem. Soc. Rev, 33 (2004), 156 [2] S. Pandya et al Dalton Trans. (2006), 2757 

[3] A. Beeby et al J. Photochem. Photobiol. 57 (2000), 83 [4] J. Yu et al J. Am. Chem. Soc. 128 (2006), 2294 

[5] R. Poole et al Org. Biol. Chem. 3, (2005), 1013 [6] Y. Bretonnie’re et al Chem.Comm. (2002) 1930 

[7] J.R. Lakowicz, in Principles of Fluorescence Spectroscopy, 3 rd ed. Springer Berlin 2006. 

206


UC2P-3 

The symmetry of two-photon excited states determined by time-resolved 

fluorescence depolarisation experiments 

Linus Ryderfors 1 , Emad Mukhtar 1 , and Lennart B.-Å. Johansson 2 

1 Department of Photochemistry and Molecular Science, Uppsala University, P. O. Box 523, S-751 20 

Uppsala and 2 Department of Chemistry; Biophysical Chemistry, Umeå University, S-901 87 Umeå, Sweden. 

E-mail: Linus.Ryderfors@fotomol.uu.se 

A quantitative method is presented for the determination and assignment of the two-photon absorption 

tensor of fluorophores dissolved in liquid solutions. From time-resolved fluorescence depolarisation 

c 

l 

experiments two linearly independent anisotropies r ( t ) and r ( t ) , as well as the two-photon polarisation 

ratio ( ! ) can be determined, when using circularly (c) and linearly (l) polarised excitation. The value of 

TP 

TP 

! is defined by the ratio between the isotropic emission measured for the c- and l-polarised excitation. In 

all experiments the excitation source was an amplified 200 kHz 800 nm Ti:sapphire femtosecond laser and 

the fluorescence emission was detected by using the time-correlated single photon counting 

technique(1).The depolarisation dynamics is ascribed to diffusive molecular reorientations but account for 

the influence of rapid unresolved reorientations (cf. Fig. 1) is also taken. The appropriate equations are 

described elsewhere(2). 

Figure 1. Schematic of the coordinate 

systems that relate the microscopic and 

macroscopic properties in fluorescence 

depolarisation experiments. The laboratory, 

the diffusion and molecular fixed Cartesian 

coordinate systems is denoted L, D and M, 

respectively. The chemical structure of 

perylene is displayed. Usually perylene is 

approximated to reorient like an oblate 

rotor. The electronic transition dipole of the 

emission transition S 1 → S 0 is polarised 

along the X M -axis. The symbols ˆ! 

ex 

and 

ˆ! 

em 

indicate the propagation direction of the 

excitation beam and direction of monitoring 

the fluorescence emission. 

The work presents the results obtained from the two-photon excited studies of perylenes (perylene, 

2,5,8,11-tetra-tert-butylperylene and 1,7-diazaperylene) which were dissolved in polar and non-polar 

solvents. The procedure used for globally analysing the various depolarisation data will be displayed 

together with the quantitative information obtained about two-photon absorption tensors of perylenes 

belonging to the point groups C 2h and D 2h . 

References: (1) L Ryderfors, E Mukhtar, LB-Å Johansson: Two-photon excited fluorescence depolarisation 

experiments: II. The proper response function for analysing TCSPC data. Chem. Phys. Letters 411 (2005) 51-60. 

(2) L Ryderfors, E Mukhtar, LB-Å Johansson: Two-Photon Excited Fluorescence and Molecular Reorientations in 

Liquid Solutions. J. Fluorescence, accepted (2007). 

207


UC2P-4 

Preparation and characterization of nanocrystalline ZrO 2 :Yb 3+ ,Er 3+ 

up-conversion phosphors 

Iko Hyppänen 1 , Jorma Hölsä 1 , Jouko Kankare 1 , Mika Lastusaari 1 and Laura Pihlgren 1,2,3 

1 University of Turku, Department of Chemistry, FI-20014 Turku, Finland 

2 University of Turku, Department of Biotechnology, Tykistökatu 6, FI-20520 Turku, Finland 

3 Graduate School of Materials Research, Turku, Finland. E-mail: laerle@utu.fi 

The field of the up-conversion luminescence where absorption of two or more low energy photons is 

followed by emission of a higher energy photon has witnessed numerous breakthroughs during the past 

decades. Most up-conversion luminescence materials operate by using the combination of a trivalent rareearth 

sensitizer (e.g. Yb, Er or Sm) and an activator (e.g. Er, Ho, Pr or Tm) ion in an optically passive 

crystal lattice [1]. Up-converting phosphors have a variety of potential applications as lasers, displays, 

quantum counters and inks for security printing (bank notes, bonds) [2]. Up-conversion luminescence 

materials may also be used in clinical diagnostic assays. 

In this work, nanocrystalline up-converting phosphors with zirconium oxide (ZrO 2 ) as the host lattice were 

prepared. In this host, the lanthanide dopants can possess a multisite position that improves the absorption 

efficiency and possibly makes the energy transfer from the sensitizer (Yb 3+ ) to the activator (Er 3+ ) easier, 

too. The ZrO 2 :Yb 3+ ,Er 3+ phosphors were obtained with combustion [3] and sol-gel [2] methods. Crystal 

structures and phase purities were analyzed with X-ray powder diffraction (XPD). Impurities were studied 

with FT-IR spectroscopy. Up-conversion luminescence was excited at room temperature with an IR-laser at 

970 nm. 

The XPD measurements revealed that the structure of the ZrO 2 :Yb 3+ ,Er 3+ phosphors was cubic. The 

crystallite sizes estimated with the Scherrer equation [4] were 5-30 and ca. 50 nm for materials prepared 

with the combustion synthesis and the sol-gel method, respectively. The materials prepared with the former 

method were found pure whereas the FT-IR spectra revealed the conventional impurities (NO - 3 , OH - ) in the 

materials prepared by the latter method. The up-conversion luminescence spectra showed red (640-690 nm) 

and green emission (535-570 nm) due to the 4 F 9/2 → 4 I 15/2 and ( 2 H 11/2 , 4 S 3/2 ) → 4 I 15/2 transitions of Er 3+ , 

respectively (Fig.). The materials prepared with combustion synthesis were found to yield the most efficient 

up-conversion luminescence intensity, however. 

Intensity / Arb. units 

200 

150 

100 

50 

0 

ZrO 2 

:Yb 3+ ,Er 3+ 

x Yb 

= 0.10, x Er 

=0.04 

Urea 

3 

2 

1 

0 

-1 

520 540 560 580 

2 

H 11/2 4 I 15/2 

4 S 3/2 

T = 293 K 

4 

I 15/2 

Semicarbazide 

+ NH 4 

NO 3 

Sol-gel, 6 h@ 400 o C+ 

20 h @ 1000 o C 

Sol-gel, 3 h@ 400 o C+ 

10 h @ 1000 o C 

AMP 

Glycine, 

1 h @ 700 o C 

4 F 9/2 

4 I 15/2 

Urea 

Figure. 

Up-conversion luminescence spectra 

(λ exc = 970 nm) of selected 

ZrO 2 :Yb 3+ ,Er 3+ materials prepared by 

sol-gel and combustion synthesis. 

500 550 600 650 700 

Wavelength / nm 

This study was supported by the Finnish Funding Agency for Technology and Innovation (Tekes). 

References: [1] Auzel, F., Chem. Rev. 104 (2004) 139. [2] Díaz-Torres, L.A., de la Rosa-Cruz, E., Salas, P., Angeles 

-Chavez, C., J. Phys. D: Appl. Phys. 37 (2004) 2489. [3] Vetrone, F., Boyer, J.-C., Capobianco, J.A., Speghini, A., 

Bettinelli, M., J. Appl. Phys. 96 (2004) 661. [4] Klug, H.P., Alexander, L.E., X-Ray Diffraction Procedure, Wiley, 

New York, 1959, p. 491. 

208


UC2P-5 

Benzothiazolestyrylcyanine dyes: application in fluorescent visualization 

of nucleic acids in vivo 

Kateryna D. Volkova, Analoiy O. Balanda, Vladyslava B. Kovalska and Sergiy M. Yarmoluk 

Institute of Molecular Biology and Genetics, NASci of Ukraine,03143 Kyiv, Ukraine. 


Previously, series of benzothiazole styrylcyanine dyes were developed and proposed as potential 

fluorescent probes for the use in 3-D DNA imaging upon TPE. [1] The main advantage of two-photon 

bioimaging in comparison with single-photon are low autofluorescence, higher signal-to-background ratio 

for fluorescence detection resulting from well-separated excitation and emission wavelengths and deeper 

penetration of the exciting light into biological tissues. 

It was shown that developed styrylcyanine dyes interact with nucleic acids with up to 1000 times 

fluorescent increasing [2], demonstrate noticeable specificity to DNA, comparing with RNA and moderate 

values two-photon absorption cross- sections value up to 7.4×10 -50 cm 4 s. Excitation and emission of these 

dyes are placed correspondingly in the range 548-568 nm and 600-610 nm. Such position of excitation 

maxima allowed to use of YAG:Nd 3+ 15 ns pulsed laser with 1064 nm radiation to obtain two-photon 

excited fluorescence. 

Next step was to determine ability of dimeric/monomeric styrylcyanines modified with spermine-like 

linkage/tail group (in order to increase affinity of dye to nucleic acids) to penetrate into living cells and to 

stain specifically DNA and RNA in vivo. 

N 

N 

S 

I 

I 

I 

I 

N 

+ (CH 2 

) + 

5 N (CH 2 

) 3 

N 

+ + 

(CH 2 

) 5 N 

S 

A 

DBos-13 

B 

Figure: Structure of dimeric benzothiazole styrylcyanine dye DBos-13 (A) and image of the living cell 

obtained with the use of this dye (B). 

In Figure transmission (left) and fluorescent (right) images of cells from rat hyppocampus in culture stained 

with dimeric styrylcyanine DBos-13 are presented, showing clearly the distribution of dye inside cell. The 

dislocation of the brightest compartments in the cell correlates with the transmission image of the nuclei. 

Studied benzothiazolestyrylcyanines were reported to be non-toxic in Paramecium toxicity assay. [3] 

It was shown that developed styrylcyanines are cell-permeating; also they give bright fluorescent staining 

of nucleic acids inside the living cell. Thus we consider these dyes could be efficiently applied for the 

fluorescent the visualization of DNA and RNA in vivo upon single- and next two-photon excitation 

experiments. 

This work was supported by the Science and Technology Center in Ukraine (STCU) grant #U3104k 

References: [1] V.P. Tokar et al., J. Fluoresc. 16 (2006) 783. [2] A.O. Balanda et al., Ukrainica Bioorganica Acta 4 

(2006) 17. [3] N.N. Nizamov et al., Ukrainica Bioorganica Acta 3 (2005) 35. 

209


UC2P-6 

Two-photon excited (TPE) fluorescence depolarisation: 

Molecular reorientation and donor-donor energy migration 

Therese Olofsson, Linus Ryderfors, Julian G. Molotkovsky 1 , Emad Mukhtar, 

Lennart B.-Å. Johansson 

Umeå University, Department of Chemistry; Biophysical Chemistry, S-90187 Umeå (Sweden), 

1 Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia 

E-mail: Therese.Olofsson@chem.umu.se 

Interestingly and contrary to one-photon excited fluorescence the TPE fluorescence depolarisation 

experiments enable the determination of two linearly independent anisotropies, which can be obtained from 

one-colour experiments. This means that more information can be obtained about spectroscopic transitions, 

as well as reorientation dynamics. Relatively few studies have hitherto been published, in which more 

elaborate theories have been applied for the analyses of data(1-4). In the study of the electronic states and 

the transition probabilities between them, however, knowledge about the absorption transition tensor is 

important, as well as in the applications that deal with molecular properties related to the reorientation 

correlation functions. 

HO 

CH 2 

O 

H 3 C 

CH 3 

CH 3 

CH 2 

O 

P 

O - 

O 

CH 3 

O 

O 

P 

O - 

CH 2 

Figure 1: The chemical 

structure of bis(3-perylenylmethylphosphonate)-bisteroid 

is 

displayed together with its 

corresponding space filling 

model. In the mono-(3-perylenylmethylphosphonate) 

bisteroid (structure not shown) 

one of the perylenylmethylphosphonates 

is replaced by an 

H-atom. 

In this work the TPE fluorescence depolarisations of two perylene derivatives have been studied. The 

derivatives, a mono- and bis-perylenyl labelled bisteriod (cf. Fig. 1) were solubilised in micelles composed 

of octa-ethylene glycol mono-dodecyl ether. The anisotropic reorienting motions of the perylenyl group 

were studied using the mono-form, while the bis-form was used to examine the intramolecular donor-donor 

energy migration process. 

References: (1) S-Y Chen, BW Van der Meer: Theory of two-photon induced fluorescence anisotropy decay in 

membranes. Biophys. J. 64 (1993) 1567-75. (2) C Wan, CK Johnson: Time-resolved anisotropic two-photon 

spectroscopy. Chem. Phys. 179 (1994) 513-31. (3) SW Pauls, JF Hedstrom, CK Johnson: Rotational relaxation of 

perylene in n-alcohols and n-alkanes studied by two-photon-induced anisotropy decay. Chemical Physics 237 (1998) 

205-22. (4) L Ryderfors, E Mukhtar, LB-Å Johansson: Two-Photon Excited Fluorescence and Molecular 

Reorientations in Liquid Solutions. J. Fluorescence, in press (2007). 

210


UC2P-7 

Time-gated detection by time-correlated single photon counting (TCSPC) 

enables separation of coherent Anti-Stokes Raman scattering (CARS) 

microscopy data from multiphoton-excited fluorescence 

Samantha Fore, Sonny Ly, Gregory McNerney, James Chan, and Thomas Huser 

NSF Center for Biophotonics, University of California Davis, Sacramento, CA 95817. 

E-mail: srfore@ucdavis.edu 

We demonstrate the time-gated detection of coherent Anti-Stokes Raman scattering (CARS) images at the 

microscopic scale. CARS is an instantaneous process, while fluorescence exhibits typical decay times of 

several nanoseconds. We show that multiphoton-excited (MPE) tissue autofluorescence, the major source 

of background contributions in CARS microscopy, can be sufficiently reduced if single photon counting 

detectors and time-correlated single photon counting electronics are employed for signal detection. Images 

similar to those obtained using fluorescence lifetime imaging (FLIM) show distinct regions with high 

CARS intensity versus those with high MPE fluorescence (Figure 1a). Furthermore, time-gating of the 

photon-arrival time is used to separate instantaneous (< 1ns) CARS photons from delayed (> 1ns) 

fluorescence photons and to generate CARS and MPE fluorescence intensity images, respectively (Figure 1 

b and c, respectively). We demonstrate how this technique allows us to image and isolate lipid-rich deposits 

surrounding the arteries of rats and mice. At the same time, multiphoton-excited fluorescence allows for 

imaging and identification of the arterial tissue. Local spectra collected in the arterial tissue at and near 

lipid-rich deposits further confirm the nature of CARS signals as well as tissue autofluorescence. 

Figure 1: a.) FLIM image obtained from a cross-section of rat artery tissue. Blue regions correspond to 

high CARS intensity (instantaneous photon arrival time) due to lipid rich deposits in the arterial wall. Green 

and red regions correspond to high multiphoton-excited fluorescence with their corresponding delayed 

fluorescence lifetime indicated on the lifetime scale shown in the inset. Regions of interest were selected 

(indicated by dashed circles) and their corresponding lifetime decays shown in the image above. b.) 

Intensity image generated from CARS photons arriving during time-gate indicated in the blue region (i.e. < 

0.5 ns) of the lifetime decay curve shown in the image above. c.) Intensity image generated from 

fluorescence photons arriving during time gate indicated in the red region (i.e. > 1.5 ns) of the lifetime 

decay. White lines drawn on images indicate the interface between lumen and vessel wall. Distance units 

are in microns. 

211

212

Part VI 

Nanomaterials 

213

214

Abstracts Poster – Part VI: Nanomaterials 

NANO-1 

Fluorescent lipid nanocontainers as label system for protein chips 

Johanna Pultar, Claudia Preininger 

Austrian research Centers GmbH, Division of Biogenetics and Natural Resources 

A-2444 Seibersdorf (Austria). E-Mail: Johanna.pultar@arcs.ac.at 

A novel and sensitive liposome immunoassay is described using fluorescent nanocontainers as marker 

molecules. Various strategies to integrate fluorescent dye molecules into lipid vesicles have been 

investigated. The liposomes were prepared from cholesterol and phospholipids including a biotin-modified 

lipid which further bears a PEG-group for stabilization [1]. In another approach, dye molecules were 

coupled via an amide-bond to a phospholipid prior to liposome incorporation [2]. The lipophilic dye R46 

was entrapped into the lipid bilayer, whereas Dy647-NH 2 and Dy647-NHS purchased from Dyomics, Alexa 

Fluor 647 from Invitrogen, and Cy5 from Amersham Biosciences were entrapped into the inner cavity. The 

various dye-conjugation and encapsulation strategies were evidenced via confocal microscopy using giant 

unilamellar vesicles with a mean size of 14 µm for visualization [3]. The size of the liposomes further used 

as marker molecule for protein biochips was about 142 nm. 

The encapsulation efficiency in % was determined as the total amount of dye molecules entrapped in or 

conjugated to the lipid vesicle versus the total initial input of encapsulant. The effects of the dyeconjugation- 

and encapsulation on the entrapment efficiency and furthermore signal enhancement for 

protein chips was investigated using a spectrofluorimeter and a non-confocal fluorescence scanner. 

For ARChip Epoxy optimized protocols for protein arrays have been developed before, resulting in high 

immobilization capacity and excellent signal-to-noise ratio. These protocols were adapted and applied using 

fluorescent lipid nanocontainers as marker molecules. 

The protein biochip presented herein consists of antibodies against markers of inflammation and sepsis. The 

capture antibodies are immobilized onto ARChip Epoxy and on commercially available polymer surfaces. 

The fluorescence signals were enhanced by a factor of thirty using fluorescent lipid nanocontainers instead 

of Dy647-Streptavidin, Cy5-Streptavidin or Alexa Fluor 647-Streptavidin as detection reagent. On that 

way, the sensitivity was as low as 10 ng/L for recombinant cytokines. To further enhance assay sensitivity 

the liposome size (tested range 50 nm to 800 nm) was tuned with respect to the assay conditions and optical 

characteristics of the detection system. 

Miniaturization and multiplexing with protein microarrays allow a reduction of sample volume, an increase 

in the number of analytes that can be measured simultaneously, and an increased throughput. 

Our results indicate that lipid nanocontainers are a feasible label system that has the potential of producing 

significantly enhanced fluorescence signals and as a consequence, is capable of measuring extremely low 

levels of analyte, which especially in medical diagnostics is of major importance. 

References: [1] Vermette, P. et al; Characterization of surface-immobilized layers of intact liposomes. 

Biomacromolecules 5 (2004) 1496-1502. [2] Baeumner, A.J. et al; A generic sandwich-type biosensor with 

nanomolar detection limits. Anal Bioanal Chem 378 (2004) 1587-1593. [3] Moscho, A. et al. Rapid preparation of 

giant unilamellar vesicles. Chemistry 93 (1996) 11443-11447. 

215


NANO-2 

Dextrin microencapsulated porphyrin: luminescent properties 

Priscilla Paiva Luz, Cláudio Roberto Neri and Osvaldo Antonio Serra 

University of São Paulo, Chemistry Department, FFCLRP – Av. Bandeirantes 3900, Ribeirão Preto SP 

14040-901 (Brazil). E-mail: pripaiva@usp.br 

Porphyrins have attracted a lot of attention because of their ability to accumulate in many kinds of cancer 

cells. Their photophysical properties allow their application in Photodynamic Therapy (PDT). The 

porphyrins are macromolecules that can be easily adjusted by modifications of the electronic distribution on 

the aromatic ring through peripheral substitutions or changes in the chemical environment. Solid-state 

porphyrins and their derivatives do not exhibit emission because of the concentration quenching 1 . So the 

aim of this work is to study the luminescence of a solid state porphyrin when it is microencapsulated or 

physically blended. 

Sodium meso-tetra(4-sulfonatophenyl)porphyrin (TPPS 4 ) loaded microspheres were prepared by spray 

drying an aqueous solution in an ultrasonic spray-dryer system developed in our laboratory 2 . To prepare the 

solution, dextrin was dissolved in water, and then solid TPPS 4 was added 3 . This solution was spray-dried 

with inlet and outlet temperatures around 300ºC. The obtained powder was morphologically investigated by 

using scanning electron microscopy (SEM). Encapsulation efficiency and the percentage of TPPS 4 in the 

dextrin microsphere were evaluated by UV-Vis absorption, and luminescent properties of the microspheres 

were also investigated. Physical blends containing different proportions of TPPS 4 in dextrin were prepared 

in order to compare their luminescence characteristics with those of the microspheres. 

Fig.: The luminescence emissions were analyzed 

as a function of the percentage of TPPS 4 

blended or encapsulated in dextrin. From the 

plot it is clear that TPPS 4 luminescence 

increases with increasing TSPP 4 percentage in 

dextrin. Indeed, TPPS 4 undergoes almost a 

100% increase in its luminecesce intensity 

when encapsulated. 

λ exc. =517 nm; λ em. =648 nm 

1,5x10 5 

1,2x10 5 

9,0x10 4 

6,0x10 4 

physical blends 

Relative Intensity (cps) 

3,0x10 4 

0,0 

TPPS 4 

encapsulated 

in dextrin 

0,2 0,4 0,6 0,8 1,0 

% of TPPS 4 in dextrin 

The average distances betwen TPPS4 ions when the porphyrin is microencapsulated are larger than when it 

is physically blended with the polymer, where some aggregation occurs. As a result, a lower amount of 

TPPS4 should be used in Photodynamic Therapy. 

Acknowledgements: CAPES, CNPq and FAPESP; Brazilian Scientific Financial Agencies. 

References: [1] R. Wiglusz et al., J. Alloy Comp. 380 (2004) 396. [2] P. P. Luz et al., Quim. Nova, (in press). 

[3] A. Synytsya et al., Spectrochim. Acta A. 66 (2007) 225. 

216


NANO-3 

Luminescent chemosensors based on silica nanoparticles 

Gionata Battistini, Sara Bonacchi, Marco Montalti, Luca Prodi, Enrico Rampazzo, 

Nelsi Zaccheroni 

Department of Chemistry “G. Ciamician”, Latemar unit, University of Bologna, 

Via Selmi 2, 40126 Bologna, Italy. E-mail: sara.bonacchi3@unibo.it 

In the field of fluorescent chemosensors, a big effort has been recently addressed towards the design of 

more sensitive and efficient systems. As far as sensitivity improvement is concerned, one of the limiting 

steps is the feeble change of the signal when it is due to the interaction between single receptor–fluorophore 

pairs obtained by complexation of the analyte. It has already been proved, in fact, that to gain a higher 

sensitivity a single binding event has to alter the properties of a large number of fluorophores. Following 

this idea, all the multifluorophoric species can be in principle employed as basic structures to design 

chemosensors featuring signal amplification. Among them, silica nanoparticles represent a very interesting 

solution, since they are relatively easy to synthesize, extremely versatile, biocompatible and inert from a 

photophysical point of view. [1,2] We have designed, prepared and characterized a few different systems 

presenting the dyes covalently linked on the nanoparticle surface or inside the core of the structure. These 

systems offer different advantages in terms of solubility, dye protection and/or interaction with the 

environment and mutual dyes communication, and all these terms will be discussed in the presentation. 

Furthermore, we will describe systems presenting amplified ON-OFF (scheme below) or OFF-ON response 

to metal ion complexation, depending on the nature of the nanoparticle 

All these features make these new materials extremely promising for applications as luminescent probes in 

many fields including cell biology. 

References: [1] L. Prodi, New J. Chem. 29 (2005) 20. [2] M. Montalti et al., J. Mater. Chem. 15 (2005) 2810. 

217


NANO-4 

Excitation energy transfer and trapping in dye-loaded solid particles 

Hernán B. Rodríguez, Enrique San Román 

University of Buenos Aires, School of Sciences, Ciudad Universitaria, Pab. II, C1428EHA Buenos Aires, 

Argentina. E-mail: esr@qi.fcen.uba.ar 

Physical or chemical attachment of dye molecules to solid particles allows the attainment of high local dye 

concentrations. Surface densities near 4 × 10 −4 dye molecules / Å 2 , resulting in average inter-molecular 

distances of about 5 nm, are easily reached. To attain this proximity in solution, concentrations in the order 

of 10 −2 M would be required. In these conditions and assuming random distribution, ca. 25 % of the 

molecules have neighbors at less than 15 Å. Therefore, the effect of interactions among dye molecules and 

singlet-singlet energy migration and transfer cannot be disregarded. Indeed, in systems containing two 

different dyes efficient energy transfer was observed [1]. For single dyes, on the other side, fluorescence 

quenching was found as the surface concentration increases [2-3]. An explanation of this behavior is 

assayed on grounds of suitable models. 

Results obtained for a rhodamine on microcrystalline 

cellulose [3] show that fluorescence 

quantum yields – corrected for inner filter effects 

– decrease somewhat more rapidly than 

fluorescence lifetimes on increasing the dye 

concentration. These effects may be attributed to 

energy trapping by a) dimers or quasi dimers or 

b) statistical traps. Energy migration and transfer 

should be responsible for the decrease in 

lifetimes [4]. 

Application of model (a) shows that nearly 20 % 

of the dye molecules should be in the dimeric 

state for the highest dye concentration. On the 

other hand, if model (b) is applied assuming a Poisson distribution of dye molecules, a quenching radius of 

nearly 15 Å is found. As no conclusive evidence on changes of the absorption spectrum with concentration 

was found, the nature of the traps cannot be ascertained. On the other hand, the trapping effect of dimers 

could be demonstrated for methylene blue adsorbed on the same support, where dimerization could be 

quantified. The application of model (a) explained quantitatively in this case the fluorescence quantum 

yield decrease with concentration. Irrespective of the nature of the traps, it is clear that concentration 

quenching is the result of both static (trap absorption) and dynamic (energy migration and transfer) nature. 

No excimer fluorescence has been detected in the so far studied systems. 

Another common observation is the occurrence of concentration dependent Stokes shifts, resulting in a 

displacement of the fluorescence spectrum – again after correction for inner filter effects – to higher 

wavelengths as the dye concentration increases, while the absorption spectrum remains unchanged. This 

effect is noticed even at average intermolecular distances in excess of 10 nm. 

The aim of these studies is the development of solid energy or charge transfer photosensitizers, exploiting 

the occurrence of high dye concentrations to afford substantial absorption of incident light and energy 

transfer among different dyes to broaden the excitation spectrum. The unraveling of energy trapping 

mechanisms in systems composed by a single dye is a key to the development of efficient systems. 

References: [1] H.B. Rodríguez et al., Photochem. Photobiol., 82 (2006) 200. [2] M.G. Lagorio et al. Phys. Chem. 

Chem. Phys., 3 (2001) 1524. [3] H.B. Rodríguez et al., to be published. [4] P. Bojarski et al., Chem. Phys. 210 (1996) 

485-499. 

218


NANO-5 

Processing and characterization of Au nanoparticles for use in plasmon probe 

spectroscopy and microscopy of biosystems 

Y. Chen 1,2 , J. A. Preece 3 , R. E. Palmer 2 

1 

Department of Physics, University of Strathclyde, 107 Rottenrow, Glasgow G4 0NG UK 

2 

Nanoscale Physics Research Laboratory, School of Physics and Astronomy, University of Birmingham, 

Birmingham B15 2TT, UK 

3 

School of Chemistry, University of Birmingham, Birmingham B15 2TT, UK 

Noble metal nanoparticles have great potential for applications in biochemical sensing and biological 

imaging because of their unique optical properties originating from the excitation of local surface plasmon 

resonances. In particular, gold nanoparticles have attracted intensive interest because they are easily 

prepared, have low toxicity and can be attached, readily, to molecules of biological interest. It is believed 

that the surface plasmon properties of Au nanoparticles are dramatically affected by their size, shape and 

surrounding surface environment. In this work, we investigated Au nanoparticles with controlled size, 

shape and passivating agents, along with a novel process of guided self-assembly to create 2D 

nanostructures from such nanoparticles. 

Au colloidal nanoparticles were synthesized with different passivating ligands including citrate, magnesium 

oleate and dialkyl sulfides. Structural characterization using high resolution TEM shows multi-twinned 

FCC structures with an average size of 16 nm in diameter. Most particles have non-spherical shapes. High 

resolution electron energy loss spectroscopy (HREELS) reveals a weak Au-S bond in the case of dialkyl 

sulfide adsorption and bond dissociation under low energy electron impact. Guided self-assembly of Au 

nanoparticles is achieved using precise surface chemical techniques. In particular, electron beam irradiation 

of a self-assembled monolayer of NPPTMS on a silicon wafer through a mask modifies the NO 2 

terminating SAM to an NH 2 terminating SAM, thus changing the functionality of molecules in the exposed 

area. This leads to the guided self-assembly of (subsequently deposited) citrate-stabilized Au nanoparticles 

onto a specific pattern through a self-recognition process. Nanowires of nanoparticles are also created 

using direct electron beam writing. Finally, Au nanoparticles with controlled size and symmetry have been 

prepared using a size-selected cluster beam source, creating the potential for the investigation of single 

nanoparticle fluorescence in dilute arrays. 

219


NANO-6 

New synthesis, characterization and photophysical properties of 

tert-butyl alkoxide-stabilized silver nanoparticles 

L. Balan a , J. P. Malval a , R. Schneider b , D. Burget a 

a Département de Photochimie Générale, UMR CNRS 7525, Université de Haute-Alsace, ENSCMu, 3 rue 

Alfred Werner, 68093 Mulhouse Cedex, France. 

Corresponding author : E-mail: Lavinia.Balan@uha.fr 

b Laboratoire de Chimie Physique et Microbiologie pour l’Environnement - UMR CNRS-UHP 7564, 

Faculté de Pharmacie, Nancy Université, 30 rue Lionnois, BP 80 403, 54001 Nancy Cedex, France. 

Nanoscale metal particles such as silver provide a very exciting research field due to their 

interesting optical, electronic, magnetic and catalytic properties [1]. We report here a new solution 

phase synthetic route to prepare silver(0) nanoparticles and their optical properties. 

Silver(0) particles were produced by a simple and efficient low temperature solution phase 

reduction of AgNO 3 using t-BuONa-activated sodium hydride in THF [2]. Gram-scale quantity of 

nearly monodisperse Ag(0) nanoparticles can be readily prepared using this method. The resulting 

t-BuONa-stabilized silver nanoparticles were characterized by transmission electron microscopy 

(TEM) (fig.1), X-ray powder diffraction (XRD) and UV-vis spectroscopy. The X-ray powder 

diffraction patterns of these particles show the cubic structure of Ag metal. The particle diameter 

of silver(0) particles in the as-synthesized material is ca. 3.4 nm. t-BuONa-coated silver 

nanoparticles are stable in solution under inert atmosphere. 

ABS. 

0.1 

(a) 

0.0 

5x10 5 

400 500 600 700 800 

FLUO. / a.u. 

(b) 

400 500 600 700 800 

! / nm 

Fig. 1. Bright-field TEM micrograph of 

silver nanoparticles. 

Fig. 2. (a) Absorption spectra of Ag(0) dispersed in THF 

under inert (dashed line) and under air (full line) 

atmosphere. (b) Fluorescence spectra of Ag(0) 

nanoparticles upon air exposure. 

The non-fluorescent silver 

nanoparticles dispersed in THF exhibit a broad and structured fluorescence band when exposed to 

oxygen. This phenomenon is accompanied by a red shift of the surface plasmon absorption band 

(fig. 2) [3,4]. This phenomenon is attributed to the generation of charged nanoclusters, Ag m 2+ , 

produced by oxidation and subsequent chemisorption of Ag + onto the metal surface [5]. Charged 

nanoclusters resulting thereof are fluorescent and present a very large structured band centered at 

550 nm. 

References: [1] M.D. Malinsky, K.L. Kelly, G.C. Schatz, R.P. Van Duyne, J. Am. Chem. Soc. 123 (2001) 1471. 

[2] L. Balan, J.P. Malval, R. Schneider, D. Burget, Mater. Chem. Phys. (2007) in press. [3] L.A. Peyser, T.-H. Lee, 

R.M. Dickson, J. Phys. Chem. B 106 (2002) 7725. [4] J. Zheng, R.M. Dickson, J. Am. Chem. Soc. 124 (2002) 13982. 

[5] M. Treguer, F. Rocco, G. Lelong, A. Le Nestour, T. Cardinal, A. Maali, B. Lounis, Solid State Sci. 7 (2005) 812. 

220


NANO-7 

Fluorescence quantum yield and stability of CdSe/ZnS quantum dots - 

influence of the thickness of the ZnS-shell 

Markus Grabolle a , Jan Ziegler b , Alexei Merkulov c , Thomas Nann b , Ute Resch-Genger a 

a Federal Institute for Materials Research and Testing (BAM), Richard-Willstaetter-Str. 11, 

D-12489 Berlin, Germany, b School of Chemical Sciences and Pharmacy, University of East Anglia (UEA), 

Norwich NR4 7TJ, UK, c Laboratory for Nanosciences, Freiburg Material Research Centre (FMF), 

Albert Ludwig University Freiburg, Stefan-Meier Str. 21, 79104 Freiburg, Germany 

Semiconductor nanocrystals (NCs) or so-called quantum dots (QDs) represent a new class of fluorescence 

markers which are of increasing importance in bioanalytical applications and biological imaging. [1] This 

luminescent nanocrystals overcome many drawbacks of organic fluorophores. They show high fluorescence 

quantum yields, a narrow and symmetric emission spectrum (FWHM < 30-40 nm), a high absorption 

coefficient over a wide wavelength range and are very stable against photobleaching. The onset of 

absorption and the emission wavelength can easily be tuned by the size of the particles. [2,3] 

These luminescent NCs usually consist of a low-bandgap semiconductor core of some nm diameter 

(typically II/VI-systems e.g. CdSe) and a shell of a high bandgap material (e.g. ZnS) of some monolayers 

thickness. This shell is crucial for the luminescence properties and photochemical stability of the 

nanocrystal. It saturates defect states and dangling bonds on the surface, which favor undesired nonradiative 

recombination and long wavelength (trapped) emission, and protects the sensitive core against 

photo-induced degradation. [4] Up to now, little work has been done to investigate the relation between the 

thickness and quality of this inorganic shell and the photochemical stability and fluorescence properties of 

the nanocrystals. 

We studied the fluorescence quantum yield and the stability of CdSe/ZnS core/shell systems in dependence 

of the thickness of the ZnS-shell. A clear correlation is found between the shell thickness, stability and 

fluorescence quantum yield. The quantum yield increases from below 5 % up to 50 % with increasing shell 

thickness. At the same time, the stability of the shell increases as revealed by a new shell test. This test is 

based on the reaction of nanocrystals with photochemically formed thiophenol radicals and communicates 

an imperfect shell by a rapid and complete loss of fluorescence due to the non-radiative deactivation of the 

luminescent state. 

References: [1] H. Y. Fan, K. Yang, D. M. Boye, T. Sigmon, K. J. Malloy, G. F. Xu, G. P. Lopez, C. Brinker, 

J. Science 304 (2004) 567-571. [2] M. Bruchez, Jr., M. Moronne, P. Gin, S. Weiss, A. P. Alivisatos, Science 281 

(1998) 2013-2016. [3] 10 Y. T. Lim, S. Kim, A. Nakayama, N. E. Stott, M. G. Bawendi, J. V. Frangioni, Molecular 

Imaging 2 (2003) 50-64. [4] E. Kucur, W. Bücking, R. Giernoth, T. Nann, J. Phys. Chem B 109 (2005) 20355-20360. 

221


NANO-8 

Photonics of polymethine dyes on silver and gold nanoparticles 

Iryna Fedyunyayeva 1 , Leonid Patsenker 1,2 , Igor Borovoy 1 , Ewald Terpetschnig 2 




The interaction of light with small, sub-wavelength particles can result in a significant increase in the local 

electromagnetic field. If a fluorescent molecule is exposed to the enhanced local field of such a metallic 

nanoparticle a substantial increase in the fluorescence intensity can be observed. In contrast at small 

distances of 50 Å or less the fluorescence intensity can be totally quenched. 

We investigated the behaviour of water-soluble squaraines 1 and 2 and the bis-polymethine dye 3 in 

presence of such metallic nanoparticles. The fluorescence of dyes 1 and 2 that contain metal-reactive 

ethylthio-groups was found to be significantly decreased (up to 13 times) in aqueous solutions in presence 

of 20-nm gold nanoparticles. Dyes of similar structure lacking the ethylthio-group did not exhibit any 

fluorescence quenching in presence of the same gold nanoparticles. 

O 

HO 3 S 

NC 

CN 

SO 3 H 

N 

CH 3 

N 

NH CH 3 

N 

N 

(CH 2 ) 5 O (CH 2 ) 5 

HN O O NH 

HSO 4 

S 

1 

S 2 

S 

O 3 S 

(H 4 

COOH 

COOH 

(CH Et 

2 ) 5 

(CH 2 ) 5 

N 

SO 3 

HC HC HC 

CH CH CH 

N 

N 

N 

(CH 2 ) 4 

Et 

2 C) 

SO 3 3 

SO 3 

We also investigated the surface plasmon enhancement effect known as “radiative decay engineering”. For 

that purpose we coated one part of a quartz slide surface with a silver island film while the other one 

remained untreated. 10–15-nm silver islands were prepared by chemical deposition. To obtain the required 

distance between the silver nanoparticles and the dye a 15-nm spacer layer was deposited onto the plate. 

The spacer covering both, the silver nanoparticles and the free quartz surface was formed by cryolite 

(AlF 3·3NaF) thermal vacuum evaporation. Dye 3 having 5% fluorescence quantum yield was deposited on 

the spacer surface layer by spin-coating its aqueous solution. In this way a two- or three-layer coating was 

obtained on the quartz plate surface: spacer–dye and nanosilver–spacer–dye. Subsequently the fluorescence 

emission spectrum was measured for both layers at excitation wavelength 600 nm. The fluorescence 

intensity of dye 3 located at a certain distance from the silver nanoparticles was found to be 2.3 times 

higher as compared to the dye deposited directly to the quartz slide. The size of silver islands and the spacer 

layer were not optimized and we expect to obtain even a higher enhancement of the spectral response after 

optimization of these parameters. 

The aforementioned experiments show the potential of these dyes to be used for the development of 

biomedical assays that are based on the enhancement of the fluorescence intensity of such dyes in proximity 

of the metal-island surfaces. 

The work was supported by the STCU grant No. P313 and the grant No. 0107U000487 of the National Academy of 

Sciences of Ukraine. 

222


NANO-9 

Multifunctionalized biocompatible microspheres for sensing 

Rosario M. Sánchez-Martín , Lois Alexander and Mark Bradley 

School of Chemistry, University of Edinburgh, Joseph Black Building, West Mains road, Edinburgh 

EH9 3JJ, United Kingdom. E-mail: rsmartin@staffmail.ed.ac.uk 

We will present our last adventures in the use of multifunctionalized microspheres (we can routinely 

prepare a variety of mono-disperse cross-linked beads from 250nm-5µm), which remarkably, and quite 

generally, are taken up by all cell types studied to date. [1] Importantly the nature of these synthetic beads 

allows multi-step solid phase chemistry and the ability to bind essentially any molecule/sensor/nucleic acid 

to them. Importantly, these microspheres have a number of advantages over other approaches. Firstly, a 

diverse range of compounds can be attached to the microspheres including small molecule inhibitors, 

sensors, peptides, RNA and DNA. Additionally, we have demonstrated that ALL these materials are 

effectively delivered into the cells, while the cellular cargo can be modulated through modification of bead 

loading. [1] At the same time, the ability to doubly label the microspheres allows the trafficking of the loaded 

beads to be continuously monitored within the cells. Additionally, they are large enough to visualise using 

standard microscopy techniques (unlike nano-particles) and their cargos are not diluted within the cell. 

Populations of cells containing beads can be readily sorted (FACS) from other cells for subsequent analysis 

with very high, but controllable uptake rates (which can be modulated through alteration of the bead size 

and incubation time). Nowadays we have been focused on the development of these microspheres as 

sensors and we have used these devices to follow intracellular calcium changes. [2] Also we are studying the 

possibility of using them as pH sensors. 

Figure: Confocal fluorescence microscopy 

image of cellular uptake of fluorescein 

labelled- microspheres. 

References: [1] R.M.Sanchez-Martin, M. Muzerellle, N. Chitkul, S.E. How, S. Mittoo, M. Bradley, ChemBio Chem. 6 

(2005) 1341. [2] R.M.Sanchez-Martin, M. Cuttle, S. Mittoo, M. Bradley, Angew. Chem. Int. Ed.. 45 (2006) 5472. 

223


NANO-10 

Acoustically levitated droplets - new sampling method for fluorescence studies 

Jork Leiterer a , Markus Grabolle a , Knut Rurack a , Ute Resch-Genger a , Jan Ziegler b , 

Thomas Nann b , Ulrich Panne a 

a Federal Institute for Materials Research and Testing (BAM), Richard-Willstaetter-Str. 11, 12489 Berlin, 

(Germany), b University of East Anglia (UEA), School of Chemical Sciences, Norwich, NR4 7TJ, UK 

E-mail: jork.leiterer@bam.de 

Many of today’s analytical problems are characterized by small sample volumes and can only be solved 

through a corresponding miniaturization of the analytical instrumentation. In principle, analytical methods 

that are based on spectroscopic techniques are sufficiently sensitive for the analysis of small sample 

amounts, but handling of small volumes is inherently difficult due to contamination and sorption processes 

on the walls of containers. Furthermore, the sample container itself can have a non-negligible influence on 

the detected signal. 

Acoustic levitation is a powerful tool to circumvent these drawbacks. In this contact-free method of sample 

handling, solid and liquid samples are suspended in a gaseous environment by means of a stationary 

ultrasonic field. Levitated samples have a typical volume of 5 nL−5 µL (corresponding to a diameter of 

0.2−2 mm). Another advantage of ultrasonic traps as sample compartments is the possibility to conveniently 

monitor chemical and physical processes as a function of concentration. Evaporation of the solvent 

during levitation gradually decreases the droplet’s volume and allows the study of phenomena such as 

aggregation in a dynamic and continuous fashion. To monitor the size and shape of levitated droplets is thus 

critical and gradually different methods have been developed lately [1] . As a first proof of the suitability of 

this technique, the crystallization of NaCl was monitored in-situ by X-ray scattering [2] . 

Acoustic levitation used as a new technique 

to study agglomeration processes of 

nanocrystals based on the evaporation of the 

solvent of the droplets. The studied 

nanocrystals are CdSe/ZnS core/shell 

systems made by a rapid microwave 

synthesis [3] . 

In a levitated droplet measurements at very 

high analyte concentrations and volumedependent 

studies over three orders of 

magnitude are possible. 

P O 

O 

P 

P 

O 

O 

P 

P 

O 

CdSe 

ZnS 

O 

P 

P 

O 

O 

P 

O P 

TOPO 

Few monolayers of 

protection shell 

Organic capping layer for 

solubility and hybridization 

In this presentation, the spectral properties of organic dyes and semiconductor nanocrystals (quantum dots) 

are studied as a function of particle concentration. The average distance of the initially dispersed 

chromophores decrease due to evaporation of the solvent and changes in the fluorescence signals are 

observed. Because of the small sample volume used in our experiments it is possible to measure high 

sample concentrations without disturbance by inner filter effects (reabsorption), which often limits such 

fluorescence studies. Changes in the fluorescence signal can be used to follow agglomeration processes and 

to analyze distance dependent interactions. Such interactions were already studied on layers on surfaces [4] . 

In contrast to these experiments based on coating techniques, in our approach the concentration is freely 

adjustable in a continuous way. Furthermore, in contrast to layer experiments, a possible influence of the 

surface on the observed spectral changes can be excluded in the trap. 

References: [1] J. Leiterer et al., Z. Anorg. Allg. Chem., 632 (2006) 2132. [2] J. Leiterer et al., J. Appl. Crystallogr., 

39 (2006) 771-773. [3] Ziegler et al., Langmuir, (2007) accepted. [4] T. Franzl et al., Nano Lett., 4 (2004) 1599- 

1603. 

224


NANO-11 

The use of semiconductor quantum dots for the fluorescent analysis of 

NAD + -dependent enzymes and intracellular metabolic pathway 

Ronit Freeman, Ron Gill and Itamar Willner 

Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel 

Semiconductor quantum dots (QDs) exhibit unique size-controlled fluorescence functions. Although QDs 

are often used as fluorescent labels for biorecognition events, their use as dynamic labels that probe 

biocatalytic transformations is scarce. We describe the design of functionalized CdSe/ZnS QDs that enable 

the optical detection of NADH and to follow NAD + - dependent enzyme activities. The functionalized QDs 

are incorporated into HeLa cells with the aim to probe the intercellular metabolism at the single cell level. 

CdSe/ZnS were functionalized with the Nile-Blue dye that quenches the fluorescence of the QDs. The 

reduction of Nile-Blue by NADH yields the reduced dye, and this activates the fluorescence of the QDs. 

This enables the quantitative detection of the NADH cofactor. The QDs were applied to analyze ethanol in 

the presence of the NAD + -dependent alcohol dehydrogenase. The ethanol mediated formation of NADH 

allows the quantitative analysis of ethanol. As a first step toward our final goal, the functionalized QDs 

were successfully incorporated into HeLa cancer cells. 

225


NANO-12 

Determination of enoxacin using synergistic enhancement of fluorescent probe 

of Tb composite nanoparticles 

Mohammad Mainul Karim 1 , Sang Hak Lee 1 , Seikh Mafiz Alam 1 , Seung Oh Jin 1 , 

Jung Kee Suh 2 

1 Kyungpook National University, Department of Chemistry, Daegu 702-701, Republic of Korea 

E-mail: moinulcd@yahoo.com 

2 Division of Chemical Metrology and Materials Evaluation, KRISS, P.O. Box 102, Yusung, Taejon, 

305-600, Republic of Korea 

Enoxacin (ENX), the second generation drug of the quinolone antibiotics is used in the treatment of 

systemic infections including urinary tract, respiratory, gastrointestinal and skin infections. It kills bacteria 

through inhibiting cell DNA-gyrase and prohibiting DNA replication. Fluroimetric method is useful for the 

determination of various drugs. [1] Determination of enoxacin was reported in several articles. [2-4] In our 

study, terbium-acetyl acetone (acac) composite nanoparticles have been prepared under vigorous ultrasonic 

radiation. The nanoparticles synthesized are water-soluble, stable and have extremely narrow emission 

bands. They were used as fluorigenic probe for the determination of enoxacin. The fluorescence intensity of 

Tb 3+ in composite nanoparticles is synergistically enhanced by the addiction of enoxacin. The observed 

synergism could be due to the energy transfer from the enoxacin to Tb 3+ -acac composites. The enhancement 

is directly proportional to concentration of enoxacin concentrations. Under the optimum experimental 

conditions, the linear working curve was obtained over the concentration range of 1×10 -4 -2×10 -6 M with a 

correlation coefficient of 0.9987. The detection limit is 2.5×10 -7 M. The relative standard deviation is 

1.75% for 1×10 -5 M (n=10). The proposed method has been applied to the determination of enoxacin in 

pharmaceutical tablet. The method reported here is interference free. 

References: [1] M. M. Karim et al., J. Fluoresc. 16 (2006) 535. [2] M. M. Karim et al., J. Fluoresc. 16 (2006) 713. 

[3] F. You et al., Anal. Commun. 36 (1999) 231. [4] L. Yi et al., Talanta 61(2003) 403. 

226


NANO-13 

Absorption and emission properties during the formation kinetics of pyrene 

doped silica nanoparticles 

Sara Bonacchi, Marco Montalti, Luca Prodi, Enrico Rampazzo, Nelsi Zaccheroni 

University of Bologna, Department of Chemistry “G. Ciamician”, Latemar unit, via Selmi 2, 

40126 Bologna (Italy). E-mail: enrico.rampazzo@unibo.it 

Fluorescent Silica Nanoparticles [1] are polymeric nanostructures with dimension spanning the range 10- 

200nm, in which the self-organisation of a large number of fluorescent dyes provides an efficient strategy 

for the realisation and optimisation of fluorescence probes. Together with Quantum Dots (QDs) [2] these 

systems have opened, in these last decades, new interesting perspectives in the development of fluorescent 

biomarkers for applications in the filds of bioimaging, labelling and sensing. 

The most valuable characteristic of fluorescent silica nanoparticles is a large enhancement of sensitivity and 

photostability in comparison with organic fluorophores. This makes modified silica nanoparticles extremely 

attractive for applications in the fields of sensors and biosciences. 

Previous investigations show that the photophysical properties of these systems [3] strongly depend from the 

position of the fluorophores with respect to the nanoparticles surface. This makes fluorescent nanoparticles 

rather complex systems since fluorophores are confined in extremely small spaces, they can interact with 

each other [4] and, therefore, they experience mediate environmental conditions. Aim of this work is to gain 

a better understanding of dye doped silica nanoparticles formation and of the fluorophores self-organization 

inside the nanoparticles. 

O 

NH 

O 

NH 

NH 

NH 

Si 

O O Si 

O O 

R O 

R R R 

R 

NH 3 

, H 2 O, 

O R 

O, EtOH 

TEOS 

We discuss here emission and absorption data collected during the synthesis of pyrene doped Silica 

Nanoparticles. A trialchoxysilane derivatised pyrene has been synthesised and used in Stöber-based 

protocols to prepare doped nanoparticles in which the fluorophores are covalently linked to the silica 

matrix, exploiting different dye concentrations. The kinetics of formation of these nanoparticles was 

followed studying the photophysical properties of the dyes jointly to DLS (dynamic light scattering) 

measurements. Pyrene was chosen as suitable dye because of its tipical excimer formation, which 

photophysical properties can give useful insights on the organization of fluorophores inside the 

nanoparticle. 

References: [1] L. Wang, et al., Anal. Chem. A-Pages, 78 (2006) 646-654. [2] I.L. Medintz, et al., Nature Materials, 

4 (2005) 435-446. Michalet, X. et al. Science 307 (2005) 538. [3] M. Montalti, et al., Langmuir, 22 (2006) 5877. 

[4] M. Montalti et al., Langmuir, 20 (2004) 2989. 

227


NANO-14 

Size distribution of CdS nanoparticles obtained from gel electrophoresis 

templating 

Teresa S. V. Reis, Cândido A. G. Mendes, Paulo J. G. Coutinho 

Physics Department, University of Minho, Campus de Gualtar, 4710-057 Braga (Portugal). 

E-mail: pcoutinho@fisica.uminho.pt 

Nanotechnology is a field of very active investigation. There is a wide scope of technological applications 

ranging from photocatalysis and sensors to fluorescence imaging. 

In this work, cadmium sulfide (CdS) nanoparticles have been prepared by templating methods using 

surfactants and gel electrophoresis. The surfactant templating protocol was based on that of Pileni, [1] using 

AOT [sodium bis(2-ethylhexyl) sulfosuccinate] water-in-oil microemulsions. In the gel electrophoresis 

procedure, cadmium and sulfide ions migrate under an electric field in opposite directions until they meet 

and react within the gel pores. This reaction is thus controlled by diffusion through the gel pores. The 

application of reaction-diffusion processes in nanofabrication involving gels has recently been reviewed. [2] 

The size dependence of CdS electronic states was obtained by a tight binding approximation. [3] Using these 

theoretical results, in conjunction with a size distribution and a Mie formalism for the scatter/absorption of 

nanoparticles of a given size, we were able to fit the experimental absorption and excitation spectra of CdS 

nanoparticles, either in AOT reversed micelles or in dried gels. In the case of AOT templating, the resulting 

particles should be spherical and the calculated average sizes can be compared to those obtained using 

empirical relations between first absorption peak and nanoparticle size proposed by Yu et al. [4] In the gel 

electrophoresis templating experiments, the effect of excess concentration of one of the ions and the 

presence of SDS surfactant were found to influence the size distribution of the nanoparticles and the 

corresponding photoluminescence spectra. 

Fits of A - absorption 

spectra of CdS nanoparticles 

in AOT reversed 

micelles 

(ω 0 =2.5). 

B - excitation spectra 

of CdS in agarose dried 

gel (λ emission =650nm). 

Obtained size distributions 

are shown as 

the inset. The absorption 

of monodisperse 

particles with 1.8nm 

and 2.0nm is also 

shown. 

Normalized Optical Density 

1 

0.9 

0.8 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

1.8nm 

2.0nm 

A 

B 

0 

250 300 350 400 450 500 550 600 650 

Probability 


1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

A 

0 1 2 3 4 

B 

Particle radius (nm) 

Acknowledgements: Financial support from Fundação para a Ciência e a Tecnologia (FCT), Portugal. 

References: [1] J. Cizeron, M. P. Pileni, J. Phys. Chem. 99 (1995) 17410. [2] B .A. Grzybowski et al., Soft Matter 1 

(2005) 114. [3] V. A. Fonoberov et al., Phys. Rev. B 66 (2002) 085310. [4] W. William Yu et.al., Chem. Mater. 15(14) 

(2003) 2854. 

228


NANO-15 

A universal platform for designing luminescent nanosensors 

Sergey M. Borisov, Torsten Mayr, Ingo Klimant 

University of Technology of Graz, Institute of Analytical Chemistry and Radiochemistry, 

Stremayrgasse 16, 8010 Graz (Austria). E-mail: sergey.borisov@tugraz.at 

Real time optical sensing and imaging of dissolved oxygen (DO), pH, temperature, cations and anions have 

become increasingly popular in recent years. Quantification of these analytes is of wide interest in various 

fields of science and technology including biotechnology, clinical medicine, marine research and others. 

Planar sensor foils are often used for imaging purposes. [1] Alternatively, luminescent nanobeads [2] are 

versatile tools since (a), they can be easily used virtually in any type of flow-through cells and bioreactors, 

(b) allow for 3D imaging, (c) show much faster response than planar sensor foils, (d) enable measurements 

in very small volumes, and, particularly, intracellular measurements, and (e) are well suitable for multyanalyte 

measurements (e). We have developed a set of nanosensors (average size 220 nm) based on 

poly(styrene-co-vinylpyrrolidone) beads. These nanospheres show no tendency to aggregation even in 

samples with complex composition such as blood or fermentation media since the hydrophilic shell bears 

no electric charges. The respective sensor „chemistries“ are incorporated either into the hydrophobic core or 

into the hydrophilic shell of a nanobead. The sensing nanoparticles are produced from polymer emulsion 

and lipophilic dyes using an unsophisticated procedure, including swelling of the beads in organic 

solvent/water mixtures and subsequent removal of the 

solvent. Addressed staining (of the core or the shell) 

is performed depending on the type of analyte. 

Nanobeads for sensing and imaging of DO, 

temperature, pH, Cl - and Cu 2+ were developed. The 

spectroscopic schemes include luminescence decay 

time measurements, ratiometric intensity 

measurements and Dual Lifetime Referencing. 

Simultaneous determination of several analytes in 

frequency or in time domain also becomes possible 

by dispersing nanobeads of different types and using 

for example modified Dual Lifetime Referencing 

scheme. [3,4] 

References: [1] G. Liebsch et al., Appl. Spectrosc. 54 (2000) 548. [2] H. A. Clark et al., Anal. Chem. 71 (1999) 4831. 

[3] S. M. Borisov et al., Appl. Spectrosc. 60 (2006) 1167. [4] C. R. Schroeder et al., Anal. Chem. 79 (2007) 60. 

229


NANO-16 

Luminescent properties of defected and erbium-doped silica nanospheres 

Francesco Enrichi, Paolo Falcaro, Giacomo Giannini 

Associazione CIVEN – Coordinamento Interuniversitario Veneto per le Nanotecnologie, 

Marghera (Venezia) – ITALY. E-mail: enrichi@civen.org 

Monodisperse nanometer silica spheres can be obtained by condensation of tetraethylortosilicate (TEOS) 

via the Stober-Fink-Bohn process [1]. Several strategies have been recently developed to make them 

luminescent by the incorporation of organic or inorganic emission centers such as common dyes [2], rare 

earths [3] or quantum dots [4]. Most of these procedures unfortunately require multiple processing steps 

and use of expensive or toxic fluorophores. 

In this work we follow a different approach for synthesizing luminescent silica spheres. It consists in the 

calcinations of hybrid aminopropylsilica spheres by using a procedure similar to that of van Blaaderen [5] 

and Jakob [6]. 

The structural and optical properties of these spheres are presented. In particular their luminescence has 

been studied in terms of excitation, emission and time-resolved spectroscopy and optimized by variation of 

the thermal treatment and of the aminopropyl-trietoxysilane (APTES) concentration. 

Moreover, the possibility to introduce rare earths like erbium in the silica spheres was also studied, pointing 

out an interaction between the defects of the silica network and the erbium ions when APTES is used in the 

synthesis. 

The obtained results show the possibility of realizing good luminescent silica spheres by following the 

described procedure. Moreover, their cheap and easy synthesis, stability in water, possible functionalization 

and bio-compatibility makes them important alternatives to the use of quantum dots or organic dyes in 

biological imaging and other applications. 

Intensity (arb. units) 

70 

60 

50 

40 

30 

20 

PL - ex 337 nm 

APTES no treat 

APTES 400°C air 

APTES 800°C air 

10 

0 

400 450 500 550 600 650 700 750 

Wavelenght (nm) 

Photoluminescence emission from hybrid aminopropylsilica spheres after thermal treatment at different 

temperatures upon excitation at 337 nm. 

References: [1] W. Stober et al. J. Colloid Interface Sci. 26 (1968) 62. [2] I. Sokolov et al. Small. 3 (2007) 419. [3] M. 

J. A. de Dood et al. Chem. Mater. 14 (2002) 2849. [4] Y. Chan et al. Adv. Mater. 16 (2004) 2092. [5] A. van 

Blaaderen, A. Vrij. J. Colloid Interface Sci. 156 (1993) 1. [6] A. M. Jakob, T. A. Schmedake. Chem. Mater. 18 (2006) 

3173. 

230


NANO-17 

Application of up-converting phosphor particles in lab-on-a-chip devices 

Hans J. Tanke, Paul Corstjens. 

Leiden University Medical Center, Department of Molecular Cell Biology; PO Box 9600; 

NL-2300 RC Leiden (The Netherlands). E-mail: h.j.tanke@lumc.nl. 

Up-converting phosphor (UCP) reporter particles are being applied in microfluidic devices developed for 

point-of-care testing of infectious diseases. In the design of the microfluidic device, three aspects are to be 

distinguished: (1) sample acquisition, (2) sample processing, and (3) analyte detection. The analyte 

detection part utilizes UCP reporter particles integrated in effective rapid lateral flow based assays. The 

overall design of the microfluidic device is modular in a way that various types of biomolecules can be 

detected when desired. 

In a model study we used HIV infection. The goal in this particular case was to demonstrate the 

simultaneous detection of human antibodies against the pathogen (to reflect the immune status of the host) 

as well as pathogen-derived proteins and nucleic acids (to obtain a measure of viral load). A prototype was 

developed to perform both reactions, including a PCR step on a single credit card size chip. UCP reporter 

particles were used as reporters for the lateral flow assay. A portable detector (in the future to be handheld) 

was used to quantitatively evaluate the lateral flow results using an IR laser. 

Assays to simultaneously detect different types of biomolecules require a multifacetted and versatile 

approach in the sample processing and analyte detection path. We discuss different aspects of the 

microfluidic device in respect to sample processing (e.g. mixing, fluid propulsion, purification and 

concentration) and analyte detection (e.g. lateral flow, UCP particles storage and functionalizing, UCP 

assay formats). 

The microfluidic device is developed in particular for infectious disease testing using non-invasive oral 

fluid (saliva). Research groups from University of Pennsylvania, New York University, Lehigh and Leiden 

University Medical Center are collaborating in this multi-disciplinary project supported by NIH grant UO1- 

DE-017855. 

Also see: http://www.nyu.edu/projects/mod/index.html 

231

232

Part VII 

Other 

Materials 

233

234

Abstracts Poster – Part VII: Other Materials 

MATR-1 ATR-235 

Using intramolecular charge transfer in conjugated polyfluorene derivatives as 

a new strategy towards white polymer LEDs 

Fernando B. Dias, Andy Monkman, Igor F. Perepichka, Martin R. Bryce 

OEM Research Group, Department of Physics, University of Durham, Durham DH1 3LE, (UK). 

E-mail: f.m.b.dias @durham.ac.uk 

New solid state light emitting panels, based in a single organic conjugated polymer layer, promises to be a 

flexible, cheap alternative to low energy consumption ligthning applications. To achieve that goal new 

organic polymers with emission spanning across the entire visible spectrum have to be sinthesized and 

characterized. Here we report the photophysical characterization of new luminescent fluorene 

dibenzothiophene-dioxide co-oligomers and co-polymers, having both balanced charge injection and 

mobility and showing dual electroluminescence with potential to give broad light emission with good 

external efficiency 1,2 . 

The strong solvatochromism observed for two fluorene–dibenzothiophene-S,S-dioxide oligomers in polar 

solvents has been investigated using steady-state and time resolved fluorescence techniques. A low energy 

absorption band, attributed to a charge transfer (CT) state, is identified showing a red shift with increasing 

solvent polarity. In non-polar solvents, emission of these conjugated luminescent oligomers shows narrow 

and well resolved features, suggesting that the emission comes from a local excited state (LE), by analogy 

with their conjugated fluorene based polymer counterparts. However, in polar solvents only a featureless 

broad emission is observed at longer wavelengths (CT emission). A linear correlation between the energy 

maximum of the fluorescence emission and the solvent orientation polarizability factor Δf (Lippert-Mataga 

equation), is observed through a large range of solvents. In ethanol, below 230 K, the emission spectra of 

both oligomers show dual fluorescence (LE-like and CT) with the observation of a red-edge excitation 

effect. The stabilization of the CT emissive state by solvent polarity is accompanied/followed by structural 

changes to adapt the molecular structure to the new electronic density distribution. Above 220 K in ethanol, 

the solvent reorganization occurs in a faster time scale (less than 10 ps at 290 K), and the structural 

relaxation of the molecule (CT unrelaxed →CT Relaxed ) can be followed independently. The magnitude of the 

forward rate constant k 1 (20°C)∼20×10 9 s –1 , and the reaction energy barrier E a ∼3.9 kcal mol –1 close to the 

energy barrier for viscous flow in ethanol (3.54 kcal mol –1 ), show that large amplitude molecular motions 

are present on the stabilization of the CT state. 

LE and ICT emission spectra of 

3,7-bis-(9,9-dihexyl-2-acetylenefluorene)-dibenzothiophene-S,Sdioxide 

. 

The study done with oligomers is extended to co-polymers with different fractions of S units randomly 

distributed along the polymer backbone (PFSx). We demonstrate that like in the co-oligomers, the 

formation of a CT state in polar solvents is also present in the PFSx copolymers, and the impact on the 

photoluminescence (PL), both in solution and solid state and electroluminescence (EL) is discussed as a 

potential strategy to conceive high efficient white Polymer LEDs. 

References: [1] Fernando B. Dias, et al.; J. Phys. Chem. B. 110 (2006) 19329. [2] I.I. Perepichka, et al. Chem. 

Commun.27 (2005) 3397. 

235



Photoluminescence study of SrBi 2 Nb 2 O 9 doped with Eu 3+ obtained by 

a soft chemistry route 

Diogo P. Volanti a , Ieda L. V. Rosa b , Laécio S. Cavalcante b , Elaine C. Paris a , Miryam R. 

Joya a , E. Longo a , José. A. Varela a 

a LIEC-IQ-Universidade Estadual Paulista, R.Francisco Degni, s/n, Bairro Quitandinha, CEP 14800-900, 

Araraquara, SP, (Brazil). 

b LIEC-DQ-Universidade Federal de São Carlos, Rod. Washington Luiz, km 235, C.P. 676, CEP 13565- 

905, São Carlos, SP, (Brazil). E-mail: ilvrosa@power.ufscar.br 

Aurivillius family is widely studied nowadays due to its ferroelectric properties which make then a 

potential component for nonvolatile memory. This kind of material has a layered structure consisting of 

interconnected [Bi 2 O 2 ], fluorite like, and [A x-1 B x O 3x-1 ], perovskite like, blocks which general formula is 

[Bi 2 O 2 ][A x-1 B x O 3x-1 ] [1] . According to the literature it belongs to the A2 1 am space group derived from the 

I4/mmm space group when A cation is displaced by the BO 6 units [2] . In this work it is described the 

synthesis of the SrBi 2 Nb 2 O 9 doped with 1.0 mol % of Eu 3+ (SrBi 2 Nb 2 O 9 :Eu 3+ ) by the polymeric precursor 

method [3] . These materials were annealed for different temperatures for 2 hours under oxygen atmosphere. 

X ray diffraction (XRD), Raman spectroscopy and the photoluminescent properties of both materials were 

used to characterize then. 

(a) 

Fig. 1. Emission spectra of the 

SrBi 2 Nb 2 O 9 :Eu 3+ at room temperature, 

λ EXC. = 488 nm annealed at (a) 400, (b) 450, 

(c) 500, (d) 550, (e) 600, (f) 650 and (g) 

700ºC for 2 hours under oxygen 

atmosphere. 

Relative Intensity (a. u.) 

(b) 

(c) 

(d) 

(e) 

(f) 

(g) 

450 500 550 600 650 700 750 800 850 


Both XRD and Raman spectroscopy data are consistent with the fact that the presence of Eu 3+ favours the 

crystallization of the perovskite phase (orthorhombic) in detriment of the fluorite one (tetragonal). As the 

annealed temperature is increased it was observed in the emission spectra of the SrBi 2 Nb 2 O 9 :Eu 3+ (see Fig. 

1) the disappearance of the broad band at around 560 nm and the appearance of the characteristic transitions 

5 D J → 7 F J’ (J= 0, 1 and 2, while J’= 1, 2, 3 and 4) of the Eu 3+ , when this material is excited at 488 nm. 

Visible photoluminescence broad band and the appearance of Eu 3+ emission are efficient to monitor the 

order/disorder during the heat-treatment process, thus allowing the short and intermediate structural range 

order analysis. 

Acknowledgements: CAPES, FAPES and CNPq. 

References: [1] B. Aurivillius, Ark. Kem. 1 (1949) 463. [2] C. H. Hervoches et al. J. Solid State Chem. 164 (2002) 

280. [3] T. Asai, et al. J. Alloys. Comp. 309 (2000) 113. 

236



Platinum phosphor incorporated into a blue luminescent polymer 

Christian Slugovc,* Fabian Niedermair, Gabriele Kremser 

Institute for Chemistry and Technology of Organic Materials (ICTOS), Graz University of Technology, 

Stremayrgasse 16, A-8010 Graz, Austria. E-mail: slugovc@tugraz.at. 

A significant research effort focuses on luminescent d 8 transition metal complexes and their potential 

applications in different fields such as chemical sensors, [1] organic light emitting devices (OLEDs), [2] or 

photovoltaics. [3] For applications often a host material is necessary, which has to be selected carefully, e.g., 

in terms of providing efficient Förster energy transfer to name one important prerequisite. Polymeric hosts 

are of great interest as they are more amenable to solution processing techniques such as spin-coating or 

ink-jet printing than small molecules. Moreover, covalent incorporation of the guest into the host-polymer 

constitutes a further step forward for an easy and reliable processing of the material. 

Statistical copolymer comprising the host-dye (blue) and the guest (orange) as well as absorbance and 

luminescence spectra of poly2/3 (blue) and the guest as monomer (orange). Insets show photographs of 

poly2/3 in solution (left vial) and in the solid state (red vial). 

Herein we wish to report our endeavors to prepare polymerizable platinum(II) complexes by discussion of 

the synthesis and photophysical properties of a Pt(II)quinolinolate derivative statistically copolymerized 

with a suitable luminescent host material. As the polymerization method Ring Opening Metathesis 

Polymerization (ROMP) was used because of its excellent functional group tolerance. [4] As revealed by 

photophysical measurements, efficient energy transfer from the host to the platinum-guest in the solid state 

occurred. As a result, red emission of the platinum complex could be obtained by exciting the host material 

at 360 nm. The energytransfer is affected by external stimuli such as temperature or organic solvent vapors. 

Corresponding sensory characterizations and the applicability of such polymers as oxygen-sensor materials 

will be disclosed. 

Financial support by the Austrian Science Fund (FWF) in the framework of the Austrian Nano Initiative (Research 

Project Cluster 0700 - Integrated Organic Sensor and Optoelectronics Technologies – Research Project 0701) is 

gratefully acknowledged. 

References: [1] (a) S. W. Thomas III, S. Yagi, T. M. Swager, J. Mater. Chem. 15 (2005) 2829. (b) Y. Kunugi, K. R. 

Mann, L. L. et al. J. Am. Chem. Soc. 120 (1998) 589. [2] W. Lu, et al. J. Am. Chem. Soc. 126 (2004) 4958. [3] J. E. 

McGarrah, R. Eisenberg, Inorg. Chem. 42 ( 2003) 42, 4355. [4] C. Slugovc, Macromol. Rapid. Comm. 25 (2004) 

1283. 

237



Nanostructured cationic platinum dyes via the self assembly of 

ROMP block copolymers 

Kurt Stubenrauch, Fabian Niedermair, Gregor Trimmel, Christian Slugovc* 

Graz University of Technology, Institute of Chemistry and Technology of Organic Materials, 

Stremayrgasse 16, 8010 Graz, Austria, e-mail: k.stubenrauch@tugraz.at 

Luminescent positively charged platinum complexes have recently attracted intense scientific interest due 

to their bright phosphorescence in the visible region of the electromagnetic spectrum. 1 It is worth 

mentioning that such platinum complexes tend to form aggregates, stabilized by platinum-platinum 

interaction, which result in significant red shifts relative to the mononuclear emission spectra. 2 

A novel cationic platinum dye was synthesised bearing 3-hexyloxy-2-phenylpyridine and 1,10- 

phenanthroline as cyclometalating ligands. Luminescence is mainly observed in the solid state where close 

proximity of the dyes is given whereas in solution no phosporescence could be observed. 

Our approach is to self assembly the cationic dye using a polyelectrolyte block copolymer. Ring opening 

metathesis polymerisation (ROMP) was used as polymerisation technique for the preparation of well 

defined homo and block copolymers. 3 As hydrophobic monomer we chose endo,exo[2.2.1]bicyclo-hept-5- 

ene-2,3-dicarboxyclic acid dimethylester and as hydrophilic building block endo,exo[2.2.1]bicyclo-hept-5- 

ene-2,3-dicarboxyclic acid. For the polymer synthesis the acid functionalities were protected with tert-butyl 

group enabeling controlled synthesis and complete characterisation. 4 

In a mixture of a block copolymer and the platinum dye the polyacid block acts as polyanion where the 

cationic platinum complexes are concentrated. Pure platinum dye solution does not show luminescence 

whereas in the prescence of a block copolymer bright red luminescence is observed due to the accumulation 

of the dyes in the polyacid block. The close proximity of the dyes leds to a switching on of luminescence. 2 

H H 

O O 

O 

O 

CF 3 COO 

Hex 

N N 

Ph 

O 

Pt 

N 

m 

n 

O O 

O O 

a) 

b) 

c) 

Figure 1: a) Cationic platinum dye: [Pt(hoppy)phen]CF 3 CO 2 (hoppy = 3-hexyloxy-2-phenylpyridine, phen 

= 1,10-phenanthroline); b) ROMP block copolymer: poly-[(endo,exo[2.2.1]bicyclo-hept-5-ene-2,3- 

dicarboxyclic acid dimethylester)-b-(endo,exo[2.2.1]bicyclo-hept-5-ene-2,3-dicarboxyclic acid)]; 

c) Schematic presentation of the accumulation of the positvely charged dye in the poly acid block due to 

ionic interaction and it`s luminescence (red) 

Films were prepared dropcasting the block copolymer – dye solution onto a substrate. This type of block 

copolymer is known to self assembly into morphologies on the nanosacle. The dye can be therefore 

nanostructured being incorporated in just one block of a for examle lamellae forming 1:1 block copolymer. 

The self assembly was studied with DLS, SAXS, TEM and Fluorescence spectroscopy. 

References: [1] F. Camerel et al, Angew. Chem. 119 (2007) 2713, [2] W. Lu et al, J. Am. Chem. Soc. 126 (2004) 

7639, [3] Riegler et al. Macromol. Symp. 217 (2004) 231, [4] K. Stubenrauch et al., Macromolecules 39 (2006) 

5865. 

238



Use of DASPMI to monitor the viscosity of sol-gel derived monoliths during 

gelation and aging processes 

Graham Hungerford 1,2 , Ana Rei 1 , M. Isabel C. Ferreira 1 



E-mail: anarei@fisica.uminho.pt 

Stillbazolium salts present remarkable potential for application on several scientific areas. This versatile 

behaviour is explained invoking the “twisted intramolecular charge transfer” (TICT) mechanism, a model 

that explains the multiple fluorescence of DASPMI (4-(4-(dimethylamino)styryl)-N-methylpyridiniumiodine) 

[1,2] . One feature of their behaviour is the sensitivity of the fluorescence lifetime to 

viscosity, thus suggesting them as adequate probes for micro-heterogeneous systems, such as sol-gel 

derived media [3] and cells [4] . 

The sol-gel process has been successfully used to produce hosts to biomolecules like proteins, for biosensor 

applications. Due to their optical transparency, sol-gel matrices are light addressable, therefore suitable for 

performing spectroscopic studies. When incorporating enzymes into host media, it is essential to ascertain 

the flow of both substrate and reaction products. Our purpose is to understand the evolution of the viscosity 

(and therefore, the mass transport of reactants) throughout the gelation and aging processes of the matrix. 

Although extensively used, silica sol-gel derived matrices are not always ideal hosts for biomolecules. In 

this study, modification of the matrices was attempted by altering the hydrophobic / hydrophilic balance of 

the interior of the pores, by capping un-reacted OH groups with alkyl and other groups, or by the addition 

of stabilisers. This was achieved, employing two routes; one, the modification of the sol preparation 

reaction; the other, by adjustment, during the matrix manufacturing procedure. The effect on the local 

viscosity induced by these modifications was monitored via time-resolved fluorescence spectroscopy 

measurements of DASPMI. Although both the fluorescence lifetime and quantum yield are influenced by 

viscosity, the lifetime measurement is not affected by the shrinkage of the matrix during the aging process 

(leading to changes in dye concentration), thus providing a good means by which to monitor modifications 

intrinsic to the host medium. 

References: [1] B. Strehmel, W. Rettig, J. Biomed.Optics, 1 (1) (1996), 98. [2] B. Strehmel et al., J. Phys. Chem. B, 

101 (1997), 2232. [3] G. Hungerford, et al., J. Fluorescence, 12 (2002), 397. [4] K. Kemnitz in: New Trends in 

Fluorescence Spectroscopy, B. Valeur, J.-C. Brochon (Eds.) Springer Berlin, 2001, p381. 

239



Effect of polymer strengtheners on the local environment of biocompatible 

glass as probed by fluorescence 

Graham Hungerford 1,2 , Mariana Amaro 1 , Pedro Martins 1 , M. Isabel Ferreira 1 , Mahesh 

Uttamlal 3 and A. Sheila Holmes-Smith 3 

1 Centro de Física, Universidade do Minho, 4710-057 Braga, Portugal 


3 School of Engineering, Science and Design, Glasgow Caledonian University, Cowcaddens Road, 

Glasgow G4 0BA, Scotland, UK. e-mail: mamaro@fisica.uminho.pt 

There are many ways to produce bioactive glass for the purpose of bone tissue scaffolds amongst other 

applications 1 . Most methods involve high temperatures when creating the matrix via a sintering phase 2 . 

However, it is possible to produce this type of material by the sol-gel technique, through a direct method at 

ambient / room temperature 3 . This is important as it avoids the problem of temperature degradation when 

incorporating probes or biological materials, such as proteins, during matrix production. It is important to 

ascertain how these materials interact with biological molecules and thus knowledge on the local matrix 

environment and how this can affect any adsorbed protein is vital. 

The samples prepared in our investigation were based on a “low” temperature (sol-gel) method, however 

they were found to be brittle and in need of reinforcement. In order to strengthen them, biocompatible 

polymers were added. The polymers used were polyethylene glycol (PEG) (several molecular weights), 

polymethyl methacrylate (PMMA) and polyethylene (PE). The control over pore size was explored and 

optimized due to its importance if the material is to be used for scaffold or drug release applications. The 

monitoring of the pores size was made using scanning electronic microscopy (SEM). 

In order to analyse and characterise the sample’s microenvironment, the probe Nile red was used. Nile red 

is a fluorescent dye that displays a spectroscopic behaviour largely dependent upon the polarity of the host 

medium 4 , thus providing elucidation on the role of polymer addition upon local environmental effects in the 

host media. This study also included the monitoring of protein adsorption to the matrices, by means of 

bovine serum albumin, making use of either intrinsic fluorescence or the addition of a covalently bound 

probe, in order to learn about the influence of the polymer addition to the matrix on the protein adsorption 

properties and any induced conformational change. 

References: [1] H. Podbielska, A. Ulatowska-Jarza, Bull. Pol. Ac.: Tech. 53, 3, (2005) 261. [2] M. Vallet-Regi, 

J Chem. Soc, Dalton trans. (2001) 97. [3] S.R. Hall, et al., J Mater. Chem. 13 (2003) 186. [4] G. Hungerford, et al., 

FEBS Journal. 272 (2005) 6161. 

240



SiO 2 -GeO 2 soot perform as a core for Eu 2 O 3 nanocoating: 

Synthesis and photophysical study 

Ieda L. V. Rosa a , Larissa H. Oliveira a , Elson Longo a,b , Edson R. Leite a , José A. Varela b 

a UFSCar, Depart. of Chemistry, Caixa Postal 676, 13560-905, São Carlos, SP, Brazil. b UNESP, 

Institute of Chemistry , Caixa Postal 355, 14801-970, Araraquara, SP, Brazil 

Nowadays solid state chemists have the possibility of work with low temperature strategies to obtain solid 

state materials with appropriate physical and chemical properties for useful technological applications [1]. 

Photonic core shell materials having core and shell domains of a great variety of compounds have been 

synthesized by different methods [2-4]. 

In this work we used silica-germania soot (SiO 2 -GeO 2 ) prepared by vapor–phase axial deposition [5] as a 

core where a nanoshell of Eu 2 O 3 was deposited. A new sol-gel like method [3] was used to obtain the Eu 2 O 3 

nanoshell coating the SiO 2 -GeO 2 particles. The photophysical properties of Eu 3+ were used to get 

information about the rare earth surrounding in the SiO 2 -GeO 2 @Eu 2 O 3 material during the sintering 

process. 

(a) 

(b) 

Fig. 1. Emission spectra of the SiO 2 -GeO 2 @Eu 2 O 3 

annealed at 100 (a), 300 (b), 400 (c), 500 (d), 800 

(e) and 1000 o C (f), excited at 394 nm, recorded at 

room temperature. 

Relative Intensity (a.u.) 

(c) 

(d) 

(e) 

(f) 

500 550 600 650 700 750 


The sintering process was followed by the Luminescence spectra of Eu 3+ obtained at room temperature 

(Fig.1). All of the samples presented the characteristic emissions related to the 5 D 0 → 7 F J transitions, where 

J=0, 1, 2, 3 and 4. The hypersensitive transition 5 D 0 → 7 F 2 is strongly dependent on the Eu 3+ surrounding 

due to its electric dipole character, while the intensity of the 5 D 0 → 7 F 1 , a magnetic dipole transition, is 

almost independent. The ratio of the 5 D 0 → 7 F 2 / 5 D 0 → 7 F 1 emission intensity for the SiO 2 -GeO 2 @Eu 2 O 3 

system was calculated and it was observed an increase in its values, indicating a low symmetry around the 

Eu 3+ as the temperature increase. 

Acknowledgements: CAPES, FAPESP, CNPq and Prof. Carlos K. Suzuki (UNICAMP) for provide us the silicagermania 

soot (SiO 2 -GeO 2 ). 

References: [1] H. Lee, L. J. Kepley, H-G. Hong, T. E. Mallouk, J. Am. Chem. Soc. 110 (1988) 618. [2] L. S. 

Cavalcante, M. F. C. Gurgel, A. Z. Simões, E. Longo, J. A. Varela, M. R. Joya, P. S. Pizani, Appl. Phys. Letters 90 

(2007 ) 011901. [3] I. L. V. Rosa, A. P. Maciel, E. Longo, E. R. Leite, J. A. Varela, Mater. Research Bull. 41 (2006) 

1791. [4] P. Schuetzand, F. Caruzo, Chem. Mater. 14 (2002) 4509. [5] E. H. Sekiya, D. Torikai, E. Gusken, D. Y. 

Ogata, R. F. Cuevas, C. K. Suzuki, J. Non-Cryst. Solids 273 (2000) 228. 

241



Suitability of modified sol-gel derived monoliths for enzyme incorporation 

monitored by fluorescence techniques and catalytic activity measurements 

Ana Rei 1 , M. Isabel C. Ferreira 1 and Graham Hungerford 1,2 


2 Physics Department, King´s College London, Strand, London WC2R 2LS, UK. 

E-mail: anarei@fisica.uminho.pt 

Silica sol-gel derived matrices have been extensively used to incorporate biomolecules, such as enzymes [1] . 

However, the properties of the host media are not always ideal for enzymes to exhibit their catalytic 

potential in full [2] . This, in part, relates to the internal pore environment, in terms of pore size and the 

presence of unreacted surface groups which can affect the local polarity. Both these factors have an 

influence over the enzyme conformation. 

An adequate compromise can be achieved by capping unreacted OH groups with alkyl and other groups, or 

by the addition of stabilisers. In the present work this strategy was adopted by employing two routes; one, 

the modification of the sol preparation reaction, the other, by incorporating additives during the matrix 

manufacturing procedure. 

Two enzymes, Subtilisin Carlsberg and cytochrome c, were encapsulated in the differently modified host 

media and effects on their conformation (in relation to a solution study) monitored, making using of a 

highly solvatochromic dye, Nile red, in conjunction with synchronous scan fluorescence spectroscopy. We 

have previously found this dye to be a suitable probe with which to follow the encapsulation process, by 

making use of spectral decomposition [3] . The synchronous scan method, however, leads to a simplification 

in ascertaining changes in the local environment sensed by this probe. Comparative measurements were 

also performed to confirm that the encapsulated biomolecules were accessible and able to exhibit their 

catalytic activity. 

References: [1] D.Avnir et al., J.Mater. Chem., 16 (2006), 1013. [2] R. Gupta, N. K. Chaudhury, Biosens. 

Bioelectron. (2007), in press. [3] G. Hungerford, et al., Biophys. Chem., 120 (2006), 81. 

242



Novel diphenylpyrrolopyrroles for electroluminescence applications 

Martin Vala (1) , Martin Weiter (1) , Miroslava Krcmova (1) , Petra Jerabkova (1) , Jan Vynuchal (2) , 

Petr Toman (3) 

(1) 

Brno University of Technology, Faculty of Chemistry, Purkynova 118, 612 00 Brno, Czech Republic. 

(2) 

E-mail: vala@fch.vutbr.cz Research Institute of Organic Syntheses, Rybitvi 296, 532 18 Pardubice 20, 

Czech Republic. (3) Academy of Sciences of the Czech Republic, Institute of Macromolecular Chemistry, 

Heyrovsky Sq. 2, 162 Praque, Czech Republic. 

Nowadays, we can see strong effort to produce Organic electroluminescent devices (OLED) that can be 

used as a completely new generation of lamps and monochromatic or even full colour flat panel displays. 

The potential advantages of these devices are high efficiency, low driving voltage, versatility in its 

application (flexibility, transparency), low weight, relatively cheap production, etc. The materials used to 

build electroluminescent device have to fulfil whole range of requirements, e.g. high fluorescence quantum 

yield good transporting properties for charges which have to recombine, good film forming properties, 

fatigue resistance, etc. It is therefore more common to incorporate fluorescent dye with high quantum yield 

to the carrier transporting host layer. Small molecular organic materials are particularly suitable mainly for 

their ability to meet the needs drawn above. 

In this study we investigated group of several derivatives of 3,6-diphenyl-2,5-dihydro-pyrrolo[3,4- 

c]pyrrole-1,4 dione, also known as DPP, see Figure 1. DPP itself has a high quantum yield of fluorescence, 

as well as a high molar decadic absorption coefficient. Although it has been already reported that several 

derivatives are potentially suitable for electro–optical applications, these materials are mainly used as high 

performance pigments. However, for the optoelectronic applications also compatibility with charge 

transporting materials is required to produce cheap devices with high performance. Several derivatives have 

been synthesised and studied with respect to this demand. The change of the structure results also in 

modification of the other required 

properties such as the fluorescence 

quantum yield, position of HOMO and 

LUMO orbitals necessary for efficient 

charge transfer, etc. In addition to 

experimental optical characterization 

also quantum chemical calculations 

were employed to determine these 

parameters and to find links between 

the structure and desired functionality. 

Figure 1: The basic structure of 3,6-diphenyl-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4 

dione, also known as DPP. 

This work was supported by the project IAA401770601 from the Academy of Sciences of the Czech Republic and by 

project No. 0021630501 from Ministry of Education, Youth and Sports of the Czech Republic. We also thank to 

Ministry of Industry and Trade of the Czech Republic for support via Tandem project No. FT-TA3/048. 

243



Eu 3+ -doped rare earth orthophosphates obtained by polymeric method 

Paulo C. de Sousa Filho, Osvaldo A. Serra 

University of São Paulo; Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto; Chemistry 

Department; Av. dos Bandeirantes, 3900, CEP: 14040-901 – Ribeirão Preto, SP (Brazil). 

E-mail: osaserra@usp.br 

Rare Earth phosphates have been widely studied as hosts for activator centers because of their favorable 

chemical and physical properties. Some orthophosphates have already been commercially employed, while 

others are potential inorganic luminescent materials. In this work, Rare Earth orthophosphates were 

obtained by a modified Pechini method. The developed synthetic route was based on the ability of the 

tripolyphosphate anion (P 3 O 10 5- ) to act both as a complexing agent and as an orthophosphate precursor. 

Heating of an aqueous solution containing RE 3+ , Eu 3+ , P 3 O 10 5- , citric acid, and ethylene glycol led to a 

polymeric resin. Ignition of this resin at different temperatures yielded a luminescent orthophosphate. The 

method is applicable to the obtention of orthophosphates of several compositions, with a satisfactory 

stoichiometric control of the reactants. 

The red phosphors Y 0.96 Eu 0.04 PO 4 , Y 0.80 Gd 0.16 Eu 0.04 PO 4 , and La 0.96 Eu 0.04 PO 4 were obtained by calcination at 

650, 750, 850, and 950ºC. The infrared spectra of these compounds display the Rare Earth orthophosphates 

3- 

characteristic bands only, thus confirming that all polyphosphates were converted into PO 4 groups. 

Scanning electron micrographies show that particles are spherical, with sizes ranging between 50 and 150. 

XRD analysis give evidence that YPO 4 :Eu 3+ and (Y,Gd)PO 4 :Eu 3+ present a Zircon/Xenotime-type pattern, 

whereas LaPO 4 :Eu 3+ presents a Monazite-type pattern. The average crystallite sizes were estimated from the 

diffractograms by applying the Scherrer method. In all cases, higher calcination temperatures led to larger 

crystallites, which is an effect of the gathering of particles at higher ignition temperatures. 

The calcination temperature led to small 

changes in the photophysical properties of the 

compounds. The excitation spectra of the 

phosphors display a main band ascribed to 

excitation ( 5 L 6 level) and a charge 

transfer band above 250 nm. In 

(Y,Gd)PO 4 :Eu 3+ , a band related to Gd 3+ 

excitation ( 6 I J in 274 nm) is present, which is 

due to an energy transfer process. In 

YPO 4 :Eu 3+ , the spectral distribution of the 

emission bands indicates that Eu 3+ may lie in 

a D 2 or D 2d symmetry site, with the following 

luminescence lifetimes: 3.4, 2.5, 3.2, and 3.5 

ms (for 650, 750, 850, and 950ºC 

respectively). The set of emissions leads to 

highly pure red colours (0.61



Fluorescence imaging of film formation from polymer latex materials 

Albert M. Brouwer, a Tanzeela N. Raja, a Koen Biemans, b Tijs T. Nabuurs, b Ronald 

Tennebroek b 

a 

Universiteit van Amsterdam, Van ‘t Hoff Institute for Molecular Sciences, Nieuwe Achtergracht 129, 1018 

WS Amsterdam (The Netherlands). E-mail: a.m.brouwer@uva.nl 

b 

DSM Neoresins, Sluisweg 12, 5140AC Waalwijk (The Netherlands) 

Organic coatings play a key role in numerous technologies, yet knowledge about the process of film 

formation from various precursors is limited. We are engaged in a project in which an attempt is made to 

gain more insight into the evolution from a wet film to a solid coating by using spatially and temporally 

resolved fluorescence measurements. More specifically, the current subjects of study are so called “latices”, 

water-borne coatings prepared by emulsion polymerization. The polymers used are random copolymers of 

acrylates and styrene, which allows a wide range of physical properties (polarity, glass transition 

temperature T g ) to be obtained. 

One of the fluorescence properties that has not been extensively used in polymer chemistry is the 

solvatochromic shift of fluorescent dyes that have a nonpolar ground state and a very polar excited state. [1] 

The emission spectrum of such a dye shifts to longer wavelengths with increasing polarity of the medium, 

provided that structural relaxation is possible on the time scale of the excited state lifetime (typically 

nanoseconds). In this work we have prepared a number of materials containing a fluorescent probe 

molecule, named “maleimidofluorotrope” (MFT) covalenly linked to the polymer backbone. MFT itself is 

not fluorescent due to the presence of a low-energy locally excited state in the maleimide unit, but after 

removal of the double bond (as in butylamine adduct 1) a strong fluorescence (Φ f up to 60%) arises. [2] Also 

copolymerization of the maleimide unit leads to effective destruction of the low-energy chromophore, and 

strongly fluorescent copolymers can be made with a ppm-level content of MFT. 

Solvent effect on the 

emission spectra of 

compound 1 which serves 

as a model for the 

solvatochromic probe 

molecule MFT when 

copolymerized in various 

polymer latices. The 

maximum intensity of 

each trace corresponds to 

the fluorescence quantum 

yield. 

In order for water-borne coatings to produce good films, organic co-solvents are still indispensible. We will 

describe experiments which employ the solvatochromic fluorescence of the copolymerized MFT to shed 

light on the dynamics of such co-solvents (e.g. partitioning, evaporation) upon mixing of different latices 

and during film formation. 

This research forms part of the research programme of the Dutch Polymer Institute (DPI), project #606. 

References: [1] J. W. Hofstraat, J. Veurink et al., J. Fluoresc. 8 (1998) 335. [2] M. Goes, X. Y. Lauteslager et al., 

Eur. J. Org. Chem. (1998) 2373. 

245



Effect of temperature and oxygen on luminescence spectra and polarization 

of dibenzoxazolylbiphenyl thin films 

Alexander V. Kukhta a , Eduard E. Kolesnik a , Elena V. Dudko a , Ivan I. Kalosha a , Vitaly A. 

Tolkachev a , Vyacheslav K. Olkhovik b , Nikolay A. Galinovskii b , Konstantin A. Osipov c , 

Vyacheslav N. Pavlovskii c 

a Institute of Molecular and Atomic Physics, National Academy of Sciences of Belarus, 220072 Minsk 

(Belarus). E-mail: kukhta@imaph.bas-net.by 

b Institute of Chemistry of New Materials, National Academy of Sciences of Belarus, 

220141 Minsk (Belarus) 

c B.I.Stepanov Institute of Physics, National Academy of Sciences of Belarus, 220072 Minsk (Belarus) 

Organic thin films have attracted wide attention not only for versatile properties but also for manifold 

technological applications, such as field effect transistors, photovoltaic cells, electroluminescence diodes, 

etc. The effect of temperature and oxygen on luminescence spectra, intensity and polarization of 

dibenzoxazolylbiphenyl thin films (4,4’-bis-[(Z)-1-(1,3-benzoxazol-2-yl-2-ethenyl) biphenyl and 4,4’-bis- 

[(Z)-1-(1,3-benzoxazol-2-yl-2-ethenyl) 2-n-hexyloxybiphenyl) thermovacuum deposited on quartz glass 

substrate with the thickness in the range of 30-150 nm has been studied. Molecular aggregation with 

chromophores dipoles arranged parallel to each other has been observed in absorption and luminescence 

spectra. The reversible molecular rearrangement resulting in the formation of partly ordered structures has 

been observed under film heating below the glass transition temperature. The addition of oxygen was found 

to cause an essential luminescence quenching. The hexyloxy group in the side chain of this molecule 

decreases molecular aggregation and temperature formation of ordered structures, and increases adsorbed 

oxygen induced luminescence quenching apparently owing to the formation of more porous film 

morphology. Dibenzoxazolylbiphenyl without side groups forms highly stable and luminescent thin films 

owing to strong molecular aggregation. These properties as well as low quenching by oxygen allow to 

conclude the prospects of these substances for molecular electronics devices, in particular for 

electroluminescent diodes. 

246

Part VIII 

Biophysics 

247

248

Abstracts Poster – Part VIII: Biophysics 

BIOP-1 

Mechanistic studies on BO and TO cyanine dyes: Self-aggregation and 

interaction with nucleic acids 

Tarita Biver, a Alessia Boggioni, a Fernando Secco, a Marcella Venturini, a Sergiy Yarmoluk b 

a University of Pisa, Chemistry and Industrial Chemistry Department, Via Risorgimento 35 -56126 PISA 

(Italy). E-mail: tarita@dcci.unipi.it b Institute of Molecular Biology and Genetics of NAS of Ukraine, 

Zabolotnogo Str. 150, 03143 Kyiv (Ukraine). 

Nowadays, molecules of the cyanine family are widely used in both the fields of biomedicine and 

biochemistry, where they find application as antitumour agents and for polynucleotides probing and 

staining respectively. This latter application is strongly related to the optical property of these dyes to 

sharply increase their fluorescence emission upon interaction with polynucleotides, [1] which they bind both 

by intercalation or groove binding. [2] 

A recent investigation on the intercalation mechanism of two cyanines containing a benzothiazole residue 

into DNA [3] has shown that the binding process occurs according to a sequential three-step mechanism 

where the first step of the sequence exhibits a binding constant much higher than expected on the basis of 

the electrostatic theory. This finding indicates that forces other than electrostatic predominate in the very 

first stage of the binding process where likely “external” complex formation is involved. This type of 

external binding was previously observed with the DNA/ethidium system and ascribed to hydrophobic 

groove interactions. [4] 

In order to get further light on the binding of intercalators to nucleic acids, we have undertaken a 

mechanistic study of the interaction with DNA of two cyanines, BO and TO, which differ for the extension 

of the hydrophobic surfaces. The kinetic method, allowing the characteristic of individual reaction steps to 

be investigated, makes it possible to analyse the details of the intercalation process and to find out which 

step is mainly affected by changes of the dye structure. 

Kinetics and equilibria of the cyanine dyes thiazole orange (TO) and benzothioazole orange (BO) selfaggregation 

and binding to CT-DNA are investigated in aqueous solution at 25°C and pH 7. Absorbance 

spectra and T-jump experiments reveal that TO molecules gives rise to H-aggregates, more stable than 

those of BO, that on the contrary prefers the staggered J-aggregate form. 

Fluorescence and absorbance titrations show that TO binds to DNA more tightly than BO. 

TO stacks externally to DNA for low polymer-to-dye concentration ratios (C P /C D ) while dye intercalation 

occurs for high values of C P /C D . T-jump and stopped-flow experiments performed at high C P /C D agree with 

reaction scheme D+SD,SDS I DS II where the precursor complex D,S evolves to a partially intercalated 

complex DS I which converts to the more stable intercalate DS II . Non-electrostatic forces were indeed found 

to play a major role in D,S stabilisation. Last step is similar for both dyes suggesting accommodation of the 

common benzothioazole residue between base pairs. 

References: [1] H.S. Rye et al., Nucleic Acids Res. 20 (1992) 2803-2812. [2] H.J. Karlsson et al. Nucleic Acids Res. 

31 (2003) 6227-6234. [3] T. Biver et al. Biophys. J. 89 (2005) 374-383. [4] H.W. Zimmermann, Angew. Chem. Int. 

Ed. Engl. 25 (1986) 115-130. 

249


BIOP-2 

Interaction of tyroxine hormone with 7-hydroxycoumarin: 

a fluorescence quenching study 

Nuriye Akbay, Canan Öztürk and Elmas Gök 

Department of Chemistry, Hacettepe University, 06800 Beytepe, Ankara, TURKEY 

Thyroxine (T4, tetraiodothyronine) is one of the most important hormones of the thyroid gland. The 

hormonal activity of the gland and the importance of its iodine content for this activity have been known 

for many years [1, 2]. Coumarins exhibit strong fluorescence in the visible region which makes them 

suitable for use as colorants, in dye lasers and as nonlinear optical chromophores. 7-hydroxycoumarin (7- 

HC), also known as umbelliferone, is a major product of coumarin family. 

In this study, the interaction of T4 with 7-HC has been studied by fluorescence quenching method (Figure). 

The experiment results indicated that the probable quenching mechanism of 7-HC fluorescence by T4 is 

static quenching with a complex formation in the ground-state and due to the presence of heavy atom in 

such a complex the intersystem-crossing rate is enhanced and thus the fluorescence quantum yield is 

decreased [3]. The binding constant and binding site of T4 to 7-HC at pH 7.4 are calculated to be 1.51x10 4 

L/mol and 0.99, respectively, according to double logarithm regression curve. In addition, the binding 

properties of T4 with 7-HC in complex are investigated based on NMR and FTIR spectroscopic results. 

Figure: The quenching effect of thyroxine on 7-HC fluorescence. The inset corresponds to the Stern- 

Volmer plot. λ ex /λ em = 334/464 nm 

Acknowledgements: This work was supported by Hacettepe University Scientific Research Fund (0302601017). 

References: [1] T. Ghous, A. Townshend, Anal. Chim. Acta 411 (2000) 45.[2] E. Gök, S. Ates, Anal. Chim. Acta 505 

(2004) 125.[3] J.R. Lakowicz, Principles of Fluorescence Spectroscopy (2006) 3 rd Edition, Springer, New York. 

250


BIOP-3 

Pyrene and 1-pyrenesulfonate probes in monitoring polarity in lipid bilayers 

containing binary cholesterol mixtures and surface potential effects in 

partitioning into zwiterionic/anionic phospholipid mixtures 

Jorge Martins a,b , Dalila Arrais a , Miguel Manuel a 

a) IBB-Institute for Biotechnology and Bioengineering − CBME-Center for Molecular and Structural 

Biomedicine and b) DQBF, Faculdade de Ciências e Tecnologia, Universidade do Algarve, Campus de 

Gambelas, P-8005-139 Faro (Portugal). E-mail: jmartin@ualg.pt 

Pyrene and its derivatives offer advantageous properties, such as high quantum yields, long lifetimes, and 

diverse solvatochromic effects, most appropriate for fluorescence studies in proteins, nucleic acids, and 

biomembranes. Therefore, they have been used extensively to probe diverse phenomena in a variety of 

biological systems, particularly in lipid bilayers biophysics [1,2]. We present here further developments in 

probing lipid bilayers interfacial and polarity properties using molecular pyrene and pyrene derivatives to 

study: the equivalent polarity properties of binary mixtures of DMPC/cholesterol and DPPC/cholesterol, in 

liquid-ordered (l o ) and liquid-disordered (l d ) phases, probed by means of the pyrene Ham Effect, and the 

partition of the anionic probe 1-pyrenesulfonate (PSA) into multilamellar vesicles composed by POPC 

(zwiterionic) and by mixtures of zwiterionic and anionic phospholipids (bilayers with negative surface 

potential), using UV derivative spectrophotometry. 

Binary mixtures of cholesterol and phospholipids in bilayers are nonideal, displaying single or phase 

coexistence, depending on chemical composition and on other thermodynamic parameters, e.g. temperature 

and pressure. There are plenty of changes in fluid phase lipid bilayer properties upon mixing cholesterol 

[3], such as reduction of water permeability, reduction by a factor of about 2–3 of the lipid lateral diffusion, 

higher conformational ordering of phospholipids aliphatic chains, which influences the modulation of the 

lateral pressure in a depth-manner and thermomechanical and elasticity properties, as well as increasing in 

bilayer thickness. We demonstrate hear an additional effect of cholesterol in the l o phase of lipid bilayers, 

indicating that in this phase, the polarity of the bilayer, and its thermal dependence, varies principally with 

the chemical composition in respect to the cholesterol proportion. We additionally discuss the potential 

implications of this effect in diverse membrane associated processes and reactions. 

The majority of water/membrane partition data is obtained using pure phosphatidylcholine bilayers since it 

is the major phospholipid in biomembranes. However, the diversity of biological membranes requires 

approximations reflecting at least some basic features of the various lipid compositions. Lipid mixtures 

have been seldom used (basically phospholipid-cholesterol systems) and mixtures of zwiterionic and 

anionic phospholipids are even more disregarded. Contrasting with recent studies [4] indicating a decrease 

in the partition of anionic probes to bilayers with negative surface potential, we find that the Nernstian 

partition constant of PSA into multilamellar vesicles (MLV) at 25ºC increases from K p =4,8×10 3 for pure 

POPC bilayers (zwiterionic), to K p =2,9×10 4 for bilayers composed by 5% POPS (anionic) and 95% POPC 

(molar proportion), and it is even higher for 10% of POPS, K p =6,3×10 4 . We propose a suitable 

interpretation, based on the interfacial properties of negatively charged bilayers. 

Acknowledgements: Work partially funded through the projects POCTI/QUI/45090/2002 and 

POCTI/BCI/46174/2002, from Fundação para a Ciência e a Tecnologia, Portugal. 

References: [1] P. Somerharju, Chem. Phys. Lipids 116 (2002) 57. [2] E. Melo, J. Martins, Biophys. Chem. 123 

(2006) 85. [3] K. Simons, W.L.C. Vaz, Annu. Rev. Biophys. Biomol. Struct. 33 (2004) 269. [4] A. Mateazik et al., 

Bioelectrochem. 55 (2002) 173 and I. Waczulikova et al., Biochim. Biophys. Acta 1567 (2002) 176. 

251


BIOP-4 

Investigations of the diffusional behavior in bacterial cells under 

disturbed cell division 

Johan Strömqvist 1 , Daniel Daley 2 , Kalle Skoog 2 , Niklas Bark 3 , Hans Blom 1 , 

Gunnar von Heijne 2 , Jerker Widengren 1 

1 Experimental Biomolecular Physics, Department of Applied Physics, Albanova University Center, Royal 

Institute of Technology, SE-10691 Stockholm, Sweden. E-mail: johan@biomolphysics.kth.se 

2 Department of Biochemistry and Biophysics, Stockholm University, SE-10691, Stockholm, Sweden 

3 Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, SE-17177, Sweden 

Bacterial cells like E.coli have many properties that makes them useful as a biological model system. They 

divide rapidly, are easy to use for synthesizing proteins/DNA and have a less complex structure than 

eucaryotes. However, their relatively small size (a few microns) makes it a bit difficult to study the 

diffusional behaviour of membrane proteins with conventional fluorescence techniques, especially FCS 

measurements [1] . In this project E. coli cells have been treated with antibiotic [2] so that daughter cells are not 

separated, causing the cells to be elongated as in Figure 1. FRAP experiments have been carried out and 

compared with simulations. This investigation has revealed that the diffusional behavior of proteins and 

lipids during formation of the septal ring [3] is highly affected. 

. 

Figure 1. Elongated E. coli labeled with 

EGFP and DiD. 

References: [1] J. Widengren, P. Thyberg, Cytometry Part A, 68A (2005) 101-112. [2] C. W. Mullineaux et al., 

J. of Bacteriology, 108 (10) (2006) 3442. [3] D. S. Weiss, Molecular Microbiology, 54 (3) (2007) 588-597. 

252


BIOP-5 

Human serum albumin (HSA)-flavonoids interactions monitored by means of 

tryptophan (Trp) kinetics 

Olaf J. Rolinski and David J.S. Birch 

University of Strathclyde, Department of Physics, John Anderson Building, 107 Rottenrow, 

Glasgow G4 0NG, UK. E-mail: o.j.rolinski@strath.ac.uk 

Non-invasive fluorescence sensing based on intrinsic fluorescence of biomolecules is a subject of extensive 

research in biology and medicine. Development of the intrinsic lifetime sensing requires adequate 

modelling of the fluorescence decay, which reflects usually complex excited-state kinetics. 

In this poster we report time-resolved studies of Trp in free HSA molecules and HSA complexes with two 

flavonoids (quercetin and morin) and propose a non-exponential model of the decay functions. 

Flavonoids are naturally present in human body, as they form a part of normal diet, and bind to 

biomolecules and biomembranes. They are known to play important biological functions (inhibit specific 

enzymes, simulate some hormones and neurotransmitters, change the cell membrane properties) and play a 

role in a wide spectrum of diseases (eg their antioxidant function by scavenging free radicals). 

0.08 

g(τ) / arbit. units 

0.06 

0.04 

0.02 

Figure 1: Fluorescence lifetime distribution 

functions g(t) obtained for Trp214 in free 

HSA (solid line) and for the 1:1 HSAquercetin 

complex (dashed line). 

[HSA] = [quercetin] = 30·10 -6 M 

0.00 

0 2 4 6 8 10 12 

τ / ns 

The fluorescence decays of the only tryptophan (Trp214) in HSA and HSA-flavonoid complexes were 

analysed by means of 1,2,3-exponential functions and by using lifetime distributions approach, the later 

with application of the maximum entropy method (MEM). 

The tryptophan decay in free HSA demonstrated a wide distribution of lifetimes g(τ) (see Fig.1), which 

becomes more specific (three well separated peaks) for flavonoid-bound HSA [1] . The results will be 

discussed in terms of rotamer model and power-like [2] fluorescence kinetics. 

References: [1] O.Rolinski, et al., J.Biomed.Optics (2007), in press. [2] G.Wilk, Z.Wlodarczyk, Phys.Rev.Letts, 84, 

13 (2000) 2770. 

253


BIOP-6 

A fluorescence analysis of ANS bound to bovine serum albumin: 

Binding properties revisited 

Denisio Togashi 1,2 and Alan G. Ryder 1,2 

1) 

Nanoscale Biophotonics Laboratory, Department of Chemistry, National University of Ireland, Galway. 

2) National Centre for Biomedical Engineering Science, National University of Ireland, Galway. 

Determination of binding parameters such as the number of ligands and the respective binding constants 

need a considerable number of experiments to be performed. These involve accurate determination of 

either free and/or bound ligand concentration independent of the measurement technique applied. Then, an 

appropriate theoretical approach is used to fit the experimental data, and to extract the binding parameters. 

In this work, the interaction between bovine serum albumin (BSA) and 1-anilino-8-naphthalene sulphonate 

(ANS) is revisited using steady state fluorescence spectroscopy. The binding parameters for the ANS 

bound to BSA were determined and reviewed using different multiple classes of independent sites models 

such as: Scatchard, Klotz, and Halfman-Nishida. Job’s plot and simulations were made in order to 

determine the scope and limitation for those methods. In addition, a new approach using the energy transfer 

from the tryptophan residues to the BSA-ANS complex is presented as a tool to help understand the binding 

mechanism of the albumin fluorescent complex. 

Acknowledgements: This work was supported by Science Foundation Ireland under Grant number (02/IN.1/M231). 

254


BIOP-7 

Spectral study of bovine and human serum albumins with two carboxy 

phenoxathiin derivatives 

Aurica Varlan, Diana Constantinescu-Aruxandei, Liliana Birla, Mihaela Hillebrand 

University of Bucharest, Department. of Physical Chemistry, Bd. Regina Elisabeta, 4-12, Bucharest, 

Romania. E-mail: mihh@gw-chimie.math.unibuc.ro 

Previous work on the photophysical properties of some phenoxathiin derivatives showed that despite the 

weak emission efficiency of the unsubstituted phenoxathiin, the presence of some substituents like formyl, 

acetyl and carboxy determines good enough emission properties to be used as fluorescence probes for 

proteins. [1] The sensitivity of the emission properties to the local environment was proved by the spectral 

study of the corresponding inclusion complexes with β-cyclodextrin. [2,3] Among the phenoxathiin 

derivatives, the 2- and 3-carboxy substituted compounds were found most suitable to be used in the protein 

study, owing to the acid-base equilibrium and their enhanced solubility in aqueous media. Previous 

experimental data on the interaction with bovine serum albumin (BSA) showed a significant albumin – 

ligand interactions. [4] 

O R 2 

S 

R 1 =COOH, R 2 =H 2-carboxyphenoxathiin; 

R 1 =H, R 2 =COOH 3-carboxyphenoxathiin; 

In the present work, the interaction of both ligands was extended to human serum albumin (HSA). Steady 

state fluorescence spectra of BSA and HSA in the presence of variable amounts of 2- and 3-carboxyphenoxathiin, 

at pH=7.4, are reported and discussed. The fluorescence changes were monitored on both the 

band of the ligand and that of the protein. The experimental results are rationalized in terms of the albumin– 

organic ligand interaction and allow for the estimation of the binding constants using either the Scatchard 

model or nonlinear regression analysis. The influence of several parameters as ionic strengths, 

temperature… on the binding parameters are also considered. The fluorescence results are correlated with 

those obtained from the circular dichroism spectra which reveal the change of the albumin conformation 

and the α-helix percent during the interaction process 

References : [1] S. Ionescu, et al, J. Photochem. Photobiol. A: Chemistry 124 (1999) 67. [2] A. Tintaru et al, J. Incl. 

Phen. Macrocyc. Chem. 45 (2003) 35. [3] M. Oana et al, J. Phys. Chem. B, 106 (2002) 257. [4] L.Birla, et al , Rev. 

Roum. Chim., 47 (2002) 769. 

255


BIOP-8 

Monitoring and modeling of protein adsorption kinetics 

Michael Rabe, Dorinel Verdes, Stefan Seeger 

University of Zurich, Institute of Physical Chemistry, CH-8057 Zurich (Switzerland). 

E-mail: m.rabe@pci.unizh.ch 

The adsorption of proteins at solid interfaces plays a key role in many natural processes and has therefore 

promoted a wide interest in the past years. In many industrial applications it is desirable to understand and 

control the mechanisms behind protein adsorption such as to enhance the efficiency of immunoassays, 

improve the biocompatibility of implants, or to prevent fouling in the food processing industry. 

Protein adsorption kinetics were recorded by means of a novel supercritical angle fluorescence (SAF) 

biosensor recently developed. [1] Exploiting the high sensitivity and surface selectivity of the SAF-technique 

we revealed a transition pathway in the adsorption mechanism of the blood plasma protein Immunoglobulin 

G (IgG) on various surfaces. [2] 

Here we present a comprehensive mechanistic study of the non-specific adsorption of β-Lactoglobulin on a 

solid interface by comparing the adsorption kinetics of this protein to theoretical predictions of reported 

models. Using the SAF-biosensor the adsorption and desorption behavior on a hydrophilic glass surface in 

citrate buffer (pH 3.0) was monitored for a large set of different bulk concentrations covering two orders of 

magnitude. Important experimental observations were increasing adsorption rates and overshootings in the 

beginning of the adsorption as well as a transition to an almost irreversibly bound state of the protein in the 

long term. Furthermore, rinsing experiments proved that adsorbed proteins abruptly change their desorption 

behavior from irreversible to reversible when a critical surface coverage is reached. The experimental 

observations were translated into mathematical concepts to propose a complete model which satisfactorily 

describes the recorded adsorption kinetics. In this way the study experimentally confirmed several 

theoretically predicted phenomena, such as cooperative effects or structural reorganizations which are 

commonly assumed to play an important role in the course of protein adsorption events. [3] 

Representation of the experimentally 

acquired adsorption kinetics of β- 

Lactoglobulin (circles) and the 

calculated kinetics using the proposed 

model (black solid line). Three distinct 

adsorption states contribute to the total 

surface coverage: an initial (green 

line), a reversible (red line), and an 

irreversible (blue line) state. 

Inset: Schematic representation of the 

adsorption mechanism involving the 

three states. 

References: [1] T. Ruckstuhl et al., Biosens. Bioelectron. 18 (2003) 1193-1199. [2] M. Rankl, et al., ChemPhysChem 

7 (2006) 837-846. [3] M. Rabe, et al., ChemPhysChem (2007), in press. 

256


BIOP-9 

Time-resolved fluorescence probing of lysozyme-lipid association 

1 Valeriya M. Ioffe, 1 Galyna P. Gorbenko, 2 Yegor A. Domanov, 2 Paavo K.J. Kinnunen 

1 Department of Biological and Medical Physics, V.N. Karazin Kharkov National University, Kharkov, 

Ukraine 

2 Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine, University of Helsinki, Finland 

E-mail: valioffe@yahoo.com 

Fluorescence spectroscopy is one of the most powerful tools providing new insights into the structural, 

dynamic and functional behavior of biological macromolecules, being particularly useful in investigating 

the molecular details of protein-lipid association. Complete and accurate information about the 

conformational dynamics of protein molecules can be obtained using tryptophan (Trp) residues as intrinsic 

fluorescence probes. [1] . Hen egg white lysozyme (Lz) is a multi-tryptophan protein which is extensively 

used in elucidating fundamental aspects of protein-lipid interactions. The present study was undertaken to 

ascertain the alterations in lysozyme structural state upon association with model membranes composed of 

zwitterionic lipid 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) and anionic lipid 1-palmitoyl- 

2-oleoyl-sn-glycero-3-phosphoglycerol (POPG). Fluorescence lifetime measurements showed that 

intensity-averaged lifetime () of Trp residues in lysozyme decreased upon the protein binding to model 

membranes. Furthermore, reduction from 1.94 to 1.74 ns was observed at decreasing lipid-to-protein 

molar ratio (L:P) from 1130 to 120. Lysozyme contains six Trp residues, three of which (Trp62, Trp63 and 

Trp108) are located in the active site. Since Trp62 and Trp108 are thought to be the major emitters, 

accounting for about 80% of lysozyme fluorescence, the changes in Lz spectral behavior can be attributed 

mainly to these residues [2] . The possible explanations for decrease of Trp lifetime in membrane-bound 

lysozyme involves: i) increased polarity of Trp microenvironment; ii) changes in the local environment of 

the indole ring (rotation about Trp side chain); iii) Trp interactions with neighboring amino acid residues 

(e.g. Cys); iv) intermolecular energy transfer from Trp62 to Trp63 [3] . The first possibility was ruled out by 

quenching and steady-state fluorescence experiments. Specifically, it was found that lysozyme association 

with lipid vesicles was followed by a decrease in Stern-Volmer constants for acrylamide quenching 

indicating the transfer of Lz fluorophores into membrane environment with lower polarity. This contradicts 

the observation that lysozyme emission maximum does not exhibit a blue shift upon membrane binding, so 

we concluded that increase in polarity could not account for the observed lifetime decrease. Rotation about 

Trp side chain and intermolecular energy transfer may occur, but these processes cannot satisfactorily 

explain dependence on L:P. Motivated by the above rationales, we suggested that Trp specific 

interactions with certain amino acid residues in its surroundings is the main factor responsible for the 

recovered decrease in tryptophan lifetime and the observed contradictions between lifetime, quenching and 

steady-state experiments. Since Lz is a stable protein whose conformation is reported to change 

insignificantly upon the formation of protein-lipid contacts, it can be assumed that the processes behind the 

drop in involve Lz self-association in membrane-bound state. Trp62 and Trp108 are located in the 

protein active site which reportedly participates in Lz aggregation. Moreover, Cys76-Cys94 disulfide 

bridge capable of efficient quenching of Trp fluorescence and reducing the lifetime of protein fluorophores, 

also resides in the active site cleft. The dependence on L:P can be explained by the fact that lysozyme 

self-association is apparently coverage-dependent process controlled by both electrostatic and hydrophobic 

protein-lipid interactions. The recovered membrane ability to modulate Lz aggregation behavior may 

largely determine the bactericidal and amyloidogenic propensities of this protein. 

References: [1] J.R. Lakowicz, Principles of fluorescent spectroscopy, Plenum Press: New York, 1999. [2] Imoto, T. 

et al., Proc. Natl. Acad. Sci. USA 69 (1971) 1151. [3] B. Valeur, Molecular fluorescence. Principles and applications, 

Wiley-VCH: New York, 2001. 

257


BIOP-10 

Fluorescent studies on cooperative binding of cationic pheophorbide - 

a derivative to polyphosphate 

Olga Ryazanova 1 , Igor Voloshin 1 , Igor Dubey 2 , Victor Zozulya 1 

1 B. Verkin Institute for Low Temperature Physics and Engineering of NAS of Ukraine, Department of 

Molecular Biophysics, 47 Lenin ave., 61103, Kharkov (Ukraine). E-mail: ryazanova@ilt.kharkov.ua 

2 Institute of Molecular Biology and Genetics of NAS of Ukraine, 150 Zabolotnogo str., 03143, Kyiv 

(Ukraine) 

The pheophorbide-a (Pheo) is an anionic porphyrin derivative. It is widely used as a photosensitizer in 

photodynamical therapy of tumors because of its high photosensitizing activity in vitro and in vivo [1-2]. 

Modification of Pheophorbide-a with the trimethylammonium group was carried out to obtain a cationic 

dye derivative (CatPheo, Fig.1) capable of polyanion binding. Spectroscopic properties of CatPheo 

complexes with polyanionic chain of DNA backbone were modelled by the dye binding to the 

polyphosphate. The investigations were carried out in buffered (pH6.9) aqueous solutions of different ionic 

strengths by methods of absorption and polarized fluorescence spectroscopy in a wide range of molar 

phosphate-to-dye ratios, P/D. Experimental investigations revealed that the character of changes in CatPheo 

absorption and fluorescence properties under its interaction with polyphosphate is similar to those for 

anionic Pheo with poly-L-lysine [3]. In particular, the strong fluorescence quenching was observed along 

with increase in the fluorescence polarization degree. The spectrum of residual fluorescence took the 

characteristic two-humped shape. The P/D dependence of the fluorescence intensity evidences that at low 

P/D values CatPheo forms continuous stacking associates on the polyanionic matrix, and at large P/D it 

binds to polyphosphate in the dimer form. The absorption and fluorescent properties of the aggregates were 

established. Such parameters of cooperative binding as the number of binding sites per monomer unit of 

polyphosphate, cooperativity parameter and the cooperative binding constant were estimated by Schwarz's 

method [4]. 

HC 

CH 3 

C H 3 

C 2 

H 5 

NH 

N 

Figure 1: Molecular structure of 

cationic Pheophorbide-a derivative. 

N 

HN 

C H 3 

CH 3 

COOCH 3 

O 

O 

NH 

CH 2 

N(CH 3 

) 3 

+ 

− 

AcO 

This work is partially supported by Science and Technology Center in Ukraine (Project #3172). 

References: [1] B. Roeder, J. Photochem. Photobiol. B 5 (1990) 519. [2] B. Roeder, Lasers Med. Sci. 5 (1990) 99. 

[3] O. Ryazanova et al., J. of Porphyrins and Phthalocyanines 10 (2006), 846. [4] G. Schwarz, Eur. J. Biochem. 12 

(1970) 442. 

258


BIOP-11 

Characterization of lipolexes using fluorescence spectroscopy – the effect of 

monoolein (monoacyl-rac-glycerol) and cholesterol (Ch) of the condensation 

efficiency of DNA 

Joao Paulo Neves da Silva, Paulo J.G. Coutinho, M.E.C.D. Real Oliveira * 

Departamento de Física, Universidade do Minho, Portugal 

*E-mail: beta@fisica.uminho.pt 

Lipoplexes are membranous structures that are capable of transducing genes into cells, eventually leading to 

expression of the genes by the process called transfection. The driving force for lipoplex formation is the 

removal of counterions from the lipids surface by the DNA. Inclusion of helper lipids in the liposomal 

formulation may facilitate this removal by weakening the binding of the ions to the cationic surface [1]. 

Moreover, inclusion of a helper lipid, provided its good miscibility with the cationic lipid, brings about a 

better matching in the density of surface charge distribution between the liposomes and DNA, resulting in a 

more complete and balanced packaging of DNA by the lipids. Apart from facilitating the complex 

formation, an additional effect of the helper lipid may rely on its effect on complex stability. 

In this study we have studied the lipoplexes formed by cationic liposomes composed of DODAB 

(dioctadecyldimethylammonium bromide) and salmon sperm DNA using two different helper lipids, 

monoolein (monoacyl-rac-glicerol) and cholesterol (Ch). 

The effect of different ratio of cationic lipid/helper on the lipoplexes, DODAB/Monoolein (1:1, 1:2, 1:1) 

and DODAB/cholesterol (1:1) on the physical properties of lipoplexes (such as, condensation efficiency of 

DNA, structural changes) was accessed by exclusion studies of EtBr (Ehidium Bromide) from DNA and 

fluorescence resonance energy transfer (FRET) using as pair donor/acceptor, the lipid probe [2-(3- 

difenilhexatrieno) propanoil-1-hexadecanoil-sn-glicero-3-fosfocolina (DPH-PC) as donor and EtBr as 

acceptor. 

The stability of lipoplex was analyzed at 37°C by the addition of salt [2] and protein (serum) [3] using the same 

fluorescence techniques. 

This study contributed to a deeper knowledge on the physicochemical characteristics of lipoplexes based on 

monoolein and for exploring its viability and potentiality as potential non-viral vectors to gene therapy. 

References: [1] M.C.P. Lima, S. Simões, P. Pires, H. Faneca, N. Düzgünes, (2001), Ad. Drug Delivery Reviews, 47, 

277-294. [2] Y. Zhang, W. Garzon-Rodriguez, M.C. Manning, T.J. Anchordoquy, (2003), Biochimica et Biophysica 

Acta, 1614, 182–192. [3] B. Wetzer, G. Byk, M. Frederic, M. Airiau, F. Blanche, B. Pitard, D. Scherman, (2001), 

Biochemic. J., 356, 747-756. 

259


BIOP-12 

Fluorescence resonance energy transfer applied to the investigation of the 

phospholipids composition of the annular region of lactose permease 

Laura Picas § , José Luis Vázquez-Ibar ¥ , M. Teresa Montero §,± , Jordi Hernández-Borrell §,±, * 

§ Departament de Fisicoquímica, Facultat de Farmacia Universitat de Barcelona (U.B.) Barcelona, 

08028-Spain. and ¥ ICREA and Institute for Research in Biomedicine, Scientific Parc of Barcelona. 

08028 Barcelona, Spain. 

± Institut de Nanociència i Nanotecnologia de la Universitat de Barcelona (IN 2 UB) 

*Corresponding author: E-mail: jordihernandezborrell@ub.edu 

It is known that the physiological activity of transmembrane proteins may be influenced by, or be 

dependent upon, the physical properties of neighbouring phospholipids. In particular, several studies have 

shown that 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), one of the most abundant 

phospholipids in prokaryotic membranes, plays a dual role in both activity and proper folding of 

transmembrane proteins (1). In addition, POPE is partially responsible for the formation of pores across the 

membranes, most likely because of its ability to form inverted hexagonal phases (H II ) (2). Recently, it has 

been suggested that other constituents of Escherichia coli inner membrane (3) such as cardiolipin (CL) (4) 

and 1-palmitoyl-2-oleoyl-sn-phosphoglycerol (POPG) (5) may play a significant role on the activity of 

membrane proteins. In this work, we investigate the proximity of different phospholipids species in the 

immediate vicinity (or annular region) of the lactose permease of Eschericia coli (LacY), using 

fluorescence resonance energy transfer (FRET) techniques. LacY, a β-galactoside/H + symporter, is often 

used as a paradigm for membrane transport proteins because its large amount of structure-function studies 

and, mainly, because its 3D crystal structure is already known. The structure reveals the residues involved 

in substrate recognition and translocation along with the side chains exposed to the lipid phase; however no 

phospholipids were present in the crystal. After reconstituted the purified protein into liposomes with 

different phospholipid composition, we measure the proximity between two single tryptophan mutants of 

LacY (W320 and 151W) and two phospholipids analogs of POPG and POPE: 1-hexadecanoyl-2-(1- 

pyrenedecanoyl)-sn-glycero-3-phosphoglycerol, and 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3- 

phosphoethanolamine, respectively. As seen in the crystal structure of LacY, W151 is located in the internal 

aqueous cavity of the protein, away from the lipid phase while position 320 lies in the interface 

phospholipids/protein. The results revealed that POPE is the most abundant phospholipid at the annular 

region and provided new evidences on the presence of POPG and CL in this particular region. The results 

are interpreted as a consequence of lateral compressibility and mixing properties of these phospholipids (4). 

References: [1] S. Merino et al., Langmuir 21 (2005) , 4642-4647. [2] Ò. Domènech et al., Biochim. Biophys. Acta 

(2006) (in press). [3] K. Matsumoto et al., Molecular Microbiology 61, (2006) 1110-1117. [4] Ò. Doménech et al., 

Biochim. Biophys. Acta, (2006) 1758, 213-221. [5] L. Picas et al., J. Fluorescence (2006) (available on line). 

260


BIOP-13 

Fluorescence study of lipid-based DNA carriers properties: 

Influence of cationic lipid chemical structure 

Laure Burel-Deschamps, Farouk Ayadi, Sondes Lounissi, Mathieu Mével, Jean-Claude 

Clément, Philippe Giamarchi 

Université de Bretagne Occidentale, UMR CNRS 6521, 6 avenue Le Gorgeu, BP 809, 29285 Brest cedex, 

France. E-mail : philippe.giamarchi@univ-brest.fr 

Gene therapy requires suitable carriers for intracellular delivery of genetic materials. Cationic lipids are a 

promising alternative to the use of viral vectors, due to their safety and versatility. Different types of 

original cationic lipids have been synthesized in our laboratory [1] and tested on various cell lines [2]. 

All of them are composed of three parts: the hydrophobic chains, the spacer and the cationic head. In an 

attempt to establish relationship between the structure of lipids and their transfection efficiency and to 

understand the mechanisms involved, we carried out different fluorescent based analysis. 

Fusogenic properties exhibited by the cationic liposome formulation can induce fusion or destabilization of 

the plasma membrane, thus facilitating the intracellular release of complexed DNA. Förster Resonant 

Energy Transfer (FRET) measurements allow to estimate these fusogenic properties through lipid mixing 

assay of membrane fusion [3]. FRET efficiency between NBD-PE and Rhod-PE included in model 

membrane was measured to quantify the fusion of cationic liposomes with that model membrane. 

We showed that the nature of the hydrophobic part and of the cationic head influence the fusogenic 

properties. The addition of different co-lipid also affect membrane fusion efficiency. 

The fluidity of the liposome cationic bilayer was also evaluated by measuring fluorescence anisotropy 

of the fluorescent probe diphenylhxatriene located in the bilayer. The influence of the structure of the 

hydrophobic chain and of the nature of the neutral co-lipid on membrane fluidity was evidenced as describe 

in the figure below. 

Fluorescence anisotropy of 

diphenylhexatriene probe 0.20 

inserted in the membrane of 

different cationic phospholipids 

(with cholesterol) record 

versus the temperature. 

0.15 

The figure shows two 

different populations corresponding 

to the type of 

0.10 

hydrophobic tail (myristyl 

C14:0, oleyl C18:1). 0.05 

Anisotropy ( r ) 

0.25 

« C18:1 chain » 

« C14:0 chain » 

15 25 35 45 55 

Temperature (°C) 

DOTAP 

KLN 47 

EG 308 

GLB 73 

MM 42 

MM 44 

Through these anisotropy and FRET measurements, we were able to establish some correlations between 

the chemical structure of the cationic and neutral lipids versus the membrane fluidity and fusion ability, 

which both may influence transfection efficiency. 

References: [1] T. Montier et al. Recent Res.Devel. Chem. 1 (2003) 41-58. [2] T. Montier et al. Biochimica et 

Biophysica Acta 1665 (2004) 118. [3] DK Sruck et al. Biochemistry 20 (1941) 4093-4099. 

261


BIOP-14 

The molecular basis of anesthesia studied by solvent relaxation technique 

Justyna Barucha 1 , Magdalena Przybyło 2 , Marek Langner 2 , Martin Hof 1 

1 J. Heyrovsky Institute of Physical Chemistry of ASCR, Dolejskova 2155/3, 182 23 Praha 8 (Czech 

Republic). E-mail: justyna.barucha@jh-inst.cas.cz 

2 Wrocław University of Technology, Wybrzeże Wyspiańskiego27,50-370 Wrocław (Poland) 

The molecular bases of both local and general anesthesia were studied by means of solvent relaxation 

technique, with respect to the type of an anesthetic molecule (either amide or ester) and “the range of a 

molecule action “(local vs. general anesthesia). As has been shown the solvent relaxation technique 

provides direct information on the hydration properties of the lipid membranes [1]. Moreover it serves also 

as an excellent tool for studying membrane dynamics on the nanosecond time scale [1,2,3]. Therefore, by 

using two fluorescent dyes of different localization in the membrane, we applied SR to examine the 

influence of the anesthetic molecules on the lipid bilayer nano-dynamics and the membrane hydration, as 

well as their precise localization within the lipid bilayer. The alteration of the membrane dynamics by the 

presence of two amino amides, namely lidocaine and bupivacaine, and two amino esters, procaine and 

benzocaine, was measured as a function of their concentration and pH. The effect of mentioned local 

anesthetics was compared to the effect induced by halothane, a common general anesthetic. 

References: [1] P.Jurkiewicz et al., J.Fluor. 15(6) (2005) 883; [2] P.Jurkiewicz et al., Langmuir 22(21) (2006) 8741, 

[3] J.Sykora et al., Langmuir 18(3) (2002) 571 

262


BIOP-15 

Observation of stable GPI-GFP clusters diffusing in the plasma membrane 

of living CHO cells 

Mario Brameshuber 1 , Manuel Moertelmaier 1 , Julian Weghuber 1 , Verena Ruprecht 1 , Hannes 

Stockinger 2 , Gerhard J. Schütz 1 

1 Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr. 69, A-4040 Linz, Austria 

2 Department of Molecular Immunology, Center of Biomolecular, Medicine and Pharmacology, 

Medical University of Vienna, Lazarettgasse 19, A-1090 Vienna, Austria. 

E-mail: mario.brameshuber@jku.at 

The current picture of cellular plasma membrane is based on the existence of small stable structures which 

enable controlled aggregation and segregation of distinct sets of proteins. These structures, commonly 

termed lipid rafts, are too small and too close to be observed directly with fluorescence microscopy, too 

mobile for high resolution scanning techniques, and too fragile for reliable chemical purification. 

We developed a novel method 1 (TOCCSL - Thinning Out Clusters while Conserving the Stoichiometry of 

Labeling) for the stoichiometric analysis of molecular aggregates in the cellular plasma membrane. We use 

selective photobleaching to erase all active fluorophores within a small region of the membrane, while 

conserving the stoichiometry of labeling in the remaining part of the membrane. At the onset of 

repopulation due to Brownian motion, single diffraction limited spots of individual aggregates can be 

resolved and quantified. 

Figure 1. Principle of TOCCSL. Anti-DNP antibodies labeled with multiple FITC molecules were used to 

mimic stable clusters. A fluid supported lipid bilayer containing a fraction of DNP-labeled lipid provided 

the matrix for the experiment. On the left, the initial equilibrium situation is shown: a surface density of 

~15 clusters per µm 2 makes direct observation of individual clusters impossible. Upon photobleaching for 

t bl =200ms, clusters were allowed to diffuse into the bleached area. To the right, three images recorded after 

distinct recovery times are shown: after t rec =0.5ms, no fluorescence signal can be observed within the 

illuminated part of the membrane; this image serves as control for complete photobleaching. After 

t rec =500ms, individual clusters were clearly resolvable in the central part of the image, indicated by the 

dashed white circle; such single cluster signals are used for subsequent stoichiometric analysis. Using a 

much longer recovery time of t rec =10s, the system has nearly reached equilibrium again 1 . 

To address the question of stable lipid rafts within the cellular plasma membrane, we applied TOCCSL to 

investigate the aggregation of a glycosyl-phosphatidyl-inositol (GPI) anchored monomeric green 

fluorescent protein stable expressed in living CHO cells. Besides monomers, we found a significant fraction 

of dimers diffusing freely in the plasma membranes. Those dimers were stable on a seconds time scale. 

With this study, one basis of the raft concept – the formation of stable platforms in the plasma membrane – 

has been confirmed. 

Reference: [1] M. Moertelmaier et al., Appl. Phys. Lett. 78, 263903 (2005). 

263


BIOP-16 

Usage of fluorescent dyes for spectroscopic investigation of bimolecular 

chemical reactions 

Alexander Schmitt, Michaela Jacob, Gregor Jung 

Biophysikalische Chemie, Universität des Saarlandes, Im Stadtwald, Geb. B2.2, 66123 Saarbrücken 

(Germany). E-mail: alex.schmitt@mx.uni-saarland.de 

The examination of chemical reactions on a single molecule level is possible by focusing on individual 

fluorescent dyes. For that purpose fluorescent dyes have to change their spectroscopic properties, like e.g. 

maximum fluorescence wavelength, during a chemical reaction. 

We synthesized and used Bor-dipyrromethene-dyes (BODIPY-dyes) [1] to investigate the oxidative 

transformation of C=C-double bonds. Bodipy-dyes are appropriate for single-molecule-detection research. 

Different oxidizing agents like MCPBA (m-Chlor-perbenzoic-acid) and KMnO 4 (Kaliumpermanganate) 

were used on an isolated C=C-double-bond in synthesized BODIPY-dyes [2] . 

During the reactions the C=C-bond is transformed and a shift in the fluorescence maximum is observed by 

fluorescence-spectroscopy. The different oxidation products are isolated by TLC (Thin Layer 

Chromatography), from which the resulting spots are scraped off and used for fluorescence spectroscopic 

analysis, like FCS (Fluorescence Correlation Spectroscopy) and TCSPC (Time Correlated Single Photon 

Counting) [fig]. 

The observation of the kinetics and the characterization of the oxidation-products of a highly fluorescent 

compound helps us to establish a reactive system on which the chemical transformation of an individual 

molecule can be studied in the future. 

TLC-color-inverted-photo of a reaction of 

the BODIPY-dye with KMnO 4 . 

The immediate formation of two products 

can be observed. 

After about 15 minutes a decay of these 

products is observed, paralleled to a decay 

of the overall fluorescence intensity. 

In another approach, we synthesize fluorescence dyes for the investigation of the catalyzed cleavage of 

phosphate esters. During the catalyzed cleavage, the fluorescence lifetime changes, which can be monitored 

by FLIM (Fluorescence Lifetime Imaging Microscopy). 

References: [1] Treibs A., Kreuzer F.; Liebigs Ann.; 718, (1968); 203; [2] Schmitt A , Hinkeldey B., Jung G.; in 

preparation 

264


BIOP-17 

Trp-Trp Energy Migration as Tool to Follow Protein Unfolding 

Nina V. Visser 1 , Adrie H. Westphal 2 , Arie van Hoek 1,3 , Carlo P.M. van Mierlo 2 , 

Herbert van Amerongen 1,3 , Antonie J.W.G. Visser 2,3 

Laboratories of Biophysics 1 and Biochemistry 2 , MicroSpectroscopy Centre 3 , Wageningen University, 

P.O. Box 8128, 6700 ET Wageningen, The Netherlands. 

E-mail: ton.visser@wur.nl 

The understanding of how a linear chain of amino acids folds into a functional protein molecule with a 

complex three-dimensional structure is one of the major challenges in structural biology today. Folding of a 

protein to its native state can go through many distinctive pathways. Here, using apoflavodoxin as a model 

protein, we follow protein unfolding with polarized fluorescence spectroscopy monitoring the emission of 

the three tryptophanyl residues at picosecond time resolution. In the native, folded state analysis of the 

fluorescence anisotropy decay reveals the presence of correlation times on picosecond and nanosecond 

timescale. We conclude by comparing simulated and experimental results that the main depolarization 

mechanism is due to homo-transfer of energy between pairs of tryptophan residues. Since the critical 

transfer distance between two tryptophan residues is rather small, ~ 1.0 nm, any change in distance and 

orientation during the unfolding process can be immediately measured via changes in fluorescence 

anisotropy decay parameters. 

265


BIOP-18 

Probing the interaction of acyl-coenzyme A with SDS 

Kell K. Andersen 1 , Peter Westh 2 , Daniel E. Otzen 1 

1 Centre for Insoluble Protein Structures (inSPIN), Department of Life Sciences, Aalborg University, 

Sohngaardsholmsvej 49, DK–9000 Aalborg 

2 Department of Chemistry and Biology, Roskilde University, DK – 4000 Roskilde 

Protein-surfactant interactions are of interest, since they shed light on the way proteins respond to changes 

in their environment [1] . Ionic surfactants can denature proteins by strong binding to charged and hydrophobic 

side-chains at millimolar concentrations, unlike chemical denaturants such as guanidinium chloride 

or urea [2] which are only effective at molar concentrations presumably due to weak binding to the protein 

backbone [3] . These interactions are of great practical interest, since the majority of industrial enzyme 

production (both in terms of value and volume) is targeted to the detergent sector, primarily for laundering 

and dishwashers [4] . 

We have studied the interactions of Acyl-coenzyme A (ACBP) with the ionic surfactant SDS using a 

variety of fluorescence methods including intrinsic tryptophan fluorescence, anisotropy and probes. These 

studies have been supplemented by investigations with Circular dichroism, Capillary electrophoresis and 

Isothermal Titration Calorimetry to give further insight to the interactions between ACBP and SDS. 

Different techniques used to study 

ACBP-SDS interactions: 

(Lower graphs) The hydrophobic 

probe ANS reveals formation of 

exposed hydrophobic patches on 

ACBP. Upon titration with SDS 

the native structure is perturbed 

and ANS fluorescence decreases. 

Pyrene is a hydrophobic probe 

that is sensitive to the polarity of 

the surrounding environment and 

reports on the formation of hemimicelles 

on the protein surface. 

(Upper graphs) Steady-state 

tryptophan fluorescence monitors 

changes in the protein structure 

while stopped-flow kinetics 

provides insight into the 

mechanism of unfolding. 

Trp fluorescence - 345nm 

Fluorescence - 500nm 

Tryptophan 

50 

45 

40 

35 

30 

25 

0 2 4 6 8 10 

SDS [mM] 

ANS 

200 

150 

100 

50 

Emission - I3/I1 

k-obs (s-1) 

Unfolding kinetics 

200 

150 

100 

50 

0 100 200 300 400 500 

SDS (mM) 

Pyrene 

0,95 

0,9 

0,85 

0,8 

0,75 

0,7 

0 

0 2 4 6 8 10 

[SDS] (mM) 

Interestingly, ACBP can be denatured both by SDS monomers and SDS micelles. SDS monomers denature 

by clustering together on ACBP forming so-called hemi-micelles which disrupt the native structure. 

According to ITC, there are up to 6-7 SDS molecules in such hemi-micelles. In contrast fully saturated 

ACBP binds 42 SDS molecules. Above the critical micelle concentration (CMC), SDS micelles can unfold 

ACBP in milliseconds but the rate of unfolding is highly dependent on the SDS concentration. An increase 

in the unfolding kinetics is observed until ~50mM SDS, after which a decline is observed that eventually 

levels out. We rationalize this by an inhibition mechanism whereby several micelles bind to ACBP at the 

same time, slowing down the unfolding kinetics at high SDS concentrations. 

References: [1] LaMesa, C. (2005) J. Coll. Int. Sci. 286, 148-157; [2] Sudhahar, C. G., and Chin, D.-H. (2006) 

Bioorg. Med. Chem. 14, 3543-3552; [3] Timasheff, S. (2002) Biochemistry 41(2948), 13473-13482; Kirk, O et al. 

(2002) Curr. Opin. Biotechnol. 13(3417), 345-351 

0,65 

0,6 

No ACBP 

2µM ACBP 

0 2 4 6 8 10 

SDS [mM] 

266


BIOP-19 

Principles and recent applications of fluorescence solvent relaxation technique 

– drug delivery systems 

Agnieszka Olżyńska 1) , Piotr Jurkiewicz 1) , Jan Sýkora 1) , Rudolf Hutterer 2) , Martin Hof 1) 

1) 

Department of Biophysical Chemistry, J. Heyrovský Institute of Physical Chemistry, Academy of Sciences 

of the Czech Republic, Dolejškova 3, CZ-18223 Prague 8 (Czech Republic). 

E-mail: agnieszka.olzynska@jh-inst.cas.cz 

2) 

Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 

D-93040 Regensburg (Germany) 

Fluorescence solvent relaxation (SR) technique, based on reconstruction of time-resolved emission spectra 

(TRES), enables to study hydration and dynamics of the lipid membranes. [1] Solvent relaxation process 

refers to dynamic reorganization of solvent as a response to a rapid change in the fluorophore electric 

charge distribution upon electronic excitation. The overall Stokes shift gives the information on the polarity 

of the vicinity of the probe and thus reporting the degree of hydration of the bilayer. Moreover, the kinetics 

of the Stokes shift reflects mobility of the probe environment. Applied to the headgoup region of fully 

hydrated lipid bilayers, the solvent relaxation technique provides quantitative information on hydration and 

mobility of the membrane on a molecular level. [1-3] 

Transferosomes® are the highly deformable lipid vesicles designed for transdermal drug delivery. We use 

SR technique to examine surface properties of Transferosomes®, which are important for the efficiency of 

the drug loading. More precisely, we have recently investigated headgroup hydration and mobility of two 

types of mixed lipid vesicles, containing nonionic surfactants; straight chain Brij 98 and polysorbat Tween 

80, with the same number of oxyethylene units as Brij but attached via a sorbitan ring to oleic acid. 

Additionally, we have studied interactions of those systems with protein Interferon alfa-2b (a candidate for 

a non-invasive drug delivery). [4] 

We also apply SR technique to characterize positively charged lipid membranes. Despite the fact that 

structure and properties of the so-called lipoplexes (nucleic acids and lipid complexes) are extensively 

investigated nowadays, biophysical description of such structures is still lacking. We compare binary lipid 

mixtures consist of a cationic lipid (1,2-Dioleoyl-3-Trimethylammonium-Propane, DOTAP [3] or 1,2- 

Dimyristoyl-3-Dimethylammonium-Propane, DMTAP) and a neutral helper lipid (dioleoylphosphatidylcholine, 

DOPC; dimyristoylphosphatidylcholine, DMPC; or dioleoylphosphatidyloethanoloamine, DOPE). 

Because SR technique is a very sensitive method, we are indeed able to see the difference between those 

systems. The obtained results are in good agreement with molecular dynamics studies. 

References: [1] P. Jurkiewicz et al., J. Fluorescence 15 (2005) 883. [2] A. Olzynska et al., Chem. Phys. Lipids (2007) 

in press. [3] P. Jurkiewicz et al., Langmuir 22 (2006) 8741. [4] K. Rieber et al., Biochim. Biophys. Acta (2006) in 

press. 

267


BIOP-20 

Investigation of DNA base flipping by adenine methyltransferases through the 

fluorescence decay of 2-aminopurine 

Eleanor Y.M. Bonnist 1 , Robert K. Neely 1 , Anita C. Jones 1 , David T.F. Dryden 1 , Thomas 

Lenz 2 , Elmar Weinhold 2 , Axel J. Schiedig 3 , Kirsten Liebert 4 and Albert Jeltsch 4 

1 

School of Chemistry and Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre, 

University of Edinburgh, Edinburgh EH9 3JJ, UK. 

2 

Institute of Organic Chemistry, RWTH Aachen University, D-52056 Aachen, Germany. 

3 

Department 2.5 Biophysics/Structural Biology, Saarland University, D-66421 Homburg/Saar, Germany. 

4 School of Science and Engineering, International University Bremen, 28725 Bremen, Germany. 

DNA methyltransferase enzymes play an essential role in important biological processes, such as gene 

expression, DNA replication, DNA repair, genomic imprinting and gene silencing. Methyltransferases 

catalyse the specific transfer of a methyl group to either cytosine or adenine, thus leaving DNA in a 

chemically modified state. For methylation to take place, the target base must enter the active site of the 

enzyme. This requires a remarkable conformational distortion of the duplex by the enzyme, which involves 

rotation of the target the base around the DNA backbone into an extrahelical position. This mechanism is 

known as base flipping. 

The crystal structure of the M.TaqI 

methyltransferase bound to 2AP 

labelled DNA and a cofactor 

analogue (2.4 angstrom resolution) 2 

Previously, time-resolved fluorescence of 2-aminopurine (2AP) has been used to unambiguously identify 

base flipping by the cytosine methyltransferase M.HhaI 1 . We now report the use of 2AP time-resolved 

fluorescence to study the mechanism of base flipping by two adenine methyltransferases, M.TaqI and 

M.EcoRV. 

As in our previous study of M.HhaI 1 , we find that the fluorescence decay of 2AP unambiguously indicates 

base flipping by M.TaqI and M.EcoRV. Moreover, the decay parameters of 2AP report on the environment 

of the flipped base in the enzyme active site. In M.HhaI, the flipped base experiences a largely unquenched 

environment and its fluorescence decay resembles that in free solution. However, in the adenine 

methyltransferases, the fluorescence of the flipped base is strongly quenched. By recording the timeresolved 

fluorescence of crystals of DNA-enzyme complexes, of which the X-ray structure has been 

determined, we have identified the quenching mechanism to be interaction with the aromatic side chain of a 

tyrosine residue in the conserved catalytic amino acid motif of the adenine methyltransferases. 

References: [1] R.K. Neely et al., Nucleic . Acids Res .33 (2005) 6953. [2] T. Lenz et al., J. Am. Chem. Soc. (in press) 

268


BIOP-21 

Test for electron transfer theories with donor-acceptor distance-dependent 

rates of photo-induced electron transfer in flavoproteins 

Fumio Tanaka a , Rong Rujkorakarn b , Haik Chosrowjan c , Seiji Taniguchi c and 

Noboru Mataga c 

a SC1-414 Department of Chemistry, Faculty of Science, Mahasarakham University, Thailand. 

b Department of Physics, Faculty of Science, Khon Kean University, Thailand. 

c Institute of Laser Technology, Japan. E-mail: fukoh2003@yahoo.com 

Remarkable quenching of fluorescence from flavin in most flavoproteins is ascribed to ET from Trp or Tyr 

to nearby excited isoalloxazine [1]. It was also demonstrated that an averaged distance (R) between the 

donor and acceptor over all pairs of atoms are important, rather than edge to edge distance or inter-planer 

angle [2]. 

R-dependent rates of ET from Trp, Tyr, and benzoate (in D-amino acid oxidase-benzoate complex) to the 

excited isoalloxazine in ten flavoprotein systems were analyzed with three kinds of electron transfer 

theories by Marcus (M), by Bixon and Jortner (BJ), and also by Kakitani, Yoshimori, and Mataga (KYM). 

The distances, R, were obtained from X-ray structures of flavoproteins. The values of deviation were 

0.0235 by M theory, 0.0177 by BJ theory and 0.0150 by KYM theory. The observed ET rates were best 

reproduced by KYM theory. 

31 

Figure 1 Analysis of ET rates in 

flavoproteins by KYM theory 

Y and YC represent the observed 

and calculated ln k ET 

, where k ET is 

ET rate. 

Y or YC 

29 

27 

Y 

25 

YC 

23 

0.3 0.5 0.7 0.9 

R / nm 

References: [1] N. Mataga, H. Chosrowjan, S. Taniguchi, F. Tanaka, N. Kido, M. Kitamura, J. Phys. Chem. B 106, 

8917 (2002). [2] Fumio Tanaka, Haik Chosrowjan, Seiji Taniguchi, Noboru Mataga, Kyosuke Sato, Yasuzo Nishina, 

and Kiyoshi Shiga, J. Phys. Chem. B, in press. 

269


BIOP-22 

Investigation of the effect of extracellular osmolarity on sodium content and 

exocytosis in presynaptic endings using different fluorescent probes 

T.V. Waseem, V.A. Kolos, L.P. Lopatina, S.V. Fedorovich 

Institute of Biophysics and Cell Engineering of National Academy of Sciences, Minsk, Belarus. 

Neurotransmitter release is dependent on both calcium and sodium influx. Hypotonic swelling and 

hypertonic shrinking of neurons evoke release of neurotransmitters into the synaptic cleft. To date, there are 

little data available on relationship between extracellular osmolarity and exocytosis of neurotransmitters in 

presynaptic endings. No direct measurements of sodium content in presynaptic endings were performed at 

changing of extracellular osmolarity. 

We investigated the effects of hypotonic swelling and hypertonic shrinking on sodium levels, as measured 

using fluorescent dyes SBFI − AM and Sodium Green in isolated presynaptic nerve endings 

(synaptosomes). 

Reduction of incubation medium osmolarity from 310 to 230 mOsm did not raise the intrasynaptosomal 

sodium concentration ([Na + ] i ). An increase of osmolarity from 310 to 810 mOsm is accompanied by a dose 

− dependent elevation of sodium concentration from 8.1±0.5 to 46.5±2.8 мМ, respectively. This effect was 

insensitive to several channel inhibitors such as: tetrodotoxin, an inhibitor of voltage − gated sodium 

channels, bumetanide, an inhibitor of Na + /K + /2Cl − cotransport, gadolinium, an inhibitor of nonselective 

mechanosensitive channels, ruthenium red, an inhibitor of transient receptor potential channel and 

amiloride, an inhibitor of epithelial sodium channel/degenerin. Additionally, using the fluorescent dye 

BCECF − AM, we have shown that hypertonic shrinking caused a dose − dependent acidification of 

intrasynaptosomal cytosol, which suggests that the Na + /H + exchanger is not involved in the effect of 

increased osmolarity on cytosolic sodium levels. 

The increase in intrasynaptosomal sodium concentrations following increases in osmolarity is likely due to 

sodium influx through another sodium channels. 

We also studied the mechanism of exocytosis induced by hypotonic swelling and hypertonic shrinkage in 

synaptosomes. 

Exocytosis was visualized by fluorescent probes acridine orange, FM 1-43 and FM 2-10. It was shown that 

lowering of incubation medium osmolarity to 230mOsm leads to increase of [ 3 H]-D-Aspartate and 

[ 3 H]GABA release. Neurotransmitters release were calcium independent. Hypotonic shock caused release 

of acridine orange and FM 2-10, but not FM 1-43 from synaptosomes. Release of acridine orange from 

swollen synaptosomes was Ca 2+ -independent, while destaining of FM 2-10 was more remarkable in Ca 2+ 

containing medium. 

Hypertonic stimulation led to Ca 2+ -independent release of [ 3 H]-D-Aspartate and [ 3 H]GABA from isolated 

presynaptic nerve endings. Quantities of neurotransmitters released under hyperosmotic shrinkage of 

synaptosomes were dependent from sucrose concentration applied. Synaptosomes shrinkage were 

accompanied by release of acridine orange from synaptic vesicles and FM 2-10 destaining, both processes 

were Ca-independent. Fluorescence intensity of FM 1-43 was not changed under hypertonic stimulation. 

To judge form our data in isolated presynaptic nerve endings osmotic shock leads to Ca 2+ -independent 

exocytosis. Osmoinduced exocytosis occurs by mechanisms known as “Kiss and Run”. 

270


BIOP-23 

Photochromic green and blue fluorescent protein mutants: 

a Raman study of the chromophore states 

Stefano Luin 1,3 , Valerio Voliani 1,2 , Giacomo Lanza 1 , Valentina Tozzini 1,2 , Ranieri 

Bizzarri 1,2,3 , Riccardo Nifosì 1,2 , Michela Serresi 1,3 , Fabio Beltram 1,2,3 

1 Scuola Normale Superiore, 2 NEST CNR-INFM, 3 IIT Research Unit; Scuola Normale Superiore, 

Piazza dei Cavalieri 7, I-56126 Pisa (Italy). E-mail: s.luin@sns.it. 

Intrinsically fluorescent proteins (IFPs) of the green fluorescent protein family are extensively used in 

molecular and cellular biology as genetically encoded fluorescent markers for monitoring protein dynamics 

and interactions. Specific mutations make it possible to tailor the protein structure and consequently their 

chemical and photophysical properties such as color, quantum yield, sensitivity to pH or other ions, and 

their photochromic properties [1-3] . Raman spectroscopy is a powerful method to investigate selectively 

conformational changes in active domains of these proteins, particularly the chromophores. Indeed by 

exciting under pre-resonance conditions it is possible to measure the vibrational spectrum of the 

chromophore without the need of crystallization. This is extremely helpful to enable a rational protein 

engineering. Moreover, Raman is a non-destructive technique that allows one to monitor on-the-flow the 

products of photoconversion. 

We used this technique in order to study photochromism in IFPs in two cases: a blue highly-stable variant, 

and a green mutant whose photochromism is fully reversible with negligible loss of active protein. 

Theoretical and experimental results on chemically synthesized isolated chromophores under different 

protonation and/or isomerization states will be presented: a very good agreement with calculations based on 

time-dependent density functional theory for resonant and pre-resonant Raman spectra will be shown. This 

will allow us to clarify the nature of the detected vibrational modes and to link the latter to the different 

ground states configurations. Based on this knowledge, we shall discuss the chromophore state when in the 

protein. These results allow us to discriminate between the effect of cis-trans isomerization and of different 

protonation of the chromophore in the photoproducts of these proteins. 

The impact of these results for the design of photocromic IFPs with improved stability and reversibility for 

a number of applications will be discussed. 

References: [1] R. Bizzarri et al., Biochemistry 46, 5494 (2007). [2] D. Arosio et al., Biophys. J., in press; available 

on-line (2007). [3] S. Habuchi et al., J. Am. Chem. Soc. 127, 8977 (2005) 

271


BIOP-24 

Time-resolved microspectrofluorimetry and fluorescence imaging techniques: 

Study of cellular uptake of modified oligonucleotides 

Petr Praus 1 , Eva Kočišová 1 , Peter Mojzeš 1 , Josef Štěpánek 1 , Franck Sureau 2 and 

Pierre-Yves Turpin 2 

1) Charles University in Prague, Faculty of Mathematics and Physics, Ke Karlovu 3, Prague 2, CZ-121 16, 

Czech Republic. E-mail: praus@karlov.mff.cuni.cz 

2) BioMoCeTi (Laboratoire de Biophysique Moléculaire, Cellulaire et Tissulaire) UPMC/CNRS UMR 7033 

GENOPOLE Campus 1, 5, rue Henri Desbruères 91030 EVRY Cedex, France 

Confocal microspectrofluorimeter has been adapted for time-resolved intracellular fluorescence 

measurements by using a phase-modulation principle and homodyne data acquisition method. This 

approach has been employed to acquire intracellular time-resolved fluorescence spectra, which enabled us 

to determine lifetimes from selected sites in the cell. 

Modified oligonucleotides (ON) as sequences of chemically prepared deoxyribo- or ribonucleotides are 

able to inhibit primarily transcription of a specific gene (antigene strategy), translation from mRNA into 

protein (antisense strategy) or the function of a key targeted protein (aptamer strategy) [1]. Potential 

successful healing properties are conditioned by effective ON uptake through the cellular membrane. 

Synthetic derivatives of porphyrins, important biological molecules, seem to be one of the promising 

candidates for this purpose. Moreover their cationic forms have been already studied and successfully 

employed in the ON uptake [2]. Cationic porphyrin (H 2 TMPYP 4 ) assisted delivery system is studied to be 

used for modified ON intracellular transport. Time-resolved fluorescence spectra can monitor the ON 

interactions with present biological molecules. Fluorescence confocal microimaging has been employed as 

a complementarily technique to observe the oligonucleotide uptake into the living cells and its intracellular 

distribution. It clearly visualizes penetration of the ONs through cellular membrane and their progress 

inside the cell. 

Fluorescence image of 3T3 cell incubated overnight 

with rhodamine labeled dT 15 phosphorothioate 

complexed with cationic porphyrin used as delivery 

agent (right) and intracellular fluorescence spectrum 

recorded on the CCD detector (up) 

Acknowledgements: The financial support from the Ministry of Education of the Czech Republic (No. MSM 

0021620835) is gratefully acknowledged. 

References: [1] J. Goodchild, Curr Opin Mol Ther, (2004), 6, 120-8. [2] L. Benimetskaya et.al., Nucleic Acids Res. 

(1998), 26, 5310-5317. 

272


BIOP-25 

Structural changes in the cell membrane induced by radiation exposure 

Inta Kalnina 1 , Tija Zvagule 2 , Natalija Gabruseva 2 , Jelena Kirilova 1 , Natalja Kurjaane 2 , 

Ruta Bruvere 3 , Andris Kesters 2 , Gunta Kizane 5 , Imants Merovics 4 

1 Daugavpils University, 13 Vienibas Str., LV-5491 Daugavpils (Latvia) E-mail: lulc@lanet.lv 

2 Riga Stradins University, 16 Dzirciema Str. , Riga (Latvia) 

3 Biomedical Research and Study Centre, 1 Ratsupites Str. Riga (Latvia) 

4 Riga Technical University, Kalku Str. Riga (Latvia). 

5 University of Latvia 19 Raina Boulv. Riga (Latvia) 

The effects of ionizing radiation on biological membranes include alterations in membrane proteins and 

unsaturated lipids accompanied by perturbations in lipid bilayer polarity [1]. The present study investigates 

several aspects of the membrane damage caused by radiation exposure in relation to the resulting 

biophysical modifications. The a study of 54 Latvian residents, who participated in the accident cleaningup 

works in Chernobyl during 1987-1988 were selected during April 2006 – June 2007. We have used the 

fluorescent probe ABM – derivative of the 3-aminobenzanthrone, developed at the Riga Technical 

University [2]. We registered the spectral characteristics of ABM in peripheral blood mononuclear cell 

(PBMC) suspension and blood plasma, determine the membrane anisotropy and plasma albumin selffluorescence. 

Screening of the ABM-labeled cell samples revealed two patterns of fluorescence spectra: 

(1) the fluorescence zone shifted (compared to the spectrum for healthy donors) by 10-50 nm to the 

shortwave region (max 580-620 nm), (2) a wide fluorescence maximum (plateau) is observed in the 625- 

650 nm. In the group (1) the shift of fluorescence maximum on passing from 620 nm to 580 nm is 

accompanied by increasing ABM fluorescence intensity from 1.3 to 4.0 times higher than that previously 

observed in healthy donors. The obtained patterns of spectra are due to ABM fluorescence originating 

from lipid-bound probe and protein-bound probe. The two groups of patients differed not only by the 

fluorescence spectra, but also by anisotropy index. Using fluorescent probes ABM and ANS lipophilic 

phase of membrane was shown to be more fluid whereas the lipid-protein interface was shown be more 

rigid in observed patients as compared by healthy donors groups. The data of ABM spectral characteristics 

in blood plasma and plasma albumin self-fluorescence showed that Irradiation exposure affect the structures 

(modifications) of membrane proteins. It is necessary to note that all investigated parameters significantly 

differ in the observed groups of patients. 

Taken together, these results evidencing that the cell membrane is a significant target of radiation. Spectral 

characteristics (patterns of spectra) correlate with immunological characteristics. Clean-up workers with 

most significant changes in spectrum also demonstrates largest changes and negative dynamics in EEG. 

References: [1] A. Berroud, A. Le-Royet et al, Radiat. Environ. Biophys. 35 (1996) 289. [2] I. Kalnina et al, 

Proceedings of the Latvian Academy of Science 60 (2006) 113. 

273

274

Part IX 

Fluorescence in Biology, 

Medicine, Bioassays and 

Diagnostics 

275

276

Abstracts Poster – Part IX: Biology 

BIOL-1 

Meso-substituted tetra-cationic porphyrins photosensitize the death of 

HeLa cells via mitochondrial target 

Christiane Pavani 1 , Adjaci U. Fernandes 2 , Maurício S. Baptista 2 , Yassuko Iamamoto 1 

1 

University of São Paulo, FFCLRP, Chemistry Department, Ribeirão Preto (Brazil). 

2 

University of São Paulo, Chemistry Institute, São Paulo (Brazil). e-mail: christp@usp.br 

Photodynamic therapy is being evaluated as a new promising modality fot the treatment of neoplasic 

diseases. This treatment is based on the administration of photosensitizing dyes, followed by exposure of 

the tumor area to light at appropriate wavelenghts [1]. The intracellular localization of photosensitizers, 

and consequently the cellular photosensitization efficiency, strongly depend on their structure. In 

particular, the distribution of polar and hydrophobic substituents around the chromophoric macrocycle 

are important. The charge of the side chains also plays a significant role [2]. Cationic photosensitizers 

are potentially effective clinical agents because, when they display appropriate structural features, they 

accumulate inside the mitochondria driven by the transmembrane potential of the inner mitochondrial 

membrane [3]. Mitochondria targetting is considered particularly important for an effective anti-cancer 

therapy inasmuch as inhibition of mitochondrial functions and/or damage to mitochondrial components 

are very critical for cell survival and may induce a rapid apoptotic response. In this work, we examined 

the subcellular localization of a series of new porphyrin photosensitizers by staining the HeLa cells with 

the porphyrin and the Rhodamine 123 probe and obtaining the fluorescence image on a confocal 

microscope (Zeiss LSM510). Dark and phototoxicity of the compounds, a series of derivatives of 

5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin whith varying degrees of lipophilicity, were also 

examined was varied through replacement of the methyl groups with alkyl chains with eight carbons 

and zinc insertion was also carried out. In the toxicity experiments (dark and light) we used 10 -6 mol L -1 

solutions of the compounds and an incubation time of 3 hours. The phototoxicity experiment were 

carried out using the Morgotron Laser 20 mW emiting at 532 nm for metal free porphyrins and the Laser 

line INOVA 300 mW emiting at 650 nm equipped with a diffuser for zincporphyrins. The cells were 

irradiated with 7 cycles of 1-minute irradiation and 1-minute waiting for oxygenation. The cell viability 

was determined by the MTT colorimetric assay as described by Mosmann [4]. 

Fluorescence images of the HeLa cells 

stained with the compounds. 

Intracellular localization of: a) 100 

nmol.L -1 Rhodamine probe b) 2,1 

μmol.L -1 5,10,15,20-tetrakis(N-octyl- 

4-piridyl)porphyrin; after 3 hours of 

incubation at 37° C. 

The image of the HeLa cells stained 

with the compound displays red 

fluorescence distributed through the 

entire cytoplasm, but this remained 

outside the nucleus. 

A very similar fluorescence distribution was observed between the porphyrin fluorescence and the 

mitochondrial probe Rhodamine fluorescence. These observations suggest that, under our experimental 

conditions, the porphyrin is mainly localizated in the mitochondria. The toxicity tests showed an 

increase in the dark toxicity with increasing porphyrin lipophilicity. This effect is more pronunced in the 

light toxicity tests than in the dark ones as a result of the increasing singlet oxygen quantum yield. We 

also observed the effect of zinc insertion into the porphyrin ring core. The zinc porphyrins are more 

toxic to HeLa Cells than the respective metal free porphyrin, both in the dark and with light activation. 

References: [1] F.C.B. Vena et al. Lasers Med Sci 19 (2004) 119-126. [2] I. Bronshtein et al. Photochem. 

Photobiol. 82 (2006) 1319-1325. [3] F. Ricchelli et al. Int J Biochem Cell B 37 (2005) 306-319. [4] T. Mosman 

J. Immunol Methods 65 (1983) 55-63. 

277


BIOL-2 

Anti-cholesterol IgG antibodies: novel probes of clustered membrane 

cholesterol (microdomains) in intact cells 

Andrea Balogh, András Lőrincz, Glória László, János Matkó 

Eotvos Lorand University, Institute of Biology, Department of Immunology, H-1117 Budapest, Hungary. E- 

mail: andi.balogh@gmail.com 

Natural autoantibodies against cholesterol are present in the sera of all healthy individuals, however, their 

function, production and regulation is still unclear. Until now only murine monoclonal IgM anti-cholesterol 

antibody (ACHA) was produced and characterized. We generated two new mouse IgG3 ACHAs (AC1 and 

AC8) reactive with cholesterol and structurally closely related sterols, by immunizing mice with 

cholesterol-rich liposomes. The 3β-OH moiety of sterols proved to be a critical motif in their binding, while 

no cross-reactivity was found with non-sterol lipids. The IgG3 ACHAs also reacted with lipoproteins, 

VLDL, LDL and HDL [1] . 

Here we further characterized the IgG3 ACHAs in terms of their binding to cellular structures and 

localization. They bound weakly to the surface of various murine and human lymphocyte and 

monocyte/macrophage (Mf) cell lines. Their binding was enhanced by limited papain digestion of 

protruding extracellular protein domains (e.g. CD44, CD45), indicating that the weak binding is likely due 

to masking of the small epitope. The membrane-bound ACHA showed a strongly patchy staining (domains 

with 2-400 nm diameter) and highly colocalized with both lipid (choleratoxin B) and protein (Thy1, 

caveolin-1) markers of non-caveolar and caveolar lipid rafts, and somewhat weaker with chlatrin-coated 

pits (CD71), as assessed by confocal microscopy. This suggests that AC8 preferentially recognizes locally 

clustered membrane cholesterol, consistent also with its intracellular colocalization with markers of ER and 

Golgi complex. That AC8 co-polarized with lipid rafts in T-cells’ membrane upon mitogenic activation 

indicates its ability to monitor raft redistribution during signal transduction. 

In addition, we have shown that AC8 augmented both antigen presentation in an APC-Th cell 

immunological synapse model and the phagocytosis of yeast cells by Mf cells. Thus, the first IgG ACHAs 

can be considered as potential modulators of several important immune functions dependent on cholesterolrich 

lipid rafts, such as pathogen internalization or lymphocyte activation. Consistent with their 

substantially enhanced binding to the surface of various immunocytes upon a moderate papain digestion, 

we propose that IgG ACHAs, in contrast to IgM type ACHAs, may act directly as modulators of immune 

functions, especially under conditions altering the epitope accessibility on their target cells (e.g. apoptosis, 

tumor, virus infection, etc.). Further studies on the mechanism of their immunomodulatory action, as well 

as, attempts to their application in ELISA or protein-chip HTS assay systems are currently running in our 

laboratory. 

This work was supported by Grant T049696 from the Hungarian National Science Foundation (OTKA). 

Reference: [1] A. Biro et al., Journal of Lipid Research 48 (2007) 19. 

278


BIOL-3 

Cytogenetic erbB-receptor gene quantification in breast cancer using the 

pseudoconfocal ApoTome TM technology 

Gero Brockhoff 1 , Andrea Sassen 1 , Justine Rochon 2 , Peter Wild 3 , Arndt Hartmann 1 , 

Stephan Schwarz 1 , Ferdinand Hofstaedter 1 

1 Institute of Pathology, University of Regensburg, Germany, 2 Center for Clinical Studies, University of 

Regensburg, Germany, 3 Institue of Pathology, University of Zurich, Switzerland 

The Her2 receptor tyrosine kinase (RTK) has been linked to 

carcinogenesis and tumor progression and consequently became 

an indispensable diagnostic marker in metastatic breast cancer 

patients. Reliable and quantitative detection of Her2 gene 

amplification via fluorescence-in-situ-hybridization (FISH) in 

tumor tissues is essential and represents evidence for targeted 

therapy using Herceptin, a humanized monoclonal antibody 

(Trastuzumab). However, only about 50% of Her2 positive and 

Herceptin treated patients benefit from this therapy either in terms 

of recurrence free survival (RFS) or overall survival (OS) 

indicating that additional molecular and/or cellular parameters 

have impact on the course of disease and therapy efficiency in 

Her2 positive breast cancer patients. We extended Her2 FISH 

analysis to all related erbB-receptor genes comprising Epidermal- 

Growth-Factor-Receptor (EGFR), Her2, c-erbB3, and c-erbB4. 

The approach is based on the rationale that the respective receptor 

proteins represent a complex signaling network and interact with 

each other depending on their individual expression density that in 

turn is primarily determined by gene copy number. 

Fluorescence-in-situ-Hybridization 

(FISH): Her2 gene amplification 

(green signals) and polysomy (red 

signals) in breast cancer. 

Here we present quantitative multiplex-FISH (M-FISH) in primary tumor tissue using fluorescent DNA 

probes targeted to four erbB-receptor genes (ZytoVysion, Bremerhaven, Germany) and quantified gene 

copy numbers related to chromosome number. 278 primary tissue specimens were analyzed in a 3- 

dimensional (3-D) manner using the pseudoconfocal ApoTome TM technology built-in an AxioImager-Z.1 

automated microscope (Zeiss, Goettingen, Germany). Stack imaging of six µm sections provide the basis 

for 3-D image construction enabling quantitative signal assessment without loss of information. 3-D 

pseudoconfocal images were constructed from stack images. The procedure was supported by elimination 

of scattered light out of focus (structured illumination). The QuantiFISH TM add-on, complementing 

AxioVision software, allows single cell identification and automatic FISH signal recognition within tissue 

organization. Additionally we assessed receptor protein expression using fluorescent and conventional 

immunohistochemistry (IH). 

Probe hybridization and quantification works reliably both in three and five color setup (FISH probes and 

DAPI nucleus staining respectively). Her3 gene amplification (gene-centromer ratio > 1.3) turned out to 

have additional negative impact on overall survival in Her2-neg. (gene-centromer ratio > 2.0) breast cancer 

patients, whereas Her1 and Her4 amplification is a rather rare event without additional significant impact 

on prognosis in terms of OS. Protein expression appeared more variable and in contrast to FISH bears less 

prognostic information. 

M-FISH of erbB-receptors based on 3D-imaging provides valuable additional information in pathological 

diagnosis of breast cancer tissues and supports understanding of related gene alteration which is responsible 

for carcinogenesis and tumor progression. 

References: Brockhoff G, Heckel B et al.: Cell Prol, 2007, Brockhoff G: Verh Dtsch Ges Path 90, 2006; Lottner C, 

Schwarz S, et al.: J Pathol. 2005; Diermeier S, Horvath G et al.: Exp Cell Res, 2005. 

279


BIOL-4 

New ratiometric fluorescent probes for apoptosis 

Dmytro A. Yushchenko, Andrey S. Klymchenko, Vasyl V. Shynkar, Vanille Greiner, 

Hugues de Rocquigny, Volodymyr V. Shvadchak, Guy Duportail, Yves Mély 

Photophysique des Interactions Biomoléculaires, UMR 7175 du CNRS, Faculté de Pharmacie, 

Université Louis Pasteur, 67401 Illkirch (France). E-mail: Dmytro.Yushchenko@pharma.u-strasbg.fr 

Apoptosis, the programmed cell death, plays a key role in tissue homeostasis 1 . New fluorescent probes for 

apoptosis can help to understand better its basic mechanisms. Moreover, they can be very useful for 

monitoring the therapeutic treatment of diseases that show imbalance between cell proliferation and cell 

loss. At the early steps of apoptosis the loss of phospholipid asymmetry of the plasma membrane results to 

the exposure of phosphatidylserine (PS) residues at the outer plasma membrane leaflet 2 . To detect this 

process we developed a fluorescent probe (F2N12S) which stains selectively the outer leaflet of the cell 

plasma membrane. The fluorescent reporter of this probe is 4’-(diethylamino)-3-hydroxyflavone, which 

exhibits excited-state intramolecular proton transfer (ESIPT), resulting in two-band emission highly 

sensitive to the lipid composition of the biomembranes. Fluorescence spectroscopy, flow cytometry and 

microscopy measurements show that the ratio of the two emission bands of the probe changes dramatically 

in response to apoptosis. 3 However, though this new dye appeared very promising, its wide application is 

still limited by its excitation wavelength. Therefore, we developed a new improved fluorophore reporter, 2- 

(2-(dialkylamino)thienyl)-3-hydroxychromone. Applying design principles of the first generation apoptosis 

probe F2N12S to this fluorophore, we synthesized the new probe TCN12S. This probe in lipid vesicles and 

cell membranes exhibit excitation maximum close to 450 nm, which is already suitable for confocal 

microscopy with common lasers He/Cd (442nm) and Ar laser (458nm line). Moreover, in lipid vesicles the 

new probe exhibits higher sensitivity to the surface charge and better resolution of the two emission bands 

compared to F2N12S. Examination of the new probe in cells using fluorescence spectroscopy and confocal 

microscopy shows that it is highly sensitive to apoptosis in HeLa cells induced by actinomycin D. The twoband 

ratiometric response of the new probe to apoptosis is considerably larger as compared to its parent 

probe F2N12S. In addition, the second generation probe stains fast the cell plasma membranes and allows 

stable measurements for at least 45 min at 37 o C. Due to their properties, the new probes appear highly 

promising for a wide application in the apoptosis research. 

I N* /I T* ratio 

I N* /I T* ratio 

0.9 

NORMAL CELLS 

0.9 

APOPTOTIC CELLS 

Ratiometric fluorescent 

images of a) normal HeLa 

cells and b) cells treated 

with actinomycin D (18h 

incubation) obtained with 

confocal microscope. 

0 0 

References: [1] G. F. Erickson, J. Soc. Gynecol. Investig. 4 (1997) 219-28. [2] V. A. Fadok, D. J. Laszlo, P. W. 

Noble, L. Weinstein, D. W. Riches, P. M. Henson, J. Immunol. 151 (1993) 4274–4285. [3] V. V. Shynkar, A. S. 

Klymchenko, C. Kunzelmann, G. Duportail, C. D. Muller, A. P. Demchenko, J.-M. Freyssinet, Y. Mely, J. Am. 

Chem. Soc., 129 (2007) 2187-2193. 

280


BIOL-5 

DNA-ZIP code based glycoarray. A dual fluorescence assay for probing 

lectin carbohydrate affinity 

Yann Chevolot 1 , Camille Bouillon 2 , Sébastien Vidal 3 , François Morvan 2 , Albert Meyer 2 , 

Jean-Pierre Cloarec 1 , D. Lallemand 1 , Anne Jochum 3 , Jean-Pierre Praly 3 , Emmanuelle 

Laurenceau 1 , Magali Phaner Goutorbe 1 , Jean-Jacques Vasseur 2 , and Eliane Souteyrand 1 

1 Institut des Nanotechnologies de Lyon, Equipe Chimie et Nanobiotechnologies, Ecole Centrale de Lyon, 

36 Avenue Guy de Collongue, 69134 Ecully, France. 

2 Institut des Biomolécules Max Mousseron UMR 5247 CNRS UM1 UM2, Laboratoires des Analogues et 

Constituants des Acides Nucléiques, Université de Montpellier II, Place E. Bataillon, CC008, 34095 

Montpellier, France. 

3 Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Laboratoire de Chimie Organique 2 – 

Glycochimie, Université Claude Bernard Lyon 1, 43 Boulevard du 11 novembre 1918, Bâtiment 308 – 

CPE, 69622 Villeurbanne, France. E-mail : yann.chevolot@ec-lyon.fr 

Glycans are information-rich molecules composed of complex carbohydrates (sugars or polysaccharides) 

that are often attached to proteins or lipids. Carbohydrates and glycoconjugates play a major role in key 

biological events [1, 2] . As a consequence, there is a need for understanding the underlying structural 

parameters governing the recognition of carbohydrates by their receptors. 

However, research in this field is slowed by the wide diversity of carbohydrate structures and by the minute 

amounts of materials available for experimentation. Carbohydrate microarrays are an attractive tool for the 

design of sensitive and high-throughput technologies for the characterisation of oligosaccharide/protein 

interactions [3] . 

However, this technology has various limitations: (1) As argued by Alvarez (Nature Methods 2006, 3(7), 

571-578), “one of the limitations of the technique (i.e. of carbohydrate array) is that there is no way of 

knowing how much sample is bound at each spot…”. (2) The surface vicinity can hinder the interaction of 

the immobilised probe. (3) The interactions of oligosaccharides with lectins are usually weak (mM range). 

(4) Finally, the syntheses of functionalized oligosaccharide ligands are labour intensive. 

We have demonstrated [4] , that some of these limitations can be circumvented using an original approach for 

the surface immobilization of oligosaccharides based on original glycoconjugate molecules presenting a 

DNA sequence for anchoring onto DNA “zip code” chips through hybridization. 

Such strategy has been shown to be very successful in the field of DNA array, but in the field of protein 

array. [5] [6, 7] . 

We have demonstrated that this approach has the following advantages: 

• An original synthesis strategy was developed for glycomimetics based on an pseudo oligonucleotide 

scaffold [8] allowing solid phase synthesis and microwave assisted click chemistry. 

• Very minute amounts of materials are necessary for immobilisation of the oligosaccharide/DNA conjugate 

(1 µM vs mM range reported in the literature) and thus with a lower detection limit of 2-20 nM. 

• DNA hybridization is reversible and we have demonstrated that our biochip is therefore reusable. 

• Our methodology allows determining the relative surface density of bound carbohydrate based on a 

double fluorescence labelling. 

• The biological lectin/oligosaccharide recognition was performed in solution before hybridisation of the 

whole complex onto the DNA chip. This process circumvents some of the limitations related to the 

vicinity of the surface. 

Future work will focus on understanding kinetic and thermodynamic parameters between the two strategies 

the so-called “on-chip” approach and the “in-solution” approach. 

References: [1] A. Varki, Glycobiology, 3 (1993) 97. [2] N. Sharon, H. Lis, Glycobiology, 14 (2004) 53R. 

[3] T. Feizi, F. Fazio, W. C. Chai, C. H. Wong, Current Opinion in Structural Biology, 13 (2003) 637. [4] Y. 

Chevolot, C. Bouillon, S. Vidal, F. Morvan, A. Meyer, J.-P. Cloarec, A. Jochum, J.-P. Praly, J.-J. Vasseur, E. 

Souteyrand, Angewandte Chemie International Edition, 46 (2007) 2398. [5] S. Weng, K. Gu, P. Hammond, W., P. 

Lohse, C. Rise, R. W. Wagner, M. Wright, C., R. Kuimelis, G., Proteomics, 2 (2002) 48. [6] Y. Seong Choi, S. Pil 

Pack, Y. Je Yoo, Biochemical and Biophysical Research Communications, 329 (2005) 1315. [7] C. Boozer, J. Ladd, 

S. F. Chen, S. T. Jiang, Analytical Chemistry, 78 (2006) 1515. [8] C. Bouillon, A. Meyer, S. Vidal, A. Jochum, Y. 

Chevolot, J. P. Cloarec, J. P. Praly, J. J. Vasseur, F. Morvan, Journal of Organic Chemistry, 71 (2006) 4700. 

281


BIOL-6 

Exploring the oligomerization state of muscarinic receptor M1 in living cells 

with FRET by cytometry and by anisotropy imaging 

Spencer Brown 4 and Gilles Mourier 1 , Catherine Marquer 1 , Carole Fruchart-Gaillard 1 , 

Emmanuelle Girard 2 , Olivier Grandjean 3 , Denis Servent 1 

1 CEA, iBiTECS, Service d’Ingénierie Moléculaire des Protéines (SIMOPRO), Laboratoire de Toxinologie 

Moléculaire et Biotechnologie, 91191-Gif sur Yvette cedex, France. E-mail : catherine.marquer@cea.fr 

2 Laboratoire de Neurobiologie Cellulaire et Moléculaire UPR 9040, CNRS, Institut Fédératif de 

Neurobiologie Alfred Fessard, 1 Avenue de la Terrasse, 91198-Gif sur Yvette cedex, France. 

3 

IJPB-Laboratoire Commun de Cytologie, UR 254, INRA, RD 10, Route de Saint-Cyr, 78026-Versailles 

cedex 

4 "Dynamique de la compartimentation cellulaire", Institut des Sciences du Végétal, UPR 2355, CNRS, 

1 Avenue de la Terrasse, 91198-Gif sur Yvette cedex, France. 

Human muscarinic receptor M1 (hM1) is a member of the G-protein coupled receptor family (GPCR). This 

receptor is characterized by at least two distinct ligand binding sites. Agonists and competitive antagonists 

bind to the orthosteric site, located inside the transmembrane domain, whereas allosteric agents induce a 

significant perturbation of the kinetics of binding of ligands to the primary site by interacting with an 

allosteric site, located more extracellularly. Due to the saturable effect of allosteric agents, to their potency 

to modulate the functional activity of endogenous ligands and to their important subtype specificity, 

considerable efforts are underway to identify and develop novel therapeutic agents targeting the allosteric 

sites of GPCRs. The most potent and specific allosteric ligand of receptor hM1 is the muscarinic toxin 

MT7. MT7 is a 65 residues peptidic neurotoxin, initially purified from the venom of African mamba 

Dendroaspis angusticeps and lately chemically synthetized. [1-2] 

Using double mutant cycle analysis and docking studies, we recently described a model of the interaction of 

MT7 on the hM1 receptor. [3] To pursue this interaction, we have developed two different fluorescent 

approaches in order to explore the oligomerization state of hM1 in living cells, in the absence or presence 

of MT7. 

The emission fluorescence anisotropy of hM1-EGFP receptors expressed at the surface of living human 

cells (TSA culture) will be reduced in the case of dimerization due to homo-FRET between EGFPs : this is 

imaged in a confocal microscope with crossed polarization analysers and off-line calculation. 

In parallel, we run FRET experiments by flow cytometry. Here, TSA cells preincubated or not with MT7 

and cotransfected with two types of receptors either linked to EGFP or indirectly labeled with a Cy3 

fluorophore [4] are assessed for EGFP-Cy3 FRET by enhanced emission of the acceptor. 

Results obtained using these complementary techniques will be presented. 

References : [1] G. Mourier et al., Mol. Pharmacol. 63 (2003) 26. [2] C. Fruchart-Gaillard et al., Mol. Pharmacol. 69 

(2006) 1641. [3] C. Marquer et al., submitted. [4] C. Weill et al., J. Neurochem. 73 (1999) 791. 

282


BIOL-7 

Understanding GFP pH-dependent ground states: a way to design tailored 

ratiometric pH biosensor targetable in vivo 

Ranieri Bizzarri,º , Caterina Arcangeli, Daniele Arosio, Stefania Abbruzzetti, ‡ Gianpiero 

Garau, § Barbara Campanini, † Cristiano Viappiani, ‡ Fabio Beltramº , 

ºScuola Normale Superiore, IIT research unit, P.za dei Cavalieri 7 I-56126 Pisa (Italy); Scuola Normale 

Superiore, NEST CNR-INFM, P.za dei Cavalieri 7 I-56126 Pisa (Italy); ‡ Dipartimento di Fisica, Università 

di Parma – NEST CNR-INFM, viale G.P. Usberti 7A 43100 Parma (Italy); § Biocrystallographic Unit- 

DIBIT, San Raffaele Scientific Institute, via Olgettina 56, 20134 Milano (Italy); † Dipartimento di 

Biochimica e Biologia Molecolare, Università di Parma, viale G.P. Usberti 23A, 43100, Parma (Italy). 

E-mail: r.bizzarri@sns.it 

Intracellular pH (pH i ) is an important modulator of cell function, as the activity of most protein is affected 

by small changes of H + concentration. Hence, protein-based fluorescent ratiometric pH indicators appear 

more advantageous than their organic counterparts, as they can be selectively targeted to subcellular 

compartments by genetic engineering. The Green Fluorescent Proteins (GFP) show a naturally optimized 

structure for fluorescent probing of environmental pH, due to the phenolic characteristics of the 

fluorophore. [1] Although some GFP-based ratiometric pH indicators have been reported, most are not 

optimized for the physiological pH range of the cellular processes or rely upon FRET couples whose 

control is difficult to achieve. [2] 

From our detailed analysis of GFP protonation photophysics, [3] we developed a ratiometric excitation and 

emission pH indicator (E 2 GFP), which shows an optimized working range between pH 6 and 8. 

Remarkably, E 2 GFP allows the selection of the proper excitation line (in the range 400-500 nm) or emission 

interval (in the range 480-600 nm) to obtain a ratiometric signal with a modulable amplitude and pHresponse 

linearity range. The presence of effectors known to bind reversibly to GFP variants in the 

intracellular environment (for example: chloride anion) [4] was demonstrated to affect neither the ratiometric 

calibration curve, nor the pH i measurement. [2] E 2 GFP and a closely related mutant designed to report on 

lower pH ranges are currently used in our laboratory to monitor pH in vivo under different physiological 

conditions and/or targeted to specific organelles by fusion with localization signals (Figure 1). Both the 

proton-dependent photophysical characteristics of GFPs and the biological relevance of the developed 

indicators will be reviewed. 

Time=0’ +15’ +18’ +19’ +21’ +24’ +36’ 

Figure 1. Time evolution of the 

transmission image (left column), 

intensity image (center column), and 

ratiometric pH map by excitation 

(right column) of one dividing CHO 

cell transfected with E 2 GFP and 

maintained in physiological medium. 

Below is reported the normalized 

frequency histograms of the spatial 

pH i maps by excitation of the dividing 

cell, at Time=0’ (black bars), +18’ 

+110’ 

References: [1] G. T. Hanson, et al., Biochemistry 41 (2002). [2] R. Bizzarri, et al., Biophys. J. 90 (2006) 3300. 

[3] R. Bizzarri, Biochemistry, in press. [4] D. Arosio, Biophys. J., in press. 

283


BIOL-8 

Properties of fluorescence dyes used for labeling DNA in 

microarray experiments 

Jens Sobek, Catharine Aquino, Ralph Schlapbach 

Functional Genomics Center Zurich, Winterthurerstrasse 190, CH-8057 Zurich (Switzerland) 

E-mail: Jens.Sobek@fgcz.ethz.ch 

In a typical microarray experiment, fluorescence dyes are used for signal detection. Hence, the performance 

of these experiments strongly depend on dye spectral properties (absorbance, fluorescence), fluorescence 

lifetime, quantum yield, and dye stability. The most common dyes used in this field are indocarbocyanines 

(trimethines, pentamethines). A general problem of many organic dyes, especially those absorbing at long 

wavelenghts, is an intrinsic instability to oxidation by atmospheric ozone which causes a fast loss of signal 

due to dye degradation at the microarray surface. In our study we have performed microarray model 

experiments using oligonucleotides labeled with CY3, CY5, and related dyes, including CY3 and CY5 

derivatives, unsymmetrical (mero-) cyanines, rhodamines, and carborhodamines. We have compared 

fluorescence intensities, photostability and environmental stability of oligonucleotide-dye conjugates 

hybridised to complementary sequences immobilised at the microarray slide surface. For some dyes we 

observed an influence of probe sequence on fluorescence intensity that is related to the overall dye charge. 

Additionally, we determined spectral data in solution including absorption, excitation, and corrected 

emission spectra, as well as fluorescence lifetimes and fluorescence quantum yields. 

284


BIOL-9 

Complexity of ceramide signals and their impact on life or death of 

lymphocytes: complex fluorescence flow- and image cytometric analysis 

Endre Kiss, Cynthia Detre, Janos Matko 

Eotvos Lorand University, Institute of Biology, Department of Immunology, H-1117 Budapest, Hungary. E- 

mail: kisse@elte.hu 

Ceramides (Cer) released from plasma membrane sphingomyelin upon cell death, stress or inflammatory 

stimuli are important mediators of various lymphocyte responses, including mitochondrial cell death 

pathway. Recently, we have shown that the fate of T-cells depends on the strength and duration of Ceraccumulation 

in the plasma membrane. In addition, below a deathful threshold, the ceramide signal 

suppressed the antigen-induced T-cell activation [1] . This immunomodulatory effect may have an interest in 

selective immunesuppressive therapy of diseases linked to cellular immune responses by autoreactive T 

lymphocytes. 

In the present work we aimed at: 1. identifying the major Cer-targets involved in this immunomodulatory 

effect, 2. analyzing the ceramide-effect on the activation signalling and cell fate of other immunocytes of 

various maturation/differentiation stage. Calcium signals of the cells were analyzed with both the 

statistically robust flow cytometric method using Fluo-3 or Fluo-4 probes, by confocal microscopic kinetic 

recording and single cell fluorescence microscopy equipped with multichannel perfusion system. We setup 

a multiparameter flow cytometric apoptosis detection panel for investigation early and late stages of 

apoptosis on the same cell sample, including fluorescent detection of Annexin-V binding, mitochondrial 

potential changes (DiOC6(3) or JC-1), caspase 3 activation, spontaneous PI uptake and DNA fragmentation 

(hypotonic extraction+PI). Localization of lipid rafts, ion channels, etc. was done by confocal laser 

scanning microscopy. Using fluorescent microscopy (+ electrophysiology) we found that ion channels 

involved in maintaining the electrochemical driving force for Ca 2+ influx (Kv1.3 potassium channel) and in 

the influx process (CRAC) are both targets of ceramide action. We also identified expression of functionally 

active voltage-dependent Ca 2+ -channels (VDCC-like channels) in T lymphocytes, the mRNA level 

expression of which was reported recently [2] . This channel also showed ceramide-sensitivity. The various 

immunocytes responded to weaker Cer-signals in a highly cell-stage specific way [3] , as shown by their 

antigen-induced Ca 2+ responses. Interestingly, Burkitt B lymphoma cells were fully resistant to low Cer 

doses. A similar heterogeneity was found in the responses of various immunocytes to strong (deathful) 

ceramide signals in terms of both the extent of cell death and its mechanism (apoptosis or primary necrosis). 

Our data suggest that Cer can differentially modulate activation responses or cell fate in lymphocytes of 

various maturation/differentiation stage. Studies on the mechanism and cell stage specificity (differences in 

their membrane composition, microdomain structure, or differential ion channel expressions?) of this effect 

are currently in progress. 

This work was supported by Grant T049696 from the Hungarian National Science Foundation (OTKA). 

References: [1] C. Detre et al., Cellular Signalling 18 (2006) 294. [2] M. F. Kotturi et al., J. Biol. Chem. 278 (2003) 

46949. [3] E. Kiss et al., Ann. N. Y. Acad. Sci. 1090 (2006) 161. 

285


BIOL-10 

Application of different fluorescent (NBD-labeled) probes for evaluation of 

transmembrane phosphatidylcholine distribution in human erythrocytes 

Dzmitry Kostsin, Ekaterina Slobozhanina, Natalia Kozlova 

National Academy of Science of Belarus, Institute of Biophysics and Cell Engineering, 

220072 Minsk (Belarus). E-mail: Kostin_dima@mail.ru 

Various analogous of phosphatidylcholine (PC) are utilized for measurement the distribution of endogenous 

PC between outer and inner leaflet at the plasma membrane in different cells. [1] They are produced by 

diverse companies («Molecular probes», «Avanti Polar Lipids») or prepared and synthesized by authors 

independently in laboratory condition. Nevertheless, not always explotable analog of PC (and used 

methodology) reflect the distribution of plasma membrane PC. [2] 

In the present work we studied distribution of two fluorescent probes of PC (short-chain and long-chain 

NBD-labeled analogous) at the plasma membrane of human erythrocytes. Experiments were performed on 

donor erythrocytes. In this study 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoylsn-glycero-3-phosphocholine 

(16:0/C 6 -NBD-PC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)- 

dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (16:0/C 12 -NBD-PC) from «Molecular Probes» 

(USA) were used. The amount of internalized probe was determined by comparing the fluorescence 

intensity associated with the cells before and after back 

exchange procedure with 2% (for 16:0/C 6 -NBD-PC) or 10% 

(for 16:0/C 12 -NBD-PC) bovine serum albumin (BSA). 

Introduction of NBD-labeled analogous into human 

erythrocytes membrane was clearly distinct for the two 

probes. In case of 16:0/C 6 -NBD-PC incorporation into the 

lipid bilayer was very fast and reached a maximum after 20 

minutes, at the same time integration of 16:0/C 12 -NBD-PC 

occurred much slowly and reached a plateau only after 23-24 

hours of cells incubation. Also the distribution of short-chain 

fluorescent analog of PC was different from that observed 

with long-chain one in human erythrocyte membranes. In 

steady-state condition approximately 80% of 16:0/C 6 -NBD- 

PC distributed into the outer leaflet that reflected the 

sequestration of endogenous PC at the plasma membrane of 

human erythrocytes. Whereas only 20-30% of 16:0/C 12 -NBD- 

PC could be extracted from the outer leaflet by utilizing 

10% BSA (maximal BSA concentration normally used for 

probes extraction from the outer leaflet). 

It was shown that the distribution of 16:0/C 6 - and C 12 -NBD- 

PC in human erythrocyte plasma membranes reliably 

differed for two probes in native condition that can be 

explained by chemical structure of probes. When necessary 

to study or to diagnose status of PC distribution at the 

0 

0 2 4 6 22 24 

hours 

plasma membrane under pathology (for research as well as for practical purposes) it is essential to use 

short-chain fluorescent analog of PC because of it adequately reflect the sequestration of endogenous PC. In 

case of long-chain fluorescent analog of PC actual question is application of other compounds for 

extraction or bleaching the fluorescence of probe incorporated in the outer membrane leaflet. 

This work was supported by Belarussian Republican Foundation for Fundamental Research, grant N B06M-116. 

References: [1] P.F. Devaux et al., Chem. Phys. Lipids. 116 (2002) 115. [2] D. Wustner et al., Biochemistry. 37 

(1998) 17093. 

% in inner leaflet 

80 

60 

40 

20 

The distribution of 16:0/C 6 -NBD-PC (1) 

and 16:0/C 12 -NBD-PC (2) in the inner 

leaflet at the plasma membrane of human 

erythrocytes. Cells were incubated in 20 

mM Hepes buffer; pH 7,4; 37°C; 

λ ex =466 nm; λ em =522 nm 

2 

1 

286


BIOL-11 

Intracellular ROS production and lipid bilayer fluidity modifications in 

human lymphocytes in response to anticancer drug treatment in vitro 

Alexander Tamashevski 1 , Ekaterina Slobozhanina 1 , Sergienko Tatiana 2 , 

Natalia Goncharova 2 , Svirnovski Arcadi 2 

1 Institute of Biophysics and Cell Engineering of National Academy of Sciences, 220072 Minsk (Belarus). E- 

mail: Tayzoe@mail.ru 

2 Republican Scientific and Practical Center for Hematology and Transfusiology, Ministry of Healthcare, 

220053, Minsk (Belarus). 

Therapeutic selectivity is one of the most important considerations in cancer chemotherapy. The design of 

therapeutic strategies is to preferentially kill malignant cells while minimizing harmful effects to normal 

cells. It depends on the biological differences between cancer and normal cells. Such fundamental processes 

as intracellular reactive oxygen species (ROS) production and lipid bilayer fluidity may be used as specific 

tests for early drug response in normal and leukemic cells. 

The aim of the study is to evaluate the intracellular ROS accumulation and lipid bilayer fluidity 

modification in normal and leukemic cell in response to drug treatment. 

Lymphocytes were separated from peripheral blood of donors and patients with chronic lymphocytic 

leukemia (CLL) by density gradient centrifugation on ficoll-urografin. Anticancer drug leucladine (2- 

chlorine -2'-deoxyadenosine) was used in therapeutic concentration of 5μg/ml. Cells were incubated with 

leucladine during 2-3 hours. Lymphocytes sensitivity was estimated by 6-dodecanoyl -2 

dimethylaminonaphtalene (Laurdan, “Sigma”) test [1] . Laurdan is localized at the lipid bilayer hydrophilichydrophobic 

region and allows to estimate membrane fluidity. Fluorescence intensity of 5'-(and-6')- 

chlorometyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA, “Molecular Probes”) assessment 

shows intracellular ROS production [2] . Laurdan fluorescence was measured by spectrofluorimeter SOLAR 

(Belarus), CM-H 2 DCFDA fluorescence was estimated by FACScan (Becton Dickinson, USA). 

Under leucladin treatment in donors lymphocytes CM-H 2 DCFDA fluorescence intensity (FI) increases up 

to 7%, laurdan generalized polarization (GP) decreases by 13%, compared to intact cells. It is observed 

CM-H 2 DCFDA FI 11% increase, and laurdan GP 22% decrease in CLL cells. 

Leucladin leads to intracellular ROS accumulation to a greater extent in leukemic cells, than in normal 

lymphocytes. This effect is the cause of different activity level of deoxycytidine kinase and 5'- 

nucleotidases. In cells with a high activity ratio of deoxycytidine kinase to 5'-nucleotidase, purine 

nucleoside analog rapidly accumulates, activates through phosphorylation, and inhibits DNA synthesis [3] . 

In the same way, laurdan GP decrease is seen in malignant lymphocytes, that is the evidence of lipid bilayer 

fluidity increase in CLL cells after leucladin treatment. Probably, membrane microviscosity changes can be 

associated with multidrug resistance proteins activity [4] . Our preliminary data shows close connection 

between multidrug resistance proteins activity, ROS production and membrane fluidity. 

These findings suggest, that intracellular ROS accumulation and lipid bilayer fluidity increase at early 

stages of CLL lymphocyte activation by leucladine in therapeutic concentrations. According to acquired 

data leucladin isn’t specific drug to leukemic cells, because it leads to intracellular ROS accumulation and 

lipid bilayer fluidity changes in donor cells too. 

References: [1] K.Gaus et al., J. Cell Biol. 171 (2005) 121. [2] J. Chandra, Blood, 102 (2003) 4512. 

[3] H. Kalinichenko, Science and innovations – Belorussian J. 9 (2004) 57. [4] J. Ferte, Biochem. – Eur. J. 267 (2000) 

277. 

287


BIOL-12 

Influence of fluorescent probes of different structure on confluent cell cultures 

Elena I. Goncharuk, Tatyana S. Dyubko., Elena V. Onishchenko, Victoriya V. Timon, 

Valentin I. Grischenko 

Institute for Problems of Cryobiology & Cryomedicine, Natl. Acad. Sci. of Ukraine, 

Kharkov, 61015 (Ukraine). E-mail: goncharuk_elena@rambler.ru 

Highly sensitive fluorescent dyes, binding with different cell organelles, allow to observe visually the 

processes occuring inside a cell. However staining of cells directly before microscopy does not permit to 

monitor cell metabolic processes in time. In this connection it would be useful to find out, whether it is 

possible to cultivate the cells in dye presence during long time. We studied the effect of fluorescent probes 

of distinguished chemical structure on confluent cell cultures at different mode of staining (cell culture 

staining immediately before observation and culture of cell with integrated probe). 

Carbocyanine probes 3,3’-diethiloxocarbocyanine bromide (С2), 3,3’-dinonyloxocarbocyanine bromide 

(С9) and JC-1 were synthesized and given by Igor A. Borovoj (Institute for Scintillation Materials Natl. 

Acad. Sci., Kharkov, Ukraine). 3-hydroxyflavone derivates F2N8, BQBF, FME, PPZ8 were synthesized 

and given by Andrey S. Klymchenko and Vasyl G. Pivovarenko (Natl. Taras Shevchenko University of 

Kyiv, Ukraine). We also have used styryl derivative DSM (4-(N-dimethylaminostyryl)-1-methylpiridine N- 

toluene-sulfonate). All dyes were used in 10 -5 М final concentration. Cell images were obtained using the 

Olympus IX71 fluorescent microscope, equipped with the digital chamber Olympus С-5060. 

At the first stage the confluent cultures of SPEV 

(recultured cell line of the pig’s embryonic 

kidney) and diploid human fibroblast line were 

stained by the standard procedure [1] . It was 

determined that the BQBF, PPZ8 and DSM 

probes the same as С2, С9 and JC-1 ones did not 

exert toxic influence on cell cultures. FME and 

F2N8 hydroxyflavone derivatives caused a toxic 

effect, producing unfastened cells from the glass 

and produced its death. FME and F2N8 dyes 

cytotoxicity was also tested on the cell 

suspension, cell viability decreased on 27±5% 

and 45±7%, accordingly. 

When introducing of dyes into the cell 

suspension and subsequent culturing during 72 

Cells of SPEV line stained by the DSM fluorescent 

probe (×600). 72 hours culturing. 

hours it was established, that carbocyanine probes did not affect cell growth, that testified to the absence of 

its toxic effect. The DSM did not decrease cell adhesive capacity, but partially reduced cell proliferation. 

Confluent culture formed 24 hours later then in control. When culturing cell lines, stained by BQBF, F2N8, 

PPZ8 and FME probes there was an essential decrease of cell capacity to adhesion and proliferation, the 

cells did not grow up to a confluent state. The morphology of cells in both lines was changed, the cells were 

of spherical shape and had cytoplasmic vacuolization. Therefore, the probes of flavone group show toxicity 

and can not be used in integrated state in cells in vitro. Nevertheless unique spectral properties of these 

probes undoubtedly can be used for characterising cells at a short-time contact. 

It was noted, that the luminescence of cells stained with carbocyanine dyes was much more intensive in 

comparison with other ones in all cases of observation. 

Thus, comparing the influence of carbocyanine, flavone and styryl derivatives on cell lines it is possible to 

assert about preferences of carbocyanine probes at long-term joint culturing and monitoring of cell state 

under normal conditions and different effects in vitro. 

Referencs: [1] V. V. Shynkar et al., BBA 1712 (2005) 129. 

288


BIOL-13 

Pyrene labeled α-synuclein: tracking the early stages of 

amyloid protein aggregation 

Shyamala Thirunavukkuarasu 1 , Elizabeth A Jares-Erijman 2 and Thomas M Jovin 1 

1 Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, 37077 Goettingen 

(Germany), 2 Department of Organic Chemistry, Faculty of Natural and Exact Sciences, University of 

Buenos Aires, 1428 Buenos Aires (Argentina). 

E-mail: tshyama@gwdg.de 

The aggregation of α-synuclein (AS), a presynaptic protein, plays an important role in the etiology of 

Parkinson’s disease. The low molecular weight oligomers or protofibrils adopting the β-sheet structure 

characteristic of amyloid proteins are presumed to be the cytotoxic species [1] . Unfortunately, currently 

employed techniques for following the kinetics of AS aggregation e.g. the fluorescence enhancement of 

thioflavin-T detect only the fibrillar species formed at the later stages of the reaction. Moreover, these 

assays are not continuous and lack reproducibility. 

We have devised a new fluorescence aggregation assay that is continuous and can detect the formation of 

oligomeric intermediates [2] . The approach is based on fluorescent tagging of functionally neutral ala-tocysteine 

mutants of AS. Pyrene conjugates of AS at three positions in the AS sequence were used: N- 

terminal (residue 18), the core NAC region (residue 90), and the C-terminal region (residue 140). Pyrene 

was selected as fluorescence probe because of its long fluorescence lifetime, environmental sensitivity, and 

high fluorescence anisotropy in the immobilized state. Different spectral properties of pyrene were 

monitored during AS aggregation: fluorescence intensity, spectral distribution of the monomer emission, 

excimer formation, steady state and time resolved anisotropy and fluorescence lifetime. All of these 

parameters changed in a systematic manner right from the onset of aggregation. The formation of lower 

molecular weight oligomers were evident in both the wild type and genetic (A53T, A30P familial 

mutations) variants of AS, but the responses differed according to the position of the pyrene tag and the 

mutation. The pyrene labeled AS assay represents the first continuous method to follow the early kinetics of 

amyloid protein aggregation. We believe that this new assay will provide a convenient platform for high 

throughput screening of potential therapeutic drugs for Parkinsons’s disease (aggregation inhibitors, 

antagonists) as well as basic science investigation. 

N-terminus NAC C-terminus 

A18C 

A30P 

A53T 

A90C 

A140C 

References: [1] F. Chiti, C.M. Dobson, Annu Rev Biochem 75 (2006) 333. [2] T. Shyamala et al., to be 

communicated 

289


BIOL-14 

Alzheimer’s β-amyloid (1-40) peptide interacts with G M1 -micelles 

Ilya Mikhalyov, Gerhard Gröbner, Lennart B.-Å. Johansson 

Umeå University, Department of Chemistry; Biophysical Chemistry, S-90187 Umeå, Sweden 

E-mail: Ilya.Mikhalyov@chem.umu.se 

Alzheimer’s disease is the most abundant age-related neurodegenerative disease, which is associated with 

progressive deposits of amyloid plaques in the brain. The principal component of amyloid deposits is the 

Aβ-peptide, which contains 39-42 amino acids. 

The interaction between a fluorescent BODIPY-FL-labelled Aβ (1-40) peptide and G M1 ganglioside 

micelles has been studied. A fluorescent ganglioside, BODIPY-564/570-C5-G M1 , which was labelled in the 

polar head of molecule, was mixed with unlabelled G M1 at various molar ratios. The molar ratio between 

peptide and total lipid varied between 1 : 12 and 1 : 325. It is known that Aβ needs negative charged lipids 

for bounding with lipid surface [1, 2]. 

Using RET we observed a strong 

interaction between the peptide and G M1 

micelles, whereby a continuous decrease 

of fluorescence and an increased 

fluorescence steady-state anisotropy of 

peptide emission (donor) occurs upon 

increasing the mole fraction of labelled 

G M1 (acceptor). 

The emission anisotropy of the labelled 

peptide,which is low in the buffer 

solution, increases upon adding the 

labelled as well as the pure G M1 micelles. 

This was observed instantaneous and the 

change was depending on the amount of 

micelles added. A maximum was reached 

at lipid/peptide ratio of 300 : 1. 

r 

0.4 

0.3 

0.2 

0.1 

0.0 

500 550 600 650 700 

nm 

Figure 1: The emission spectrum (normalised, solid line) and the emission anisotropy (dotted) of BODIPY- 

FL-Aβ in buffer. The emission from liposomes of DOPC/DOPG/BODIPY-564/570-G M1 at the lipid/peptide 

ratio of 100 : 1 (short dashed), emission with BODIPY-564/570-GM1/GM1 micelles, lipid/peptide ratio, 

100 : 1 (dot-dot-dashed). The emission anisotropy for the corresponding system (long dashed) and emission 

anisotropy of labelled peptide with non-labelled G M1 micelles (dot-dash). 

After mixing the peptide and micelles the anisotropy slowly increased to a maximum reached after 10 – 12 

days. Interestingly, this maximum was very similar for all the peptide/lipid ratios studied. Furthermore, the 

emission of the donor and acceptor decreased with the time. This suggest, that after a fast initial association 

between peptides and micelles a gradual growth of aggregate takes place. 

References: [1] M. Bokvist, F. Lindström, A. Watts and G. Gröbner J. Mol. Biol. 335 (2004) 1039. [2] E. Y. Chi,. 

L. Frey and K. Y. C. Lee, Biochemistry 46 (2007) 1913. 

290


BIOL-15 

Development of novel Cy3-labeled glucose bioprobe and its application in 

bioimaging and screening for anticancer agents 

Hyang Yeon Lee┼; Jongmin Park┼; Myung-Haing Cho; Seung Bum Park* 

Department of Chemistry, Seoul National University, Seoul 151-747 (Korea). 

E-mail: sbpark@snu.ac.kr 

Glucose is the most important energy source for cell growth, therefore fast-growing cancer cell requires 

more Glucose than normal cell. Based on this phenomenon, diagnosis of various cancers has been 

performed by PET(Positron Emission Tomography) in these days. FDG(2-fluoro-2-deoxy-D-glucose), 

which can be detected by PET, allows us imaging exact positions of tumors in our body. Another N- 

glycosylated glucose analog, 2-NDBG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-Dglucose), 

is a glucose-mimicking bioprobe which can be detected by fluorescence instead of using radio 

isotope. 2-NBDG has been widely applied in various researches, such as tumor imaging and GLUT related 

cell metabolism. However, there are several limitations of this bioprobe, therefore we designed and 

synthesized a novel fluorescent labeled glucose analog, Cy3 linked O-glycosylated glucose. The first 

generation of fluorescent-labeled glucose was based on Cellobiose labeled with FITC (Fluorescence 

Isocynate) through reductive amination. But this approach has several disadvantages, which is low yield 

and accessible to only β anomer. The second generation of fluorescent-labeled glucose was designed to 

achieve the asymmetric synthesis of two anomers (α, β) with higher yields including the potential study of 

the linker effect. Especially, previously labeled Fluorescene showed extensive photobleaching, therefore 

Cy3 fluorescent dye was synthesized and labeled on the glucose. Under these goals, we successfully finish 

the synthesis of α and β anomer of D-glucose labeled with Cy3 dye. 

This novel glucose-based fluorescent bioprobes were 

examined for the application of bioassay system and 

high-throughput screening through the measurement of 

glucose uptake of cells by CLSM (Confocal laser 

scanning microscope). Behaviors of our bioprobes are 

superb to previous glucose analogs, most importantly, 

it does not require the glucose starvation of media, 

which is critical to observe glucose metabolism in 

cell’s normal physiology. With these results, we tried 

to establish assay system for the evaluation of bioactive 

small molecules by the measurement of glucose uptake in cancer cells. For instance, cancer cells were pretreated 

with anticancer agent and measured the reduced uptake of our bioprobes. We expect that our assay 

can be used for HTS (High Throughput Screen) and bioresearch on glucose uptake-related disease. 

References: [1] M.Zhang et al., Bioconjugate Chem. 14 (2003) 709-714. [2] P. Som et al., J. Nucl. Med. 21 (1980) 

670-675. [3] P. S. Conti et al. Nucl. Med. Biol. 23 (1996) 717-735. [4] K. Yoshioka. et al., Biochim. Biophys. Acta 

128 (1996) 5-9. 

291


BIOL-16 

Different way of membrane permeabilization by two RTX toxins: 

HlyA and CyaA 

Radovan Fiser and Ivo Konopasek 

Department of Genetics and Microbiology, Faculty of Science, Charles University, 

CZ-128 44, Prague 2, Czech Republic 

The adenylate cyclase toxin (CyaA, ACT, 177kDa) of Bordetella pertussis and α-hemolysin (HlyA, 

117kDa) of Escherichia coli belong to the RTX toxin family and their C-terminal hemolysin portion is 

highly homologous. This part of both molecules is known for its ability to damage biological membranes 

even without requirement for the specific cellular receptors. Our study clarifies membrane disruption 

mechanisms of these two RTX toxins. We employed so called fluorescence requenching method [1] using 

liposomes of varying diameter with encapsulated fluorescent dye/quencher pair ANTS/DPX. 

In principle, there are two basic ways of membrane disruption: 

1) “All-or-none” leakage via a large-diameter pore that allows a rapid release of an inner vesicle content by 

diffusion, in less than 1s. After such leakage, there are two distinct populations of vesicles: a) completely 

“empty” vesicles without ANTX/DPX inside, and b) the “unaffected” vesicles having an initial 

concentration of ANTX/DPX inside. The extent of quenching inside unaffected vesicles (Q in ) remains 

constant because there is no loss of DPX during the release of ANTS (f out ) from the whole vesicle 

population. 

2) “Graded” leakage means that all vesicles affected by toxin are continuously losing some part of their 

inner contents and that Q in increases with f out . This corresponds to the formation of transient narrow pores 

which do not allow an immediate release of vesicles content. This release can theoretically be more 

effective for cationic DPX + (selectivity of the leakage α > 1) or for anionic ANTS - (α < 1). 

In our study, CyaA caused graded leakage 

ofencapsulated material with high selectivity 

forDPX + . Pore selectivity of “wild type” CyaA 

was much higher than that of mutant toxin CyaA- 

E509K+E516K, the mutated form with 

substitutions within predicted amphipatic α-helix 

and decreased channel selectivity (α~15.70±2.01 

and α~4.97±0.12, respectively, see figure on the 

right). In contrast, HlyA induced non-preferential 

leakage (α~0.80±0.01). Lines in the graph 

[1] 

represent the best fit of the model with 

respective value of selectivity α. 

Using two membrane disrupting RTX toxins, HlyA and CyaA, we observed different DPX/ANTS 

selectivity (α) of the pores depending of the toxin used. Moreover, we found that mutated form of CyaA 

with decreased channel selectivity [2] due to substitutions in predicted transmembrane segment (CyaA- 

E509K+E516K) shows much lower selectivity for the cationic quencher. This correlation suggests that the 

observed leakage rather corresponds to the disruption via natural toxin channels than to some non-specific 

disruption of the membrane. 

References: [1] A. S. Ladokhin, et al., Biophys. J. 69 (1995) 1964. [2] A. Osickova, et al. J. Biol. Chem. 274 (1999) 

37644. 

292


BIOL-17 

Sensitive determination of rutin in pharmaceutical preparation and human 

urine by its enhancement on chemiluminescence from lucigenin 

Seung Oh Jin 1 , Jong Ha Choi 2 , Sang Hak Lee 1 , Hye Young Chung 1 , S M. Wabaidur 1 

1 Department of Chemistry, Kyungpook National University, Daegu 702-701, Republic of Korea. 

E-mail: azure1979@daum.net 

2 Department of Chemistry, Andong National University, Andong, 760-749, Republic of Korea. 

Rutin (3,3',4',5,7-pentahydrohyflavone-3-rhamnoglucoside) is a flavonoid of the flavonol type, consisting 

of the flavonol quercetin and disaccharide rutinose (rhamnose and glucose). It is exists in many typical 

nutrimental plants (especially in buckwheat, apple and black tea) and is an important dietary constituent of 

food and plant-based beverages. Rutin exhibits antioxidant, antiinflammatory, anticarcinogenic, 

antithrombic, cytoprotective and vasoprotective activities. A number of analytical techniques have been 

reported for the determination of rutin. [1-4] An economic and environment friendly chemiluminescent 

method for the determination of rutin was described. It was based on the enhanced chemiluminescent 

emission of alkaline lucigenin–H 2 O 2 system by rutin. The difference of chemiluminesent intensity of the 

alkaline lucigenin–H 2 O 2 in the presence of rutin from that in the absence of rutin was linear with the 

concentration of rutin in the range from 4.58 - 305.27 μg/ml with a detection limit of 0.350 μg/ml. The 

correlation coefficient of the working curve was 0.9984.The relative standard deviation of eleven 

determinations of 7.5 × 10 -5 M rutin was 0.75%. All experimental parameters were optimized. The method 

was successfully applied to the determination of rutin in pharmaceutical preparation and human urine. The 

recovery results obtained by the method were satisfactory. 

References: [1] J.P.V. Leite et al., J. Agric. Food Chem. 49 (2001) 3796. [2] W.K. Li, J.F. Fitzloff, J. Chromatogr. B 

765 (2001) 99. [3] C. Queija et al., J. Chem. Educ. 78 (2001) 236. [4] L. Bramati et al., J. Agric. Food Chem. 51 

(2003) 7472. 

293


BIOL-18 

Rolling circle amplification (RCA) – a new detection method for the sensitive 

measurement of interactions on biochips 

Elke Mayer-Enthart a , Julien Sialleli a , Knut Rurack a , Ute Resch-Genger a , Daniela Köster b , 

Harald Seitz b 

a Federal Institute for Materials Research and Testing (BAM), Div. I.5, D-12489 Berlin (Germany). 

E-mail: elke.mayer-enthart@bam.de 

b Max Planck Institute for Molecular Genetics, Functional Protein Analysis Group, D-14195 Berlin 

(Germany). 

The use of DNA-microarray technology in biological and medicinal applications is constantly increasing. 

Although it is very attractive due to the possibility for the fast detection of many genetic parameters in 

multiplexing applications, the sensitivity of detection is often insufficient. Since sample quantities are often 

considerably small, e.g. few nanograms of genetic material from biopsies, preamplification of the samples 

is necessary. The most frequently used technique is the polymerase chain reaction (PCR). PCR however has 

several inherent problems. Above all, the additional enzymatic step has to be done in solution before the 

real detection step. It is also very sensitive to cross-contamination and irregular amplification events, which 

strongly limits the reliability and comparability of results from different assays. 

Our idea to improve DNA-BioChips is the use of rolling circle amplification (RCA) as an alternative for 

signal amplification. RCA is a very fast isothermal enzymatic DNA-polymerisation reaction, which needs 

circular templates. In progress of the primer elongation the strand displacement activity of certain 

polymerases leads to a more than 10 kbp-long single-stranded DNA bearing hundreds of copies of the 

original target sequence. [1-4] As it is possible to perform target hybridisation, signal amplification and 

detection under isothermal conditions on a single immobilised sample spot, problems that are typical for 

PCR amplified assays can be avoided. 

Our goal is to find optimum conditions for performing nucleic acid detection with RCA on a microarray 

format. Therefore both the biochemical conditions for the enzymatic reaction on the microarray surface and 

the fluorescence detection of the amplified single-stranded RCA product have to be optimised. Methods for 

the systematic incorporation of various fluorophore-modified nucleotides in RCA reactions have to be 

developed to facilitate the application-oriented choice of chromophores. For instance, to ensure strongest 

signal enhancements (i.e. smallest sample amounts), the optical properties of the fluorescently labeled 

nucleic acids have to be investigated under several assay-relevant conditions. Additionally it is necessary to 

avoid or account for nonlinear effects at high labeling densities when attempting to quantify the 

fluorescence signals of such an assay. Moreover, systematic investigations of these RCA-related behaviours 

of fluorescent dyes have not yet been done. 

Scheme: Principle of microarrays based on RCA. (A) 

ssDNA (red) is immobilised on surface. (B) Circularized 

DNA hybridises to complementary sequence. (C) Addition 

of special enzyme starts RCA on circular template, and (D) 

many fluorescently labeled nucleotides are incorporated and 

enhance the optical signal. 

References: [1] J. Baner et al., Nucl. Acids Res. 34 (1998) 5073. [2] P. M. Lizardi et al., Nat. Genet. 19 (1998) 225. 

[3] G. Nallur et al., Nucl. Acids Res. 29 (2001) e118. [4] D. Y. Zhang et al., Gene 274 (2001) 209. 

294


BIOL-19 

New analytical developments of fluorescence polarization immunoassay 

M.L. Sánchez-Martínez, M.P. Aguilar-Caballos, A. Gómez-Hens 

Department of Analytical Chemistry. University of Córdoba. Campus of Rabanales. Marie-Curie Annex 

building. 14071-Córdoba (Spain). E-mail: qa1agcam@uco.es 

The versatility of fluorescence polarization immunoassay (FPIA) is increased by using two long 

wavelength labels, Nile Blue and a ruthenium chelate. The first label has been used to study the potential of 

FPIA on solid surface using dry reagent technology. The aminoglycoside antibiotic amikacin has been used 

as model analyte and the method has been applied to the analysis of serum samples. The second label has 

been used to show the practical application of FPIA to the determination of macromolecules, using gliadins 

as model analyte, which have been determined in gluten-free foods. For the development of the first 

immunoassay method, very low amounts of anti-amikacin antibodies and tracer were immobilized onto 

nitrocellulose membranes, being the consumption of reagents lower than in conventional FPIA. Only the 

addition of the standard or sample at the adequate pH is required at the analysis time. The analyte displaces 

the tracer from the tracer-antibody immunocomplex, obtaining a decrease in the fluorescence polarization 

proportional to the analyte concentration. The gliadin tracer shows a relatively long lifetime, which allows 

the observation of the differences in fluorescence polarization values between the tracer-antibody complex 

and the tracer alone. The dynamic range of the calibration graphs for both analytes is 0.5-10 μg ml -1 and the 

detection limits are 0.1 μg ml -1 and 0.09 μg ml -1 for amikacin and gliadins, respectively. The study of the 

precision gave values of relative standard deviations lower than 5% and 1.5% for amikacin and gliadin 

methods. Amikacin was determined in human serum samples using a previous deproteinization step with 

acetonitrile, obtaining recovery values in the range 83.4-122.8%. The gliadin method was applied to the 

analysis of gluten-free food samples by using a previous extraction step. The recovery study gave values 

between 94.3-105.0%. 

295


BIOL-20 

Characterization of new near infrared dyes for molecular imaging 

Jutta Pauli 1 , Tibor Vag 2 , Romy Haag 2 , Werner A. Kaiser 2 , Ingrid Hilger 2 , 

and Ute Resch-Genger 1 

1 Federal Institute for Material Research and Testing, D-12489 Berlin (Germany). 

E-mail: jutta.pauli@bam.de 

2 

Friedrich-Schiller-University Jena, Institute for Diagnostic and Interventional Radiology, D-07747 Jena 

(Germany). E-mail: tabor.vag@med.uni-jena.de 

The sensitivity of near-infrared fluorescence (NIRF) imaging depends to a strong extent on the 

spectroscopic properties of the chosen fluorescent reporters. Suitable dyes are characterized by e.g. a high 

molar absorption coefficient at the excitation wavelength and a high fluorescence quantum yield under 

application-relevant conditions. Aiming at the introduction of new fluorescent tools for medical diagnostics, 

we spectroscopically studied the NIR hemicyanine dyes DY-676, DY-681, DY-731, DY-751, and DY-776 

in phosphate buffered saline solution (PBS) and in a solution of bovine serum albumin (BSA) in PBS 

modelling body fluid and compared their absorption and fluorescence properties to that of indocyanine 

green (ICG), the only clinically approved fluorescent dye until now. 

The absorption and fluorescence properties of the DY dyes and ICG are controlled by dye hydrophilicity, 

dye aggregation, dye-protein interactions, and the energy gap rule. The fluorescence quantum yields of all 

the hemicyanine dyes in PBS and in PBS/BSA are always higher than the φ f values of ICG rendering the 

DY dyes attractive diagnostic reagents. In all cases, the fluorescence quantum yields of the dyes in 

PBS/BSA exceed those in PBS suggesting specific dye-albumine interactions. [1,2] This is supported by 

corresponding spectral shifts in absorption. These shifts, that can be most likely used as an indicator of dye 

hydrophility, point e.g. to an increased hydrophilicity of DY-676, DY-681, DY-731, and DY-751 as 

compared to ICG. The maximum fluorescence quantum yields in PBS/BSA were found for DY-681 and in 

PBS for DY-681, DY-731, and DY-751. The reduced values of φ f resulting for DY-676 and DY-776 in 

PBS are caused by aggregation of the dye molecules as also indicated by the broadening of the absorption 

spectra. 

References: [1] P. Czerney, et al., Biol. Chem. 382 (2001)495. [2] T. Vag , et al., submitted to Invest. Radiology. 

296


BIOL-21 

NADH fluorescence as a measure of bacterial metabolic activity 

Petri Koponen, Marja Palmroth, Harri Huttunen, Ilpo Niskanen 

University of Oulu, Measurement and Sensor Laboratory, Technology Park 127, FI-87400 Kajaani 

(Finland). E-mail: petri.koponen@oulu.fi 

Fluorescence of NADH, the reduced form of nicotinamide adenine dinucleotide (NAD), is a good indicator 

of microbial metabolic activity. The fluorescence signal is a measure of the intracellular redox state of the 

micro-organisms. NADH/NAD plays a key role in the electron transfer from electron donor to electron 

acceptor inside living cells. During actively functioning metabolism, NADH is quickly oxidized into 

NAD + , whereas accumulation of NADH indicates inefficiency. The reduced form NADH is capable of 

fluorescent emission at 445 nm when excited at 340 nm, while the oxidized form NAD + is not. Therefore, 

the amount of NADH and the activity of microbial metabolism can be measured with fluorescence 

spectroscopy. In waste water treatment plants, for instance, the fluorescence techniques can be used to 

detect the transition from anoxic to anaerobic conditions and to identify the situation when nitrate is 

depleted. 

Our aim is to develop and build a versatile NADH fluorescence measurement unit that can operate in harsh 

environments. The main idea is that the excitation is done via suitable LED and emission is detected via 

PMT. The biggest challenge is to build the sampling unit. This unit is designed to separate or collect 

bacteria form different mediums, count the number of bacteria and extract intracellular NADH for 

detection. 

We are finishing our fluorescence unit and the sampling unit is under development. It is expected that this 

detection and measurement unit will enable simple and accurate detection of bacterial metabolic activity in 

different environments. 

297


BIOL-22 

Flow cytometric, single cell based FRET analysis of erbB receptor tyrosine 

kinase interaction in breast cancer cell lines 

Simone Diermeier, Mark Plander, Andrea Sassen, Ferdinand Hofstaedter, Gero Brockhoff 

University of Regensburg, Institute of Pathology, D-93051 Regensburg (Germany). 

E-mail: simone.diermeier@klinik.uni-r.de 

Flow Cytometric Fluorescence Resonance Energy Transfer (FRET) is a powerful tool to study protein 

interaction on a vital cell by cell basis. In breast cancer, the interaction of erbB receptor tyrosine kinase 

family members is of peculiar impact on the initiation and progression of the disease. C-erbB2 

overexpression is associated with poor clinical outcome and worse prognosis. However c-erbB2 is not a 

stand-alone receptor. Instead its malignant potential is conducted and amplified by homodimerization and 

heterodimerization with cognate family members to form potent signaling complexes that drive cell 

proliferation, tumor progression and malignancy. 

Herceptin and Omnitarg (both Roche Diagnostics, Penzberg, Germany) are humanized therapeutic 

monoclonal antibodies that target c-erbB2 at different epitopes. Herceptin significantly improves survival of 

c-erbB2 overexpressing breast cancer patients. Omnitarg inhibits cell proliferation in breast cancer cell lines 

and also exerts therapeutic efficiency. But the mechanisms by which Herceptin and Omnitarg mediate their 

anti-proliferative and anti-tumor effect are incompletely understood and need to be elucidated on receptor 

level in more detail in order to render more precisely antigen targeted therapeutic strategies. 

We examined the effects of Herceptin and Omnitarg on c-erbB2 homodimerization in c-erbB2 

overexpressing BT474 and SK-BR-3 breast cancer cell lines and the final impact of antibody treatment on 

cell proliferation. FRET was measured on a FACSCalibur two-laser flow cytometer on a cell by cell basis 

after cell treatment and harvest. Donor (Cyanine-3) and acceptor dye (Cyanine-5) labeled Herceptin and 

Omnitarg were used as staining reagents for Omnitarg and Herceptin 

treated cells, respectively. The ratio of donor to acceptor dye was 1:2. 

Control experiments using Fab labeled reagents served to exclude any 

artificial crosslinking effect. Four samples were run for each individual 

experimental setup: i) unlabeled cells ii) donor-dye labeled cells iii) 

acceptor-dye labeled cells and iv) donor- and acceptor-labeled cells. 

The single labeled samples allowed to determine the spectral overspill 

and cross excitation caused by the two laser instrument we used for 

Herceptin 

Cyanine-3 (488 nm) and Cyanine-5 (635 nm) excitation, respectively. Energy Transfer Efficiency (E) was 

calculated by quantification of donor dye quenching and acceptor sensitized emission of the double labeled 

sample using the ReFlex Software (provided by the Inst. of Biophysics and Cell Biology, Debrecen, 

Hungary). Dynamic cell proliferation assessment was performed by propidium iodide/Hoechst double 

staining and BrdU based Hoechst quenching. 

Both in BT474 and SK-BR-3 breast cancer cell lines treatment with Herceptin results in an increase of 

Energy Transfer Efficiency indicating an induction of c-erbB2 homodimerization. In contrast, cell treatment 

with Omnitarg abrogates c-erbB2 homodimerization but also inhibits tumor cell proliferation although to a 

lower extend than Herceptin does. Fab-based control experiments verified the observation that antibody 

induced interaction of the c-erbB2-receptor oncoprotein is associated with inhibited tumor cell 

proliferation. 

In contrast to conventional biochemical approaches, flow cytometric FRET measurements allow 

quantitative assessment of receptor interaction in vital cells on a cell by cell basis, hence taking receptordynamics 

and cell heterogeneity into account. Herceptin and Omnitarg might be complementarily 

administered and thereby inhibit cell growth more efficiently than in a separate treatment setting. Flow 

cytometric FRET analysis is a powerful tool and will significantly contribute to untangle complex patterns 

of potentially interacting molecules, an essential approach to identify relevant therapy targets and the 

efficiency of therapeutic treatments. 

References: [1] G. Brockhoff et al., Cell Prolif. (2007), in press. [2] S. Diermeier et al., Exp.Cell Res. 304 (2005) 

604. [3] G. Szentesi et al., Comput. Methods Programs Biomed. 75 (2004) 201. 

P 

P 

Cy3-Fab- 

Omnitarg 

Cy5-Fab- 

Omnitarg 

298


BIOL-23 

Chemiluminometric determination of vitamin B9 by a flow injection 

analysis assembly 

Seikh Mafiz Alam 1 , Mohammad Mainul Karim 1 , Sang Hak Lee 1 , Jung Kee Suh 2 , Hye Young 

Chung 1 , Hyun Woo Park 1 

1 Kyungpook National University, Department of Chemistry, Daegu 702-701, Republic of Korea 

E-mail: seikh_alam@hotmail.com 

2 Division of Chemical Metrology and Materials Evaluation, KRISS, P.O. Box 102, Yusung, Taejon, 

305-600, Republic of Korea 

Vitamin B9, also known as folic acid, is an important component of the haemapoietic system and is the 

coenzyme that controls the generation of ferrohaeme [1] . Lack of folic acid gives rise to the gigantocytic 

anemia, associating with leucopoenia, devolution of mentality and psychosis etc. Determination of folic 

acid is often required in pharmaceutical, clinical and food samples. Methods used for it are generally 

spectrophotometry [2] , chromatography [3] and electrochemical methods [4] . In this work, we proposed a 

chemiluminescence method based on the enhancement of folic acid to the CL intensity of tris(2,2’- 

bipyridyl) ruthenium(II) - Ce(IV) system. Under optimal conditions, the linear relation is in the range of 

2.5× 10 -5 -3.1 × 10 -7 mol/L with the detection limit of 2.3 × 10 -8 mol/L. The recovery was higher than 95.3 

%. The method was accurate, sensitive, highly selective and effective for assay of folic acid. This CL 

method can be successfully applied to the determination of folic acid in pharmaceutical preparations. The 

mechanism of CL reaction was also studied. 

References: [1] H.X. Luo et al., Anal. Chem. 73 (2001), 915–920. [2] G.J. Volikakis et al., Talanta 51 (2000), 775– 

785. [3] K. Ishii et al., J. Chromatogr. B 759 (2001), 161–168. [4] S.M. Lunte et al., Analyst 113 (1988), 99–102. 

299


BIOL-24 

The mechanism of benzothiazole styrylcyanine dyes binding with dsDNA 

Mykhaylo Yu. Losytskyy 1,2 , Nuriye Akbay 3 , Vladyslava B. Kovalska 2 , Anatoliy O. Balanda 2 , 

and Sergiy M. Yarmoluk 2 

1) Kyiv Taras Shevchenko National University, Physics Department, 2 Glushkov Ave., Build. 1, 03680 Kyiv, 

Ukraine; E-mail: m_losytskyy@svitonline.com; 

2) Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150 Zabolotnogo 

St., 03143 Kyiv, Ukraine; Теl/fax: 380 44 252 24 58; 

3) Hacettepe University, Department of Chemistry, 06800, Ankara, Turkey 

Styrylcyanines were reported to be efficient fluorescent dyes for double-stranded (ds) DNA detection, 

particularly in applications using the two-photon excitation [1]. Thus the detailed study of the styryl dyes 

interaction mode with dsDNA is very important, both for understanding processes taking place in the dyedsDNA 

solution, as well as for the development of novel dsDNA probes based on styryl dyes. 

In the presented work the binding of two monomer and two homodimer benzothiazole styryl dyes (Figure) 

with dsDNA was studied. For these dyes, equilibrium constant of dye binding to dsDNA (K), as well as the 

number of dsDNA base pairs occupied by one bound dye molecule (n) were determined. The values of K 

and n were obtained as parameters of approximation of the data of fluorescent titration of dye by dsDNA 

with the equation obtained by McGhee and von Hippel [2]. Besides, the dyes sensitivity to the presence of 

AT- and GC-containing polynucleotides was studied. 

N + 

N 

Sbt 

S 

S 

I 

S 

N + 

DBsu-10 

I 

N 

I 

N + 

Bos-5 

I 

N + 

O 

N 

N + N+ N 

O 

I 

I 

Chemical structures of the studied styryl dyes. 

N 

S 

N + 

I 

N 

S 

N + 

I 

N 

I 

N 

DBos-13 

I 

N + 

N + 

S 

N + 

I 

Basing on the results of our studies, the intercalation mechanism of binding to dsDNA is proposed for the 

monomer dyes Sbt and Bos-5, as well as for the homodimer dye DBos-13 with the linkage group attached 

to nitrogen atoms of benzothiazole ring. This assumption is supported with the comparable fluorescent 

response of these dyes on the presence of both poly(dA-dT) 2 and poly(dG-dC) 2 polynucleotides. Besides, 

the obtained values of K and n are in agreement with the intercalation mechanism on dye-dsDNA 

interaction. It should be mentioned that the presence of spermine-like tail group in the structure of Bos-5 

leads to the increasing of equilibrium constant of dye-dsDNA binding value in more than 3 times as 

compared to the parent dye Sbt. 

At the same time, for the homodimer dye DBsu-10 with linkage group bound in 6-positions of 

benzothiazole heterocycle the groove-binding mechanism of interaction with dsDNA was proposed. The 

evidences for such binding mode are the strong AT-binding preference demonstrated by this dye, as well as 

the high value of the number of dsDNA base pairs occupied by one bound dye molecule. 

Thus it was shown that the position of linkage group significantly affects the mode of homodimer dyes 

interaction with dsDNA. 

Acknowledgement: This work was supported by the Science and Technology Center in Ukraine (STCU) grant 

#U3104k 

References: [1] V.P. Tokar et al., J. Fluorescence 16 (2006) 783. [2] J.D. McGhee, P.H. von Hippel, J. Mol. Biol. 86 

(1974) 469. 

300


BIOL-25 

Coralyne self-aggregation and affinity for DNAs and RNAs: 

Analysis and solvent Effects 

Tarita Biver, a Alessia Boggioni, a Fernando Secco, a Marcella Venturini, a Begona Garcia, b 

Josè Maria Leal, b Rebeca Ruiz b 

a University of Pisa, Chemistry and Industrial Chemistry Department, 56126 Pisa (Italy). 

E-mail: ferdi@dcci.unipi.it 

b University of Burgos, Chemistry Department, 09001 Burgos (Spain). 

Coralyne is a fluorescent synthetic alkaloid, analogous of the 

natural alkaloid berberine, that has been found to exhibit 

antileukemic activity. [1] This activity, together with the low 

toxicity, is the basis of the high interest aroused by this molecule in 

recent years. Coralyne strongly binds to polynucleotides of both 

DNA and RNA type, showing high affinity also for 

poly(dA)·2poly(dT) triple helices and for poly(A) single 

strands. [2,3] The binding follows an intercalative mode but, for high 

dye concentrations, molecular aggregation, induced by the DNA 

template, is also found to occur. [4] 

Coralyne chloride 

Despite the high number of studies performed on this molecule, an 

in depth understanding of the binding mechanism is lacking, this 

being principally due to the fact that experiments are difficult to 

carry out due to the high tendency of coralyne to self-aggregation. 

Fluorescent measurements constitute an important tool to overcome such a problem, grace to the very low 

dye concentration that can be used with this technique. 

We have performed a kinetic analysis (T-jump technique) of coralyne self-aggregation, in water (0.1M 

NaCl, pH 7) and in the presence of increasing amount of ethanol (0÷20%). Then, spectrofluorometric, 

spectrophotometric, viscometric and circular dichroism titrations were performed on 

Coralyne/polynucleotide systems, where as polynucleotides calf-thymus DNA, poly(dA-dT)·poly(dA-dT), 

poly(dG-dC)·poly(dG-dC), poly(A), poly(A)·poly(U), poly(A)·2poly(U) were taken into account. The 

experiments concerned with polynucleotide binding were also carried out both in water and water-ethanol 

mixtures. 

The results obtained indicate that coralyne self-aggregation is indeed strong, ethanol affecting both the 

forward and backward aggregation rates, but scarcely modifying the aggregation constant. Concerning 

polynucleotides, it was found that binding to DNAs differs from that to RNAs, as shown by the sharply 

different viscosimetric and dichroic behaviours. Among DNA sequences, A-T base pairing was found to be 

preferred, whereas concerning RNA higher affinity for the triple helix respect to the double was found to 

occur. Further details of this analysis will be presented. 

References: [1] B. Gatto et al., Cancer Res. 56 (1996) 2795-2800. [2] M. Polak and V. Hud, Nucleic Acids Res. 30 

(2002) 983-992. [3] J. Ren and J.B. Chaires, Biochemistry. 38 (1999) 16067-16075. [4] W.D. Wilson et al. J. Med. 

Chem. 19 (1976) 1261-1263. 

301


BIOL-26 

Investigation of mechanisms of biomembrane cryopreservation using the 

fluorescent dicyanomethylene-squaraine Probe 

Oksana Sokolik 1 , Tatyana Dyubko 1,2 , Tamara Linnik 2 , Anatoliy Tatarets 1 , Leonid 

Patsenker 1 


60 Lenin Ave., UA-61001 Kharkov (Ukraine). E-mail: ksenaksena@mail.ru 

2) Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine, 

23 Pereyaslavskaya Str., UA-61015 Kharkov (Ukraine). E-mail: tdyubko@mail.ru 

Long-term low temperature storage of cells is of great importance for biomedical research and 

transplantation medicine. Biomembranes are known to be most unstable structures in cells towards freezing. 

To protect the cells against low temperature, special organic compounds, so-called cryoprotectants (CPs), 

are used. However, the molecular mechanism of CP protective action is not established well. One of the 

approaches to study this mechanism is based on observation of fluorescent probe interaction with cell 

membranes in presence of CP. Fluorescent response of the probe gives information on strength and 

peculiarity of CP interaction with cell membrane. This work investigates mechanism of interaction of CPs 

such as ethylene glycol (EG), 1,2-propanediol (PD) and DMSO with surfaces of natural membranes by 

using fluorescent dicyanomethylene-squaraine probe. This probe was recently found to be very sensitive to 

a change of structure and hydratation of lipid bilayer polar region [1]. Chicken liver microsomes were 

utilized as the model natural membranes. 

O 3 S 

NC 

CN 

1.2 

N 

(CH 2 ) 5 

COOH 

O 

N 

F/F max 

1.0 

0.8 

PD 

EG 

The figures show structure of the dicyanomethylene-squaraine 

probe and relative 

fluorescence intensity of the probe in chicken 

liver microsomes vs. concentration of 

cryoprotectants PD, EG and DMSO (excitation 

wavelength 650 nm). 

0.6 

0.4 

0.2 

DMSO 

0 2 4 6 8 

Concentration of Cryoprotectant, M 

CP interaction with biomembranes is found to depend on the CP nature. The fluorescence maximum and 

intensity of the probe bound to microsomal membranes change non-linearly vs. CPs concentrations. The 

observed changes of fluorescence spectra of the stained microsomes are conditioned by processes of CP 

sorption at the membrane surface followed by competitive replacement of the dye molecules with CP. 

DMSO causes more pronounced decrease of fluorescence intensity compared to EG and PD, which is an 

evidence of its stronger interaction with membrane binding sites. High performance of DMSO interaction 

with microsomal membranes is supposed to be connected with amphiphilic nature of its molecule. DMSO 

molecule can form Н-bound with microsome surface area but also has a nonspecific hydrophobic attraction 

to the non-polar frontier area of lipid bilayer. At the same time, the smaller displacement efficiency of the 

dye molecules with PD and EG evidences that these CPs interact predominantly with microsomes surfaces. 

Apparently, specific H-binding predominates in this interaction. We found a clear correlation between the 

displacement rate (substitution of dye molecules with CP in surface area of natural lipid-protein 

membranes) and the cryopreservation efficiency of cryoprotectants applied to low-temperature storage of 

cells. The obtained data demonstrate also that the dicyanometylene-squaraine dye allows obtaining useful 

information on molecular mechanism of biomembranes and cells cryoprotection. 

Reference: [1] V. M. Ioffe, G. P. Gorbenko et al. J. Fluorescence., 16 (2006) 47. 

302


BIOL-27 

New time resolved-FRET systems for homogeneous assay formats 

Th. Enderle*, D. Roth*, H. Matile*, H.-P. Josel, R. Herrmann, D. Belik#, B. Koenig**, 

F. Mueller*** 

Roche Centralized Diagnostics, Rare Reagent Development, Penzberg 

F. Hoffmann-La Roche Ltd., Pharmaceuticals Division, *Assay Development/HTS Basel, 

**Biology Penzberg, ***Structure Research, Basel 

Roche Applied Sciences#, Penzberg 

Fluorescence is a key detection technology in diagnostics and bio/molecular screening due to its high 

sensitivity and the versatility of different readout modalities (intensity, lifetimes, polarization, energy 

transfer, etc). 

Fluorescence Resonance Energy Transfer (FRET) systems offer great advantages and are intensively used 

for state of the art DNA testing. In combination with time resolved fluorescence techniques such systems 

are applied also in other homogenous assay formats, e.g. for ultra high throughput screening systems in 

drug discovery. 

We have developed a new Time Resolved-FRET system using Ruthenium complexes with lifetimes in the 

µs time domain as donor or acceptor in combination with organic acceptor/donor dyes. Data of the 

evaluation of these systems in model assays and applications in HTS assays will be presented. 

303


BIOL-28 

Single Beads, single molecules, single cells - a fully integrated synthesis, 

screening and mechanistic profiling system for chemical biology 

Martin Hintersteiner, Thierry Kimmerlin, Volker Uhl, Mario Schmied, Geraldine Garavel, 

Janmarcus Seifert, Christof Buehler, Nicole-Claudia Meisner and Manfred Auer 

Novartis Institutes for Biomedical Research, Discovery Technologies A-1230 Vienna (Austria); 

E-mail: Martin.Hintersteiner@novartis.com; Manfred.Auer@novartis.com. 

The modern drug discovery process is perceived as an increasingly cost-intensive, lengthy and complex 

multi-step process [1] . Despite of the progress made, the still unchanged classical concept of purifying 

several mgs of LMW compounds and building up of large solution- or solid compound-archives for testing 

in HTS is associated with extensive storage, liquid handling and maintenance costs. All currently marketed 

drugs act on less than 250 proven target proteins from only 6 major target classes [2] . The sequencing of the 

human genome followed by several years of functional genomics and proteomics research has so far failed 

to show the expected impact in increasing the number of successfully tackled drug targets. A good target 

protein needs to fulfill two requirements. Its up or down regulation must ameliorate or cure a disease and it 

must be drugable i.e. susceptible to functional modulation by chemical or biological agents. Chemical 

Biology was born as a new scientific discipline to investigate the drug target potential of the entire 

proteome with small molecules [3] . With thousands of virgin proteins it is key to follow target platform 

processes instead of single targets, and integrated technology platforms for higher throughput and higher 

mechanistic quality of analysis. We have developed a miniaturized on-bead screening (OBS2) platform 

which integrates chemistry, HTS, and the quantitative mechanistic profiling of hits in living cells to identify 

new target/compound pairs with quantitative and mechanistic characterization. Our current OBS process 

combines automated confocal on-bead screening and quantitative analysis of bead/protein interactions with 

a PostSynthesis/PostScreening (PS/PS) labeling step of single hit beads. The PS/PS labeling step generates 

fluorescently tagged hit compounds and allows for a direct off-bead confirmation of target/ligand 

interactions in solution by confocal spectroscopy in multi-well formats up to 1536-well plates. 

General process flow: 

confocal on-bead screening 

results in a series of hit 

beads. The material from 

individual, beads is subjected 

to PS/PS labeling, single 

molecule confirmation in 

solution and cellular testing. 

Single hit bead 

PS/PS 

labeling 

Confirmation & quantification 

in solution 

Fluorescence anisotropy [a.u.] 

0.12 Bead C6-72 vs Grb2-SH2 

0.1 

0.08 

0.06 

0.04 

Kd (Grb2-SH2 vs ß-pept.) = 243 +/- 11 nM 

0 2 4 6 8 10 12 14 16 18 20 

Concentration of unlabeled target [µM] 

Cellular testing of 

labeled/unlabeled cpd 

20 µm 

Our on-bead screening approach extracts quantitative affinity information on target/compound interactions 

from only 10-50 pmoles of individual compounds. Thereby resources are shifted towards profiling of active 

hits. As a major benefit this OBS2 process provides fluorescent ligands, directly applicable in various 

miniaturized in-vitro or cellular assay systems. 

References: [1.] Federsel, H. Drug Discov. Today 11 (2006) 966. [2.] Imming, P. et al. Nat.Rev. Drug Discovery 5 

(2006) 821. [3.] Meisner, N-C. et al. Curr.Opin.Chem.Biol. 8 (2004) 424. 

304


BIOL-29 

Application of carbocyanine probes to estimate cryopreservation effect on 

functional state of cell cultures 

V.V. Timon, E.I. Goncharuk, * S.O. Gurina, N.A. Volkova, * I.A. Borovoj, * Yu.V. 

Malyukin, V.I. Grischenko 

Institute for Problems of Cryobiology & Cryomedicine of the National Academy of Sciences of Ukraine, 

23 Pereyaslavaskaya str., 61015, Kharkov, Ukraine, e-mail: goncharuk_elena@rambler.ru 

*Institute for Scintillation Materials of the National Academy of Sciences of Ukraine, 60 Lenin ave, 61001, 

Kharkov, Ukraine 

Assessment of morphological and functional integrity of cryopreserved material is an important aspect for 

its further use in biology and medicine. Carbocyanine probes are successfully used to estimate the state of 

plasma and mitochondrial cell membranes. In our research we applied JC-1, C2 (3,3- 

diethyloxocarbocyanine bromide) and C9 (3,3’-dinonyloxocarbocyanine bromide) for characterization of 

cryopreserved cells of diploid line of human fibroblasts. 

At the first stage there were tested the optimal concentracion of probes and then its toxicity in vitro. The 

cells were removed from a glass and stained with fluorescent probes C2 (10-6, 15 min) and IC-1 (10-5M, 

60 min). When the dyes were introduced into suspension of cells their viability did not change. 

Microscopically there were characterized with a roundish shape with no formation of membrane vesicles. 

When culturing stained cells if compared with the control no alterations in adhesive ability, proliferative 

index and morphology were found. During luminescent microscopy (Olympus IX71 microscope, Olympus 

C5060 camera) in the cells stained with probes C2 and C9 there was observed green luminescence of the 

structures, representing filamentous branched formations, evenly distributed in a cell, with the size of 60 

nm and higher. This is a classic picture of fibroblast chondriome. Under FCCP effect on cells of the culture 

stained with probes C2 and C9 there was quenching of mitochondria luminescence. Their contours became 

dimmer, discontinuous, the preciseness was lost, there was found a dye release into cytoplasm and into 

pericellular space. This points to mitochondrial character of probe binding. During staining of the cells with 

probe JC-1 there was observed a typical for this dye picture. 

The suspension of labeled cells was cryopreserved with 10% DMSO protection. After warming and 

cryoprotectant removal the cells were inoculated into Petri 

dishes and cultured in the medium 199 with adding 10% 

FBS in CO 2 incubator. During culturing of stained cells after 

thawing there was established that the presence of probes in 

cells did not affect the growth parameters of the culture. 

Adhesability, proliferative activity, terms of monolayer 

formation were similar in cell culture with the probes and 

without them. Localization and character of luminescence 

for probes in cells did not change (Fig. 1). Thus 

cryopreservation does not affect localization of the studied 

probes in cells. 

It is known that homologue of the studied probes DiOC6(3) 

may response both to the ΔΨ changes and the ones of plasma 

membrane potential. This complicates the use of 

carbocyanine probes for studying the state of cell plasma 

membrane. 

Fig. 1. Culture of fibroblasts, stained 

with probe C2 in 48 hrs after thawing 

and seeding (x1,000) 

Cryopreservation obviously affects this parameter, however the researches in this direction show that 

recovery of cell membrane electric potential occurs within 2-3 hrs of rehabilitation. Thus these probes may 

be used in an integrated state during cryopreservation of cell lines with an aimed monitoring of their 

functional state after thawing. 

305


BIOL-30 

Visualization of localized protease activity at sub-cellular resolution in real time 

Veronica Olsson, Ricardo Figueroa 

Södertörns University, Institute of Science, 141 89 Huddinge (Sweden). 

E-mail: ricardo.figueroa@sh.se 

Proteases are active in several processes of the cell. The most recognized is perhaps the processes of 

apoptosis that is mediated by protease cascades. We have developed a reporter that allows visualization of 

protease activity at sub cellular resolution in real time. The reporter consists of a FRET pair that are 

connected by a short linker containing a protease consensus sequence. The reporter will FRET as long as 

the linker remains intact. However upon protease activity the linker is readily proteolysed and FRET is lost. 

Additionally the reporter has a N-terminally fused microtubule binding domain. The microtubule binding 

domain localizes the reporter to the microtubule and limits the sped of diffusion. The lowered sped of 

diffusion allows localized detection of protease activity. Utilizing the reporter we are working to gain new 

insight in to the processes of apoptosis in Alzheimer’s disease. 

Pseudo-color ratio image (I FRET /I CFP ) of differentiated SH-SY5Y cells expressing the caspase 3 variant of 

the reporter. The cells are triggered to enter apoptosis by localized photoinduced ROS production at the 

mitochondria using the KillerRed-mito system. 

306


BIOL-31 

Genetically encoded calcium- and hydrogen peroxide-sensitive indicators based 

on circularly permuted yellow fluorescent protein 

Ekaterina Suslova, Vsevolod Belousov, Dmitry Chudakov 

Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia. 

E-mail: souslova@gmail.com 

One of the most promising approaches for developing genetically encoded fluorescent sensors implies 

fusing circularly permuted fluorescent proteins (cpFPs) to or inserting them into sensitive domains that 

undergo structural rearrangements in the presence of an analyte. These rearrangements, in their turn, induce 

conformational changes of cpFP resulting in its altered fluorescent properties. 

In most cases it was shown that spectral changes of the cpFP-based sensors occur through the chromophore 

transition from the neutral (protonated) to the charged (anionic) form. Noteworthy, the same mechanism 

leads to 100-400-fold increase of fluorescence after photoactivation for photoactivatable proteins [1] . 

To construct calcium sensor we used a well-studied model of cpFP-based calcium-sensitive indicators (such 

as G-Camp [2] and Pericams [3] ) consisting of circularly permuted fluorescent “core”, calmodulin and its 

target peptide M13. Here we report a significant progress in the development of Ca 2+ -sensitive indicators of 

high contrast. 

We generated two Ca 2+ -sensitive indicators that were characterized with particular high brightness and 

superior dynamic range, up to 16.5-fold increase of green fluorescence in the presence of Ca 2+ . We 

demonstrated the high potential of these sensors on various examples, including monitoring of calcium 

response to a prolonged glutamate treatment in cortical neurons. 

Our novel calcium sensors are more pH-stable and have an approximately 3-fold higher dynamic range 

compared to G-Camp [2] and thus are more reliable for in vivo microscopy, comparable with the 

commercially available chemical Ca 2+ -sensitive probes. At the same time, genetically encoded sensors 

provide more opportunities, allowing to be targeted to any chosen cellular compartment, to generate stable 

cell lines and transgenic animals, to be expressed in a particular tissue and/or in a temporary controlled 

manner under a specific promoter. 

Using the same fluorescent core (cpYFP) as in described above Ca 2+ -sensors, we developed the first 

genetically encoded, highly specific fluorescent indicator for detecting hydrogen peroxide (H 2 O 2 ) in living 

cells. This probe, named HyPer [4] , consists of cpYFP inserted into the regulatory domain of the prokaryotic 

H 2 O 2 -sensitive protein OxyR. Hyper was characterized with two exitation peaks at 420 and 500 nm and one 

emission peak at 516 nm. Upon exposure to H 2 O 2 it turned out to be ratiometric: the exitation peak at 420 

nm decreased proportionally to the increase in the peak at 500 nm. An apparent advantage of such a 

ratiometric indicator is that its readout is dependent on the amount of the protein expressed. 

HyPer demonstrated submicromolar affinity and high specificity to H 2 O 2 . Our experiments showed that it 

was a powerful tool for investigating the effect of various stimuli on the amount of H 2 O 2 in different cell 

compartments. Using Hyper we monitored H 2 O 2 production at the single-cell level in the cytoplasm and 

mitochondria of HeLa cells treated with Apo2L/TRAIL. We also observed local brusts in mitochondrial 

H 2 O 2 production during the oscillations of the transmembrane potential (Δψ) and changes in cell shape. 

We believe that HyPer provides a good alternative to the existing synthetic probes for detecting 

intracellular hydrogen peroxide. 

References: [1] Chudakov et al., Nature Biotechnol. Vol.22 NO.11 (2004), 1435-1439. [2] J. Nakai et al., Nature 

Biotechnol. 19 (2001), 137-141. [3] T. Nagai et al., PNAS 98 (2001), 3197-3202. [4] V. Belousov et al., Nature 

Methods Vol.3 NO.4 (2006), 281-286. 

307


BIOL-32 

Usage of chemiluminescence methods for researches of ozonotherapy influence 

on total antioxidant activity of blood plasma of patients with a urogenital 

infection contamination 

Tatyana Dyubko 1,2 , Yurij Kozin 3 , Alexander Roshal 4 , Oksana Sokolik 1 , Vasyl Zinchenko 2 , 

Karol Krzyminski 5 

1 

State Scientific Institution "Institute for Single Crystals" National Academy of Sciences of Ukraine; 

Kharkov, 61001 (Ukraine); E-mail: tdyubko@mail.ru; 

2 Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine; 

Kharkov, 61015 (Ukraine); 

3 Kharkov State Medical University, Kharkov, 61022 (Ukraine); 

4 

Institute of Chemistry at V. N. Karazin Kharkov National University, Kharkov, 61077, (Ukraine); 

5 University of Gdańsk, Faculty of Chemistry, Gdańsk 80-952 (Poland) 

The last decade, the most serious problem is the intensive expansion of urogenital infection contaminations, 

which were found in case of 50-70 % of the sexual partners. As the oxidative stress is first stage, which 

proves development of the secondary immunodeficiency, the analysis of dynamics of plasma total 

antioxidant activity (TAA) for this category of patients is the most actual problem. Taking into account a 

negligible number of standardized chemical and organic immunocorrectors, a method of physico-chemical 

immunocorrection – ozonotherapy, was successfully used during last 15 years. 

In the present work, the influence of ozonotherapy procedures (intravenous introducing ozonized 

physiological solution (ОFS) and extracorporal administration of ozonized whole blood (GAHT)) on TAA 

level was investigated. 

Analysis of TAA of blood plasma was conducted by chemiluminescence method using recently synthesized 

phenyl ester of acridiniumcarbonic acid. The TAA detection was carried out in carbonate buffer (рН – 9,93) 

in the presence of hydrogen peroxide [1, 2] . The value of partial rate constant of chemiluminescence 

quenching (K CL ) was used as parameter characterizing plasma TAA level. 

It has been found that K CL parameter of patients having a urogenital infection contamination was lower than 

that of healthy patients. It was also demonstrated that the procedures OFS, GAHT, as well as their 

combination exert similar influence on TAA level. In all the cases, first ozonation procedure leaded to 

maximal decrease of K CL , and following reiteration of the procedures resulted in the growth of this value. 

After the end of treatment course, K CL reached a reference level or exceeded it. 

It was found that K CL parameter depended on sex and age of patients, and also on the disease painful. Thus, 

the relation between K CL and the doze of ozone was more pronounced for men. 

The obtained results demonstrate that products of ozone destruction and of biomolecule oxidation lead to 

recovery of antioxidant protection of defeated organs and tissues. At the end of ozonotherapy course (10-12 

procedures), when organ antioxidant protection is recovered, we observed following growth of TAA 

parameters. 

It was demonstrated that used chemiluminescence method for plasma TAA definition might be used for 

effective express examination of pathological process dynamics. 

References: [1]. Weeks I., Behehti I., McCapra F. et al. Clin. Chem. 29(1983)1474; [2]. Krzymiński K., Roshal A. D., 

Synchykova O. P., Sandomirsky B. P. Patent P-381661, Poland, prior. date: 01.02.2007 

308


BIOL-33 

Novel polydiacetylene (PDA)-based living cell fluorescent biosensor 

Sofiya Kolusheva, Zulfiya Orynbayeva, Raz Jelinek 

Ilse Katz Center for Meso and Nanoscale Science and Technology, Ben Gurion University of the Negev, 

POB 653, 84105 Beer-Sheva (Israel). E-mail: kolushev@bgu.ac.il 

The medical relevance of the membrane activity of biological substances such as toxins, viruses, drugs and 

others is evident, and it is important to focus on early events occurring in the membrane level. A new cell 

biosensor technique was developed in our laboratory for studying membrane events on the plasma 

membrane. We have constructed new chemically engineered cells through attachment of chromatic PDA 

nano-patches onto the plasma membrane of living cells - thereby functioning as localized membrane 

sensors on the cell surface. Conjugated PDA assemblies exhibit unique chromatic and fluorescent 

properties. PDA vesicular aggregates and films have been shown to undergo distinct blue-red colorimetric 

changes owing to conformational transitions in the conjugated polymer backbone induced by external 

structural perturbations. The fluorescent PDA patches do not report upon specific biomolecular targets 

within the cell surface but rather respond to processes and surface interactions that give rise to structural 

and dynamic modifications of the plasma membrane. These cell hybrids facilitated quantitative 

spectroscopic analysis and microscopic visualization and investigation of surface phenomena in living cells 

occurring in real time. 

309


BIOL-34 

Quantum dot-labeled antimicrobial peptides reveal real-time dynamics of 

membrane disruption of Gram-negative bacteria 

Sebastian Leptihn 1,# , Jia Yi Har 1,# , Jianzhu Chen J 1,2 , Bow Ho 3 , Thorsten Wohland 1,4 

and Jeak Ling Ding 1,5 

1 

Singapore-MIT Alliance, E4-04-10, 4 Engineering Drive 3, Singapore 117576 

2 Massachusetts Institute of Technology, Center for Cancer Research, 40 Ames St, E17-132, 

Cambridge, MA 02139 USA 

3 Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 

Singapore 117597 

4 Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 

5 Department of Biological Sciences, National University of Singapore, Science Drive 4, Singapore 117543 

# These authors have contributed equally to this work as first authors 

The mechanism how antimicrobial peptides (AMPs) exploit their action has been widely investigated and 

several prominent models, called carpet, barrel-stave and torroidal pore have been proposed. To date most 

of the experiments and simulations have been done on artificial membranes and not on live bacteria and the 

majority of measurements were done on an ensemble scale. However, recently it has been shown that 

leakage experiments on artificial membranes are not always a valuable indicator for the prediction of 

antimicrobial activity in vivo. In addition, most experiments have been done up to now on an ensembles and 

single molecules have not been tracked in vivo to observe the action of AMPs on live bacteria. The 

principal objective of this work were, first, to characterize the movements of the Factor C derived AMP 

Sushi 1, and second, to give a simple interpretation for the observed behaviors leading to a consistent model 

of membrane perturbation by AMPs. We therefore developed a live bacteria leakage assay by using GFP 

expressing E.coli and confocal optics for sensitive detection of the fluorophore. In addition, we developed 

in vivo single molecule experiments by using quantum dot labeled AMPs for the live tracking of AMP 

action over time at different AMP concentrations to distinguish individual steps in the bactericidal process 

from binding, to aggregation, leading to lysis and finally membrane disruption, To our knowledge this is 

the first time that the real time process of bacterial killing by AMPs was observed with single molecule 

resolution in vivo. 

310

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Author Index 

Author Index 

A 

Abbruzzetti, S. 283 

Abdul Jalil, R.B. 25 

Aberger, F. 186 

Abyan, M. 126 

Acuña A.U. 172 

Ádány, R. 28 

Afonso, C.A.M. 197 

Aguilar-Caballos, M.P. 295 

Aizawa, S. 146 

Ajdarzadeh, O.A. 205 

Akbay, N. 250, 300 

Aker, J. 72 

Alam, S.M. 136, 226, 299 

Aleksandrova, D. 129, 130 

Alexander, L 223 

Alfonso, C. 181 

Allonas, X. 68 

Almeida, P. 145 

Almonasy, N. 80 

Altmeier, S. 127 

Álvarez, M.J.G. 88 

Amaro, M. 240 

Amat-Guerri, F. 172 

Ameloot, M. 183 

Anand, S. 159 

Andersen, K.K. 266 

Andrade, L.H.C. 71 

Andrade, S.M. 196 

Aquino, C. 284 

Arcadi, S. 287 

Arcangeli, C. 283 

Arden-Jacob, J. 89, 143 

Arlt, J. 103 

Arosio, D. 283 

Arrais, D. 251 

Audugé, N. 17 

Auer, M 15 

Auer, M. 304 

Ayadi, F. 261 

B 

Balan, L. 220 

Balanda, A.O. 167, 168, 209, 300 

Balázs, M. 28 

Balbuena, P. 84 

Baleizão, C. 78, 93 

Balogh, A. 278 

Baptista, M.S. 277 

Bark, N. 252 

Barucha, J. 262 

Baselga, J. 111 

Battistini, G. 217 

Beeby, A. 206 

Bégány, A. 28 

Belik, D. 303 

Bell, T.D.M. 180 

Belousov, V. 307 

Beltram, F. 271, 283 

Benda, A. 34 

Benesch, J. 150 

Béni, S. 148, 158 

Bento, A. 145 

Berberan-Santos, M.N. 31, 78, 93 

Bernhardt, I. 185 

Berni, E. 160 

Biemans, K. 245 

Bilenca, A. 24 

Birch, D.J.S. 81, 102, 187, 253 

Birla, L. 255 

Biver, T. 249, 301 

Bizzari, A. 162 

Bizzarri, A. 120 

Bizzarri, R. 271, 283 

Blackburn, E.H. 179 

Blamey, N. 64 

Blokhin, A.P. 94 

Blom, H. 252 

Blum, C. 58 

Boggioni, A. 301 

Boggioni,A. 249 

Bonacchi, S. 217, 227 

Bonnist, E.Y.M. 56, 268 

Borisov, S.M. 99, 139, 229 

Borovoj, I.A. 305 

Borovoy, I. 222 

Borst, J.W. 72, 196 

Bouillon, C. 281 

Bouma, B.E. 24 

Bradley, M. 223 

Bräm, O. 205 

Brameshuber, M. 263 

Brändén, M. 190 

Bräuchle, C. 200, 201 

Brédas, J-L. 52 

Brochon, J-C. 184, 191 

Brockhoff, G. 279, 298 

Brotosudarmo, T. 201 

Brouwer, A.M. 245 

Brown, S. 282 

Bruno, A. 69 

Bruvere, R. 273 

Bryce, M.R. 235 

Brzezinski, P. 190 

Buehler, C. 15, 304 

Bünzli, J-C.G. 49 

Burel-Deschamps, L. 261 

Burger, D.M. 155 

Burget, D. 220 

Buschmann, V. 195 

Bustamante, N. 152 

Buzády, A. 65, 83, 119 

C 

Cabanelas, J.C. 111 

Caires, A.R.L. 71 

Cajlakovic, M. 120 

Campanini, B. 283 

Cannizzo, A. 205 

Carayon, K. 191 

Cavalcant, L.S.e 236 

Chaix, D. 181 

Chan, J. 211 

Chatterjee, D.K. 25 

Chaudhury, N.K. 159 

Chauvin, A-S. 49 

Chen, Y. 219 

Chergui, M. 205

Author Index 

Chevolot, Y. 281 

Chmyrov, A. 89 

Cho, M-H. 21, 291 

Choi, J.H. 141, 293 

Choi, M.T.M. 161 

Chosrowjan, H. 269 

Chudakov, D. 307 

Chung, H.Y. 141, 293, 299 

Clément, J-C. 261 

Cloarec, J-P. 281 

Comby, S. 49 

Connally, R. 107 

Constantinescu-Aruxandei, D. 255 

Coppey-Moisan, M. 17 

Corral, I. 70 

Costa, S.M.B. 196, 197 

Coutinho, P.J.G. 228, 259 

Crenshaw, B.R. 46 

Crut, A. 179 

D 

D’Alessio, A. 69 

Daley, D. 252 

de Bruijn, H.S. 106 

De Clercq, B. 183 

de Francisco, R. 87 

de Lisio, C. 69 

de Rocquigny, H. 188, 280 

de Sars, V. 112 

de Sousa Filho, P.C. 244 

Debyser, Z. 183 

Declerck, N. 181 

Dekker, N.H. 179 

Delelis, O. 191 

Delgado, J. 172 

Deprez, E. 184, 191 

Dertinger, T. 37 

Descalzo, A.B. 134 

Detre, C. 285 

Dias, F.B. 235 

Diaspro, A. 23 

Didier, P. 188 

Diermeier, S. 298 

Domanov, Y.A. 257 

Dong, C. 19, 59 

Doroshenko, A. 74 

Douhal, A. 197 

Drexhage, K.H. 143 

Drexhage, K-H. 89 

Dryden, D.T.F. 56, 268 

Dubey, I. 258 

Dudko, E.V. 246 

Duerkop, A. 129 

Duportail, G. 41, 132, 280 

Dvořák, M. 86 

Dyubko, T. 153, 302, 308 

Dyubko, T.S. 288 

E 

Ecsedi, S. 28 

Eichberger, T. 186 

Emri, G. 28 

Enderle, T. 303 

Enderlein, J. 37 

Engelborghs, Y. 183 

Enrichi, F. 230 

Erdmann, R. 195 

Ermilov, E. 79 

Ermilov, E.A. 70 

Erostyák, J. 65, 83, 119 

Ewers, B. 195 

F 

Fahrni, C.J. 52 

Falcaro, . 230 

Falkenroth, A. 101 

Fedorovich, S.V. 270 

Fedyunyayeva, I. 121, 154, 222 

Fernandes, A.U. 277 

Ferreira, I.M.C. 242 

Ferreira, J.A.B. 197 

Ferreira, M.I. 240 

Ferreira, M.I.C. 239 

Fery-Forgues, S. 126 

Fidler, V. 80, 86 

Figueroa, R. 306 

Fiser, R. 292 

Fore, S. 211 

Fouassier, J-P. 68 

Freeman, R. 225 

Freschi, G.P.G. 71 

Friedrich, J.F. 90 

Frischauf, A. 186 

Fritz, J. 188 

Fruchart-Gaillard, C. 282 

Fürtbauer, E. 198 

G 

Gabruseva, N. 273 

Gahlaut, N. 171 

Galay, O. 95 

Galinovskii, N.A. 246 

Gaplovsky, M. 194 

Garau, G. 283 

Garavel, G. 15, 304 

García Fernández, J.M. 84 

Garcia, B. 301 

Gelin, M.F. 94 

Gerritsen, H.C. 106, 189 

Ghiggino, K.P. 180 

Giamarchi, P. 261 

Giannini, . 230 

Gielen, E. 183 

Gijsbers, R. 183 

Gill, J. 38 

Gill, R. 225 

Girard, E. 282 

Glover, P.B. 29 

Gohil, N.K. 159 

Gojak, C. 110 

Gök, E. 250 

Gomes, L. 189 

Gómez-Hens, A. 295 

Goncharova, N. 287 

Goncharuk, E.I. 288, 305 

González Álvarez, M.J. 84 

Gonzalez, L. 70 

González, M. 111 

Gorbenko, G.P. 257 

Gosse, I. 160 

Goutorbe, M.P. 281 

Grabolle, M. 221, 224 

Graham, E.M. 104 

Grandjean, O. 282 

Granö-Fabritius, H. 73 

Green, M. 150

Author Index 

Gregor, I. 37 

Greiner, V. 280 

Grischenko, V.I. 288, 305 

Gröbner, G. 290 

Gros, P. 189 

Guiot, E. 184, 191 

Gull, S.F. 184 

Gurina, S.O. 305 

Guzow, K. 131, 147 

H 

Haag, R. 163, 296 

Haase, M. 50 

Hamers-Schneider, M. 143 

Hammond, S.P. 29 

Han, L-F. 135 

Hania, R. 193 

Hänninen, P.E. 47 

Hapala, P. 80 

Harinen, R-R. 73 

Hartmann, A. 279 

He, H. 19, 59 

Hebling, J. 65 

Heilemann, M. 55 

Hell, S.W. 16, 89 

Hemmilä, I. 53 

Henary, M. 44 

Henary, M.M. 52 

Herman, P. 85 

Hermetter, A. 43 

Hernández-Borrell, J. 260 

Herrmann, A. 39 

Herrmann, R. 303 

Herten, D-P. 176 

Herzog, A. 101 

Hesse, J. 186 

Hilger, I. 163, 296 

Hillebrand, M. 92, 255 

Hinkeldey, B. 177 

Hintersteiner, M. 15, 304 

Hischemöller, A. 50 

Hof, M. 34, 262, 267 

Hoffmann, A. 144 

Hoffmann, K. 90, 144 

Hofstaedter, F. 279, 298 

Hoh, S.V. 91 

Holmes-Smith, S. 240 

Hölsä, J. 208 

Hornillos, V. 172 

Hornyák, I. 119 

Hötzer, B. 127 

Howorka, S. 186 

Huang, X. 19, 59 

Huang, Z. 179 

Humpolíčková, J. 34 

Hungerford, G. 150, 239, 240, 242 

Huser, T. 211 

Hutterer, R. 63, 267 

Huttunen, H. 297 

Hyppänen, I. . 208 

I 

Iamamoto, Y. 277 

Ilk, N. 137 

Ioffe, V.M. 257 

Ionescu, S. 66 

Ismailov, Z. 151 

Iwai, K. 104 

J 

Jaakkola, L. 53 

Jacak, J. 186 

Jacob, M. 264 

Jager, W.F. 157 

Jähne, B. 101 

Jančář, L. 75 

Jani, C. 180 

Janssen, B.J.C. 189 

Jares-Erijman, E.A. 289 

Järvenpää, M-L. 51 

Jażdżewska, D. 131 

Jelinek, R. 309 

Jeltsch, A. 268 

Jeon, C.W. 136, 142 

Jerabkova, P. 243 

Jiang, Y-B. 135, 169 

Jin, S.O. 226, 293 

Jochum, A. 281 

Johansson, L.B.-A. 182, 192, 207, 210, 290 

Jones, A.C. 56, 103, 104, 268 

Josel, H-P. 303 

Jovin, T.M. 289 

Joya, M.R. 236 

Jung, G. 114, 127, 177, 185, 264 

Jurkiewicz, P. 267 

K 

Kainz, B. 137, 162 

Kaiser, W.A. 163, 296 

Kalnina, I. 273 

Kalosha, I.I. 246 

Kankare, J. 208 

Karasyov, A.A. 166 

Karim, M.M. 226, 299 

Karolin, J. 102, 187 

Kasper, M. 186 

Kasper, R. 55 

Kele, P. 148, 158 

Kemnitz, K. 108 

Kemnitzer, N.U. 143 

Kesters, A. 273 

Ketola, J. 53 

Khabuseva, S. 122, 149, 153 

Khodjayev, G. 151 

Kiel, A. 176 

Kim, E. 21, 140 

Kim, J. 21 

Kim. W.H. 141 

Kimmerlin, T. 15, 304 

Kinnunen, P.K.J. 257 

Кiriiak, A.V. 76 

Kirilova, E.M. 138 

Kirilova, J. 273 

Kiss, E. 285 

Kiyose, K. 146 

Kizane, G. 273 

Klimant, I. 139, 229 

Klučiar, M. 93 

Klymchenko, A.S. 41, 280 

Knall, A.C. 124, 125 

Knight, A.E. 199 

Koberling, F. 195 

Kočišová, E. 272 

Koenig, B. 303 

Kojima, H. 146 

Kolesnik, E.E. 246 

Kolos, V.A. 270

Author Index 

Kolosova, O. 121, 122, 123, 154 

Kolusheva, S. 309 

Kömpe, K. 50 

Konopasek, I. 292 

Konrad, C. 120, 125, 162 

Koponen, P. 297 

Kornowska, K. 147 

Korovin, Y. 79 

Korppi-Tommola, J. 83 

Koskinen, J.O. 47 

Köster, D. 294 

Koster, D.A. 179 

Kosterin, S.O. 132 

Köstler, S. 109, 137, 162 

Kostsin, D. 286 

Kotschy, A. 148, 158 

Kovacs, J. 176 

Kovalska, V.B. 145, 167, 168, 209, 300 

Kozin, Y. 308 

Kozlova, N. 286 

Kozma, I.Z. 65 

Krämer, B. 195 

Krämer, R. 170, 176 

Krcmova, M. 243 

Kremser, G. 237 

Kröschel, R. 194 

Krzyminski, K. 308 

Kuchinsky, A. 95 

Kudryavtseva, Y. 122, 154 

Kuhl, J. 65 

Kukhta, A.V. 246 

Kuningas, K. 51 

Kunzelman, J. 46 

Kurjaane, N. 273 

Kurtaliev, E. 151 

Kwak, J.H. 142 

L 

Lagunas, M.C. 45 

Laitala, V. 53 

Lakowicz, J.R. 30 

Lallemand, D. 281 

Lamère, J-F. 126 

Lampinen, J. 73 

Lang, M.J. 40 

Langford, S.J. 180 

Langhals, H. 22 

Langner, M. 262 

Lanza, G. 271 

Laptenok, S.P. 72 

Lastusaari, M. 208 

László, G. 278 

Laurenceau, E. 281 

Lawson, P. 52 

Lázár, V. 28 

Leal, J.M. 301 

Lee, H.K. 136 

Lee, H.Y. 291 

Lee, S.H. 136, 141, 142, 226, 293, 299 

Leite, E.R. 241 

Leiterer, J. 224 

Lenferink, A. 178 

Lenz, T. 268 

Lewis, D.J. 29 

Li, A-F. 169 

Li, Q. 105 

Li, Z. 25 

Lichtblau, H. 137 

Liebert, K. 268 

Lillo, P. 181 

Lima, S.M. 71 

Lin, H. 193 

Lin, J. 179 

Lincoln, C.N. 115 

Link, M. 156 

Linnik, T. 302 

Litwinski, C. 70 

Loman, A. 37 

Longo, E. 236, 241 

Lopatina, L.P. 270 

Lőrincz, A. 278 

Losytskyy, M.Y. 167, 168, 300 

Lounissi, S. 261 

Lövgren, T. 51 

Luin, S. 271 

Lukatskaya, L. 74 

Luz, P.P. 216 

Ly, S. 211 

M 

M’Baye, G. 41 

Mackowski, S. 200, 201 

Macmillan, A.M. 102, 187 

Maeda, H. 42 

Magennis, S.W. 103, 104 

Makarov, S. 70 

Malval, J.P. 220 

Malval, J-P. 68 

Malyukin, Y.V. 305 

Mano, J.F. 150 

Manuel, M. 251 

Marcelo, G. 87 

Margeat, E. 181 

Markova, L. 123 

Marquer, C. 282 

Martins, J. 251 

Martins, P. 240 

Martsinko, E. 79 

Marushchak, D. 182 

Maskevich, A.A. 91 

Mataga, N. 269 

Matei, I 92 

Matile, H. 303 

Matko, J. 285 

Matkó, J. 278 

Mayer-Enthart, E. 294 

Mayr, T. 133, 229 

McArthy, A. 103 

McGuinness, C.D. 102, 187 

McNerney, G. 211 

Meirovics, I. 138 

Meisner, N-C. 15, 304 

Meixner, A. 58 

Mellet, C.O. 84 

Mély, I. 41, 188, 280 

Mély, Y. 132 

Mendels, D.A. 104 

Mendes, C.A.G. 228 

Mendicuti, F. 84, 87, 88 

Merkulov, A. 221 

Merovics, I. 273 

Meshkova, S.B. 76 

Mével, M. 261 

Meyer, A. 281 

Michl, M. 80 

Mikhalyov, I. 290 

Millar, D. 38 

Mille, M. 126

Author Index 

Miller, L.W. 171 

Minutolo, P. 69 

Mirzov, O. 193 

Mix, R. 90 

Moertelmaier, M. 198, 263 

Mojzeš, P. 272 

Mokhir, A. 176 

Molotkovsky, J.G. 210 

Monkman, A. 235 

Montalti, M. 217, 227 

Montero, M.T. 260 

Morlet-Savary, F. 68 

Morvan, F. 281 

Mourier, G. 282 

Mouscadet, J-F. 191 

Mueller, F. 303 

Mukhtar, E. 207, 210 

Mukkala, V-M. 53 

Müllen, K. 39 

Mulvaney, P. 33 

Murari, B.M. 159 

Muresan, L. 186 

Murray, B.S.L. 206 

Myllyperkiö, P. 83 

N 

Nabuurs, T.T. 245 

Nagano, T. 146 

Nagy, K. 148, 158 

Nann, T. 221, 224 

Ndao, A.S. 119 

Neely, R.K. 56, 268 

Nepraš, M. 80 

Neri, C.R. 216 

Neves da Silva, J.P. 259 

Niedermair, F. 237, 238 

Nifosì, R. 271 

Nikolova, R. 66 

Niskanen, I. 297 

Nizamov, S. 151 

Nizomov, N. 151 

Noormofidi, N. 124, 125 

Norlin, N. 192 

Noszál, B. 148, 158 

Nuhiji, E. 33 

O 

O’Riordan, T.O. 164, 165 

Obukhova, Y. 121, 123, 151 

Oheim, M. 112 

Oliveira, L.H. 241 

Olkhovik, Y.K. 246 

Olofsson, T. 210 

Olsson, V. 306 

Olżyńska, A. 267 

Onishchenko, E.V. 288 

Oosterveld-Hut, R. 113 

Opanasyuk, O. 182 

Orellana, G. 152 

Organero, J.A. 197 

Ortega, A. 181 

Ortmann, U. 195 

Orynbayeva, Z. 309 

Osipov, K.A. 246 

Ossler, F. 69 

Otto, C. 178 

Otzen, D.E. 266 

Owens, P. 64 

Öztürk, C. 250 

P 

Pal, R. 206 

Palero, J. 106 

Palmer, R.E. 219 

Palmroth, M. 297 

Pålsson, L-O. 206 

Pánek, D. 102, 187 

Panne, U. 224 

Papkovsky, D.B. 164, 165 

Paris, E.C. 236 

Park, H.W. 299 

Park, J. 291 

Park, S.B. 21, 140, 291 

Parker, D. 206 

Pasula, A. 185 

Patonay, G. 44 

Patsenker, L. 121, 122, 123, 149, 151, 153, 154, 222, 302 

Patting, M. 195 

Pauli, J. 163, 296 

Pavani, C. 277 

Pavlovich, V.S. 77 

Pavlovskii, V.N. 246 

Pein, A. 124, 125 

Perepichka, I.F. 235 

Perry, J. 52 

Persson, G. 36 

Peuralahti. J. 53 

Picas, L. 260 

Picken, S.J. 157 

Pickup, J.C. 187 

Pihlgren, L. 208 

Pikramenou, Z. 29 

Pinet, S. 160 

Piper, J. 107 

Pischel, U. 93 

Pivovarenko, V.G. 132 

Plander, M. 298 

Pond, S. 38 

Povrozin, Y. 121, 123, 154 

Praly, J-P. 281 

Prasanna de Silva, A. 104 

Praus, P. 272 

Preece, J.A. 219 

Preininger, C. 215 

Prodi, L. 217, 227 

Prokopets, V.M. 168 

Przybyło, M. 262 

Ptacek, P. 50 

Pultar, J. 215 

Pum, D. 137, 162 

Q 

Qian, H. 19, 59 

R 

Rabe, M. 110, 256 

Raja, T.N. 245 

Rajapakse, H. 171 

Rákosy, Z. 28 

Rampazzo, E. 217, 227 

Rantanen, T. 51 

Real Oliveira, M.E.C.D. 259 

Regl, G. 186 

Rei, A. 239, 242

Author Index 

Reichwein, J. 89 

Reis, L.V. 145 

Reis, R.R. 150 

Reis, T.S.V. 228 

Reischl, M. 109 

Ren, J. 19, 59 

Resch-Genger, U. 90, 144, 163, 221, 224, 294, 296 

Reymond, J-L. 27 

Riaplov, E. 105 

Ribitsch, V. 109, 120, 125, 162 

Richardson, P.R. 103 

Rivas, G. 181 

Rochon, J. 279 

Röder, B. 70, 161 

Rodríguez, H.B. 218 

Rolinski, O.J. 81, 253 

Román, E.S. 218 

Rosa, I.L.V. 236, 241 

Roshal, A. 308 

Roth, D. 303 

Royer, C.A. 54, 181 

Ruan, Y-B. 169 

Rudorfer, A. 109 

Ruiz, R. 301 

Rujkorakarn, R. 269 

Rünzler, D. 137 

Ruprecht, V. 263 

Rurack, K. 134, 224, 294 

Rusakova, N. 79 

Rüttinger, S. 195, 199 

Ryazanova, O. 82, 258 

Ryder, A. 64 

Ryder, A.G. 100, 254 

Ryderfors, L. 207, 210 

Ryu, J. 140 

S 

Sánchez-Martín, R.M. 223 

Sánchez-Martínez, M.L. 295 

Sandén, T. 190 

Santos, P.F. 145 

Sassen, A. 279, 298 

Sauer, M. 55 

Schäfer, H. 50 

Schäferling, M. 99, 128 

Schaffenberger, M. 109 

Schaub, E. 188 

Scheblykin, I. 193 

Scheer, H. 201 

Scheicher, S. 137, 162 

Schiedig, A.J. 268 

Schlapak, R. 186 

Schlapbach, R. 284 

Schmeisser, U. 166 

Schmied, M. 15, 304 

Schmitt, A. 177, 264 

Schneider, R. 220 

Schroeder, J. 86 

Schütz, G.J. 26, 186, 198, 263 

Schwarz, S. 279 

Schwille, P. 48 

Scripinets, Y. 129, 130 

Secco, F. 249, 301 

Seeger, S. 105, 110, 194, 256 

Segers-Nolten, I. 178 

Seifert, J. 304 

Seifert, J-M. 15 

Seifullina, I. 79 

Seitz, H. 294 

Seok, J-K. 44 

Serra, O.A. 216, 244 

Serrano, B. 111 

Serresi, M. 271 

Servent, D. 282 

Shen, Z. 134 

Shvadchak, V.V. 280 

Shynkar, V.V. 280 

Sialleli, J. 294 

Sidorov, V. 123 

Siegel, N. 52 

Skoog, K. 252 

Sleytr, U.B. 137 

Slobozhanina, E. 286, 287 

Slugovc, C. 124, 125, 237, 238 

Smisdom, N. 183 

Smith, T.A. 115 

Smola, S. 79 

Soare, L. 92 

Sobek, J. 284 

Soini, J.T. 47 

Sojic, N. 160 

Sokolik, O. 153, 302, 308 

Solomons, M. 29 

Sommer, L. 75 

Song, B. 49 

Sonnleitner, M. 186 

Soukka, T. 51 

Souteyrand, E. 281 

Spangler, C.M. 128 

Spangler, Ch. 128 

Spinelli, N. 69 

Stelzer, F. 125 

Stenholm, T. 47 

Štěpánek, J. 272 

Sterenborg, H.J.C.M. 106 

Stich, M.I. 99 

Stockinger, H. 198, 263 

Stortelder, A. 189 

Strano, M.S. 18 

Strekowski, L. 44 

Strömqvist. J. 252 

Stsiapura, V.I. 91 

Stubenrauch, K. 238 

Subramaniam, V. 58, 167, 178 

Suh, J.K 226 

Suh, J.K. 299 

Suh, Y.S. 141, 142 

Suhling, K. 150 

Sumalekshmy, S. 52 

Sureau, F. 272 

Suslova, E. 307 

Sutter, J-U. 81 

Suvorova, O. 70 

Svechkarev, D. 74 

Sýkora, J. 34, 267 

Szakács, Z. 148, 158 

T 

Tablet, C. 66, 92 

Tamashevski, A. 287 

Tan, W. 32 

Tanaka, F. 269 

Tanke, H.J. 231 

Taniguchi, S. 269 

Tannert, S. 70, 161 

Tari, O. 111 

Tatarets, A. 121, 122, 123, 149, 154, 302 

Tatiana, S. 287

Author Index 

Tauc, P. 191 

Tearney, G.J. 24 

Teixeira, M.R.O. 71 

Tennebroek, R. 245 

Terpetschnig, E. 121, 123, 149, 154, 222 

Thirunavukkuarasu, S. 289 

Thompson, N.L. 35 

Thomsson, D. 193 

Thyberg, P. 36 

Timon, V.V. 288, 305 

Tinnefeld, P. 55 

Tirri, M. 47 

Togashi, D. 100, 254 

Tokar, V.P. 168 

Tolkachev, V.A. 246 

Toman, P. 243 

Tormo, L. 152, 172 

Tortschanoff, A. 205 

Tozzini, V. 271 

Tramier, M. 17 

Trimmel, G. 238 

Tscherner, M. 125 

Tsvirko, M.P. 76 

Turpin, P-Y. 272 

U 

Uchiyama, S. 104 

Uhl, V. 15, 304 

Uray, G. 125 

Usero, R. 88 

Uttamlal, M. 240 

V 

Vadzyuk O.B. 132 

Vag, T. 163, 296 

Vala, M. 243 

van ‘t Hoff, M. 112 

van Amerongen, H. 265 

van den Berg, O. 157 

van den Heuvel, D.J. 189 

van der Ploeg van den Heuvel, A. 106 

van Hoek, A. 72, 196, 265 

van Mierlo, C.P.M. 265 

van Mourik, F. 205 

vandeVen, M. 183 

Vandevyver, C.C.B. 49 

Varela, J.A. 236, 241 

Varlan, A. 255 

Vasseur, J-J. 281 

Vázquez-Ibar, J.L. 260 

Vecer, J. 85 

Veettil, S.K. 185 

Venturini, M. 249, 301 

Verdes, D. 110, 256 

Vermeij, R.J. 167 

Viappiani, C. 283 

Vidal, S. 281 

Vila, P.M.B. 197 

Visser, A.J.W.G. 72, 196, 265 

Visser, N.V. 72, 265 

Vitali, M. 108 

Vityukova, E. 129, 130 

Vízkeleti, L. 28 

Vojta, Š. 75 

Volanti, D.P. 236 

Voliani, V. 271 

Volkova, K.D. 145, 167, 168, 209 

Volkova, N.A. 305 

Voloshin, I. 82, 258 

von der Hocht, I. 37 

von Heijne, G. 252 

Vynuchal, J. 243 

W 

Wabaidur, S.M. 136, 293 

Wagener, P. 86 

Wagenknecht, H-A. 57 

Wahl, M. 195 

Wahlroos, R. 47 

Walter, C. 50 

Wang, Q-L. 135 

Waseem, T.V. 270 

Weder, C. 46 

Weghuber, J. 263 

Weinhold, E. 268 

Weiss, M. 202 

Weiter, M. 243 

Westh, P. 266 

Westlund, P-O. 192 

Westphal, A.H. 72, 265 

Wichta, P. 115 

Wiczk, W. 131, 147 

Widengren, J. 36, 89, 190, 252 

Wieser, S. 198 

Wild, P. 279 

Willner, I 225 

Willner, I. 20 

Wiltsche, H. 133 

Wöhrle, D. 70 

Wolfbeis, O.S. 99, 155, 156, 166 

Wörmke, S. 200, 201 

Wu, Y. 52 

X 

Xu, H-J. 134 

Y 

Yarmoluk, S. 249 

Yarmoluk, S.M. 145, 167, 168, 209, 300 

Yegorova, A. 129, 130 

Yermolenko, I. 153, 154 

Yersin, H. 67 

Yoon, T. 21 

Yu, K. 21 

Yushchenko, D.A. 132, 280 

Z 

Zaccheroni, N. 217, 227 

Zalesskaya, G. 95 

Zhang, D. 178 

Zhang, H. 135 

Zhang, Y. 25 

Zhdanov, A.V. 164, 165 

Ziegler, J. 221, 224 

Zilles, A. 89, 143 

Zinchenko, V. 308 

Zorrilla, S. 181 

Zozulya, V. 82, 258 

Zuschratter, W. 108 

Zvagule, T. 273

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