Huge Images? - GIT Verlag

69721 

VOLUME 9 

NOVEMbER 2007 

4 

3D Orientation Microscopy 

FRET, FRAP and FISH 

Wide-field CARS-Microscopy 

Series: Digital Materials Analysis 

Imaging 

Microscopy 

&RESEARCH • DEVELOPMENT • PRODUCTION 

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& Microscopy 2/2007 •

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Nobel Prize for Surface Scientist Gerhard Ertl 

Dear Reader, 

Some days ago the Royal Swedish Academy 

of Sciences announced the 2007 Nobel 

Prize in Chemistry for Gerhard Ertl, 

professor emeritus at the Fritz-Haber Institute 

in Berlin. 

Imaging & Microscopy congratulates 

Gerhard Ertl for this most prestigious 

honour in science that is awarded due to 

his thorough studies of chemical reactions 

on solid surfaces. When having a 

closer look on fundamental molecular 

processes at the gas-solid interface, small 

gas molecules may either be adsorbed or 

bounce back at the solid surface, according 

to a note by the Royal Swedish Academy 

of Sciences (www.kva.se). The first 

case includes the most interesting possibilities: 

The gas molecule can dissociate 

at the interface, the chemical properties 

of the surface can be changed, or the absorbed 

molecule can chemically react 

with a previously absorbed one. 

Ertl’s giant step forward in the understanding 

of these scenarios led to various 

breakthroughs in the development of catalysts 

being invaluable across industry. 

Carbon monoxide and hydrocarbons are 

converted to carbon dioxide in vehicles 

exhaust gasses today, and even the content 

of nitrous gasses can significantly be 

reduced. Car manufactures around the 

globe are producing vehicles that are less 

harmful to the environment and more 

fuel efficient. 

Very early, in the mid seventies, Ertl 

unravelled the surface mechanism of ammonia 

synthesis, a reaction that was first 

discovered by Fritz Haber, reaching such 

a technical and economical significance. 

By applying new surface science methods 

he showed, that the active species is not 

molecular but dissociatively adsorbed 

atomic nitrogen. The nitrogen is hydrogenated 

in a step-process. 

In terms of a more general description 

of related applications, Ertl’s findings 

had impact on the microelectronics industries 

where thin semiconductor layers 

are formed by CVD, chemical vapour 

deposition. Corrosion protection is yet 

another area that crucially depends on 

knowledge in surface science. Ertl’s findings 

help solving problems caused by 

corrosion both in daily life and in industry, 

for example, related to aeronautics 

or nuclear power plants. 

Ertl’s surface studies have opened a 

wide span of new techniques. A citation 

from the Royal Swedish Academy of Sciences 

says that “Gerhard Ertl had been 

one of the first to see the potential of 

these new techniques. Step by step he 

had created a methodology for surface 

chemistry by demonstrating how different 

experimental procedures can be used 

to provide a complete picture of a surface 

reaction”. 

Ertl recognized the significance of a 

microscopy related methodology in surface 

science very early. Quite interestingly 

Ertl and his group have set up a 

scanning tunneling microscope, STM, for 

imaging surface reconstructions in the 

presence of adsorbates at a time where 

many others were skeptical about STM, 

after its invention by Binnig and Rohrer 

in 1982. Some years later Ertl and coworkers 

succeeded in directly visualizing 

diffusion processes using high speed STM 

in order to verify macroscopic laws. Another 

example demonstrating Ertl’s ambition 

to take use out of modern microscopy 

methods in heterogeneous catalysis 

research is the observation of adsoption 

patterns in case of CO oxidation on Pt by 

Photo Emission Electron Microscopy, 

PEEM. Some excellent illustrations of 

adsoption patterns are published by the 

Surface Imaging Group, Dept. of Physical 

Chemistry, Fritz-Haber Institute of the 

Max-Planck-Society, www.fhi-berlin. 

mpg.de/surfimag. Field Ion Microscopy, 

FIM, is another important experimental 

approach to study catalytic reactions at 

the nanometer scale. This unique microscopy 

method has been further developed 

and applied to imaging and in-situ 

chemical probing in heterogeneous catalysis 

by Norbert Kruse, former Fritz- 

Haber Institute staff member and scientific 

advisor of Imaging & Microscopy 

[1, 2]. 

In this issue of Imaging & Microscopy 

some excellent scientific articles are published, 

which give a view to recent research 

in Scanning Probe Microscopy, 

Compositional Analysis, Electron- and 

Light Microscopy. Furthermore, we like 

to steer the reader’s attention to Imaging 

& Microscopy’s conference reports and 

announcements. 

Enjoy your reading this issue 

Martin Friedrich Thomas Matzelle 

[1] Visart de Bocarmé T., Imaging & Microscopy 

8 (1), 19–21 (2006). 

[2] News & People, Imaging & Microscopy 9 (3), 8 

(2007). 

E d i t o r i a l 

[3] Freund H.-J., Knözinger H., J. Phys. Chem. B, 

108, 38, 14183–14186 (2004). 

G.I.T. Imaging & Microscopy 4/2007 •

C o n t e n t s 

E D I TO R I A L 

NObEL PRIzE fOR SuRfAcE ScIENTIST 

GERhARD ERTL 

Dr. T. Matzelle, Dr. M. Friedrich, 

GIT VERLAG, DE 1 

c OV E R S TO RY 

ThE “f” WORDS 

fRET, fRAP, and fISh 

– Technology and 

Techniques 

K. Garsha, Photometrics, 

AZ, USA 46 

P R O D u c T S 70 

I & M S h OW c A S E 

Bruker AXS 65 

FEI Company 65 

JPK 66 

Leica 66 

Nikon 67 

Olympus 67 

c O M PA N Y P R O f I L E 

AGAR ScIENTIfIc 64 

E V E N T c A L E N DA R 4 

I & M N E W S T I c K E R 6 

c O M PA N Y N E W S 1 4 

Index Inside Back Cover 

Imprint Inside Back Cover 

business Partner Inside Back Cover 

• G.I.T. Imaging & Microscopy 4/2007 

N E W S f R O M E M S 

EMS NEWSLETTER 20, OcTObER 2007 

Prof. Dr. D. Schryvers, University of 

Antwerp, BE 10 

R M S I N f O c u S 

ThE RMS – AccESS AND PROGRESSION ... 

A. Winton, Royal Microscopical Society, UK 12 

ORGANIzING ELMI 2008 hAS ALREADY 

STARTED 

Dr. M. Friedrich, GIT VERLAG, DE 13 

A N N O u N c E M E N T 

fOcuS ON MIcROScOPY 2008 

Prof. Dr. G.J. Brakenhoff et al., University of 

Amsterdam, NL 16 

E V E N T R E P O RT 

SEE YOu LATER ALLIGATOR 

Dr. M. Friedrich, GIT VERLAG, DE 18 

AT ThE REGION Of fORMER 

IRONWORKS 

Prof. Dr. P. Mestres-Ventura, Prof. Dr. U. 

Hartmann, University of Saarland, DE 22 

E L E c T R O N M I c R O S c O P Y 

ENAbLING 3D TEM/STEM Of 

NANOPARTIcLES 

Dr. K. F. Jarausch, Hitachi High 

Technologies America, Inc., CA, USA 

Dr. D.N. Leonard, Appalachian State 

University, NC, USA 24 

VERIfYING ENGINEERING AT ThE 

NANOScALE 

Dr. I. F. Uchegbu, University of London, UK 28 

cRYO ELEcTRON TOMOGRAPhY 

M. Harris, FEI Company, NL 31 

S c A N N I N G 

S E c T I O N 

uLTRAfAST cONfOcAL 

RAMAN IMAGING 

O. Hollricher et al., Witec, DE 34 

uLTRASONIc MAchINING AT ThE 

NANOMETER ScALE 

Dr. M. T. Cuberes, University of 

Castilla – La Mancha, ES 36 

3D ORIENTATION MIcROScOPY 

S.I. Wright, EDAX-TSL, Draper, Utah, USA, 

S. Zaefferer, Max-Planck-Institute for Iron 

Research, DE 40 

cOMbINING OPTIcAL uPRIGhT 

MIcROScOPY AND AfM 

J. Barner, JPK Instruments, DE 42 

M I c R O S c O P Y fAc I L I T I E S 

cANcER RESEARch IN GLASGOW 

Dr. K. I. Anderson, Beatson Research 

Center, UK 44 

L I G h T M I c R O S c O P Y 

SYSTEMATIc ANALYSIS Of fRAP 

ExPERIMENTS 

Dr. S. Seiffert et al., Clausthal University of 

Technology, DE 48 

WIDE-fIELD cARS MIcROScOPY 

Prof. Dr. M.A.M. Ritsch-Marte et al., Innsbruck 

Medical University, AT 52 

NExT GENERATION LIGhT SOuRcES 

fOR IMAGING 

Dr. J. Clowes, Fianium, UK 55 

MIcROScOPY SERIES ON DIGITAL 

MATERIALS ANALYSIS – PART 1 

Esther Ahrent, Olympus, DE 58 

I M AG E P R O c E S S I N G 

AIR QuALITY cONTROL fOR hAzARDOuS 

bIO-MATERIAL 

Dr. P. Perner, Institute of Computer Vision 

and Applied Computer Sciences, DE 62 

N OT E S f R O M N I KO N 

cONTROLLED LIGhT ExPOSuRE 

MIcROScOPY 

Dr. M. Balzar, Nikon Instruments 

Europe BV, NL 68

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E v E n t C a l E n d a r 


EVENT CALENDAR 

EVENT WhEN WhERE SouRCE of INfoRmATIoN 

Celebrating 50 Years of Multislice – Symposium November 6 Monash University, Brighton, Australia www.conferences.monash.org/multislice/index. 

cfm?p=455 

Scottish Microscopy Symposium, The Scottish 

Microscopy Group 

November 14 Dundee, Scotland, UK www.gla.ac.uk/ibls/II/em/SMG/smgnew.html 

Cryo Microscopy Group Meeting November 21 University of Birmingham, UK www.cryomicroscopygroup.org.uk 

MRS Fall Meeting 2007 November 

26–30 

Symposium on Quantitative Electron Microscopy for 

Materials Science 

November 

26–30 

Hynes Convention Center and Sheraton 

Boston Hotel 

Boston, MA, USA 

Hynes Convention Center and Sheraton 

Boston Hotel 

Boston, MA, USA 

www.mrs.org/s_mrs 


American Society for Cell Biology Annual Meeting December 1–5 Washington DC, USA www.ascb.org 

4 th International Conference of MRS-Africa December 

10–14 

Advanced TEM & TOM December 

17–18 

2008 

2008 Winter Conference on Plasma Spectrochemistry 

Winter School on Microstructural Characterization 

Focused on Electron Microscopy 

20 th Australian Conference on Microscopy and 

Microanalysis 

Dar es Salaam, Tanzania www.mrs.org/s_mrs 

Manchester, UK www.rms.org.uk/event_temtom.shtml 

January 7–12 Temecula, CA, USA www.uc.edu/plasmachem/taormina/Documents/2008_ 

Winter_Conference_Information.pdf 

January 14–18 Thessaloniki, Greece pam1.physics.auth.gr 

pam1@physics.auth.gr 

February 9–15 Perth, Western Australia, Australia www.microscopy.org.au/ACMM20 

MRS Spring Meeting 2008 March 24–28 Moscone West and San Francisco 

Marriott, San Francisco, CA, USA 

Electron Backscatter Diffraction Meeting March 31– 

April 1 

The Microscopic Ice Age – A Course in Cryo 

Techniques for Electron Microscopy 


Sheffield, UK www.rms.org.uk/event_EBSD.shtml 

April 7–11 Rothamsted Research, Harpenden, UK www.rms.org.uk/event_cryo08.shtml 

Spring School in Electron Microscopy April 14–18 University of Birmingham victoria@rms.org.uk 

Jeels 2008 May 14–16 Laboratoire de Métallurgie Physique 

Poitiers, France 

7 th European Conference on Nonlinear Optical 

Spectroscopy (ECONOS 2008) 

1 st European Conference on CARS microscopy 

(microCARS 2008) 

May 25–28 Igls, Austria Igls2008.org 

May 25–28 Igls, Austria Igls2008.org 

jeels2008.sp2mi.univ-poitiers.fr 

ELMI Meeting 2008 May 27-30 Davos, Switzerland elmi08.unibas.ch/index.html 

EM2008: 8 th International Conference on Electron 

Microscopy of Solids 

June 8–11 Cracow-Zakopane, Poland kusinski@uci.agh.edu.pl 

MRS International Materials Research Conference June, 9–12 Chongqing, China www.mrs.org/s_mrs 

13 th International Conference on Alkali-Aggregate 

Reaction in Concrete 

June 16–19 Trondheim, Norway www.icaar2008.org

MICROSCIENCE 2008 June 23–26 ExCeL, London, UK www.microscience2008.org.uk 

BIAMS 08–9 th International Workshop on Beam 

Injection Assessments of Microstructures in Semiconductors 

June 29– July 3 Toledo, Spain www.biams08.org 

Light Microscopy Summer School July 7–9 University of York www.rms.org.uk/event_lmschool06.shtml 

Getting the most from your Confocal July 10 University of York www.rms.org.uk/event_Confocal.shtml 

Intern. Conference on Mass Data Analysis of Signals 

and Images in Medicine, Biotechnology, Chemistry 

and Food Industry, MDA 

July 14 Leipzig, Germany mda-signals.de 

Microscopy & Microanalysis 2008 August 3–7 Albuquerque, New Mexico, USA www.msa.microscopy.com 

14 th European Microscopy Congress (EMC 2008) September 1–5 Aachen, Germany www.eurmicsoc.org/emc2008.html 

Microscopy of Oxidation 7 September 

15–17 

The University of Chester, UK www.liv.ac.uk/engdept/conferences/moo_07.htm 

APMC9: 9 th Asia-Pacific Microscopy Conference November 2–7 Jeju Island, Korea www.apmc9.or.kr 

huchul@snu.ac.kr 

ASCB Annual Meeting 2008 December 

13–17 

THROUGH 

YOUR WORLD 

IN SECONDS 

Bridging the gap between optical and electron microscopy 

www.fei.com/phenom 

San Francisco, USA www.ascb.org 

G.I.T. Imaging & Microscopy 4/2007 •

Lasers Fabricated by Nanoimprint 

Lithography 

The team of V. Reboud at the Tyndall National Institute, 

Cork, Ireland, report on the fabrication and 

characterization of two-dimensional polymer photonic 

crystal band-edge lasers operating in the visible 

range. The components have been fabricated in 

a dye chromophore-loaded polymer matrix by nanoimprint 

lithography and high-symmetry bandedge 

modes are used to generate laser emission. 

Their work demonstrates the potential of nanoimprint 

lithography for the fabrication of two-dimensional 

planar photonic crystal structures in an active 

medium in a one-step process. 

» Appl. Phys. Lett. 91, 151101 

» doi:10.1063/1.2798250 

Optical Coherence Computed 

Tomography 

L. Li and L.V. Wang from the Washington University 

in St Louis, Missouri, USA, propose a device to 

bridge the gap between diffuse optical tomography 

and optical coherence tomography. Both ballistic 

and multiple-scattered photons are measured at 

multiple source-detection positions by low-coherence 

interferometry providing a temporal resolution 

smaller than 100 fs. A light-tissue interaction 

model was established using the time-resolved 

Monte Carlo method. The optical properties were 

then reconstructed by solving the inverse transient 

radiative transport problem under the first Born approximation. 

Absorbing inclusions of 100 µm diameter 

were imaged through a 2.6-mm-thick (~ 30 

scattering mean-free-paths) scattering medium. 

» Appl. Phys. Lett. 91, 141107 

» doi:10.1063/1.2793625 

Frequency Response of an AFM: 

Magnetic Versus Acoustic Excitation 

E.T. Herruzo and R. Garcia from the CSIC, Madrid, 

Spain, discuss the dynamics of an amplitude modulation 

AFM in different environments such as water 

and air, and show that the resonance curves depend 

on the excitation method used to drive the 

cantilever, either mechanical or magnetic. This dependence 

is magnified for small force constants 

and quality factors, i.e., below 1 N/m and 10, respectively. 

They also show that the equation for the 

observable, the cantilever deflection, depends on 

the excitation method. Under mechanical excitation, 

the deflection involves the base and tip displacements, 

while in magnetic excitation, the cantilever 

deflection and tip displacement coincide. 

» Appl. Phys. Lett. 91, 143113 

» doi:10.1063/1.2794426 


NEwS TICkER 

Influence of Sample Conductivity on 

Oxidation by the Tip of AFM 

V. Cambel and J. Soltys at the Slovak Academy of 

Sciences, Bratislava, Slovakia analyzed the role of 

the electric field distribution in the nano-oxidation 

process realized by the tip of AFM experimentally 

and theoretically. Authors show the importance of 

the sample conductivity and the water bridge in the 

process applied to bulk GaAs and Ga[Al]As heterostructures 

in both contact and noncontact AFM 

modes and the consequences for the lines witten. 

They show that the electric field distribution in the 

system tip-sample is controlled by the sample conductivity. 

In the case of low-conductive samples, 

maximum field is located apart from the tip apex 

for both contact and noncontact AFM modes. 

» J. Appl. Phys. 102, 074315 

» doi:10.1063/1.2794374 

High-resolution Microscope for Tipenhanced 

Optical Processes in UHV 

J. Steidtner and B. Pettinger in Berlin, Germany 

present an optical microscope based on tip-enhanced 

optical processes that can be used for studies 

on adsorbates as well as thin layers and nanostructures. 

It provides chemical and topographic 

informations with a resolution of a few nanometers 

and can be employed in ultrahigh vacuum as well 

as gas phase. The central idea is to mount, within 

an UHV system, an optical platform with all necessary 

optical elements to a rigid frame that also carries 

the scanning tunneling microscope unit and to 

integrate a high numerical aperture parabolic mirror 

between the scanning probe microscope head 

and the sample. Authors present the first results of 

Raman measurements using the device and the experimentally 

determined requirements of the parabolic 

mirror in terms of alignment accuracy. 

» Rev. Sci. Instrum. 78, 103104 

» doi:10.1063/1.2794227 

An UHV Fast-scanning and Variable 

Temperature STM for Large Scale 

Imaging 

B. Diaconescu and co-workers from the University 

of New Hampshire, USA describe the design and 

performance of a fast-scanning, variable temperature 

scanning tunneling microscope (STM) operating 

from 80 to 700 K in ultrahigh vacuum (UHV), 

which routinely achieves large scale atomically resolved 

imaging of compact metallic surfaces. The 

vertical resolution of the instrument was found to 

be about 2 pm at room temperature. The total 

scanning area is about 8 × 8 µm 2 . The sample tem- 

perature can be adjusted by a few tens of degrees 

while scanning over the same sample area. 


» doi:10.1063/1.2789655 

Suppression of Spurious Vibration of 

Cantilever in AFM 

T. Tsuji and colleagues from Tohoku University, Japan, 

developed a simple but effective method for 

suppressing spurious response (SR) to improve the 

precision of dynamic AFM using cantilever vibration 

spectra. The dominant origin of SR was identified 

to be the bending vibration of the cantilever 

substrate, but while a rigid cover pressing the 

whole surface of the substrate suppressed SR, the 

utility was insufficient. Then, a method of enhancing 

the bending rigidity of the substrate by gluing a 

rigid plate (clamping plate, CP) to the substrate 

was developed. The CP method will particularly 

contribute to improving dynamic-mode AFM, in 

which resonance spectra with a low quality factor 

are used, such as noncontact mode AFM in liquid or 

contact resonance mode AFM. 


» doi:10.1063/1.2793498 

High Resolution Gamma Ray 

Tomography Scanner 

U. Hampel and co-workers at Forschungszentrum 

Dresden-Rossendorf e.V., Dresden, Germany, report 

on the development of a high resolution gamma 

ray tomography scanner that is operated with a Cs- 

137 isotopic source at 662 keV gamma photon energy 

and achieves a spatial image resolution of 0.2 

line pairs/mm at 10 % modulation transfer function 

for noncollimated detectors. It is primarily intended 

for the scientific study of flow regimes and phase 

fraction distributions in fuel element assemblies, 

chemical reactors, pipelines, and hydrodynamic machines, 

but it is applicable to nondestructive testing 

of larger radiologically dense objects. They also 

built a computed tomography scanner gantry for 

measurements at fixed vessels or plant components. 


» doi:10.1063/1.2795648 

Correction of Axial Geometrical Distortion 

in Microscopic Images 

H.J. Van Elburg and colleagues from the University 

of Antwerp, Belgium, discuss the extraction of 

quantitative data from microscopic volume images 

when imperfectly matched immersion and mounting 

media result in axial geometrical distortion. Linear 

correction of the axial distortion using the

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I & M N e w s T I c k e r 

paraxial estimate of the axial scaling factor yields 

results that may differ as much as 4 % from the actual 

values. From calculations based on a theoretical 

expression of the 3-D point-spread function in 

the focal region of a high-aperture microscope authors 

derived axial scaling factors that result in 

quantitative results accurate to better than 1 %. 

From a non-linear correction procedure, an improved 

formula for the paraxial estimate of the axial 

scaling factor is derived. 

» Journal of Microscopy 228 (1), 45–54. 

» doi:10.1111/j.1365-2818.2007.01822.x 

A white Light Confocal Microscope for 

Spectrally Resolved Multidimensional 

Imaging 

J.H. Frank and co-workers from the University of 

Cambridge, UK, demonstrate spectrofluorometric 

imaging microscopy in a confocal microscope using 

a supercontinuum laser as an excitation source and 

a custom-built prism spectrometer for detection. 

This microscope system provides confocal imaging 

with spectrally resolved fluorescence excitation and 

detection from 450 to 700 nm, and authors present 

the device performances. The speed of the spectral 

scans is suitable for spectrofluorometric imaging of 

live cells. Effects of chromatic aberration are modest 

and do not significantly limit the spatial resolution 

of the confocal measurements. 

» Journal of Microscopy 227 (3), 203–215. 

» doi:10.1111/j.1365-2818.2007.01803.x 

In Vivo Imaging of Hydrogen Peroxide 

with Chemiluminescent Nanoparticles 

D. Lee from the Georgia Institute of Technology, Atlanta, 

Georgia, demonstrate that nanoparticles formulated 

from peroxalate esters and fluorescent 

dyes can image hydrogen peroxide in vivo with 


high specificity and sensitivity and discuss possible 

applications. The peroxalate nanoparticles image 

hydrogen peroxide by undergoing a three-component 

chemiluminescent reaction between hydrogen 

peroxide, peroxalate esters and fluorescent dyes. 

The peroxalate nanoparticles have attractive properties 

for in vivo imaging, such as tunable wavelength 

emission (460–630 nm), nanomolar sensitivity 

for hydrogen peroxide and excellent specificity 

over other reactive oxygen species. 

» Nature Materials 6, 765–769 

» doi:10.1038/nmat1983 

3-D Imaging of Liquid Crystals by CARS 

Microscopy 

B.G. Saar and colleagues used Coherent anti-Stokes 

Raman scattering (CARS) microscopy to provide 

three-dimensional chemical maps of liquid crystalline 

samples without the use of external labels. 

CARS is an optical imaging technique that derives 

contrast from Raman-active molecular vibrations in 

the sample, that offers more rapid chemical characterization 

without the use of external dyes or contrast 

agents. The use of CARS to image chemical 

and orientational order in liquid crystals is demonstrated 

using several examples, and the limitations 

and benefits are discussed. 

» Opt. Express 15, 13585–13596 

» http://www.opticsinfobase.org/abstract. 

cfm?URI=oe-15-21-13585 

Direct Measurement of Hydrophobic 

Forces on Cell Surfaces Using AFM 

D. Alsteens and co-workers at the Université 

Catholique de Louvain, Louvain-La-Neuve, Belgium 

present chemical force microscopy (CFM) with hydrophobic 

tips to measure local hydrophobic forces 

on organic surfaces and on live bacteria. On organic 

surfaces, they found an excellent correlation 

between nanoscale CFM and macroscale wettability 

measurements, demonstrating the sensitivity of 

the method toward hydrophobicity and providing 

novel insight into the nature of hydrophobic forces. 

Authors also studied hydrophobic forces associated 

with mycolic acids on the surface of mycobacteria, 

and discuss the importance of these compounds. 

» Langmuir, ASAP Article 10.1021/la702765c 

S0743-7463(70)02765-8 

Multicolor STORM Imaging 

Using photoswitchable fluorescent probes with distinct 

colors, M. Bates and his colleagues demonstrate 

the feasibility of multicolour stochastic optical 

reconstruction microscopy (STORM). The pairing 

of different activator dyes with a range of photoswitchable 

reporter fluorophores allowed iterative, 

color-specific activation of distinct color subsets 

and STORM imaging with a resolution of 20–30 

nm. 

» Science, 317, pp. 1749–1753 

Fluorescence Nanoscopy 

A. Egner and co-workers report nanoscopy imaging 

in intact cells through reversible photoswitching of 

individual fluorophores at fast recording speeds 

and demonstrate this by imaging the microtubular 

network of a mammalian cell at 40 nm resolution. 

» Biophys. Journal, 93, pp. 3285–3290 

Correlative 3D LM-EM Imaging of 

Mitochondria During Apoptosis 

M. Sun and colleagues studied the behaviour of mitochondria 

during apoptosis using fluorescence microscopy 

in combination with 3D electron tomography 

of the same cells. After locating apoptotic cells 

in the light microscope, they could subsequentially 

study the remodelling of the inner mitochondrial 

membrane into separate vesicular compartments at 

the ultrastructural level. 

» Nature Cell Biology, 9, pp. 1057–1065 

Even Illumination Field in TIRF Imaging 

with a Laser 

R. Fiolka and co-workers describe a method to overcome 

the problem of the scattering of coherent laser-light 

that normally causes uneven illumination 

of the image field during TIRF imaging by azimuthal 

rotation of the illumination laser beam. The incidence 

angle of the laser can still be changed quickly 

so that fast switching to epifluorescence illumination 

is still possible. 

» Microsc. Res. Tech. (advance on-line publication) 

Cryo-fluorescence Microscopy 

The use of a novel cryo-light microscope stage enabled 

C. Schwartz and her colleagues to perform correlative 

light and electron microscopy of vitreous 

samples prepared for cryo-EM. In addition to the 

correlative imaging aspect, photobleaching of the 

fluorophores is reduced the cryogenic temperatures 

(–140 °C) of the setup. 

» J. Microsc., 227, pp. 98–109 

white Light Confocal with a Supercontinuum 

Laser 

J.H. Frank and co-workers describe the use of a supercontinuum 

laser as the excitation light source 

for a confocal microscope. Throught the use of an 

AOTF, up to eight excitation wavelengths can be selected 

and be freely positioned along the spectrum, 

thus allowing to closely match the excitations to 

the spectral requirements of the selected fluorophores. 

In combination with spectral detection fluorescence 

excitation and emission spectra can be 

taklen at every position in the image. 

» J. Microsc., 227, pp. 203–215

Understand the 

Dynamic Processes of Life. 

Reach Out for Experience. 

Axio Observer LSM 5 DUO PALM MicroBeam 

Carl Zeiss: Living Cells 

www.zeiss.de/FluoresScience

Ueli Aebi 

EMS President 

During the summer months no EMS 

Newsletter was published, so we do have 

quite a few items to cover in this autumn 

issue. As you know, supporting European 

microscopy activities is one of the primary 

goals of EMS. In this spirit the EMS 

Board at its meeting in Prague decided to 

offer six scholarships of 500 Euro each to 

young researchers to participate at Symposium 

C on “Quantitative TEM for Advanced 

Materials” that will be held during 

the Boston Fall Meeting of the US 

Materials Research Society and is organized 

by Etienne Snoeck, Rafal Dunun- 

Burkowski, Johan Verbeeck and Uli Dahmen. 

This support was made possible by 

a special 2500 Euro grant from FEI. The 

successful applicants are Leonardo LARI 

(Liverpool), Wouter van den Broek (Antwerp), 

Lang-Yun (Shery) Chang (Cambridge), 

Sandra Van Aert (Antwerp), 

Florent Houdellier (Toulouse) and Magnus 

Garbrecht (Kiel); we wish them all a 

very fruitful meeting. 

By the closing of the first round of applications 

for sponsored events taking 

place during the first six months of 2008, 

four applications have been received and 

will be evaluated by the Board during the 

coming weeks. Since in 2008 EMS will 

Nick Schryvers 

EMS Secretary 

EMS Newsletter 20, October 2007 

Dear EMS member, 

We also like to announce the following 

training courses 

Network of Excellence (EU-NOE) 

for 3D-electron microscopy (3D-EM) 

(http://www.3dem-noe-training.org): 

Transmission Electron Microscopy in Life Science 

February 4 th –8 th , Eindhoven, The Netherlands, 

and 

N e w s f r o m e m s 

Single Particle Analysis, February 25 th –29 th 2008, 

Madrid, Spain 

10 • G.I.T. Imaging & Microscopy 4/2007 

organize the quadrennial European Microscopy 

Congress EMC 2008, no EMS 

extension was granted. The next Extension 

will be held in 2009 with the deadline 

for applications being June 30, 

2008. 

Most exciting for EMS during the past 

summer period has been the formal decision 

of the Portuguese Microscopy Society 

SPMicros to join EMS as an en-bloc 

member, which brings the total number 

of EMS members to over 5000 and a coverage 

of the continent that is close to 

complete! The Portuguese Society has 

gone through some important organizational 

changes lately and we do warmly 

welcome SPMicros to the EMS family 

hoping that we can be of some assistance 

to help increasing the visibility of microscopy 

in Portugal and, most importantly, 

improving communication channels with 

the rest of Europe. 

And now a quick update on EMC 2008 

in Aachen: during the past three months 

chair persons for symposia and regular 

sessions have been selected, and at 

present names for keynote and invited 

speakers are being solicited. Most importantly, 

a first flyer has been mailed, and 

potential exhibitors have been contacted. 

Last but not least, the first few pages of 

the EMC 2008 website have recently been 

made available at www.emc2008.de. 

Please allow us to remind you that we 

are presently looking for candidates 

poised to organize the 15 th European Microscopy 

Congress in 2012. With the 

growing interest in microscopy, EMC is 

looking for congress sites capable of accommodating 

up to 1500 participants 

following up to 10 parallel sessions plus 

a commercial exhibitor space of around 

2000 m 2 (incl. walking space). Applications 

have to be sent to the EMS Secre- 

tary by January 30 th , 2008, according to 

the stipulations listed in point F.2 of the 

By-Laws of the Constitution (see www. 

eurmicsoc.org). The final venue will be 

decided by the General Council which 

will meet in Aachen during EMC 2008. 

At EMC 2008 the quadrennial FEI 

Awards (one in the Life Sciences and one 

in Materials Science/Physics) will be presented. 

Candidates should be proposed 

by a Microscopy Society, a group of scientists, 

or an individual scientist. Applications 

for these prestigious awards 

should be sent to the EMS Secretary by 

regular mail and reach him by no later 

than April 1 st , 2008. More details on the 

qualification criteria and application procedures 

can be found on the EMS website 

www.eurmicsoc.org under the 

header “funding”. 

We have just been informed that Dr. 

Charles (Chuck) Garber, chairman of SPI 

Supplies and one of our early-day ECMA 

members, died on September 19 th , 2007. 

We all valued Chuck for his candid remarks 

and ever constructive suggestions 

aimed at improving the performance of 

scientists and exhibitors alike, so we will 

all miss Chuck at future microscopy 

meetings, be this at his booth, in the sessions 

or at the social gatherings. The 

EMS Board members would like to express 

their sincere condolences to Babszy 

Garber, his wife, and his family. 

Contact: 

Prof. Dr. D. Schryvers, Ph.D. 

Electron Microscopy for Materials Science (EMAT) 

Department of Physics 

University of Antwerp, Belgium 

Tel.: +32 3 2653247 

Fax: +32 3 2653257 

nick.schryvers@ua.ac.be

R M S I n F o c u S 

The RMS – Access and Progression … 

… From the Cradle (Well Almost!) 

“ When planning for a year, plant corn. 

When planning for a decade, plant trees. 

When planning for life, train and educate people.” 

Chinese proverb: Guanzi (c.645 B.C.) 

The European Commission has recognised 

the inherent wisdom of this Chinese 

proverb through the instigation this 

year of the Lifelong Learning Programme. 

Billed as the flagship European funding 

programme in the field of education and 

training, this is the first time a single programme 

will cover learning opportunities 

from childhood to old age. It aims to 

support projects and activities that foster 

interchange, cooperation and mobility 

between education and training systems 

within the EU, so that they become a 

world quality reference. 

The Royal Microscopical Society 

wholeheartedly endorses this ethos and 

is a keen advocate of learning and professional 

development. “As a professional 

body we see that one of our core rolls is 

to provide educational resources, not 

only to our members but to the community 

as a whole, in order to develop careers 

and support a wider understanding 

of science and microscopy at all levels”, 

explains Rob Flavin, Executive Director 

of the RMS. 

Training and Events 

In support of the above, the RMS publishes 

The Journal of Microscopy and a 

series of microscopy books, as well as 

Date Event Title Location 

17–18 December 2007 Aberration Corrected & Quantitative 

(S)TEM & Advances in Tomography II 

Manchester 

31 March–01 April 2008 Electron Backscatter Diffraction Conference 

Sheffield 

07–11 April 2008 The Microscopic Ice Age – A Course in 

Cryo Techniques for Electron Microscopy 

Rothamsted Research, Harpenden 

14–18 April 2008 Spring School in Electron Microscopy University of Oxford 

23–26 June 2008 MICROSCIENCE 2008 – International 

Conference and Exhibition 

ExCeL, London 

07–09 July 2008 Light Microscopy Summer School University of York 

10–11 July 2008 Getting the most from your Confocal University of York 


helping young scientists through bursaries. 

The Society further demonstrates 

its commitment to professional development 

by offering qualifications in microscopy, 

and organising annual 5 day training 

courses in light, electron and confocal 

microscopy, flow cytometry and cell imaging. 

In addition, it stages its own scientific 

meetings that address topics at the 

cuttingedge of microscopy. 

Financial Assistance in order to support 

their continuing education, RMS 

members are entitled to a substantial 

discount on all course and meetings fees. 

Offering further assistance to young scientists 

who are members, the RMS encourages 

them to attend conferences in 

both the UK and abroad through a generous 

bursary scheme. 

RMS Learning Zone at Microscience 

2008 

One of the unique features of Microscience 

is the RMS Learning Zone, which 

provides a free ‘taster’ to RMS courses. 

The Learning Zone aims to provide a 

friendly environment for absolutely anyone 

to drop in and get help with all aspects 

of electron and light microscopy. 

This makes it an ideal place to visit for 

those just starting out and for others 

wishing to learn more about unfamiliar 

techniques.

News from the RMS 

RMS endorses university courses: 

The first Imaging MSc kicked off at Oxford Brookes 

University this September. As it is RMS endorsed, 

all students on the course gain free membership. 

Any post-graduate microscopy/imaging related 

courses are eligible for RMS endorsement and the 

Society would be happy to discuss such backing 

with any interested universities and institutes. 

Call for Microscience 2008 papers: 

Building on the success of Microscience 2004 and 

2006, London‘s ExCeL will once again host Europe‘s 

premier international conference and exhibition 

on the science of microscopy, imaging and 

analysis on 23–26 June 2008. 

First call for papers October 2007 – Deadline for 

contributed oral papers 23:00 GMT February 29 th 

2008 

“Having a good grasp of the basic theories 

and practices of microscopy is a vitally 

important step to carrying out 

meaningful research, and we are especially 

keen to support researchers in the 

early stages of their careers,” says Debbie 

Stokes, RMS Honorary Secretary Science 

(Physical). “Our internationally renowned 

Learning Zone volunteers will be 

on hand to generously pass on their experience 

and knowledge to the future 

generation of microscopists. This is such 

a valuable opportunity and, with the introduction 

of Microscience Early Stage 

Researcher Bursaries, we hope to see 

many academic and industrial researchers 

from Europe and around the world.” 

Contact: 

Allison Winton 

Royal Microscopical Society 

St Clements, United Kingdom 

Tel.: +44 1865 254760 

Fax: +44 1865 791237 

allison@rms.org.uk 

www.rms.org.uk 

www.microscience2008.org.ukv 

Organizing ELMI 2008 Has Already Started 

The next venue for the annual ELMI meeting will be the congress center in Davos, Switzerland from May 27 

to 30, 2008. Basis for this decision was not only the nice environment as a promotion for the Swiss countryside 

but the very good experience in running the Microscopy Conference late summer in 2005 (see Imaging 

& Microscopy November 2005 issue). 

In respect to the strong interest of the organizers 

that the ELMI meeting will keep 

its very special attitude as a workshop 

based unique event with a lot of handson 

sessions, they invited their partners to 

a planning meeting directly at the venue. 

The purpose was to inspect the congress 

center concerning the needs for appropriate 

space for manufacturers system 

installations and further infrastructure. 

In agreement with the attendant companies, 

Carl Zeiss MicroImaging, Leica Microsystems, 

Life Imaging Services, Nikon, 

and Olympus the whole congress center 

will be rent for the 2008 event. 

To make your own planning to attend 

the conference or to save your company’s 

workshop slot we like to recommend to 

get in contact with the organizers as listed 

in the box below. We are looking forward 

to meeting you in springlike Davos. 

Martin Friedrich 

GIT VERLAG, A Wiley Company 

Secretary: 

Gabriele Gruber 

Gabriele.gruber@fmi.ch 

Industry Contacts: 

Patrick Schwarb 

Patrick.schwarb@fmi.ch 

ELMI Board Member Contacts: 

Jens Rietdorf 

jens.rietdorf@fmi.ch 

Scientific Committee: 

Nathalie Garin 

Nathalie.garin@epfl.ch 

Gabor Csucs 

csucs@bc.biol.ethz.ch 

Finances: 

Gianni Morson 

Gianni.morson@unibas.ch 

R M S I n F o c u S 

Meeting Contact: 

Markus Dürrenberger 

markus.duerrenberger@unibas.ch 

Media Partner Imaging & Microscopy: 

Martin Friedrich 

m.friedrich@gitverlag.com 

G.I.T. Imaging & Microscopy 4/2007 • 13

C o m pa n y n e w s 

Nanosolution Center Opening 

Invited by Carl Zeiss AG and the Photonics 

BW technology network, German Federal 

Minister for Education and Research Dr. 

Annette Schavan visited on 7 th of September 

Oberkochen. While there, she opened 

the Nanosolutions Center, the largest and 

most advanced demo center for cuttingedge 

microscopy in Germany. During her 

address to the 250 guests, she emphasized 

the value of optical technologies for the 

competitiveness of the location: “These 

technologies drive important innovations 

and are clearly among the future technologies 

of the 21 st century. They provide key 

impulses for Germany. The Federal Ministry 

of Education and Research (BMBF) 

recognized the significance of optical technologies 

at an early stage and have specifically 

promoted their expansion. These 

technologies are a central element in the 

government’s high-tech strategy.” 

www.smt.zeiss.com 

Improving Europe’s Image 

The European Science Foundation calls for greater 

collaboration across Europe on research in medical 

imaging. New imaging technologies will result in 

improved and cost-effective healthcare, the ESF 

says, but there needs to be closer cooperation between 

doctors, scientists and industry if Europe is 

to realise the full potential of new developments 

and remain competitive globally. The call comes in 

a new ESF Science Policy Briefing (SPB) released 

this week. The briefing is the result of a workshop 

attended by key experts in the field organised by 

ESF’s medical section, the European Medical Research 

Councils (EMRC). Imaging is one of the fastest 

growing areas within medicine. 

www.esf.org 

Harvard Apparatus Acquires 

PanLab 

Harvard Apparatus has announced its acquisition of 

Panlab, a distributor and manufacturer of products 

and software for the life sciences researcher, primarily 

in the neuroscience research market. Harvard 

Apparatus now offers a full line of products for: activity 

and exploration, depression studies, motor 

function and coordination, anxiety studies, learning 

and memory, pain and inflammation , video tracking, 

respiratory metabolism, food and liquid monitoring, 

social interactions and phenotyping. 

www.harvardapparatus.com 


From left to right: Dr. Hermann Gerlinger, Member of the Board at Carl Zeiss SMT AG, German 

Federal Minister for Education and Research Dr. Annette Schavan, President and CEO of Carl Zeiss AG 

Dr. Dieter Kurz, and Dr. Dirk Stenkamp, Member of the Board at Carl Zeiss SMT AG. 

JPK in Asia-Pacific Region 

JPK Instruments has strengthened its marketing position 

in the booming Asia-Pacific region: The company 

has granted the exclusive distribution rights 

for its products in India to Inkarp Instruments, and 

in Australia to Scitech. In addition to Canada, Brazil 

and the European market, JPK Instruments’ market 

presence now covers Australia, India, Taiwan, China, 

Korea, Japan, Singapore and Thailand. 

www.jpk-instruments.de 

Palm Merged with Carl 

Zeiss Microimaging 

On 24 September 2007 Palm Microlaser Technologies, 

a 100 % subsidiary of Carl Zeiss Microimaging, 

was merged with Carl Zeiss Microimaging. All current 

activities in the field of laser microdissection 

will be continued on an ongoing basis. Under the 

company name Carl Zeiss Microimaging, all former 

contacts can be reached under the same address 

data from the Bernried location in the Microdissection 

business field. With its Microdissection business 

sector the company is now the sole manufacturer 

of laser microdissection and micromanipulation 

systems using patent protected LMPC technology. 

The systems are used both in biomedical and clinical 

research as well as in routine applications. 

www.zeiss.de/microbeam 

Gold Medal for Donal Denvir 

Dr. Donal Denvir, co-founder of Andor Technology 

has been recognized with a gold medal from the Institute 

of Physics. The Business and Innovation 

medal of the Institute of Physics is awarded to individuals 

for outstanding contributions to the organisation 

or application of physics in an industrial or 

commercial context. Dr Denvir’s award in recognition 

for his role in founding Andor Technology 

which manufactures high-performance digital cameras, 

and for leading an R&D programme. 

www.andor.com

R&D 100 Award for Zeiss Electron Microscope 

Carl Zeiss SMT was honoured with a coveted R&D 100 Award for one of the 100 

most technologically significant new products in 2007. R&D Magazine recognized 

the company’s Scanning Transmission Electron Microscope (STEM) for its 

ability to advance the field of materials science and research. The advanced version 

of the Libra 200 STM enables scientists and researchers to analyze materials 

at the atomic level. Nanostructures can be viewed with imaging resolutions 

and analytical capabilities never before possible in one single instrument. 


JPK Fastest Growing Nanotech Company 

JPK Instruments is the fastest growing company of the nanotech industry on the 

Deloitte Technology Fast 50 ranking, which has been established for the fifth 

consecutive year. The company develops, produces and markets essential base 

technologies enabling unique analytical access at an atomic and molecular level. 

The leading companies in Germany’s high-tech industries were determined on 

the basis of their compounded percentage sales growth rates of the past five 

years. JPK achieved a growth rate of over 970 %. 

www.jpk-instruments.de 

Leica Selling BioSystems Products in Sweden 

Leica Microsystems announced that it has signed a purchase contract for the 

rights to sell products for the former Vision BioSystems into the Swedish market 

thereby establishing a stronger European sales organization for the efficient 

support of ist pathology and diagnostics business. Leica Microsystems and the 

Swedish company Immunkemi announced the signing of this purchase contract 

on October 1, 2007. For the last seven years, Immunkemi had represented the 

former Australian Vision BioSystems as the Swedish distributor for its specimen 

preparation instruments as well as reagents and antibodies. 

www.leica-microsystems.com 

Leica to Sell Expression Pathology’s Slides 

Rapid, gentle and contamination free laser microdissection, directly into sample 

buffer, is now possible with the combination of Leica Microsystems’ laser microdissection 

system Leica LMD6000 and Expression Pathology’s Director glass 

slides. A sales agreement between the two companies now gives scientists the 

opportunity to buy both the instrument and the slides from Leica Microsystems 

worldwide. The slides bring a new level of speed and accuracy to laser microdissection, 

allowing collection directly into a vial, while eliminating the need for 

plastic membrane-coated slides or sticky caps. These membrane-free slides employ 

a proprietary energy transfer coating bonded to a glass support. UV-laser 

energy is converted to kinetic energy upon striking the energy transfer coating, 

vaporizing it, thereby propelling selected features into the vial. 


Image Reconstruction Software Successful 

in Hospital 

Syncroscopy has announced Auto-Montage Pro, its 3D image reconstruction 

software, has been successfully used at a major UK teaching hospital, Addenbrooke’s 

Hospital, part of the Cambridge University Hospitals group, (UK), to 

help confirm diagnosis of schistosomiasis, a parasitic disease rarely seen in the 

UK. Pathologists in the Department of Histopathology used the software as a diagnostic 

tool to produce in-focus microscope images of the abnormality within 

the brain biopsy from an adult patient who had recently developed focal epileptic 

seizures. With the help of these images, the eggs of Schistosoma mansoni, 

one of several species of flatworm that cause schistosomiasis, could be identified 

and characterised. Schistosomiasis is a common chronic disease in Africa 

and Asia, where the patient had extensively traveled. 

www.syncroscopy.com 

Microscope Automation 

at Every Stage 

If you demand speed, accuracy and 

precision from your microscope, take a 

look at Prior’s world leading microscope 

automation systems for effective (and 

cost-effective) solutions. We design and 

manufacture the widest range of 

scanning stages, filter wheels, shutters, 

automated focus units, motor controllers 

and illumination systems for end users, 

system integrators and OEM’s worldwide. 

For further information visit the web at 

www.prior.com or email a brochure 

request to uksales@prior.com 

Focussed on microscopy 

Prior Scientific Instruments Cambridge CB21 5ET UK 

Telephone +44 (0)1223 881711 www.prior.com 


A n n o u n c e m e n t 

Focus on Microscopy 2008 

April 13–16, Osaka-Awaji, Japan 

After the successful 2007 FOM conference this year in Valencia, Spain the next conference in the FOM series 

will take place in Osaka, Awaji Island, Japan from Sunday, April 13 to Wednesday, April 16, 2008. It will start 

around 6 o‘clock in the afternoon on Sunday the 13 th with a plenary opening session followed by a welcome 

reception. The program schedule and general information of the conference can be found at the conference 

website: FocusOnMicroscopy.org. 

The conference location is the Awaji 

Yumebutai International Conference 

Center/Resort near Osaka. The location 

can easily be reached from Kobe and Osaka 

airports. All details around registration, 

abstract submission and deadlines 

etc. is present or will come shortly available 

on this website. 

A wide range of microscopy and microscopy 

related subjects will be addressed. 

These range from the advanced 

use of fluorescent probes in – live – cellular 

biophysics, specialized spatial image 

analysis of the resulting images to the 

physics of sub-resolution spatial image 

formation. 

With its origin and focus in sectioned 

confocal/2-photon microscopy the conference 

also signals important developments 

in neighboring fields. 

Topics of the FOM conference series 

include: 

� Confocal and multiphoton-excitation 

microscopies 

� 3D and 4D live cell and tissue imaging 


� 

� 

� 

� 

� 

� 

� 

� 

� 

� 

Novel illumination and detection strategies 

– selective plane extended depth 

of focus, 4pi, structured illumination 

Fluorescence – new labels, fluorescent 

proteins, quantum dots, single molecule, 

excitation-emission spectroscopy 

Time-resolved fluorescence – FRET, 

FRAP, FLIM, FCS 

Coherent non-linear microscopies – 

SHG, THG, SFG, CARS 

Scattering processes: Raman, light 

scattering spectroscopy, second harmonic 

Multi-dimensional imaging 

Sub-wavelength resolution – near field 

microscopy, total internal reflection 

Laser manipulation, ablation and 

microdissection, photoactivation 

3D Image processing and visualization 

Whole tissue imaging – optical coherence 

tomography, endoscopy, whole 

animal fluorescence 

A technical exhibition will accompany 

the Osaka-Awaji conference. 

Welcoming you to the FOM2008 conference 

and exhibition, on behalf of the 

FocusOnMicroscopy society 

Satoshi Kawata, Katsumasa Fujita, Osaka 

University, RIKEN, Wako City, Japan 

Fred Brakenhoff, University of Amsterdam, 

The Netherlands 

The present Focus on Microscopy 

2008 conference incorporates 

21st International Conf. on 3D Image 

Processing in Microscopy 

20th � 

� International Conf. on Confocal 

Microscopy 

Contact: 

Satoshi Kawata 

Katsumasa Fujita 

Osaka, Japan 

fom2008@ap.eng.osaka-u.ac.jp 

Prof. Dr. G.J. Brakenhoff 

Section of Molecular Cytology 

Centre for Advanced Microscopy 

Swammerdam Institute for Life Sciences 

University of Amsterdam, The Netherlands 

Tel.: +31 20 525 5189 

Fax.: +31 20 525 6271 

brakenhoff@science.uva.nl

Accessories for Microscopy 

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disciplines of microscopy. 

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and fast, efficient, friendly service 

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Request a catalogue or visit our website to 

see our extensive range of products. 

www.agarscientific.com 

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E v E n t R E p o R t 

See You Later Alligator 

Traditionally, one of the most important events for presenting new developments in high end microscopy instrumentation is the Microscopy & Microanalysis Congress 

and Fair in the United States. Reason enough for the Imaging & Microscopy team to join this year’s exhibition at the Broward County Convention Center in 

Fort Lauderdale, Florida. In contrast to the very hot temperatures and the incredible humidity outside, the Convention Center provided an appropriate cooled exhibition 

floor. Following the challenge to increase visibility in nanostructures multitude up-to-date products in hardware, software and accessories were offered 

by 100 manufacturers. In this section we would like to take you on our trip through this tradeshow by introducing some products and the people behind them. 

Dr Stefan Scherer, President of Alicona Imaging 

presenting the InfiniteFocus; an optical 3D measurement 

device for quality assurance in the micro- and 

nano range. 

Eric C. Ambrose, Carestream Healths Media Product 

Manager for Molecular Imaging Systems with the In- 

Vivo Imaging System FX. This instrument combines 

high-sensitivity Optical Molecular Imaging and high 

resolution Digital X-ray to deliver anatomical 

localization of molecular and cellular biomarkers. 

Mike Sousa and Nicole Lackey from Evex presenting 

the LN-free violin-shaped QD Detector to be applied 

for spectral acquisition and elemental mapping. 


Alex Vogt (left), President, and Dr Andres Kaech 

(sitting), Scientific Director of Life Science Applications 

of Bal-Tec introducing the HPM 100, a high 

Pressure Freezing work station for cryo fixation. 

Stacie G. Kirsch, Managing Director of Diatome U.S., 

showing the Lynx II; an automated tissue processor 

for histology and microscopy from Electron Microscopy 

Science. 

The desktop electron microscope Phenom with touch 

screen control from FEI Company introduced by JJ 

Blackwood (left), Senior Application Development 

Engineer and Matthew Harris (right), Vice President 

and General Manager NanoBiology Market Division. 

Bruker AXS Microanalysis Marketing Communications 

Manager Stefan Langner in front of the poster 

of HyperMap/PTS, a software interface for fast 

spectral mapping based on “position-tagged 

spectrometry” technique. 

Christine Meehan, Marketing Communication 

Specialist and Mark Massey European Director of 

Sales flanking Edax WDS TEXS HP, a parallel beam 

spectrometer that employs capillary optics enabling 

the spectrometer to have an energy range from 

100 eV to 10 keV. 

President Paul Fischione (right) and Director of Market 

Development Alan Robins (left) from Fischione Instruments 

with their 1040 NanoMill device used to create 

thin specimens needed for advanced transmission 

electron microscopy imaging and analysis.

Huge Images? No Problem 

Introducing Imaris® 6.0 

Interactive Visualization, Quantification, Tracing and Tracking 

Imaris® 6.0 shatters the barriers that cause other 

programs to fail by allowing users to visualize and 

process 3D and 4D microscopy data sets of up to 

30 GB in size. Through constant innovation, Bitplane 

has shaped the way microscopists work with 

images for the past 12 years. The feature-filled 

Imaris® version 6.0 release will be no exception. 

For more information visit: www.bitplane.com 

T h e i m a g e r e v o l u t i o n s t a r t s h e r e . 

Bitplane‘s suite of software modules provides users 

with an opportunity to build a package based on 

their specific needs. Imaris® is available for Windows 

x32 and x64 systems and both Intel and 

PowerPC based Macs. There is no longer a need to 

improvise. Bitplane offers customers a complete 

solution.


Dr Christel Genoud, 3View Product Specialist and 

John Hyun, Marketing Communications Manager 

from Gatan in front of application images derived 

from their 3View-system. An ultra-microtome, stage 

and imaging system, which allows serial block face 

scanning electron microscopy within a variable 

pressure field emission SEM. 

Kenny Witherspoon (right), IXRF Systems Vice 

President of Marketing demonstrating their fully 

automated EDS systems including quantitative analysis, 

high resolution digital imaging, X-ray mapping, 

X-ray linescans, and particle analysis. 

Ian Lamswood (left), Marketing Manager EM 

Products and Ann Korsen (mid), Director of Sales 

and Marketing Ultramicrotomy from Leica Microsystems 

explaining their automatic microwave tissue 

processor for electron microscopy EM AMW to Judi 

Stasko (right), National Animal Disease Center, 

Ames, IA, USA. 

Joel Silfies (left), Senior Applications Manager and 

Marty Whitted (right), Sales Representative Industrial 

Microscopy & Metrology flanking the AZ100 

Multizoom Macro System from Nikon. 


Chris Haig (left), Regional Manager USA Midwest 

and Bill Moore (right), Business Manager of System 

Division Hamamatsu presenting their electron 

multiplier CCD camera ImageEM suitable for high 

dynamic range applications to dim fluorescence in 

living cells. 

Toshiyuki Kanazawa (sitting), Senior SEM Application 

Specialist and Vernon E. Robertson (standing), Field 

Emission SEM Product Manager from Jeol introducing 

the JSM-7500F, an analytical Field Emission SEM 

achieving a resolution of 1.4 nm at 1 kV. 

Claudia Dunphy, Marketing & Communication 

Manager and Brian Graydon, Business Development 

Manager Scientific Division from Lumenera showing 

the Infinity CCD Camera series for colour and monochrome 

images with resolution array from 1,3 to 21 

megapixel. 

President Dr Thomas Moore (left) and Senior 

Mechanical Designer Rocky Kruger (right) from 

Omniprobe surrounding OmniGIS the multiple gas 

injection device for FIB & SEM. 

Michael Dixon, Sales & Business Development 

Manager from Hitachi showing the HD-2700, a 

STEM which is equipped with a spherical aberration 

corrector. 

The VHX digital microscope from Keyence containing 

54 million pixel 3CCD handheld camera presented 

by Thomas Takao (right), Product Sales 

Director and Katz Muta (left), Japanese Account 

Manager. 

Roy Opie (mid), Publisher and Julian Heath (right), 

Head Editor from the Wiley Journal Microscopy & 

Analysis while talking with Debbie Stokes (left) 

from the Royal Microscopical Society. 

The 2 k x 2 k pixel side-mounted TEM CCD camera 

Veleta presented by Heidi Mills from Olympus Soft 

Imaging Solutions Sales Support/Logistics.

Sales Manager Mike Wombell showing Quorum 

Technologies PP2000/PP2000T Cryo-SEM System 

featuring rapid sample freezing, vacuum transfer, 

freeze etching, and sputter coating. 

Jack Vermeulen, Head of Sales and Marketing from 

Ted Pella offering a broad range of Specimen 

Mounts for SEM, including the SEMClip series. 

The World’s highest resolution camera for TEMs (8 k, 

16 µm, 14 bit) TemCam-F816 presented by TVIPS 

President Dr Hans R. Tietz (right) and Dr Matthias 

Stumpf (left), Product Development Manager. 

Sales Representative Erica Byrd from Wiley USA 

offering the broad range of the Publisher’s textbooks 

and journals concerning Microscopy and 

Microanalysis. 

Tim Prusnick, Application and Support Engineer for 

raman products introducing fast chemical imaging 

using StreamLine technology for Renishaw’s inVia 

Raman microscopes 

TESCAN’s FESEM series Mira with a Schottky Field 

Emission electron gun demonstrated by Tony Owen 

(left), and William J. Mershon (right), Application 

Manager. 

Martin Klein President of Visitec Microtechnik 

introducing the large chamber scanning electron 

microscope Mira used for non-destructive testing. 

Doug D’Arcy, USA Sales Representative from Witec 

introducing the Alpha 300 series of scanning nearfield 

optical microscopes (SNOM) that combine 

SNOM, confocal microscopy and AFM in single 

instruments. 


Vice President Eugene E. Rodek from SPI Supplies 

presenting the Osmium Plasma Coater for SEM and 

FESEM samples. 

Fran Ebert, Senior Marketing Communication 

Specialist Scientific Instruments and David Rohde, 

Product Manager X-Ray Microanalysis Scientific 

Instruments showing the Thermo Fisher Scientific 

UltraDry Silicon Drift Detector. 

Confocal Product Manager Layla Billowitz from VTI 

Visitech in front of the VT Eye poster. Using Acousto 

Optical Deflector (AOD) technology this confocal 

point-scanner is designed for Multi-colour acquisition 

for FRET, FRAP and FLIP applications. 

Nick Economou (left), Board Member from ALIS, a 

subsidiary from Carl Zeiss AG and Dr. Peter Fruhstorfer 

(right) Director International Sales, Service & 

Business Development from Carl Zeiss NTS presenting 

the Helium ion microscope Orion. 


E v E n t r E p o r t 

At the Region of Former Ironworks 

Microscopy Conference (MC) 2007 

At the university campus of Saarbrücken, the capital of Saarland, Germany’s smallest federal state, well 

known in the past for its iron producing industry, with the world cultural heritage “Völklinger Hütte”, the 

33 rd conference of the German Society of Electron Microscopy (DGE) took place between 2 nd and 7 th of September. 

In total 450 delegates were offered to attend 

21 oral sessions consisting of 130 

talks, four poster sessions with 120 contributions, 

one technical lecture including 

16 exhibitors presentations and several 

workshops. The exhibition floor, 

connecting the lecture halls, areas for 

poster sessions, coffee bar and registration, 

presented 30 companies introduc 

ing their product novelties in Hardware, 

Software and consumables to the audience. 

Following the welcome and followed 

by the first plenary lecture one of the 

most prestigious Prizes for outstanding 

scientific developments in electron microscopy, 

the Ernst Ruska Prize was 

awarded this year by Paul Walter (Ulm, 

Fig. 1: Ernst Ruska Prize 2007 Awarding. Paul A. Midgley, Hiroshi Jinnai, Richard J. Spontak, and Paul 

Walter. 


World cultural heritage “Völklinger Hütte” 

Germany) president of the DFE to Richard 

J. Spontak (Berkeley, USA), Hiroshi 

Jinnai (Kyoto, Japan) and Paul A. Midgley 

(Cambridge, UK) (Fig. 1). The Jury 

honoured the three Scientists and MC 

2007 plenary lecturers for their work on 

novel and quantitative uses of electron 

tomography in the 3D study of nanostructured 

materials. Further plenary 

talks were given by: 

� Andrew Bleloch, Cambridge, UK (Applications 

of Aberration Corrected 

STEM: Imaging and EELS) 

� Hans Cerva, Munich, Germany (Application 

of Selected Electron Microscopy 

Methods to Materials Analysis Problems 

from an Industrial Environment) 

� Marek Cyrklaff, Martinsried, Germany 

(CryoTomography of Whole Cells) 

� Jacques Dubochet, Lausanne, Switzerland 

(Cryoelectron Microscopy of 

Vitreous Sections) 

� Gareth Griffiths, Heidelberg, Germany 

(State of the Art Microscopy for Cell 

Biology with a Focus on Mycobacteria 

and Phagocytosis) 

� Othmar Marti, Ulm, Germany (Nanomechanical 

Characterization from 

� 

Living Cells to the Cytoskeleton) 

Andreas Thust, Juelich, Germany 

(HighResolution Transmission Electron 

Microscopy Entering the Sub 

Angstrom Era)

� 

Roger Wepf, Zurich, Switzerland (Correlative 

Microscopy in Focus: Shortcuts, 

Prerequisites and Future Needs 

in Diagnostic and Analytical Biomedical 

Structure Research) 

For the social programme delegates were 

first invited on September 4 th to the FEI 

Company and Carl Zeiss SMT customers 

evening event at the Congress Centre 

Saarbrücken and “Völklinger Hütte”, respectively. 

The following evening was reserved 

for the congress dinner at the 

“Alte Schmelze” (eng. Old Smeltery) in 

St. Ingbert a further monument to former 

ironworks close to the venue. But before 

the participants could enjoy the delicacies 

of the region, a fantastic organ performance 

in St. Hildegards Church took 

the audience on a trip through compositions 

of Johann Sebastian Bach, Charles 

Marie Widor, Louis Vierne, Wolfgang 

Amadeus Mozart, and Naji Hakim. 

To all who enjoyed this successful and 

very informative event hosted by the 

DGE, the next Microscopy Conference coorganised 

by them is the European Microscopy 

Congress in Aachen, Germany 

2008 and the “Dreiländertagung” in 

Measurement of steep flanks and complex geometries 

True color information registered to 3D data 

Traceable results even on highly sculptured surfaces 

Usable on highly reflective and 

inhomogeneous surfaces 

Highest resolution across measurement 

areas of several mm 

Comprehensive roughness 

measurement capabilities, conforming 

to the latest ISO standards 

Usable in the lab and as 

Inline measurement sensor 

Fig, 2: Heads of local organizing committee: Pedro Mesters-Ventura and Uwe Hartmann 

Graz, Austria 2009. Hopefully see you all 

there! 

Contacts: 

Prof. Dr. Uwe Hartmann 

Institute of Applied Physics 

University of Saarland, Saarbrücken, Germany 

Tel.: +49 681 302 3798 

Fax: +49 681 302 3790 

u.hartmann@mx.uni-saarland.de 

OPTICAL MEASUREMENT AND INSPECTION 

E v E n t r E p o r t 

Prof. Dr. Pedro Mestres-Ventura 

Institut für Anatomie und Zellbiologie 

Zentrum für Elektronenmikroskopie 

Universitätsklinikum, Homburg/Saar, Germany 

Tel.: +49 6841 1626141 

Fax: +49 6841 1626293 

anpmes@uniklinikum-saarland.de 

www.dge-homepage.de 

www.uni-saarland.de/mc2007 

Alicona Imaging GmbH 

headquarters 

Austria, Teslastraße 8 

A-8074 Grambach/Graz 

phone: +43 316 4000-700 

fax: +43 316 4000-711 

e-mail: info@alicona.com 


E l E c t r o n M i c r o s c o p y 

Enabling 360 degree TEM/STEM 

of Nanoparticles 

Functionalized Holders for 3D Electron Microscopy 

A new protocol for functionalizing sample holders has been developed for 360° TEM/STEM observation of 

nanoparticles and nanostructures. The three step process includes FIB milling to customize sample stub geometry, 

thin film deposition for substrate selection and subsequent chemical functionalization for nanoparticle 

adhesion. This protocol was used to determine the morphology and local material properties of individual 

Au/SiO 2 core-shell nanoparticles used in a DNA detection assay. 

Nanoscience Imaging & Spectroscopy 

Tools for characterizing nanoparticles 

with complex three-dimensional (3D) 

geometrics are required to further nanoscience 

research and enable the continuing 

development of viable nanotechnologies. 

In this manuscript we report a 

protocol which combines focused ion 

beam milling (FIB), thin film deposition 

and solution chemistry to customize a rotation 

holder [1] for 3D structural and 

chemical analysis of nanoparticles with 

transmission, and scanning transmission 

electron microscopy (TEM and STEM). 

By using this protocol the geometry, sub- 

Fig. 1: A. A novel sample 

holder allowing for 360° 

rotation in a TEM/STEM [6]. 

The location of the sample 

to be analyzed is indicated 

by the arrow. B. Illustrated 

geometry of rotation 

holder sample, incident 

electron beam and analytical 

detectors. 


strate material and chemistry of the 

STEM or TEM rotation holder can be optimized 

for the nanoparticle system of interest. 

To illustrate this protocol, 3D 

STEM imaging rotation holders were 

modified for analysis of core-shell nanoparticles 

used in a DNA detection assay. 

The details of the Au/SiO 2 core-shell nanoparticle 

synthesis and resulting increased 

DNA detection efficiency are reported 

elsewhere [2]. 

Electron microscopy techniques are 

widely used to characterize morphology 

and electronic properties of materials at 

the near-atomic scale but are not well 

suited for visualizing complex structures 

Keywords: 

nanoscience, nanoanalysis, 3D STEM, functionalized 

holder, bionanotechnology 

in 3D [3, 4]. A nanoparticle must be either 

tilted or rotated with respect to the 

electron beam to enable multiazimuthal 

observation and 3D analysis. This requires 

a “nanoparticle holder” which can 

be either rotated or titled in a TEM or 

STEM. A 3D rotation holder, with sample 

location indicated by the arrow in Figure 

1A, allows analysis from a full 360 degrees 

as opposed to traditional tomographic 

tilt-series based analysis which 

suffers from the missing wedge [5]. Unfortunately 

the scanning electron microscopy 

(SEM) and focused ion beam (FIB) 

based nano-manipulation techniques traditionally 

used for attaching samples to 

such rotation holders are not well suited 

for mounting individual nanoparticles 

[6–8]. Due to the small particle size and 

the practical difficulties of attaching and 

depositing individual particles with nanomanipulators 

a new technique is 

needed. 

To enable 3D STEM or TEM analysis 

of nanoparticles a three step process was 

developed to functionalize the brass needle 

stubs used in the rotation holder. In

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2007


Fig. 2: A. Brass sample stub before modification. 

B. Sample stub after FIB modification. 

the first step a FIB is used to customize 

the geometry of a brass needle stub. 

Then a ion beam sputter-deposition system 

(Gatan 682 PECS) is used to coat the 

surface of the needle stub with a substrate 

material ‘matched’ to the nanoparticle 

of interest. In the final step, the 

surface chemistry of the stub or nanoparticle 

is optimized to control the adhesion 

between these two surfaces. 

To demonstrate this protocol, functionalized 

rotation holders were created 

to enable 3D STEM imaging and analysis 

of core-shell nanoparticles used in a DNA 

detection assay [2]. The functionalized 

holders were used to rotate individual 

Au/SiO 2 nanoparticles a full 360° with respect 

to the electron beam of the STEM 

as illustrated in figure 1B. High angular 

annular darkfield (HAADF or Z-contrast 

(ZC)), bright-field (BF) and secondary 

electron (SE) STEM signals were recorded 

at regular angular intervals to 

study the 3D morphology and composition 

of the nanoparticle. Electron energy 

loss spectroscopy (EELS) spectrum images 

[9] were recorded at several angular 

intervals to map optoelectronic and 

material properties of the core-shell nanoparticle. 

Functionalized Holder Preparation 

The sample stub used for 360° rotation in 

a STEM/TEM is composed of brass and 

measures 3 mm in length along the rotation 

axis, ~300 um in diameter and is tapered 

to a point at the tip with a 30 um diameter 

flat (fig. 2A). The flat at the apex 

is well suited for attaching large micronsized 

samples with FIB or SEM based 

nano-manipulation techniques. However 

these techniques are not well suited for 

nanoparticles and the roughness of the 

30 um flat can easily obscure the nanoparticles 

from the electron beam at certain 

angles of rotation. To improve the 

sample stub geometry, a FIB was used to 

customize the shape of the holder apex. 

For holding the 100 nm core-shell nanoparticles 

used in the DNA assay, the 

30 um flat was FIB milled into a 5 x 5 array 

of pillars. This array of pillars minimizes 

potential shadowing from the substrate 

during rotation and increases the 

number of potential sites well suited for 

TEM/STEM analysis of individual nanoparticles. 

The apex of the rotation holder 

before and after FIB modification is 

shown in figures 2A and B. A variety of 

custom holder geometries can created 

using FIB modification and application 

specific solutions are only limited by creativity. 

The nanoparticle system of interest 

dictates which substrate materials are 

best suited for controlling of the adhesion 

and disperion of the nanoparticles. 

Fortunately there are a variety of thin 

film deposition techniques and materials 

which can be used to coat the FIB modified 

brass sample stub with a well 

‚matched‘ substrate material. For the silica 

core-shell particles, a 10–20 nm thick 

silica film was deposited on the stub using 

a Gatan 682 PECS ion beam sputter 

coater. By coating the stub with silica, solution 

chemistry could be varied in a 

predicatible way to control the adhesion 

and dispersion of the core-shell particles 

used in the DNA assay. 

The nanoparticles were deposited 

onto the coated and FIB modified sample 

holder by placing a 20 uL droplet of the 

Fig. 3: A. and B. STEM SE 

and ZC micrographs 

showing many nanoparticles 

adhered onto a 

functionalized sample 

holder. The large number 

of nanoparticles make 

STEM/STEM analysis of an 

individual nanoparticle 

difficult. 

nanoparticles in solution onto the apex of 

the customized sample holder. Several 

nanoparticle solution chemistries, from 

Ref. [2], were tested to determine which 

solution resulted in the best adhesion, 

dispersion and produced minimal beam 

induced carbon deposition. For some solutions, 

several layers of nanoparticles 

adhered to apex, or analysis region of 

the pillars as shown in figures 3A and B. 

For other solutions, the nanoparticlesubstrate 

adhesion was reduced, and individual 

nanoparticles were found near 

the apex of several pillars as shown in 

Figure 4A. The effect of solution chemistry 

on the surface charge of the nanoparticle 

and substrate silica is thought to explain 

these differences in adhesion and 

dispersion, though this has not yet been 

independently verified. This example of 

functionalizing sample holders for a particular 

nanoparticle system can be 

adapted for many different types of nanoscience 

applications by customizing 

the holder geometry, the composition of 

the thin film coating and/or by modifying 

the surface chemistry of the sample stub 

for the system of interest. 

360° Rotation STEM Imaging 

The ability to rotate individual nanoparticles 

360° with respect to the incident electron 

beam greatly simplifies nanoscale 

structural characterization. The micrographs 

of figure 4A-D show a single silica 

core-shell nanoparticle which adhered 

near the apex of a functionalized 3D 

holder. The size, shape, surface features 

and chemical composition of this core-sell 

nanoparticle was mapped in 3D by acquiring 

STEM ZC, BF, and SE images 

Fig. 4: A. Functionalized sample holder with individual nanoparticles adheared near the apex, well suited for STEM/TEM analysis. B. Single core-shell nanoparticle 

(arrow) attached to apex of functionalized stub (STEM BF). C. STEM SE. D. STEM ZC. 

26 • G.I.T. Imaging & Microscopy 4/2007

while rotating this nanoparticle a full 

360°. Z-contrast images from another 

core-shell nanoparticle, containing two 

Au cores, acquired at 20° intervals are 

shown in figure 5. The dual core nanoparticle 

shown in figure 5A could have been 

mistaken for a single core nanoparticle 

(fig. 5F) if rotation was not possible. Using 

this same approach, images of yet another 

nanoparticle were acquired in 5° intervals 

and then used to create animations and 

tomographic reconstruction (to be published). 

EELS spectrum images were also 

acquired at several angular increments to 

determine the optoelectronic properties 

of the sol-gel silica shell. The EELS data 

showed the silica to have a band gap of 

8.9 eV and a chemical composition ratio 

of 1:2 for silicon to oxygen (to be published), 

similar values to those reported in 

the literature for evaporated microelectronics 

grade silica [9, 10]. 

Summary 

A new electron microscopy protocol has 

been developed for functionalizing sample 

holders to enable 360° TEM/STEM 

nanoanalysis of individual nanoparticles. 

This protocol can be easily adapted for a 

wide variety of nanoparticle systems, and 

was demonstrated by preparing and then 

using functionalized holders for the analysis 

of core-shell nanoparticles used in a 

DNA detection assay. The functionalized 

holders were used for 360° STEM/EELS 

imaging and analysis of individual nanoparticles 

cantilevered over vacuum. Local 

material properties responsible for 

the increased DNA detection efficiency of 

these core-shell nanoparticles were 

mapped in 3D by combining information 

taken at regular angular intervals. By 

controlling the sample holder‘s geometry, 

coating and surface chemistry this 

three-step protocol can be adapted to enable 

the 360° TEM/STEM analysis of 

many nanoparticle systems. 


Fig. 5: STEM ZC micrograph series showing an individual nanoparticle with a double 

Au core at different angles of rotation. A. 0° rotation. B. 20° rotation. C. 40° rotation. 

D. 60° rotation. E. 80° rotation. F. 100° rotation. 

Acknowledgments 

We would like to thank Drs. M. Cerruti 

and S. Franzen from North Carolina State 

University’s Dept. of Chemistry for synthesis 

of the core-shell nanoparticles and 

supporting our attempt to elicit the materials 

properties responsible for increased 

DNA detection efficiency. 

References: 

[1] Koguchi, M., et al., J. Of Electron Microscopy 

50(3), 235–241 (2001). 

[2] Cerruti, M. G., et al., Analytical Chemistry 78, 

3282–3288 (2006). 

[3] deJong, K., et al., Chem. Phys. Chem. 3, 776– 

780 (2002). 

[4] Holzer, L., et al., J. of Microscopy 216, 84–95 

(2004). 

[5] Kawase, N., et al., Ultramicroscopy 107, 8–15 

(2007). 

[6] Kamino, T., et al., J. Of Electron Microscopy 

53(6), 583–588 (2004). 

[7] Kamino, T., et al., J. Of Electron Microscopy 

53(5), 563–566 (2004). 

[8] Ozasa, K., et al., Ultramicroscopy 101, 55–61 

(2004). 

[9] Barranco, A., et al., J. of Applied Physics 

97(11), (2005). 

[10] Keranen, J., et al., J. of Applied Physics 

84(12), 6827–6831 (1998). 

Contact: 

Dr. Konrad F. Jarausch 

Product Development Manager 

Hitachi High Technologies America, Inc. 

Nanotechnology Systems Division 

Pleasanton, CA, USA 

Tel.: +1 925 218 2830 

konrad.jarausch@hitachi-hta.com 

www.hitachi-hta.com 

Dr. Donovan N. Leonard 

Research Assistant Professor 

Appalachian State University 

Dept. of Physics & Astronomy 

Boone, NC, USA 

leonarddn@appstate.edu 

I II 

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Verifying Engineering at the Nanoscale 

Electron Microscopy and Drug Delivery 

Packaging drugs and genes into nanoparticles enables drug or gene biodistribution to be 

favourably altered, with an ultimate therapeutic benefit [1–3]. To acquire such control on 

the in vivo fate of drugs and genes requires that such particles be precision engineered and 

electron microscopy is one of the techniques used to visualise and confirm the results of 

such engineering. 

Methods 

Pharmaceutical nanosystems in our laboratory 

have been prepared from the self 

assembly of: a) comb type polymers [4–6] 

and b) dendrimers [7, 8] (fig. 1). By exercising 

control on the chemistry of these 

self assembling molecules, a variety of 

functional nanosystems may be prepared. 

Applying physical characterisation techniques 

including imaging to the resulting 

self assemblies and studying their in vivo 

behaviour ultimately enables robust correlations 

to be made between polymer 

chemistry, nature of the self assembly 

and drug delivery performance. 

Results 


Self assembling comb type polymers have 

been prepared from carbohydrates [2, 

5], polyamino acids [4] and polyamines 

[1, 6] and altering the level of hydropho 


Fig. 1: Amphiphilic polymers and 

dendrimers used to make self 

assembling drug and gene delivery 

systems respectively. 

Fig. 2: Polymeric dense nanoparticles, 

vesicles and micelles 

produced by cetyl 

poly(ethylenimine). Increasing 

the level of hydrophobic substitution 

(cetyl chains) gives rise, in 

turn, to polymeric micelles, 

polymeric bilayer vesicles and 

dense nanoparticles. The polymer 

coat is clearly visible on the 

surface of the polymeric vesicles 

(arrowed). 

Ijeoma F. Uchegbu

Electron microscopes from Hitachi. 

www.hitachi-hitec-uk.com 

You provide 

the challenges 

- we’ll help you 

find the answers. 

Hitachi High-Technologies Corporation 

Tel: +44 (0) 800 316 1500


Fig. 3: The linear relationship between the level of cetyl substitution on cetyl 

poly(ethylenimine) and polymeric bilayer vesicle size. Vesicles were prepared 

by probe sonicating cetyl poly(ethylenimine) (4 mg mL –1 ) and cholesterol 

(2 mg mL –1 ). 

Fig. 4: Pharmacodynamic activity (sleep time, mean ± s.d., n ≥ 4) of propofol formulations after tail 

vein dosing to male MF1 mice. Mice were dosed as described below. 0.2 mg animals were dosed with 

0.2 mg propofol in a 100 µL volume administered as either propofol emulsion (10 mg mL –1 , Fresenius, 

Germany) diluted to 2 mg mL –1 with phosphate buffered saline (PBS, pH = 7.4) or a filtered amphiphilic 

chitosan formulation (amphiphilic chitosan – 5 mg mL –1 , propofol – 1.9 mg mL –1 in PBS – pH 

= 7.4). 0.2 UF animals were dosed with 0.2 mg propofol in a 100 µL volume administered as either 

propofol emulsion (10 mg mL –1 , Diprivan) diluted to 2 mg mL –1 in glycerol – 0.24M or an unfiltered 

amphiphilic chitosan formulation (amphiphilic chitosan – 5 mg mL –1 , propofol – 1.9 mg mL –1 , lecithin - 

2 mg mL –1 , in glycerol – 0.24 M). 0.4 mg animals were dosed with 0.4 mg of propofol in a 100 µL 

volume administered as either Diprivan diluted in glycerol (0.24 M – TEM of formulation shown in 

inset i) to a concentration of 4 mg mL –1 or a filtered amphiphilic chitosan formulation of propofol 

(amphiphilic chitosan – 5 mg mL –1 , propofol – 4.2 mg mL –1 , lecithin - 2 mg mL –1 , in glycerol – 0.24 M – 

TEM of formulation shown in inset ii). 0.5 mg animals were dosed with 0.5 mg propofol administered 

in a 50 µL volume as either Diprivan (10 mg mL –1 ) or propofol emulsion (Fresenius, 10 mg mL –1 ). A loss 

of righting reflex time was only observed in animals receiving a low dose of the commercial emulsion 

formulations (0.37 ± 0.19 min in the 0.2 Fresenius animals). No sleep times were recorded in animals 

receiving the polymer alone. * = statistically significantly different (p < 0.05). 

bic substitution alters the nature of the 

self assembly (fig. 2). Additionally we 

have identified two chemical features of 

these amphiphilic polymers which control 

nanosystem size (and ultimately nanosystem 

biodistribution) and these are 

the molecular weight of the polymer [5] 

and the level of hydrophobic substitution 


of the polymer [6], both of which are positively 

and linearly correlated with nanosystem 

size (e.g. fig. 3). 

Carbohydrate micellar clusters, in 

which one micelle is linked to another, 

presumably via trailing polymer chains 

are able to form nanoparticles in the 

presence of hydrophobic drugs [2]. These 

drug loaded nanoparticles increase drug 

bioavailability across the blood brain 

barrier by up to ten fold when compared 

to the current state of the art commercial 

emulsion system (fig. 4) [2]. Amphiphilic 

polyamine drug loaded nanoparticles increase 

drug absorption via the oral route 

by up to three fold when compared to the 

drug suspension in water [1]. Additionally 

polypropylenimine dendrimers form 

colloidal particles with DNA which are 

able to transfer an antiproliferative 

gene, the tumour necrosis alpha gene, 

into mouse tumours and produce a 100 % 

response in all tumours studied, due in 

part to the additional antiproliferative 

activity of the dendrimer [3]. 

Conclusions 

It is possible to correlate polymer chemistry 

with the nature of the resulting self 

assembly and ultimately link a range of 

morphologically distinct nanosize self 

assemblies with specific drug delivery 

function. With these nanosystems, a ten 

fold increase in drug activity may be obtained 

[2] and an effective anticancer 

gene medicine is achievable [3]. 

References: 

[1] Cheng, W., et al., Biomacromolecules, 7, 

1509–1520 (2006) 

[2] Qu, X., et al., Biomacromolecules, 7, 3452– 

3459 (2006) 

[3] Dufes, C., et al., Cancer Research, 65, 8079– 

8084 (2005) 

[4] Wang, W., et al., Langmuir, 16, 7859–7866 

(2000) 

[5] Wang, W., et al., Langmuir, 17, 631–636 

(2001) 

[6] Wang, W., et al., Macromolecules, 37, 9114– 

9122 (2004) 

[7] Zinselmeyer, B. H., et al., Pharmaceutical Research, 

19, 960–967 (2002) 

[8] Schätzlein, A. G., et al., J. Control. Rel., 101, 

247–258 (2005) 

Contact: 

Ijeoma F. Uchegbu 

University of London, United Kingdom 

Department of Pharmaceutics, School of Pharmacy 

Tel.: +44 20 7753 5997 

Fax: +44 20 7753 5946 

ijeoma.f.uchegbu@pharmacy.ac.uk 

www.pharmacy.ac.uk

Cryo Electron Tomography 

Unique Capability for Structural Biology Investigations 

Cryo electron tomography’s (CET) ability to visualize 

three dimensional biological structures – ranging 

in size from molecular to cellular – fills a critical 

gap between techniques with atomic resolution, 

such as x-ray diffraction (XRD) and nuclear magnetic 

resonance (NMR), and conventional light microscopy. 

However, it is CET’s ability to investigate 

biological structures in their unperturbed, native 

context that makes it an indispensable tool in the 

currently exploding field of structural biology. 

Introduction 

The recent decoding of the human 

genome, and the realization that we can 

now read the entire blueprint of the 

vastly complex biological machine that is 

a living organism-human or any other, 

has had a tremendous symbolic and 

practical impact on the public consciousness 

in general, and the scientific community 

in particular. Since that time, 

biologists have focused increasing attention 

on understanding the structure and 

function of the machine’s components – 

the proteins encoded by the genetic blueprint. 

Fig. 1: The image shows a surface rendered 

representation of a segment of the nucleus from 

a eukaryotic organism (Dictyostelium discoideum). 

The nuclear membranes are in blue, and 

the Nuclear Pore Complexes (NPCs) are in red. 

The NPCs are 125 nm in diameter. Structures of 

individual NPCs have been substituted by the 

averaged structure. Images courtesy of: Martin 

Beck, Friedrich Förster, Mary Ecke, Jürgen M. 

Plitzko, Frauke Melchior, Günther Gerisch, Wolfgang 

Baumeister and Ohad Medalia, Max- 

Planck-Institute of Biochemistry, Martinsried, 

Germany. We gratefully acknowledge help with 

the design and visualization by Julio Ortiz. Details 

published in Science 306:1387–1390, 2004. 

The Strength of CET in 

Structural Biology 

One approach, call it brute force, in many 

ways analogous to the approach taken in 

the decoding effort itself, simply seeks to 

determine the structure of each of the 

large but finite number of individual proteins 

that constitute the entire proteome, 

perhaps one to two million in a human. 

Though this basic structural information 

is certainly fundamental to a complete 

understanding, it is only the lowest level 

in the hierarchy of structural complexity. 

Few proteins work alone. Most function 

in concert with others as components of 

supramolecular complexes that may be 

persistent or quite transient. At a still 

higher level, the cellular context in which 

these complexes function cannot be 

regarded as a simple sack of cytoplasm 

filled with randomly colliding complexes. 

It is itself a highly structured environment 

throughout which components are 

manufactured, regulated, transported 

and consumed among at myriad functional 

localities. 

Clearly, the operation of the machine 

can be more easily understood by observations 

of the assembled components. 

Compare it to building a watch. We have 

the parts list, the genetic code. With sufficient 

effort we may eventually describe 

each component in atomic detail. But 

how much easier is it to comprehend the 

workings of the entire machine if we can 

look at a fully assembled watch. This is 

the strength of CET in structural biology. 

Alone among experimental techniques, it 

permits observations of fully assembled 

biological machines from the macromolecular 

cogs and pulleys to the cellular 


systems for synthesis and distribution (as 

shown in figures 1 – 3). 

Vitrification Essential for CET 

As its name implies, cryo electron tomography 

uses electron images of frozen 

samples to construct a three dimensional 

model of the imaged volume. The images, 

provided by a high resolution transmission 

electron microscope (TEM), are two 

dimensional projections acquired as the 

sample is rotated incrementally about an 

axis perpendicular to the viewing direction. 

A computer creates the 3D model 

from the projections with reconstruction 

techniques familiar to most of us from 

their use in medical imaging applications 

such as CAT scans and MRI. Key to the 

value of CET is its use of a cryogenic 

freezing process known as vitrification. 

The process freezes the hydrated sample 

so rapidly that water molecules do not 

crystallize, instead forming an amorphous 

solid (vitreous ice) that does little 

or no damage to delicate molecular 

structures. 

CET can resolve structures down to a 

few nanometers, sufficient for tertiary 

and quaternary protein morphology. In 

hybrid approaches to structural analysis, 

atomic scale structures determined by 

XRD and NMR are fitted to larger scale 

CET models to enhance the level of structural 

detail. CET samples may be pristine 

biological material, or selectively stained 

with electron dense materials to emphasize 

particular features. More specific 

emphasis to particular proteins or 

domains can be achieved with immuno 

labeling techniques using gold or other 

high contrast nanoparticles. 



Fig. 2: The image shows a 3D rendering of a 

mitochondrion from a mouse neuron. Tomograms 

obtained from thick mouse brain sections 

were selectively segmented to highlight the 

mitochondrial membranes (in green), cristae (in 

blue) and surrounding neurofilaments and microtubules 

(in grey). 

Investigation of Morphological 

Transformations 

The ability of XRD and NMR to achieve 

atomic resolution is due in large part to 

its use of a composite signal acquired 

from a collection of presumably identical 

structures. In contrast, CET examines an 

individual structure. Since the sample is 

quite literally frozen in time, CET can 

distinguish morphological differences 

among structures with identical atomic 

compositions, such as protein molecules, 

with identical peptide sequences. By 

examining a collection of frozen molecular 

instances, CET can be used to investigate 

morphological transformations that 


occur as a result of interactions, the 

dynamic behavior of flexible proteins, 

the formation of transient complexes in 

signaling pathways, and more. 

CET eliminates XRD’s requirement for 

crystalline samples, avoiding the possibility 

of structural distortions induced by 

crystallization, and allowing the examination 

of virtually any specimen, including 

flexible and insoluble proteins that 

are notoriously difficult to crystallize. It 

also does not suffer the increasing difficulty 

of NMR analysis with larger molecular 

size. 

CET does face at least two significant 

challenges. Because the signal originates 

from a single molecule it is weak relative 

to random variations in its intensity (shot 

noise). The delicate nature of molecular 

structures and the high energy of the 

interrogating radiation do not allow 

increases in beam intensity or exposure 

time without inflicting significant damage 

in the sample. Careful dose management 

and automated acquisition procedures 

can help, but this remains a 

fundamental limitation. 

Correlative Microscopy Increasingly 

Important 

The second issue relates to the crowded 

environment of the cell. We would like to 

look at distinct structures clearly contrasted 

against a featureless background. 

However, in an intact biological system 

there is no empty space and little fundamental 

difference between the atoms 

that compose the structures of interest 

and those that surround it. It is rather 

like looking into a very big bag of marbles, 

or perhaps like looking for specific 

configurations of bubbles in a bucket of 

foam. There are techniques that reduce 

the difficulty. We have mentioned staining 

and immuno labeling. Another area 

of growing interest is correlative microscopy, 

using an optical technique such as 

Fig. 3: Reovirus-Polymerase. Cross-section 

of a reovirus shows features down to 7.6- 

Ångström resolution, a scale that reveals 

the inner features of the viral particle. 

Visible for the first time within the virus 

are several tiny ‘factories’, shown here in 

red, which convert raw materials from a 

victim cell’s interior into RNA messages 

instructing the cell to begin manufacturing 

more viruses. Photo by Purdue University/ 

Department of Biology, sample courtesy of 

Xing Zhang, Stephen B. Walker, Paul R. 

Chipman, Timothy S. Baker, Department of 

Biological Sciences, Purdue University and 

Max L. Nibert, Department of Microbiology 

and Molecular Genetics, Harvard 

Medical School. 

fluorescence microscopy to locate labeled 

structures for subsequent high resolution 

CET analysis. Finally, the digital 

nature of tomographic data lends itself 

well to computational search and fit routines 

using previously defined templates 

to locate specific structures within the 

sample volume. 

TEM technology has advanced dramatically 

in recent years. Perhaps the 

greatest development has been the 

incorporation of aberration correctors, 

which has pushed image resolution well 

below the Angstrom barrier – 0.5 Angstrom 

resolution was just announced by 

the TEAM (Transmission Electron Aberration-corrected 

Microscope) project, a 

joint effort by the U.S. Department of Energy, 

FEI Company and CEOS GmbBH. 

Improvements in TEM 

A significant contribution to improved 

performance has also come from better 

instrument stability – mechanical, electronic 

and environmental. Sophisticated 

automation at all levels from setup and 

alignment, through operation, data 

acquisition, and sample preparation has 

made TEM much faster, easier, more 

repeatable and more reliable. Some very 

interesting progress has been made in 

sample preparation. FEI’s Vitrobot provides 

automatic vitrification fluid suspensions 

on a TEM sample grid. Focused ion 

beam (FIB) based preparation procedures 

have greatly improved the ease, 

speed, and reliability of preparing thin 

samples from bulk specimens, including 

frozen material. Another area of interest 

is the FIB based preparation of cylindrical 

tomography samples that permit the 

acquisition of 2D images through a complete, 

360 degree range of rotation. 

The sheer volume of information 

potentially available in a high resolution 

tomogram is mind boggling. Theoretically, 

one could capture the entire proteome 

of a cell, including all of its functional 

complexes and higher level 

structures. As one researcher put it, one 

good tomogram can make a whole career 

– or perhaps several. With this kind of 

potential, there is little doubt that CET 

will play a critical role in the future of 

structural biology. 

Contact: 

Matthew Harris 

Vice President NanoBiology Market Division 

FEI Company 

Eindhoven, NL 

Tel.: +31 40 23 56184 

Fax: +31 40 23 56634 

matthew.harris@fei.com 

www.fei.com

SCANNING SECTION

Ultrafast Confocal Raman Imaging 

Acquiring Spectra in a Few Milliseconds with Improved Sensitivity 

In Confocal Raman imaging the acquisition time for 

one Raman spectrum is a crucial value, as it influences 

the acquisition time of the image which typically 

consists of tens of thousands of Raman spectra. 

This article describes how the use of a 

spectroscopic EMCCD as the detector can significantly 

reduce the acquisition time down to a few 

milliseconds per spectrum, as well as tremendously 

improve sensitivity. 

Introduction 

S c a n n i n g S e c t i o n 

In confocal Raman imaging, as in the 

WITec alpha300 R system which was 

used for the experiment described here, 

only light from the image focal plane can 

reach the detector, which strongly increases 

image contrast and slightly increases 

resolution. Special filters are 

used to suppress the reflected laser light 

while enabling the Raman scattered light 

to be detected with a spectrometer/CCD 

camera combination. To obtain an image, 

thousands of spectra are acquired in 

a very short time with typically less than 

100 ms integration time per spectrum 

using a normal CCD camera. In order to 

further improve the overall sensitivity of 

the system a EM-CCD can be used. 

Spectroscopic EMCCD 

An electron multiplying CCD (EMCCD) is 

a normal CCD with an additional readout 

register which is driven with a much 

higher clock voltage than a normal CCD 

readout register. Due to this high clock 

voltage, an electron multiplication 

through impact ionization is achieved 

with an adjustable total amplification of 

the signal of up to 1000 times. With this 

setup, it is always possible to amplify the 

signal above the readout noise so that 

the S/N ratio is always limited by the 

Poisson noise of the signal, even if a very 

fast readout amplifier is used. As an example, 

a 1600 x 200 pixel EMCCD with a 

2.5 MHz readout amplifier, as used for 

the experiments in this article, can be 

read out in only 1.7 ms. 


Fig. 1: a) Confocal Raman Image of a PS-PMMA blend acquired with the WITec Ultrafast Raman Imaging 

Option. 120 x 120 pixel = 14,400 spectra. Acquisition time for one spectrum: 2,3 milliseconds; total 

acquisition time for the image 67 seconds. Red: PS; green: PMMA. b) Corresponding Raman spectra 

The following calculations show the 

improvement in S/N that can be expected 

for different signals. It is assumed that 

the quantum efficiency (QE) of the CCD is 

90 % and that the amplification of the 

signal is set to a value at which one A/D 

count equals the number of electrons of 

the readout noise (1 A/D count = 30 electrons 

for a 2.5 MHz readout amplifier). 

If 100 photons fall on a CCD pixel in a 

given integration time, 90 electrons will 

be generated and converted to 3 A/D 

counts. The readout noise will be 1 A/D 

count and the Poisson noise will be 9.5, 

which is approximately 0.3 A/D counts. 

With these numbers, the S/N ratio is 

about 2.6. 

In an EMCCD, the signal will be multiplied 

by the electron gain factor which 

can be as high as 1,000. A smaller amplification 

factor would generally be used, 

but for the calculation it does not make a 

difference. 90 electrons will be amplified 

to 90,000 electrons resulting in 3,000 A/ 

D counts. The Poisson noise is 9,500 electrons 

which translates to 317 counts, 

while the 1 count readout noise is completely 

negligible. S/N is 9.5, which is an 

improvement of a factor of 3.6. 

If the signal is only 10 photons, this 

will result in a signal of only 0.3 counts 

for a normal CCD. Poisson noise can be 

neglected in this case. With 1 count readout 

noise, S/N is 0.3, hardly a detectable 

signal. 

For an EMCCD, the signal is 333 

counts and Poisson noise is 100 counts 

which gives a S/N of 3.3, an improvement 

of 11 times over a normal CCD. 

In reality the electron multiplying 

process itself adds an additional, so 

called excess noise factor of about 1.4, so 

that the real improvements in S/N are reduced 

to 2.6 and 7.9 respectively for the 

above examples. 

For higher signals, in which the signal 

intensity is no longer readout limited, the 

excess noise factor of the EM process reduces 

the S/N ratio of an EMCCD to below 

that of a normal CCD. In this case, 

the EM register can be switched off and 

then the “normal” readout register is 

used. Thus, the EMCCD behaves just as a 

normal back-illuminated CCD. 

Application Examples 

Figure 1 a) shows a confocal Raman image 

of a PS-PMMA polymer blend spin- 

coated onto a glass slide. It was imaged 

with the WITec Ultrafast Raman Imaging 

Option for the alpha300 R Confocal Raman 

Microscope using a spectroscopic 

EMCCD as the detector. The scan range 

was 50 x 50 µm with 120 x 120 image 

pixels = 14,400 spectra. The acquisition 

time per spectrum was only 2.3 milliseconds, 

resulting in a total acquisition time 

for the complete image of 67 seconds (including 

retrace at the end of the line). 

The image was obtained by evaluating 

the different peak characteristics of PS 

and PMMA as shown in figure 1b. with 

the WITec Project software package. This 

results in a color-coded image of the distribution 

of the two compounds (red: PS; 

green: PMMA).

Fig 2: a) Overview scan of carbon nanotubes on 

a silicon substrate, 25 x 25 µm, 120 x 120 pixel = 

14,400 spectra. b) Zoom-in of the marked area. 

Scan Range 3.5 x 3.5 µm, 120 x 120 pixel = 

14,400 spectra. Total acquisition time of both 

images was 96 seconds. c) Typical measured 

spectrum obtained by averaging some of the 

14,400 spectra which showed a clear signal from 

the CNTs. 

In a second experiment, carbon nanotubes 

(CNTs) on a silicon substrate were 

imaged using the WITec Ultrafast Raman 

Imaging Option. As the first step, a large 

overview scan with a scan range of 25 x 

25 µm with 120 x 120 pixels = 14,400 

spectra was performed. The acquisition 

time was 4 ms per spectrum, so the 

measurement required only 96 seconds 

to complete the image shown in figure 

2 a). It clearly reveals the heterogeneous 

distribution of the CNTs on the substrate. 


In a second step, a zoomed-in image of 

the marked area with a scan range of 3.5 

x 3.5 µm and 120 x 120 pixels was obtained 

as shown in figure 2 b). Due to the 

optimized readout of the EMCCD, the acquisition 

time per spectrum was again 4 

ms and 96 seconds for the complete image 

(including retrace at the end of the 

line). Figure 2 c) shows a typical measured 

spectrum of the CNTs also showing 

peaks from the Si-Substrate. It was obtained 

by averaging some of the 14,400 

spectra which showed a clear signal from 

the CNTs. 

Summary 

It was demonstrated that the use of an 

EMCCD camera can greatly increase detection 

efficiency and speed, especially 

for the short integration times necessary 

with a confocal Raman microscope. For 

very small signals that are dominated by 

the CCD’s readout noise, the use of an 

EMCCD can improve the S/N ratio by a 

factor of 5 10 compared to the best available 

standard CCD’s, while for larger signals 

the electron multiplying circuit can 

simply be switched off and all properties 

of a standard (back-illuminated) CCD are 

maintained. 

Contact: 

Olaf Hollricher 

Harald Fischer 

Andrea Jauss 

Thomas Dieing 

WITec GmbH 

Ulm, Germany 

Tel: +49 731 14070 0 

Fax: +49 731 14070 20 

Harald.Fischer@witec.de 

www.witec.de 



Ultrasonic Nanofabrication with an AFM 

Ultrasound Facilitates Nanolithography and Nanomanipulation 

Ultrasonic AFM may improve fabrication technologies on the nanometer scale. In the presence of ultrasonic 

vibration, hard surfaces can be indented and scratched with the tip of a soft cantilever, due to its inertia. Ultrasound 

reduces or even eliminates friction, and hence modifies the tip-nanoparticle-surface interactions in 

AFM manipulation. The subsurface sensitivity of the technique makes feasible the purposed manipulation of 

subsurface nanoscale features by ultrasonic actuation. 

Ultrasonic Atomic Force Microscopies 

Information of ultrasonic vibration on 

the nanoscale has recently become 

accessible by a new family of Scanning 

Probe Microscopy techniques based on 

the use of Atomic Force Microscopy 

(AFM) with ultrasound excitation [1]. 

Among them, the techniques of Ultrasonic 

Force Microscopy (UFM) [2] and 

Heterodyne Force Microscopy (HFM) [3] 

rely in the so-called „mechanical-diode“ 

effect [4], in which a cantilever tip is in 

contact with the sample surface and normal 

ultrasonic vibration is excited at the 

tip-sample contact (see fig. 1). If the 

excitation frequency is high enough, or is 

not coincident with a high-order cantilever 

contact resonance, the cantilever 

will not be able to linearly follow the surface 

vibration due to its inertia. Nevertheless, 

if the ultrasonic excitation am- 

Keywords: 

atomic force microscopy, ultrasonic force 

microscopy, nanomanipulation, nanolithography, 

nanoscratching 


plitude is sufficiently high that the 

tip-sample distance varies over the nonlinear 

tip-sample force interaction 

regime, the cantilever experiences a 

static force during the time that the 

ultrasonic excitation is acting. This force 

is the so-called “ultrasonic force”, and 

can be understood as the net force that 

acts upon the cantilever during a complete 

ultrasonic cycle, due to the nonlinearity 

of the tip-sample interaction force. 

The cantilever behaves then as a 

mechanical diode and deflects when the 

tip-sample contact vibrates at ultrasonic 

frequencies of sufficiently high amplitude. 

The magnitude of the ultrasonic 

force or of the ultrasonic-force-induced 

additional cantilever deflection (UFM signal) 

is dependent on the details of the 

tip-sample interaction force, and hence 

on material properties such as elasticity 

and adhesion. Therefore, UFM allow us 

to discern nanoscale topographic regions 

with distinct elastic contrast, as shown in 

figure 2, where images of Sb nanoparticles 

on Highly Oriented Pyrolitic Graphite 

(HOPG) are displayed. Remarkably, in 

M. Teresa Cuberes 

this case, the UFM contrast reveals stiffness 

variations even within individual Sb 

particles [5]. Recently, a novel ultrasonic 

AFM mode, namely Mechanical-Diode 

Ultrasonic Friction Force Microscopy 

(MD-UFFM) based on the lateral 

mechanical diode effect has additionally 

been proposed for the study of friction 

and lubrication on the nanoscale, in the 

presence of surface shear ultrasonic 

vibration [6]. 

Ultrasonic Nanofabrication 

Ultrasonic AFM techniques provide a 

means to monitor ultrasonic vibration at 

the nanoscale, and open up novel opportunities 

to improve nanofabrication technologies 

[7]. 

In the presence of ultrasonic vibration, 

the tip of a soft cantilever can dynamically 

indent hard samples due to its inertia. In 

addition, it has been demonstrated that 

ultrasound reduces or even eliminates 

nanoscale friction [8]. Typical top-down 

approaches that rely in the AFM are 

based on the use of a cantilever tip that

acts as a plow or as an engraving 

tool. The ability of the 

AFM tip to respond inertially 

to ultrasonic vibration excited 

perpendicular to the sample 

surface and dynamically indent 

hard samples may facilitate 

the nanoscale machining 

of semiconductors or engineering 

ceramics in a reduced 

time. Figure 3 demonstrates 

the machining of nanotrenches 

and holes on a silicon sample 

in the presence of ultrasonic 

vibration. Interestingly, no debris 

is found in the proximity 

of lithographed areas. Figure 

3 (a) refer to results performed 

using a cantilever with nominal 

stiffness comprised between 

28–91 Nm –1 and a diamond-coated 

tip. Figure 4 (b) 

refer to results achieved using 

a cantilever with nominal 

stiffness 0.11 Nm –1 and a SiN 

tip; in the absence of ultrasound, 

it was not possible to 

scratch the Si surface using 

such a soft cantilever. In the 

machining of soft materials, 

as for instance plastic coatings, 

the ultrasonic-induced 

reduction of nanoscale friction 

may permit eventual finer 

features and improved surface 

quality in quasi-static approaches. 

In [9], an in-plane 

acoustic wave coupled to the 

sample support was used to 

enhance the intermittent force 

exerted by the tip in dynamic 

AFM nanomachining of thin 

polymer resist films. 

In bottom-up approaches, 

ultrasound may assist in the 

self-assembly or AFM manipulation 

of nanostructures [7]. 

Effects such as sonolubrication 

and acoustic levitation 

have been studied at the microscale. 

These phenomena 

may facilitate a tip-induced 

motion of nano-objects. In the 

manipulation of nanoparticles 

(NPs) on surfaces with 

the tip of an AFM cantilever, 

when ultrasound is excited at 

a sample surface both tipparticle 

and particle-surface 

frictional properties change 

[10]. Moreover, the excitation 

of NP high-frequency internal 

vibration modes may also 

modify the NP dynamic re- 

sponse, and introduce novel 

mechanisms of particle motion. 

Some of the opportunities 

in ultrasonic-assisted 

AFM manipulation are illustrated 

in figure 4, which show 

images of Au NPs on a silicon 

surface. The surface was covered 

by poly-l-lysine to prevent 

that the Au NPs were 

swept away by the AFM tip. 

Nevertheless, when scanning 

in contact mode in the absence 

of ultrasound, most NPs 

were inevitably swept away 

by the tip. Scanning in the 

same conditions that led to a 

NP displacement but in the 

presence of surface ultrasonic 

vibration, with an appropriate 

election of the ultrasonic 

amplitude, the NPs remained 

undisturbed. In figure 4, two 

NP were displaced while recording 

the images, which 

have been pointed out by blue 

arrows. A close inspection of 

the data in figure 4 reveals 

traces of the UFM and LFM 

responses from those moving 

particles while being in motion 

(see areas enclosed by ellipses 

and rectangles). The 

study of the UFM response of 

a moving NP may allow us to 

learn about the dynamic 

mechanisms of NP displacement 

across surfaces. Consistently 

with the fact that ultrasound 

eliminates friction, 

no frictional contrast is distinguished 

on the undisturbed 

Au NPs in the LFM images. 

However, from the comparison 

of the traces of the two 

moving NPs in the forward 

and backward LFM scans, 

friction during the tip-induced 

NP motion is apparent. Controlled 

and accurate measurements 

of lateral and ultrasonic 

forces exerted by 

individual NPs when in motion 

under tip ultrasonic actuation 

may bring about a wealth of 

information about the dissipated 

energy, ultrasonic lubrication 

effects, NP dynamics, 

etc. 

Eventually, it should be 

pointed out that the sensitivity 

of ultrasonic-AFM to subsurface 

features makes feasible 

to monitor subsurface 

The New BioMAT TM 

Workstation AFM. 

Perfect Imaging 

Results for 

Opaque Samples. 

The BioMAT TM Workstation combines upright optical 

microscopy with AFM and opens up a wide range of 

applications for the study of non-transparent specimens. 

Even in liquids. 

www.jpk.com 




Fig. 1: Detection of surface vibration with the tip 

of an AFM cantilever. (a) At low frequencies, the 

tip follows the surface vibration. (b) In the highfrequency 

regime, for sufficiently high vibration 

amplitudes, tip experiences an ultrasonic force 

F us. (c) Tipsample force F ts versus tipsample distance 

d curve. 

Fig. 2: Sb NPs on HOPG. (a, b) Simultaneously 

recorded contactmode AFM topography (a) and 

UFM image (b). (c, d) Simultaneously recorded 

highresolution images from the areas enclosed 

by white squares in (a), (b). Set point: 1 nN; Kc: 

0.11 Nm –1 ; UFM parameters: 2.2 MHz, 8 Vpp. 

Contrast in UFM indicates stiffness variations. 

modifications [7]. We have recently demonstrated 

that actuation with an AFM tip 

in the presence of ultrasonic vibration 

can produce stacking changes of extended 

grapheme layers, and induce permanent 

displacements of buried dislocations 

in Highly Oriented Pyrolytic 


Fig. 3: AFM nanomachining of trenches and holes 

on Si(111) in the presence of normal surface 

ultrasonic vibration of ~5 MHz. (a) Scratch and 

indentation with a diamondcoated cantilever 

tip. Rt: 35 nm. Kc: 2891 Nm –1 . (b) Nanotrenches 

formed in 50, 75 and 100 cycles respectively, at 

a load of ~40 nN, with a pyramidal SiN cantilever 

tip. Kc: 0.11 Nm –1 . 

Fig. 4: Displacement of Au NP induced by the tip 

of an AFM cantilever in the presence of normal 

surface ultrasonic vibration of ~ 2.6 MHz. (ad) 

were simultaneously recorded. (a) Contactmode 

AFM topography. (b) UFM; (c) LFM forward scan; 

(d) LFM backward scan 

Fig. 5: Lateral displacement 

of a subsurface dislocation 

in HOPG by ultrasonic tip 

actuation (a) Topography of 

the HOPG surface in AFM 

contact mode: (700 x 700) 

nm; Fo=105 nN. (b,c) UltrasonicAFM 

images recorded 

in sequence over nearly the 

same surface region: 

a=2.15 MHz (b) A=3.5 Vpp 

(c) A=2.4 Vpp. (d–f) LFM 

forward scan, simultaneously 

recorded with (a–b) 

respectively, with the same 

parameters. 

Graphite (HOPG). This effect is illustrated 

in figure 5. In the presence of normal 

surface ultrasonic vibration, both AFM 

and LFM images reveal subsurface features 

[1]. Subsurface modification was 

brought about in this case by scanning in 

contact mode, with high set-point forces, 

and high surface ultrasonic excitation 

amplitudes [7]. 

Summary 

Ultrasonic AFM techniques provide a 

means to monitor ultrasonic vibration at 

the nanoscale, and open up novel opportunities 

in nanofabrication technologies. 

The use of ultrasound may improve both 

down-top and bottom-down approaches 

in nanofabrication, facilitating the patterning 

of nanoscale surface features, 

the manipulation or self-assembly of nanostructures, 

and possibly the controlled 

subsurface manipulation of buried nanoobjects. 

Acknowledgments 

The author thanks J. J. Martinez and A. 

Lusvardi for assistance in the UFM lab. 

The samples of Sb NPs were provided by 

C. Ritter and U. Schwarz. The Au NPs 

were provided by M. A. Gonzalez and 

M.P. Morales. Financial support from the 

JCCM (Junta de Comunidades de Castilla-La 

Mancha) under project PBI-05- 

018 is gratefully acknowledged. 

References: 

[1] Gnecco, E. and Meyer, E. (Eds.), Fundamentals 

of Friction and Wear on the nanometer 

scale, Springer, 2007, 49-71. 

[2] Yamanaka K., et al., Appl. Phys. Lett. 64, 

178–180 (1994). 

[3] Cuberes, M. T., et al., J. Phys. D.: Appl. Phys. 

33, 2347–2355 (2000). 

[4] Rohrbeck, W. and Chilla, E., Phys. Status 

Solidi (a) 131, 69–71(1992). 

[5] Cuberes, M. T., et al., Ultramicroscopy 107, 

1053–1060 (2007). 

[6] Cuberes, M. T. and Martínez, J. J., J. of Phys.: 

Conf. Ser. 62, 224–228 (2007). 

[7] Cuberes, M. T., J. of Phys.: Conf. Ser. 61, 219– 

223 (2007). 

[8] Dinelli, F., et al., Appl. Phys. Lett. 71, 1177– 

1179 (1997). 

[9] Rubio-Sierra, F. J., et al., Phys. Stat. Sol. (a) 6, 

1481–1486 (2006). 

[10] Cuberes, M. T., Proc. of the 30th Annual Meeting 

of the Adhesion Society, 430–432 (2007). 

Contact: 

Dr. M. Teresa Cuberes 

Engineering School Professor 

University of Castilla-La Mancha 

Applied Mechanics and Project Engineering 

Laboratory of Nanotechnology 

Almadén, Spain 

Tel.: +34 902 204100 ext. 6045 

Fax: +34 926 264401 

teresa.cuberes@uclm.es 

www.uclm.es

Shhh. It’s the Innova AFM. 

No noise. Higher resolution. This kind 

of news doesn’t stay quiet for long. 

www.veeco.com/innova


3D Orientation Microscopy 

Electron Backscatter Diffraction in a Combined FIB/SEM 

Combining electron backscatter diffraction (EBSD), a scanning electron microscope (SEM) and a focused ion 

beam (FIB) together into a single instrument enables three dimensional (3D) characterization of microstructure 

in crystalline materials. Combining these techniques together has enormous potential in materials science. 

Electron Backscatter Diffraction 

Traditionally, microstructure refers to 

features that are visually evident in an 

optical or electron microscope. However, 

many critical aspects of microstructure 

are not visually evident. For example, 

the crystallographic orientation of the 

constituent grains can not be observed in 

basic microscopic imaging. While some 

indirect evidence may be observed, other 

techniques are required to gain true 

quantitative information. If we consider 

the microstructure at the scale revealed 


Keywords: 

electron backscatter diffraction (EBSD), 

3D microstructure characterization Stuart I. Wright Stefan Zaefferer 

in the SEM then the crystallographic orientation 

is best characterized using 

EBSD. An EBSD pattern is formed when 

a stationary electron beam is focused on 

a highly-tilted and properly-prepared 

sample in the SEM. Electrons are scattered 

as the incident electron beam interacts 

with the crystal lattice within the 

Fig. 1: A schematic showing 

how EBSD is used to obtain 

orientation from a crystalline 

material and an example 

of an orientation map generated 

from the data. 

sample. Electrons satisfying physical 

laws governing the interaction of electrons 

with the crystal lattice are coherently 

diffracted out of the sample. The 

diffracted electrons form a pattern on a 

phosphor screen positioned near the 

sample. The pattern provides information 

about the structure of the crystal lattice 

within the interaction volume such 

as the orientation of the crystal lattice 

with respect to the sample reference 

frame. Orientation microscopy [1] is a 

fully automated technique where the patterns 

are imaged using a CCD camera 

and then analyzed to determine the corresponding 

orientation. The system controls 

the electron beam enabling the procedure 

to be repeated at each point on a 

scan grid superposed over the sample. 

By mapping the measured orientations 

to various color scales it is possible to 

visualize orientation aspects of the micro-

structure. Orientation microscopy has 

proven to be a valuable tool for characterizing 

crystalline materials as evidenced 

by the large number of papers 

published where the technique has been 

used in the research. 

3D Data Acquistion 

As with traditional metallographic techniques, 

EBSD is usually performed on 2D 

planes cut through a sample. 3D microstructural 

characterization can be 

achieved through serial sectioning. The 

technique simply comprises the cutting 

of material slices, recording of the structure 

of these slices and then reconstructing 

the 3D structure by stacking of the 

recorded images. Serial sectioning is applicable 

to a wide range of materials and 

material problems with the only serious 

disadvantage that it is destructive. For 

serial sectioning a large number of different 

methods can be imagined, e.g. mechanical 

cutting, grinding or polishing, 

chemical polishing, or laser or electrical 

discharge ablation. The main challenge 

associated with these methods is controlling 

the sectioning depth, obtaining flat 

and parallel surfaces and correctly redetecting 

and aligning the observation 

area. Also, these serial sectioning methods 

tend to be extremely laborious. A 

technique that avoids these problems is 

serial sectioning with an ion beam in a 

combined FIB-SEM. The impact of the 

ion beam onto the sample leads to localized 

removal of the target material and 

can therefore be used to mill parallel serial 

sections through the material a few 

nanometers in depth. Irradiating a material 

surface in grazing incidence allows 

preparation of smooth surfaces that show 

little damage [2]. Recent progress has led 

to a fully automated technique for alternately 

sectioning and subsequent scanning 

of the new section using EBSD 

[3–5]. 

Data Visualization 

Software for analyzing and visualizing 

the wealth of information in the 3D EBSD 

data is still in its infancy. Basic functions 

based on the analysis of 2D EBSD data 

are being extended into 3D [6]. Visualization 

software is being built upon existing 

tools developed for general 3D rendering. 

Functions for rotating and slicing 

through the data cube as well as the ability 

to extract individual grains from the 

3D data have been developed. Grain facets 

can be colored according to their orientation 

relative to the associated crystal 

Fig. 2: Schematic of the instrument combining 

the EBSD, SEM and FIB techniques and micrograph 

of a milled section. 

lattice of the grain [7]. Figures 3 and 4 

show examples from steel and nickel. 

Conclusions 

A system for 3D orientation microscopy 

based on fully automated serial sectioning 

and EBSD based orientation microscopy 

has been developed in a FIB-SEM. 

The technique yields the crystal orientation 

at each voxel of the measured volume. 

A spatial resolution 50 x 50 x 50 nm³ 

can be achieved. Volumes on the order of 

50 x 50 x 50 μm³ can be practically observed. 

The technique works on a wide 

variety of materials but some exceptions 

have been found, namely materials that 

adversely react to the Ga+-ion beam irradiation. 

The technique has been applied 

to several different materials and 

has yielded information that gives insight 

into microstructures that cannot be realized 

without the 3 rd dimension. 

References: 

[1] Adams, B. L., et al., Met. Trans. A 24, 819– 

831 (1986). 

[2] Michael, J., et al., Microscopy and Micoranal- 

ysis 13, 926–927 (2007). 

[3] Mulders, J. J. L., et al., Mat. Sci. Forum 495– 

497, 237–242 (2005). 

[4] Uchic, M. D., et al., Scripta Mat. 55, 23–28 

(2006) 

[5] Groeber, M. A., et al., Mat. Char. 57, 259–273 

(2006). 

[6] Rollett, A. D., et al., Annu. Rev. Mater. Res. 

37, 627–658 (2007). 

[7] Rowenhorst, D. J., et al., Scripta Mat. 55, 11– 

16 (2006). 

Further references are available from the au- 

thors. 


Fig. 3: A 3D EBSD scan from pearlite structure in 

a steel sample showing a 3D data cube reconstructed 

from EBSD scans on serial sections. 

Fig. 4: A 3D EBSD scan from a nickel sample and 

a twinned grain extracted from the data and 

rendered in 3D. 

Contact: 

Stuart I. Wright 

Director of Applications Science and Software 

Engineering 

EDAX-TSL 

Draper, Utah, USA 

Tel.: +1 801 4952750 

Fax: +1 801 4952758 

stuart.wright@ametek.com 

www.edax.com 

Stefan Zaefferer 

Group Head – Diffraction and Microscopy 

Max-Planck-Institute for Iron Research 

Department of Microstructure Physics and Metal 

Forming 

Düsseldorf, Germany 

Tel.: +49 211 6792 803 

s.zaefferer@mpie.de 

www.mpie.de 



Combining Optical Upright 

Microscopy and AFM 

Working with the Biomaterial Workstation BioMAT 

The atomic force microscope (AFM) is a flexible instrument that can be used for imaging, measuring forces 

and elastic properties and manipulating a variety of samples, at high resolution. The applicability of AFM is 

further extended in combination with light microscopy as optics deliver more bulk details and by fluorescence, 

compositional contrast. To date, the combination of AFM and light microscopy has been limited to 

samples on transparent substrates, where AFM has top-down access to the sample and an inverted light microscope 

has bottom-up access to the same area [1, 2]. 

A Combination of Techniques 

However, there are many areas of research 

in which the combination of AFM 

with optical microscopy on opaque samples 

would be a powerful tool. Topics 

such as bacterial growth on metallic surfaces, 

bionics and surface chemistry, fluorescent 

polymers and coatings, i.e. areas 

from both life and material science 

could be addressed with such a combination 

of techniques. 

The main problem limiting the effective 

combination of these techniques on 

non-transparent surfaces has been providing 

access for both techniques at the 

same spot on the sample surface. To reach 

the full capabilities of optical microscopy, 

objective lenses with an extremely short 

working distance are required, leaving no 

space for AFM access to the same position. 

JPK Instruments has developed a solution 

to allow integration of upright optical 

microscopy and AFM, named the 

BioMAT Workstation (fig. 1). 


Portable Shuttle Stage 

The workstation spatially separates the 

upright optical microscope from the AFM 

to assure that neither of the two techniques 

is compromised. The key element 

of the BioMAT Workstation design is the 

portable shuttle stage on which the sample 

is loaded. The transfer of this shuttle 

stage from the upright optical microscope 

to AFM and vice versa allows precise 

positioning of the sample on both 

microscopes such that the same area is 

imaged by both systems. This transfer 

can be repeated as often as necessary, 

allowing the sequential measurement of 

optics and AFM. 

For combining upright light microscopy 

with AFM first the BioMAT Workstation 

has to be aligned, matching the 

AFM scan rage to the field of view of the 

optical microscope. To do this, a transparent 

reference sample displaying a 

cross-hair structure, which can be imaged 

with both systems is used (fig. 2). 

With the workstation’s integrated inverted 

optics, the AFM tip can be coarsely 

aligned with respect to the cross hair on 

the reference sample (fig. 3). This coarse 

alignment involves adjusting the position 

of the AFM head on top of the BioMAT 

Workstation so that the AFM tip position 

matches the center of the reference cross. 

By exchanging the reference sample with 

the sample of interest, the same area of 

the sample can be imaged sequentially 

with the two separate microscopes. 

Applications 

One area of research where combining 

light microscopy and AFM on opaque 

substrates would be useful is the investigation 

of bacteria with metal surfaces. 

Thiobacteria can leach mineral sulfides 

from various metals. During this process 

of bioleaching the bacteria, which mostly 

belong to species of thiobacillus ferrooxidans 

are in close contact to the mineral 

sulfides, forming a monolayered biofilm. 

AFM is particularly suitable for investigation 

of such biofilms as it allows the 

visualization and characterization of biological 

samples under physiological conditions 

with high spatial resolution. The 

option to combine AFM with fluorescence 

microscopy is a powerful means to correctly 

interpret and validate the topographic 

images obtained by AFM with

Fig. 1: NanoWizard II integrated into the BioMAT Workstation 

the help of corresponding images of fluorescently 

labelled structures. Since almost 

all sulphur containing minerals are 

opaque such samples are the perfect application 

for the BioMAT Workstation. 

Here, thiobacillus ferrooxidans was 

grown on a piece of compressed sulphur 

(sample courtesy Prof. Sand, University 

Duisburg-Essen). For fluorescence microscopy 

the bacterial DNA was stained 

using DAPI. Fluorescence images were 

acquired with a Zeiss AxioImager A1m 

equipped with a 100 x Acroplan water 

immersion objective. After imaging on 

the AxioImager, the specialized sample 

holder was transferred to the BioMAT 

Workstation and AFM imaging was conducted 

in intermittent contact mode in 

fluid using the NanoWizard II. 

As contrast in AFM is based on structure, 

the images of bacteria on the surface 

of the elementary sulphur are complex. 

One can see steps and structures in 

the sulphur substrate, as well as groups 

of bacteria on the surface. By comparing 

the AFM image with the fluorescence image 

of the same area it is clear which 

surface features correspond to bacteria 

(fig 4). When it is clear exactly which region 

of the surface contains bacteria the 

surface properties of the metal and the 

biofilm can be characterised in high resolution 

with the AFM. 

Conclusion 

With such a tool many new possibilities 

for combined imaging of biological samples 

on opaque surfaces are now possi- 

Fig. 2: To demonstrate imaging of the same area 

a mixture of 80 nm fluorescence PMMA and 

1 µm silica beads were first imaged in the upright 

microscope (left) and then with the AFM 

(right). 

Fig. 3: The cantilever of the AFM can be aligned 

with the chrome reference cross using the alignment 

optics integrated into the BioMAT workstation. 

This allows the user to establish common 

reference points in both AFM and optical 

space that can be applied to a sample of interest. 

ble. The investigation of structural and 

elastic properties of various types of cells 

on patterned surfaces can provide valuable 

information about the interaction of 

cells with potential implant surfaces. As 

seen here, the effect of biofilm formation 

on a metal or crystalline surface can be 

investigated. Polymer formation on 

opaque surfaces can also be investigated, 

using both fluorescence microscopy and 

AFM. Such access to a sample with the 

two forms of microscopy further extends 

the applicability of the AFM for characterization 

of samples on opaque surfaces. 

References: 


Fig. 4: Imaging of bacteria on an opaque sulphur 

surface. In (a) DAPI stained bacteria have been 

imaged using fluorescent microscopy. The 

marked regions correspond to the AFM images 

in (b) and (c). While the fluorescent image makes 

it clear where the bacteria are located, high 

resolution structural information can only be 

derived from the corresponding AFM images. 

[1] Chiantia, S., Kahya, N., Schwille, P., Lang- 

muir. 21(14) 6317–23 (2005). 

[2] Poole, K., Müller, D., Br J Cancer. 92(8):1499– 

505 (2005). 

Contact: 

Jörg Barner 

application scientist 

JPK Instruments AG 

Berlin, Germany 

Tel.: +49 30 5331 12070 

Fax: +49 30 5331 22555 

nanowizard@jpk.com 

www.jpk.com 


M i c r o s c o p y Fa c i l i t i e s 

Cancer Research in Glasgow 

The Beatson Advanced Imaging Resource (BAIR) 

Cancer Research UK is a major funder of biomedical research in Europe. In 2006/2007 they spent £315 million 

to support over 500 research groups in the UK through a variety of funding mechanisms including research 

institutes, clinical centers, and grants. The Beatson Cancer Research Institute is one of four corefunded 

CR-UK research institutes. Recently, CR-UK invested £22 million towards a new building for the 

Beatson (Fig. 1) located on the University of Glasgow campus and scheduled for occupation in December of 

2007. This expansion will double the size of the institute and is associated with substantial new investment 

in research infrastructure. 

New Facility of the Beatson Cancer 

Research Institute 

The Beatson Advanced Imaging Resource 

(BAIR) is the new imaging facility of the 

Beatson Cancer Research Institute. The 

Bair occupies 280 m 2 of space in eleven 

rooms located primarily in the basement 

of the new building, with two additional 

rooms located in the biomedical unit to 

facilitate mouse in vivo imaging (see below). 

Smooth operation is ensured by two 

full time scientific officers who train users, 

assist with advanced applications, 

troubleshoot equipment, and perform 

quality control. Staff also maintain an internal 

website which includes tutorials, 

operating procedures, protocols, and 

equipment specifications in addition to 

the scheduling database through which 

both equipment and assistants are 

booked. 

The BAIR is managed by Dr Kurt I. 

Anderson, who brought to Glasgow the 

experience of having set up the light mi- 


croscopy facility of the Max Planck Institute 

for Cell Biology and Genetics in Dresden. 

Dr. Anderson also leads a research 

group at the Beatson Institute, which investigates 

actin dynamics in cell migration 

using advanced imaging techniques 

including TIRF, FRAP, photo-activation/ 

-switching (PA/PS), and FLIM-FRET. The 

rational behind this joint appointment is 

to promote the use of advanced imaging 

techniques among Beatson research 

groups by introducing opportunities for 

substantive collaborations which might 

not be possible within the context of a 

service alone. 

Imaging Systems 

The imaging systems are roughly grouped 

according to applications (Fig. 2). At the 

south end of the facility (room 122) are 

three systems for medium throughput, 

high content, long term time lapse microscopy 

consisting of a Nikon TE2000 stand 

with Perfect Focus, ASI stage with linear 

Fig. 1: The new Beatson Institute for Cancer 

Research in Glasgow 

encoder, Sutter illumination system, and 

okolab incubator, all driven by Meta- 

Morph software. BAIR staff maintain 

FACS analyzers throughout the building, 

in addition to a BD FACSAria in room 123 

with 561 nm diode laser for use with red 

fluorescent proteins. Three confocal microscopes 

are housed in room 124, including 

a Leica TCS-SP2 with AOBS and 

Olympus FV1000 with 405 nm diode and 

SIM scanner. The latter system is especially 

powerful for FRAP and the use of 

PA/PS probes. A second TCS-SP2 is fitted 

with a Becker and Hickl module (SPC- 

730) and pulsed IR laser for time domain 

detection of FLIM-FRET. A small room 

(room 125) is centrally located for the 

placement of microscope laser modules, 

which promotes operational stability of 

the equipment and a more comfortable 

user environment. A workshop is located 

in room 126 along with a demo space for 

testing of new equipment. Room 127 

houses several custom systems for live 

cell imaging. A system for TIRF microscopy 

has been built based on a Nikon 

TE2000 stand and custom condensor incorporating 

405 and 473 nm diode lasers, 

which enables FRAP and PA/PS in 

TIRF. Integration of a 561 nm diode laser 

is currently under way, which will enable 

simultaneous GFP/RFP imaging in conjunction 

with an Optical Insights Dual- 

View and Roper 512 x 512 EMCCDFT

camera. A frequency domain system for 

FLIM-TIRF is under development together 

with Lambert Instruments based 

on their LIFA system, which will offer enhanced 

time resolution compared to the 

TCSPC approach and sensitive detection 

of protein-protein interactions at the cell 

surface due to the optical sectioning of 

TIRF. A LIFA system coupled to a 

Yokogawa CSU22 spinning disk scanhead 

is also planned. Room 130 houses several 

upright stands for imaging fixed 

specimens, including histological stains, 

immunofluorescence, and FISH. Offline 

Beatson Advanced Imaging Resource (BAIR) 

rm 122 rm 123 rm 124 

rm 125 / rm 126 rm 127 rm 130 rm 129 rm 131 

C 

D E 

E 

Fig. 2: Floor plan of the Beatson Advanced Imaging Resource (see text for details) 

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workstations for image processing and 

analysis are located in room 129, including 

Volocity, MetaMorph, the Olympus 

and Leica confocal packages, and of 

course ImageJ. Finally, staff share office 

space in room 128. 

Focus at the Beatson 

rm 128 

The development and use of murine cancer 

models to better understand and treat 

human disease is an important focus at 

the Beatson. Mouse in vivo imaging involves 

a wide range of magnification, sen- 

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2 m 

M i c r o s c o p y Fa c i l i t i e s 

sitivity, and resolution depending on the 

experiment. For imaging groups of fluorescent 

cells down to the level of single 

cells an Olympus OV100 is used. With this 

system tumor progression can be monitored 

in a variety of tissues, including skin, 

colon and pancreas. For high resolution 

imaging of cells and sub-cellular structure 

is currently used an Olympus FV1000 confocal 

microscope. However a multi-photon 

TRIM scope from LaVision Biotec is under 

installation. This system incorporates both 

a Coherent Chameleon laser for excitation 

of GFP and an APE optical parametric oscillator 

for excitation of red fluorescent 

proteins. Finally, an Ivis 50 is used for detection 

of chemi-luminescence. 

The Beatson Institute looks forward to 

introducing the BAIR and their new research 

facility to the imaging community 

by hosting the European Light Microscopy 

Initiative meeting in Glasgow together 

with the University of Strathclyde in 

2009. 

Contact: 

Dr. Kurt I. Anderson 

Beatson Institute for Cancer Research 

Glasgow, Scotland, United Kingdom 

Tel.: +44 141 330 2864 

Fax: +44 141 942 6521 

k.anderson@beatson.gla.ac.uk 

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C o v e r S t o ry 

The “F” Words 

FRET, FRAP, and FISH – Technology and Techniques 

The use of fluorescence microscopy in the life sciences 

runs the gamut from basic photodocumentation 

to dynamic single-molecule fluorescence (SMF) 

studies. Recent advances in digital imaging technology 

are helping to expand the utility and popularity 

of many fluorescence microscopy techniques, 

including Förster resonance energy transfer (FRET), 

fluorescence recovery after photobleaching (FRAP), 

and fluorescence in situ hybridization (FISH). The 

newly created Microimaging Applications Group, 

which comprises Photometrics, Media Cybernetics, 

QImaging, Gatan, and MAG Biosystems, offers a 

broad range of innovative imaging solutions designed 

to enhance life science research capabilities. 

FRET Imaging 

Förster resonance energy transfer is a 

phenomenon in which nonradiative 

transfer of energy occurs between donor 

and acceptor molecules in close proximity 

(2–7 nm). Since FRET efficiency decays 

as a function of the inverse sixth 

power of the distance between the donor 

and acceptor, this phenomenon can be 

leveraged to provide solid evidence of an 

interaction between the donor and acceptor 

in a FRET pair. 

In FRET, the donor molecule is returned 

to a ground state without fluorescence 

emission while the acceptor molecule 

is raised to an excited state. Upon 

decay of the acceptor’s excited state, fluorescence 

emission may be witnessed. 

Thus, an increase in FRET between label 

molecules will result in a decrease in donor 

emission and a simultaneous increase 

in acceptor emission. Using FRET detection, 

interactions between molecules can 

be monitored in subcellular compartments 

and tracked as a function of time. 

FRET applications include evaluating the 

structure of proteins, determining the 

spatial distribution and assembly of protein 

complexes, monitoring receptor/ligand 

interactions, and sensing the presence 

of small molecules in living cells. 

FRET experiments are often performed 

using standard ratio imaging 


techniques. Depending on the application, 

FRET is used to qualitatively or 

quantitatively investigate experimental 

phenomena. While qualitative experiments 

focus on simply determining the 

presence or absence of FRET as an indicator 

of interaction, quantitative experiments 

require a more methodical strategy. 

To help ensure accurate results, 

next-generation Photometrics electronmultiplying 

CCD (EMCCD) cameras provide 

exceptionally high quantum efficiency, 

quantitative stability across 16 

bits, and linear EM gain up to 1000x. 

Even with superior camera performance, 

sequential imaging techniques 

(e.g., using an emission filter wheel or 

switching microscope filter cubes) can 

make proper data calibration and correction 

very difficult, if not impossible, 

when dynamic samples are used. Therefore, 

many quantitative FRET applications 

require that the donor and acceptor 

emissions be simultaneously imaged. To 

meet this criterion, MAG Biosystems offers 

easy-to-use instrumentation that 

splits the incident beam from the microscope 

into independent beams. Each of 

the resultant emission channels is projected 

onto a region of a CCD or EMCCD 

array. A precision optical and mechanical 

design allows subpixel image registration 

and minimizes light loss for simultaneous 

multichannel acquisition. 

FRAP Imaging 

Fluorescence recovery after photobleaching 

is useful for examining intracellular 

molecular variables such as nuclear 

protein complex dynamics, 

diffusional mobility of membrane proteins, 

and cytoskeletal dynamics. FRAP is 

a powerful mode of fluorescence light 

microscopy in which a specialized illumination 

strategy is implemented in order 

to permit perturbation of the steady-state 

fluorescence distribution by bleaching 

fluorescence in selected regions of a sample. 

After the bleaching step, researchers 

can observe and analyze how the fluorescence 

distribution returns to the 

steady state. Because the photobleaching 

of fluorophores is permanent, changes in 

the fluorescence intensity in both the 

bleached and unbleached regions are attributable 

to the exchange of bleached 

and unbleached fluorescent molecules 

between those regions. FRAP microscopy 

is typically geared towards dynamic, lowlight 

endeavors. 

Recently, MAG Biosystems introduced 

a widefield imaging system designed to 

study the intracellular dynamics of proteins 

and other macromolecular complexes 

via FRAP and iFRAP (inverse 

FRAP) experiments in 2D plus time and 

3D plus time. Photoactivation and photo-

conversion studies with fluorescent proteins 

such as PA-GFP, EOS, KFP, Kaede, 

and Dronpa can be performed. 

Several technological innovations, including 

Burst mode and a custom optical 

path, provide a combination of speed, 

sensitivity, and ease of use not found in 

other FRAP systems. When run in Burst 

mode, the delay between the end of the 

bleach pulse and the first recovery image 

is minimized, enabling fast dynamic 

analyses. The FRAP-3D system also lets 

researchers photobleach-on-the-fly by 

simply clicking within a live image display 

window to bleach the region appearing 

under the cursor. 

FRAP-3D includes a galvanometerbased 

FRAP head, an advanced laser 

launch module, high-speed I/O circuitry 

to control all system components, acquisition 

and analysis software with an intuitive 

graphical user interface (GUI), and a 

configured workstation. The head can be 

mounted to many inverted microscopes 

through the epi-illumination port in order 

to enable simultaneous laser and widefield 

illumination. A versatile optical design 

allows researchers to switch seamlessly 

between FRAP studies and standard 

widefield applications without reconfiguring 

the system’s hardware. 

To meet user-specific quantum efficiency, 

spatial resolution, and frame rate 

requirements, the FRAP-3D system utilizes 

high-performance quantitative CCD 

and EMCCD cameras from Photometrics. 

To ensure the utmost instrumentation 

utility, FRAP-3D allows future upgrades 

for spinning-disk confocal microscopy 

and other imaging modalities. 

FISH Imaging 

Fluorescence in situ hybridization is a biochemical 

means of labeling specific nucleic 

acid sequences in cell preparations 

for the purposes of confirming the presence 

of certain genes and for spatial localization 

of sequences of interest within 

a cell or on chromosomes. Essentially, 

FISH provides a way to visualize and map 

genetic material in single cells. FISH has 

been instrumental in elucidating a variety 

of chromosomal abnormalities and 

genetic anomalies. The technique is used 

heavily in the basic research arena as 

well as the clinical arena. The use of 

FISH continues to grow quickly in such 

areas as genetics, cytogenetics, prenatal 

research, and tumor biology. 

The first step in FISH is the production 

of sequence-specific probes, which 

is accomplished by synthesizing antisense 

strands to sequences of interest 

and conjugating these antisense strands 

to fluorescent probes so that they can be 

detected using fluorescence microscopy. 

The power of FISH is greatly enhanced 

by the simultaneous use of multiple fluorescent 

probes. By using a multiplexing 

strategy, numerous nucleic acid sequences 

of interest can be detected and 

mapped. 

There are three basic types of FISH 

probes: (1) locus-specific probes, (2) alphoid 

or centromeric repeat probes, and 

(3) whole-chromosome probes. Locusspecific 

probes, which bind to a particular 

region of a chromosome, are useful 

for determining which chromosome a 

gene is located on once a small sequence 

of a particular gene has been isolated. 

Centromeric repeat probes, which are 

generated from repetitive sequences 

found in the middle of each chromosome, 

are utilized to determine whether a cell 

has the correct number of chromosomes. 

Whole-chromosome probes, which are 

collections of genetic sequences common 

to a particular chromosome, can be used 

to map individual chromosomes as well 

as to identify different chromosomes in 

respect to one another. 

Many researchers utilize two- to fourcolor 

FISH analysis on fixed samples. 

The main requirement for imaging of this 

kind is an ultra-high-resolution detector 

that allows all of the spatial information 

to be preserved under high magnification. 

Since the preparations most often 

contain fixed cells, more intense illumination 

can be used to produce stronger 

signals. A midrange-performance camera 

may be deemed adequate for fixedsample, 

moderate-light FISH studies, 

provided it offers sufficiently high spatial 

resolution. 

With the maturation of techniques 

such as spectral imaging, many researchers 

are now enhancing their FISH experiment 

capabilities by using a far greater 

number of fluorescent probes at one time. 

Spectral imaging enables the identification 

of probes based on their spectral 

curves, allowing differentiation of closely 

overlapping fluorophores. When a camera 

is utilized in conjunction with a spectral 

imaging system, a specimen’s fluorescent 

emission can be split into 

component wavelengths prior to reaching 

the detector. Thus, excellent camera performance 

and sensitivity become critical. 

To facilitate the use of FISH, QImaging 

has engineered a wide selection of easyto-operate 

CCD cameras that address 

myriad resolution, speed, and sensitivity 

requirements. QImaging also offers several 

color options, including solutions 

that combine the use of Bayer mask CCDs 

and innovative data interpolation meth- 

ods to deliver high-fidelity color rendition. 

Software Considerations 

Fluorescence microscopy techniques are 

evolving at a rapid pace. New fluorescent 

probes, camera technologies, and optics 

offer researchers an expansive set of investigative 

capabilities. The importance 

of using intelligent software for image 

acquisition, analysis, and management 

cannot be overstated. 

Powerful packages designed specifically 

for life science experiments, such as 

the Media Cybernetics line of software 

solutions, provide researchers the latest 

image analysis tools for object tracking, 

3D rendering, image deconvolution, and 

a broad diversity of fluorescence-based 

techniques. These programs integrate 

extensive hardware automation support 

and flexible image acquisition and 

processing into an easy-to-use GUI. 

Media Cybernetics also offers an image-asset 

management solution that lets 

life science researchers store, query, and 

share a large number of images (as well 

as data) via a client-server database application 

or the internet. Simple clickand-choose 

tools streamline archiving, 

searching, displaying, customizing, and 

reporting. 

Future Trends 

C o v e r S t o ry 

As imaging instrumentation simultaneously 

becomes more sophisticated yet 

simpler to use, the list of applications for 

FRET, FRAP, and FISH gets longer every 

day. Other fluorescence-based techniques 

benefiting from these technological advances 

include total internal reflection 

fluorescence (TIRF), fluorescence lifetime 

imaging microscopy (FLIM), and fluorescence 

anisotropy and polarization. 

Instrumentation providers such as the 

Microimaging Applications Group will 

continue to introduce high-performance 

cameras, versatile software, and complete 

imaging systems that offer life science 

researchers increasingly powerful 

and efficient capabilities for fluorescence 

microscopy. 

Contact: 

Karl Garsha 

Applications Scientist 

Microimaging Applications Group 

Photometrics 

Tucson, AZ, USA 

Tel.: +1 520 5472704 

Fax: +1 520 5731944 

kgarsha@photomet.com 

www.magworldwide.com 


Systematic Analysis of FRAP Experiments 

An Approach for the Evaluation of Spatially Resolved Data 

Introduction 

L i g h t M i c r o s c o p y 

Sebastian Seiffert 

Fluorescence Recovery after Photobleaching (FRAP) 

is a versatile technique to study dynamic phenomena. 

Performing FRAP on a confocal laser scanning 

microscope documents the recovery process with 

high spatial resolution. This enables a consistent 

determination of the diffusion coefficient and the 

dimensionality of diffusion in calibration-free manner. 

Moreover, experiments representing multi-component 

diffusion can be analyzed as well, thus 

yielding the distribution of diffusion coefficients. 

In the last 30 years, Fluorescence Recovery 

after Photobleaching (FRAP) has 

evolved as a versatile technique to determine 

diffusion coefficients of suitably labeled 

species in fields like pharmaceutical 

research, biophysics or polymer 

chemistry [1]. Basically, a FRAP experiment 

is realized by bleaching a certain 

area of a sample by short and intense laser 

irradiation. Afterwards, the diffusion 

of unbleached molecules from the surroundings 

leads to a temporal recovery 

of fluorescence intensity in the bleached 

region that is monitored with a highly attenuated 

beam. The diffusion coefficient 

can be deduced from the rate of recovery 

after suitable calibration. This procedure 

forms the basis for a variety of classical 

approaches to evaluate FRAP experiments 

[1]. 

Analysis of Spatially Resolved FRAP – 

Theoretical Background 

When FRAP is performed on a confocal 

laser scanning microscope (CLSM), the 

recovery process can be followed with 

high spatial resolution on a µm-scale besides 

temporal resolution. Utilizing this 

aspect for a systematic analysis of FRAP 


data enables the estimation of the diffusion 

coefficient and the dimensionality of 

the diffusion process without any need 

for calibration [2]. 

Basically, each FRAP experiment 

makes use of the diffusion equation, 

which is also known as Fick’s second 

law: 

(1) 

where C represents the concentration of 

the substance under consideration, while 

D is the translational diffusion coefficient. 

Eq. (1) describes diffusion phenomena 

in an isotropic medium for the one-, 

two- or three-dimensional case. Solutions 

can be derived for certain initial 

and boundary conditions [3]. We focus 

on the following simple case which is relevant 

to FRAP experiments on a CLSM: 

(2) 

Herein, r represents the generalized (radial) 

coordinate, and M denotes the total 

amount of the diffusing species in the 

three-dimensional case, while in the two- 

or one-dimensional case, it stands for the 

amount of substance per unit length or 

unit area, respectively. d symbolizes the 

diffusion dimension. 

The shapes of the functions according 

to Eq. (2) are Gaussians with an e- 1/2 -radius 

of . They broaden and become 

shallower with increasing time. Since the 

decrease of the prefactor with time depends 

on the dimensionality and the 

amount of diffusing substance, it is suited 

to estimate either of these parameters. 

On the other hand, the diffusion coefficient 

can be derived from the course of 

the widths. 

If we consider FRAP processes, the 

situation is in essence just inversed, since 

bleaching takes away a certain amount 

of the fluorescent molecules, while the 

considerations above dealt with a local 

excess of a substance. This means no 

more than a change of sign combined 

with a baseline shift. 

By utilizing a CLSM and an objective 

with a low NA value, it is readily possible 

to bleach geometries into the sample that 

correspond closely to the cases of diffusion 

from a plane (one-dimensional) or a 

line source (two-dimensional). Hence, 

the solutions of Fick’s second law are applicable 

to FRAP processes too. One obtains

eady for 

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where I represents the fluorescence intensity 

at the position r and the time t after 

bleaching. M now is formally a fluorescence 

intensity (per length or area for 

d = 2 or 1) corresponding to the amount 

of fluorophore destroyed by bleaching. w 

is the e- 1/2 -radius of the Gaussian function 

and I 0 denotes the background intensity 

at r → ∞. 

The quantity D appears in the prefactor 

and the exponent of the Gaussian, 

hence both terms can be used to determine 

it. Comparison of the exponential 

terms of Eq. (3) yields 

(4) 

and therewith the possibility to deduce 

the diffusion coefficient by plotting w² vs. 

t for a series of intensity profiles obtained 

from images taken during the recovery 

process. A straight line with slope 2D 

passing through the origin should be expected. 

On the other hand, we obtain an algebraic 

decay for the time dependence of 

A(t): 

(5) 

As an alternative, Eq. (5) can be written 

in logarithmic form: 

(6) 

This means that a plot of log A versus log 

t should give a straight line with a slope 

of –d/2, forming the basis for the experimental 

determination of the dimensionality 

of the diffusion process. 

Analysis of Spatially Resolved FRAP – 

Practical Realization 

Figure 1 shows some intensity profiles 

taken from FRAP measurements on solutions 

of rhodamine B in glycerol. One observes 

Gaussians as expected from Eq. 

(3). Plots of the fit parameters w² vs. t 

thus yield straight lines. However, they 

do not pass through the origin but show 

a notable intercept as illustrated in figure 

2a. Moreover, double-logarithmic 

plots of A vs. t do not give straight lines 

but curves as depicted in figure 2b. 

These findings can be addressed to 

the fact that in a real experiment, the initial 

conditions used so far to obtain ex- 


(3) 

Fig. 1: Images and corresponding intensity profiles obtained from FRAP experiments 

on rhodamine B in glycerol for one-dimensional diffusion after bleaching a line (a) 

and two-dimensional diffusion after bleaching a point (b) into the focal plane. They 

show the sample before bleaching and at 1s (�), 30 s (�), and 90 s (�) after bleaching, 

respectively. The image dimensions are about 80 × 80 μm² in each case. Full 

lines in the diagrams represent fits to Gaussians like Eq. (3). Adapted from [2]. 

Fig. 2: Plots of w² vs. t and log A vs. log t for two examples of FRAP experiments on 

rhodamine B in glycerol. �: one-dimensional line-bleaching, �: two-dimensional 

point-bleaching. (a) & (b) represent the real case accompanied by deviations from 

ideality. Introduction of an appropriate time shift t 0 transforms the situation into 

the ideal case as illustrated in (c) & (d). Adapted from [2]. 

act solutions of the diffusion equation are 

never met perfectly. Instead of starting 

from a Dirac function (in one, two, or 

three dimensions) for the concentration 

profile at t = 0, the initial state will be 

characterized by some finite spatial 

width. Furthermore, some time is required 

to achieve bleaching. This both 

means that it is not possible to exactly 

define the zero point of the time scale. 

Since in an experiment the initial conditions 

are only approximated, one should 

be aware that deviations from Eq. (2) are 

expected when such approximations cannot 

be neglected. That is the case when 

the diffusion occurs fast in comparison 

with the duration of the bleach pulse, or 

when concentration profiles are analyzed 

on a length scale comparable to the spatial 

width of the bleaching beam. 

However, with the aid of a procedure 

described recently [2] one is able to take 

the deviations from ideality into account 

by a proper shift of the experimental 

time scale. Advantageously, the two independent 

criteria requiring 

i the intercept of the plot of w² vs. t to 

vanish 

ii the curvature of the plot of log A vs. 

log t to be minimized 

can be consistently employed to determine 

the appropriate shift t 0 . For convenience, 

we proceed in two steps: after 

deriving a first estimate for t 0 from an 

extrapolation of the w² vs. t plot (crite-

Fig. 3: D-distributions estimated from mixtures of differently sized 

fluorescent polystyrene microspheres (Molecular Probes, Eugene) 

in aqueous suspension. The shading below the signals visualizes 

their intensity in an alternative form. 

rion i), fine-tuning is achieved 

by plotting log A vs. log (t + t 0) 

and varying t 0 iteratively with 

respect to fulfill criterion ii. 

Therewith, the graphs depicted 

in figure 2a and 2b 

turn into the ones shown in 

figure 2c and 2d, which now 

correspond to the expectations 

according to Eq. (4) and 

(6), i.e., the ideal case. 

A number of experiments 

analyzed by the method 

shows that the diffusion coefficient 

and the expected dimensionality 

are obtained 

with high accuracy. Some results 

from measurements on 

rhodamine B in glycerol as 

well as aqueous suspensions 

of differently sized fluorescent 

microspheres are compiled 

in table 1 and 2, illustrating 

that both, D and d, can 

be estimated in consistent 

manner. Note that these analyses 

do not require any kind 

of previous calibration. 

Multi-component FRAP 

Processes 

Besides the possibilities for 

the evaluation of simple FRAP 

experiments as discussed 

above, the usage of temporal 

and spatial information also 

enables the estimation of multiple 

diffusion coefficients 

from experiments where 

more than one species is involved. 

The principle for this 

is to superimpose Eq. (3) for 

many independently diffusing 

components with diffusion coefficients 

D i and corresponding 

fractions M i and to fit the 

entire dataset of fluorescence 

intensity profiles as a function 

of time and space I(r,t). 

This yields a set of D i;M i-pairs, 

i.e., the distribution of diffusion 

coefficients. Figure 3 depicts 

several examples for 

such distributions as derived 

from mixtures of the differently 

sized spherical probes 

mentioned above (cf. table 2). 

Ratios of 10:90, 30:70, 50:50, 

70:30 and 90:10 were employed 

in this respect. The results 

illustrated show that one 

accurately finds both, the Dvalues 

of the pure compounds 

as well as the correct compositions 

when analyzing the 

mixtures. Note that these 

types of analyses can again 

be performed without any 

need for calibration. Moreover, 

they also allow one to determine 

the accompanying 

distributions of dimensionalities 

(results not shown) and to 

account for effects that sometimes 

penetrate a FRAP experiment, 

e.g., a continuous 

loss of fluorescence intensity 

due to bleaching during the 

scanning process. 

Closing Remark 

All procedures for the systematic 

evaluation of spatially 

resolved FRAP data mentioned 

here are fully implemented 

in a MATLAB software, 

which is available upon 

request from the authors. 

Note that in principle, one 

could also imagine to modify 

them with regard to allow for 

the analysis of anisotropic 

FRAP experiments, what is 

presently in progress in our 

group. 

References: 

[1] Meyvis, T. K. L., et al., Pharm. 

Res. 16, 1153–1162 (1999). 

[2] Seiffert, S., Oppermann W., J. 

Microsc. 220, 20–30 (2005). 

[3] Crank, J., The Mathematics of 

Diffusion, Clarendon Press, Ox- 

ford, 1975. 


experiment D/(μm s 2–1 ) d 

line-bleaching (one-dimensional diffusion) 0.58 ± 0.04 1.02 ± 0.03 

point-bleaching (two-dimensional diffusion) 0.57 ± 0.11 2.00 ± 0.09 

Tab. 1: Translational diffusion coefficients (D) and dimensionalities (d) obtained from 

FRAP measurements on solutions of rhodamine B in glycerol. Adapted from [2]. 

method r (24 nm microspheres)/nm r (100 nm microspheres)/nm 

FRAP 17.2 50.2 

DLS 16.7 49.5 

UA 14.0 51.7 

AFM 12.2 49.1 

Tab. 2: (Hydrodynamic) radii of fluorescently labeled microspheres with diameters of 

about 24 nm and 100 nm as estimated by FRAP in comparison with dynamic light 

scattering (DLS), ultracentrifugal analysis (UA) and atomic force microscopy (AFM). 

�� 

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A publication focusing on the analy- 

sis of multi-component FRAP exper- 

iments is presently in preparation. 

Contact: 

Sebastian Seiffert 

Gerald I. Hauser 

Wilhelm Oppermann 

Clausthal University of Technology 

Institute of Physical Chemistry 

Clausthal-Zellerfeld, Germany 

Tel.: +49 5323 72 3642 

Fax.: +49 5323 72 2863 

sebastian.seiffert@tu-clausthal.de 

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Wide-field CARS-Microscopy 

Functional Imaging at a Glimpse 

Coherent anti-Stokes Raman scattering (CARS) microscopy is a branch of nonlinear microscopy that allows 

chemical imaging of targeted vibrational transitions in unstained samples. A resonantly enhanced blueshifted 

CARS signal is generated from NIR or visible light, thus the method is more sensitive than normal Raman 

microscopy and offers better resolution than IR microscopy. CARS microscopes are mostly set up as 

confocal scanning microscopes, but wide-field approaches are possible as well. 

Brief Introduction to CARS-microscopy 

The beating between two laser beams 

overlapping at a sample in space and 

time can excite vibrations of molecules in 

the sample, if their difference in frequency 

matches a vibrational transition. 

In this case inelastic coherent anti-Stokes 

Raman scattering (CARS) takes place and 

Fig.1: Fluorescence-label-free differentiation of 

polymer beads by wide-field CARS microscopy. 


a blue-shifted (anti-Stokes) signal beam 

is emitted. As this is a nonlinear optical 

process, high intensities, i.e. pulsed lasers, 

are required. The CARS signal grows 

proportional to the Stokes intensity and 

quadratically with the pump intensity and 

the number of resonant scatterers. CARSmicroscopy 

allows labelling-free mapping 

of spatial distributions of organic molecules 

as shown in figure 1. As no staining 

with fluorescent dyes is necessary, one 

avoids problems of photobleaching and 

phototoxicity alltogether. 

Since pioneering work in the early 

eighties [1], over the last 10 years CARS 

microscopy has developed into a powerful 

new microscopic tool, triggered especially 

by the work of Zumbusch, Holtom, 

and Xie [2] in the late nineties. Many 

technical variations of CARS microscopy 

have been developed (see [3] for an overview) 

most of them motivated by the necessity 

to increase the signal to noise ratio, 

which is often affected by a nonlinear 

CARS background that is not chemically 

selective. 

Keywords: 

coherent anti-Stokes Raman scattering, vibrational 

imaging, labelling-free imaging, nonlinear 

microscopy, wide-field microscopy 

Setup and Instrumentation 

Most CARS microscopes are based on 

confocal scanning microscopy, working 

with tightly focussed beams of NIR laser 

pulses in the pico- or femtosecond regime 

and with oil-immersion objectives of high 

numerical aperture (NA). The confocal 

(CF) setup has the advantage of high spatial 

resolution, but requires scanning of 

the sample, which is avoided in wide-field 

(WF) CARS microscopy where one can 

take an image of the whole sample “all at 

once”. In contrast to scanning CARS-microscopy, 

where the tight focussing of the 

lasers leads to large momentum uncertainty 

and thus makes momentum conservation 

rather uncritical, in wide-field 

CARS the phase-matching condition imposes 

a restriction that has to be met. In 

our case a special excitation geometry, 

which satisfies momentum conservation 

among the beams in the sample, enables 

efficient build-up of a CARS signal from 

the scatterers [4–5]. The idea for the implementation 

of wide-field CARS in the

“extremely-folded box-CARS geometry” 

is schematically shown in figure 2. We 

use nanosecond pulses and wavelengths 

in the NIR, for example 1064 nm for the 

Stokes pulse and a wavelength around 

800 nm for the pump pulse which is tuned 

by an optical parametric oscillator (OPO). 


Fig. 2: Schematic sketch of a wide-field CARS-microscope: The excitation laser beams distribute the 

intensity of ns pulse trains homogeneously over the whole sample region of interest. The pump beam is 

coupled through a high numerical aperture dark field condenser delivering a cone of light from above, 

while the Stokes beam is guided in from below through the objective of the inverted microscope. This 

gives rise to an anti-Stokes signal beam that counter-propagates with respect to the Stokes beam, and 

which can conveniently be read out through the microscope objective. Note that the high numerical 

aperture of the dark-field condenser provides a narrow “sheet of light” illumination which reduces the 

non-resonant CARS background. 

Fig. 3: Selective imaging of a mix of polymer beads at two CARS resonances. The 1,44 µm large particles 

are excited at two different CARS resonances which are selected by tuning the NIR pump beam. 

The PS and the PMMA CARS images were overlayed in the image on the left, the measured Raman 

spectra of the Raman-active resonances that were used are shown on the right. 

Fig. 4: Snapshot image of an olive-oil droplet that was imaged with 3 ns, with a single pair of nanosecond 

pulses. 

This gives access to the aliphatic stretching 

vibrations between 2850 cm –1 and 

2950 cm –1 , which are abundant in organic 

molecules and give a particularly 

strong CARS signal. Other non-scanning 

apporaches [6] are based on collinear geometries 

and rely on the scattering struc- 

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tures themselves to redirect the beams to 

satisfy phase-matching. As phase-matching 

is not satisfied in the bulk solvent, the 

nonlinear background from the solvent is 

effectively prevented. 

Performance 


As a consequence of the photon energy 

conservation ω_ aS = 2ω_ P – ω_ S, the anti- 

Stokes wavelength can be considerably 

shorter than the beams exciting the sample. 

The combination of NIR excitation and 

detection at shorter wavelength makes it 

possible to have deep penetration depth 

into the sample volume and good optical 

resolution at the same time. The lateral 

spatial resolution of a WF-CARS microscope 

is on the same order as for an ordinary 

WF-microscope. The NA of the 

microscope objective and the CARS-wavelength 

determine the diffraction-limit of 

the spatial resolution, whereas the NA of 

the dark-field condensor is adapted to fulfil 

phase-matching, and thus determines 

the efficiency, rather than the resolution. 

The actually achieved imaging performance 

depends not only on the wavelengths 

and the NA of the microscope objective 

and the dark-field condensor, but also on 

the details of the coherent nonlinear interaction 

in the sample (e.g.on the local refractive 

index and its influence on the 

phase-matching). In practice, we have 

been able to image spherical polymer particles 

as small as 500 nm in diameter. Axially 

one can achieve optical sectioning, 

since the region where all interacting 

beams have sufficiently high intensity is 

only a few µm wide. The nonlinear dependence 

on the pump field favors optical 

sectioning, as in other types of nonlinear 

microscopes. We have measured the optical 

sectioning power of our system to be 

on the order of 3–4 µm. 

The sensitivity of the imaging depends 

on the coupling strength of the trageted 

Raman-active resonance and quadratically 

on the number of scattereres in the 

sample. Presently the sensitivity of CF- 

CARS microscopy lies around 10 5 vibrating 

molecules per focal volume for probing 

the symmetric CH 2 stretching mode 

[7], which allows to image single lipid bilayers 

and cellular membranes [8]. 

Figure 3 examplifies the typical performance 

of our wide-field system. A mix 

of two species of 1.44 µm polymer beads 

in water is imaged. For polystyrol (PS) 

the frequency difference between Stokes 

and pump beam was tuned to a CARS 

resonance at 3052 cm –1 shown in the upper 

spectrum on the right in figure 3. Another 

image was recorded while selec- 


Fig. 5: WF-CARS images of small pulmonary surfactant vesicles inside living lung cells from rats. 

tively exciting the polymethyl methacrylate 

(PMMA) beads at 2943 cm –1 (see lower 

spectrum). The beads are spatially and 

chemically well resolved, with the spectral 

resolution of our WF-CARS microscope 

being limited by the bandwidth of 

the OPO, which is about 5 cm –1 . 

A specialty of our WF-CARS microscope 

is the option to take snapshots with 

only a single pair of pump- and probe 

pulses of 3 ns duration (see fig. 4). This 

enables real-time imaging of micrometer-sized 

structures, which could be useful 

to study transport phaenomena in biological 

systems. However, the speed of 

acquisition is limited by the repetition 

rate of the OPO which is typically less 

than 50 Hz. 

Applications 

Lipids give a particularly strong CARS 

signals, thus CARS-microscopy may become 

an established tool in lipid metabolism 

research, which is a very important 

topic at present. Many groups are currently 

setting up CARS experiments with 

living cells, e.g. trying to visualize 

changes in the location, distribution, or 

concentration of fatty acids. 

Our group is interested in functional 

imaging of phospholipid-rich vesicles inside 

living alveolar cells. These cells, 

called alveolar type II cells, are specialized 

to generate cell vesicles (the “lamellar 

bodies”) that contain pulmonary surfactant. 

These vesicles are released from 

the cells by exocytosis to provide the 

monomolecular lipid film of surfactant 

that is required to lower the surface tension 

in the alveoli. Figure 5 shows CARS 

images where these small intracellular 

vesicles selectively light up inside living 

alveolar cells. 

Since CARS-microscopy is a labellingfree 

method it may prove the ideal 

method for risk assesment of nanoparticles, 

which in modern nanomedicine are 

becoming increasingly popular, e.g. as 

contrast agents or for drug delivery. To 

date there often exists only limited or unsatisfactory 

information on the “fate” of 

the nanoparticles in the human body. Be- 

ing an optical method, diagnostics deep 

within the body is impossible – but CARS 

microscopy can provide information on 

the up-take or clearance of the nanoparticles 

and their degradation products in 

cell or tissue studies, for instance, 

whether they build aggregates anywhere 

in the cells or tissues. The fact that the 

no prior marking of the particles with 

fluorescent dyes is necessary – which 

might change the behavior in the tissue 

– could be of special importance. 

We expect CARS-microscopy to reach 

its full potential in the next years, developing 

a user-friendly and compact optical 

tool that provides answers for many 

of tomorrow‘s urgent questions in the life 

and material sciences. 

Acknowledgements 

This work was supported by the Austrian 

Science Fund (FWF-Project Nr. P16658_ 

N02). 

References: 

[1] Duncan, M., et al., Opt. Lett. 7, 350–51 

(1982). 

[2] Zumbusch, A., et al., Phys. Rev. Lett. 82, 

4142–45 (1999). 

[3] Volkmer, A., J. Phys. D.: Appl. Phys. 38, R59– 

81 (2005). 

[4] Heinrich, C., et al., Appl. Phys. Lett. 84, 816– 

18 (2004). 

[5] Heinrich, C., et al., New J. Phys. 8, 36 (2006). 

[6] Toytman, I., et al., Opt. Lett. 32, 1941–43 

(2007). 

[7] Potma, E. O., et al., Optics Letters 31, 241– 

243 (2006). 

[8] Potma, E. O., Xie, X. S., Journal of Raman 

Spectroscopy 34, 642–50 (2003). 

Contact: 

Prof. Dr. M.A.M. Ritsch-Marte 

Chair of Biomedical Physics 

Prof. Dr. S. Bernet 

Dr. C. Heinrich 

Innsbruck Medical University 

Innsbruck, Austria 

Tel./Fax: +43 512 9003 70870 

monika.ritsch-marte@i-med.ac.at 

www2.i-med.ac.at/medphysik/

Next Generation Light Sources for Imaging 

Fibre Lasers – Compact, Cost-Effective, Turnkey Solutions 

Lasers continue to be increasingly important components within biological imaging and analysis systems – 

from Argon-Ion and HeNe lasers used within flow cytometry and confocal microscopy, to femtosecond Ti: 

Sapphire and DPSS femtosecond sources in multi-photon microscopy. However, many commercial imaging 

systems are still limited in performance by the availability of suitable laser sources – both in the limited 

availability of wavelengths and in the size, cost and reliability of conventional laser technologies. Ultrafast 

fibre lasers offer turnkey, compact and reliable solutions for next generation biomedical imaging systems. 

Here we introduce the basic concept of the ultrafast fibre laser and describe some of the applications and 

benefits of this technology within biophotonics research and imaging systems. 

Ultrafast fibre lasers, with their compact 

form, inherent reliability and low cost of 

ownership are becoming the laser of 

choice for many biomedical imaging applications, 

challenging the Ti:Sapphire 

laser within multi-photon excitation microscopy 

and diode, HeNe and Ar-Ion lasers 

within fluorescence imaging. 

The Master Oscillator, Power Amplifier 

(MOPA) architecture of Fianium’s ultrafast 

fibre lasers provides simplicity 

and flexibility by design [fig. 1]. The 

MOPA comprises two independent modules 

– a low-power femtosecond or picosecond 

fibre laser followed by a high 

power diode-pumped fibre amplifier. By 

independently changing the parameters 

of the oscillator or the amplifier modules, 

one can achieve very different performance 

parameters from the laser, tailored 

to a given application. This approach has 

quickly enabled ultrafast fibre lasers to 

meet the growing demands of a wide 

range of applications. 

The master oscillator is an all-fibre, 

passively modelocked laser which is both 

turnkey operated and self-starting without 

the need for any adjustment. The 

pulse repetition rates of the laser (from 

less than 1MHz to several hundred MHz) 

are ideally suited to quasi-cw laser applications 

and also for lifetime imaging applications. 

For applications within microscopy, ultrafast 

lasers are typically associated 

with multi-photon fluorescence microscopy, 

where femtosecond Ti:Sapphire lasers 

have been the historic laser of choice. 

While not offering the broad wavelength 

tunability of the Ti:Sapphire, Fianium’s 

FP1060-s femtosecond lasers do offer a 

low-cost, compact solution at discrete 

wavelengths from 980 nm to 1100 nm 

and offer potential for incorporation into 

any existing microscope system. 

Delivering average powers up to 5 

Watts and with pulse durations shorter 

than 250 femtoseconds, the long wave- 


John Clowes 

lengths of the FP1060, extending beyond 

the tuning limits of most Ti:Sapphire 

sources, offer many benefits for Two Photon 

Fluorescence (TPF) and Second Harmonic 

Generation (SHG) microscopy [1, 2]. 

The FP1060 high power lasers from 

Fianium operate within the picosecond 

or femtosecond regime and can provide 

pulse energies from a few pico-joules to 

ten microJoules, particularly important 

for materials processing, both of devices 

and of tissues. 

Extension of the FP1060 laser source 

from the near Infra Red to the visible and 

UV region of the spectrum, is achieved 

through nonlinear frequency conversion 

techniques including harmonic generation 

to 532 nm, 355 nm and 266 nm and 

in the generation of ultra broad band 

“supercontinuum” spectra – a phenomenon 

that will have a huge impact on next 

generation biomedical imaging systems. 

The supercontinuum fibre laser is 

based on a high power picosecond source 



Fig. 1: The all-fibre MOPA (Master Oscillator Power Amplifier) – flexibility to meet 

the demands of a huge application space. 

(FP1060) and highly nonlinear photonic 

crystal fibre. The nonlinear interaction 

between the high intensity pulsed optical 

field within the tight confinement of a silica 

optical fibre waveguide, results in the 

generation of a continuous spectrum 

spanning from the visible (below 400 nm) 

extending in the IR to beyond 2 um. 

Fianium’s SC400 and SC450 supercontinuum 

fibre lasers, utilising high 

pulse repetition rates in the MHz range, 

deliver high spectral brightness in the 

range of several milli-Watts per nm 

across the entire optical spectrum. Filtration 

of several nm of this spectrum 

can deliver tens of mW optical power at 

any wavelength required – a vast improvement 

over the discrete wavelengths 

offered by conventional diode and HeNe 

laser sources. 

Furthermore, the repetition rates of 

the supercontinuum make them ideally 

suited to fluorescence imaging applications 

requiring either a quasi-continuous 

wave or a pulsed regime for time-resolved 

measurements. 

The remainder of this article focuses 

on examples of the use of Fianium SC400 

and SC450 supercontinuum fibre lasers 

within fluorescence imaging. 

Single Source Solution for Fluorescence 

Imaging 

Flow cytometers (or fluorescent activated 

cell sorters) are critical tools for biomedical 

research. These complex instruments 

measure the properties of individual 

cells, often by detecting fluorescent 

molecules (fluorophores) attached to 

their surface. The fluorescent molecules 

act as probes for analyzing the identity of 

cells for studying the immune system, 

identifying cancer cells, and diagnosing 

disease. 


Flow cytometers rely almost exclusively 

on lasers for exciting fluorophores. 

While the coherence and power level of 

lasers makes them ideal light sources for 

illuminating individual cells, their discrete 

wavelengths limits the types of fluorescent 

probes that can be analyzed by 

flow cytometry. 

Although solid state laser technology 

has increased the variety of discrete laser 

wavelengths available, there are still significant 

gaps in the excitation capabilities 

that put limitations of the fluorescent 

probes used for biomedical analysis. 

Confocal fluorescence microscopy has 

similar laser source requirements to 

those of flow cytometry and is one of the 

most powerful techniques for probing a 

wide range of phenomena within biological 

sciences. 

Almost all commercially available 

confocal microscope systems use a combination 

of lasers to excite fluorescence 

at a few discrete wavelengths within the 

visible region of the spectrum. As with 

flow cytometry, only a small number of 

excitation wavelengths are available by 

using fixed wavelength lasers. Many 

fluorophores are therefore unusable because 

of this limitation. 

A recent development, which further 

aids fluorophore excitation, is the combination 

of multi-channel AOTF’s (acoustooptic-tunable-filters) 

with the supercontinuum 

fibre laser. An SC400 

supercontinuum laser in conjunction 

with an 8-channel AOTF, enables the delivery 

of up to 8 laser lines within the visible 

region of the spectrum, each individually 

tunable across the entire spectrum 

and all within the same, diffraction limited 

collinear beam. 

The supercontinuum and AOTF provides 

the flexibility required for optimal 

excitation and detection of a wide range 

Fig. 2: The visible spectrum of the SC400 spatially dispersed 

through a transmission diffraction grating demonstrates the continuous 

nature of the spectrum from violet to near Infra-Red. 

of fluorophores within flow cytometers 

or fluorescence confocal microscopes. 

The laser can be tuned precisely to the 

excitation peak of the fluorescent probes 

needed for analysis. 

Furthermore, the supercontinuum not 

only improves performance but is also 

cost-effective. For example, with the exception 

of the 355 nm frequency tripled 

Nd:YVO4 laser, all laser sources and 

beam combination optics installed within 

the flow cytometer platform shown in 

fig. 3 can be replaced by a single SC400- 

AOTF. Furthermore, with continued advances 

in supercontinuum fibre laser 

technology, it is only a matter of time before 

the spectrum is further expanded 

down to the UV, enabling the replacement 

of all lasers within even the highest 

specification imaging systems. 

While commercial vendors of confocal 

microscopes will soon incorporate supercontinuum 

fibre lasers within their imaging 

systems, it is often research groups 

who are first to investigate the technology. 

Dr. Clemens Kaminski and his group 

at the Laser Analytics Group in Cambridge 

(UK) have recently incorporated a 

fibre supercontinuum laser within an Olympus 

Fluoview microscope scanning 

unit to demonstrate 2-D, 3-D and livecell 

imaging. 

Figure 4 demonstrates an example of 

2-D high resolution fluorescent confocal 

imaging using an SC450 supercontinuum 

fibre laser and AOTF system. In this example 

a Convallaria Majallis (Lily-of-the- 

Valley) specimen is stained with Safranin 

and Fast Green, having peak excitation 

wavelengths of 530 nm and 620 nm respectively. 

Since the two dyes have affinities 

to different regions of the sample, 

scanning the excitation wavelength highlights 

different regions of the sample. 

Fast Green stains the cellulosic cell walls

present over the whole sample (620 nm 

and 640 nm images) where the Safranin 

dye stains lignin – present within the endodermis 

and xylem, highlighted within 

[figure 3] at 540 nm to 560 nm excitation 

wavelengths. 

Looking Forward 

The need for suitable illumination 

sources across a wide range of biomedical 

applications including Flow Cytometry, 

Optical Coherence Tomography, 

CARS Microscopy and various guises of 

Confocal Fluorescence Microscopy continues 

to drive the development of laser 

technology. 

Over the past 5 years, ultrafast fibre 

lasers have started to challenge solidstate, 

diode and gas lasers across numerous 

markets and we expect the ongoing 

rapid development in performance and 


Fig. 3: Example high-spec. flow cytometry development platform showing up to 8 discrete laser lines 

and associated beam combination optics – can be replaced by a single supercontinuum laser. Image 

courtesy of Dr. William Telford, National Institute of Health / National Cancer Institute, USA. 

Fig. 4: Image courtesy of the Laser Analytics Group, University of Cambridge, UK2-D Fluorescent confocal 

imaging of the rhizome of Convallaria Majalis (Lily-of-the-Valley) stained with Safranin and Fast 

Green dyes (peak excitation wavelengths of 530 nm and 620 nm respectively). 

reduction in laser cost to further establish 

this technology as the illumination 

source for next generation imaging systems. 

References: 

[1] Sacconi, L., et al., PNAS, V.103, No.9, 3124– 

3129 (2006) 

[2] Dombeck, D. A. et al., J Neurophysiology, 

3628–3636, 94 (2005) 

[3] Kapoor, V. et al., Nature Methods, V.4, N.9, 

678–679 (2007) 

[4] Frank, J. H. et al., Jnl of Microscopy, V.227, 

Issue 3, pp. 203, (2007) 

Contact: 

John Clowes, PhD 

R & D Manager 

Fianium Ltd 

Hamble, Southampton, UK 

Tel.: +44 2380 458776 

Fax: +44 2380 458734 

john.clowes@fianium.com 


Fundamental Knowledge 

Part 1 

D i g i ta l M at e r i a l s a n a ly s i s 

Ever since scientists started investigating our world, 

two aims have had the highest priority. One simply 

understands how everything around us is organised. 

The other is using this knowledge to make our 

lives more productive and comfortable. Much of 

our mental potential is developed by applying our 5 

senses in direct day-to-day practice. Of the 5 senses, 

sight is a critical tool with very high potential and 

bandwidth for extracting information making it a 

powerful interface for gathering knowledge. 

“I saw it with my own eyes!” – think 

about the significance of that expression. 

It provides a hint as to why optical microscopy 

is such a popular method for 

exploring the structures of materials. All 

other information received by instrumental 

sensory technologies in materials science 

research enters our minds via some 

secondary visualisation process. Though 

optical microscopy itself makes increasing 

use of integrated digital technology 

for interfacing image information, it is 

still closest to the actual process of seeing 

with the naked eye – i.e., receiving 

image information directly. 

How do people actually receive information 

about their surroundings? How 

does a person receive the image information 

offered via a microscope? The information-bearing 

medium is visible light 

and the information it carries is as a result 

of its various interactions with physical 

matter. The electromagnetic radiation 

of visible light is the point of 

departure for light microscopy in general, 

as well as in the metallography 

field. It is necessary to understand terms 

such as reflection, polarization and interference, 

alongside fundamental quantities 

closely linked to them such as amplitude, 

radiation and phase shift. This 

facilitates grasping the various light-microscopical 

contrast and imaging procedures 

for metallography, which involves 

a good deal of fundamental knowledge 

on the physical nature of light. This part 

of the chapter is from one of our “Digital 

Materials Analysis” series, and therefore, 

deals with the optical fundamentals of 

light and the interaction with a sample. 

Subsequently, the light microscopy that 

is commonly used in metallography is introduced 

and explained in part two. The 


Fig. 1: Schematic display of the visible spectrum of electromagnetic radiation and the resulting colour 

ranges. 

final segment deals with the contrasts 

that play an important role in imaging 

metallographic samples. 

Optical Fundamentals 

The fundamentals described below on 

light properties focus on the information 

needed to understand the material presented 

and are not exhaustive. For more 

advanced detail and more sophisticated 

theoretical fundamentals, we would advise 

interested readers to consult the extensive 

web pages of Florida State University, 

for example, on microscopy and 

the related physical background information 

(www.microscopy.fsu.edu/primer/ 

index.html). 

Visible Light 

In everyday language, light is understood 

as the range of electromagnetic radiation 

that we can see with the naked eye. This 

comprises a wavelength spectrum of ca. 

400 nm–750 nm. The colours of light that 

can be seen by the eye occur due to varying 

wavelengths and their blending (fig. 

1). The adjoining spectra from the deepred 

and infrared range (to 1200 nm) as 

well as the near ultraviolet light (200– 

380 nm) are used in light microscopy. 

Even though these segments of the spectrum 

are not visible to our eyes, these 

segments can be acquired using suitable 

detectors, CCD cameras for example, and 

made available via a monitor for imaging 

or analysis. 

Wavelength, Frequency, Energy 

The smallest distance between two points 

of the same phase of a wave is referred 

to as wavelength λ (fig. 2). The two points 

are precisely in the same phase when 

they have the same deflection, ie, amplitude 

(A), and move in the same direction 

through time. The wavelength of light 

has a direct correspondence with the frequency, 

as does any other form of electromagnetic 

radiation (ν): i.e. it is inversely 

proportional. When entering or 

traversing a medium, the speed and 

wavelength of the light are changed. The 

frequency, however, remains unchanged. 

Alongside its wave properties, light is 

quantified in photons. A photon (quantum 

of electromagnetic energy) is considered 

the smallest amount of energy of 

any frequency of electromagnetic radiation. 

The energy of a photon is directly 

proportional to frequency and thus indirectly 

proportional to wavelength. This 

means that energy increases as frequency 

increases and wavelength shortens. 

This is why shortwave radiation is 

dangerous, since it has high energy in a 

narrow range. It can have dangerous effects 

on organisms, for example there is 

a well known correlation between UV 

light and skin cancer. 

The brightness of light perceived by 

an animal depends on the number of 

photons that reach the eye per unit time. 

It can be described, albeit simply, as the 

amount of wave deflection or amplitude 

(fig. 2). Other factors are the colour or 

wavelength and contrast (to the background) 

as well as its expansion. The 

eye‘s sensitivity to brightness is greatest 

with green-yellow colour tones (500– 

560 nm). In this case, small points of 

light are interpreted as brighter than 

larger ones of other colours, even though 

they have the same physical light intensity. 

The eye can discern 50–60 levels of

Fig. 2: Schematic diagram of waves with the 

wavelength (λ), of the smallest distance of two 

points of the same phase of a wave and of the 

deflection height (amplitude: A). The lower 

example shows how a wave of light is phase 

delayed when passing through an optically 

denser medium (eg, glass or a cell). The amplitude 

remains practically the same. 

Fig. 3: Reflection is where electromagnetic 

radiation is deflected and “bounced back“. This 

takes place either at the boundary between two 

media (surface reflection) or within the interior 

of a medium (volume reflection). Transmission is 

the expansion of electromagnetic radiation 

passing through a medium. Diffusion can take 

place in both phenomena. Diffusion is where a 

ray of light with a certain dispersive direction is 

deflected in many different directions (diffuse 

reflection or diffuse transmission). If there is no 

scattering upon reflection or transmission, the 

ray of light is deflected in a certain direction in 

accordance with the physical laws of optics 

(directed light reflection or transmission). Reflection, 

transmission and scattering do not alter 

the frequency of a wave. Absorption is the 

conversion of radiation into a different form of 

energy (usually heat) that occurs upon the interaction 

of radiation with matter. 

brightness in gray-scale images. A computer 

monitor on the other hand shows 

images with 256 gray scales (8 bits) and 

digital cameras, depending on the model, 

can acquire up to 4096 gray scales (12 

bits) – inasmuch these are actually 

present in the image. These numbers 

show how digital image acquisition exponentially 

expands what image analysis 

can do – especially compared with viewing 

a sample with the naked eye alone. 

Light – Object Interactions 

As soon as light encounters an object, 

three phenomena can take place: reflection 

(ρ), absorption (α) and transmission 

(τ). These processes, and their respective 

interrelationships, vary depending on the 

object’s parameters – such as material 

(refraction index n), surface, thickness 

(absorption coefficient µ) and colour. Important 

effects such as polarization or interference 

are direct results of these phenomena, 

which are decisive for contrast 

formation, imaging and material detection 

in the incident light microscope used 

for metal microscopy. 

Absorption and Scatter 

In our day-to-day lives we experience 

how colourless light, also frequently referred 

to as white light, can diminish in 

brightness due to absorption. This explains 

how much brighter a room looks 

after the windows have been cleaned. 

When a wave is absorbed by an absorbent, 

homogeneous material, the absorption 

probability (per unit of length) is the 

same at any penetration depth. According 

to the Beer-Lambert law, the amount 

of photons non-absorbed or scattered in 

the process depends on the layer-thick- 


Fig. 4: When photons encounter a surface there are various interactions with the electrons 

and the material. Fluorescence may occur, which is the absorption of light of a particular 

wavelength (excitation light) involving various molecules and the simultaneous emission of 

light at longer wavelengths. 

ness of the material. This explains why 

the sky seems darker and the sun appears 

brighter in the mountains. 

But it’s not just the intensity that decreases. 

The light absorption of surfaces 

is usually dependent upon frequency and 

also varies in strength according to the 

surface colour. This kind of frequencydependent 

absorption alters the remaining 

wavelength characteristic of the light, 

which results in us seeing colours. 

Absorption is caused by scatter, i.e. 

the interactions of the light quanta with 

the free and bonded electrons of the material. 

Elastic and inelastic scattering are 

of decisive significance here. Elastic scattering 

is where the sum of energy of the 

incident photons is too small to excite the 

atom. The energy of the scattered photon 

remains unchanged but its direction 

changes (fig. 4). Inelastic scattering is 

where the photon loses a part of its energy 

as excitation energy of an atom or 

during ionization processes (fig. 4). After 

scatter, the scattered radiation usually 

has an altered diffusional direction. Inelastic 

scattering also means changed energy 

(frequency ν) and sometimes altered 

polarization direction. 

Table 1 describes how materials science 

visual effects are linked to both 

elastic and inelastic scattering. 

Refraction and Refraction Index 

Various transparent media such as air, 

water and glass slow down the light passing 

through them (phase shift) to differ- 



Table 1: Overview of interaction of photons and matter 

Visual effect Typical materials Physical origin Images 

Total transparency accompanied 

by gradual reflection 

Most gasses, some liquids, some 

mineral substances like glass (SiO 2), 

quartz and other crystalline structures 

of similar physical behaviour with 

smooth surfaces and homogeneous 

structure. 

Depending on their refractive index 

they are used for optical imaging 

lenses. 

Diffuse transparency As above, but contaminated with 

scattering substances or diffused 

interfaces. 

Total reflectance All metals and other materials whose 

physical behaviour does not bind 

electrons by strong individual forces to 

an atom or molecule. 

Electrons behave like a free moving 

gas only weakly bound to the entire 

material structure. 

(Depending on their reflectance 

behaviour these materials are used for 

producing mirror devices for optical 

instruments.) 

Remittance Non-polished, clean, metal surface 

without oxidation contamination. 

Absorption Most organic and inorganic materials 

show strong absorption and are 

termed opaque. 

To obtain information about their 

interaction with photons, these materials 

can be prepared as thin locally 

homogeneous layers so they still 

transmit relevant numbers of photons 

to describe the absorption behaviour. 

Fluorescence/ 

Luminescence 

Complex organic molecules and semiconducting 

crystalline structures 

(quantum dots) are typically used for 

showing fluorescence. 

Polarization Clean, smooth, metal surfaces show a 

dependency on photon interaction 

(dependent on their polarization). 

The parameter is the angle of the 

photon incidence towards the surface. 

Most natural crystalline minerals show 

anisotropy in absorbing or transmitting 

to the photon polarization as well. 

(birefringent materials such as quartz 

or calcite are familiar examples). 


Elastic scattering of photons in interaction with 

electrons, which are bound by electromagnetic 

forces in an atomic /molecular system. 

The system structure must either be completely 

homogeneous or of high crystalline order. 

Smooth interfaces to other matter or vacuum, like 

polished glass to air, cause almost no irregular 

effect on interference. 

Interfaces between media with different refraction 

indexes result in correspondingly clear reflectivity. 

Colourless, transparent matter with statistically 

irregular interfaces of different refractive indices 

(changes the speed of light) cause elastic scattering 

in all spatial directions due to interference effects 

Elastic scattering of photons in interaction with 

electrons which form a common electrical field. The 

electrons are just weakly bound to an atomic or molecular 

structure; this explains their absorbent 

behaviour towards the photon, though the photon 

is immediately remitted (repelled). Surfaces need to 

be perfectly smooth. 

Same as in clear reflectance but with a surface 

design which causes diffuse reflection due to 

interference effects. 

Various effects of inelastic scattering of photons in 

interaction with electrons, whereby the absorbed 

photon energy typically dissipates inside matter as 

thermodynamic energy. 

Absorption is typically energy dependent, whereas it 

is often that just bands of photon energy are absorbed 

by individual matter. 

Specific inelastic scattering effects may leave photons 

with reduced energy (Raman scattering). 

Inelastic scattering of photons which leads to 

energy exited status of electrons in matter. Depending 

on the type of molecule the absorbed photon 

energy is released after some characteristic time 

period in form of lower energy photons which are 

remitted or absorbed in secondary processes. 

Portions of absorbed photon energy may dissipate 

to the matter as thermodynamic energy. 

The most tricky, as the photon force acts dynamically, 

changing symmetrically along an axis perpendicular 

to the propagation direction and kept in this 

state as long as the photon exists. 

Any anisotropy of matter in terms of polarity in 

interacting with electromagnetic forces will determine 

whether one of the interactions named above 

with a polarized photon will take place at all.

Fig. 5: Diagram of the refraction of light. Light that enters into an 

optically denser medium at an angle is refracted toward the perpendicular. 

When it exits, it is subject to the reverse deflection and is 

thus parallel shifted. 

ing extents, dependent on their optical 

density. When light from a medium (e.g., 

air) enters into another medium with a 

higher optical density (e.g., glass) at an 

angle, the light is not only slowed down, 

but also refracted. This means that the 

light is deflected in an angle specific to 

the two media. When the light exits and 

encounters a medium with a lower density 

(e.g., air) the speed is once again specific 

to this medium and it is subject to a 

reverse deflection. The ray of light in this 

example is thus parallel shifted (fig. 5). 

There is a value that indicates the extent 

of refraction for transparent media 

– the refractive index n. This is 1 for air 

and increases with the optical density of 

the medium (e.g., water n = 1.33). Oils 

that are used in microscopy for high-resolution 

or powerful magnifying objectives 

(for immersion of the frontal lens) 

have a refraction index of ca. 1.51. 

Reflection 

If electromagnetic waves are deflected 

back from the material, this is termed reflection. 

With smooth surfaces, the light 

reflects according to the physical principle: 

entry angle equals exit angle. This 

means that every ray of light is reflected 

in precisely one direction. Boundary layers 

with significant roughness relative to 

the wavelength, reflect diffusely. If the 

material contains many scatter centres, 

the reflection follows the Beer-Lambert 

Law, whereby the main back scatter takes 

place perpendicular to the material, independent 

of the incident direction. Thus, if 

a ray of light encounters a rough surface, 

it is reflected in all directions spatially. 

Interference 

Various waves of light can interact with 

one another. These waves can also overlap 

each other. If the waves‘ frequencies 

and wavelengths are roughly the same, a 

new wave emerges. The new wave is amplified 

or weakened in comparison to the 

original waves – depending on the phase 

correspondence between the overlapping 

waves. This phenomenon is called interference. 

The waves are amplified when 

the phase shift corresponds to an entire 

wavelength. This effect is referred to as 

constructive interference. Wave traits 

are erased at a phase shift of half a wavelength 

(destructive interference). Any 

wave capable of interference is designated 

as a coherent wave. These are 

wavelengths with a constant phase ratio 

and equal frequency. 

Polarization 

Polarization is a property of transverse 

waves, and thus of the electromagnetic 

waves, which describe the direction of 

the amplitude vector. No polarization 

phenomenon, on the other hand, can occur 

with longitudinal waves, whose oscillation 

takes place in the direction of dispersion. 

A transverse wave has two 

directions. Firstly, the wave vector, which 

points in the direction of dispersion – secondly, 

the amplitude vector. This is always 

perpendicular to the wave vector in 

transverse waves. The third degree of 

freedom in three-dimensional space is 

rotation around the wave vector. 

There are three distinct kinds of polarization. 

They differ in direction and 

magnitude of the amplitude vector (at a 

constant point in space): 

linear polarization: The amplitude 

vector always points in a constant direction 

and the deflection changes its magnitude 

and its sign periodically (with constant 

amplitude) with the progression of 

the wave. 

circular polarization (also referred to 

as rotational polarization): The amplitude 

vector rotates at constant angle 


Fig. 6: The law of reflection applies to smooth surfaces. This states that the incident 

ray, the perpendicular of incidence and the reflected ray are all within one 

incident plane and the incident angle is always exactly the same as the reflection 

angle. Boundaries with a greater roughness relative to the wavelength, on the 

other hand, reflect diffusely. 

speed around the wave vector and does 

not change its magnitude with the progression 

of the wave. 

elliptical polarization: The amplitude 

vector rotates around the wave vector 

and alters its magnitude periodically. The 

peak of the field vector follows an ellipse 

in this instance. 

Polarization methods facilitate the application 

of highly qualitative contrast 

methods such as Differential Interference 

Contrast (DIC). Polarization microscopy 

is of critical importance in microscopy 

of double refractive sample 

structures such as crystals. 

Diffraction 

Light that shines through a small aperture 

generates a pattern of bright rings 

referred to as a diffraction pattern. Patterns 

can range from a centrally bright 

segment (direct, unrefracted light or 

main maximum), followed by a number 

of rings with significantly less brightness 

(refracted light or secondary maxima). 

This occurs due to series of destructive 

and constructive interferences. According 

to the Abbé theory of resolution, image 

formation only takes place once, at 

least one secondary maximum interacts 

with the main maximum in the intermediary 

image plane. The more secondary 

maxima contributing to image formation, 

the higher the resolution. 

Contact: 

Esther Ahrent 

Department Manager Marketing Communication 

Microscopy and Diagnostics 

Olympus Life and Material Science Europa GmbH 

Hamburg, Germany 

Tel.: +49 40 23773 5426 

Fax: +49 40 23773 4647 

esther.ahrent@olympus-europa.com 

www.olympus-europa.com 


Photo: Pixelio 

I m a g e P r o c e s s I n g 

Air Quality Control for Hazardous 

Bio-Material 

Automatic Probe Handling, Image Acquisition and Image Analysis 

Airborne microorganisms are ubiquitously present in various indoor and outdoor environments. The potential 

implication of fungal contaminants in bio-aerosols on occupational health is recognized as a problem in 

several working environments. There is a concern on the exposure of workers to bio-aerosols especially in 

composting facilities, in agriculture, and in municipal waste treatment. The European Commission has therefore 

guiding rules protecting employees in the workplace from airborne biological hazards. In fact, there are 

an increasing number of incidents of building-related sickness, especially in offices and residential buildings. 

Some of these problems are attributed to biological agents, especially in relation to airborne fungal spores. 

However, the knowledge of health effects of indoor fungal contaminants is still limited. One of the reasons 

for this limitation is that appropriate methods for rapid and long-time monitoring of airborne microorganisms 

are not available. 

Fig. 1: Top view of the mechanical unit for moving 

object slides, indicating also the position of 

the cover-glass storage, the dosing pump for 

lactophenol, the slit impactor or air collector, 

and the storage for the object slides. The numbers 

1–5 indicate the sequences of the movements; 

axis No. 6 is not shown. 


Airborne Microorganisms 

Besides the detection of parameters relevant 

to occupational and public health, 

in many controlled environments the 

number of airborne microorganisms has 

to be kept below the permissible or recommended 

values, e.g. in clean rooms, in 

operating theatres, and in domains of the 

food and pharmaceutical industry. Consequently, 

the continuous monitoring of 

airborne biological agents is a necessity 

for the detection of risks of human health 

as well as for the flawless operation of 

technological processes. 

At present a variety of methods are 

used for the detection of fungal spores. 

The culture-based methods depend on 

the growth of spores on an agar plate 

and on the counting of colony-forming 

Dr. Petra Perner 

units. Culture-independent methods are 

based on the enumeration of spores 

under a microscope, the use of a polymerase 

chain reaction or on DNA hybridization 

for the detection of fungi. However, 

all these methods are limited by timeconsuming 

procedures of sample preparation 

in the laboratory. 

Automated Detection of Dangerous 

Bio-Substances 

We have developed an automated imageacquisition 

and probe handling unit of 

biologically dangerous substances and 

the automated analysis and interpretation 

of microscope images of these substances. 

In the system contaminated air 

containing bio-aerosols is collected in a 

defined volume via a carrier agent. They

are recorded by an image-acquisition 

unit, counted, and classified. Their nature 

is determined by means of an automated 

image-analysis and interpretation 

system. Air samples are automatically 

acquired, prepared and transferred by a 

multi-axis servo-system to an imageacquisition 

unit based on a standard 

optical microscope with a digital color 

camera. By a novel image analysis methods 

fungi spores are recognized in the 

image, described by features, and classified 

in one of the different fungi spore 

classes. The following parameters are 

calculated by the image analysis: 

� 

� 

� 

� 

� 

� 

� 

� 

� 

� 

Total number of airborne particles 

Classification of all particles according 

to the calculated image features 

Classification of biological particles, 

e.g. spores, fragments of fungal mycelia, 

and fragments of insects 

Number of respirable particles 

Total number of airborne particles of 

biological origin 

Number of dead particles of biological 

origin 

Number of viable and augmentable 

particles of biological origin 

Identification of species or geni 

Proportion of airborne abiotic and 

biotic particles 

Proportion of dead and viable airborne 

microorganisms. 

The probe handling and image acquisition 

unit works as follows: In the slit 

impactor the air (fig. 3), potentially containing 

airborne germs, is guided on the 

sticky area of the object slide by the air 

stream generated by an air pump. After 

a few tens of seconds which can be 

adjusted accordingly, the pump is 

switched off and the object slide is transported 

to the pipetting unit driven by the 

dosing pump. To this aim it has to change 

its transporting axis and thus its direction 

of movement. From a thin nozzle 

one drop of lactophenol is deposited on 

the sticky area of the object slide which 

is afterwards transported via the axis 

crossing to the cover-slip gripper unit. 

This gripper acts as a low-pressure 

sucker and takes one cover glass from 

the deposit and puts it with one edge first 

on the object slide. Then the cover glass 

falls down on the object slide and flattens 

the drop so that it will be distributed all 

over the sticky area forming a thin layer. 

In this way the airborne germs collected 

in the sticky layer are immersed in the 

lactophenol. In lactophenol living germs 

get a blue color. The object slide is then 

transported back to an axis crossingpoint 

where it again changes its direction 

of movement by 90° and is transported to 

Fig. 2: System architecture 

Fig. 3: Recognized objects in the image 

the xy-table of the microscope which 

takes over the slide and transports it 

directly under the lens. After the object 

slide has reached the image acquisition 

position, the microscope camera then 

grabs the images at the programmed 

slide positions after auto-focusing of the 


Fig. 4: Screenshot of 

the final system 

microscope lens at each position. After 

having finished the imaging sequence, 

the slide is transported away from the 

xy-table with a special arm and falls into 

a box. When the image grabbing procedure 

by the microscope unit is still under 

way, the object-slide preparation unit 



already starts with the preparation of a 

new object slide. Once an image has been 

taken it is given to the image-analysis 

unit for further processing. 

Objects are recognized in the microscopic 

image by a novel case-based 

object-recognition unit. This unit has a 

case-base of shapes for fungi spores and 

determines on a similarity-based inference 

if there are objects in the image that 

have a similar shape as the ones stored 

in the case base. In this case the objects 

get labeled and are transferred for further 

processing to the feature-extraction 

unit. To ensure proper performance of 

this unit, the general appearance of the 

shapes of the fungi spores have to be 

learned. To this end we have developed a 

semi-automated procedure that allows 

one to acquire the shape information 

from the raw image data and to learn 

groups of shape-cases and general 

shape-cases. The feature-extraction procedures 

are based on the knowledge of 

an expert. Note that a particular application 

requires special feature descriptors. 

Therefore not all possible feature-extraction 

procedures can be implemented into 

c o m Pa n y P r o f I l e 

Established in 1972, Agar Scientific has 

grown into an internationally respected 

supplier of consumables and accessories 

for all fields of microscopy, with agents 

worldwide and an enviable reputation 

for service and quality. The extensive 

experience and knowledge of 

our staff enables us to provide 

technical support and advice to 

microscopists from all disciplines. 

It is this unique understanding of 

the needs of our customers that 

has enabled us to grow our product 

range to over 4000 items, offering 

a single supplier for most 

needs. We offer products manufactured 

onsite by our skilled technicians. 

We also provide products from 

well-established suppliers such as Kodak, 

British Biocell International, Dumont 

and Fischione Instruments. 

High quality sample preparation is essential 

which is why we market a wide 

range of instruments, tools, accessories 

and consumables. In addition, we offer 

such a system from the beginning. Our 

aim was to develop a special vocabulary 

and the associated feature-extraction 

procedures for application on fungi identification. 

Application in Health Monitoring and 

Process Control 

Based on the feature description, the second 

case-based reasoning unit decides 

about the type of the fungi spore. This 

unit employs a novel prototype-based 

classifier. It starts its performance on 

prototypical cases that were selected or 

created by the expert. It can learn with 

time the different appearances of the 

fungi spores. The special features of this 

unit ensure its proper performance. It 

can learn the relevant prototypes from 

the subjectively selected set of prototypes, 

as well as create new prototypes. 

It can also learn the importance of the 

features of the cases. The final result of 

the system will be the identification of 

the fungi spores that appear in the image 

and the number of these spores. This is 

shown on the display of the system and 

Images courtesy of David McCarthy, School of 

Pharmacy, University of London; John Runions & 

Chris Hawes, Oxford Brooks University 

in a file, together with the date and the 

time when the data were acquired. 

The system allows automatic on-line 

monitoring of environments. The final 

information can be used to determine its 

contamination with biological hazardous 

material. It can be used for health monitoring 

as well as for process control. 

References: 

Sklarczyk, Ch., et al., In: P. Perner and O. Salvetti, 

Mass Data Analysis of Signals and Images in Med- 

icine, Biotechnology, and Chemistry MDA, 

Springer Verlag 2007. 

Contact: 

Dr. Petra Perner 

Director 

Institute of Computer Vision and Applied Computer 

Sciences, Leipzig, Germany 

Tel. +49 341 8612 273 

Fax: +49 341 8612 275 

pperner@ibai-institut.de 

www.ibai-institut.de 

www.mda-signals.de 

Agar Scientific for all your microscopy 

accessories and consumable needs 


the Agar branded range of vacuum coating 

systems and add-ons including targets, 

carbon rods and film thickness 

monitors. This ensures that users have 

a single source for all their sample 

preparation needs. 

A core part of our business is the 

manufacture and supply of replacement 

filaments and specialist 

apertures for the majority of electron 

microscopes. We also produce 

grids, support films and calibration 

standards. Furthermore, 

we have the capability to provide 

customised solutions. 

Full details of our extensive range 

may be found on our website. Register 

for a free copy of our catalogue. 

Contact: 

Agar Scientific Limited 

Stansted, Essex, UK 

Tel: +441279 813519 

sales@agarscientific.com 

www.agarscientific.com

4th generation XFlash ® silicon drift detectors (10, 30 and 40 mm2 � 

) 

� LN2 free, vibration free 

� Unmatched acquisition speed 

� Excellent results at both low and high count rates 

� Precise light element analysis (FWHM C-Kα = 48 eV) 

� New HyperMapping function (high speed PTS) 

� Modular, customizable ESPRIT software 

Technical Specifications 

sales-ma@bruker-axs.de 

www.bruker-axs-microanalysis.com 

� Long lifetime filament (guaranteed 1000 hrs) running at 5kV 

� Specially designed Backscattered Electron Detector (BSD) allowing compositional 

and topographical imaging 

� High depth of focus with an maximum image resolution of 30nm 

� Fast time-to-image; optical image < 10 seconds and electron image 

< 30 seconds 

� Robust design; no risk of damaging the lens due to sample-container design 

www.fei.com/phenom 

infophenom@fei.com 

I & M S H o w c a S e 

QUANTAX – fast, reliable and 

convenient microanalysis 

Bruker AXS Microanalysis’ QUANTAX EDS system delivers fast 

reliable analyses across a broad range of applications for all 

kinds of samples ranging from powders and metals to coatings. 

Its LN 2-free XFlash ® Silicon Drift Detectors (SDD) are vibration 

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at high count rates (125 eV Mn Kα 100,000 cps). 

The SDD technology makes real time spectrometry for instant 

element preview, high speed element mapping or Colorscan 

possible. Just one click and Colorscan turns the black and white 

SEM image into a full color image. While the SEM is scanning 

the image is superimposed in color with the element information 

gathered by the detector. In this way the user can assess 

interesting areas and the homogeneity of a sample at a glance. 

Especially noteworthy is the QUANTAX HyperMap function. It 

stores a complete spectrum at every pixel of an element map. 

This “database” can be reanalyzed anytime later for additional 

elements or features of interest. Only the XFlash ® detector‘s 

high count rate detection capability (>750,000 cps for the 

XFlash ® 4010 and >3,000,000 cps for the XFlash ® QUAD 

4040) allows for efficient and fast HyperMapping. 

PHENOM; Closing the Imaging 

Gap between Optical and Electron 

Microscopy 

The Phenom is a new tabletop scanning electron microscope (SEM) 

which combines the high magnification of electron microscopy with the 

ease of use of optical microscopy to improve performance in a benchtop 

instrument.The Phenom, a tabletop or benchtop SEM provides useful 

magnifications up to 20,000 x and is easy to use as the typical laboratory-grade 

optical microscopes. Operating an optical microscope 

requires little more than placing the sample on the stage and focusing 

the image and is usually accomplished in a matter of seconds. In conventional 

SEMs, the time required to get an image can easily become 

many minutes to hours. The Phenom cuts away the time, difficulty, and 

expense of the conventional SEM. The operator simply places the sample 

in the specially designed holder on the microscope. The automatically 

focused image is displayed in less than 30 seconds later, with the 

resolution and depth of focus typical belonging to SEMs. Some of the 

key features of the Phenom are; 

� Imaging power: Up to 20,000 x magnification with superior depth 

of focus and superb picture quality 

� Ease of use: Intuitive control system and interactive touch screen 

reduces operator training and increases the number of users 

� Fastest Time to Image: Through patented vacuum technology 

and never lost navigation 

� Low cost of ownership: Easy to install and maintain and no 

special operator training needed 



Features: 

Completely integrated system comprising upright optical microscope and JPK NanoWizard ® � 

II AFM system 

� Developed for the investigation of opaque samples in life and materials sciences 

� 100 % performance of both techniques, without any limitations 

� Unique capability to investigate the ROI precisely with optics and AFM 

� Outstanding reproducibility of the focussed position (ROI) with both systems 

� Also perfect during operation in liquids 

� Wide range of applications: Biochips, cell chips or patterned substrates for cell adhesion, nanostructured 

surfaces from PDMS or other imprints, lipid bilayers, biomarkers such as quantum dots, rods, CNT ... 

� Flexible concept with shuttle stage 

� Both techniques can be operated even in different laboratory rooms 

Technical Specifications 

� Lateral resolution 90 nm (full width half maximum) 

� Easy access to STED resolution by a mouse click 

� STED fully integrated into TCS SP5 platform 

� All Leica TCS SP5 systems upgradable to STED 

� Full functionality of Leica TCS SP5 Multiphoton included 


christine.ludwig@leica-microsystems.com 

www.confocal-microscopy.com 

BioMAT Workstation 

– Uncompromising AFM and 

Optical Imaging of Opaque 

Samples 

JPK Instruments introduces a new technical breakthrough that 

enables the combination of upright microscopy with atomic 

force microscopy (AFM) on the same sample spot – the BioMAT 

Workstation. This clears the way for a new range of applications 

where the full capabilities of both AFM and advanced optical 

imaging can be realized even on opaque samples. A patented 

calibration procedure ensures that the region of interest can be 

found and re-imaged with micron precision, so exactly the same 

area can be investigated optically and with the AFM. 

Beyond the Limits! 

nanowizard@jpk.com 

www.jpk.com 

Superresolution Microscope Leica TCS STED 

The new Leica TCS STED opens up the access to resolve the finest 

structures beyond the diffraction limit – without any image 

computation! 

With the integration of the groundbreaking STED concept into 

the approved broadband confocal platform Leica TCS SP5 a new 

class of microscope has been created. The superresolution 

capacity of the Leica TCS STED allows confocal imaging with a 

resolution 2 to 3 times higher than could ever be achieved in a 

conventional scanning microscope – without compromising on 

usability. This means: superresolution at a mouse click! 

The Leica TCS STED breaks the diffraction limited resolution of a 

light microscope by downsizing the diameter of the fluorescence 

spot, that determines the resolution, by a doughnut shaped 

pulsed depletion laser beam, tightly synchronized with the excitation 

light pulse. 

With this technique it is possible to resolve protein complexes 

smaller than 90 nm and to gain new information for instance 

about the construction of synapses and membrane domains.

‘Cellogy’, a term coined by Nikon, describes the company’s strategic 

move into Complete Cell Care: 

� Maintaining live cells in an optimum environment, aiding researchers to obtain a true 

picture of what is happening in vivo 

� Developing optical hardware and analysis software to help researchers accurately track 

and minimise stress imposed on cells, reduce phototoxicity and extend cell life 

� Focusing R&D activity on ways of integrating imaging solutions with other advanced 

technologies to simplify experimental protocols and enhance results 

info@nikoninstruments.eu 

www.nikoninstruments.eu 

Moving further into 

‘complete live cell care’ 

BioStation CT represents next stage in Nikon’s 

product development plans 

In a major extension of its traditional involvement in cellular 

imaging, Nikon Instruments is expanding further into the field of 

live cell care with the launch of the new BioStation CT. By combining 

the precise environmental control capabilities of a highperformance 

incubator with the advanced optics needed for 

drift-free live-cell imaging, the BioStation CT creates conditions 

that are as close to in vivo as possible. 

Nikon’s commitment to Live Cell Care was originally founded on 

the TE2000-PFS system for time lapse recording, the C1si microscope 

for spectral confocal applications, and the BioStation IM, 

a bench-top integrated incubation and monitoring system, but 

has recently been enhanced by the introduction of Controlled 

Light Exposure Microscopy (CLEM) and the LiveScan Swept Field 

Confocal (SFC) Microscope. CLEM reduces photobleaching and 

enhances cell survival thus allowing time-lapse studies of protein 

interactions to be undertaken. In addition to reduced crosstalk 

and noise, the fast scan speed of the LiveScan SFC microscope 

dramatically minimises phototoxicity and photobleaching 

during image acquisition and is ideal for live samples. 

Olympus FV1000: Taking cLSM 

to another level 

Flexible confocal microscopy 

Confocal laser scanning microscopy provided scientists with the 

tools to see a much greater level of detail in cells. The Olympus 

FV1000 has taken this to an even higher level, with the added 

functionality and flexibility of the multi-laser combiner and SIM 

scanner. 

� Multiple lasers: The multi-laser combiner enables the easy integration and more flexible use of an unmatched number of lasers, providing a wider range of 

available wavelengths for high-resolution, confocal live cell imaging. The system supports up to six diode and gas lasers, enabling the selection of a large 

number of wavelengths from near UV to far red. Presently, laser diodes provide 405, 440, 473, 559 and 635 nm and the gas lasers provide 457, 488, 

514.5, 543 and 594 nm. 

� SIM scanner: The FV1000 incorporates 2 laser scanners in a single compact design for simultaneous confocal fluorescence observation and independent 

laser light stimulation. Synchronization of these two functions ensures that rapid cellular reactions that occur during or immediately following stimulation 

are not overlooked. 

� Regions of interest: Any region of interest can be specified for stimulation and scanning independently, with unrestricted control of variations in timing, 

duration and intensity. The circular “Tornado” scan provides highly efficient photobleaching and photoactivation in contrast to standard raster-scan patterns. 

� Application flexibility: The SIM scanner and multi-laser combiner make the FV1000 the most suitable confocal microscope for a variety of applications, 

including FRAP, FLIP, photoactivation, photoconversion, uncaging, laser ablation and many others. 

microscopy@olympus-europa.com 

www.microscopy.olympus.eu 



N o t e s f r o m N i k o N 

Controlled Light Exposure Microscopy 

Minimising Phototoxicity and Photobleaching in Live Cell Fluorescence Imaging 

The labelling of cellular targets with fluorescent probes is used widely in live cell imaging to identify and 

monitor intracellular events. Excitation of probes with light, however, can result in phototoxicity and photobleaching 

– factors that can seriously compromise fluorescence timelapse recordings especially in confocal 

microscopy. Laserinduced phototoxicity and photobleaching can be reduced with an innovative technology 

known as ‘Controlled Light Exposure Microscopy (CLEM)’. Here, photobleaching and cell survival are compared 

when imaged with a Nikon C1 confocal system configured with, and without, CLEM. 

Fluorescence Imaging in Living Cells 

The ability to observe dynamic events in 

living cells is one the greatest challenges 

in biological research. Fluorescent 

probes, such as green fluorescent protein 

(GFP) and its derivatives, help researchers 

view specific cell targets and 

track protein localisation and distribution 

in living cells. The variety of probes 

now available (Miyawaki, 2004) allows 

researchers to study interactions between 

proteins tagged with different fluorescent 

proteins over time. However, 

problems associated with light-induced 

probe bleaching and phototoxicity can 

seriously limit time-lapse experiments 

especially when laser light is used. Controlled 

Light Exposure Microscopy 

(CLEM) regulates laser illumination so 

that photobleaching is reduced and cell 

survival increased (Hoebe et al., 2007). 

Imaging System 

In a conventional laser scanning confocal 

microscope system every pixel is illumi- 

Author’s 

background 

Maarten Balzar is product manager for Nikon 

Instruments Europe BV. He is responsible for 

biological research applications such as TIRF, 

confocal and advanced fluorescence microscopy. 

His current activities are focussed on the 

bioscience product line. Maarten joined Nikon 

in 1999 after finishing his PhD at the Department 

of Pathology, Leiden University Medical 

Centre (LUMC), The Netherlands. 


nated individually by laser light (typically 

in the micro-second range) and fluorescence 

emission detected simultaneously. 

A confocal image consists of many of 

these individual pixels and typically displays 

high (bright) and low (dim) intensity 

pixels. Generally, each pixel is acquired 

with the same laser power and 

detector sensitivity setting. In contrast, 

when CLEM is used, exposure to laser 

light is determined on a per pixel basis 

(i.e. when and where required). Excitation 

light is reduced using two strategies: 

1. The first is based on the principle 

that if there is no signal, then no illumination 

is required (for example, the background). 

2. The second detects whether there is 

sufficient signal to acquire an image. If 

so, illumination can be stopped. 

A schematic of the CLEM principle is 

shown in figure 1A. In “non-CLEM microscopy” 

an object is illuminated uniformly 

and, consequently, the detected 

image is a direct representation of the 

object. In “CLEM” microscopy the nonuniform 

illumination is controlled by the 

detection signal via a feedback system. 

The final image is created by combining 

the illumination image and the detection 

image. Figure 1B shows CLEM configured 

with the Nikon C1 confocal system. 

CLEM electronics use the pixelclock and 

the detector signal as inputs. An acousto 

optical modulator (AOM) is used as a fast 

shutter. Modulation of a solid-state (diode) 

laser may also be used. 

The Investigations 

Three investigations were carried out: 

1. CLEM was used firstly to image fluorescence 

in pollen grains to ensure that 

there were no differences in cell mor- 

phology when imaged with and without 

CLEM. 

2. BY-2 tobacco cells expressing GFP- 

MAP4 associated with microtubules were 

used in a time-lapse experiment to determine 

the effect of CLEM on photobleaching. 

Identical settings were used for the 

time-lapse experiment (a 3D acquisition 

in time) in the presence and absence of 

CLEM. 

3. To assess the impact of CLEM on 

cell survival, HeLa cells expressing GFPtagged 

histone-2B were plated on a Petridish 

and multiple areas on the dish imaged 

in a multi-dimensional time-lapse 

experiment in the presence or absence of 

CLEM. Both transmitted light and (GFP) 

fluorescence images were acquired to 

obtain optimal morphological information 

on the cells. 

Results 

The morphology of the pollen grain is 

similar with both non-CLEM and CLEM 

imaging (fig. 2). However, exposure of 

the sample to light in the absence of 

CLEM is much higher than in the presence 

of CLEM. 

In the absence of CLEM, the microtubule-associated 

GFP-MAP4 expressed by 

BY-2 tobacco cells is bleached to 50 % of 

its original fluorescence intensity (arbitrary 

units) after just five 3D scans (fig. 

3A). In contrast, in the presence of CLEM, 

the GFP-MAP4 fluorescence intensity is 

reduced by 50 % after twenty five 3D 

scans (fig. 3B). 

Time-lapse acquisition of HeLa cells 

expressing GFP tagged histone-2B in the 

presence or absence of CLEM shows that 

the intensity levels of the GFP tagged histone-2B 

remain approximately equal in 

both cases (fig. 4). However, in the absence 

of CLEM, the HeLa cells reveal 

membrane blebbing at an early stage followed 

by cell death (A: top panel). Cell 

survival is greatly prolonged in the presence 

of CLEM (B: bottom panel). 

It has been shown that CLEM reduces 

photobleaching and phototoxicity two- to 

tenfold, depending on the fluorophore 

distribution in the sample (Hoebe et al.,

Fig. 1: A) Comparison of non- 

CLEM and CLEM microscopy. 

B) Schematic overview of the 

C1 system equipped with the 

CLEM module. 

Fig. 3: (A) Time-lapse acquisition (XYZ in time) of BY-2 tobacco cells expressing 

GFP-MAP4 associated with microtubules. The XY image at the middle 

of the image stack is presented in time. The sequential images were acquired 

in the absence of CLEM. (B) Time-lapse acquisition (XYZ in time) of 

BY-2 tobacco cells expressing GFP-MAP4 associated with microtubules. The 

XY image at the middle of the image stack is presented in time. The sequential 

images were acquired in the presence of CLEM. 

2007). By spatially controlling the lightexposure 

time, CLEM reduces the excitation-light 

dose without compromising image 

quality. CLEM directly affects the 

pixel-by-pixel laser excitation light dose. 

While CLEM was created for qualitative 

live cell imaging, it may also have an impact 

on quantitative studies. The observation 

that CLEM reduces photobleaching 

and phototoxicity means that results 

are less likely to be affected by artefacts 

related to the processes of cell death. 

Since the illumination image, detection 

image and CLEM image are all available; 

it should be possible to completely correct 

the CLEM image for accurate quantitative 

imaging. As bright pixels are only 

illuminated for a limited time, it should 

also be possible to extrapolate the detected 

fluorescence emission intensity. 

This may contribute to greatly increased 

dynamic range and provides an additional 

advantage in addition to reduced 

photobleaching and improved cell viability. 

Conclusions 

Cells expressing fluorescent protein are 

sensitive to photobleaching and phototoxicity. 

In live cell imaging, it is important 

to minimise these effects to prolong 

cell survival especially for time-lapse 

studies. The use of CLEM in a confocal 

Fig. 2: Confocal image of pollen grain autofluorescence 

in the absence / presence of CLEM. 

Top panel shows the illumination image (left) 

and confocal image (right) in the absence of 

CLEM. Bottom panel shows the illumination 

image (left), detected image (middle) and CLEM 

image (right). 

microscope system can reduce the effects 

of both photobleaching and phototoxicity. 

CLEM is, therefore, an essential tool for 

live cell imaging studies. 

Fig. 4: Time-lapse acquisition of HeLa cells expressing GFP tagged histone- 

2B. The transmitted light and fluorescence images were simultaneously 

acquired in the absence (A) or presence (B) of CLEM. 

References: 

[1] Hoebe, R. A., Van Oven, C. H., Gadella, T. W. Jr, 

Dhonukshe, P. B., Van Noorden, C. J., Manders, 

E. M., Nat. Biotechnol. 25(2) 249–53 (2007). 

[2] Miyawaki, A., Nat. Biotechnol. 22, 1374–1376 

(2004). 

N o t e s f r o m N i k o N 

Contact: 

Maarten Balzar (PhD) 

Application manager biological microscopes 

Nikon Instruments Europe BV 

Badhoevedorp, The Netherlands 

Tel.: +31 2044 96273, Fax: +31 2044 96298 

balzar@nikonbv.nl 


P r o d u c t s 

Creating Own Filtersets 

Optical filters made by the 

hardcoating technique from 

AHF Analysentechnik show 

extrem high transmission, 

high thermal stability and are 

easy to handle. The company 

Nikon Instruments Europe 

and Cool-LED have agreed 

that Nikon will offer the “Precis 

Excite” LED light-source 

with their fluorescence microscopes 

in Europe. Following 

product evaluation at 

their European headquarters 

in Amsterdam, Nikon identified 

the benefits and potential 

of the light-source to provide 

stable, homogeneous control 

of fluorescence excitation. 

Applications such as Confocal, 

time-lapse and live cell 

imaging all benefit from the 

high level of control. The 

modular light source enables 

the user to select the wavelength 

of excitation and control 

the intensity and period 

presents a wide program also 

of single filters and beamsplitters 

which can be combined to 

individual filtersets, avoiding 

high costs for custom specification. 

The company offers to 

handle the detailed requests 

of difficult optical setups. 

Wherever possible, Demofilters 

will be offered for testing 

at the customers’ samples. 

AHF Analysentechnik AG 

Tel.: +49 7071 970901-0 

info@ahf.de 

www.ahf.de 

LED Light-source Used for Microscopes 

Catalog Released 

Omega Optical has released a 

new catalog of fluorescence 

filter sets. The 2007 catalog, 

Precision Optical Filters for 

Fluorescence Microscopy, includes 

fifteen high performance 

sets for fluorescent proteins, 

and ten new sets for 

Fret applications. These products 

complement an extensive 

selection of dye-specific filter 

sets for all single and multilabel 

microscopy applications, 

including Quantum Dots, M- 

FISH, Pinkel, Sedat, Ratio Imaging, 

Confocal, and Multiphoton. 

In addition, there are 

resources such as a fluorophore 

reference table, light 

source and detector spectral 


of excitation very precisely. 

As new wavelengths are required, 

and intensity of LEDs 

continues, additional LED Array 

Modules can be added 

and interchanged in the unit. 

CoolLED 

Tel.: +44 1264 320989 

sales@coolled.com 

www.precisexcite.com 

data, an explanation of filter 

nomenclature, guidelines for 

choosing the optimal filter 

sets, and a list of components 

organized by wavelength. 

Omega Optical, Inc. 

Tel.: +1 802251 7342 

rjiraskova@omegafilters.com 

www.omegafilters.com 

Optical Filters 

Semrock has announced new 

filters for the efficient isolation 

of individual lines of highpower 

mercury arc lamps. 

Each filter obtains maximum 

throughput of the desired 

mercury line through passband 

transmission and optimization 

of the filter design. 

The non-absorbing blocking 

and fused silica substrate ensures 

that undesired mercury 

The Fusion Microslides from 

BTX Harvard Apparatus are 

designed to fit on a microscope 

and allow observation 

of the dimer formation during 

electrofusion. They are composed 

of a glass slide and two 

strips of stainless steel (wire 

or bar) and are available in 

0.5 mm, 1.0 mm, 3.2 mm and 

10 mm gap sizes. The slides 

work with the company’s ECM 

2001 Electrofusion device. 

lines and other background 

light are effectively eliminated 

without the risk of filter 

solarization. The new Max- 

Lamp family has been 

launched with mercury arc 

lamp filters to isolate the popular 

365 nm i-line and the 

critical 254 nm UV line. These 

bandpass filters offer an average 

transmission of > 93 % 

and > 65 % over the 365 nm 

and 254 nm bands respectively, 

yet with higher and 

wider out-of-band blocking 

than incumbent filters. 

Semrock, Inc. 

Tel.: +1 585 594 7003 

amacdonald@semrock.com 

www.semrock.com 

Microslides for Hybridoma Production 

Machine Vision Illuminator 

Dolan-Jenner Industries has 

introduced its Fiber-Lite 

DC950H Machine Vision Fiber 

Optic Illuminator for machine 

vision integrators. This is a 

150-watt quartz halogen regulated 

illuminator, which has 

been improved to produce a 

higher output with greater 

reliability, and is now RoHS 

compliant. It features DC regulated 

output, fast lamp response, 

and a 0-5 VDC remote 

intensity control interface 

with linear voltage adjustment 

(8 bit D/A module available). 

The instrument can be 

operated remotely and offers 

remote notification of lamp 

failure to decrease downtime. 

It is made of stackable heavy 

duty steel housing with a 

BTX Harvard Apparatus 

Tel.: +1 800 272 2775 

Techsupport@btxonline.com 

www.btxonline.com 

mounting capability. The front 

panel can be accessed easily 

for fast lamp changes. Several 

illuminating options are 

available, including color filtering, 

manual iris, and an 

analog or digital remote interface. 

Dolan-Jenner 

Tel.: +1 800 626 8324 

bgagnon@donal-jenner.com 

www.dolan-jenner.com

Confocal Raman Imaging 

Option 

Witec introduces the „Ultrafast Raman 

Imaging Option“ for the Alpha300 R Confocal 

Raman Microscope. With this option 

the acquisition time for a single Raman 

spectrum can be as low as 1.7 milliseconds. 

As a Confocal Raman image typically 

consists of tens of thousands of spectra, 

the option reduces the total acquisition 

time for a complete image to only a few 

minutes. For example, a complete hyperspectral 

image consisting of 250 x 250 

pixels = 62,500 Raman spectra can be recorded 

in less than two minutes. The latest 

spectroscopic EMCCD detector technology 

combined with the high throughput 

optics featured system are the keys to this 

improvement. The new option reduces 

the overall experiment duration and delivers 

more valuable Raman data in a 

given time, thereby reducing the total cost 

of ownership of the system. 

Witec GmbH 

Tel.: +49 731 140 700 

info@witec.de 

www.witec.de 

Photo-Bleaching and Photo- 

Activation 

Andor Technology launches a new tool 

for its Revolution laser microscopy solutions 

aimed at live cell imaging. Revolution 

Frappa is the company’s latest innovation 

in laser microscopy using a 

computer-steered laser beam to photobleach 

or photo-active a user-defined region 

in a live cell specimen. It is a photobleaching 

module using a dual 

galvanometer scan head. It can be configured 

in line with a CSU and/or camera. 

Under Andor iQ software control, the 

user commands the instrument to bleach 

or activate regions of interest with userdefined 

times, laser lines and powers. 

Laser switching is tightly synchronized 

using the company’s proprietary laser 

combiner multi-port switch (MPS). 

Andor Technology 

Tel.: +44 289023 7126 

www.andor.com 

Continuous Wave Laser 

Newport Corporation’s Spectra-Physics 

Laser Division introduced the two newest 

members of its Excelsior low power, continuous 

wave (CW) laser family: The Excelsior-1064 

OEM-laser with 500 mW of 

output power, and the Excelsior-1064 


Scientific laser with both 500 and 800 

mW of output power. Using Vanadate as 

the gain medium, these two lasers operate 

at the 1064 nm wavelength. The company 

has showcased the latest technology 

advancements in its 488 nm Excelsior 

and Cyan laser product lines during the 

“Laser” show in Munich. These demonstrations 

included a 100 mW Excelsior- 

488, a 75 mW Cyan laser, and a 20 mW 

Cyan power adjustable laser that customers 

can test on the show floor. 

Newport Spectra-Physics GmbH 

Tel.: +49 6151 708 0 

germany@newport.com 

www.newport.com 

Image Asset Management 

Solution 

Media Cybernetics announced the release 

of IQbase 2.5 scientific image management 

software. This image management 

software enables organizations to 

effectively store, query, and share large 

numbers of images and related data. Users 

can collaborate within their organization 

and with outside partners by sharing 

image data through their network or 

via the Internet. With the software, users 

can perform powerful image searches 

based on keywords or using more detailed 

context sensitive search parameters. 

Images and data can be easily 

shared with others through the use of 

automatic PDF report templates, onestep 

export to PowerPoint tools, and webbased 

image searching and downloading. 

The software allows users to further 

explore their images with qualitative visualization 

tools like image overlays and 

annotations, as well as with quantitative 

graphs and charts for interactive data 

analysis. 

Media Cybernetics, Inc. 

Tel.: +1 301 495 3305 260 

www.mediacy.com 

� � � � � � � �� 

�� 

�� 

Interference 3D microscope 

and vibrometer 

� 

� 

� 

� 

� 

� 

Fast and accurate optical 3D profiling 

Phase shifting (PSI) and white light 

vertical scanning (VSI) interferometry 

Sub-nanometer rougness analysis 

MEMS option for oscillating surfaces 

up to 3 MHz, also Laser Doppler 

Vibrometer module 

Optional vacuum/pressure chamber 

Top quality at affordable price 

We also supply 

Confocal and other 3D microscopes, AFM’s 

for research, industry and teaching, Nano-/ 

Micro- Indenter and Tribology Tester, 

Nanoparticle size measurement, LEED/AES 

Contact your nearest office 

Germany · info@schaefer-tec.com · +496103300980 

France · info@schaefer-tech.com · +33164496350 

Italy · italia@schaefer-tec.com · +39 0425460218 

Switzerland · ch@schaefer-tec.com · +41344237070 

www.schaefer-tec.com 



Intuitive Smart Camera/Software Package 

– Without Programming 

The Matrox Iris-E with Design 

Assistant is a powerful 

smart camera/software package 

and allows OEMs and systems 

integrators to develop 

vision and imaging applications 

without programming. 

In the Matrox Design Assistant 

environment, users create 

a flow chart of the application 

that instructs the 

Matrox Iris E-Series camera 

to grab, process and display, 

perform measurements, analyze 

image data, read machine 

codes, and more. The 

intuitive nature of the Matrox 

Iris E-Series with Design Assistant 

eliminates the need 

for programming and scripting. 

Users benefit from simplified 

application development 

that offers the same 

reliability and robustness as 

the field-proven Matrox Imaging 

Library. The Matrox 

Digital Pathology Solution 

Image Solutions has launched 

“Tissue-Mine”, a user friendly 

web based digital pathology 

solution. It has been designed 

by pathologists to automate 

the pathology workflow for 

research and development. 

Improving efficiency, accelerating 

the workflow and minimising 

bottlenecks in an otherwise 

manual process. It is 

now possible to reduce analysis 

time from weeks to minutes 

without the process being 

subject to user variability. 

The system provides a supe- 

TEM Camera at M&M 

At this year’s Fort Lauderdale 

M&M show, Olympus Soft Imaging 

Solutions is launching 

the Cantega G2, the 2 nd generation 

Olympus Soft Imaging 

Solutions 2k x 2k, bottommounted 

CCD TEM camera 

for all TEM brands. The fiberoptically 

coupled and watercooled 

camera is equipped 

with an optimized YAG scin- 


Iris-E is a smart camera that 

support the flexibility of PCbased 

machine vision systems, 

and features hardware 

for image sensing as well as 

software for image capture, 

processing, and analysis. It is 

powered by an Ultra Low 

Power (ULP) Celeron, an embedded 

Intel architecture 

processor, and runs the Windows 

CE .NET real-time operating 

system. 

Rauscher 

Tel.: +49 8142 44841 0 

info@rauscher.de 

www.rauscher.de 

rior, highly customisable alternative 

to the traditional 

manual analysis of tissue 

slides using a microscope. I 

scan, which is included in the 

package, is a new high speed 

tissue slide scanner which 

can acquire images with great 

speed and image quality capable 

of auto loading up to 

160 slides. 

Image Solutions (UK) Ltd. 

Tel.: +44 1772663 140 

andy@imsol.co.uk 

www.imsol.co.uk 

Board Level Camera 

Hamamatsu Photonics introduce 

the C10000 TDI (time 

delay integration) board level 

CCD camera featuring the latest 

high speed, high sensitivity, 

back thinned CCD image 

sensor. The newly developed 

CCD sensor features 2048 

pixel length by 128 pixel 

height, with 90 % peak quantum 

efficiency, high UV sensitivity 

and a very high speed 

data readout of up to 50 KHz 

line rates. Ordinarily high 

speed imaging systems suffer 

from a lack of available light 

and special illumination systems 

need to be constructed. 

However, using the TDI image 

acquisition method the 

C10000 board camera synchronises 

charge transfer 

with the moving object. This 

Excellent Test Results 

In a test commissioned by IDS 

and conducted according to 

the EMVA 1288 Standard, the 

two cameras UI-1220-M and 

UI-2220-M from the U-Eye 

series of the German machine 

vision specialist achieved excellent 

results. The cameras 

under test were the model UI- 

1220-M, a monochrome camera 

with a 1/3“ CMOS sensor 

and a resolution of 752 x 480 

pixels, and the UI-2220-M, a 

monochrome camera with a 

1/2“ CCD sensor and a 768 x 

576 pixel resolution. With an 

excess housing temperature 

of only 3 °C and 4 °C, respectively, 

the cameras shine with 

measurement values that are 

among the lowest achieved so 

far. The design as well as the 

tillator. On customer request, 

the company supplies a phosphor 

scintillator as well. The 

highly sensitive CCD chip provides 

2048 x 2048 pixel resolution 

with a 14-bit dynamic 

range. With a pixel size of 

14 µm x 14 µm, the full frame 

CCD offers an active area of 

28.7 mm x 28.7 mm. The CCD 

sensor possesses a uniquely 

gives 128 times the image integration 

of a conventional 

line sensor, and when combined 

with the high sensitivity 

of the back thinned CCD chip 

results in a greater magnitude 

output of two or three 

orders compared to a regular 

line scan camera. 

Hamamatsu Photonics UK Ltd. 

Tel.: +800 80080088 

Europe@hamamatsu.com 

www.sales.hamamatsu.com 

optical density inside the Cmount 

lens connector, which 

effectively eliminates stray 

light in the flange, are exemplary 

and the dark noise is 

very low. 

IDS Imaging Development 

Systems GmbH 

Tel.: +49 7134 96196 0 

t.schmidgall@ids-imaging.de 

www.ids-imaging.de 

high quantum efficiency. The 

camera supports different 

frame rates. 

Olympus Soft Imaging Solutions 

GmbH 

Tel.: +49 251 79800 160 

Manfred.Kaessens@olympus-sis. 

com 

www.olympus-sis.com

Ultra-high Resolution 

Fei Company has added the 

Titan3 80-300 to its Titan 

product family. Aptly named 

the Titan “Cubed” because of 

its fully enclosed profile, the 

system is designed to deliver 

the highest stability and performance 

in a commercial 

scanning/transmission electron 

microscope (S/TEM). The 

ultra-high resolution S/TEM 

performance of this new system 

is achieved by its all-new 

design that allows the combination 

of two C s-abberation 

correctors and a monochromator 

on a single instrument. 

The system’s enclosure significantly 

reduces environmental 

interference providing 

greater stability and eliminating 

the need for many expensive 

lab improvements. 

FEI Company 

Tel.: +1 503 726 2695 

dzenka@fei.com 

www.fei.com 

Omniprobe Acquires Assets of Ascend 

Instruments 

Omniprobe announced that 

they have acquired the assets 

of Ascend Instruments. This 

company provides hardware 

and software for sample preparation 

and manipulation in 

the Focused Ion Beam (FIB) 

Microanalysis Software 

Edax has introduced the latest 

generation of its Genesis 

EDS microanalysis software. 

“The 5.2 version of the software 

includes features such 

as Auto Shape – an automatic 

collection routine with a free 

drawing capability,” explains 

Del Redfern, Product Marketing 

Manager. “The Auto 

Shape is an automatic spectrum 

collection routine that 

allows the user to select 

points or shapes from an image 

to collect spectrum. The 

microscope. In the near term, 

its product line will be manufactured 

at Omniprobe‘s Dallas, 

Texas, facility. In addition, 

Omniprobe has been 

selected by Entrepreneur 

magazine for their “Hot 500” 

Fastest Growing Companies 

in the U.S. The Hot 500 rankings 

are based on company 

performance and sales 

growth. The listing appears in 

the August 2007 issue of Entrepreneur 

magazine. 

Omniprobe, Inc. 

Tel.: +1 214 572 6800 

www.omniprobe.com 

software saves and labels the 

spectrum collected from the 

multiple positions along with 

their position that is then laid 

over the image.” The new features 

also include a software 

interface for the company’s 

Apollo 40 SDD and Apollo 10 

SDD silicon drift detectors. 

Edax, Inc. 

Tel.: +1 201 529 4880 

info.edax@ametek.com 

www.edax.com 

Transmission Electron Microscope 

The Nano Technology Systems 

Division (NTS) at Carl 

Zeiss SMT will introduce the 

new Centra 100, a transmission 

electron microscope with 

up to 100 kV accelerating 

voltage, at the MC Saarbrücken. 

Specially designed 

as a sophisticated “imaging 

system”, the highly compact 

and robust instrument offers 

maximum resolution down to 

PELCO ® Silicon Nitride Membranes 

Next Generation Si 3N 4TEM Support Films with 

many advantages: 

• Durable and chemically 

inert planar 50nm 

substrate 

• 3.0mm circular frame 

compatible with standardTEM 

holders 

• EasyGrip micro rough 

edges for ease of handling 

• Free from debris - no broken edges 

• Large area support film: up to 0.5 x 1.5mm 

• Complimented with Aperture Frames and Blank 

Disks for nanotech experiments 

Aperture 

Frame 


0.2 nm. The ease-of-use and 

fast specimen exchange capability 

make this microscope 

particularly well-suited for 

biomedical or clinical laboratory 

environments. A key 

technical feature of the system 

is the option of choosing 

between two different imaging 

modes: high resolution 

and high contrast. This is particularly 

important for investigating 

low-contrast biological 

specimens. The specially 

developed mini-lens design 

leads to a very compact size 

where the electron-optical 

lens elements exhibit a minimum 

aberration level only. 

Carl Zeiss SMT AG 

Tel.: +49 736420 2194 

info@zeiss.de 


Blank Disks 

TED PELLA, INC. 

Microscopy Products for Science and Industry 

+1-530-243-2200 www.tedpella.com 



New Stereo Microscope 

Carl Zeiss Microimaging introduces 

the Stereo Discovery 

V20 stereomicroscope, designed 

for dissection and documentation, 

research and development, 

and quality 

assurance applications in a 

number of industries. The optical 

performance of the instrument 

boasts the largest 

available field of view (23 mm 

at 10x), the highest available 

zoom range (20 to 1) and the 

highest available resolution 

in the industry, combined in 

one stereomicroscope. These 

features allow users to visualize 

both large specimen and 

their small details on a single 

microscope without needing 

to change objectives or eyepieces. 

Thanks to the step 

motor control of the zoom op- 

LED light sources 

Olympus offers a range of 

LED sources for all fluorescent 

work, from entry level 

single colour units to advanced 

high speed multi-colour 

systems. It provides the 

right illumination solution, 

even for ‘in field’ fluorescence. 

LEDs now offer users 

an excellent alternative to arc 

burners since they have lifetimes 

in excess of 10,000 

hours, much longer than even 

the most advanced arc burner 

system. The Fluo-LED product 

range introduces additional 

flexibility to fluorescence 

microscopy. Each of the 

three models is designed to fit 

to the company’s CX microscopes 

making them ideal for 

routine as well as educational 

purposes. Further extending 

this application area is the 

ability to power the LEDs by 


tics, the stereomicroscope enables 

continuous increases in 

magnification with precise 

zoom levels to create a welldefined, 

high contrast image 

throughout the entire zoom 

range. 

Carl Zeiss MicroImaging GmbH 

Tel.: +49 551 5060 276 

mikro@zeiss.de 

www.zeiss.com/micro 

battery or even solar power 

ensuring that fluorescence 

microscopy can be conducted 

‘in field’. 

Olympus Life and Material 

Science Europa GmbH 

Tel.: +49 4023773 5426 

microscopy@olympus-europa. 

com 


Inverted Microscope for Live Cell Imaging 

Leica Microsystems presents 

the new generation Leica 

DMI3000 B inverted microscope, 

specifically designed 

for live cell research applications. 

It offers convenience 

and configuration possibilities 

that are unparalleled in 

this class of manual microscope. 

The company’s new, 

integrated incident light fluorescence 

axis produces brilliant 

images for all fluorescence 

techniques. The 

microscope also offers integrated 

modulation and phase 

contrast methods that do not 

require the use of special objectives. 

It is ideal for all manual 

fluorescence techniques. 

The system features a 5-position 

fluorescence turret for 

the fluorescence filter cubes. 

The company’s Fluorescence 

Intensity Manager (FIM) reg- 

Live Cell Care 

Nikon has launched 

its BioStation CT. By 

combining the precise 

environmental control 

capabilities of a highperformanceincubator 

with the advanced 

optics needed for driftfree 

live-cell imaging, 

it removes the need 

for culture dishes to 

be transported from 

one location to another for observation, 

and represents a 

‘hands-free’ approach to managing, 

observing and recording 

cells in culture. The company’s 

commitment to Live 

Cell Care was originally 

founded on the TE2000PFS 

system, for time lapse recording, 

the C1si microscope, for 

spectral confocal applications, 

and the BioStation IM, a single 

ulates the illumination, as 

well as the aperture and field 

diaphragm and their centering. 

Leica Microsystems GmbH 

Tel.: +49 6441 29 2550 

kirstin.henze@leica- 

microsystems.com 


user, single experiment incubator, 

but has recently been 

enhanced by the introduction 

of Controlled Light Exposure 

Microscopy (CLEM) and the 

LiveScan Swept Field Confocal 

(SFC) Microscope. 

Nikon Instruments Europe 

Tel.: +44 208247 1718 

info@nikoninstruments.eu 

www.nikoninstruments.eu

Company page 

Agar Scientific 17, 64 

AHF Analysentechnik 70, 74 

Alicona Imaging 23 

Andor Technology 14, 71 

Bitplane 19 

Bruker-AXS Microanalysis 65 

BTX Harvard Apparatus 14, 70 

Coolled 70 

CRC Beatson Laboratories Garscube Estate 44 

Dhs Solution 51 

Digital Surf 8 

Dolan-Jenner Industries 70 

EDAX 40, 73 

European Science Foundation 14 

FEI Company 5, 31, 65, 73, Outside Back Cover 

Fianium 55 

Ges. f. Biotechnologische Forschung (GBF) 73 

Halcyonics 27 

Hamamatsu Photonics 72 

Hitachi High Technol. 24, 29 

IBaI Solutions Dr. Petra Perner GbR 62 

IDS Imaging Development Systems 72 

Image Solutions 72 

Imagic Bildverarbeitung 53 

JPK Instruments 14, 15, 37, 42, 66 

Leibniz-Institut für Altersforschung 15 

Leica Microsystems 15, 66, 74, Inside Front Cover 

Media Cybernetics 71 

MPI f. Eisenforschung 40 

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Company page 

Newport Spectra- Physics 71 

Nikon Instruments Europe 7, 67, 68, 74 

Olympus Life and Material Science Europa 3, 58, 67, 74 

Olympus Soft Imaging Solutions 45, 72 

Omega Optical 70 

Omniprobe 73 

PCO 35 

Photometrics 46, 49, Front Cover 

Physik Instrumente (PI) 17 

PicoQuant 57 

Prior Scientific Instr. 15 

Rauscher 72 

Royal Microscopical Society 12 

Schaefer- Tec 71 

Semrock 70 

SkyScan 11 

Syncroscopy 15 

Techn. Univers. Clausthal 48 

Ted Pella 73 

UCLM Laborat. de Nanotecnicas 36 

Unesco-Kommission 74 

Univers. Amsterdam 16 

Univers. Innsbruck 52 

Univers. Klinikum des Saarlandes 22 

Univers. of Antwerpen 10 

Univers. of London 28 

Veeco Instruments 39 

WiTec Wissenschaftl. Instr. u. Technologie 34, 71 

Carl Zeiss MicroImaging 9, 14, 74 

Carl Zeiss NTS 14, 15, 25, 73 

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Prof. M. Gu, Swinburne Univ., Australia 

Prof. B. Hecht, Univ. of Wuerzburg, Germany 

Prof. F.-J. Kao, Nat. Sun Yat-Sen Univ., Taiwan 

Prof. N. Kruse, Univ. of Brussels, Belgium 

Priv.-Doz. M. Hegner, Univ. of Basel, Switzerland 

Dr. D. Nicastro, Brandeis Univ., MA, USA 

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Dr. P. Schwarb, FMI, Basel, Switzerland 

Prof. G. A. Stanciu, Univ. of Bucharest, Romania 

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Active Vibration Isolation for high resolution 

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Optical microscopes, digital cameras for 

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Optical microscopy systems for life science 

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