Imaging Microscopy & 2.03 GIT
VOLUME 5 MAY 2003
RESEARCH • DEVELOPMENT • PRODUCTION
Official Partner of the EMS
Scanning Electrochemical Microscopy Scanning Electron Microscopy Scanning Microradiography DualBeam Microscopy
Diagnostica Medical Endoscopy Microscopy Surgical Endoscopy Software
“Taking Shape” The body is a work of art. The upright routine microscopes of the BX2 series aren’t simply innovative enhancements to existing standards in the field of life science: the BX45 completely redefines usability standards, offering peerless ergonomic flexibility. Users no longer have to adapt – the BX45 conforms to their needs. From 45 mm tube extension through variable eye level configuration to the exceptionally low stage, the BX45 combines outstanding versatility and efficiency. Physical stresses on users are reduced to a minimum, even over long working periods. Defying the notion that great works of art can never be comfortable. For more information, contact: Olympus Optical Co. (Europa) GmbH Phone: +49 40 23 77 33 26 E-Mail: [email protected]
Easy Info • 101
To be continued
Dear Reader, after the great success and the enormous
award for his calculations of an optical
A device operating on this principle
system for error correction in electron
was developed in a joint project with
EMBL, Heidelberg, and the Jülich Re-
demand last year, we are again issuing
As with glass lenses, ‘spherical aber-
search Centre, and has already been suc-
our special supplement on image-captur-
ration’ is a problem with electron lenses.
cessfully used. Professor Kurt Urban’s
ing scanning procedures. In this edition
Rays which pass through the lens near
research group at Jülich has recently ob-
of ‘The Scanning Issue’, we have reports
the edge are markedly deflected, and the
tained the first images of oxygen atoms
for you on application and products rang-
image becomes blurred. In the light mi-
in high-temperature supraconductors.
ing from confocal laser scanning mi-
croscope and cameras, therefore, several
Over the past two years, Professor
croscopy to scanning electrochemical mi-
converging and diverging lenses are
Rose has developed a device which can
croscopy. We have considerably widened
arranged in series, which compensate
be used for the imaging of atomic
the spectrum of topics compared with last
for the aberrations. With electron micro-
processes, supported by a grant of 30
year’s issue – reflecting the clear trend
scopes, corrective measures of this sort
million dollars from the Department of
within this expanding area of microscopy.
have so far not been possible, since di-
verging lenses for the electrons have not
The ‘Imaging & Microscopy’ editorial
been available. This problem has been
team would like to congratulate Profes-
ald Rose will be presented with the ‘2003
sor Rose very warmly for receiving the
Distinguished Scientist Award for the
which combines the classic round elec-
award and wishes him every success in
Physical Sciences’ by the ‘Microscopy So-
tron lens with non-round elements – six-
his further work.
ciety of America’ (MSA) in San Antonio,
pole magnets. In a similar way to a pair
Texas. This university professor for ap-
of spectacles, this corrects the refractive
plied physics at the Technical University
error, thus creating a largely error-free
of Darmstadt is receiving this significant
In August of this year, Professor Har-
I hope you enjoy reading this issue.
Martin Friedrich G.I.T. Imaging & Microscopy 2/2003 • 1
CONTENTS EVENT REPORT
‘Learn from Nature!’
The Use of a SEM/FIB DualBeam Applied to Biological Samples
SCANNING TUNNELING MICROSCOPY PRODUCTS
Ultraflat Gold Surfaces
Microscopes ..................................................19 Laser ............................................................21 Software ......................................................37 Miscellaneous Products ..........................36, 50
I&M SHOW CASE: SCANNING MICROSCOPY FEI Company ................................................26 LEO ..............................................................26 Nikon ............................................................27 point electronic ............................................27
M. HASSELBLATT, M. HEIDECKER, ET AL.
ATOMIC FORCE MICROSCOPY
Studies of Erythrocyte Changes Caused by the Viruses Using Atomic Force Microscopy
B. ZAITSEV, A. DURYMANOV, ET AL.
I M AG E A N A LYS I S
The SEM as a Profile Measurement Device
S. SCHERER, A. PIFFER
Scanning Microradiography – A Digital 2-D X-Ray Imaging Technique
P. ANDERSON, J. C. ELLIOTT, ET AL.
SCANNING ELECTRON MICROSCOPY
SEM Imaging on Uncoated Insulators
D. BRAGA, G. BLAISE, ET AL.
Different Approaches to Visualize Cells with the Scanning Electron Microscope
SCANNING PROBE MICROSCOPY
Scanning Probe Microscopy – Enhanced Contrast by a New Mode of Operation
Microscopy Applications in Nanotechnology
CONFOCAL LASER SCANNING MICROSCOPY
Confocal Analysis of Microstructures and Micromaterials
ELECTRON TRANSMISSION ELECTRON MICROSCOPY
Electron Microscopy of Compound Materials Using Low Voltage STEM Mode
Rapid Confocal Microscope for Multidimensional Live Cell Imaging
I N S E R T: FA X R E P LY F O R M
SCANNING ELECTROCHEMICAL MICROSCOPY
High-Resolution Constant-Distance Scanning Electrochemical Microscopy on Immobilized Enzyme Micropatterns 46 A. SCHULTE, M. ETIENNE, ET AL.
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All trademarks or registered trademarks are the property of PerkinElmer Life and Analytical Sciences. Image kindly donated by Dr. David Sharp, Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY, USA. © 2002 PerkinElmer Life and Analytical Sciences.
www.liveCELLimaging.com Easy Info • 102
Events · Events · Events CONFERENCES AND MEETINGS ISPMB 2003: 7th International Congress of Plant Molecular Biology
12th International Workshop on Stereology, Stochastic Geometry and Related Fields
Jun, 23–28, Barcelona, Spain
Aug, 25–29, Prague, Czech Republic
MCM-6: Multinational Congress on Microscopy
BIO 2003, International Convention and Exhibition on Biotechnology
ICSMA-13 13th International Conference on the Strength of Materials
Jun, 1–5, Pula, Croatia
Jun, 24–26, Washington D.C., USA
Aug, 25–30, Faculty of Science of the Eötvös Loránd University, Budapest, Hungary
Scanning Probe Microscopy Short Course
CYTO 2003: Fluorescent Proteins
Jun, 2–6, Univ Surrey, Guildford, UK
Jun, 29 – Jul, 1, Oxford, UK
Surface Analysis ‘03
IV National Meeting of Material Science and Technology
Jun, 3–5, Urbana, IL, USA
Jun, 29 – Jul, 2, Naples, Italy
2003 Microscopical Society of Canada Meeting Jun, 4–6, University of British Columbia, Vancouver, Canada
Frank Rowntree Meeting, Leeds Microscopical Society Jun, 7, Morley, Leeds, UK
SCANDEM: 54th Ann Meeting of Scandinavian Society for EM Jun, 10–13, Oslo, Norway
E-MRS 2003: European Materials Research Society Spring Meeting Jun, 10–13, Strasbourg, France
EMBO Workshop on Advanced Light Microscopy 3rd international meeting of the European Light Microscopy Initiative (ELMI), Jun, 11–13, Barcelona, Spain
Scanning Probe Microscopy Short Course Jun, 16–20, Univ Surrey, Guildford, UK
ECBO 2003, European Conference on Biomedical Optics Jun, 22–25, International Conference Center, Munich, Germany
INF Meeting 2003 (National Institute for Material Physics) Jun, 23–25, Genova, Italy
NANOCOM 2003, First International Symposium on Nanotechnology in Construction Jun, 23–25, Paisley, Scotland, UK
Immunocytochemistry Course FEMS 2003, Congress of European Microbiologists
Sep, 1–5, Oxford, UK
Jun, 29 – July, 3, Ljubliana, Slowenia
EUROMAT 2003: European Congress on Advanced Materials and Processes
1st Stanislaw Gorczyca Summer School on Advanced Transmission Electron Microscopy
Sep, 1–5, Lausanne, Switzerland
Jun, 30 – July, 5, Krakow, Poland
16th National Electron Microscopy Congress (with International Participation)
JULY 2003 IBRO 2003, 6th IBRO World Congress of Neuroscience Jul, 10–15, Prague Congress Center, Prague, Czech Republic
Developments in FEGTEM V Jul, 14, Leeds, UK
Light Microscopy Summer School Jul, 14–18, Leeds, UK
Third International Conference on Scanning Probe Microscopy of Polymers (SPMP 2003) Jul, 15–18, Rolduc Abbey in Kerkrade, The Netherlands
InterOpto, International Optoelectronics Exhibition Jul, 15–18, Chiba, Japan
STM2003: 12th International Conference on Scanning Tunneling Microscopy/Spectroscopy and Related Techniques Jul, 21–25, Eindhoven University of Technology, The Netherlands
Sep, 2–5, Zmir, Turkey
EMAG 2003: Electron Microscopy and Analysis Group Conference Sep, 3–5, Oxford, UK
MC 2003: Microscopy Conference of German Society for Electron Microscopy Sep, 7–12, Dresden, Germany
9th Euroseminar on Microscopy Applied to Building Materials (EMABM) Sep, 9–12, Trondheim, Norway
Flow Cytometry Course Sep, 15–19, Sheffield, UK
2nd International Workshop Scanning Probe Microscopy in Life Sciences Sep, 18, University Clinic Charité, Berlin, Germany
ESEM 2003: European Society for Engineering and Medicine Sep, 18–21, Halle (Saale), Germany
ELSO Meeting Sep, 20–24, Dresden, Germany
DGM (German Mineralogical Society) LASER, World of Photonics Jun, 23–26, Munich, Germany
MiMeA 2003, 8th congress of the French Society of Microscopies Jun, 23–26, Toulon, France
4 • G.I.T. Imaging & Microscopy 2/2003
Sep, 22–25, Bochum, Germany
Microscopy & Microanalysis 2003
ICXOM XVII: International Congress on X-ray Optics and Microanalysis
Aug, 3–7, San Antonio, Texas, USA
Sep, 22–26, Chamonix Mont Blanc, France
Events · Events CONFERENCES AND MEETINGS
3 Dimensions THERMINIC 2003, 9th International Workshop on Thermal Investigations of ICs and Systems
Frits Zernike 50th Anniversary of Nobel Prize RMS/Zeiss Meeting
Sep, 24–26, Aix-en-Provence, France
Oct, 15, London, UK
7th European Biotech Crossroads, ‘Biotech Nantes 2003’
APHYS-2003, 1st International Meeting on Applied Physics
Sep, 25–26, Nantes, France
Oct, 15–18, Badajoz, Spain
EMBO Practical Course on “Modern Methods in Cell Biology”
POLYTRONIC 2003, 3rd International IEEE Conference on Polymers and Adhesives in Microelectronics and Photonics
Sep, 25 – Oct, 4, EMBL, Heidelberg, Germany
Oct, 20–23, Hotel Eden au Lac, Montreux, Switzerland
Spanish Microscopy Society (SME) Meeting Sep, 28 – Oct, 1, Cadiz, Spain
OCTOBER 2003 EMBL/EMBO Minisymposium on “Modern Methods in Cell Biology” Oct, 1–4, EMBL, Heidelberg, Germany
Robert Hooke Commemoration Symposium Oct, 2, Oxford, UK
ECASIA 2003, 10th European Conference on Applications of Surface and Interface Analysis Oct, 5–10, Berlin, Germany
FEMMS 2003, Frontiers of Electron Microscopy in Materials Science Oct, 5–10, Claremont Resort and Spa, Berkeley, CA, USA
BIOTECHNICA 2003, International Trade Fair for Biotechnology Oct, 7–9, Hanover, Germany
9th Congress of the French Association on Cytometry (AFC) Oct, 7–10, Strasbourg, France
OMIBS 2003, Optical Microscopy & Imaging in the Biomedical Sciences Oct, 7–16, Marine Biological Laboratory, Woods Hole, MA, USA
PHOTONEX, Trade Fair for Photonics, Optics, Fibres, Imaging and Display Oct, 8–9, Stoneleigh, Uk
4th ASEAN Microscopy Conference and 3rd Vietnam Conference on Electron Microscopy Oct, 9–10, Hanoi, Vietnam
BCEIA 2003, Beijing Conference and Exhibition on Instrumental Analysis Oct, 14–17, Beijing, China
Principles and Applications of Time-Resolved Fluorescence Spectroscopy Oct, 20–24, Wista-campus Adlershof, Berlin, Germany
VISION 2003, International Fair for Image Analysis Oct, 21–23, Stuttgart, Germany
MESUREXPO, Exhibition on Measurement and Instrumentation Solutions Oct, 21–23, Paris, France
SEMT Half-Day Meeting, The School of Pharmacy Oct, 29, London, UK
HIGH-RESOLUTION CONTACT-FREE Surface Topography and Flatness Measurement The TopMap 50 utilizes scanning white light interferometry to generate high resolution, 3D topographical images on rough and shiny surfaces. sample area of about 5 x 6 mm2 und 30 x 40 mm2 with resolution up to 0.01 micron in Z-direction
GSA Meeting, Geological Society America Nov, 2–5, Seattle, WA, USA
MICROTECH, Electrooptics and Microelectronics Nov, 4–6, Tel Aviv, Israel
MEDIPHAR, Medical Equipment and Pharmaceuticals Show Nov, 7–10, Taipei, Taiwan
Society for Neuroscience Annual Meeting Nov, 8–12, New Orleans, LA, USA
Cryo-Microscopy Group Annual Meeting Nov, 19, The EM Centre, University of Birmingham, UK
· Test stration · Demon g in lt 4 7 su Con 43 604-1 9 (0) 72 4 + e e .d n c o Ph @polyte topmap m pmap.co www.to
BioTech Forum, International Forum for Biotechnology Nov, 26–28, Stockholm, Sweden
BIONOVA Nov, 26–29, Padua, Italy Easy Info • 103 2
POLYTEC GMBH Polytec-Platz 1-7 D-76337 Waldbronn Phone +49 (0) 72 43 604-0 Fax +49 (0) 72 43 6 99 44
Laser 2003 • Halle C1 • Stand C1.375 Advancing Measurements by Light
News · News Prior Scientific Announce Internal Promotion Prior Scientific are pleased to announce that with effect from April 1st 2003, Gavin Mills has been promoted to the position of Product Manager with responsibility for both Microscopy and Automated Microscopy products. Gavin will be working closely with Prior’s Automated Microscopy Product Specialist, who will be providing pre- and post-sales customer support while contributing to the marketing planning for our product portfolio. Previously Gavin worked for Carl Zeiss and was responsible for the sale of automated microscope systems. www.prior.com
Microscopy for Harvard Medical School Prior Scientific announced the installation of a number of ProScan and OptiScan microscope automation systems at the Nikon Imaging Centre on the Campus of Harvard Medical School in Boston. From training in light and confocal microscopy to consulting on specific microscope projects, the Imaging Centre utilises the latest in technology to provide solutions to the many challenges in cell biology research and other related fields. With several types of motorised stages, focus controls, high speed filter wheels, shutters and custom configurations supplied, the company is an integral part of the multi level capabilities offered in the Imaging Centre. www.prior.com
British Subsidiary of Soft Imaging System Founded As of March 1st 2003, Soft Imaging System, product provider in the digital image-analytical field, has its own subsidiary in Great Britain. Opening this office is a reflection of the company’s business growth as well as the steadily rising demand. Executive manager Johannes Rosenmöller: „Locating in Britain provides us with the opportunity to optimise communication
with our existing customers and with prospective customers. We’ve got competent assistance for questions regarding our products, for installations, training courses and support services. And then there’s the close cooperation with our sales and OEM partners in the UK. We can make necessary sales-related decisions more quickly and move on to immediate implementation“. www.soft-imaging.net
Dr Wolf-Otto Reuter Takes Charge of Leica Microsystems Dr Wolf-Otto Reuter has been appointed the new CEO of Leica Microsystems. Reuter’s succession to Dr Gerhard Kleineidam is immediately effective. Kleineidam, who joined the international enterprise in February 2002 and became CEO in September 2002, had decided to pursue his career outside the company. Reuter is taking the helm at a time of general economic weakness which has not left even Leica unscathed. Reuter assesses his new role as follows: “My paramount tasks are to increase our customer orientation, to strengthen the company’s innovation power and to continue standardising and improving in-house processes.” www.leica-microsystems.com
Award for Service For the third consecutive year, Jeol has received the Omega Northface Award in recognition for its commitment to providing exemplary service and exceeding custumer expectation. Through quarterly surveys of 150 customers, Omega Management Group computed the company’s customer satisfaction score to be 4.0 or above out of a possible 5.0 in the categories of technical support, field service, account management, and training. The company was rated on product reliability, service response time, call center assistance, and service expertise. This year 75 companys and over 275 projects throughout the United States were judged.
Design Award for Microscope The new MIC-D digital microscope from Olympus has won a prestigious design award for its innovation and functionality. The “red dot” Award is presented annually by the Design Zentrum Nordrhein Westfalen, Germany. It is one of the biggest design competitions worldwide. Winners are selected by 26 independent jurors from nine countries, and all jurors are international design experts. The microscope offers user friendly solutions for schools, universities and laboratories. Specimen views can be enlarged with the freely adjustable zoom and immediately displayed on an USB-connected PC, without the need for timeconsuming configuration. www.olympus-europa.com
The Royal Microscopical Society has opened exhibitor and attendee registration for its microscopy event, MicroScience 2004. The international conference and exhibition of the science of microscopy and in situ analysis will take place from 6th to 8th July 2004. For three days, visitors to London’s Excel centre will be able to hear the world’s leading microsocopy experts, see the latest product developments and participate in MicroSciences workshops. The conference sessions will cover twelve key topics from materials and life science microscopy, such as nanostructures, dynamic events, neuroscience and advanced SEM. www.microscience2004.org.uk
Easy Info • 104 6 • G.I.T. Imaging & Microscopy 2/2003
‘Learn from Nature!’ The Microscopy Business Division of the Carl Zeiss Group staged the ‘7th Microscope Day at Zeiss’ in Jena on 19th March with the motto ‘Bionics – in the footsteps of nature’. With more than 400 visitors from the areas of research, technology and academia, the event achieved a new visitor record. The programme had something to everyone, from those new to the field to experts. There was plenty of information on trends and innovations for visitors in lectures, workshops and expert discussions, and the latest technologies and software developments were presented in hands-on demonstrations with devices. As guest speaker, Professor Andreas Offenhäuser of the Institute for Thin Films&Interfaces (ISG2) – Bio and Chemosensors, Jülich Research Center, reported amongst other things on the development of cell-based hybrid systems. The objective of these projects is the controlled formation of networks made of nerve cells, which are then used – together with electronics – to investigate neuronal information processes. One focus of the event was the presentation of innovative microscope solutions for the material sciences, amongst other things, the optical imaging of uneven surfaces in the nanometre range, and the measurement of the depth of focus in quality-real time. This accelerates inspection procedures and also improves their quality. A new development in the area of fluorescence microscopy puts users in a position to obtain optical sections of biological fluorescence samples that are not masked. This increases the degree of contrast in the images captured and collocations of marked substances can be clearly demonstrated. In the past business year, the Microscopy Business Division of the Carl Zeiss Group achieved an increase in turnover of 21% compared to the previous year. This meant that this division had the highest growth rate within the group for two years running.
Dr Ulrich Simon , General Manag er Microscopy
Business Divisio n Group at Carl Zeiss
Instrument de monstrations
Dr Martin Friedrich
For more info circle no.
Hands-on sess ion G.I.T. Imaging & Microscopy 2/2003 • 7
The Use of a SEM/FIB DualBeam Applied to Biological Samples Hans Mulders
A DualBeam instrument using both a focusing electron and ion column, can effectively be applied to biological samples using cryogenic temperatures to ensure compatibility with the vacuum conditions of the instrument. A cryo stage as commonly used for SEM can be made compatible with a DualBeam geometry taking into account the requirements for both techniques. Experiments on biological samples have shown that milling into the frozen substrate to create site-specific cross-sections is fast and easy and that the obtained cross-sections show very good information that is comparable or better than obtained with non-site-specific cryo fracturing. This opens the way to three-dimensional local analysis of the sample.
Introduction In the world of Scanning Electron Microscopy (SEM) the use of a cryo stage is a very common way to accommodate wet samples and colloidal solutions or mixtures of water and fat, such as present in many food and cosmetic products. By freezing the sample to a low temperature, the vapor pressure becomes so low that it is well below the operating pressure of the instrument and thus the sample is stable. For example, at a temperature of –140°C the vapor pressure is in the range of 10–10 mBar, whereas the working chamber pressure is between 10–4 and 10–6 mBar. In regular use the practical operating temperature of the cryo system is between an upper limit and a lower limit of liquid nitrogen temperature (77 K) and for this specific range the evaporation of ice can be neglected. The very low temperature can be used to prevent local heating of the sample by electron beam irradiation to a temperature well above the upper limit where the evaporation of ice starts to be noticeable. Note that within the chamber it is very common to have at least one surface at an even lower temperature then the sample and this surface will act as a collector: it is generally referred to as anti-contaminator.
System Components A DualBeam Strata 235M makes use of the following geometry for the two columns (Fig. 1). The two beams have an 8 • G.I.T. Imaging & Microscopy 2/2003
intersection point at 5 mm below the electron column, which coincides with the eucentric point of the stage (at this point the tilt axis is perpendicular to the electron optical axis and hence the image does not move when tilting the sample). It can be seen from this set-up that it is necessary to be able to mill into the sample when the stage is tilted to 52 degree and as a consequence the cryo stage that is mounted on top of the standard stage will be tilted as well. A Gatan Alto 2500 cryo stage was used with a facility for cryo fracture and thin film coating. The cryo stage and the anti-contaminator and its supplies take up a considerable space in the DualBeam chamber. Following detailed engineering discussions a configuration was found where all components fit well and uncompromised operation was guaranteed. It was particularly important to ensure that the required tilt of 52 degree can be obtained and that the anti-contaminator could be sited to ensure optimal cryo performance with minimal geometric disturbance. For loading of samples into the chamber use is made of the transfer valve of the Alto 2500 cryo system and standard procedures for cooling of the sample from room temperature to cryogenic temperature are followed. It is important that during the freezing of the sample, care is taken to avoid ice formation on the top surface, as this will obstruct selection of the proper sample area and decent imaging of the sample. In the standard operation of the DualBeam it is common practice to apply a
Fig. 1: Schematic set-up of the cryo stage on the standard stage and the position of the two columns
Fig. 2: Milling a square hole into pure, frozen water helps to understand parameters such as dwell time, overlap, beam current and allows to quantify the milling rate.
Fig. 3: Bakers yeast showing both a cryo fracture part and a FIB created cross-section part (below the drawn line). Image is slightly scan rotated.
Experiment Prior to using real samples, some experiments were done with milling into pure water. A simple square was created by top down milling as shown in Fig. 2. With a simple experiment like this it was confirmed that the milling rate in ice actually is very high and around 10 um3/nC. This is a big advantage as making relatively large holes and cross-sections or multiple sections in the third dimension is now possible in an acceptable time frame. Another consequence is that an ion beam, when used for imaging, will rapidly remove sample layers even in low current mode: as a consequence a DualBeam, where the electron column can be used for non-destructive imaging is far more ideal for this application then a single beam FIB. As many biological systems have a very high water content, the milling rate measured here can be used as a good reference value for setting up automated milling jobs. Another characteristic of the milling process is the smoothness of the crosssection. Using pure ice as a reference sample helps to define the overlap and the milling strategy for optimal smoothness of the cross-section.
Results One of the first tests was done using bakers yeast as a reference sample. This sample is commonly used as a test sample. In this case it was cryo fractured in the preparation chamber, passed through the transfer valve and loaded onto the stage. A cross-section was then made with the ion beam, followed by imaging with the electron beam. The result is
shown in Fig. 3. It should be noted that in an image such as this, the actual start of the cross-section does not show up as a straight transition line from top surface to vertical section. This is due to the original uneven fracture surface. This would easily be seen if the sample top surface was flat. The image in Fig. 3 has a slight scan rotation and the actual milling direction is perpendicular to the solid line drawn in the image. The cell in the upper left corner is opened by the cryo-fracture, whereas the cell in the lower right section is opened up by the FIB cut. It is clear that the information revealed by the FIB cut is very good and that the internal bodies in the cell are not damaged, but nicely sectioned in a straight plane. Details like cell membrane and nucleus are very visible. It would be possible to make another section using an image like this, by cutting just a little deeper in the material and so revealing the third dimension. Another example is a food product such as liquid margarine: this basically is an emulsion of water droplets in oil that is stabilized by emulsifiers and proteins. In material combinations such as these, interest in the mechanisms at the interface between water droplets and oil lead to a better understanding of the structuring mechanism of emulsions. A complete milled section is presented in Figs. 4a and 4b, showing individual water droplets in the cross-section and a higher magnification image showing more fine- detail. This example shows again that milling into biological samples such as emulsions of fat, oil and water can be done easily, and that interface studies at localized positions can be performed very well. Most important in this respect are the absence of milling artifacts (due to different material behavior) and good control over the final milling steps and the geometric stability of the system. Another biological system is presented by a bacteria culture or cell culture in a rich nutrition and friendly environment. If the dilution of the cells / bacteria is low enough and the growth conditions are favorable, then it will be possible to create a sample with individual and separated organisms, in a thin water film on a substrate. Such cells can be presented as monolayers on a flat substrate such as a microscope cover glass (eucaryotic cells) and covered in a thin water film only. Bacterial cells can be presented as isolated colonies from petri dish culture or dispersed within cryopreservative storage gel as here. In this way, once the sample is frozen, individual cells / bacteria can be selected with the electron beam when looking top/down and cut at specific sites with the FIB to reveal the internal struc-
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G.I.T. Imaging & Microscopy 2/2003 • 9
Easy Info • 105
coating on the sample prior to milling, especially for non-conductive samples. This very local coating is created by ion beam or electron beam induced deposition from a local gas supply of an organometallic vapor. This commonly used system, referred to as Gas Injector System or GIS, however cannot be applied to a very cold sample: gas molecules from the GIS system will immediately condense on the cold surface and form a (thick) film of frozen precursor gas rather then a thin film of decomposed organo-metallic material. As a consequence all operation of the DualBeam with cold samples has been realized without use of any GIS system. Note that the use of the coating equipment of the cryo system is primarily applied for the purpose of high-resolution imaging and not for enhancing milling properties: as such this kind of coating is too thin (nm range instead of µm range).
Figs. 4 a, b: Cross-section into liquid margarine, showing individual water droplets (left) and higher detail about the interface (right) at 100 kx.
cut faces contains several profiles through the eucaryote cell nucleus. Within this nucleoplasm the DNA exhibits molecular coiling to differing complexities. Many such supercoils can be seen in Fig. 6b. In some occasions a thin metal coating deposited on the sample, after it has been cut with FIB, is very helpful to increase the image quality: this coating is created in the preparation chamber of the cryo system so it means re-loading and repositioning of the sample. In practice, using stage automation of the instrument, this can be done very rapidly. The coating not only helps to reduce charging of the sample, but also enhances the edges in the image. For any further milling the coating is not a problem as it will be milled away easily.
Figs. 5a, b: Single bacterium completely cross-sectioned by FIB after the wall has been stripped away. In Fig. 5a a loop of mesosomal membrane can be seen to penetrate the cell at the approximate 9 o’clock position. Attached to the mesosome is the bacterial circular chromosome. Much of the bacterial cytoplasm is occupied by densely packed ribosomes (Fig. 5b).
For samples that are commonly used in cryo SEM observation (such as biological and cosmetic / food samples) an added FIB capability for cutting into the sample is a very valuable extension. The FIB can be used as a micro-surgery tool, that can open up the third dimension. Milling is very fast and precise and most important, structures that show up are sharply cut without noticeable damage. A DualBeam is ideal for such type of investigation as the electron column is used for imaging without cutting and the ion beam for cutting without imaging: the two beams are fully complementary to each other.
Figs. 6a, b: 90 degree „double FIB-cut“ on a single gut epithelial cell. The edge on the right of Fig. 6a has not been cleaned up. Higher magnification on the right (Fig. 6b) shows a greater detail of what is thought to be supercoils of DNA exposed by ice sublimation.
tures. So it is possible to directly use living material to work from and examine it without staining or chemical fixation. Its internal structure can be investigated in a cross-section at a very local site, created by the ion beam. As an example a Ractobacillus bacterium has been cut perpendicular to its long axis, similar to macro analogue situation of a sausage being cut with a knife. The FIB really acts now as a micro-surgical knife, that creates a very smooth and straight cut. 10 • G.I.T. Imaging & Microscopy 2/2003
Vertebrate gut epithelial cells were chosen as model systems from more complex higher organisms where the cell not only contains complexly coiled chromosomes within a distinct nucleus but also many sub cellular cytoplasmic organelles. In the figures shown the cell has been milled on two faces at right angles to each other (Fig. 6a). This double cut was employed to follow the projection of different features into the bulk of the sample. The area framed within the two
The experimental work was realized by good teamwork within the FEI applications lab in Acht, The Netherlands. In particular the author would like to thank Mike Hayles (FEI) and Marilyn Carey (Gatan) for their knowledgeable support on many aspects of the cryo system and for helping to optimize the system. Both the Unilever Research group in Vlaardingen, The Netherlands and Dr G. McKerr from the School for Bio-medial Sciences of the University of Ulster are kindly acknowledged for providing prepared samples used in the experiments described above. Dr Hans Mulders Application Development Manager FEI Electron Optics 5600 MD Eindhoven, The Netherlands [email protected]
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SCANNING TUNNELING MICROSCOPY
Ultraflat Gold Surfaces Markus Hasselblatt, Manfred Heidecker, Bryan Jackson, Peter Kernen, Peter Wagner
Substrates used for microscopy are generally desired to be flat. This is especially important for ultra high resolution microscopy such as scanning tunneling microscopy (STM)  or atomic force microscopy (AFM) . Some flat substrates such as mica, highly oriented pyrolytic graphite (HOPG), or glass are readily available and widely used.
However, obtaining substrates of any elemental composition with flat topography is not straightforward. A common strategy to make flat substrates of a given elemental composition is to use thin film growth of the desired element or compound on a flat carrier. Topographic features resulting from such thin film growth may still be large enough to significantly interfere with the dimensions and properties of adsorbed objects under microscopic observation. As an example, studies of the immobilization of biological molecules using SPM typically face such challenges when the roughness of the un-
derlying substrate is on the same order of magnitude as the size of the biological molecules. In particular, gold surfaces have been used to immobilize biological molecules [3, 4]. More sophisticated approaches make use of thin organic films on gold surfaces that serve as immobilization interfaces for biological molecules. The roughness of thermally evaporated gold surfaces is about 2 nm RMS with typical zranges between 10–30 nm (Fig. 1, bottom right). This poses a limitation to SPM studies on regular gold surfaces that catalyzed the discovery of template stripped gold (TSG) surfaces [5, 6].
Fig. 1: STM micrographs of thermally evaporated (lower right) and TSG (left) gold surfaces. Section along the black line in TSG micrograph (top left) shows a total height variation of less than 1 nm across 4000 nm. TSG surfaces more accurately represent the substrate topography often depicted in protein adsorption models (top right). The STM used in this work is a commercial instrument from RHK Technologies. A high gain preamplifier combination was used to enable ultra-low current or ultra high resistance measurements. 12 • G.I.T. Imaging & Microscopy 2/2003
In this approach, mica as a flat carrier substrate is coated with gold very much like regular gold surfaces. However, instead of using the gold surface that is left after deposition, the entire sandwich is glued onto a secondary carrier with the gold surface facing the glue. This allows the stripping of the mica off the gold film leaving it intact. A pristine gold surface that mimics the flat topography of the mica template is exposed. In the last decade, TSG surfaces have become popular among SPM users desiring ultraflat gold surfaces. While its advantages are straightforward, two limitations remain: The template formed gold surface needs to be exposed by stripping mica off its surface. In practice, it is rather tedious to ensure that no thin mica films remain on the gold since mica itself cleaves rather easily. Also, the resulting gold-epoxy-substrate complex is susceptible to degradation by some common solvents. This narrows the types of buffers or solvents that can be used for biological experiments on this substrate. Recently, we have developed a bonding technique, in which indium solder is used to bond the gold film to the supporting substrate. Such sandwiches resist all common organic solvents and aqueous buffers typically used in biological applications. Also, stripping the mica off the gold surface was found to be easier in comparison to the epoxy based gluing.
Discussion The gain in substrate quality when using TSG is illustrated in Fig. 1. A thermally evaporated plain gold substrate is compared to a TSG substrate with a topo-
SCANNING TUNNELING MICROSCOPY
graphic section of the latter across four micrometers showing less than 1 nm in height variation. It is only on such substrates that the model depicted in Figure 1 comes close to representing the actual state of a protein bound to a surface. Following is a brief summary of important aspects of TSG formation. Mica used as a template is in muscovite form. Mica sheets can be purchased in various formats and grades. We used square muscovite sheets of the highest grade from Structure Probe Inc. in 25 mm x 25 mm format. Freshly cleaved mica is degassed and dried overnight in vacuum at elevated temperature. Since substrate heating and temperature measurement in a vacuum chamber is not trivial, we found that each deposition setup requires individual calibration. We determined a temperature of 350ºC for both annealing and thin film deposition. This value was found to yield the flattest and most defect free TSG surfaces when comparing STM micrographs of TSG samples grown at different temperatures. Also, our particular setup incorporated slow cooling of the finished gold film to room temperature at about 2ºC/s. Goldcoated mica sheets can be cut to size with a sharp knife. The resulting pieces are glued (for instance with 377 Epoxy from Epotek) to supporting substrates smaller than the mica pieces. This keeps glue from creeping onto the sides of the mica sheets which would make subsequent stripping impossible. Once the glue is cured, mica is carefully lifted off the gold surface. Also, solvent based weakening of the gold mica interface may facilitate the stripping process. A convenient way to check for remaining mica sheets is a resistance measurement across the surface. Two drawbacks arise when using TSG as previously described . The use of epoxy limits the types of solvents that can be used in subsequent experiments with TSG. Also, stripping the mica sheets off the gold surface is not always complete. While the latter limitation can be alleviated by good cleaving practice and visual as well as conductance validation of mica and epoxy removal, the first constraint remains a fundamental one when using epoxy glue for bonding. A non-glue based bonding strategy has been developed by the authors to address the second restriction. In this approach, indium is used to solder the gold film to the supporting silicon substrate prior to mica stripping. Indium is a highly malleable metal with a low melting temperature that readily forms an alloy with gold at low temperatures. XPS  data in Fig. 2 illustrates why a multi-layered thin film structure is needed to successfully produce indium bonded
TSG. Titanium is used as a diffusion barrier to protect the gold surface in contact with the mica from the indium when soldering the mica sheet to the gold coated silicon substrate. Sputter depth profiles clearly show the difference induced by the diffusion barrier. The top part shows a schematic (left) and the corresponding profile (right) of indium soldered gold. Clearly, indium and gold have formed an alloy. Due to the significantly higher amounts of indium in relation to gold, the atomic concentration of the latter is very small in the alloy. Also, indium Fig. 2: Indium forms an alloy with gold (schematic top left) as deduced seems to aggregate in from sputter depth profiling (top right). A titanium diffusion barrier higher concentration at the (schematic lower right) effectively blocks indium from reaching the gold film in contact with the mica (lower left). XPS measurements were permica alloy interface. Fiformed using a Physical Electronics Quantum 2000 X-Ray Microprobe. nally, the observed indium The XPS was used to investigate the elemental composition of TSG mica interface showed sig- surfaces. Repeated 4kV argon ion bombardment and spectral acquisinificantly higher adhesion tions were used to perform elemental depth profiling of samples. in comparison to just gold other noble metals, metals, or metal oxiand mica. Stripping the mica off was exdes, and polymers. We encourage further tremely difficult. The bottom part of Fig. 2 investigation of said technology. illustrates how a titanium diffusion barrier effectively blocks the alloying process from reaching the mica interface. The profile Literature (left) shows areas of distinct elemental  G. Binnig, H. Rohrer, C. Gerber, E. Weibel: Physicomposition with the corresponding diacal Rev. Letters 49(1) 57 (1982) gram shown on the right.  G. Binnig, C. Quate, C. Gerber: Indium bonded TSG has been successPhysical Rev. Letters 56(9) 930 (1986) fully stripped off the mica and has re M. Hegner, P. Wagner, G. Semenza: mained inert even after prolonged expoFEBS Letters 336 452-456 (1993) sure to solvents such as chloroform.  O. Medalia, J. Englander, R. Guckenberger, Indium bonding also made stripping easier J. Sperling: Ultramicroscopy 90 103-112 (2001) than epoxy gluing. These substrates have  M. Hegner, P. Wagner, G. Semenza: Surface Sciopened up the opportunity to use organic ence 291, 39-46 (1993) thin film coating protocols that previously  P. Wagner, M. Hegner, H. J. Güntherodt, failed on the epoxy based TSG. G. Semenza: Langmuir 10, 3867 (1995)
 K. Siegbahn, et. Al.: ESCA – Atomic Molecules,
As resolution in microscopy has improved greatly over time, the need for ultraflat substrates gains importance. The availability of such substrates greatly depends on the elemental composition needed at the surface. While thin film deposition on flat substrates expands the variety of substrates available in flat form, thin film growth morphology can still yield unacceptable roughness. In the case of gold, template stripping has proven to be an effective way to elude this problem over the past decade. We have shown that alternative bonding strategies can improve some shortcomings of the TSG approach. We believe that template stripping technology may be expanded to encompass
of Electron Spectroscopy, Nova Acta Regia Soc. Sci.
and Solid State Structure Studies by Means Ups. (1967)
Manfred Heidecker Bryan Jackson Peter Kernen Peter Wagner Markus Hasselblatt, Ph.D. Manager R&D Support ZYOMYX, Inc. 26101 Research Road Hayward, CA 94545, USA Tel.: +1 510 266 7500 [email protected]
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ATOMIC FORCE MICROSCOPY
Studies of Erythrocyte Changes Caused by the Viruses Using Atomic Force Microscopy Boris Zaitsev, Aleksandr Durymanov, Ludmila Bakulina, Vladimir Generalov
Erythrocytes or red blood cells (RBC) are one of the favorable objects for atomic force microscopy (AFM). The membrane of RBC has a relatively simple structure and is stable during preparation of the samples for AFM. The membrane has been well characterized at the biochemical level . The AFM was applied to study RBC in air , and in saline solution , after chemical influences [4–6], mechanical deformation , and after infection with malaria plasmodium [8, 9]. AFM has also been used for direct diagnostics of some blood diseases .
14 • G.I.T. Imaging & Microscopy 2/2003
Interaction of virus particles with cell membrane is one of the key problems of the virology. Method of AFM could give a unique opportunity for direct observation of the processes of virus adsorption and penetration through a membrane. We tried to apply AFM to examine the interaction of virus particles with a membrane of RBC, which serves as a simple model for studies of virus-cell interaction.
Materials and Method Suspensions of the animal viruses: (1) influenza virus A strain H7N7, in a concentration 64 hemosorbtion units (HU); (2) canine parvovirus (CPV) strain ”Layka” prepared as a 10% homogenate of a dog small intestine, in a concentration 128 HU; (3) rubella virus strain P-1965 prepared on Vero cells, in a concentration 256 HU. Fresh RBC were obtained from rhesus monkey, chicken and goose. Viral suspensions were mixed with erythrocytes at 4–6°C and incubated at the same temperature during different periods from 1 to 90 min. Then the incubation was stopped by addition of the paraformaldehyde to final concentration 2%. The samples were fixed during 2 h at 4°C. At the next step fixed erythrocytes were immersed in distilled water (pH 6.8) to reach their concentration to
108 cell/ml. Suspension 5µml-aliquots were placed on a slide and dried in the air to be ready for examination in atomic force microscope. The samples were examined in atomic force microscope SolverP47BIO (NT-MDT, Russia), using cantilevers NSG11 (NT-MDT, Russia). Intermittent contact mode in air was used, which provide simultaneous registration of two signals: Height and Mag (Error Mode). First signal represents an altitude of the sample in each point of scanned surface, while the second signal detects cantilever deflection. This mode of the observation provides visualization of fine Z features having sizes of 10–20 nm on a sharp slope of a surface, when height difference is about 1 µm.
Results Incubation of the RBC with viruses caused the changes in a shape and fine structure of membranes of RBC. While changes of the shape may be recognized in light microscope, high resolving capacity of the atomic force microscope provides visualization of tiny structural details. Thus, we observed the spectrin skeleton of the surface of native erythrocytes, which was described previously . Intact monkey RBC have toroidal shape (Fig. 1a). Examination of monkey RBC incubated with influenza virus and
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ATOMIC FORCE MICROSCOPY
Fig. 1: Rhesus monkey RBC before (a) and after (b) incubation with influenza virus during 1 min at 4°C. Intermittent contact mode. Scan size 20x20 µm
Fig. 2: Goose RBC before (a) and after (b) incubation with rubella virus during 10 min at 4°C. Intermittent contact mode. Smudgy strips on (b) show scanning artifacts. They are associated with the distortion of erythrocyte membrane and appear at the sites to which the probe clings. This effect can be applied to detect changes occurring in the membrane. Scan size 17x17 µm (a); 20x20 µm (b).
Fig. 3: a – A cavity on the surface of monkey RBC after incubation with canine parvovirus; b – surface of the goose RBC after incubation with rubella virus. MAG image (Error mode). Height of particles of the chains are 20-30 nm, length of chains – to 1500 nm. Linear dimensions of blocks are about hundreds of nanometers. These features were absent on native cells. Scan 600 x 600 nm (a); 5x6 µm (b).
canine parvovirus during 1 min showed development of several protrusions looking as a knob or crest (Fig. 1b). Incubation of RBC with these viruses during 90 min resulted in loosing of the shape by erythrocytes, which looked like conglomerates of formless sacs. Incubation of monkey RBC with rubella virus did not alter erythrocyte shape. Avian RBC are ellipsoidal cells showing central bulge related to the nucleus (Fig. 2a). Influenza virus and parvovirus did not alter the shape of avian RBC during incubation for 90 min. Incubation of the goose RBC with rubella virus during 10 min did not alter erythrocyte shape, while the central bulge disappeared (Fig. 2b). Scanning artifacts are seen on the Fig. 2b as smudgy strips lying parallel to scan direction. These artifacts could be related to the appearance of sites to which the probe clings, and reflect a distortion goose erythrocyte membrane caused by the interaction with rubella virus. Incubation of monkey RBC with influenza and canine parvovirus, as well as incubation of goose RBC with rubella virus, resulted in the development of tiny changes of the surface, which could be examined only in atomic force microscope. The pits of various contours and knobs were observed on the surface of RBC membrane (Fig. 3a). Microparticles having heights of 20–30 nm were found. The microparticles formed the chains lying parallel to each other. These bands of chains were located at various angles to the direction of the scanning. The blocklike structures having a height of 200 nm and lateral dimensions about hundreds of nanometers also were found on goose erythrocyte surface incubated with rubella virus (Fig. 3b). The same blocklike structures were observed on monkey RBC after incubation with influenza virus and parvovirus. Examination of chicken RBC incubated with influenza virus in atomic force microscope found virus-like structures on erythrocyte surface (Fig. 4). The structures had a height of 25–30 nm, which corresponded to average value of the size of dried influenza virus particles on the flat surface of mica.
Fig. 4: Influenza virus particles on the membrane of chicken RBC. Intermitted contact mode. Two pictures represent two simultaneously detected signals: a – Height image three-dimensional view; b – MAG (error mode) image. Scan size 3x3 µm. Height image Fig. 4a is able to show virions only on the limited portion of the erythrocyte. In the error mode (Fig. 4b), virions can be observed on the whole surfaces of erythrocyte. Artifacts in right bottom corner were caused by the probe excitation at sharp boundaries during scanning. The pictures illustrate the advantage of Error Mode for examination of a small details on abrupt slope of erythrocyte (about 1 µm height). 16 • G.I.T. Imaging & Microscopy 2/2003
Our studies showed that the incubation of RBC with viruses resulted in different changes of erythrocyte shape and features of the structure of their membrane depending on the origin of RBC and the type of the virus. The most prominent changes of erythrocyte shape were detected in case of the incubation of mon-
ATOMIC FORCE MICROSCOPY
ies of the process of virus interaction with RBC using atomic force microscope showed a possibility to visualize the virus on erythrocyte surface. Obviously, further elaboration of the experimental technique and usage of other types of the cells are needed for resolving the task of direct observation of the viruscell interaction.
changes under viral influence. The method provides possibility of the simultaneous observation of the shape deformation of RBC and fine structure of their membrane. Different viruses cause a different pattern of the RBC changes. A correlation of the results of the reaction of hemagglutination and changes of the shape of RBC treated by the same virus has been found.
Conclusions Atomic force microscopy is a simple and fast method for examination of RBC and their
Acknowledgements This study was supported by the ISTC grant #1802.
Literature A list of references can be obtained from the authors.
Boris Zaitsev, Ph.D. Aleksandr Durymanov Ludmila Baculina, Ph.D. Vladimir Generalov, Ph.D. State Research Center of Viropogy and Biotechnology “Vector” Koltsovo, Novosibirsk region 630559, Russia [email protected]
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MFP-3D: Topography in three Dimensions
Poly(styrene(ethylene-r-butadiene)-styrene) triblock copolymer (SEBS) spuncoat onto a silicon wafer. Sample courtesy Rachel Segalman and Alexander Hexemer, Kramer Group, UCSB
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key RBC with influenza virus and canine parvovirus. Similar changes were reported after the application of MayGrundwald Giemsa to human RBC . The shape of avian nucleated RBC appeared to be more resistant to the influence of the viruses. No visible changes of the shape were detected after the incubation of avian RBC with influenza and parvovirus even for 90 min. However, rubella virus altered the shape of goose RBC. In contrast, rhesus monkey RBC did not change after the incubation with the same virus. It is interesting, that the results of AFM were in a good correlation with the data of hemagglutination studies of the same virus-RBC pairs. The changes of the shape of RBC correlated with a positive reaction of hemagglutination, while negative reaction were observed for virus-RBC pairs did not show the changes of the shape. Incubation of the RBC with the viruses altered fine structure of their membrane, which resulted in the appearance of the block-like structures and microparticles forming sheets of chains on RBC surface. We couldn’t explain their nature yet. It is easy to suggest that these structures represent a kind of artifacts of the scanning. However, the angles between the parallel structures in chains and the scanning direction varied on the surface of single cells, evidencing for their relation with the membrane changes. Our studies revealed that AFM is able to visualize the viruses on surface of mica. We could not detect any virus on the surface of monkey RBC even after incubation during 1 min. It may be suggested that the process of virus-erythrocyte interaction was very fast, and the viruses “disappeared” during this period of the incubation at 4°C. Chicken RBC were resistant to influence of the influenza virus, and it was possible to visualize the virus on erythrocyte surface (Fig. 4). Thus, our stud-
ATOMIC FORCE MICROSCOPY
The SEM as a Profile Measurement Device Stefan Scherer, Anton Piffer The scanning electron microscope is widely used in observing microscopic objects with a large depth-of-focus. Unfortunately, no measurements concerning the 3D structure of the observed objects are possible. The software package MeX (see also G.I.T. Imaging & Microscopy 3/2002, pp. 45–46) by Alicona allows to extend the SEM towards a 3D measurement device. In this report the roughness measurements performed by MeX are compared to those performed by a Taylor Hobson device.
Two Different Measurement Techniques A profile measurement device is usually based on a tactile measurement principle. The surface is measured by moving a stylus across the surface. As the stylus moves up and down along the surface, a transducer converts these movements into a signal which is then transformed into a roughness number and usually a visually displayed profile. Multiple profiles can often be combined to form a surface representation. MeX, however, as a non-tactile measurement method is based on the analysis of stereoscopic images. From the object observed in the SEM two images are captured from two different viewpoints. This is usually performed by a simple tilting of the specimen holder in the order of 3 to 7 degrees. The resulting two images are imported into the MeX software and a 3D surface representation is instantly calculated. The user is then enabled to visualize and analyse the 3D dataset
in various forms such as a profile, area and even volume analysis.
Experimental Results In order to evaluate the applicability of MeX as a roughness measurement method, comparative measurements on the same specimen were performed. Fig. 1 : Roughness measurement with Taylor Hobson device As a test sample the surface of a ground glass rod was examined. The field of view was approximately 500 µm times 375 µm at an image resolution of 1024 times 768 pixel. Fig. 1 shows the roughness measurement performed with the Taylor Hobson device. Fig. 2 shows the measurement performed with the SEM and the MeX software. The profilometer, however, only allows the visualization of the profile measurement without the viFig. 2 : Roughness measurement with SEM and MeX sual link to the surface, whereas MeX offers the visualization of the profile Table 1: Numerical comparison between profilometer and MeX measurement and the visual appearance of the surface. The numerical comparison Ra Rq Rz Rp is summarized in Table 1. Taylor Hobson 0,7585 0,985 3,7076 1,7735 The average roughness of the profile MeX 0,7573 0,995 5,6163 2,5968 (Ra) and the Root-Mean-Square roughness of the profile (Rq) are measured with very little deviation. Within the measurement accuracy of both methods roughness measurement device from the results can be classified as identical. Taylor Hobson and non-tactile measureThe maximum height of the roughness ments performed by MeX. Both methods profile (Rz) and the maximum peak value showed similar results. Compared to conof the roughness profile (Rp) show a deventional tactile roughness measurement viation of 35% and 30% respectively. As devices the non tactile MeX method comthese values represent a single measurebines all advantages of the SEM with the ment along the profile these deviations possibilities of a 3D measurement device. are caused by measuring the profile at Additional analysis capabilities as area non identical positions on the specimen. and volume calculations are also feasible. This in general is one major drawback of Dr Stefan Scherer measurement principles that do not capHe has been working in image processing now for ture the visual appearance of the surface over 15 years and is CEO of Alicona Imaging GmbH. at the same time. MeX allows the direct Alicona Imaging GmbH link between the visual appearance and Parkring 2 · 8074 Grambach · Austria the 3D measurement. Besides the greatly [email protected]
· www.alicona.com eased identification of measurement positions including artifacts on the speciAnton Piffer He is head of the R&D Microscopy-Laboratory at D. mens that can only be detected by a viSwarovski & Co, the global market leader in crystal sual inspection, all measurements can be jewellery stones. interpreted much better.
Summary Two different roughness measurement principles were compared: A tactile 18 • G.I.T. Imaging & Microscopy 2/2003
D. Swarovski & Co Swarovskistraße 30 · 6112 Wattens · Austria [email protected]
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objective use and axial-shifting that provides 3D image stacking for truly infocus photos. www.leica-microsystems.com Easy Info no
Olympus Optical and Palm Microlaser Technologies announce an OEM agreement providing Olympus Europe with the right to distribute a new MicroBeam system for laser microdissection throughout Europe. The system is based on Palm technology and Olympus IX71 and IX51 microscopes. The company is incorporating this micromanipulation tool into its portfolio to provide new and complete system solutions to its customers for a wide range of biological analyses in molecular biology, in genetic and proteomic research, in oncology, pathology and in many other areas in biological science. www.olympus-europa.com Easy Info no
Something is missing
Result of 20 Years AFM Experience
Based on the long time experience of its designers, the NanoR-AFM from LOT Oriel has been designed from the ground up to meet the needs of its users and to make life for them easier. It is easy to use and with its high performance surprisingly affordable. In this price category it is surely the only AFM with this high performance. Thanks to its closed loop system and its durable calibration, the NanoRAFM is ideal for routine measurements. www.lot-oriel.com Easy Info no
FRET Application for Confocal Microscopy
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Leica Microsystems has introduced the Fluo Combi, an innovative accessory for the MZ FLIII fluorescence stereomicroscope that merges high resolution imaging and 3D stereo viewing at the same workstation. Users will no longer have to switch between microscopes to achieve sufficient working distance for specimen manipulation and high quality documentation imaging. The accessory offers easy, in-focus, and parcentric switching from a 1x Plan and 1x Planapo stereo objective to a 10x or 20x compound objective. It has an integrated beam-splitter for binocular viewing during compound
Leica Microsystems Heidelberg GmbH has released a FRET (Fluorescence Resonance Energy Transfer) application for Confocal Microscopy. FRET measurements give insights into molecular distances and interactions. Molecules are labelled with two fluorophores with the emission spectrum of the donor overlapping the absorption spectrum of the acceptor. Non-radiative energy can be transferred from the excited donor molecule to the acceptor molecule which emits then fluorescent light. In the new FRET application, the researcher is guided step by step by an intuitive user interface through the experiment up to the final analytical results. www.leica-microsystems.com 208 G.I.T. Imaging & Microscopy 2/2003 • 19
Closer to the Whitefly Pest
the same advanced UIS optics as the company’s top level microscopes. In addition, Plan Achromat objectives give sharp high contrast images throughout the field of view. The system is specified for use in schools or clinics, with safety and security features to match. Eyepieces, objectives and condenser are all factory attached to the microscope body so that they can not become detached or lost. [email protected]
, www.olympus-europa.com Easy Info no
A range of ergonomically superior microscopes from Vision Engineering are now being used by entomologists within the European Whitefly Studies Network to assist their studies into insect pests. The Tobacco Whitefly, Bemisia tabaci, is one of the world’s worst agricultural insect pests and poses a significant threat to agriculture. To underpin research into this pest the European Whitefly Studies Network (EWSN) was established. It involves scientists and industrialists from all over Europe who together lead the multi-disciplined research to find ways of combating this destructive insect pest. Using the company’s microscopes, researchers are studying the effects of a range of crop protection products on whiteflies. www.visioneng.com Easy Info no
Materials Research and Nanotechnology
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Tool for Micro-Applications
Top Optic for Schools Microscope
Witec has introduced the Mercury 100 AFM, a new Atomic Force Microscopy (AFM) system, designed especially for materials research and nanotechnology. The integrated scientific-grade research microscope and the Digital Pulsed Force Mode, a measurement mode for AFM, allows nondestructive imaging of various material properties along with the topography. State of the art components for scanning, beam deflection or vibration isolation are used to ensure ease of operation and optimized sample investigation. A modular design ensures upgrade possibilities from AFM to confocal / Raman microscopy as well as Scanning Near-Field Optical Microscopy (SNOM). www.WITec.de Easy Info no
New Modes of Operation for AFM/SPM The latest budget microscope from Olympus is the CX21. For the first time in an educational microscope, it incorporates 20 • G.I.T. Imaging & Microscopy 2/2003
tems. The FMControl provides enhanced surface and materials contrast by implementing the frequency detection technique to the contactless SPM operation. The new PMControl enables Scanning Surface Potential and Kelvin probe measurements with the S.I.S. instruments. Applications for the FMControl will be in the field of polymer science, biological applications, soft materials, coatings, for the PMControl in conducting polymers, microelectronics, material science. www.sis-gmbh.com
Surface Imaging Systems (Herzogenrath, Germany) releases two new modes for their Scanning Probe Microscope sys-
The Bruker Optics Hyperion is a measurement tool for challenging micro-applications in areas such as forensics, biomedical, polymers and engineering. It features full automation, infrared chemical imaging, crystal-clear sample viewing and a wide variety of IR and visible objectives. The series can be upgraded from the base configuration FT-IR microscope Hyperion 1000, to the Hyperion 2000 which features full automation, and ultimately the Hyperion 3000, which utilizes state-of-the-art chemical imaging technology. The high quality optics and hardware, together with the integrated Opus/Video, Opus/Map, Opus/FPA and Opus/3D software packages, provide high system performance. www.brukeroptics.de Easy Info no
UV Laser Diode for Microscopy
ally controlled, enabling simultaneous imaging of multi-labelled specimens, a precondition for fast live cell experiments. The orange line optimally excites multiple dyes, such as Texas Red, Alexa 594 and CTC. Optimal excitation means lower excitation power and results in better sample protection and increased cell viability. www.confocal-microscopy.com Easy Info no
PicoQuant announces the release of a 375 nm picosecond pulsed diode laser. The photonics company is a manufacturer of picosecond pulsed diode lasers, covering a rapidly growing section of the time-resolved fluorescence research market. With pulse duration’s of less than 70 ps, their pulsed diode lasers match the time resolution of mainstream detectors, yet at a price ten times lower than that of commonly used TiSa or Argon ion lasers. As in the red and infrared range, the pulsed diode laser sources offer the benefits of cheap and compact integrated turn-key systems together with the high repetition rates desired for fast Time-Correlated Single Photon Counting (TCSPC). www.picoquant.com Easy Info no
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With AOBS and Orange laser, Leica users have a unique spectrum of 8 excitation lines in the visible wavelength at their fingertips: 456, 476, 488, 496, 514, 543, 594 and 633 nm. Any combination of these lines can be selected and individu-
Using a series of high precision optics supplied by Optical Surfaces the Japan Atomic Energy Research Institute (JAERI) in Kyoto has demonstrated the possibility of a compact pumping system for x-ray lasers in the shorter wavelength region. Scientists at the institute have demonstrated gain saturation of nickel-like ion x-ray lasers at wavelengths of 13.9 and 12.0 nm using a compact chirped pulse amplification Nd glass laser with an input energy of c. 14J. Employing a quasitraveling-wave pumping system using a special six-step mirror enabled the pumping energy to achieve gain saturation to be reduced to 2.5J / mm. The result demonstrates the possibility of a compact pumping system for xray lasers in the shorter wavelength region. www.optisurf.com Easy Info no
IMAGING & MICROSCOPY coming up August
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Orange Helium-Neon Laser
3/03 G.I.T. Imaging & Microscopy 2/2003 • 21
Scanning Microradiography – A Digital 2-D X-Ray Imaging Technique Paul Anderson, James C. Elliott, Stephanie E. P. Dowker, Frédéric Bollet-Quivogne
Stephanie E. P. Dowker, James C. Elliott, Paul Anderson, Frédéric Bollet-Quivogne
Scanning microradiography is a
digital form of microradiography in
Scanning microradiography (SMR) was originally developed in order to overcome many of the problems associated with contact microradiography, particularly non-linear response and saturation of the photographic emulsion . SMR is a digital X-ray absorption technique in which sections of thickness 0.1–2 mm (~1 x 0.5 cm typical area) are XY stepscanned past a 15 µm X-ray beam using a high-precision computer-controlled micro-positioning system. The transmitted intensity at each scan point is measured using an electronic X-ray detection system to achieve accurate quantitative measurements of X-ray attenuation, and hence mass of absorbing material. SMR is sensitive to small changes in the X-ray attenuation of specimens. The sections
which the photographic emulsion is replaced by a solid-state X-ray detector. This novel technique has allowed us to carry out a range of dissolution studies, particularly in relation to the physico-chemical mechanisms of dental caries, which hitherto have not been possible.
Keywords Scanning, microradiography, X-ray absorption, diffusion, dissolution 22 • G.I.T. Imaging & Microscopy 2/2003
do not have to be in contact with the X-ray detector and can be located within environmental cells through which reacting solutions are pumped. This allows studies of changes with time of a sample, e. g. the kinetics of acidic dissolution of the mineral in enamel and compressed hydroxyapatite (HAP) aggregates in artificial carious lesion models.
SMR Apparatus Fig. 1 is a schematic of the current SMR apparatus. X-rays are generated by a Hilger and Watts Y33 microfocus generator modified with a highly stabilised high voltage power supply. The generator is usually run at 1.5 mA and 45 kV with an Ag target. A 10 µm aperture is created from crossed pairs of Ta slits located 30 mm from the source, to provide a narrow
SCANNING MICRORADIOGX RA XX PH XX Y
X-ray beam. The XY scanner (Micromech, UK) is built with two high precision translation stages (lengths – 600 mm horizontal, and 200 mm vertical) each fitted with a 0.1 µm resolution linear encoder, driven by stepper motors using a programmable controller connected to a PC. The XY scanner can carry up to 30 SMR cells, which can be scanned in any programmed sequence, and with different scanning parameters (time, step size etc.) for each specimen. Transmitted X-ray photons are counted by a solidstate Ge photon detector outputting to a combined digital amplifier and multi-channel analyser (MCA) (Ametek, UK) connected to the same PC as the XY scanner. This equipment enables spectrum capture, spectroscopic analysis, and electronic monochromatisation of the AgKα peak (22.1 keV) at a series of preprogrammed X and Y positions within each specimen. The projected mass (in the direction of the beam) of absorber at each point is calculated using published mass attenuation coefficients of the absorbing material at the selected energy, and the measured incident and transmitted X-ray intensities. The thin sections are located in SMR cells and test solutions flow through that can be alternated using a computer controlled valve system. This enables studies of the behaviour of specimens exposed to different chemical environments.
SMR Can Be Run in either: i) perpendicular (profiling) mode; in which the direction of diffusion, acid attack, or other physical chemical process, is perpendicular to the direction of the X-ray beam. This is usually used for line or area scans of plano-parallel thin sections of a specimen, which are taken repeatedly in order to observe spatial changes in mass within the section during exposure to the test solution(s).
Fig. 1 : Schematic of SMR apparatus with cells located on XY stage.
ii) parallel (integrating) mode; in which the direction of diffusion, or acid attack, or other physical chemical process, is parallel to the direction of the X-ray beam. This is used to measure temporal changes in mass at one, or series of points in a block (which need not be a planoparallel section) of specimen during exposure to the test solution(s). SMR-MEXA (multiple energy X-ray absorptiometry) can be used when specimens or test solutions contain elements with accessible X-ray absorption edges. Transmission spectra are collected using the MCA, and the projected masses of two or more species can be calculated . This technique is used to measure the mass of two or more species simultaneously using either mode of SMR. Applications include studies of changes in diffusion coefficients of solutions within permeable solids, even as the solid partially dissolves .
µm thick section cut from a tooth, recorded at 100 h intervals. The section was varnished except for one thin
edge and located into the SMR cell through which buffered acid was circulated to simulate dental caries. The
Examples of the Use SMR: Fig. 2 (perpendicular mode SMR) shows a series of projected mass profiles of a 200
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G.I.T. Imaging & Microscopy 2/2003 • 23
Fig. 2: Results from perpendicular mode SMR showing a time series of projected mass profiles (at 100 h intervals) during the dissolution of a thin section of enamel in 0.1 mol l-1 acetic acid buffered to pH = 4.0.
Fig. 3: Results from parallel mode SMR showing change in projected mass of HAP at a single point during the acidic dissolution of a block of compressed HAP aggregate.
Fig. 4a: Results from perpendicular mode SMR-MEXA of projected mass of KI during dissolution of a ~50 % by volume porous HAP aggregate with a mixture of 1.0 mol l-1 KI solution and buffered acid.
Fig. 4b: Results from parallel mode SMR-MEXA of projected mass of HAP during dissolution of a ~50 % by volume porous HAP aggregate with a mixture of 1.0 mol l-1 KI solution and buffered acid.
top curve (at time t = 0 h) shows the initial mass profile of the section. Subsequent profiles show changes in the distribution of mass within the section at later times. The later profiles show the progress of subsurface demineralisation, which is a characteristic of these studies [4, 5]. Fig. 3 (parallel mode SMR) shows the almost linear change with time in the projected mass (in the direction of acid attack) in a block of compressed HAP (the principal inorganic component of teeth) aggregate [6, 7]. Fig. 4 (profiling mode SMR-MEXA) shows profiles of the mass of KI and the mass of HAP within a thin section of a highly porous HAP aggregate exposed to a mixture of acid buffer and KI solution. The time-series KI mass profiles (a) show the diffusion of KI into the permeable solid. The time-series of HAP mass profiles (b) are similar to those seen in Fig. 2. 24 • G.I.T. Imaging & Microscopy 2/2003
Dr Paul Anderson He is Reader in Biophysics in relation to Dentistry.
Supported by MRC Programme Grant G9824467 and The Wellcome Trust Grant No. 062850.
Prof James C. Elliott He is Professor of Biophysics in relation to Dentistry.
Literature  J. C. Elliott, S. E. P. Dowker, R. D. Knight: J. Microscopy 123, 89–92 (1981)  N. Kozul, G. R. Davis, P. Anderson, J. C. Elliott: Meas. Sci. Technol. 10, 252–259 (1999)  S. E. P. Dowker, P. Anderson, J. C. Elliott: Microsc. Microanal. Microstruct. 7, 49–63 (1996)  P. Anderson, J. C. Elliott: Caries Res. 34, 33–40 (2000)  S. E. P. Dowker, P. Anderson, J. C. Elliott: J. Mater. Sci. Mater. Med. 10, 379–382 (1999)  P. Anderson, M. Levinkind, J. C. Elliott: Archs. Oral Biol. 43, 649–656 (1998)  J. C. Elliott, F. S. L. Wong, P. Anderson. G. R.
Dr Stephanie E. P. Dowker She is Clinical Senior Lecturer in Adult Oral Health. Frédéric Bollet-Quivogne He is completing his PhD. The research interests of the Dental Biophysics Section at QMUL include the physical chemistry of dental hard tissue destruction, and the development and application of X-ray microscopies for these studies. Dental Biophysics Section Dept. of Oral Growth and Development Medical Sciences Building Mile End Road Queen Mary, University of London London E1 4NS, Great Britain [email protected]
Davis, S. E. P. Dowker: Connect. Tiss. Res. 83, 61–72 (1998)
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Powerful Performance Extreme Productivity
The new Quanta™ FEG takes the advantage of Any Sample, All Data™ in situ imaging to a new level, delivering the ultimate in analytical performance. The latest addition to the field-proven Quanta family, Quanta FEG delivers the versatility inherent in the Quanta SEM platform in a more powerful package with improved imaging, stability, and functionality. Gain the flexibility of conducting low-vac, ESEM™, and high-vac investigations on the same tool, all with the high resolution of a powerful FEG system, and without having to prepare your samples. Quanta systems allow hydrated samples to be introduced into the chamber while maintaining 100 percent relative humidity during pump down. The Quanta FEG's benefits also include: seamless “point and click” transition between imaging modes; superior low-vacuum, low kV imaging; beam stability superior to cold FEG or tungsten; simultaneous SE and BE imaging in low-vac mode; a large sample stage with 150 x 150 mm travel, en-abling sample weights up to 3 kg; STEM, WDS, EBSP and EDS; and the ability to save live action .avi files. • Failure Analysis • Drug Development • In-situ Dynamics • Material Science • Structural Research Compare the new Quanta FEG to other systems and discover how powerful performance and extreme productivity can deliver enhanced value to your lab.
Introducing Quanta™ FEG – The Ultimate Analytical System for Any Sample, All Data™
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I&M SHOW CASE: SCANNING MICROSCOPY
High-Resolution Analysis on any Material Quanta Series Field Emission SEM FEI Company introduces the Quanta environmental scanning electron microscope (ESEM) with field emission gun (FEG) technology. The Quanta FEG offers high-resolution, noncharging imaging with high and stable beam current, making it an advanced analytical tool for a wide range of applications including material and life science, automotive metallurgy, and pharmaceutical studies. With its ability to image samples without charging, the Quanta FEG is also well suited for analysis of low-k dielectrics in semiconductors and is an enabling technology for analysis of advanced photomasks. Ease of operation
Superior low vacuum, low kV imaging Ultra stable analytical operation (EDX, EBSD, WDX) Ability to save live action avi files Simultaneous SE and BSE imaging in low vacuum mode STEM imaging In-Situ dynamic experiments
The Quanta series software is robust and makes the Quanta very easy to operate. In addition to 4-quad imaging, the Quanta FEG has auto-focus, auto-stigmator, auto-contrast and brightness, and an extensive on-line help function. The multi-user software allows every operator to store and create individual preferences and protocols. Regions of interest can be dragged, centered and zoomed with a mouse-click. A digital video (AVI) facility in 3 (live) quads enables the user to record experiments. The Quanta FEG also features an image archiving system that can be fully networked.
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Supra Ultra High Resolution Analytical FESEM Supra FESEM Leo has developed the versatile ultra high resolution Supra range to combine four instruments in one; ultra high resolution FESEM over the complete voltage range, FESEM for handling large awkwardly shaped specimens, fully analytical FESEM and FESEM with variable pressure (VP) technology to investigate non conducting specimens without prior preparation. The new LEO Supra combines high resolution imaging and analysis into one single instrument, something which was previously beyond expectations has now been made possible.
Ultra high resolution over the complete voltage range Superior resolution in VP mode: 2 nm @ 30KV VPSE detector for true SE imaging in VP mode Unique In-lens detector for true surface imaging Enhanced depth of field Distortion free imaging for EBSD applications Fully motorised 5 axes stage Easy operation through Windows XP based Leo 32 SEM control
Combined Ultra High Resolution and Analysis Semiconductor and nanotechnology applications benefit from 20% resolution improvement in the low voltage range (now 1.7 nm @ 1kV and 4 nm @ 1kV). Especially for material analysis applications LEO has introduced two new models offering 20% improvement in resolution and 100% improvement in beam current without any compromises. High resolution imaging with large depth of focus can be combined with high resolution elemental analysis (EDS and WDS) and texture analysis (EBSD). The VP mode enables full investigation of non conducting specimens in their original state without intrusive preparation.
26 • G.I.T. Imaging & Microscopy 2/2003
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Modular Confocal Microscope System Digital Eclipse C1 A confocal microscope that produces images of the highest quality in its class and supports almost all imaging applications of today and tomorrow.
Nikon introduces a universal confocal microscope system that is ultra-compact and lightweight yet delivers confocal images of unrivalled quality. All the main components are modular, including the world’s smallest and lightest scanning head, making expansion and maintenance easy. Furthermore, 3-channel detection is possible by minimum upgrade, and operation is facilitated by intuitive software. With the C1, confocal microscopy is now an affordable mainstream technique.
3-channel simultaneous observation Interchangeable filters Modular design saves space and facilitates upgrading Intuitive software promotes multifaceted microscopy analysis Easy to configure, easy to operate
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Digital Image Acquisition for every SEM DISS 5
USB 2.0 interface for command and data transfer 16,384 x 16,384 pixels max, free image format 4 analogue + 12 counter signals synchronously Up to 32,000x oversampling for noise-reduction Line averaging, frame averaging Reduced Area Scan with zoom TWAIN interface to integrate as OEM product
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DISS 5 is an active image acquisition and image processing system for all scanning electron microscopes and microprobes. An upgrade with DISS 5 enhances the capabilities and adds features known only from the most modern instruments. Beyond that, DISS 5 with all its unique features is also a good choice for new microscopes.
G.I.T. Imaging & Microscopy 2/2003 • 27
SCANNING ELECTRON MICROSCOPY
SEM Imaging on Uncoated Insulators Delfin Braga, Guy Blaise, Bertrand Poumellec
f.l.t.r.: Bertrand Poumellec, Delfin Braga, Guy Blaise
A scanning electron microscope has been equipped to study the fundamental aspects of charge trapping in insulating materials, by measuring the secondary electron emission yield δ as a function of energy, electron current density and dose. The control of these three parameters allows good quality images to be taken on uncoated surface. The imaging of insulating surface materials with a scanning electron microscope (SEM) is usually performed with a thin conducting coating layer. More recently, the environmental microscope  was successful in masking the charge effects which perturbed the quality of images. But in both cases, the artifice used to prevent the nuisance of electric charges, remove any possibility to investigate the intrinsic physical characteristics of surfaces. This paper is an abridged presentation of a thorough investigation on the physical phenomenon of insulators charging and on the methodology for obtaining a physical image of an uncoated insulator surface that we can call a charge mapping . Two categories of insulating materials have to be distinguished. Those that do not contain an intrinsic mobile charge density sufficient to neutralize the deep traps produced during the elaboration of the material in which, positive and negative charges released beneath the surface, under electron injection, are trapped and their trapping is stable in time below a certain temperature Ts. This
Keywords SEM, insulator, charging regimes, poling 28 • G.I.T. Imaging & Microscopy 2/2003
Fig. 1: Scheme of the bottom part of the SEM LEO 440 equipped with the Electron Beam Blanking Unit (EBBU) and the two detectors. QIC is the induced charge, QT the trapped charge and QSB the secondary and backscattered electron charge.
category of insulators is called “trapping insulators”. At room temperature, the material representative of this category is SiO2 (amorphous and crystallised). In the second category, the mobile charge density is high enough to neutralise the deep traps so that, positive and negative charges released beneath the surface, relax in time. There is no apparent stable trapping in a certain temperature range below Ts. At room temperature, many complex materials as composite ceramics, glasses and oxides with specific additives are representative of this category, which is named “conductive insulators”. Of course above Ts a “trapping insulator” can be transformed into a conductive one because stable trapping has become ineffective. Conversely, by cooling the material, the density of stable traps increase
and the density of mobile charges reduces, so that a “conductive insulator” can become a trapping one below a certain temperature Ts’ < Ts.
Experimental Set-Up and Procedure The secondary electron emission (SEE) yield δ is the fraction of secondary and backscattered electrons released from the surface under the impact of an energetic electron beam. The measurement of this yield allows the parameters which govern the images quality of uncoated insulator (a trapping or a conductive one) to be drawn because it is very sensitive to the internal charge state of the materials. This control requires an accurate measurement of δ with a precision of a few % at least.
SCANNING ELECTRON MICROSCOPY
To carry out these measurements, the SEM LEO 440 (Fig. 1) has been equipped with the optibeam optical system  which, in the conjugate mode, allows the beam to be focused at a fixed point A positioned below the second condenser lens, for any energy and current intensity requirement. Combined with the Electron Beam Blanking Unit (EBBU) which allows the injected dose to be fixed, it is possible to perform experiments at any energy (100 eV-40 keV) with a constant beam current adjusted from a few pA to µA, in a defocused spot mode, which diameter can vary from 5 µm up to 500 µm. The specimen chamber is equipped with two detectors, which collect respectively the charge QIC induced by the charges QT left in the sample (QIC = – QT) and the charge QSB of secondary and backscattered electrons released from the surface into the vacuum. Then, the SEE yield δ is given by: δ=QSB/(QIC+QSB). Thanks to this tool, we observed that the two parameters which determine the quality of an image, are the energy E and the current density J of the primary electron beam. The effect of the electron dose is just a macroscopic consequence of the effect of J.
Fig. 2: Variation of the SEE yield δ with the injected charge density in a-SiO2, at different primary beam energies. The current density was 6·10+3 pA/cm2. Numbers associated with arrows indicate the surface potential VS when the SRR is reached (δδst = 1).
Energy Dependence of Charging at Low Current Density When positive or negative charges are trapped in a “trapping insulator”, at low current density (J 5 keV in amorphous silica (Fig. 2) and for E ≤ 5 keV, the surface potential does not exceed a few tens of volts. As EC is higher than the energy E2 of the second crossover for which δ0 = 1, the sample charges negatively at EC. More and more negative trapped charges are required for reaching the SRR as the primary electron beam energy E increases beyond EC. It results that the high negative surface potential which appears, perturbs the incident beam so much strongly that a mirror effect can be observed . Clearly the energy to use to make good images on uncoated “trapping insulators” must be less than EC. Due to the relaxation of charges in “conductive in-
sulators”, there is not a strict upper limit for EC. Charging effects in those materials are mainly dependent on J and the ability to evacuate the charges out of the sample. The metal-insulator contact is of a primary importance for that.
Current Density Dependence of the Charging Effect When the current density J exceeds a typical value ≈ 10+5 pA/cm2, the steady state regime obtained departs from the SRR. It is characterised by a steady value δst = 1 ± ε(J) where ε(J) is of the order of a few %. The fact that the system regulates at δst ≠ 1 implies that charges spread deeply and deeply in the bulk as irradiation proceeds. The insulator is laterally invaded with electrons. This corresponds to the ageing regime . In “conductive insulators”, the competition between the incoming flux of electrons and the dispersion of charges in the bulk is regulated through a layer of charges which density increases with J. Thus, there is a critical current density JC beyond which accumulation of charges in the layer is such that the image is blurred by the charging. This is exempli-
fied in Fig. 3 in a slightly conductive composite ceramic. In Fig. 3a where the current density was high, J = 9·10+4 pA/cm2, the charging effect appears rapidly on the image (σ = 2.2·10+5 pC/cm2). In Fig. 3b, the current density has been lowered to the value J = 2.10+4 pA/cm2. Although the charge density (σ = 10+6 pC/cm2) is higher than in the first case, a correct image has been obtained. When δ stabilises at a value different from unity, in both categories of insulators, the material is progressively filled with charges in proportion to the injected dose. The consequence is that a macroscopic charging effect can be observed if charges are not evacuated to the ground properly. Furthermore, increasing the magnification imposes a reduction of the current density to maintain a current density compatible with a good quality of images.
Imaging of Poled and Unpoled Silica  An amorphous silica layer (1.1 µm) was deposited by PECVD on a Si wafer. Comb shaped aluminium electrodes (80µm width, 200 µm mesh) were deposited on the glass layer and subjected to a thermal poling  under a field of 30 MV/cm. G.I.T. Imaging & Microscopy 2/2003 • 29
SCANNING ELECTRON MICROSCOPY
Fig. 3: Images of an insulating technologic composite ceramic showing charging effects (a) and the cauliflower structure (b). Operating conditions : a: E = 3 keV, J = 9·10+4 pA/cm2, σ = 2.2·10+5 pC/cm2; b: E =10 keV, J = 2.10+4 pA/cm2, σ = 10+6 pC/cm2.
Fig. 4: Contrast between poled strips (P) and unpoled silica ones (UP). E = 1100 eV. Images were taken at two different charge densities: a: σ = 3.8·10+4 pC/cm2 ; b: σ = 2.2·10+5 pC/cm2. See text for further details.
Fig. 5: Contrast between poled strips (P) and unpoled silica ones (UP). E = 5000 eV. Images were taken at two different charge densities : a: σ = 2.3·10+5 pC/cm2; b: σ = 1.9·10+6 pC/cm2. See text for further details.
Secondary electron images were taken by scanning a zone containing poled and unpoled regions at an energy of 1100 eV with an intensity of 6 pA. At E= 1100 eV, the intrinsic SEE yield δ0 is larger than one so that the sample charges positively. After a single scan (700 pC/cm2), a very little contrast appears between the two regions. Then, as the number of scans increases, the contrast becomes more and more pronounced until a maximum is reached for a dose density of about 9000 pC/cm2 (Fig. 4a). Finally, the contrast decreases slowly and practically vanishes for a dose density σ = 2.2·10+5 pC/cm2 (Fig. 5b). This behaviour has been quantitatively related to the difference of the evolution of δ with the charging kinetics in the two regions : the more rapid decrease of δ with the injected dose in the unpoled regions make them appear darker than the poled regions. Then, when δ approaches unity at high dose the contrast vanishes. The difference in the charging process kinetics is due to the fact that positive charging is stable in unpoled silica whereas a slow time relaxation is observed in poled silica.
The same experiments as those reported above were carried out at 5000 eV for a negative charging (δ0 < 1). Two spots on the unpoled (spot 1) and poled (spot 2) areas received the same charge density 1.2·10+6 pC/cm2. The image in Fig. 5a was taken just after irradiation with an additional low charge density of 2.3·10+5 pC/cm2. The poled strip is darker than the unpoled one consistently with the lower d coefficient. The two spots are clearly visible because they received a higher dose than the background and consequently their δ coefficient is closer to unity. In Fig. 5b, the image was taken with a charge density of 1.9·10+6 pC/cm2. The contrast between poled and unpoled areas is still consistent with the difference of δ. But, although spots 1 and 2 have received the same charge density (1.2+1.9)·10+6 pC/cm2, the first one is brighter in the unpoled area whereas the second one melted into the background of the poled region. This is consistent with the fact that, at a high charge density the SEE yield reaches a steady value δst which is close to unity in the unpoled areas and equals to δst = 0.84 in the poled ones .
30 • G.I.T. Imaging & Microscopy 2/2003
This experiment clearly demonstrates that poled and unpoled regions do not exhibit the same charging properties. There is a stable negative charge accumulation in unpoled silica and a charge relaxation in poled silica.
Conclusion Provided the primary electron beam energy, current density and dose are properly adjusted, it is possible to obtain good quality images on uncoated insulators. In complement, high spatial resolution could be obtained by using a field emission gun which current would be reduced to a few pA. The comparison of the charging process of poled and unpoled silica has revealed that a “trapping insulator” can be transformed into a “conductive insulator” by poling. This new feature can explain why silica is a so high-performance material in MOS structures. It is noticed that this physical study of poled / unpoled silica should not be run with a conductive coating on the surface.
Literature A list of references can be obtained from the authors. Delfin Braga Graduated in 2000 from the Ecole Universitaire D’Ingénieur de Lille and from the Université des Sciences et Technologies de Lille, he joined the Laboratoire de Physique des Solides in Paris XI University (Orsay) to investigate the physical properties of charge trapping and transport in insulating materials thanks to a scanning electron microscope. Guy Blaise Professor of Physics at the Paris XI University (Orsay) where, since 1990, he has been conducting studies on charge trapping and transport in insulators and related phenomena. Laboratoire de Physique des Solides Université Paris-Sud · Bât. 510 91405 Orsay, France [email protected]
Bertrand Poumellec agrégation Physical Sciences in 1975, Dr in Geophysics in 1981, State Doctorate in 1986. Work for CNRS since 1981, also consultant. From 1986 to 1997, he studied experimentally and theoretically in oxides the EXAFS of transition elements of the first series (Ti and V, specifically). Then, from 1988, he studied the interaction between irradiation beam and silica based glasses with the aim of refractive index change mastering. Coordinator of research training network ODUPE (Optical Devices Using Photosensitivity in their Elaboration, www.odupe.dk) from the European Commission. Laboratoire de Physico-Chimie de l’Etat Solide Université Paris-Sud 91405 Orsay, France For more info circle no.
SCANNING PROBE MICROSCOPY
Scanning Probe Microscopy Enhanced Contrast by a New Mode of Operation
small nuclei of sucrose crystals can be seen. The size of the crystals is in the range of a few tens of nanometers. In Fig. 2 the cantilever’s resonant frequency shift during the scanning is displayed. Here, we see a strong contrast on the crystal positions and some additional features are visible: The brighter areas are interpreted as island of monomolecular growth of sucrose layers. These layers are barely visible in the topographic data. Therefore, the frequency detection technique could become a valuable tool for identifying varying compositions of materials or surface layers. It is expected to find more interesting applications in the fields of polymer science, biological applications, soft materials, coatings, etc.
Dr Frank Saurenbach Managing Director Surface Imaging Systems (S.I.S.) GmbH Tel.: +49 2407 56420 Fax: +49 2407 5642100 [email protected]
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Scanning Probe Microscopy (SPM) and its contact mode version Atomic Force Microscopy (AFM) have become a standard tool for high resolution surface imaging. In addition, during the past decade several SPM modes have been developed: magnetic force, lateral force, or variable force microscopy, etc. The combination of these methods provides widening access to materials properties in the nanometer range. Now, with the new FMControl electronics of Surface Imaging Systems, enhanced material contrast can be obtained. During the standard non-contact measurement of the SPM the actual resonant frequency of the tip-holder, a small cantilever, is measured. This frequency is mainly affected by material dependant forces between the surface and the tip. Thus, simultaneously to the topography imaging process a mapping of these forces can be performed. We have imaged the sucrose crystallization during an evaporation process with the NANOStation II of Surface Imaging Systems. In Fig. 1
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223 G.I.T. Imaging & Microscopy 2/2003 • 31
Microscopy Applications in Nanotechnology Sadao Aoyagi What is Nanotechnology? Nanotechnology is attracting an increased attention in the world today. Encouraged by the National Nanotechnology Initiative (NNI) established by former US President Clinton in 2000, research and development on nanotechnology has accelerated as a national strategy of many countries. The author attended the Micro & Nano Engineering (MNE) Conference 2001 in Grenoble, France, in September 2001. Since the first meeting held at the University of Cambridge in 1975, this annual meeting has been held in various European countries and marked the 27th last
year. At the meeting, more than 450 scientists from around the world discussed and exchanged information on nanoscience and nanoengineering. Researchers also presented numerous reports on the creation, analysis, and evaluation of nanostructures. In October 2001, the author attended the 14th International Microprocesses & Nanotechnology Conference 2001 in Matsue, Japan, which drew more than 300 participants from throughout the world. Researchers here have also presented and exchanged information on nanotechnology. Although the word “nanotechnology” has become popular recently, it is not an entirely new field as one can see from these conferences.
What is meant by “nano”? “Nano” is a prefix that means one billionth of something like a second or a meter. One nanometer is one billionth of a meter (10–9 m). Fig. 1 shows the scale of lengths. We can see how small one nanometer is. We often use a phrase used to express an industrial trend by referring to a specific field. As an example, “the information technology (IT) revolution” is a revolution in communication technology. In contrast, the word “nano” does not refer to a specific field. This means that everything in the cosmos can be the target for nanotechnology. “Nanotechnology” means nanostructure engineering, research, and development in creation of useful materials, devices, and systems at nanometer-size scale. The smallest unit of substances known to us is the atom, but it is not functional by itself. We may say that nanotechnology produces the smallest functional unit (approximately one nanometer in size) by building groups of atoms.
Approaches to Nanotechnology: “ Bottom Up“ and “Top Down“ Approaches to nanotechnology research and development are grouped into two categories, “bottom up” and “ top down”. The bottom-up approach ingeniously controls the building of nanoscale structures. This approach shapes the vital functional structures by building atom by atom and molecule by molecule. The bottom-up approach researchers are work-
32 • G.I.T. Imaging & Microscopy 2/2003
Fig. 1: Scale of lengths
semiconductor device is now coming to the 50 nanometer level. This is an achievement of the top-down approach. The word “micro electro mechanical system” (MEMS) is not so popular as nanotechnology, but is well known to nanoscience and nanotechnology researchers. MEMS is a system assembled at micrometer size by a combination of electrical and mechanical technology. MEMS technology has already provided many things of practical use to society. Fig. 2 shows an example of MEMS. It is a scanning electron microscope (SEM) micrograph showing part of a gear wheel made using lithography technology (field of view: 300 micrometers). In my understanding, the final goal of MEMS is to create nanoscale structures (NEMS: nano electro mechanical system). There are, however, many hurdles to overcome in its research and development, and many scientists around the world are continuing to make hard efforts.
Nanotechnology Will Make Our Life More Comfortable
Fig. 2: SEM micrograph of a micromachine. Courtesy of Prof Fujita of the Institute of Industrial Science, University of Tokyo.
ing to find the mechanism of “self-assembly”. “Self-assembly” is like the most basic ingredients of a human body reproducing the most basic structures by themselves. “Self-assembly” covers the creation of the functional unit by building things using atoms and molecules, growing crystals and creating nanotubes. “Top down” is an approach that downsizes things from large-scale structures into nanometer-scale structures. As an example, vacuum tubes yielded to transistors, they then gave way to ICs (integrated circuits) and eventually LSIs (large scale integrated circuits). The way of creating things by downsizing from millimeter size to micrometer size is called “microtechnology”. The top-down approach is an extension of microtechnology. The narrowest line pattern on a
Why is nanotechnology so important? Former President Clinton said, “In the future, all information in the Library of Congress can be stored in the size of one sugar cube”. There are many practical issues to overcome before reaching this target even if it is theoretically possible. Thinking of our daily life, for example, since the outer diameter of a gastroscope, 15 mm in the past, was downsized to 2 to 3 mm, the patient’s pain has been greatly decreased during a stomach examination. Let us recall the 1966 movie “Fantastic Voyage”. This is a science-fiction movie in which four miniaturized doctors entered a human body to conduct a brain surgery. Today, a technique similar to this movie, called a “drug delivery system”, has become a reality. This application of nanotechnology delivers medicine to a specific body part, allowing us to use the minimum amount of medicine necessary. Nanotechnology makes our life more convenient and comfortable. ICs are already incorporated in most of our daily-use appliances and exhibit a variety of capabilities. ICs and LSIs are greatly downsized and multifunctional, making our daily life more efficient. These chips enable us to use handy cellular phones, and automatically pay for train tickets and commodities. Microtechnology has brought about such achievements. Now, nanotechnology has emerged. In the upcoming years when practical ap-
plications of nanotechnology prevail, our daily life will become much more convenient and comfortable.
Development of Nanotechnology Nanomaterials are in the Production Stage The following is a comment made by a professor at Nagoya University when the author visited him in August 2001. “We have nanomaterials, but no nanotechnology. We have techniques to produce nanomaterials like carbon nanotubes (CNT: carbon tubes 1 nm in diameter and several micrometers in length) and nanofullerene (spherical carbon approximately 1 nm in diameter) with a production yield of approximately 90%, but we have no techniques to cut and assemble these nanomaterials”. Scientists engaged in materials research have been using the transmission electron microscope (TEM) for more than 30 years to observe nanometer-size materials. The professor pointed out that observing nanomaterials is not new, but the technology needed to manipulate and control nanoscale substances is not yet fully developed. The author will introduce two examples of nanomaterials research. Figs. 3a to 3c are TEM images that show the creation process of xenon (Xe) nanoparticles. When injecting Xe ions into an aluminum (Al) substrate, Xe microcrystals combine with each other to form Xe nanoparticles. Fig. 3a shows the state before the microcrystals combine: Fig. 3b shows the state during the microcrystals merge: and Fig. 3c shows the state after the microcrystals have combined. The resulting Xe nanoparticles are arranged in the Al substrate in the form of solid octahedrons. When viewed from a certain direction, they look hexagonal. The two microcrystals isolated from each other (Fig. 3a) combine with each other to form a single large solid octahedron (Fig. 3c). The TEM images clearly visualize a nanoscale change of a nanomaterial. The next example is a nanowire. Fig. 4 shows a schematic diagram of a helical multishell gold nanowire and a micrograph taken with a TEM (magnification: 20,000,000). This micrograph shows the gold nanowire approximately 1 nm in diameter. We have great expectations for the fabrication and creation of nanodevices, which will contain nanotubes, nanoparticles, nanowires, and other materials.
G.I.T. Imaging & Microscopy 2/2003 • 33
Fig. 3: Creation of xenon nanoparticles. Courtesy of Dr Furuya of the Nanomaterials Laboratory, National Institute for Materials Science.
Nanotechnology is in the Development Stage The suggestion of the professor at Nagoya University has provided a hint to what instrument suppliers must do. Namely, we have to meet the demands from top-level researchers engaged in nanotechnology. To achieve this objective, we have to further improve the performance and capabilities of our products by enhancing our technology. After attending several meetings on nanotechnology, the author also recognized that in addition, scientists have many issues for the creation of nanostructures. A leading industry employing the topdown approach is the semiconductor industry. Integration of semiconductor devices is an application of nanotechnology. The narrowest line pattern of a semiconductor device now reaches below 100 nm; therefore, controlling the patterns at the nanometer level is essential. In addition, the semiconductor roadmap (International Roadmap for Semiconductor Technology) projects the miniaturization of the semiconductor design rule in the future. However, many difficulties confront us, and research and development are being pushed forward aggressively to solve these problems. When downsizing a large substance, the function of this substance also changes. Therefore, we must overcome many challenges that take place. From the viewpoint of the bottom-up approach, research and development efforts are made based on the following mechanism: A single atom has no function on its own, but when atoms gather together to become the size of DNA (approximately 1 nm), they suddenly have novel functions. In research on metals, ceramics, semiconductors, polymers, and biology, scientists are now evaluating physical properties of aggregates of 34 • G.I.T. Imaging & Microscopy 2/2003
atoms and molecules. However, we still have a wide range of unknown obstacles, which have to be overcome.
Nanotechnology is Making Progress in Various Fields Nanotechnology is now regarded as the core scientific technology. Many researchers have reported that ceramic, metallic, and other materials enhance their physical properties when created using nanoscale control. We can expect that materials can be improved in many ways, and large devices become compact while improving their capabilities. For example, many people are tackling the creation of single electron devices using a single CNT. Utilizing nanotechnology, we can downsize and enhance devices. Nanotechnology is expected to progress in various fields, including information/ communication, life science, environmental conservation, energy saving and medical care. Fig. 5 shows an example of the applications of “nanotube tweezers” that are created by means of CNTs. The schematic diagram at left shows the construction of a molecular device using the nanotube tweezers, and the SEM micrograph at right makes the nanotube
tweezers visible. The tips of the nanotube tweezers can be opened and closed by switching on and off a voltage between the two CNT probes. Nanotube tweezers are expected to be applied in various fields of research and development on nanoscience and nanotechnology, in terms of the bottom-up approach. In biological studies, TEM and SEM have contributed to the progress of medical care by offering imaging of virus particles several nanometers in size. Worldwide efforts are being made to create artificial human organs including eyes and ears. We may say that good news will be brought to people with visual or hearing difficulties in the near future. Many efforts in leading research and development start with the development of new technology and products. This is where Microscopy Applications to nanotechnology lies ahead.
Jeol’s Challenge to Nanotechnology Jeol’s Continuing Challenge to Nanoscience and Nanotechnology Fig. 6 shows a composite of a TEM micrograph (replica of a single crystal aluminium surface) and a photograph of mountain climbers, taken in the 1960s. This photograph is a message depicting Jeol’s challenge to the microworld. Since its establishment, Jeol has been actively engaged in research and development of products for research at the micro and nano level. Our variety of products has contributed to nanoscience research around the world. Many Jeol customers have been awarded the Nobel Prizes, and six Nobel Prize laureates planted commemorative trees when they visited our company. The information boards placed at these trees convey their outstanding accomplishments. The author will explain some examples of applications of Jeol products related to nanotechnology.
Fig. 4: Helical multi-shell gold nanowire. Courtesy of the Takayanagi Particle Surface Project, Japan Science and Technology Corporation.Fig. 4: Helical multi-shell gold nanowire. Courtesy of the Takayanagi Particle Surface Project, Japan Science and Technology Corporation.
“Characterization”: Observation of Nanomaterials and Nanostructures
Fig. 5: Application of nanotube tweezers mounted on nanomanipulator. Courtesy of Prof Nakayama of Osaka Prefecture University.
Fig. 7 shows a defect-review SEM (DRTSEM) micrograph of nanostructures of an LSI. Jeol’s DRT-SEM is used as a tool for monitoring processes and detecting defects in the semiconductor production lines. This is an example of the image of surface nanostructures. Accurate observation of nanoareas is the starting point to understand substances, just as “seeing is believing”. Jeol’s TEM, SEM, scanning probe microscopes (SPM), electron probe microanalyzers (EPMA) and other instruments can observe and analyze nanostructures of materials. These instruments are used for structural characterization and materials research at the microscale and nanoscale.
“Nanometrology”: Metrology and Analysis of Materials at the Nanoscale
Fig. 6: Composite of a TEM micrograph (replica of a single crystal aluminium surface) and a photograph of mountain climbers.
Fig. 7: LSI’s nanostructures
We use a ruler to measure the size or length of visible substances. If the substance to be measured is too small, we cannot measure it with our eyes or using an optical microscope. The TEM, SEM, and SPM are powerful tools for measuring such small substances. Our EPMA, nuclear magnetic resonance spectrometers (NMR), mass spectrometers (MS), and other instruments are used for the analysis of substances. Prof Noyori at Nagoya University, the Nobel Prize laureate in 2001, used Jeol’s NMR in the development of catalysts and evaluation of synthesis results, as an important tool in his research of asymmetric synthesis. NMR is used in threedimensional structure analysis and molecular movement evaluation at a molecular level. The NMR allows us to evaluate the distance between atoms at the 0.1 nm level and the movement of atoms inside molecules. This clarifies the three-dimensional structures of proteins. Jeol’s MS is an effective tool for mass analysis and chemical structural analysis of trace components at the picogram (pg) or femtogram (fg) level. The pico level or femto level is below the nano level. In recent years, the mass spectrometer combined with electrophoresis has been used for structural and functional analysis of genes, genomes and proteins.
“Fabrication”: Creation of Nanostructures and Nanodevices
Fig. 8: “Nanopanda” figure
One of the cutting-edge technologies for “creation” in the nanoworld is pattern writing using a SPM. Fig. 8 shows a “nanopanda” figure fabricated by moving atoms one by one on the silicon sub-
strate surface, using an ultrahighvacuum scanning tunneling microscope (UHV-STM). Nanotechnology offers such fine fabrication and creation. Semiconductor-device fabrication is a leading technology for creating nanodevices. To produce semiconductor devices, 200 to 300 processes are necessary. The production of photomasks is very important in the front-end process. Jeol has been supplying electron beam lithography systems (EBX) that are indispensable tools for the fabrication of photomasks. These systems are also used for writing micropatterns for MEMS and system on a chip (SOC). In the ultra-microscopic world, defects or foreign matter of nanometer scale can cause fatal defects in semiconductor devices. Jeol also offers systems that detect, classify and analyze defects that occur in the semiconductor fabrication process. Today, large-volume information, including images and motion pictures, is being transferred via networks at high speed. Conventional electronic-transfer becomes difficult at such large volume and high speed. As a replacement, optical communication technology that utilizes the high speed of light is being developed. In developing a variety of optical communication devices such as band-pass filters, highly sophisticated deposition technology is required to form thin films on surfaces of glasses and metals at the nanometer level. Jeol’s high density reactive ion plating systems (HDRIP) can meet this requirement and contribute to the creation of nanoscale, high-quality, multi-layer thin films. Literature  Nanotechnology (Shaping the world atom by atom): National Science and Technology Council of USA  Nanotechnology Research Directions: M. C. Roco, R. S. Williams, P. Alivisatos  Micro & Nano Engineering 2001 abstracts: 9/16-9/19 2001, Grenoble, France  Microprocesses and Nanotechnology 2001 digest of papers: 10/31-11/2 2001 Matue, Japan
Dr Sadao Aoyagi Engineering Management Division JEOL Ltd. Shin-Suzuharu Bidg. 3F 2-8-3 Akebono-cho 2-chome Tachikawa, Tokyo 190-0012 Japan www.jeol.com
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G.I.T. Imaging & Microscopy 2/2003 • 35
X XI SX CX EX L L A N E O U S P R O D U C T S M
where the parts being inspected are subject to fluctuations in position, the highly telecentric design of the fixed magnification MML`s provide vision engineers with the best possible image for repeatable and accurate processing. www.moritex.com Easy Info no
Cedip Infrared Systems has announced the launch of its newly redesigned and expanded website. Located at www.cedip-infrared.com the website uses a design format that is rapidly downloaded by web search engines and provides visitors with direct access to a comprehensive range of information and news relating to Infrared Thermal Imaging. Intuitively structured, the site provides access to an expanding applications database, an attractive image gallery and the latest information on technological and product range developments. Further information on the company, its products, representatives around the world and company’s quality accredited production and testing facilities is given. Links are included to a growing range of associated websites. www.cedip-infrared.com Easy Info no
Machine Vision and Inspection
Science and Art
Easy Info no
Syncroscopy announced Auto-Montage, an imaging system producing images that have been shown as works of art. The system was used to capture and analyse many partially focused digital images of three-dimensional hard tissues such as bones and teeth. Many of the resulting infinite depth of focus images were considered so extraordinary they were featured in an art exhibition entitled, “The Microscope and The Skeleton: A Digital Photomicrography of Hard Tissues” held at the acclaimed, Karl Leubsdorf Art Gallery in New York. Curator Professor Timothy Bromage: “The software demonstrates the important relationship between science and new trends in graphic imaging”. www.syncroscopy.com Easy Info no
Machine Micro Lens (MML) from Moritex Europe provide the high resolution and sharp contrast required by the most demanding machine vision and inspection applications. Available in a wide choice of working distance and magnification combinations the compact, low mass design of the the product delivers a range of optimised solutions for wide ranging applications. These include recognition of chip mounter parts, 2D codes and integrated circuit patterns, component placement and automated alignment and inspection of machine tools, silicon wafers and LCD substrates. In operations 36 • G.I.T. Imaging & Microscopy 2/2003
Surfaces is able to supply reference flats for high precision applications including astronomy, laser beam steering and optical flatness testing. A thermally stable manufacturing environment enables the company to routinely achieve a surface accuracy of l/30 p.v. and surface roughness of 10 Angstroms on individual reference flats up to 600 mm in diameter. All reference flats from the company come with full quality testing assurance. Optics up to 450 mm diameter are provided with a Fizeau interferometric test report, larger flats are quality assured using the Ritchey-Common test procedure. www.optisurf.com
Working with high stability materials including glass, zerodur and silica, Optical
Tunable Optical Filter
Optical Surfaces has announced that it is a key component supplier to Scientific Solutions for their award winning NIR High Resolution Liquid Crystal FabryPerot Etalon (HR-LCFP), a next-generation tunable optical filter optimised for use in the near infrared. Using proprietary manufacturing techniques in a thermally stable production environment has enabled the company to routinely supply batches of fused silica etalon pairs with industry leading accuracy to Scientific Solutions. The specifications required of the etalon pairs, to deliver the high performance of the HR-LCFP, was better than lambda / 200 matching accuracy and surface roughness of less than 10 Angstroms. www.optisurf.com Easy Info no
trolled. For high-speed slide scanning or image mapping, users can remotely control the X, Y and Z-axes with SyncroScan RT, to scan specimen at up to 20 fields per second. www.syncroscopy.com Easy Info no
Software for X-ray Microanalysis Syncroscopy introduced an integrated version of SyncroScan, its automated stage and focus control system. The extensively upgraded software offers automated, motorised control of leading optical microscopes, ensuring more accurate, efficient and fatigue-free sample inspection. The company added options to SyncroScan to allow automatic, motorised control of the XY stage, Z-axis, illumination, condenser aperture diaphragm and top lens, as well as objective turret control of an Olympus BX61 or Leica DMR microscope. In addition, the focus mechanism of a range of Leica stereomicroscopes can also be con-
Edax has released E-Sig, a U.S. FDA 21 CFR Part 11 compliant software application for the Genesis X-ray Microanalysis System (XMS). It is the first x-ray microanalysis software package that provides electronic signatures as required by 21 CFR Part 11 and is able to satisfy US FDA security and audit trail requirements. The application provides users with the ability to electronically archive spectra collected using the Genesis software along with the user information and a date and time stamp that are added to the files saved within the user-defined projects. www.edax.com Easy Info no
The Future of EDS
Media Cybernetics announced the release of Iqbase, a way to archive, manage, and extract knowledge from all images and associated data. Most technical professionals accumulate a tremendous number of images in the course of their work. The need to manage and extract information from this wealth of image data is increasing in importance. Unlike other database products that are designed for business applications, the solution provides accessibility from the pixel level to the meta-data level. Images and informations can be viewed and managed together, in one place. Users can conduct searches based on image data to locate other similar archived images and drive discovery. www.mediacy.com Easy Info no
Highly precise analysis of all sample surfaces LN2-free XFlash® Detectors (3rd Generation) now with light element detection World’s best resolution of 127 eV Brilliant compositional ColorScans within seconds Fastest element mapping system available
The worlds fastest and easiest analyzer is here now! RÖNTEC GmbH [email protected]
RÖNTEC USA, Inc. [email protected]
RÖNTEC France S.A.R.L. [email protected]
G.I.T. Imaging & Microscopy 2/2003 • 37
Easy Info • 116
Software for Automated Microscopy
CONFOCAL LASER SCANNING MICROSCOPY
Confocal Analysis of Microstructures and Micromaterials Gerald Kunath-Fandrei
This is the age of microsystems and nanotechnology, of new and biocompatible materials, microtools and bio-MEMS. An age that challenges materials testing and analysis to look for new approaches. Confocal microscopy is one of the most promising methods because it provides a fast optical and non-contact 3D analysis of such structures and materials. With the LSM 5 Pascal confocal laser scanning microscope Carl Zeiss has risen to the challenge – with multimode analysis, innovative scanning strategies, unrivaled optics and comprehensive measurement functions.
Confocal Principle Laser light coupled into the microscope hits the sample at the objective focus. The light reflected or emitted by the sample surface passes the objective and is collected by a tube lens. In this way, the laser beam converges at a second focal point, which is optically conjugate to the first. A pinhole arranged in this confocal plane ensures that only light from
Fig.1: Beam path in the LSM 5 PASCAL confocal laser scanning microscope (schematic)
the focal plane reaches the detector, whereas light reflected or emitted by regions above or below is dramatically reduced. This enhances contrast and improves lateral and axial resolution.
Optical Section – Image Stack – Topography
Keywords Confocal microscopy, optical profilometry, roughness analysis
The laser beam is made to scan the sample in X and Y directions - point-by-point, and line-by-line. As the laser focus trav-
els along the X and Y coordinates, it generates an optical section of the sample. After the sample has been shifted along the optical axis (Z), the laser beam creates another optical section of a different sample plane. With the Z position changed successively, a three-dimensional stack of digital images is generated. It contains the digital brightness levels (intensity values) for each individual point defined by the laser focus coordinates Xi, Yj, Zk. From this data
Fig. 2: From confocal section to topography: From a succession of optical slices, the software computes an intensity projection with extended depth of focus (a), a 2D topographic map (b), or a 3D surface topography (c).
38 • G.I.T. Imaging & Microscopy 2/2003
CONFOCAL LASER SCANNING MICROSCOPY
record, the software quickly computes intensity projections with extended depth of focus, intensity or height profiles, topographic maps, or surface topographies.
Fast Overview – Efficient Navigation – Highly Resolved Details Besides confocal slices and 3D image stacks a variety of innovative scanning
cumbersome sample reorientation, the scanning area and orientation can be matched to the structures under investigation. Changes of the sample in time can be observed with fully automated time series sequences – either after a definite „schedule“ or „guided“ by external trigger events. This saves valuable time and creates vacancies for further important tasks.
possible number of optical sections - up to a maximum of 2048, depending on the application. Roughness and waviness measurements with the LSM 5 Pascal can now be carried out with improved comparability: The traversing distance captured by the 10x objective is > 12.5 mm. The 20x objective covers > 4 mm, and with the 100x it is still an impressive >1.25 mm. In
Fig.3: Microcomb drive (photoresist structure). Sample courtesy: Korean Institute of Science and Technology, Seoul, South Korea. a) Non-confocal overview image of a complete chip. Tile Scan assembled from 8x8 partial images. 14,497.1 µm x 14,497.1 µm, 4,096 x 4,096 pixels. b) Non-confocal sectional re-enlargement. Single exposure with rotated scanning field. 2,562.7 µm x 2,562.7 µm, 2,048 x 2,048 pixels. c) 3D surface topography from confocal stack. 115.5 µm x 115.5 µm x 26.7 µm, 604 x 604 pixels x 100 sections.
modes can be performed with the LSM 5 Pascal confocal laser scanning microscope from Carl Zeiss. Direct acquisition of single profiles – either along a straight line or a free hand tracking curve (Spline Scan) – mosaic-like large overview images (Tile Scan – Fig. 3a), free rotatable scan fields (Fig. 3b) and scanning fields of variable size and shape define a new state-ofthe-art in laser scanning microscopy. Only structures and features of interest are investigated. There is no need for
Scan Field Size: XXL – Accuracy: Nano In conjunction with the StitchArt option and a motorized XY scanning stage, the LSM 5 Pascal also acquires assembled height profiles and image stack arrays. This allows to image large-area segments, to measure long distance profiles and so to broaden the microscope’s horizon. In the Multiple Profile Mode up to 16,384 data points can be acquired. Higher axial resolution means a greater
Fig 4: Multiple Profile Scan from 10 single profiles. 3685.5 µm x 15.5 µm, 8192 pixels x 155 sections. a) Profile, roughness-filtered, cutoff frequency 0.8 mm, b) Profile, waviness-filtered, cutoff frequency 0.8 mm
Fourier terms, broadened microscopical horizon means the detection of very low spatial frequencies. The combination of the StitchArt and Topography options enables the LSM 5 Pascal to perform also optical waviness analysis. Confocal laser scanning microscopy is a highly beneficial complement to classical light microscopy or to scanning electron microscopy (SEM). It has become the method of choice for those applications that require fast, direct and accurate quantitative analysis of 3D microstructures. Equipped with alternative contrast techniques such as confocal fluorescence (see also image on back cover) and polarization imaging, a high degree of motorization and comfortable 3D display and processing software packages, the modern day confocal Laser Scanning Microscope has paved the way for new and exciting research as well as for routine applications in industry.
Dr Gerald Kunath-Fandrei Carl Zeiss Jena GmbH Advanced Imaging Microscopy Division Carl-Zeiss-Promenade 10 07740 Jena, Germany [email protected]
www.zeiss.de/lsm-mat For more info circle no.
G.I.T. Imaging & Microscopy 2/2003 • 39
SCANNING TRANSMISSION ELECTRON MICROSCOPY
Electron Microscopy of Compound Materials Using Low Voltage STEM Mode Various combinations of materials which have different compositions or hardness have been studied for development of advanced (functional) new materials. For microscopy of these materials, specimen preparation is a mandatory requirement. In addition to conventional ultra-microtomes, focused ion beam (FIB) systems have been extensively used. The FIB systems are advantageous in that they allow specimen preparation without giving mechanical forces to the specimen. A low voltage scanning transmission electron microscopy technique has been applied for oberservation of high polymer materials and it has been proved to be useful due to high angle scattering of electrons which allow good imaging contrast. This technique has also been used for microscopy of semiconductor devices in combination with FIB systems and microscopy of biological sections. We have studied some functional compound materials including high polymer or organic materials using the S-5200 ultra-high resolution SEM. We report here on some microscopy results of inner structures made visible with the low voltage STEM technique.
Microscopy of Toner Particles Fig. 1 shows microscopy results of polymerized toner(cyan-color) used on laser color printers. The specimen was thinned at about 1 µm using the FIB system. The toner particle is designed about 6.5 µm in diameter and contains wax inside. Fig. 1a
Fig. 1: Observation of toner, Low magnification SEM image (b) Low magnification STEM image (c) High magnification STEM image, X-ray mapping image
is an SEM image which shows the toner particle has been thinned without deformation. Fig. 1b is a STEM image recorded at 15 kV and it shows resin, wax and dispersion materials clearly separated. Inner structures are clearly visible. The low voltage STEM images are useful for observation of organic materials such as resin and
wax which have only a slight difference in specimen density. Fig. 1c is an enlargement of a portion of Fig. 3b showing the dispersion material. Magnification is X50,000. It shows the dispersion material very closely. Fig. 1d is an X-ray mapping image showing that the toner contains copper.
Microscopy of DVD-RAM Fig. 2 shows a commercial 9.4Gb DVDRAM, FIB thinned to about 0.1 µm thick. Fig. 2(a) is a STEM image recorded at x50,000 and at an accelerating voltage of 30 kV. It shows 7-layer structures thinned without damage. The masked area was magnified at about 400,000 times which is shown in Fig. 2(b). It shows a layer structure of about 5 ~ 6 nm which is in the recording layer. Fig. 2(c) is an X-ray mapping image. It shows a structure of a few nm recognized by X-ray analysis even with a low voltage STEM technique.
Fig. 2: Microscopy of DVD-RAM specimen Low magnification image High magnification image X-ray mapping image
40 • G.I.T. Imaging & Microscopy 2/2003
Nadine Baumgärtner Hitachi High-Technologies Europe GmbH Nanotechnology Equipment Europark Fichtenhain A 12, 47807 Krefeld, Germany [email protected]
, www.hht-eu.com For more info circle no.
New Rapid Confocal Microscope for Multidimensional Live Cell Imaging UltraView RS Colin Blackmore
Fig. 1 : High resolution multicolour confocal reconstruction of HEK293 cells expressing mitochondrially targeted Mito-DsRed (Red) and ER-GFP. The nucleus was labelled with DRAQ5. Images courtesy of Prof P. Lipp. For more images visit www.livecellimaging.com
Obtaining the maximum information from biological specimens requires a combination of advanced biological and chemical techniques, coupled with sophisticated detection technology. Living specimens are often regarded as the most demanding for microscopy, as not only does the sample move, but the imaging process can damage the specimen or prematurely terminate the experiment. Nevertheless, in order to acquire useful data, the technology must provide excellent resolution at acquisition speeds rapid enough to follow dynamic cellular processes in multiple dimensions. All of these must be achieved while keeping the samples alive and not affecting fluorochromes.
Keywords confocal, live cell imaging, multi-dimensional, resolution 42 • G.I.T. Imaging & Microscopy 2/2003
Modern biology increasingly relies on imaging techniques to gain insight into its mysteries, and advances in labelling technologies (fluorescent dyes and genetically coded fluorescent proteins) have enabled great progress. However, as the biological questions we ask become more searching, the demand on the technology grows. Examined below are some of the key requirements for successful multidimensional live cell imaging and how we have addressed these with the new PerkinElmer UltraView RS.
Lateral and Axial Resolution Accurate structural information as well as localisation of proteins to specific organelles requires the generation of sharp images, which can be reconstructed to give 3D representations of the sample (Fig. 1). Arguably the most widely used technique for removing out-of-focus haze seen in widefield microscope images is confocal microscopy in one of its many forms (laser scanning single and multiphoton, structured illumination, Nipkow Disk). Modern systems allow discrimination of points close to theoretical optical limits, but in order to extract the maximum possible information from the sample, the signal: noise ratio (S/N) must also be optimised. Most point-scanning confocals using photomultiplier tubes (PMT) to construct an image. The Nipkow-diskbased UltraView RS uses cooled CCD camera technology to generate images, which has a number of advantages over
PMTs. Firstly, the quantum efficiency (turning photons into electrons) of CCDs is typically double that of PMTs, while S/N in the RS system can be improved by increasing the effective cumulative dwell time per pixel. The flexibility gained with CCD detection over that of PMTs is advantageous for many live cell applications.
Temporal Resolution Traditional laser scanning techniques, where the beam scans the sample pixelby-pixel, do not allow capture of more than a few high resolution images per second, while faster resonant systems typically generate noisy images, due to low pixel dwell times. The UltraView RS essentially consists of “parallel” confocals, where over 1000 micro-laser beams scan the sample at once. This generates a “real-time” image which can be read by the CCD, with typical outputs at supra-video rate while maintaining resolution and a high S/N ratio. Examining fast moving samples (e.g. bacterial or vesicular motion) requires whole volumes being captured with subsecond resolution (Fig. 2). Additional demands are placed on the system, as synchrony between all components is crucial for successful imaging. UltraView RS uses external microprocessors to control fast AOTF wavelength switching (for multi-wavelength studies), camera, confocal unit and piezo-Z drives to optimise the acquisition process. A big advantage
of increased speed is that acquisition time is decreased, thereby increasing experimental throughput.
Phototoxicity and Photobleaching
Fig. 2: Rapid high-resolution confocal acquisition of bacteria labelled with Syto 9 from a human oral biofilm analogue. Each volume (30 slices) was acquired in 1 second. Images courtesy of Dr R. Palmer. For more images visit www.livecellimaging.com
Certain cellular processes (e.g. cell division) are especially sensitive to fluorescence illumination, and all experiments can be terminated prematurely if fluorescent dyes or proteins bleach. Traditional confocal microscopy focuses the entire beam at one point in the sample and can lead to enhanced phototoxicity and bleaching. Multibeam UltraView RS technology can reduce photobleaching substantially when compared to conventional confocal illumination , allowing prolonged experimental acquisition (Fig. 3).
Multi-D Live Cell Imaging Multidimensional live cell imaging requires an un-compromised resolution to accurately localise multiple cellular structures and high temporal resolution to follow changes in time and space, while maintaining cellular viability and fluorophore integrity. The UltraView RS provides a unique solution for such challenging applications.
Acknowledgements Many thanks to Prof Peter Lipp, Molecular Cell Biology, Saarland University, Homburg, Germany for advice on the manuscript and use of Figs. 1 and 3, and Dr Robert Palmer, NIDCR, NIH, USA, for samples used in Fig. 2.
Literature  S. Inoué, T. Inoué: Methods Cell. Biol. 70, 87-127 (2002)
Dr Colin Blackmore Applications Scientist PerkinElmer Life & Analytical Sciences 204 Cambridge Science Park Cambridge, CB4 0GZ, Great Britain [email protected]
Fig. 3: Prolonged high resolution, four-dimensional capture of HEK293 cells expressing Mito-dsRed. Image stacks were taken at 30s intervals for 70 minutes, representative stacks shown. Images courtesy of Prof P. Lipp. For more images visit www.livecellimaging.com
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G.I.T. Imaging & Microscopy 2/2003 • 43
SCANNING ELECTRON MICROSCOPY
Different Approaches to Visualize Cells with the Scanning Electron Microscope Scanning electron microscopes (SEMs) are built to image the physical surface of a solid and dry sample. Elaborated specimen preparation methods are, therefore, requested to provide useful information from wet biological specimens. When imaging cell surfaces, the surrounding water layer must be removed without destroying the delicate cell surface structures. With special specimen preparation techniques, even the inner parts of cells can be made amenable to the electron beam.
Introduction During the last years, SEM has been boosted by the enhanced resolution power due to the introduction of field emission cathodes. Since the electron beam is the probe for the scanning process, resolution is ultimately limited by the primary beam diameter. which can be less than 1 nm in
a modern field emission SEM under ideal conditions. To take use of this resolving power, the preparation techniques must also be improved, in order to preserve the small structural details of interest. Preparation techniques similar to the transmission electron microscopy (TEM) have, therefore, to be used when imaging biological samples. The advantage of the SEM is, that the sample does not need to be transparent for the electron beam; therefore bulk samples can be imaged, in contrast to TEM, where the samples need to be extremely thin. This often makes preparation easier, since no thin sections or replica need to be produced. In this work three different methods are explained to prepare cells for the SEM: • a classical SEM preparation protocol to visualize the cell surface, • a protocol for the investigation of the cytoskeleton, • and a freeze-fracturing cryo-SEM protocol to visualize the cell compartments.
Fig. 1 : Surface preparation of cultivated HeLa cells infected with a XXX virus. Fig. 1a is an overview. The cells are covered with microvilli. Fig. 1b is a high magnification of a cell surface area of the same sample. Two prominent structures are present: the microvilli and the viral particles (arrows) that are leaving the cell or are attached to the cell surface (arrows), after having left the cell. The small globular structures covering the cell surface represent membrane proteins or the sugar chains of membrane glyco proteins. This Fig. is a stereo pair and can be viewed with red green or red blue stereo goggles in order to become a three-dimensional impression of the cell surface. Fig. 2: A cytoskeleton preparation of cultivated smooth muscle cells (Fig. 2a is an overview and Fig. 2b a higher magnification). Mainly F-actin (filamentous actin) and intermediate filaments are retained after the applied protocol. 44 • G.I.T. Imaging & Microscopy 2/2003
Materials and Methods: For Fig. 1 cultivated HeLa cells infected with a modified vaccinia virus (that is not infectious to human) have been prepared following standard procedures: Fixation in 2% of glutaraldehyde in PBS was followed by dehydration in graded series of propanol, afterwards the organic solvent was removed by critical point drying. After coating with platinum-carbon (unidirectional) and carbon, the sample has been imaged in a Hitachi S-5200 in-lens field emission SEM at an accelerating voltage of 10 kV, using the backscattered electron signal, as explained in Walther and Hentschel . Fig. 2 is a cytoskeleton preparation of cultivated smooth muscle cells. The cultivated cells were detergent extracted with 1% of Triton X-100 as described by Svitkina and Borisy . In addition, actin filaments were stabilized by adding phalloidin to the extraction medium. Afterwards cells were glutaraldehyde fixed, dehydrated and critical point dried as described above. This sample was rotary coated by electron beam evaporation with a platinum layer of 3 nm thickness. The samples were investigated in an Hitachi S-5200 in-lens field emission SEM at an accelerating voltage of 4 kV using the secondary electron signal. Fig. 3, a freeze fractured bakers yeast cell saccharomyces cerevisiae imaged in the cryo SEM, was prepared as follows: A cell pellet of the yeast was frozen by high pressure freezing (to prevent the formation of structure destroying ice crystals during freezing) and then cryo-fractured  as described by Walther and Müller  at a temperature of -130°C in a BalTec BAF 300 freeze etching device (BalTec, Balzers, Principality of Liechtenstein), and afterwards coated with 3 nm of platinum-carbon from an angle of 45°C and then with 5 nm of carbon perpendicularly. The sample was removed from the freeze-etching device and cryotransferred onto a special Gatan cryoholder, which enables imaging of the frozen hydrated sample in the cryo-SEM. The yeast sample was imaged at a temperature of -130°C in a Hitachi S-900 inlens field emission SEM at an accelerat-
SCANNING ELECTRON MICROSCOPY
Concluding Remarks A modern SEM is a very versatile tool to investigate biological samples. The obtained information heavily depends upon the preparation methods used.
Acknowledgment The author thanks Dr Gustavo Vargas, Eberhard Schmid, and Wolfgang Fritz for helping with sample preparation. Fig. 2 is a result of a collaborative project with Andrea Meiser, EMBL Heidelberg; Fig. 3 is from a collaborative project with Dr Martin Müller, ETH Zürich. Literature  Moor, H., Mühlethaler, K.: Fine structure in frozen-etched yeast cells. J. Cell Biol. 17, 609628, (1963)  Resch, G. P., Goldie, K. N., Krebs A., Hoenger, A., Small, J. V.: Visualisation of the actin cytoskeleton by cryoelectron microscopy. J. Cell Sci. 115, 1877-1882, (2002)  Steere, R. L.: Electron microscopy of structural detail in frozen biological specimens. J Biophys Biochem Cytol 3, 45-60, (1957)  Svitkina, T. M.,Borisy., G. G.: Arp2/3 complex
Fig. 3: Cryo-SEM image of a freeze-fractured yeast cell. This technique, originally developed for the TEM replica technique, gives a unique view of the cell ultrastructure. The fracture plane often follows the hydrophobic inner part of the phospholipid membranes, and since all organelles are covered with a membrane, an impressive view of the different compartments of an eukaryotic cell can be achieved. The thick white arrow depicts to a vesicle captured in the moment of fusing with the vacuole.
and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia. J. Cell Biol. 145:1009–1026, (1999)  Walther, P., Hentschel, J.: Improved representation of cell surface structures by freeze substitu-
ing voltage of 10 kV using the backscattered electron image as described by Walther et al. [6, 7].
Results and Discussion The art of specimen preparation is to visualize the structures of interest and to prevent the formation of artifacts. The three Figs. shall demonstrate, that different structures of an eukaryotic cell can be visualized with different preparation methods. Fig. 1 is a classical SEM preparation to visualize cell surface structures. The basic problem of specimen preparation in this case is the removal of the overlaying water layer, to make the cell surface amenable to the electron beam. Drying the cell bears the risk of shrinking artifacts leading to the collapse of sensitive structures such as the microvilli. Chemical fixation, dehydration and critical point drying is the classical well established preparation protocol to minimize these structure destroying effects, although this method is not free of artifacts as shown in many studies. The present sample was made to investigate, how vaccinia viruses leave their host cell. Several viruses (arrows) can be seen on
the host cell surface. Fig. 2 shows the cytoskeleton of the cultivated smooth muscle cell after detergent extraction. With the applied preparation protocol mainly F-actin (actin filaments or microfilaments) and intermediate filaments are conserved, whereas lipid membranes and most of the soluble proteins are removed. Obviously, also this preparation method cannot be free of artifacts, which are heavily discussed in the recent literature . The basic problem  is, that the cytoskeleton is originally surrounded by membranes and other proteins, which have to be removed. This removal by extraction may also affect the structure of the cytoskeleton. Fig. 3 is a freeze fractured yeast cell, imaged in the frozen-hydrated state with a cold stage that allows us to investigate the sample at cold temperatures (-120°C to -150°C). The results look similar to the freeze etch replica method introduced by Steere  and by Moor and Mühlethaler . The fast freezing protocol immobilizes a cell within a few milliseconds under defined physiological conditions. This produces flashlight-like images of dynamic events in a cell; the thick white arrow, e.g. depicts to a vesicle fusing with the vacuole.
tion and backscattered electron imaging. Scanning Microsc. 3, Supplement 3: 201-211, (1989)  Walther, P., Wehrli, E., Hermann, R., Müller, M.: Double layer coating for high resolution low temperature SEM. J Microsc. 179, 229237, (1995)  Walther, P., Müller, M.: Biological ultrastructure as revealed by high resolution cryo-SEM of blockfaces after cryo-sectioning. J. Microsc. 196, 279-287, (1999)
Paul Walther Central Electron Microscopy Facility, University of Ulm, Germany [email protected]
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High-Resolution Constant-Distance Scanning Electrochemical Microscopy on Immobilized Enzyme Micropatterns Albert Schulte, Mathieu Etienne, Florin Turcu, Wolfgang Schuhmann
Keywords Albert Schulte
Scanning electrochemical microscopy, SECM, microelectrodes, nanoelectrodes, enzyme microstructure
The development of micro- and nanostructures with integrated biological recognition elements calls for suitable techniques for a well-controlled preparation and characterization of the obtained biosensor architectures. We recently established a method for fabricating complex micropatterns of different enzymes using a flow-through liquid microdispenser in combination with a micropositioning system. Enzyme microstructures could be patterned [1, 2], and scanning electrochemical microscopy (SECM) was successfully used for localized visualization of the activity of enzyme-containing polymer lines  and for the characterization of multi-enzyme grids . However, SECM imaging was carried out in the constant-height mode without controlling the tip-to-sample distance and clear interpretation of measurements was difficult due to the lack of additional topographic information about the microstructures. Moreover, spatial resolution was limited by the size of the microelectrodes used as scanned tip, actually 10 µm at that time. The introduction of constant-distance mode SECM with an optical detection scheme for shear forces between the tip electrode and the surface permitted the operation of probe tips with electroactive diameters below a micrometer and simultaneous imaging of both, the topography and the local reactivity of sample surfaces became possible [3, 4]. Here, we present the fabrication of 46 • G.I.T. Imaging & Microscopy 2/2003
enzyme-containing polymer microstructures by means of microdispensing as well as the high-resolution imaging of the topography and locally immobilized enzyme activity by means of constant-distance SECM with a nonoptical readout of shear forces and operated in the substrate generator/tip collector mode.
Fig. 1: Schematic of the operational principles of scanning electrochemical microscopy (SECM).
Technical Concept of Scanning Electrochemical Microscopy (SECM) SECM is a compelling analytical imaging technique offering an advantage over other available scannedprobe methods for it is capable not only to look insitu at the topography of surfaces immersed into electrolytes, but also to visualize microscopically small local variations in the (electro) chemical reactivity of the solid/liquid interface . The basic concept behind SECM is to use amperometric or potentiometric ultramicroelectrodes with active
Fig. 2: Schematic of the flow-through microdispensers as used for the fabrication of enzyme/polymer microstructures.
SCANNING ELECTROCHEMICAL MICROSCOPY
radii, r, in the order of a few µm or less as electrochemical scanning probes. Disk-shaped amperometric SECM tips are made of small-diameter noble metal wires or conductive carbon fibres sealed into glass or polymers , whereas potentiometric SECM tips are typically the tiny tips of pulled glass capillaries filled with ion-selective ionophors or pH-sensitive metal oxides . Spatial resolution of SECM is primarily determined by the size of the SECM tip and for that reason not on the atomic scale as with scanning tunneling and atomic force microscopy. However, the electrochemical nature of the scanning probe offers an exceptional chemical selectivity and SECM therefore serves as an excellent tool for studying interfacial (electro)chemical properties and reactions. Mapping the local activity of immobilized enzyme microstructures [2, 8], spotting microscopic superficial phenomena such as localized corrosion  or the flux of ions through pores of semi permeable membranes  and imaging the activity of living biological cells  are just a few out of an increasing number of applications demonstrating the potential of SECM. The amperometric feedback mode of SECM relies on the detection of faradaic currents caused by the electrochemical conversion of redoxactive dissolved species (the mediator) at the tip electrode polarized to a suitable constant potential. With the SECM tip far above the surface, the current is controlled by hemispherical diffusion of the mediator towards the electroactive disk and a diffusion-limited steady-state current is measured (Ieim, Fig. 1A). However, the vicinity and the nature of a substrate heavily perturb the tip current. Close to a conductor, electrochemical recycling of consumed mediator molecules becomes possible leading to an increase in the tip current (I > Ieim, positive feedback, Fig. 1B). In contrast, proximity to insulating surfaces is physically hindering diffusion
of species towards the sensing disk and the tip current decreases (I < Ieim, negative feedback, Fig. 1C). Both feedback effects are highly distance-dependent, and approach curves (I/Ieim vs. tip-to-sample separation d) allow placing the SECM tip at appropriate working distances within the regime of the electrochemical “near field" (typically a few times the SECM tip radius or less). Image acquisition is achieved by rastering the SECM tip at a user-defined fixed height and simultaneously recording the tip current as a function of position (constant-height feedback mode). In the generator/collector mode of SECM, readily oxidizable or reducible species possibly released from active spots at the sample surface are detected by the SECM tip (Fig. 1D), or vice versa. The tip-generation/substrate-collection mode is very suitable for studying the kinetics of interfacial redox processes. Alternatively, the substrategeneration/tip-collection mode is an excellent approach for monitoring for example enzymatic reactions, localized corrosion, or chemical release from secretory cells. Constant-height SECM has limits on heterogeneous surfaces displaying variations in both conductivity and topography because current changes arising from distance variations cannot easily be differentiated from the ones originating from alterations in conductivity. Furthermore, tip crash is at high risk on tilted or rough surfaces, especially when decreasing the size of the SECM tip for imaging at higher resolution. On the contrary, constant-distance SECM offers the advantage of a thorough control of the tip-to-sample distance since its shear force-sensitive feedback loop forces the SECM tip to follow the contours of the surface during scanning. This allows simultaneous acquisition of the electrochemical tip response along with the sample topography and effectively prevents against tip crash.
Fabrication of Enzyme/Polymer Micropatterns Different polymer microstructures were produced using a flow-through microdispenser as previously described . This device can emit small droplets of solutions based on rapid movements of a flexible silicon membrane (Fig. 2). The membrane is coupled to a piezoelectric actor, which expands and contracts upon excitation with alternating voltages. That way short pulses of pressure are generated, each of them ejecting a single droplet of roughly 100 pl. The droplets are released through a pyramidal nozzle and directed towards the target surface where the deposition leads to spots with diameters of about 75 µm. A precise movement of the target substrate relative to the nozzle can be used to deposit either rows of individual spots or lines and grids from overlapping spots. pl-droplets of aqueous solutions of glucose oxidase (GOD, 1 mg/ml) and Vinnapas EP 16 (2 mg/ml) were dispensed in different geometries on the clean surface of glass cover slips. Vinnapas EP16 is a suspension of a vinyl acetate-ethylene co-polymer and was chosen for codeposition with the enzyme to enhance immobilization by entrapping the biomolecules into the polymer films formed during drying. Exposed to electrolytes, the polymer swells and a hydrogel-like polymer matrix keeps hold of the immobilized enzyme. Fig. 3 demonstrates the capability of the method in fabricating well-aligned and adherent enzyme/polymer micropatterns by displaying scanning electron micrographs (SEM) of arrays of spots and lines of a GOD/Vinnapas mixture on gold.
High-Resolution SECM Imaging of Enzyme/Polymer Micropatterns Using constant-distance SECM in the generator-collector mode, the integrity of microstructures was inspected and local
Fig. 3: Scanning electron micrographs of arrays of enzyme-containing polymer spots (right) and lines (middle and left) on gold. They were made by dispensing a mixture of 1 mg/ml glucose oxidase and 2 mg/ml of the polymer suspension Vinnapas EP 16. The scale represents 200 µm.
Fig. 4: Schematic of the set-up used for constant-distance mode SECM with an integrated non-optical (piezoelectric) detection of the distance-dependent shear forces. G.I.T. Imaging & Microscopy 2/2003 • 47
SCANNING ELECTROCHEMICAL MICROSCOPY
Fig. 5: Scanning electron micrograph (A) and shear-force topography image (B) of a 76-µm-diameter spot of GOD/Vinnapas on gold.
Fig. 6: Line scans of the topography (blue) and amperometric tip current (red) of a polymer microstructure consisting of three lines of Vinnapas EP 16, 75 µm in width, and with glucose oxidase entrapped only in the middle line. They were simultaneously acquired with the constant-distance mode of SECM in solutions containing 50 mM glucose and a 2 µm Pt-disk nanoelectrode as SECM tip.
enzyme activity visualized by the localized detection of enzymatically produced H2O2. Of note, a most recently introduced non-optical detection scheme for shear forces (Fig. 4) was employed to establish scanning the SECM tip in a constant distance . With this method, two piezoelectric plates are glued to the body of highly flexible, needle-like SECM tips. One plate vibrates the tip at resonance while the other serves as a detector of the vibration amplitude and shear forces, respectively. During tip approach, shear forces appear in proximity to the surface leading to a damping of tip vibration and a phase shift, both well registered by connecting the piezoelectric detector to a lock-in amplifier. The shear-force and hence distance-dependent signal of the lock-in amplifier provides the input of a computer-controlled feedback loop accurately controlling the tip-to-sample distance all through scanning. 48 • G.I.T. Imaging & Microscopy 2/2003
Topographic resolution of constantdistance SECM largely depends on the sharpest part at the very end of the probe tips, which is responsible for the shear force contact while the current resolution is related to the size of the electroactive area of the tip. In this study, we used glass-insulated Pt-disk nanoelectrodes to image enzyme-containing polymer microstructures. They can be made by pulling quartz capillaries together with inserted Pt wires using a Laser-based micropipette puller . Pulling typically produces needle-like tips with a Pt core of reduced diameter tightly sealed into quartz glass. Grinding the tapered tips on polishing pads leads to the exposure of Pt-disks with diameters of far below a micrometer. To demonstrate the performance of a non-optical shear-force detection system and to evaluate limits for topographical imaging, spots of GOD/Vinnapas mixtures were imaged in air with the unpol-
ished tip of a pulled capillary as scanning probe, as this offered much smaller tip dimensions as polished, disk-shaped nanoelectrodes. Fig. 5A shows the SEM image of a GOD/Vinnapas spot on gold. The spot morphology is not homogeneous but displays variations in the local density of the enzyme/polymer film and apparently, most of the two compounds deposit upon drying in a rim at the edge of the spot. Fig. 5B presents the topography of the same spot but obtained with the shearforce mode of SECM. As with SEM, local micrometer-sized topographical variations are clearly visible in the shear-force image underlining unequivocally the ability of shear-force based constant-distance SECM to resolve surface topography at high resolution. High-resolution SECM imaging of the topography and localized GOD activity was then performed in solutions containing 50 mM glucose on polymer micropatterns consisting of three lines of Vinnapas, about 75 µm in width and with GOD entrapped only in the middle line. Scanning probes used for the measurements were Pt-disk nanoelectrodes (φ 200 nm – 2 µm). Before imaging, the feedback control continuously adjusting the tip-to-sample separation was established. In brief, the Pt-disk mikro – and nanoelectrode was excited to vibration at resonance and slowly moved towards the surface while continuously monitoring the response of the shear-force detecting piezoelectric plate. Tip approach was automatically stopped at a user-defined degree of damping of the vibration amplitude, i.e. at 70% of the value with the tip far above surface. The slope at the end of an approach curve (lock-in amplifier output vs. distance) allows adjusting the sensitivity of the feedback loop. SECM imaging in the constant-distance mode of operation was carried out at scanning speeds of 0.1–1 µm s-1 for x and y displacements while the tuned feedback loop guaranteed a constant tip-to-sample distance of about 100–200 nm. For local detection of enzymatically generated H2O2 in the generator/collector mode, the SECM tip was poised to a potential of 0.6 V vs. an Ag/AgCl reference electrode, which is sufficiently high for the diffusion-controlled oxidation of H2O2. Fig. 6 represents, respectively, line scans of topography, and amperometric SECM tip current both simultaneously acquired by scanning across a GOD/Vinnapas microstructure using the shearforce based constant-distance mode of SECM and recording the two parameters as a function of tip position. Although the topographical resolution with diskshaped Pt nanoelectrodes was found to
SCANNING ELECTROCHEMICAL MICROSCOPY
Conclusion Needle-type Pt-disk nanoelectrodes were successfully implemented as miniaturized scanning probes in a specially designed set-up for constant-distance mode SECM. The combination of nanometresized SECM tips with the non-optical shear force positioning of this system allowed simultaneously to image the
topography and the local chemical activity of enzyme-containing polymer microstructures with high spatial resolution. That way, the non-homogenous distribution of enzyme/polymer mixtures within the circular area of a spot formed by a dispensed droplet could be visualized.
Acknowledgements The authors thank Wacker Chemie GmbH, Germany for kindly providing the polymer suspension Vinnapas EP16. Further we would like to acknowledge financial support from the DFG (Schu929/5-1 and SFB459-A5). The authors are grateful to Prof T. Laurell, Department of Electrical Measurements, University Lund, Sweden, for the cooperation concerning the piezo microdispenser.
Dr Albert Schulte He received his PhD in Applied Electrochemistry from the University of Münster, Germany. After postdoctoral research fellowships in Göttingen, and Edinburgh, he joined Prof Wolfgang Schuhmann's group at Bochum University in 2000. In the position as senior scientist he focuses on Microelectrochemistry and Surface Science. Current work includes studying the process of localized corrosion of NiTi shape memory alloys as well as applications of scanning electrochemical microscopy to examine enzyme microstructures and local chemical release from living cells. Florin Turcu Dr Mathieu Etienne Prof Dr Wolfgang Schuhmann Analytische Chemie – Elektroanalytik & Sensorik Gebäude NC 04/788 44780 Bochum, Germany Tel.: +49 234 3226202 Fax: +49 234 3214683 [email protected]
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be not as good as with the fine tips of unpolished pulled capillaries, small lateral variations in the topography of the enzyme/polymer structure are still visible. On the other hand, an increase in the amperometric tip current was observed only just above the middle polymer line. This was expected as only the middle line contained active enzyme and thus, the local production of H2O2 is limited to the area covered by that line.
G.I.T. Imaging & Microscopy 2/2003 • 49
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Halcyonics presents a new generation of compact active vibration isolation systems. The systems of the Mod-1 plus series isolate vibration-sensitive measuring equipment from these effects, such as atomic force microscopes, interferometric measuring systems, etc. The new generation combines active vibration isolation with a high level of operating convenience. The systems now feature automatic load adjustment. They also eliminate the need for manual removal of the transport locking devices. The Vibration Analysis Software enables the system connected via a USB port to a Windows-based PC to perform relative, comparative measurements of the available vibration at various work areas in a laboratory. www.halcyonics.de Easy Info no
The latest products from Cedip Infrared Systems will be in evidence at important European exhibitions this summer. At Laser 2003 (23rd-26th June), the company will showcase on stand 174 (Hall B2) the Jade Swir, a camera designed to meet the demanding infrared analysis requirements of spectroscopic test equipment for telecomms and process monitoring applications. The camera is sensitive from 0.8 – 2.5 microns and uses a MCT based focal plane array sensor cooled with a thermoelectric cooler to provide extremely high sensitivity and hence to capture in real-time high-speed dynamic events in snapshot readout mode. Coupled with the PC-based analysis and reporting software Altair, the sys50 • G.I.T. Imaging & Microscopy 2/2003
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Carl Zeiss presents two new digital cameras for photomicrography in biomedical research: The AxiCam HRm digital camera with optimum resolution, 14-bit digitization and microscanning feature, and the AxioCam MRm high-resolution, scientific digital camera with 12-bit digitization. The new technology of highly sensitive cameras permits short exposure times. The resulting reduction in exposure to light protects living biological objects and decreases the bleaching of fluorescence specimens. Furthermore, specimen-protecting fluorescence dyes in near IR can be used, which is particularly beneficial in neurobiology. www.zeiss.de Easy Info no
The users of the LSM software release for the Carl Zeiss LSM 5 Pascal, LSM 510 Meta laser scanning microscopes, the high-resolution AxioCam HR digital camera is now available as a new detection modality in biomedical research. The Peltier-cooled camera offers high image quality, even where light intensities are very low. The camera is particularly useful also for work with low objective magnifications and with transmitted light. To best suit the experimental requirements, users can choose from various resolution levels up to 3900 x 3090 pixels, and frame rates up to 20 frames per second. www.zeiss.de Easy Info no
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Chemical Metallurgy – Principles and Practice
Edited by ALEXANDER V. KOLOBOV, AIST, Japan
Photo-induced Metastability in Amorphous Semiconductors A review summarizing the current state of research in the field, bridging the gaps in the existing literature. Readers benefit from the inclusion of both experimental and theoretical results on photo-induced metastability, obtained using novel approaches, as well as from the detailed discussion of current and potential applications.
The field of nanocomposites has the attention and imagination of scientists and engineers in recent years. The reason for this is based on the simple premise that by using building blocks with dimensions in the nanosize region, it is possible to design and create new materials with unprecedented flexibility and improvements in their physical properties.
This book provides you with detailed information about the first steps of the separation of the desirable minerals and also describes the mineral processing operations. The complex chemical processes of extracting various elements through hydrometallurgical, pyrometallurgical or electrometallurgical operations are explained.
3527-30359-6 · June 2003 · approx 260pp with approx 194 figs, 4 in color · Hbk · approx € 149.00
3527-30376-6 · June 2003 · approx 650pp with approx 120 figs · Hbk approx € 209.00
Edited by JIRI MAREK, Reutlingen, Germany, et al.
Edited by HANS-RAINER TREBIN, ITAP, University of Stuttgart, Germany
Edited by LOTHAR WAGNER, TU Clausthal, Germany
Sensors in Automotive Technology
Structure and Physical Properties A comprehensive and up-to-date overview on quasicrystals. This complete presentation of the authors’ most successful results is vital for the understanding of the current status of the science of this outstanding class of materials among intermetallic alloys. 3527-40399-X · April 2003 · 668pp with 384 figs and 30 tabs · Hbk € 139.00
Shot peening has been proved to be a powerful instrument in enhancing the resistance of materials to various kinds of stress-induced damage, particularly against damage due to cyclic loading (fatigue) in air or in aggressive environments. 3527-30537-8 · April 2003 · approx 560pp with approx 250 figs and approx 20 tabs · Hbk · € 199.00
3527-40370-1 · May 2003 · approx 424pp with approx 20 figs · Hbk approx € 119.00
Sensors Applications Vol. 4 SERIES: Sensors Applications Microelectronics have become indispensable in measurement and control technology and especially in modern automobiles, all sorts of electronic and mechanical sensors find many important applications.This book shows the different types of sensors, their applications for particular tasks, and the reasons for their use.
INGO DIERKING, University of Manchester, UK
Edited by PIERRE RUTERANA, ISMRA, France, MARTIN ALBRECHT, University of Erlangen, Germany, and JÖRG NEUGEBAUER, Fritz Haber Institute, Germany
3527-29553-4 · May 2003 · approx 562pp with 450 figs and 50 tabs · Hbk · € 259.00 · Series price € 229.00
Textures of Liquid Crystals
Edited by GUIDO TSCHULENA, SGT Sensorberatung, Wehrheim, Germany, and ANDREAS LAHRMANN, Fa. Moulinex-Brandt, Lyon, France
A unique compendium of knowledge on all aspects of the texture of liquid crystals, providing not just detailed information on texture formation and determination, but also an in-depth discussion of different characterization methods. 3527-30725-7 · May 2003 · approx 314pp with approx 248 figs, 120 in color, and approx 25 tabs · Hbk € 159.00
Handbook on Materials and Devices A systematic and in-depth overview of ceramic semiconductors based on nitrides, on both a high and current level. This collection of review articles offers information on the physical basics as well as the latest results in a compact yet comprehensive manner. A highly pertinent book for anyone working in applied materials research or the semiconductor industry. 3527-40387-6 · March 2003 · 686pp with approx 387 figs and approx 40 tabs · Hbk · € 169.00
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Sensors in Household Appliances Sensors Applications Vol. 7 SERIES: Sensors Applications Microelectronics have become indispensable in measurement and control technology. Thus, there is an increasing demand for suitable sensor systems and a growing need for comprehensive information on their potentials and limitations. 3527-30362-6 · June 2003 · approx 400pp with approx 450 figs and approx 50 tabs · Hbk · approx € 259.00 · Series price approx € 209.00
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Micro fiber fabric, 422.5 µm x 422.5 µm x 330.5 µm.
Know the Ropes ...
Highly resolved detail
The LSM 5 PASCAL confocal laser scanning microscope looks below the surface. Accurate. Free of artefacts. Innovative. www.zeiss.de/lsm-mat Easy Info • 121