UK MA Nov 2011 by Andy Alldridge

November/December 2011 UK Issue 149

Plasma FIB Analysis of Through Silicon Vias p9

Variable Pressure SEM of Plant Stigmata p21

Secondary Electron Atomic Imaging in S/TEM pS5

High Throughput Automated S/TEM pS11

Keeping your slides, exactly as you know them.

The Olympus VS120 virtual slide system. Digital slides at the highest resolution. Ensure your slides are never again lost, damaged or forgotten. With the Olympus VS120 slide scanning and archiving system, you can turn your entire slide collection into a digital data mine, creating high resolution images of each slide that can be browsed â&#x20AC;&#x2DC;virtuallyâ&#x20AC;&#x2122; just as if you were sat at the microscope. You can share information with colleagues across the world or in the next room, at any time. And with the new fluorescence module you can permanently capture your data before your work can fade away.

For further information please visit: www.microscopy.olympus.eu microscopy@olympus-europa.com

CIRCLE NO. 2

ONLINE: www.microscopy-analysis.com

CONTENTS

www.microscopy-analysis.com

FEATURES

CIRCLE NO.

1 OR ONLINE www.microscopy-analysis.com

COVER STORY The cover shows a confocal image (top) of a Drosophila embryo at stage 11, expressing the tracheal marker trh-LacZ (Cy3, red) and the cell membrane marker Dlg (Alexa 488, green). The enlarged view (below) shows invaginating tracheal placodes in X-Y (left) and Y-Z (right) projections. (Courtesy of Dr Takefumi Kondo and Dr Shigeo Hayashi at the Laboratory for Morphogenetic Signalling, RIKEN Centre for Developmental Biology, Japan.) The new UPLSAPO30xS and UPLSAPO60xS silicon oil-immersion objectives from Olympus are ideal for live cell experiments investigating thick samples or requiring long-term imaging. Users can generate bright images at higher resolutions, since silicon oil significantly improves optical performance compared to water-based methods and offers larger working distances than oil immersion objectives. This maximises the effectiveness of brightfield, differential interference contrast, fluorescence, confocal laser scanning and multiphoton studies. Silicon oil allows users to capture bright, high resolution images of living samples, even deep into cells and tissues. This can be achieved because the refractive index of silicon oil (n = 1.40) is almost identical to that of living biological samples (n =1.38 on average) so silicon oil-immersion objectives minimise refractive index mismatch and spherical aberration. This is a key factor for improving the focal spot, which can be even further optimised to correct for temperature changes by adjusting the available correction collar. The properties of silicon oil also make it ideal for long-term, time-lapse studies since it is stable, viscous and of low volatility; it will not dry out or absorb moisture over time. With its 30x magnification and high NA of 1.05, the UPLSAPO30xS delivers highly resolved images of an extensive sample area. The Olympus UPSLAPO60xS provides increased magnification (60x) and NA (1.30), allowing researchers to produce highly detailed live sample images using fluorescence, confocal laser scanning and multiphoton excitation techniques. This objective is also ideal for high resolution 3D imaging applications by offering improved spherical aberration correction and a shorter working distance of 0.3 mm.

Please contact: OLYMPUS EUROPA HOLDING GMBH Katja Ansmann Marketing Communications Manager Tel: +49 40 2 37 73 - 5913 Fax: +49 40 2 37 73 - 4784

Bonding and TSV in 3D IC Integration: Physical Analysis with a Plasma FIB M M V Taklo, A Klumpp, P Ramm, L Kwakman and G Franz

Fracture Surface, Impact Energy and Hardness of Ni-Free High-Mn Steels W Sha, H Haji Talib, E Wilson, R Rajendran, S Malinov, H Charlesworth, L Ibbitson

Variable Pressure Scanning Electron Microscopy of Vicia faba Stigmatic Papillae W Chen, F Stoddard and T Baldwin

Atomic Resolution Secondary Electron Imaging in Aberration Corrected STEM H Inada, M Konno, K Tamura, Y Suzuki, K Nakamura, Y Zhu

S11

Development of a High Throughput Electron Microscope for Nanoscale Analysis M Matsushita, S Kawai, T Iwama, K Tanaka, T Kuba, N Endo, T Isabell

S19

Low Beam-Energy Energy-Dispersive X-Ray Spectroscopy for Nanotechnology P Camus

S23

Product Focus - Nanotechnology and Electron Microscopy

REGULARS

DIARY

NOVEMBER 2011

8-11

Dates for the Diary

10 12-16 14-18 16 22-23 28-2/12

People & Places

Gatan EELS School, Austrian Centre for Electron Microscopy and Nanoanalysis, TU Graz, Austria www.felmi-zfe.tugraz.at www.gatan.com/company/news/news08291102.php Electron Microscopy in Materials Science, Eindhoven University of Technology, The Netherlands http://nvvmmaterials2011.chem.tue.nl/ Neuroscience 2011: Washington DC, USA www.sfn.org NvVM European School on Nanobeams , Centre de Recherche Public Gabriel Lippmann, Belvaux, Luxembourg www.nanobeams.org Cryo Microscopy Group Annual Meeting, Boots Science Building, School of Pharmacy, University Park, Nottingham University, UK www.cryomicroscopygroup.org.uk/CMG2011.html Bruker Scanning Probe Microscopy Conference and Users’ Meeting, University of Manchester, UK www.bruker-axs.com drew.murray@bruker-nano.com 2011 MRS Fall Meeting, Boston, Massachusetts, USA www.mrs.org

DECEMBER 2011 3-4 3-7

5-9

Winter School on High-Resolution Electron Microscopy, Arizona State University, Tempe, AZ, USA wintershcool@asu.edu http://le-csss.asu.edu/winterschool

Literature Highlights

25-29

18-23

1-4

9-13

What’s New

17-20

9-11

24-25

Pittcon 2012, Orlando, FL, USA www.pittcon.org Course in Cryotechniques for Electron Microscopy, Rothamsted Research, Harpenden, UK www.rms.org.uk/events/Forthcoming_Events/CoolRunnings

3-15

MICROSCOPY & ANALYSIS ISSN 0958-1952 - UK ISSN 2043-0655 - Europe © 2011 John Wiley & Sons, Ltd Issued in: January, March, May, July, September, November Published by: John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, UK Tel: +44 (0)1243 770257 Fax: +44 (0)1243 770432 Email: editor@microscopy-analysis.com Website: www.microscopy-analysis.com Associate Editorial Director: Dr Ray J. Boucher Publisher: Roy Opie Account Management Americas & Asia-Pacific : Stephen Parkes Account Management EMEA : Charlotte Redfern Assistant Sales Executives: Jackie Sheppard and Michelle Paice Reader Services Controller: Sue Turner Reader Services Assistant: Craig Godfrey REGISTRATION: The journal is free of charge, worldwide, to users who pur chase, specify or approve microscopical, analytical or imaging equipment at their place of work, and marketing exe cutives who make advertising decisions. To register or report address changes on-line go to www.microscopy-analysis.com/ reg.htm, call the Circulation Office on +44 (0)1243 770351, or complete the card inside the back cover of this issue. You need to re-register every three years unless your address changes. We do not mail to home addresses. Subscription charges for non-qualifying readers: $110 per annum (UK) or $195 (Europe); all other countries $280 by airmail.

2-6

29-2/8

26-30

Electron Microscopy Summer School, University of Leeds, UK RMS: www.rms.org/events Light Microscopy Summer School, University of York, UK RMS: www.rms.org/events Optics Within Life Sciences (OWLS), Genoa, Italy Organised by Italian Institute of Technology www.owls2012.org Inter/Micro: 63rd Annual Applied Microscopy Conference, Chicago, IL, USA www.mcri.org CryoElectron Microscopy Short Course, University of Minnesota, MN, USA Nanostructural Materials and Processes Program of IPrime: mccormic@umn.edu www.iprime.umn.edu Microscopy & Microanalysis 2012, Phoenix, Arizona, USA www.microscopy.org

13-17

European Microscopy Congress, Central Convention Complex, Manchester, UK www.rms.org.uk Neuroscience 2012, New Orleans, Louisiana, USA www.sfn.org

NOVEMBER 2012 26-30

2012 MRS Fall Meeting & Exhibition, Boston, Massachusetts, USA www.mrs.org/fall2012

DECEMBER 2012 15-19

ASCB 52nd Annual Meeting, San Francisco, CA, USA www.ascb.org Pittcon 2013, Philadelphia, PA, USA www.pittcon.org

1-5

2013 MRS Spring Meeting & Exhibition, San Francisco, CA, USA www.mrs.org/spring2013

AUGUST 2013 4-8

Microscopy & Microanalysis 2013, Indianapolis, IN, USA www.microscopy.org

NOVEMBER 2013 9-13

Neuroscience, San Diego, California, USA www.sfn.org

See the Microscopy and Analysis online Diary for a full listing. Entries for the January issue are due 1 December. Email to: editor@microscopy-analysis.com

Microscopy School, Lehigh University, Bethlehem, PA, USA Sharon Coe: sharon.coe@lehigh.edu www.lehigh.edu/microscopy

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

A large array of sub-10-nm single-grain gold nanodots for use in nanotechnology is described by Nicolas Clément and colleagues at the Institut d’Electronique Microélectronique et Nanotechnologie, CNRS, University of Lille, France [Small 7(18):2607-2613, 2011]. A uniform array of singlegrain gold nanodots, as small as 5-8 nm, was be formed on silicon (a) STEM image showing the bulk silicon (Si), five annealed dots (Au), carbon layer (C), and platinum layers. using e-beam lithography. The (c) Coloured STEM image of a single annealed nanodot (260°C, 2 h). Reproduced with permission, Copyright © as-fabricated nanodots were 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. tify the critical size that determines whether a dot is amorphous, and thermal annealing converted them composed of single or multiple crystal domains. to pure Au single crystals covered with a thin SiO2 Moreover, they showed that annealing at moderate layer. These findings were based on physical meatemperature can convert Au dots from amorphous to surements by AFM, atomic-resolution STEM, and single-crystalline, and then they were covered with a chemical techniques using energy dispersive X-ray thin SiO2 layer. After easy removal of the SiO2 (dilute spectroscopy. The authors demonstrated the formaHF etching), these nanodots can be used as electrodes tion by e-beam lithography of sub-10-nm Au dots for the characterization of organic self-assembled with small dispersion and perfect alignment. Such monolayers (SAMs) with less than 200 molecules. precise formation of small dots enabled them to iden-

# "

The three-dimensional point spread function of an aberration-corrected scanning transmission electron microscopy (STEM) has been simulated and experimentally tested by Andrew Lupini and Niels de Jonge at the Oak Ridge National Laboratory, TN [Microscopy and Microanalysis 17:817-826, 2011]. Aberration correction reduces the depth of field in STEM and thus allows three-dimensional imaging by depth sectioning. This imaging mode offers the potential for sub-Ångstrom lateral resolution and nanometer-scale depth sensitivity. For biological samples, which may be many µm across and where high lateral resolution may not always be needed, optimizing the depth resolution even at the expense of lateral resolution may be desired, aiming to image through thick specimens. Although there has been extensive work examining and optimizing the probe formation in two dimensions, there is less known about the probe shape along the optical axis. The authors examined the probe shape in 3D in an attempt to better understand the depth resolution in

A fractal dimension analysis and mathematical morphology of structural changes in actin filaments imaged by electron microscopy is reported by Yoshitaka Kimori et al at the National Institutes of Natural Sciences, in Tokyo [Journal of Structural Biology 176(1):1-8, Oct 2011]. The authors examined structural changes of actin filaments interacting with myosin visualized by quick freeze deep-etch replica EM by using a new method of image processing and analysis based on mathematical morphology. To quantify the degree of structural changes, two characteristic patterns were extracted from the EM images: the winding pattern of the filament shape (WP) reflecting flexibility of the

APRIL 2013

AMTC3: 3rd International Symposium on Advanced Microscopy and Theoretical Calculations, Nagaragawa Convention Center, Japan www.congre.co.jp/amtc3/ IFES2012: 53rd International Field Emission Symposium, University of Alabama, Tuscaloosa, AL, USA www.ifes2012.au.edu SCUR 2012: 39th Annual Meeting of Society for Cutaneous Ultrastructure Research, Lyon, France http://orgs.dermis.net/content/e04scur/e03meetings/e770/e1074/index_ger.html

'! !

MARCH 2013 17-21

# "#

A miniature stage device to overcome resolution anisotropy in fluorescence light microscopy is described by Florian Staier and colleagues at the Kirchhoff Institute for Physics, University of Heidelberg, Germany [Rev. Sci. Instrum. 82:093701, 2011]. To overcome the limitation of fluorescence microscopes in anisotropic optical resolution or point localization precision micro-axial tomography was used which allowed object tilting on the microscope stage and led to an improvement in localization precision and spatial resolution. A glass fiber was placed in the object space of the microscope lens and its rotation controlled by a miniaturized stepping motor. By Test particles were fixed onto the glass fiber, optically localized with high precision, and automatically rotated to obtain views from different perspective angles from which distances of corresponding pairs of objects were determined. From these angle dependent distance values, the real 3D distance was calculated with a precision in the ten nanometer range (corresponding here to an optical resolution of 10-30 nm) using standard microscopical equipment. As a proof of concept, the spindle apparatus of a mature mouse oocyte was imaged during metaphase II meiotic arrest under different perspectives.

ULTRAPATH XVI: Conference on Diagnostic Electron Microscopy, Basic Research and Oncology, Regensburg, Germany www.ultrapath.org 14th International Congress of Histochemistry and Cytochemistry, Kyoto, Japan www.acplan.jp/ichc2012/

SEPTEMBER 2012 16-21

A. Beltrán and colleagues at the Department of Materials Sciences, University of Cadiz, Spain, report that three dimensional atom tomography resolves the quantum ring morphology of self-assembled GaSb buried nanostructures [Ultramicroscopy 111(8):10731076, July 2011]. Unambiguous evidence of ring-shaped self-assembled GaSb nanostructures grown by molecular beam epitaxy is presented on the basis of atom-probe tomography reconstructions and darkfield transmission electron microscopy imaging. From atom-probe tomography compositional distribution has been obtained. The GaAs capping process causes a strong segregation of Sb out of the center of GaSb quantum dots, leading to the self-assembled GaAsxSb1-x quantum rings of 20-30 nm in diameter with x~0.33.

OCTOBER 2012

Focus on Microscopy 2012; 25th International Conference on 3D Image Processing in Microscopy; 24th International Conference on Confocal Microscopy, Singapore www.focusonmicroscopy.org MRS 2012 Spring Meeting and Exhibition, San Francisco, CA, USA www.mrs.org/spring2012 Analytica 2012: 23rd International Trade Fair for Laboratory Technology, Analysis and Biotechnology and analytica Conference, Munich Trade Fair Centre, Munich, Germany www.analytica.de

JUNE 2012

Reader Enquiry form

2-6

9-13 11-13

6-10

MAY 2012

21-25

3 Introduction to SEM and EDS for the New Operator 4-8 SEM and X-Ray Microanalysis 11-14 Focused Ion Beam: Instrumentation and Applications 11-15 Problem Solving with SEM, X-Ray Microanalysis and Electron Backscatter Patterns 11-15 Quantitative X-Ray Microanalysis: Problem Solving with EDS and WDS Techniques 11-15 Scanning Transmission Electron Microscopy: From Fundamentals to Advanced Applications Abercrombie Cell Biology Symposium, Oxford, UK BSCB: www.bscb.org CARS 2012: Computer Assisted Radiology and Surgery, 26th Intl Congress and Exhibition, Joint Congress of CAR / ISCAS / CAD / CMI / EuroPACS, Congress Palace, Pisa, Italy www.cars-int.org

JULY 2012

MARCH 2012 11-16

literature highlights

AUGUST 2012

APMC 10: 10th Asia-Pacific Microscopy Conference; ICONN 2012: International Conference on Nanoscience and Nanotechnology; ACMM 22: Australian Conference on Microscopy and Microanalysis, Perth, Australia www.apmc-10.org www.iconn-2012.org www.acmm-22.org 56th Biophysical Society Annual Meeting, San Diego, California, USA www.biophysics.org/2012meeting/Main/tabid/2386/Default.aspx

APRIL 2012

27-30

JANUARY 2012 4-7

25-27

4-6

Functional Optical Imaging, Univeristy of Nottingham Ningbo Campus, China www.nottingham.ac.uk/ibios/index.php?page=foi-11 American Society for Cell Biology 51st Annual Meeting, Denver, Colorado, USA www.ascb.org

FEBRUARY 2012

L I T E R AT U R E H I G H L I G H T S

dates for the diary

filament, and the surface pattern of the filament (SP) reflecting intramolecular domain mobility of the actin monomers in the filament. EM images were processed by morphological filtering followed by box-counting to calculate the fractal dimensions for WP (DWP) and SP (DSP). The result indicated that DWP was larger than DSP irrespective of the state of the filament (myosin-free or bound) and that both parameters for myosin-bound filaments were significantly larger than those for myosin-free filaments. This work is the first quantitative insight into how conformational disorder of actin monomers is correlated with the myosin-induced increase in flexibility of actin filaments along their length.

this mode. They present examples of how aberrations change the probe shape in three dimensions, and it was found that off-axial aberrations may need to be considered for focal series of large areas. It was shown that oversized or annular apertures theoretically improve the vertical resolution for three-dimensional imaging of nanoparticles. When imaging nanoparticles of several nanometers in size, regular scanning transmission electron microscopy can thereby be optimized such that the vertical full-width at half-maximum approaches that of the aberration-corrected STEM with a standard aperture.

% $

# !

A technique for the use of two fluorophores in stimulated emission depletion (STED) microscopy of living cells is reported by Patrina Pellett and co-workers at the Department of Cell Biology, Yale School of Medicine, CT [Biomedical Optics Express 2(8):2364-2371, 2011]. Current applications of STED microscopy have been limited to single colour imaging of living cells and multicolour imaging in fixed cells. However, to study active processes, such as protein interactions, a two-colour STED imaging technique is needed in living cells. This was achieved for the first time by the authors: the key to their success was in overcoming the challenges in labeling target proteins in living cells with dyes optimal for two-colour STED microscopy. By incorporating fusion proteins, the researchers were able to improve the targeting between the protein and the dye, effectively bridging the gap. This allowed the researchers to achieve resolutions of 78 nm and 82 nm for 22 sequential twocolour scans of epidermal growth factor and its receptor in living cells.

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

Editor: Dr Julian P. Heath editor@microscopy-analysis.com Managing Editor: Nick J. Taylor ntaylor@wiley.com Assistant Editor: Samantha Moore samamoore@wiley.com EDITORIAL BOARD Jim Bentley, Oak Ridge National Lab, TN, USA Ed Boyes, University of York, UK Ray Carpenter, Arizona State University, AZ, USA Christian Colliex, CNRS Lab.de Physique des Solides, France Alby Diaspro, University of Genoa, Italy Peter Hawkes, CNRS, Toulouse, France Colin Humphreys, University of Cambridge, UK Cornelis van Noorden, University of Amsterdam, The Netherlands Jim Pawley, University of Wisconsin, WI, USA John Spence, Arizona State University, AZ, USA Nestor Zaluzec, Argonne National Lab, IL, USA NON-USA returns should be sent to Microscopy and Analysis, Reader Services, John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, United Kingdom US POSTMASTER: Send address corrections to Microscopy and Analysis, Reader Services, c/o Mercury Airfreight International Ltd, 365 Blair Road, Avenel NJ 070019871 USA. Periodicals Postage Paid at Rahway NJ. ©2011 John Wiley & Sons, Ltd. While every effort is made to ensure accuracy, John Wiley & Sons, Ltd and its agents cannot accept responsibility for claims made by contributors, manufacturers or advertisers. Search Microscopy and Analysis

@ microscopyviews

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

DIARY

dates for the diary NOVEMBER 2011 8-11 10 12-16 14-18 16 22-23 28-2/12

Gatan EELS School, Austrian Centre for Electron Microscopy and Nanoanalysis, TU Graz, Austria www.felmi-zfe.tugraz.at www.gatan.com/company/news/news08291102.php Electron Microscopy in Materials Science, Eindhoven University of Technology, The Netherlands http://nvvmmaterials2011.chem.tue.nl/ Neuroscience 2011: Washington DC, USA www.sfn.org NvVM European School on Nanobeams , Centre de Recherche Public Gabriel Lippmann, Belvaux, Luxembourg www.nanobeams.org Cryo Microscopy Group Annual Meeting, Boots Science Building, School of Pharmacy, University Park, Nottingham University, UK www.cryomicroscopygroup.org.uk/CMG2011.html Bruker Scanning Probe Microscopy Conference and Usersâ&#x20AC;&#x2122; Meeting, University of Manchester, UK www.bruker-axs.com drew.murray@bruker-nano.com 2011 MRS Fall Meeting, Boston, Massachusetts, USA www.mrs.org

DECEMBER 2011 3-4 3-7

25-27 27-30

JULY 2012 2-6 2-6 4-6 9-13 11-13

JANUARY 2012 4-7

Winter School on High-Resolution Electron Microscopy, Arizona State University, Tempe, AZ, USA wintershcool@asu.edu http://le-csss.asu.edu/winterschool

FEBRUARY 2012 5-9

25-29

29-2/8

18-23

Pittcon 2012, Orlando, FL, USA www.pittcon.org Course in Cryotechniques for Electron Microscopy, Rothamsted Research, Harpenden, UK www.rms.org.uk/events/Forthcoming_Events/CoolRunnings

APRIL 2012 1-4

9-13 17-20

MAY 2012 9-11 21-25 24-25

JUNE 2012 3-15

Microscopy School, Lehigh University, Bethlehem, PA, USA Sharon Coe: sharon.coe@lehigh.edu www.lehigh.edu/microscopy

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

AUGUST 2012 6-10 26-30

SEPTEMBER 2012 16-21

MARCH 2012 11-16

European Microscopy Congress, Central Convention Complex, Manchester, UK www.rms.org.uk

OCTOBER 2012 13-17

Neuroscience 2012, New Orleans, Louisiana, USA www.sfn.org

NOVEMBER 2012 26-30

2012 MRS Fall Meeting & Exhibition, Boston, Massachusetts, USA www.mrs.org/fall2012

DECEMBER 2012 15-19

ASCB 52nd Annual Meeting, San Francisco, CA, USA www.ascb.org

MARCH 2013 17-21

Pittcon 2013, Philadelphia, PA, USA www.pittcon.org

APRIL 2013 1-5

2013 MRS Spring Meeting & Exhibition, San Francisco, CA, USA www.mrs.org/spring2013

AUGUST 2013 4-8

Microscopy & Microanalysis 2013, Indianapolis, IN, USA www.microscopy.org

NOVEMBER 2013 9-13

Neuroscience, San Diego, California, USA www.sfn.org

See the Microscopy and Analysis online Diary for a full listing. Entries for the January issue are due 1 December. Email to: editor@microscopy-analysis.com

CIRCLE NO. 3

ONLINE: www.microscopy-analysis.com

Optical Properties at the Nanoscale

2 µm

5 µm

1 µm Top left: Top right:

Dislocations and growth zonation revealed in free standing GaN wafer by panchromatic imaging. Surface plasmon resonance modes along vertices and at apex of pyramid structures revealed using monochromatic imaging. Pyramids formed by 200nm Au film deposited on patterned substrate. Overlay of secondary electron image (grey) and, 650nm (green) and 750nm (red) monochromatic images. Bottom left: Irregularity in emission of GaAs nanowires. In-lens secondary electron image overlaid with panchromatic CL image (green); images acquired simultaneously. Bottom right: Four AlGaN concentrations revealed in graded AlxGa1-xN on GaN film using spectrum-linescan of sample cross section. Blue shift in spectrum from 365 to 320nm associated with change in Al fraction. Spectra acquired at rate of 20 spectra/s.

www.gatan.com

MonoCL 4

™

High Resolution Cathodoluminescence Imaging and Spectroscopy ANALYTICAL TEM DIGITAL IMAGING SPECIMEN PREPARATION TEM SPECIMEN HOLDERS SEM PRODUCTS SOFTWARE X-RAY MICROSCOPY

Complement Confocal with Ultra Resolution

Top: A 3D reconstruction of a dendrite from a 15,625 μm³ (25 x 25 x 25 μm) volumetric data set containing 500 serial images of mouse cerebellum generated by Gatan 3View®. Dendrite structure (green), buttons (yellow), and vesicles (red). Bottom Left: Confocal image of a dendrite. Middle left: 3View® image showing wire frame traces. Middle right: Wire frame traces rendered into a volumetric model. Bottom right: Ultra resolution dendritic spine model with synapses. Sample courtesy of Tom Deerinck and Dr. Mark Ellisman, National Center for Microscopy and Imaging Research, University of California, San Diego. Serial images were segmented using Imaris to create a 3D model of a neuron of interest.

3View

Serial Block Face Imaging in the SEM

www.gatan.com

ANALYTICAL TEM DIGITAL IMAGING SPECIMEN PREPARATION TEM SPECIMEN HOLDERS SEM PRODUCTS SOFTWARE X-RAY MICROSCOPY

Please visit us at the Society for Neuroscience 41st Annual Meeting and the 2011 American Society for Cell Biology Annual Meeting or www.gatan.com/3View

CIRCLE NO. 4

ONLINE: www.microscopy-analysis.com

Laser Free Confocal Microscopy Andor’s Revolution DSD is an innovative imaging technology that brings an affordable confocal solution to your laboratory, offering you less dependency on laser-based solutions often restricted to core facilities. Whilst laserfree, the Revolution DSD can still achieve the optical sectioning you expect of a complex laser scanning confocal system, but with low maintenance costs.

Features & Benefits • Highly cost effective • Excellent confocality • Unique design for easy filter exchange • Affordable for individual labs • Real-time control and viewing • Suitable for live and fixed specimens • High throughput

“The key benefit is that at a relatively low cost we have access to a powerful microscopy system that allows optical, wide field and confocal fluorescence in combination with our TIRF and Raman microscopy. In the future we can easily change the system to a different excitation emission combination - something that would be prohibitively expensive with lasers” Dr. Wesley R. Browne, University of Groningen

www.andor.com/dsd

CIRCLE NO. 5

Revolution DSD

ONLINE: www.microscopy-analysis.com

Simply Confocal

Taklo_Layout_Final_Euro_CMYK_ArticleTemplate_Jan2008.qxd 10/25/2011 9:48 AM Page 9

PFIB

MICROELECTRONICS

Bonding and TSV in 3D IC Integration: Physical Analysis with a Plasma FIB Maaike M. V. Taklo,1 Armin Klumpp,2 Peter Ramm,2 Laurens Kwakman 3 and German Franz 3 1. SINTEF, Oslo, Norway. 2. Fraunhofer EMFT, Munich, Germany. 3. FEI Company, Eindhoven, The Netherlands BIOGRAPHY Maaike M. V. Taklo received her PhD in physical electronics from the University of Oslo in 2002 for her thesis entitled ‘Wafer bonding for MEMS’. From 1998 until 2010 she was employed at the Department of Microsystems and Nanotechnology within SINTEF ICT in Norway where she worked on MEMS processing and was responsible for their wafer level bonding activities. Maaike is now a senior scientist at SINTEF ICT at the Department of Instrumentation and is the group leader for ‘Advanced Packaging and Interconnects’.

ABSTRACT 3D integration schemes connect stacked integrated circuits using through silicon vias (TSV) and special bonding techniques. Physical characterization of these TSVs and bonds is essential, but their relatively large size (tens or hundreds of micrometers) requires prohibitively long milling times in the conventional focused ion beam (FIB) systems typically used for this work. A new plasma-based FIB system can remove material more than 20 times faster, providing the speed and precision required to ensure robust processes and reliable products.

KEYWORDS focused ion beam, scanning electron microscopy, plasma ion source, ion beam milling, 3D integration, through silicon vias

INTRODUCTION 3D integration schemes, which stack integrated circuits and other microelectronic or MEMS devices and interconnect them using through silicon vias (TSV), are likely to be the next revolution in electronic fabrication. They can be used to continue the increases in speed and density of microelectronic systems described by Moore’s Law (More Moore), but they may offer even greater benefits when used to connect devices of different technologies (More than Moore), packing more performance and functionality into smaller volumes. In either case, the ability to physically characterize the TSVs and mechanical bonds used in 3D integration is essential for developing robust manufacturing processes and fabricating reliable products. Focused ion beam (FIB) systems have long provided physical analysis in the manufacture of integrated circuits, but conventional FIB cannot remove material fast enough to analyze these relatively large structures used in 3D integration. The launch of a new plasma-based FIB system now provides the speed and precision needed to develop and deploy these exciting new technologies.

JEMSiP_3D The Joint Equipment and Materials for Systemin-Package and 3D Integration (JEMSiP_ 3D) is a project undertaken by a consortium of European manufacturers to validate technological

solutions for the fabrication of high valueadded heterogeneous components and systems including memories, logic, sensors, actuators and wireless communications. Participants in the program include material providers, laboratories, research centers and manufacturers of equipment, components and systems. The project is structured around five themes: Methodology and evaluation tools to integrate elementary components in 3D systems. 3D technologies and integration processes onto materials and non-silicon substrates. 3D technologies and integration processes onto silicon, using processes closely deriving from microelectronics. Reliability methodologies and analysis for integrated 3D systems. Performance evaluation and equipment validation for volume production equipment and generic manufacturing. SINTEF, The Fraunhofer Institute and FEI are participants in JEMSiP_3D, and the work presented here was funded in part by the project.

· · · · ·

3 D I N T E G R AT I O N While the performance and productivity of microelectronics have increased continuously over more than four decades due to the enormous advances in lithography and device technology, it has now become questionable if advances in these areas alone will be able to

ACKNOWLEDGEMENTS A part of this work has been performed in the project JEMSiP_3D, which is funded by the public authorities in France, Germany, Hungary, The Netherlands, Norway and Sweden, as well as by the ENIAC Joint Undertaking.

A U T H O R D E TA I L S Dr Maaike M. Visser Taklo, SINTEF ICT, Department of Instrumentation, PO Box 124 Blindern, N-0314 Oslo, Norway Tel: +47 2206 7300 Email: MaaikeMargrete.VisserTaklo@sintef.no www.sintef.no Microscopy and Analysis 25(7):9-12 (EU), 2011

Figure 1a: Schematic of a xenon plasma focused ion beam (PFIB) system. A PFIB uses an inductively coupled plasma to deliver high beam current. The source is larger than a liquid metal ion source (LIMS), but delivers a more collimated beam, enabling better beam spot performance at high beam currents. M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

Taklo_Layout_Final_CMYK_ArticleTemplate_Jan2008.qxd 10/20/2011 11:16 AM Page 10

Figure 1b: The PFIB maintains excellent spot size performance over a broad range of beam currents. overcome the predicted performance and cost problems of future IC fabrication. The ITRS roadmap predicts 3D integration as a key technology to overcome this so-called ‘wiring crisis’ and the solution will most likely be based on TSV technology. The most promising 3D integration schemes currently under consideration involve the vertical stacking of integrated circuits and other devices. These schemes vary in their details but all must solve two central problems: how to bond the integrated layers together and how to create electrical connections among them. Bonding and TSV technologies each have their own unique set of considerations which often center around how the structure will hold up during subsequent processing, such as the addition of another layer: Will the stresses induced by additional thermal processing cause debonding or shifting of the existing bonds? Will the stress and strain cause cracks or delamination in the TSVs? What are the best materials and processes to use to minimize these negative effects?

Figure 1c: At high beam currents the PFIB can remove material twenty times faster than a liquid metal ion source.

(LMIS). A LMIS is essentially a point source 50 nm in diameter with a low angular intensity. The Vion system’s plasma source is larger, 15 µm, but has a much higher angular intensity. Because of its small virtual size, the LMIS is easy to focus into a small spot at low beam currents, but at beam currents above 10 nA spherical aberration effects severely degrade performance. The plasma source can deliver currents in excess of a µA (>20⫻ greater than a typical LMIS based system) while still maintaining a well focused beam. Since material removal rates are primarily a function of beam current, the PFIB has an advantage of 20⫻ or more over conventional FIB at high currents, while still preserving excellent milling precision and imaging resolution at low beam currents.

The xenon ion beam emitted by the plasma source has high sputtering yield, high brightness and low energy spread. In addition, by introducing various gases, the PFIB can selectively etch specific materials or deposit patterned conductors and insulators (similar to conventional FIB systems). The plasma source also offers the potential to use different ion species to enhance performance in specific applications.

C U R TA I N I N G The difference in FIB milling rates of the various materials present in a device (Cu, Si, Sn, dielectrics, polyimides and mold compounds) can cause ‘curtaining’ when milling cross-sections. This milling artifact can make detailed

· · ·

PLASMA FOCUSED ION BEAM FIB systems, which use an ion beam to cut and image cross sections through subsurface structures with nanoscale precision and imaging resolution, have long been a mainstay of physical analysis for integrated circuits. Although the structures used in 3D integration can be expected to decrease in size as the technologies evolve, they are much larger than the dimensions of the transistors and interconnects used in current integrated circuits, and the cutting speed of FIBs designed for ICs is generally inadequate for TSVs and bonding structures. A typical 10 µm ⫻ 10 µm IC crosssection requires the removal of 1000 µm3 of material and takes a few minutes. A 100 µm ⫻ 100 µm TSV cross-section requires the removal of 1,000,000 µm3 of material and would take most of a day with conventional FIB. The Vion PFIB system (FEI Company, Hillsboro, Oregon, USA) uses an inductively coupled plasma source [1-3] (Figure 1) to provide material removal rates 20⫻ faster than conventional FIBs that use liquid metal ion sources 10

Figure 2: Curtaining artifacts (upper left), caused by variations in milling rate for different materials, can be effectively suppressed (right) by rocking the sample to mill in a sequence of alternating angles (lower left).

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

Taklo_Layout_Final_Euro_CMYK_ArticleTemplate_Jan2008.qxd 10/25/2011 9:53 AM Page 11

PFIB analysis of the structures difficult or even impossible. Figure 2 shows typical curtaining effects on the silicon substrate as well as the TSV itself, caused by milling through the overlying rough poly crystalline metal film. These curtaining effects can be effectively suppressed by rocking the sample during the FIB milling process. Milling in a sequence of alternating incidence angles creates a clean cross section free of curtaining artifacts without the need for timeconsuming low current polish steps.

MICROELECTRONICS

E X A M P L E S O F A P P L I C AT I O N S O F PLASMA FOCUSED ION BEAM Through Silicon Vias TSVs are themselves subject to a number of effects that can result in defects and failures. For example, the large differences in thermal expansion between copper via fill and the surrounding silicon substrate can cause cracking within the copper and delamination from the via sidewall during thermal processing. ‘Keyholing’ results from incomplete filling of vias (Figure 3).

Figure 3: (a, b) Differing thermal expansion between copper via fill and silicon substrate caused delamination shown in this via before (a) and after (b) annealing. (c) Keyholing occurred when this via was not filled completely with tungsten.

Solid Liquid Interdiffusion Bonding One of the most difficult issues to address is the behavior of bonds between chips during subsequent processes (Figure 4). For example, it is critical that a bond between the first chips in the stack not be disturbed by the subsequent bonding of an additional chip. Solidliquid-interdiffusion (SLID) [4] is a unique direct metal bonding technology that avoids remelting of existing bonds during the formation of new bonds by using high melting intermetallic phases. During bond formation, solid metal diffuses into the liquid phase of a lower melting metal resulting in high melting point final phase that remains solid during subsequent bond forming processes. Anisotropic Conductive Adhesives Anisotropic conductive adhesives (ACA) can be used [5] to bond wafers together physically and electrically using an organic bonding compound (benzocyclobutene, BCB) filled with 4-µm sized metal covered polymer spheres (MPS). The BCB assures mechanical strength whereas the MPS provide the required electrical conductivity at interconnection points. The concentration of MPS must be high enough to ensure good electrical contact between opposed pads and at the same time low enough to guarantee electrical insulation where pads are not present. To study the bonding in detail, samples were cleaved, then milled with the plasma-FIB to reveal the bonding region and finally inspected with plasma-FIB imaging. The plasma-FIB milling speed makes it possible to prepare the sample (~200 ⫻ 50 ⫻ 600 µm3 material removed) within 30 minutes. The metal layer covering the polymer spheres could be observed at the bonding interface with sufficient resolution to estimate both the local MPS density and their compression state between the bond pad metal layers. In Figure 5 the bonding process is illustrated in the top

Cu Cu 3Sn Cu 6Sn5 FIB debris

Figure 4: The void between these pads is the result of an incomplete SLID bonding process. The various intermetallic phases are clearly visible above, below and to the right of the void. M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

Taklo_Layout_Final_CMYK_ArticleTemplate_Jan2008.qxd 10/20/2011 11:16 AM Page 12

images and the bottom plasma-FIB images show details of the bonding interface and compressed spheres. 3D Test Chip The 3D integrated reliability test chip shown in Figure 6 is a 3-level-stack with a modular layout designed to permit evaluation of assembly processes between two initial layers and, subsequently, the effects of adding a third layer [6]. The PFIB can mill a cross section through the entire three layer stack showing critical details of both upper and lower bonding regions and the complete TSV through the middle layer.

CONCLUSIONS By combining high-speed milling and deposition with precise control and high quality imaging, the plasma focused ion beam provides critically needed physical analysis for TSV and bonding processes that are essential to current 3D integration schemes. At high beam currents, cross-sections with dimensions of hundreds of micrometers can be completed in less than an hour, fast enough to provide effective feedback on process performance. At low beam currents, the same system delivers high resolution imaging for accurate structural analysis. The PFIB provides an effective, practical tool for a variety of 3D integration applications, including failure analysis of bumps, wire bonds, TSVs, and stacked die; site specific removal of package and other materials to enable failure analysis and fault isolation on buried die; circuit and package modifications to test design changes without repeating the fabrication process or creating new masks; process monitoring and development at the package level; and defect analysis of packaged parts and MEMS devices.

Figure 5: Anisotropic conductive adhesives provide mechanical bonding and electrical conductivity. (a,b) Images show metal coated spheres before mixing with BCB (a), and a schematic of how TSVs can be electrically connected to pads on another wafer using BCB filled with such spheres (b). (c-f) The lower four images are a clockwise sequence of increasing magnification with the compressed metal coated spheres clearly visible in the two bottom images.

REFERENCES 1. Smith, N. S., Skoczylas, W. P., Kellogg, S.M., Kinion, D.E., Tesch, P.P., Sutherland, O., Aanesland, A., Boswell, R.W. High Brightness Inductively Coupled Plasma Source for High Current Focused Ion Beam Applications. J. Vac. Sci. Technol. B24(6):2902-2906, 2006. 2. Kellogg, M., Schampers, R., Zhang, S.Y., Graupera, A.A., Miller, T., Laur, W.D., Dirriwachter, A.B. High Throughput Sample Preparation and Analysis using an Inductively Coupled Plasma (ICP) Focused Ion Beam Source. Microsc. Microanal. 16(Suppl 2):222-223, 2010. 3. Kwakman, L., Franz, G., Taklo, M. M. V., Klumpp, A., Ramm, P. Characterization and Failure Analysis of 3D Integrated Systems using a novel plasma-FIB system. Proc. International Conference on Frontiers of Characterization and Metrology for Nanoelectronics, Grenoble, France, 2011. 4. Ramm, P. Method of making a three-dimensional integrated circuit. US Patent 5,563,084; P. Ramm, A. Klumpp. Method of vertically integrating microelectronic components. US Patent 6,548,391. 5. Taklo, M. et al. Anisotropic Conductive Adhesive for Waferto-Wafer Bonding, Proceedings of 7th Intl Conference and Exhibition on Device Packaging, March 2011. 6. Ramm, P., Klumpp, A., Franz, G., Kwakman, L. Failure Analysis and Reliability of 3D Integrated Systems. Proc. IMAPS Device Packaging Conf., Scottsdale, Arizona, 2011. ÂŠ2011 John Wiley & Sons, Ltd

Figure 6: The high milling speed of PFIB permits cross-sections through the full three layer stack of the test chip, revealing both upper and lower bonding regions and the entire TSV.

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

JSM-7800F FEG Scanning Electron Microscope

Featuring our latest Super Hybrid Lens

CIRCLE NO. 6

ONLINE: www.microscopy-analysis.com

Search and observe specimens with ease, and with zero rotation.

Semi in-lens

Giving you better resolution at the same working distance.

Field free objective lens

Improved performance for imaging magnetic materials.

Superior low kV resolution

Producing better images of insulating materials.

Gentle Beam mode

Extremely high resolution at very low kV

Optional Electron Detectors

Available with four different Electron Detectors, all operating simultaneously.

www.jeol.com +44 (0)1707 377117 euro.sales@jeol.com www.harlequincreative.com

The future of TEM is here…

The HT7700 Digital TEM

Simple & seamless imaging up to 64mp.

The first TEM designed from the ground up to be fully digital. • High sensitivity digital imaging at all times - with fully-integrated screen and capture cameras

Patented dual focal length objective lens – high contrast mode (unstained sciatic nerve).

• Quick and easy imaging at up to 64 megapixels - with Hitachi’s precise multi-frame capture system • Perfectly suited to electron tomography - with Hitachi’s calibration-free and marker-less tilt acquisition system

• The highest contrast and resolution in one instrument – with the patented dual-focal-length objective lens

Hitachi High-Technologies Europe GmbH Tel. +44 (0)1628 585 200 Tel. +49 (0)2151 6435310 eminfo@hht-eu.com www.hht-eu.com

CIRCLE NO. 7

• Flexibility assured - options for 3d visualisation, cryo imaging, EDX microanalysis and more If you utilise TEM for biomedical research, pathology, neuroscience, pharmaceuticals, nanotechnology, polymers or plant science contact us now to see how the HT7700 can transform your lab.

ONLINE: www.microscopy-analysis.com

Hitachi’s unique tomographic algorithms (left image) ensure precise reconstruction compared to conventional methods (right image).

Patented dual focal length objective lens – high resolution mode (carbon nanotube)

Sha_Layout_Final_Euro_CMYK_ArticleTemplate_Jan2008.qxd 10/25/2011 9:56 AM Page 15

A N A LY S I S

STEELS

Fracture Surface, Impact Energy and Hardness of Ni-Free High-Mn Steels Wei Sha,1 H. H. Haji Talib,1 Eric A. Wilson,2 Raj Rajendran,3 Savko Malinov,4 Harvey R. Charlesworth,5 Lee Ibbitson6 1. School of Planning, Architecture and Civil Engineering, Queen’s University Belfast, UK. 2. Faculty of Arts, Computing, Engineering and Sciences, Sheffield Hallam University, UK. 3. School of Mechanical and Building Sciences, B. S. Abdur Rahman University, India. 4. School of Mechanical and Aerospace Engineering, Queen’s University Belfast, UK. 5. London & Scandinavian Metallurgical Co. Ltd., UK. 6. Tata Steel Speciality, UK BIOGRAPHY Wei Sha obtained a BEng at Tsinghua University in 1986. He was awarded in 1992 a PhD by Oxford University and in 2009 a DSc by Queen’s University Belfast. He previously worked at Imperial College and Cambridge University. He is presently Professor of Materials Science, with research interests in phase transitions and SEM.

ABSTRACT Two manganese steels were investigated: Fe-19.7%Mn (VM339A) and Fe-19.7%Mn stabilized with 0.056%C, 0.19%Ti and 0.083%Al (VM339B). The toughness of VM339A was higher than VM339B, but VM339B had higher hardness. Tempering does not affect the toughness of the alloys. SEM images of the fracture surface for both the alloys revealed ductile fractures. A further alloy with a lower manganese content, Fe-8.46%Mn-0.24%Nb-0.038%C, and thus even lower cost than the conventional 3.5Ni cryogenic steel, was tested for its impact toughness after heat treatment at 600°C, giving promising results.

KEYWORDS scanning electron microscopy, hardness measurement, mechanical characterization, steel, fracture

A U T H O R D E TA I L S Professor Wei Sha, School of Planning, Architecture and Civil Engineering, Queen’s University Belfast, Belfast BT7 1NN, UK Tel: +44 28 90974017 Email: w.sha@qub.ac.uk

Microscopy and Analysis 25(7):15-19 (EU), 2011

INTRODUCTION Nickel raises the yield strength of iron, as found many decades ago by Roberts and Owen [1] by measuring the strength of many Fe-Ni alloys. Nickel also lowers the ductile brittle transition temperature (DBTT, cleavage) of iron, as proved by Floreen et al. [2] by measuring the impact properties of Fe-Ni alloys with different Ni contents. Both results have been verified in the extensive steel research that followed, so nickel is an established alloy method of raising strength and increasing toughness. As shown by Avery and Parsons [3] with some real examples, nickel has been widely used in cryogenic steels, but the high cost of nickel now demands a second thought on the actual amounts of this element required in these steels. Oshima et al. [4] have vividly illustrated the sharp rise of nickel price on the London Metal Exchange. Oshima et al. [4] also explored the ways of nickel saving, by a detailed property comparison between low nickel stainless steels and conventional stainless steels. Based on their findings, nickel-free high-manganese steels could have a combination of good tensile strength and ductility and so could provide a great potential in applications for structural components in industry. In recent years, numerous attempts have been made to improve the performance of manganese steels by modifying their composition and by applying heat or thermomechanical treatments. For example, by examining the microstructure and the performance [5] it was concluded that the capability of work hardening and impact abrasion resistance were enhanced greatly after rolling. The cold asynchronous rolling technique taken was only one way of achieving potential property improvement. In their classical work, Holden et al. [6] reported that 15-20%Mn steels, with around 0.02% carbon, contain mostly epsilon marten-

site and austenite, as measured by optical microscopy. Nikbakht et al. [7] showed that a stabilized alloy 193 (Fe-8%Mn with 0.17%Ti, 0.18%Al and 0.018%C) exhibited brittle cleavage on ice brine quenching from 900°C, based on clear fracture surface examination. This is in agreement with a much earlier but convincing fracture profile analysis [8] and thermodynamic calculations on alloy 193 showed that there is less than 0.03 ppm N in solid solution. On tempering alloy 193 for 6 min at 450°C, the DBTT rose from 27 to 125°C, giving intergranular failure. This indicated that embrittlement is due to segregation of Mn per se to prior austenite grain boundaries. In the last five years, there have been many creditable studies in related subjects. For example, Vaynman et al. [9] have found that the increase in strength in a low-carbon, FeCu–based steel was derived from a large number density of copper-iron-nickel-aluminummanganese precipitates, characterized by state-of-the-art atom-probe tomography. Sathiya et al. [10] found using cross-section imaging that the shape of the fusion zone, generally characterized by a few geometrical features, namely bead width, bead height and depth of penetration, depended upon a number of parameters such as gas flow rate, voltage, travel speed and wire feed rate. Recent research in ferrous materials in general has been well reviewed in a recent book by Berns and Theisen [11]. However, there has been little research reported on the steels studied here.

M AT E R I A L S A N D M E T H O D S Steels In the present research, two cryogenic steels with around 20% manganese were investigated: Fe-19.7%Mn (VM339A) and Fe19.7%Mn stabilized with 0.056%C, 0.19%Ti

Heat Treatment

VM339A

VM339B

850oC 1h, air cooled

222±2

281±12

850oC 1h, water quenched (WQ)

226±2

288±2

WQ + 450oC 0.1 h

230±2

299±11

WQ + 450oC 1 h

263±17

–

WQ + 450oC 10 h

270±7

294±9

WQ + 450oC 100 h

274±9

–

Table 1: Hardness (HV1) of the experimental alloys under various heat treatment conditions.

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

CALL FOR PAPERS PERTH CONVENTION & EXHIBITION CENTRE, WESTERN AUSTRALIA, AUSTRALIA SUNDAY 5TH TO THURSDAY 9TH FEBRUARY 2012 On behalf of the Organising Committee it is our pleasure to extend an invitation to the 10th Asia-Pacific Microscopy Conference (APMC-10), the International Conference on Nanoscience and Nanotechnology (ICONN 2012) and the 22nd Australian Conference on Microscopy and Microanalysis (ACMM-22) to be held in Perth, Western Australia, 5th – 9th February 2012. The Event will include Short Courses, Workshops and a concurrent major Equipment Exhibition. The combined event will be the largest microscopy and nanotechnology-related event in Australiaʼs history. Over 2,000 delegates, from more than 30 nations, are expected to provide a unique science and technology forum. The traditional scientific programs of each Conference will be run in parallel, with some joint sessions. Delegates may attend any they choose through the single registration process. The Exhibition and social events will be fully shared, to enable our communities to network extensively. The Event is being conducted under the auspices of the Council of Asia-Pacific Societies for Microscopy (CAPSM), The Australian Nanotechnology Network (ANN), the Australian Microscopy and Microanalysis Society Inc (AMMS) and the International Federation of Societies for Microscopy (IFSM). ANN /ICONN are funding registrations for eligible ANN students and ECR; AMMS and IFSM have travel bursaries for microscopy students and ECR. Confirmed plenary speakers include Professor Knut Urban, Juelich, Germany, joint winner of the 2011 Wolf Prize in Physics. Professor Urban will give the inaugural “Cockayne Lecture”, Professor Frank Caruso, Melbourne, Australia , a 2010 Top 100 most-cited researcher in nanotechnology, and Zhores Alferov , Ioffe Institute, Russia, the winner of the Nobel Prize in Physics, 2000. The Conference is dedicated to the memory of Professor David Cockayne FRS. Brendan Griffin & Lorenzo Faraone Co-Chairs

For more information see: www.apmc-10.org or www.iconn-2012.org

CRITICAL DATES • 31st Oct 2011

CLOSE of:

• 28th Nov 2011

CLOSE of:

- paper submission - scholarship & bursary applications - early registration rates

CIRCLE NO. 8

ONLINE: www.microscopy-analysis.com

Sha_Layout_Final_CMYK_ArticleTemplate_Jan2008.qxd 10/20/2011 9:32 AM Page 17

A N A LY S I S and 0.083%Al (VM339B). The Mn content was selected based around commercial higher manganese content steels, summarised from industrial data by Subramanyam et al. [12], but its particular value was not thought to be critical, if it was, say, 1% higher or lower. Carbon was kept low to improve toughness. Ti and Al were added to tie up residual C, N and O. These alloys cost about half as much as a 3.5%Ni steel. The alloys were austenitised at 850oC, water quenched or air cooled and also tempered for 0.1, 1, 10 or 100 h at 450oC. Tempering in the range of 300-500oC increases the ductile-brittle transition temperature (DBTT), as found by Bolton et al. [13] when measuring this property in a series of Fe-Mn alloys, albeit without detailed microstructural characterisation work back then. Nasim et al. [14] noted a rapid grain boundary segregation of Mn, P, and N, on ageing, based on Auger electron spectroscopy measurements. The control group was in a water-quenched condition. Embrittlement was the most severe at 450oC. Further, as a continuation of a previous work, where Nikbakht et al. [7] identified the embrittlement mechanism in Fe-8%Mn as well as Fe-8%Mn stabilised with Ti and Al, a third alloy was studied, to investigate the effect of Nb. A Fe-8.46%Mn-0.24%Nb-0.038%C alloy was vacuum-melted as a 20 kg cast and upset forged at 1200°C into a 32⫻76 mm plate. This alloy costs about one third of a 3.5%Ni steel. The heat treatment at 600°C on the homogenised alloy was carried out to introduce reverted austenite to improve impact toughness. Excess C over stoichiometry was about 70 ppm, which lowers the DBTT (see Figure 9 of ref. [15]). The alloy was homogenised as two blocks 32⫻60⫻76 mm for 50 h at 1100°C, air cooled, and followed by two different heat treatment routes for each block. The first consisted of 1.5 h at 850°C, air-cooling, and then 4 h at 600°C, air-cooling. The second consisted of 1.5 h at 850°C, water quenching, and then 4 h at 600°C, water quenching. Longitudinal V notch Charpy specimens 5⫻10⫻55 mm were machined from the heattreated blocks. Vickers Hardness and Impact Testing Vickers hardness was measured using a load of 1 kg, at room temperature in the unstrained part of the impact-tested specimens. Impact tests were conducted on a Charpy machine with maximum impact energy of 300 J. Samples of size 5⫻10⫻55 mm with a V-notch were used. The temperatures of impact tests were 20°C, -20°C, -100°C, and -196°C. Testing temperatures below the room temperature were obtained by using a chamber cooled by flow of liquid nitrogen. Scanning Electron Microscopy The steel specimens were polished using a 0.05 µm colloidal suspension of silica after mechanical polishing down to 1 µm. The microstructure of the steels was examined in a Jeol 6500 FEG scanning electron microscope. The SEM was operated at 5-20 kV. The fracture surfaces of the samples were

STEELS

Alloys

Heat Treatment

20oC

-20oC

-100oC

-196oC

VM339A

850oC 1h, air cooled

–

850oC 1h, water quenched (WQ)

WQ + 450oC 0.1 h

–

WQ + 450oC 1 h

WQ + 450 C 10 h

–

WQ + 450oC 100 h

–

850oC 1h, air cooled

850oC 1h, WQ

WQ + 450oC 0.1 h

–

VM339B

WQ + 450 C 1 h

Table 2: Impact energy (in joules J) absorbed by the experimental alloys under various heat treatment and impact test temperatures. examined under the SEM to determine whether the sample surface had brittle cleavage type or ductile type failure.

R E S U LT S A N D D I S C U S S I O N Table 1 shows the hardness values of the experimental alloys VM339A and VM339B under various heat treatment conditions. Alloy VM339B is harder than VM339A because it contains 0.056%C, 0.19%Ti and 0.083%Al. It was found that deep quenching the alloys down to -20, -100 and -196°C does not change the hardness after the specimens were returned to room temperature before the hardness measurements were taken. Table 1 includes the hardness values for both alloys tempered at 450°C with increasing time and then impact tested. In the case of VM339A, the hardness increased significantly after tempering for 1-100 hours, compared to without tempering or tempering for 0.1 h. On the other hand, little change of hardness has been found when the tempering time varies from 1 to 100 h. However, there exists an obvious difference between tempering for 0.1 h and 1 h. There could be two reasons contributing to this effect: 1. The effect of tempering reaches a saturation point after around one hour or some time before one hour. Here, a saturation point means no further increase in hardness with increasing tempering time. 2. Experimental limitations meant that heating for 6 minutes did not lead to effective tempering, because the specimens were sealed in silica tubes in vacuum, inserted into the preheated furnace, and kept for the specified length of time. There might be a heating-up period that could take minutes. Impact testing was carried out on both alloys to estimate the ductile-brittle transition temperature, which gives the change in behaviour from ductile at high temperature to brittle at lower temperature (Table 2). As a general statement, ductile fracture initiates at a particular toughness value Micrographs of fracture surface of alloy

VM339A and VM339B (1 h at 850°C, WQ) impact tested at 20°C are shown in Figures 1 and 2, respectively. The uneven or rough surface of the fracture can be seen at low magnification (Figures 1a and 2a). High magnification shows a ductile type failure for both alloys (Figures 1b and 2b). Tearing and cone shaped dimples were observed in alloy VM339A (Figure 1), which had the higher impact toughness indicating it is more ductile than alloy VM339B. Micrographs of fracture surfaces for both alloys at different conditions and tempering time were examined which showed ductile type fracture with no obvious difference. Micrographs of the surface structure for both alloys at different conditions and tempering time were examined but there were no visible differences in the structures or grainsize. It is possible in Fe-20%Mn to get peculiar stress-strain curves at room temperature, due to gamma and epsilon phases, like in the thermally cycled Fe-8%Mn alloy [16]. Nikbakht et al. [7] summarised the formation of these phases from relevant literature. The Charpy impact test results of the heat treated Fe-8.46%Mn-0.24%Nb-0.038%C alloy are shown in Figure 3. It should be noted that the linear regression lines are used to only illustrate the difference between specimens after the two cooling treatments. The linear regression may or may not represent the real variation of the impact energy with testing temperature. These impact test results show that the water quenched alloy has higher impact energy. The reason may be that air cooling resulted in the formation of precipitates during the slower cooling process, which increases strength but decreases toughness. In the quenched condition, this third alloy had a higher impact energy than both VM339A and VM339B, showing the beneficial effect of Nb stabilisation. In the air cooled condition, however, the Nb-containing alloy had comparable impact energy with VM339A and

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

Applications, Labeling Labeling Strategies Strategies and and Applications, Fluorophores for for Super-Resolution Super-Resolution Fluorophores Great insights can be obtained from conventional fluorescence microscopy, but studying the architecture and protein dynamics of Great insights can be obtained from conventional fluorescence microscopy, but studying the architecture and protein dynamics of sub-cellular compartments can be challenging, since a major portion of information concerning the structural organization is lost sub-cellular compartments can be challenging, since a major portion of information concerning the structural organization is lost due to the light diffraction limit. Several approaches to overcome this limitation have been developed and super-resolution has due to the light diffraction limit. Several approaches to overcome this limitation have been developed and super-resolution has proven an extremely valuable tool. proven an extremely valuable tool. Speakers: Speakers: Dr. Marko Lampe, Leica Microsystems CMS GmbH, Wetzlar, Germany Dr. Marko Lampe, Leica Microsystems CMS GmbH, Wetzlar, Germany Dr. Wernher Fouquet, Leica Microsystems CMS GmbH, Mannheim, Germany Dr. Wernher Fouquet, Leica Microsystems CMS GmbH, Mannheim, Germany

Online Seminar/Webinar on Applications for Super-Resolution Online Seminar/Webinar on Applications for Super-Resolution Register at http://www.microscopy-analysis.com/leicawebinars Register at http://www.microscopy-analysis.com/leicawebinars and join on Tuesday 15th November 2011 at 17:00h (CET), 16:00h (UK) and join on Tuesday 15th November 2011 at 17:00h (CET), 16:00h (UK) Recently, super-resolution microscopy has made its way into research labs and is addressing more and more scientific questions. Recently, microscopy hasand made its how way into research and is addressing more scientific questions. Today, wesuper-resolution will highlight STED and GSDIM show these methodslabs have been optimized in the and triedmore and proven commercially Today, wesolutions will highlight GSDIM and show how these methods have been optimized in the tried and proven commercially available fromSTED Leicaand Microsystems. available solutions from Leica Microsystems. In addition to a review of the principles of method and system architecture, we will expand on specific guidelines for sample prepaIn addition to a fluorophores review of the and principles of strategies. method andWe system architecture, we will expand on specific guidelines for super-resolution sample preparation, suitable labeling will also highlight selected application examples, including ration, fluorophores and labeling strategies. We willelements also highlight selected application examples, super-resolution imagingsuitable of intracellular substructures such as cytoskeleton and associated proteins, nuclear poreincluding complexes and cellular imaging of intracellular substructures such as cytoskeleton elements and associated proteins, nuclear pore complexes and cellular compartments. In particular for STED imaging, we will consider applications requiring multi-color and live imaging. compartments. In particular for STED imaging, we will consider applications requiring multi-color and live imaging. 2 color STED 2 color STED

Image taken with a Leica TCS STED CW Image takenof with a LeicaHeLa TCScells STEDstained CW microscope wildtype microscope of wildtype HeLa cells stained against Histone H3/Chromeo505 and against Histone H3/Chromeo505 and Tubulin/V500. Tubulin/V500. Courtesy: Samples were kindly provided Courtesy: Samples were kindly provided by Active Motif Chromeon by Active Motif Chromeon

GSD application image GSD application image

Ptk2-cells. NPC-staining: anti-NUP153/ ® Ptk2-cells. anti-NUP153/ Alexa FluorNPC-staining: 532 | Microtubule-staining: ® Alexa Fluor 532 | Microtubule-staining: anti-ß-tubulin/Alexa Fluor® 647. anti-ß-tubulin/Alexa Fluor® 647. Courtesy: Wernher Fouquet, Leica Courtesy: Wernher Fouquet, Leica Microsystems in collaboration with Anna Microsystems in Jan collaboration Anna Szymborska and Ellenberg,with EMBL, Szymborska and Jan Ellenberg, EMBL, Heidelberg, Germany Heidelberg, Germany

Register at http://www.microscopy-analysis.com/leicawebinars Register at http://www.microscopy-analysis.com/leicawebinars This webinar will be available on demand under the registration link above. This webinar will be available on demand under the registration link above.

CIRCLE NO. 9

ONLINE: www.microscopy-analysis.com

Sha_Layout_Final_CMYK_ArticleTemplate_Jan2008.qxd 10/20/2011 9:32 AM Page 19

A N A LY S I S VM339B, depending on the testing temperature. The reason is perhaps, as explained earlier, air cooling has more detrimental effect on toughness, most likely related to Nb precipitation.

STEELS

cone-shaped dimple

CONCLUSIONS In conclusion, the mechanical properties and microstructure were investigated to find out the suitability of the nickel-free manganese steels to be used at low temperatures. The experimental results are summarised below. 1. It is clear that VM339A is tougher than VM339B. It has higher impact resistance, for example 61 J at -20°C in the as quenched condition. The alloying elements such as carbon, titanium and aluminium in alloy VM339B make the alloy more brittle. These elements have significant effect on the hardness of the alloy. 2. The tempering at 450oC up to 100 hours does not affect the toughness of the alloys significantly. This is an added advantage when compared with other cryogenic alloys that need a precise heat treatment and tempering. A further study is required to substantiate that the nickel free steels form a potential candidate for cryogenic applications. These alloys are cheaper than the present 9% nickel alloys. It is found from this research that tempering has no effect on the alloys, which may be useful for welding works. Therefore, welding effects can be investigated under hot and cold working condition to study the suitability for the applications.

tearing

Figure 1: Scanning electron microscope images of the fracture surface of alloy VM339A, 850oC for1 h, water quenched, and then impact tested at 20oC: mainly ductile failure, impact energy 71 joules. (a) Low magnification. (b) High magnification.

Figure 2: Scanning electron microscope images of the fracture surface of alloy VM339B, 850oC for1 h, water quenched, and then impact tested at 20oC: mainly ductile failure, impact energy 54 joules. (a) Low magnification. (b) High magnification.

REFERENCES 1. Roberts, M. J., Owen, W. S. The strength of martensitic ironnickel alloys. ASM Trans. Quart. 60:687-692, 1967. 2. Floreen, S., Haynes, H. W., Devine, T. M. Cleavage initiation in Fe-Ni alloys. Metall. Trans. 2:1403-1406, 1971. 3. Avery, R. E., Parsons, D. Welding stainless and 9% nickel steel cryogenic vessels. Welding Journal 74(11):45-50, 1995. 4. Oshima, T., Habara, Y., Kuroda, K. Efforts to save nickel in austenitic stainless steels. ISIJ Int. 47, 359-364, 2007. 5. Qiu, C. M., Wang, Y. F., Yu, J. Effect of asynchronous rolling on wear-resisting performance of high manganese steel. Adv. Mater. Res. 146-147:340-344, 2011. 6. Holden, A., Bolton, J. D., Petty, E. R. Structure and properties of iron-manganese alloys. J. Iron Steel Inst. 209, 721-728, 1971. 7. Nikbakht, F., Nasim, M., Davies, C., Wilson, E. A., Adrian, H. Isothermal embrittlement of Fe–8Mn alloys at 450°C. Mater. Sci. Technol. 26:552-558, 2010. 8. Roberts, M. J. Effect of transformation substructure on the strength and toughness of Fe-Mn alloys. Metall. Trans. 1:3287–3294, 1970. 9. Vaynman, S., Isheim, D., Kolli, R.P., Bhat, S.P., Seidman, D.N., Fine, M.E., 2008. High-strength low-carbon ferritic steel containing Cu-Fe-Ni-Al-Mn precipitates. Metall. Mater. Trans. A 39:363-373, 2008. 10. Sathiya, P., Aravindan, S., Ajith, P. M., Arivazhagan, B., Haq, A. N. Microstructural characteristics on bead on plate welding of AISI 904 L super austenitic stainless steel using gas metal arc welding process. Int. J. Eng. Sci. Technol. 2(6):189-199, 2010. 11. Berns, H., Theisen, W. Ferrous Materials: Steel and Cast Iron, Springer, Berlin, pp. 1-418, 2008. 12. Subramanyam, D. K., Swansiger, A. E., Avery, H. S. Austenitic manganese steels. In Properties and Selection: Irons, Steels, and High-Performance Alloys, vol. 1, ASM Handbook. ASM

Figure 3: Charpy impact energy as a function of specimen temperature at testing for the Fe-8.46%Mn-0.24%Nb-0.038%C alloy. International, Materials Park, OH, pp. 822-840, 1990. 13. Bolton, J. D., Petty, E. R., Allen, G. B. The mechanical properties of ␣-phase low-carbon Fe-Mn alloys. Metall. Trans. 2:2915-2923, 1971. 14. Nasim, M., Edwards, B. C., Wilson E. A. A study of grain boundary embrittlement in an Fe–8%Mn alloy. Mater. Sci. Eng. A 281:56-67, 2000. 15. Wilson, E. A., Ghosh, S. K., Scott, P. G., Hazeldine, T. A.,

Mistry, D. C., Chong, S. H. Low cost grain refined steels as alternative to conventional maraging grades. Mater. Technol. 23:1-8, 2008. 16. Nasim, M., Wilson E. A. The effect of thermal cycling on the impact toughness of an Fe-8Mn alloy. In Phase Transformations, Vol. 1, The Institution of Metallurgists, York, pp. v21-v25, 1979. ©2011 John Wiley & Sons, Ltd

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

For further information: info.osis@olympus-sis.com, www.soft-imaging.net

Multiple TEMs and TEM cameras are available to contend with the many diverse and demanding tasks in todayâ&#x20AC;&#x2122;s life and materials science. Only when the TEM and camera system are paired correctly you will be able to easily complete your tasks and overcome your challenges. All of our side-mounted and bottom-mounted TEM cameras can be easily attached to virtually any TEM. The camera itself, and also most remote-controlled TEMs and stages, can be operated via iTEM â&#x20AC;&#x201C; our TEM imaging platform. With these well aligned TEM image acquisition solutions, your TEM work flow can become the most efficient work flow possible.

CIRCLE NO. 10

TEM camera systems for Life and Materials Sciences

ONLINE: www.microscopy-analysis.com

Aligned with your application on any TEM

Baldwin_Layout_Final_Euro_CMYK_ArticleTemplate_Jan2008.qxd 10/25/2011 9:59 AM Page 21

VPSEM

PLANT SCIENCES

Variable Pressure Scanning Electron Microscopy of Vicia faba Stigmatic Papillae Wen Chen, Fred Stoddard and Timothy C. Baldwin School of Applied Sciences, University of Wolverhampton, UK BIOGRAPHY Timothy Baldwin has a PhD in botany from the University of Reading. After a post-doctoral position at the John Innes Centre, Norwich, he became a lecturer in the Botany Department at Universiti Sains Malaysia. In 1999 he returned to the UK and was appointed a post-doctoral research associate in the Department of Biochemistry at the University of Cambridge. Since 2001, Tim has been senior lecturer in plant sciences at the University of Wolverhampton. His research interests include the role of the plant cell wall in plant growth and development, pollination biology, orchid conservation and plants used in traditional Chinese medicine.

ABSTRACT The cell walls of the stigma and style are important zones for cell-cell recognition, nutrition, guidance and protection of the pollen tube along the transmitting tract. The SEM data presented here was a component of a larger study the main objective of which was to investigate pistil development and pollination in the crop species Vicia faba L. (the faba bean). The data demonstrate that there is a developmentally regulated difference in the structural integrity of the stigmatic surface in autofertile (K25) and autosterile (D07) lines of the faba bean. In the autofertile lines the stigmatic surface ruptures two days prior to flower opening (anthesis), whereas in the autosterile plants the stigma remains intact, until anthesis. The VPSEM technique used in the current study shows this difference to great effect and as such is an invaluable tool for use in plant cell biology.

INTRODUCTION

M AT E R I A L S A N D M E T H O D S

Grain legumes are extremely important in world agriculture yet information on the structure, composition, and functioning of their solid stigma and open style is limited. Global food production is underpinned by plant breeding programmes centred on important crops including the faba bean (Vicia faba L.) (Figure 1), soybean, pea, and the common bean. Understanding the mechanism of pollination in such species enhances the effectiveness of these programmes. The faba bean is particularly suitable for study because it produces numerous large flowers with straight styles that are easy to dissect and is partially dependent upon bees for pollination. For the faba bean there are both autofertile and autosterile lines. Autofertile lines do not require a pollinating insect but do not have very large yields. Autosterile lines do need a pollinating insect and have relatively high crop yields. The aim for plant scientists is to compare autosterile and autofertile lines so that crosses between the two lines can be developed. The overall aim is increased food production. In this article the development of the faba bean stigmatic papillae was observed in the days leading up to anthesis (flower opening) using low-temperature variable pressure scanning electron microscopy (VPSEM).

Preparation of Plant Tissues Stigmas were harvested from both autosterile (D07) and autofertile (K25) lines of Vicia faba in the days leading to anthesis. Samples were rapidly frozen in liquid nitrogen and attached to the cold stage (kept at -20oC) with carbon sticky pads. Scanning Electron Microscopy Plant specimens were examined in a Carl Zeiss EVO LS 15 SEM with variable pressure capability at 20 kV and imaged using a variable pressure secondary electron (VPSE) detector. In addition, the SEM was equipped with a Coolstage from Deben UK Ltd, Suffolk, and the images presented in this note were obtained with the stigmas cooled to approximately -20°C. In order to reduce charging artifacts, a low pressure of about 30 Pa of air was used. This very low pressure is sufficient to compensate for specimen charging and to provide a gas phase scintillation signal for the VPSE detector. The Coolstage, shown in Figure 3, is a Peltier device that is able to reduce the temperature of the specimen. Extended periods of SEM imaging are then possible as hydration of the specimen is maintained. This approach offers an alternative to the full environments SEM if dynamic processes involving water are not required.

KEYWORDS scanning electron microscopy, variable pressure, cryo electron microscopy, plant sciences, pollination

A U T H O R D E TA I L S Dr Timothy C. Baldwin, Senior Lecturer – Plant Sciences, School of Applied Sciences, University of Wolverhampton, Wolverhampton WV1 1SB, UK Tel: +44 (0) 1902 322142 Email: t.baldwin@wlv.ac.uk Microscopy and Analysis 25(7):21-22 (EU), 2011

Figure 1: Vicia faba. From Prof. Dr Otto Wilhelm Thomé Flora von Deutschland, Österreich und der Schweiz 1885, Gera, Germany. www.biolib.de

Figure 2: Photomicrograph showing the general anatomy of the faba bean pistil. Key: (b) – stigma, (c) – apical region, (d) – middle region, (e) – basal region, ov – ovary, sh – stylar hairs, stg – stigma, sty – style. Scale bar = 500 µm.

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

Baldwin_Layout_Final_CMYK_ArticleTemplate_Jan2008.qxd 10/20/2011 3:23 PM Page 22

Figure 3 (above): The cold stage used on the VP SEM. Courtesy of Carl Zeiss NTS. Figure 4 (right): Structure of stigma from K25 (a-e) and D07 (f-j) at d-4 (a, f), d-3 (b, g), d-2 (c, h), d-1 (d, i) and anthesis (e, j). The stigmatic cuticle started to become ruptured (arrowed) at 2 days pre-anthesis in K25 whilst remaining intact until anthesis in D07. Scale bars = 40 µm.

R E S U LT S A N D D I S C U S S I O N Figure 2 shows the general anatomy of the faba bean pistil and the positions of the stigma, style, stylar hairs and ovary. Figure 4 shows the structure of faba bean stigmas from autofertile K25 (a-e) and autosterile D07 (f-j) lines at d-4 (a, f), d-3 (b, g), d-2 (c, h), d-1 (d, i) and anthesis (e, j). The stigmatic cuticle started to become ruptured (arrowed) at 2 days pre-anthesis in K25 whilst remaining intact until anthesis in D07. Figure 5 shows close ups of faba bean stigmatic papillae. These data show the differences in stigmatic morphology in autofertile and autosterile lines of faba bean in the days prior to flower opening and pollination which clearly indicate why each line is autofertile or autosterile respectively. They also demonstrate the utility of VPSEM for similar studies in other plants species where changes in surface morphology correlate with biological function.

REFERENCES 1. Chen, W., Stoddard, F. L. and Baldwin, T. C. Int. J. Plant Sci. 167(5): 919-932, 2006. 2. Chen W. and Baldwin T. C. An improved method for the fixation, embedding and immunofluorescence labelling of resin-embedded plant tissue. Plant Molecular Biology Reporter, 25:25-37, 2007.

ACKNOWLEDGEMENTS We are very grateful to Professor W. Link, University of Göttingen, for supplying the seeds of the inbred lines; to Mr Robert Hooton, University of Wolverhampton, who assisted with the cultivation of the faba beans and to Ms Barbara Hodson for help and advice with the SEM. W. C. was financially supported by a University of Wolverhampton PhD studentship, grant number RS 328, ‘A molecular and structural investigation of autofertility and autosterility in Vicia faba (faba bean)’. The project was associated with European Commission grant QLK5-CT-2002-02307, ‘Faba bean breeding for sustainable agriculture in Europe’ (acronym EU-Faba). ©2011 John Wiley & Sons, Ltd

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

Figure 5: Close up of faba bean stigmatic papillae (a) and (b). Scale bars = 20 µm.

PEOPLE

AND

PLACES

people and places SALVE Project enters Phase II Following the successful completion of a two-year evaluation phase, the University of Ulm, the Heidelberg-based company CEOS GmbH and Carl Zeiss Nano Technology Systems have signed an agreement to embark on the next phase of the SALVE (sub-angstrom low-voltage electron) microscopy project. SALVE is one of the most ambitious research projects in the field of electron microscopy to be undertaken in Germany in recent years. The objective of the project is to develop and build a transmission electron microscope capable of imaging samples with atomic resolution at very low acceleration voltages. The advantages offered by this approach are clear: Unlike the current generation of TEMs with accelerating voltages of between 200 and 300 kV, which destroy radiation-sensitive samples before researchers can record usable images or perform material analysis, the SALVE project will keep specimens stable long enough to perform experimental work. The the first phase of the co-operation project conducted between 2009 and 2011 – in which researchers analyzed the feasibility of the key principles involved – has produced some spectacular results, with the scientists successfully generating atomic-resolution images at accelerating voltages well below 80 kV. During celebrations to mark the start of the project’s second phase, Project Manager Professor Ute Kaiser from the University of Ulm and a number of guest speakers, including Nobel Prize winner Klaus von Klitzing, presented some of the fascinating ways in which the SALVE system could potentially be used. Ranging from studies of superconductors and semiconductors to research into lithium-ion batteries, plastics and biological materials, some of the examples they highlighted have already yielded preliminary results. While Carl Zeiss presses ahead with development of the system itself, the University of Ulm will be working on application development and conducting research into sample preparation methods. Meanwhile, the third project partner, CEOS, having a lot of expertise in the development of advanced electron optical systems, is focusing its efforts on a new optimized corrector to compensate the chromatic and the spherical aberration for low voltages.

Synoptics Technology Group Synoptics has set up the Advanced Technology Group, a new division which will work with life science companies and academic clients to deliver bespoke imaging equipment to improve bench-based research, quality control or clinical development processes. Synoptics, comprising the Syngene, Synbiosis and Syncroscopy divisions, provides innovative products that life scientists need to enhance the quality and speed of their research. Syngene produces the G:BOX image analyser to analyse gels and blots in the fields of genomics and proteomics. Synbiosis provides the ProtoCOL 2, a colony counting and zone sizing system used in many major pharmaceutical companies, and Syncroscopy offers Auto-Montage software for producing focused images of 3D samples, which is marketed by major microscope manufacturer, Leica. Richard Maskell, Synoptics’ new head of the Advanced Technology Group explained: “We are increasingly being approached to develop novel systems and software to bring about a step change in biological quality control and research processes. We have experience in solving challenges associated with imaging a wide variety of materials, as well as taking those solutions to market and are delighted to offer access to this expertise via our new Advanced Technology Group.” Paul Ellwood, Managing Director of Synoptics added: “We encourage academic researchers experiencing issues with their biological imaging processes, or technology providers that are looking to design an imaging system to bring to market, to contact us today to discuss how working with our new Advanced Technology Group could help them make a strategic impact on their research or quality control objectives.”

Romanian National Institute

Left: Dr Zadrazil of Tescan. Right: TESCAN Lyra 3 at NIMP. The National Institute of Materials Physics (NIMP) in Magurele, near Bucharest, Romania, has opened new labs equipped with state-of-the-art microscopes for the complex examination and characterization of the microstructure of materials. The suppliers of these systems were JEOL (Europe), Shimadzu Handelsgesellschaft and TESCAN. Together they offered their best instruments to the lab: a TESCAN LYRA3 field-emission scanning electron microscope equipped with a focused ion beam and a JEOL JEM-ARM 200F Cs-corrected field emission atomic resolution analytical transmission electron microscope. The inauguration programme included a workshop with presentations by representatives of several important European scientific and academic institutions such as the University of Antwerp, Belgium, the University of Caen, France, Claude Bernard University in Lyon, France, and the Institute of Physics and Chemistry of Materials in Strasbourg, France. TESCAN and JEOL representatives then followed with presentations of their most advanced equipment including the two instruments delivered to NIMP in Magurele.

Nobel Prize for Shechtman The Royal Swedish Academy of Sciences has awarded the Nobel Prize in Chemistry for 2011 to Daniel Shechtman Technion – Israel Institute of Technology, Haifa, Israel “for the discovery of quasicrystals”. On 8 April 1982, an image counter to the laws of nature appeared in Daniel Shechtman’s electron microscope. In all solid matter, atoms were believed to be packed inside crystals in symmetrical patterns that were repeated periodically over and over again. For scientists, this repetition was required in order to obtain a crystal. Shechtman’s image, however, showed that the atoms in his crystal were packed in a pattern that could not be repeated. Such a pattern was considered just as impossible as creating a football using only six-cornered polygons, when a sphere needs both five- and six-cornered polygons. Aperiodic mosaics, such as those found in medieval Islamic mosaics have helped scientists understand what quasicrystals look like at the atomic level. In those mosaics, as in quasicrystals, the patterns are regular – they follow mathematical rules – but they never repeat themselves. Following Shechtman’s discovery, scientists have produced other kinds of quasicrystals in the lab and discovered naturally occurring quasicrystals in mineral samples from a Russian river. A Swedish company has also found quasicrystals in a certain form of steel, where the crystals reinforce the material like armor. Scientists are currently experimenting with using quasicrystals in different products such as frying pans and diesel engines. Daniel Shechtman was born 1941 in Tel Aviv, Israel. He has a PhD 1972 from the Technion - Israel Institute of Technology in Haifa, Israel, and is now a distinguished professor holding the Philip Tobias Chair, at that university.

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

Register now at www.microscopy-analysis.com and join the worldwide microscopy community today for FREE Our website is designed to help you find content quickly and easily wherever you are located. Visit today and discover: ◗ Current news, feature articles, webinars and blogs ◗ Latest microscopy jobs ◗ Nancy’s blog – written by an expert science writer passionate about microscopy ◗ Three dedicated community channels, with monthly e-newsletters: • Light • Electron, Ion and X-Ray • Scanning Probe ◗ An event directory covering meeting reports, event blogs and interviews from major international events like M&M, Microscience and Neuroscience

◗ A “Learn & Develop” section with tutorials, book reviews and literature highlights ◗ International commercial news and a supplier directory ◗ How to receive a print issue of “Microscopy and Analysis” read by over 120K scientists worldwide Additionally, by joining the M&A community you can submit your own images, events, articles and jobs and keep informed with up-to-the-minute news and views via the our Facebook page and @MicroscopyViews on Twitter.

Registering for the first time?

Need to re-register?

All these interactive features and content are completely FREE! Download our M&A App from iTunes or the App store.

www.microscopy-analysis.com The leading media for microscopy worldwide

CIRCLE NO. 24

ONLINE: www.microscopy-analysis.com

PEOPLE

Andor Insight Imaging Awards

Andor Technology, a world leader in scientific imaging and spectroscopy solutions, has announced that two visually stunning and scientifically captivating entries have won the Andor Insight Awards Scientific Imaging Competition. The winning entry in the Physical Sciences category was submitted by Dr Robert Marshall of Boston University and the winning entry in the Life Sciences category by Dr Satoshi Nishimura of the University of Tokyo. Dr Satoshi Nishimura of the Department of Cardiovascular Medicine at the University of Tokyo entered a series of confocal captures entitled ‘Inflammatory cellular dynamics in obese adipose tissue revealed by in vivo imaging technique’. This imaging technique allows scientists to work at a cellular level and will provide the basis for future clinical usage of in-vivo imaging for humans. It was captured using an Andor iXon3 EMCCD camera and a Confocal Spinning Disk Unit.

AND

PLACES

Institute of Photonic Technology in Jena At the Institute of Photonic Technology (IPHT) in Jena, Germany, light is the central focus of research and development. The new IPHT sees photonics as the most important key technology of the 21st century. It is guaranteed to play a leading role in the fields of information technology and communications, security, material science, life science and health. Dr Volker Deckert is the head of the Nanoscopy department which utilizes instrumental methods in the development of molecular spectroscopic methods with the highest spatial resolution. Central to this program has been the use of tip-enhanced Raman scattering, TERS, where the NanoWizard systems and Tip-Assisted Optics module from JPK Instruments have provided a platform in the development of these experimental methods. In many cases, the structural sizes of components are below the capabilities of normal optical microscopic or spectroscopic techniques. Optical near-field microscopy in combination with Raman spectroscopy pushes the achievable resolution significantly below the diffraction limit of standard instruments. The goal of their work is the

advancement of TERS to become an accessible and sensitive tool for the analysis of surfaces and boundaries under ambient conditions. Applications are carefully selected. For example, heterogeneous catalytic reactions are studied because such application-oriented experiments may be used to verify and improve the functionality of the instrument and to demonstrate its practicality for ‘real’ problems.

World Microscopy Market Grows According to a recently published report from Research and Markets Ltd in Dublin, the world microscopes market, encompassing light, confocal, electron and scanning probe microscopes, generated an estimated $5.6 billion in revenues in 2010. The market is expected to grow to over $9 billion by 2017. Increased government and corporate funding in life sciences, materials research and nanotechnology is leading to sustained growth in microscopy. Light microscopes currently account for the majority of the

microscopy market but will lose market share to electron and scanning probe microscopes in the coming years. The largest end-user market for microscopy is semiconductors, followed by life sciences, and nanotechnology and nanomaterials research. The industry was adversely affected the global recession overall, especially in the semiconductors end-user sector but witnessed growth in the life sciences and healthcare. Electron and scanning probe are the fastest growing microscopy markets.

CIRCLE NO. 40 OR ONLINE: www.microscopy-analysis.com

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

LiteratureHighlights_Euro_Nov2011_Page26_LiteratureHighlights_Nov08_Page17.qxd 10/24/2011 2:57 PM Page 26

L I T E R AT U R E H I G H L I G H T S

literature highlights Atom Probe Imaging of GaAsSb Quantum Rings A. Beltrán and colleagues at the Department of Materials Sciences, University of Cadiz, Spain, report that three dimensional atom tomography resolves the quantum ring morphology of self-assembled GaSb buried nanostructures [Ultramicroscopy 111(8):10731076, July 2011]. Unambiguous evidence of ring-shaped self-assembled GaSb nanostructures grown by molecular beam epitaxy is presented on the basis of atom-probe tomography reconstructions and darkfield transmission electron microscopy imaging. From atom-probe tomography compositional distribution has been obtained. The GaAs capping process causes a strong segregation of Sb out of the center of GaSb quantum dots, leading to the self-assembled GaAsxSb1-x quantum rings of 20-30 nm in diameter with x~0.33.

A Stage for Micro-Axial Fluorescence Tomography A miniature stage device to overcome resolution anisotropy in fluorescence light microscopy is described by Florian Staier and colleagues at the Kirchhoff Institute for Physics, University of Heidelberg, Germany [Rev. Sci. Instrum. 82:093701, 2011]. To overcome the limitation of fluorescence microscopes in anisotropic optical resolution or point localization precision micro-axial tomography was used which allowed object tilting on the microscope stage and led to an improvement in localization precision and spatial resolution. A glass fiber was placed in the object space of the microscope lens and its rotation controlled by a miniaturized stepping motor. By Test particles were fixed onto the glass fiber, optically localized with high precision, and automatically rotated to obtain views from different perspective angles from which distances of corresponding pairs of objects were determined. From these angle dependent distance values, the real 3D distance was calculated with a precision in the ten nanometer range (corresponding here to an optical resolution of 10-30 nm) using standard microscopical equipment. As a proof of concept, the spindle apparatus of a mature mouse oocyte was imaged during metaphase II meiotic arrest under different perspectives.

Gold Nanodot Arrays for Nanofabrication A large array of sub-10-nm single-grain gold nanodots for use in nanotechnology is described by Nicolas Clément and colleagues at the Institut d’Electronique Microélectronique et Nanotechnologie, CNRS, University of Lille, France [Small 7(18):2607-2613, 2011]. A uniform array of singlegrain gold nanodots, as small as 5-8 nm, was be formed on silicon (a) STEM image showing the bulk silicon (Si), five annealed dots (Au), carbon layer (C), and platinum layers. using e-beam lithography. The (c) Coloured STEM image of a single annealed nanodot (260°C, 2 h). Reproduced with permission, Copyright © as-fabricated nanodots were 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. tify the critical size that determines whether a dot is amorphous, and thermal annealing converted them composed of single or multiple crystal domains. to pure Au single crystals covered with a thin SiO2 Moreover, they showed that annealing at moderate layer. These findings were based on physical meatemperature can convert Au dots from amorphous to surements by AFM, atomic-resolution STEM, and single-crystalline, and then they were covered with a chemical techniques using energy dispersive X-ray thin SiO2 layer. After easy removal of the SiO2 (dilute spectroscopy. The authors demonstrated the formaHF etching), these nanodots can be used as electrodes tion by e-beam lithography of sub-10-nm Au dots for the characterization of organic self-assembled with small dispersion and perfect alignment. Such monolayers (SAMs) with less than 200 molecules. precise formation of small dots enabled them to iden-

3D Point Spread Function in Cs-Corrected STEM The three-dimensional point spread function of an aberration-corrected scanning transmission electron microscopy (STEM) has been simulated and experimentally tested by Andrew Lupini and Niels de Jonge at the Oak Ridge National Laboratory, TN [Microscopy and Microanalysis 17:817-826, 2011]. Aberration correction reduces the depth of field in STEM and thus allows three-dimensional imaging by depth sectioning. This imaging mode offers the potential for sub-Ångstrom lateral resolution and nanometer-scale depth sensitivity. For biological samples, which may be many µm across and where high lateral resolution may not always be needed, optimizing the depth resolution even at the expense of lateral resolution may be desired, aiming to image through thick specimens. Although there has been extensive work examining and optimizing the probe formation in two dimensions, there is less known about the probe shape along the optical axis. The authors examined the probe shape in 3D in an attempt to better understand the depth resolution in

Image Analysis of Deep Etched Actin Filaments A fractal dimension analysis and mathematical morphology of structural changes in actin filaments imaged by electron microscopy is reported by Yoshitaka Kimori et al at the National Institutes of Natural Sciences, in Tokyo [Journal of Structural Biology 176(1):1-8, Oct 2011]. The authors examined structural changes of actin filaments interacting with myosin visualized by quick freeze deep-etch replica EM by using a new method of image processing and analysis based on mathematical morphology. To quantify the degree of structural changes, two characteristic patterns were extracted from the EM images: the winding pattern of the filament shape (WP) reflecting flexibility of the

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

Enabling Two-Colour STED of Live Cells A technique for the use of two fluorophores in stimulated emission depletion (STED) microscopy of living cells is reported by Patrina Pellett and co-workers at the Department of Cell Biology, Yale School of Medicine, CT [Biomedical Optics Express 2(8):2364-2371, 2011]. Current applications of STED microscopy have been limited to single colour imaging of living cells and multicolour imaging in fixed cells. However, to study active processes, such as protein interactions, a two-colour STED imaging technique is needed in living cells. This was achieved for the first time by the authors: the key to their success was in overcoming the challenges in labeling target proteins in living cells with dyes optimal for two-colour STED microscopy. By incorporating fusion proteins, the researchers were able to improve the targeting between the protein and the dye, effectively bridging the gap. This allowed the researchers to achieve resolutions of 78 nm and 82 nm for 22 sequential twocolour scans of epidermal growth factor and its receptor in living cells.

Free Educational Educational Webinar: Webinar: Free Sample Preparation Preparation for for Sample Scanning Electron Electron Microscopy Microscopy (SEM) (SEM) Scanning State of the art Critical Point Drying with the New fully automated Leica EM CPD300 State of the art Critical Point Drying with the New fully automated Leica EM CPD300 Speaker: Dr. Ruwin Pandithage, Leica Mikrosysteme GmbH, Vienna, Austria Speaker: Dr. Ruwin Pandithage, Leica Mikrosysteme GmbH, Vienna, Austria

Online Seminar/Webinar on Sample Preparation for SEM Online Seminar/Webinar on Sample Preparation for SEM Register at http://www.microscopy-analysis.com/leicawebinars Online Seminar/Webinar on Sample Preparation for SEM Register at http://www.microscopy-analysis.com/leicawebinars th and join on Tuesday 25 October 2011 at 17:00 h (CET), 16:00 h (UK) Register http://www.microscopy-analysis.com/leicawebinars and join onatTuesday 25th October 2011 at 17:00 h (CET), 16:00 h (UK) Watch this webinar if you are responsible for Watch this webinar if you are responsible for • •• •

EM sample preparation of delicate biological specimens such as pollen, tissue, plants or insects EM specimens such as pollen,Mechanical tissue, plants or insects EM sample sample preparation preparation of of delicate industrialbiological samples for MEMS (Micro Electro Systems) applications EM sample preparation of industrial samples for MEMS (Micro Electro Mechanical Systems) applications

Specimens that can be damaged due to surface tension when changing from the liquid to gaseous state, need special treatment Specimens thatpreparation. can be damaged to surface whenmethod changing thesuch liquid to gaseous state, treatment during sample Criticaldue point drying istension an efficient forfrom drying delicate samples forneed SEMspecial applications because during sample preparation. Critical point drying is an efficient method for drying such delicate samples for SEM applications because it preserves the surface structure. Before drying, many biological samples are additionally prepared through fixation and dehydration itand preserves the surface structure. drying, aretoadditionally fixation and dehydration then coated after drying with aBefore metal such asmany gold, biological platinum orsamples palladium make theirprepared surfaces through electrically conductive for the and coated after drying structure with a metal such as gold, the platinum palladium makeoftheir conductive for the SEMthen analysis. If the surface is altered during dryingorprocess the to results the surfaces followingelectrically SEM application will provide SEM analysis. If the surface structure is altered during the drying process the results of the following SEM application will provide incorrect results. incorrect results. In the past, critical point drying was a time consuming process with low sample In the past, critical dryingoperation. was a time consuming process with low sample reproducibility due point to manual reproducibility due to manual operation. This webinar will show you how the New Leica EM CPD300 dries delicate biological or This webinar will show you automated how the New EM CPD300 dries delicate biological or industrial samples in a fully andLeica controlled process to preserve your samples industrial samples in a fullyand automated for subsequent treatment analysis.and controlled process to preserve your samples for subsequent treatment and analysis. Register at http://www.microscopy-analysis.com/leicawebinars Register at http://www.microscopy-analysis.com/leicawebinars This webinar is available on demand under the above registration link. This webinar is available on demand under the above registration link. Critical Point Drying with Leica EM CPD300 Critical Point Drying with Leica EM CPD300

CIRCLE NO. 25

ONLINE: www.microscopy-analysis.com

Make Every Electron Count ChemiSTEMTM Technology Introducing the newest member of the Titan family: Titan™ G2 80-200 with ChemiSTEM™ Technology. Combining FEI’s groundbreaking ChemiSTEM Technology with Titan’s market-leading sub-atomic resolution imaging, Titan G2 80-200 offers highly sensitive, fast elemental mapping, superior ultra-low concentration detection and the highest analytical probe current available. Even EDX tomography is easily achieved with stunning results.

GaAs Atomic EDX showing 1.4 Angstroms dumbbells with Titan G2 80-200 with ChemiSTEM Technology

La1-xSrxMnO3/SrRuO3 multilayer/quantum well systems in [100] projection

Sample courtesy of Ionela Vrejoiu and Eckhard Pippel, Max Planck Institute of Microstructure Physics, Halle/Saale, Germany.

Discover how to make every electron count at fei.com/chemistem. CIRCLE NO. 26

ONLINE: www.microscopy-analysis.com

Whatsnew_Nov11_US_Page29_Whatsnew_Sept07_Page39.qxd 10/19/2011 11:09 AM Page 29

W H AT ’ S N E W

what’s new Magnetic Field Cancellation

Technical Manufacturing Corporation (TMC) has improved upon the original design of its Mag-NetX magnetic field cancellation system to allow for easier installation and service and to make Mag-NetX more adaptable to customers’ changing needs. The stainless steel struts are now modular and interchangeable and available in a variety of lengths to accommodate virtually every commercial SEM. In addition, a new modular corner piece is designed with integrated electronics for easy installation and service. TMC developed Mag-NetX to actively compensate for magnetic field fluctuations caused by nearby machinery, elevators, power lines, and other external sources. Such sources can diminish the performance of scanning and transmission electron microscopes, electron beam lithography systems, focused ion beam instruments, and other tools that incorporate a charged beam. The open cube-shaped Mag-NetX detects magnetic fields and sends out an equal and opposite field to cancel the interference. The system consists of a dedicated controller with automated calibration and self-test features, AC and DC magnetic sensor, and Helmholtz coils in a structural casing. It can be floor-,wall-, or tool-mounted and sized to user-specific requirements. The system is dynamic, continuously monitoring and achieves 35-40 dB of attenuation. Contact: Technical Manufacturing Corporation www.techmfg.com

3D Imaging Dual Beam

Sample Vice

FEI, a leading instrumentation company providing electron microscope systems for applications in research and industry, has released its new Versa 3D DualBeam system, which provides highresolution, three-dimensional (3D) imaging and analysis on a wide range of sample types. The Versa 3D’s highly configurable platform allows customers to adapt the system’s capabilities to their specific requirements. “The flexible configuration of the Versa 3D meets the demands of today’s researchers who study a wide variety of materials,” said Trisha Rice, vice president and general manager of FEI’s Research Business Unit. “FEI’s pioneering leadership in ion beam and electron beam techniques and methodologies are well matched to give researchers information from even the most challenging samples. Last year, FEI introduced the latest generation Helios NanoLab, the highest resolution DualBeam in the world that incorporates industry-leading electron and ion beam technologies, and today we are unveiling the most flexible DualBeam, the Versa 3D.” Versa 3D is available with either high vacuum-only or high and low vacuum electron imaging hardware. Low vacuum electron imaging capabilities allows the system to accommodate contaminating or outgassing samples that are incompatible with high vacuum operation. Low vacuum also provides the ability to compensate for charge build up in non conductive samples even at the high currents required for analysis techniques, such as energy dispersive (X-ray) spectroscopy (EDS) and electron backscatter diffraction (EBSD). The Versa 3D combines FEI’s leadership in Schottky field emission electron beam and high throughput ion beam technologies into a configurable DualBeam system, setting a new standard for 3D characterization and analysis, sitespecific sample modification and advanced sample preparation for transmission electron microscopes (TEMs) and atom probes. The high-performance platform can also be configured with FEI’s impressive low vacuum capabilities and even environmental scanning electron microscopy (ESEM) for in situ analysis. Advanced SEM scanning and FIB patterning yield powerful imaging and milling performance. New features, such as FEI’s SmartSCAN and Drift Corrected Frame Integration (DCFI), facilitate electron beam imaging of sample types with a range of different properties. Advanced backscattered electron, as well as secondary electron and ion detectors, collect a wide variety of topographic, elemental and compositional information ‘from every angle’. The combination of the latest AutoSlice & View G3 software option, the versatile electron imaging hardware and high throughput ion column enables researchers to capitalize on the charge balancing capabilities of ions and electrons. Milling (with positive ions) and imaging or drift suppression (with electrons) provides a unique synergy for automation of 3D serial slicing, imaging and analysis of both electrically conductive and non-conductive samples. When combined with EDS or EBSD, FEI’s EDS3 and EBS3 software options can also be used to reconstruct elemental maps or crystallographic orientation data in 3D. The Versa 3D addresses the diverse needs in materials research, life sciences, electronics and geosciences. It is available for ordering immediately. Contact: FEI Company www.fei.com

The Micro Vice sample holder from ST Japan is a simple yet versatile tool to adjust and hold samples accurately for analysis with light microscopes, stereomicroscopes and FTIR- and Raman microscopes. It efficiently supports microscope analysis of difficult shaped samples, fibers and films and all kind of samples up to a diameter of 40 mm. The Micro Vice: holds round and unevenly shaped samples such as tablets, minerals, etc. in the desired position; stretches accurately and holds polymer films, fibers, hair, etc.; and tilts samples for correcting oblique sample orientation. In microscopical analysis it is often challenging to securely place your samples, such as tablets, gem stones, films or fibers in the exactly desired position. With the MicroVice it is easy to hold round samples such as tablets under compression in the desired position, hold films and other flexible materials under tension and even stretch them. The MicroVice sample holder is designed to be easily attached to the stages of light microscopes, stereoscopes and also on infrared and Raman microscopes. You can place your sample in the exactly desired position and tighten the two screw handles. The sample will be hold securely under compression. Even round samples can be easily held and won’t slip. The clamps are cushioned and protect your sample from damage. The sample holder is supplied with two small plates that are used for clamping taut and holding films, fibers, yarns, etc. By using the two screw handles you can stretch the samples using tension by user defined increments. Due to the design of the MicroVice, exact parallel stretching of the samples is ensured. Additionally, the fixed sample can also be tilted up to a certain angle for correcting oblique sample orientation. Contact: S.T. Japan-Europe GmbH www.stjapan.de

FOR FREE PRODUCT INFORMATION VISIT OUR WEBSITE: www.microscopy-analysis.com M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

Quantitative Cathodoluminescence Attolight AG debuted the first-ever quantitative cathodoluminescence microscope featuring nanoscale resolution and picosecond timing in an easy-to-use platform at the recent M&M meeting in Nashville, TN. “This is really the beginning of a whole new revolution in cathodoluminescence,” said Attolight CEO, Dr Samuel Sonderegger. “There is no need to compromise with this system: we run the full spectrum from UV to IR while maintaining 10 nm spatial resolution, and the full temperature range from 15K to 300K, all with 100⫻ larger field of view and up to 100⫻ more collection efficiency than any prior CL technology. Scientists can now have nanoscale imaging and pico-second time resolved spectroscopy, all in one instrument, with no compromises.” At the heart of the Attolight CL line is a newly designed scanning electron microscope (SEM) containing an embedded optical microscope, a 9-axis cryo nano-stage, and a fully integrated (not merely interfaced) cathodoluminescence system. Available in two versions, the continuous wave CL 10-Infinity can be field-upgraded to the picosecond, time-resolved CL10-10.

The specifications are: spatial resolution: 10 nm across the full spectrum; spectral range: UV to IR; field of view: 300 µm; optical NA of 0.78 with 100⫻ the collection efficiency; positioning: 9-axis cryo stage with pivot point lock; pulsed operation: picosecond speed with no electron dispersion; drift and vibration: minimized; beam blanking: proprietary laser driven, ps photoelectron gun; no compromise in spatial (10 nm) or temporal (ps) resolution across the spectrum from UV to IR; thermal range: 20K to 300K. Contact: Attolight AG www.attolight.com

Xylene-Free Tissue Processor Thermo Fisher Scientific has announced that its new Thermo Scientific STP 420ES Tissue Processor for highthroughput tissue processing has been independently validated for xylene-free protocols. Scientists at the UK’s Newcastle upon Tyne Hospitals NHS Foundation Trust confirmed the successful application of a standard xylene-free protocol on the STP 420ES. The tissue processor delivered quality results using existing protocols, with the exception of the requirement for a lower temperature of 65°C, not 85°C, for the initial wax step. Elimination of xylene from tissue processing can cut costs, save time and improve the laboratory environment. Using isopropyl alcohol (IPA) as an alternative wax miscible dehydrant removes the risks of cumulative exposure and the high disposal costs associated with xylene, which is a hazardous chemical. Using IPA also shortens cycle times and enables leaner workflows so laboratories can deliver patient results faster. Contact: Thermo Fisher Scientific www.thermoscientific.com/pathology

CIRCLE NO. 27 OR ONLINE: www.microscopy-analysis.com

Plasma FIB-FESEM Workstation

TESCAN, a world leading manufacturer of scanning electron microscopes and focused ion beam workstations has introduced the FERA3 XMH – a high-resolution Schottky field-emission scanning electron microscope with a fully entegrated plasma source focused ion beam. The system has been developed in co-operation with the French company Orsay Physics. In addition to electron and ion columns, the FERA3 XMH Plasma FIB-FESEM can be configured with gas injection systems, nanomanipulators, and a wide variety of detectors including SE detector, BSE detector, SI (secondary ion) detector, CL (cathodoluminescence) detector, EDX and EBSD microanalyzers, etc. The use of a xenon plasma source for the focused ion beam allows the FERA3 to satisfy high-resolution FIB requirements (imaging, fine milling/polishing), as well as achieving high ion currents needed for ultrafast material removal rates. The resolution of the plasma ion beam is <100 nm and the maximum Xe ion current is >1 µA. Compared to existing FIB technologies with gallium sources, the material removal rate achievable for silicon with the PFIB (plasma FIB) is over 30x faster. For this reason the FERA 3 XMH is well suited for applications requiring the removal of large volumes of material, particularly in the semiconductor packaging corridor where TSV technology is being utilized.

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

TESCAN will deliver the first system to the MiQro Innovation Collaborative Centre (C2MI) in Canada this year. The system will be used for the inspection of integrated circuit (IC) packaging. The FERA 3 FIB-SEM workstation’s integration of both an electron and a focused ion beam places this tool in a class all its own, affording the end user the benefits of electron beam analysis and characterization. Generally, systems of this kind will be used for circuit editing, 3D metrology, defect analysis and failure analysis. Contact: TESCAN www.tescan.com

FREE WEBINAR REGISTER TODAY!

Advanced Phase ID Using Combined EBSD & EDS on SEM Date: 11 January 2012, 16.00 GMT, 17.00 CEST, 11.00 EDT Duration: 1 hour Presenters: Dr. Daniel Goran, EBSD Application Scientist Dr. Laurie Palasse, EBSD Application Scientist

Electron backscatter diffraction (EBSD) and energy dispersive X-ray spectrometry (EDS) are common analytical methods used on the scanning electron microscope (SEM). They are complementary techniques and provide structural and compositional information respectively. Building on its recent developments in integrating EBSD and EDS, Bruker is now releasing an advanced phase identification feature. This new method significantly increases efficiency when dealing with multiphase materials and allows experts as well as less experienced users to acquire the best quality results.

e â&#x20AC;&#x201C;Flash

HR+

EBSD Detector

Volcanic rock - phase map

The webinar will focus on describing the new phase identification procedure and its advantages compared to the common phase identification method. Our experts will present numerous materials and earth science application examples.

Who should attend? Researchers working in electron microscopy labs studying crystalline materials Materials and earth science lecturers and students EBSD users interested in advanced applications of the method

www.microscopy-analysis.com/brukerwebinars

Innovation with Integrity CIRCLE NO. 28

ONLINE: www.microscopy-analysis.com

EBSD/EDS

FREE WEBINAR REGISTER TODAY!

The Dimension FastScan AFM: SEM like user experience, True 3D nanometrology, and quantitative material property mapping in ambient, fluid, and controlled environments DATE: December 7th 4 pm BST, 5 pm CET, 11:00 am EDT Presenting:

Dr. Johannes Kindt

In many applications Atomic Force Microscopy (AFM) can provide unique and preferred sample information, however its slow speed and high complexity have often offset these benefits in favor of Electron Microscopy. The latest generation of Bruker’s AFM, the Dimension FastScan™, enables nanometer resolution imaging, in a fraction of a minute, on a large-sample, fully automatable stage. The included ScanAsyst™ algorithm provides robust, intuitive, self-optimizing work-flow based operation. Combined these two capabilities create a highly productive nano-imaging solution akin to current Scanning Electron Microscopes (SEM). While both techniques provide surface imaging on the nano scale, the insights gained from each technique are also complimentary: •

AFM can be performed, at nm-resolution, in ambient and fluid environments, and typically requires no alterations of the sample surface chemistry prior to imaging. This enables non-destructive sample prep, convenient (multi-) sample loading, easy sample access, and imaging of dynamic sample changes over time.

•

AFM provides true nano-metrological information in all three sample dimensions

•

While SEM techniques can provide contrast based on elemental analysis, the latest-generation AFM mode, Bruker’s proprietary PeakForce-QNM, provides quantitative nanoscale mapping of surface mechanical properties, such as modulus, adhesion, or dissipation, in addition to the standard topographical information

Please join us for this focused review of the recent advances of the “other” nanoscale surface imaging technology.

To register for this webinar and for further information, please go to: http://www.microscopy-analysis.com/brukerafmwebinars http://www.microscopy-analysis.com/brukerwebinars?c=electron

Innovation with Integrity

Atomic Force Microscopy

CIRCLE NO. 29 OR ONLINE: www.microscopy-analysis.com

W H AT â&#x20AC;&#x2122; S N E W

Hybrid Light and Electron Microscope Topcon Positioning Systems (TPS), a global leader in precision optical, GPS, and automation systems for geodetic, engineering and construction applications, has introduced the Aquila hybrid microscope to the North American market. The microscope magnifies from 30âŤť in optical mode, to 50,000âŤť as an SEM. This unique hybrid design provides key advantages that should expand the use of SEM technology. Ray Oâ&#x20AC;&#x2122;Connor, TPS president and CEO, said, â&#x20AC;&#x153;The Aquila hybrid scope puts the versatility of an optical microscope and the power of an scanning electron microscope (SEM) into a simple, portable, and affordable package.â&#x20AC;? The simplicity of the Aquila, both in design and functionality â&#x20AC;&#x153;creates an instrument with virtually no learning curve,â&#x20AC;? he said. â&#x20AC;&#x153;With an array

Mini-SEM Holders

of features that make it attractive to both the experienced and new SEM user, it is the most versatile and easy-to-use microscope on the market.â&#x20AC;? â&#x20AC;&#x153;Topcon has a long and respected history in the optical market,â&#x20AC;? Oâ&#x20AC;&#x2122;Connor said. â&#x20AC;&#x153;Even though Aquila is not our typical positioning instrument, it has incredible application opportunities in several markets TPS already serves â&#x20AC;&#x201C; mining, forensics, education, and water management in particular.â&#x20AC;? In addition to the areas pointed out by Oâ&#x20AC;&#x2122;Connor, he noted the instrument has practical usage in air and water monitoring, metallurgy and metal analysis, materials analysis, food industry, quality control and micro-mechanical systems. Contact: Topcon Positioning Systems, Inc. www.topconsem.com

3LFR4XDQW IRU 0LFURVFRS\ 3LFR4XDQW IRU 0LFURVFRS\

)/,0 )5(7 DQG )&6 IRU \RXU FRQIRFDO /60 In close co-operation with its preferred development partner Deben, PhenomWorld has developed a Temperature Controlled Sample Holder to study vacuumsensitive and vulnerable samples on its Phenom G2 desktop SEMs. This active sample holder is designed to control the temperature of the sample between -25Â°C and +50Â°C. With the use of the temperature controlled holder, the temperature of the sample is manipulated, therefore the humidity around it can be controlled. This enables imaging of moistures and water containing samples as well as reducing the effect the electron beam has on beam sensitive samples. This results in an extended viewing time, without noticeable vacuum artifacts. The holder can be retrofitted to all versions of the Phenom G2 system. The improved Fibermetric application allows measurements and analysis on complicated fiber structures, ranging from spunbond and electrospun fibers to the melt blown type of fibers. Fibermetric provides accurate size information from micro and nano fiber samples. Through further automization of several important features, the Fibermetric has become even more user-friendly and guarantees a fast return on investment. The automated features that have the most effect on this are the high number of measurements (max. 1000 per image), the automated feature and fiber size detection, and the analysis of the data points. Contact: Phenom-World BV www.phenom-world.com

3URWHLQ SURWHLQ LQWHUDFWLRQ Â&#x2021; (Q]\P NLQHWLFV Â&#x2021; &HOO LPDJLQJ Â&#x2021; (QYLURQPHQWDO VHQVLQJ S+ LRQ HWF Â&#x2021; 3URWHLQ DQG PROHFXOH G\QDPLFV Â&#x2021; )XUWKHU PHWKRGV 3XOVHG ,QWHUOHDYHG ([FLWDWLRQ 3,( )5(7 Â&#x2021; )/&6 Â&#x2021; 6XLWDEOH IRU DOO PDMRU PDQXIDFWXUHUV

LRQV

SOLFDW

$ S RXQWLQJ & Q WR R K /HDGLQJ LQ 6LQJOH 3

3LFR4XDQW *PE+ %HUOLQ *HUPDQ\ 7HO LQIR#SLFRTXDQW FRP ZZZ SLFRTXDQW FRP

0HHW XV DW 63,( 3KRWRQLFV :HVW -DQXDU\ 6DQ )UDQFLVFR &$ 86$ ERRWKV DQG

CIRCLE NO. 30 OR ONLINE: www.microscopy-analysis.com M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

High Resolution FE-SEMs

Hitachi High-Technologies has launched the SU8000 family of ultrahigh-resolution field-emission scanning electron microscopes (FESEMs) for investigating the fine surface structure of materials in a wide range of nanotechnology fields. The new SU8000 series features a common, high performance electron optical platform to provide excellent imaging performance, and offers a variety of stages, chambers and signal detection systems. The new SU8010, SU8020 and SU8030 join the existing SU8040 to form this comprehensive family of ultrahigh-resolution microscopes. A high-brightness cold cathode field-emission source is used in combination with the latest generation of Hitachi’s patented super ExB in-lens detection systems for energy filtering, charge suppression, and contrast control. All microscopes offer excellent imaging performance at low accelerating voltage to minimize sample damage, and enhanced electron deceleration technology has improved resolution at ultralow landing voltages to just 1.3 nm at 1.0 kV. The SU8010 is the entry level model with dual (upper and lower) secondary electron detectors with secondary and backscattered electron signal mixing capabilities for versatile imaging. A three-axis motorized stage is provided as standard, capable of accommodating samples up to 100 mm in diameter. The SU8020 offers the same sample handling capabilities using a 5-axis motorized stage as standard but benefits from Hitachi’s unique triple detector system to extend the capability to collect secondary electrons and low energy backscattered electrons. The SU8030 features a large chamber with large specimen stage for samples up to 150 mm in diameter. Contact: Hitachi High-Technologies Corporation www.hht-eu.com

Combined Confocal and AFM

CIRCLE NO. 31 OR ONLINE: www.microscopy-analysis.com 34

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

PicoQuant GmbH has announced the successful combination of PicoQuant’s time-resolved confocal fluorescence microscope MicroTime 200 with Bruker’s BioScope Catalyst atomic force microscope. The combination of these two systems enables simultaneous recordings of AFM and optical images of the same sample region and makes new investigation schemes in the field of live-cell imaging feasible. The combined setup of the MicroTime 200 and the Bioscope Catalyst is straightforward without the need of larger modifications of the two systems. The synchronized data acquisition enables scientists to analyze, e.g., the impact of protein changes on cell shape and structure. It also allows high-resolution imaging by merging of sub-nm topography with optically encoded functionality as well as investigations of inter- and intramolecular distances using force spectroscopy. The Bioscope Catalyst AFM including its sample stage is mounted onto the inverted microscope body of the MicroTime 200, which is configured for objective scanning. In this way, precise overlay of the confocal volume and the AFM tip can be realized. Electronic communication between the sample-scanner of the AFM and the data acquisition electronics of the MicroTime 200 enables simultaneous recordings with the two instruments. Contact: PicoQuant GmbH www.picoquant.com

W H AT â&#x20AC;&#x2122; S N E W

Lift-Out Shuttle for TEM

Nano Optics on AFM

Working with leading German manufacturer of precision manipulation products, Kleindiek Nanotechnik, Agar Scientific are pleased to offer the Lift-Out Shuttle for the UK and Irish markets. In-situ lift-out techniques have become more reliable methods for preparation of samples requiring TEM and atom probe inspection. However, despite their new-found popularity, they remain considerably more expensive than ex-situ lift-out techniques and require lots of valuable time in the focused ion beam (FIB). Time and cost factors call for a faster, simpler procedure while further improving the reliability of the technique. By combining a precision sub-stage with a microgripper system and their novel SemGlu, Kleindiek have developed a new, fast, easy and efficient tool: The Lift-Out Shuttle. This device is small enough to fit through the load lock of most common electron microscopes. Mounting the sample and the TEM-grid that has been treated with a small amount of SemGlu to a single microscope stub puts all the pieces necessary for lift-out in place. The stub is then attached to the four-axis (XYZR) sub-stage. Additionally, the Microgripper is fixed to same mounting plate on which the sub-stage is positioned. In this configuration, the gripper remains stationary above the sub-stage and therefore is always centred in the field-of-view of the microscope. The stub containing the sample and grid is positioned under the gripper using the sub-stage. With a minimal amount of practice, a sample can be fixed to a grid in five minutes or less by gripping the previously cut sample, lifting out of the substrate, moving the stage to the TEM-grid (which has been pre-treated with SEMGLU by simply applying some glue to the grid using a needle), and mounting the sample to the grid by making contact between the two and focussing the e-beam on the contact point. Contact: Agar Scientific Limited www.agarscientific.com

JPK Instruments has expanded its family of high performance research AFM systems with the announcement of the availability of the NanoWizard 3 NanoOptics AFM system. Over the past decade, optical phenomena on the nanoscale have developed into an exciting area of research. To study light on the nanoscale and especially its interaction with matter, researchers look for methods with nanometer spatial resolution. The combination of light microscopy-derived techniques and scanning probe microscopy is a powerful solution. This so-called near-field optical microscopy delivers optical information from sample surfaces with sub-wavelength resolution. JPK strongly believes in combining techniques, in particular AFM, with optics. This has opened up a field of new applications including TERS/SERS, tip-enhanced fluorescence, nanomanipulation with light, chemical surface analysis and compound detection, metamaterials, developments of optically active components such as dyes, markers, light sources and switches. A large number of user publications underscore the success of this technology approach. Now, JPK introduces their latest platform for AFM and optics - the NanoWizard3 NanoOptics system. The NanoWizard NanoOptics head comes with excellent physical and optical access to the sample from top and bottom as well as from front and side, even when the head and condenser are in place. Additionally, it has an integrated port for fiber SNOM applications. The new system is ready for a broad range of applications from nanoscale optical imaging by aperture and scattering-type SNOM to experiments involving interactions of light with the sample such as absorption, excitation, nonlinear effects and quenching; these include aperture fiber SNOM experiments using an intePage 1 grated fiber SNOM port in the NanoOptics head and the tuning fork module. Contact: JPK Instruments AG www.jpk.com

SURFACE COMPOSITION ANALYSIS FOR ION MICROSCOPY

1 S F D J T J P O 4 U B C J M J U Z 1 F S G P S NB O D F " 4 * T Q S P E V D U T I B W F C F F O B U U I F G P S F G S P O U P G D V U U J O H F E H F M J G F T D J F O D F S F T F B S D I G P S P W F S Z F B S T 8F T Q F D J B M J [ F J O D M P T F E M P P Q % $ T F S W P NP U P S Q J F [ P C B T F E T Z T U F NT XJ U I B D U J W F G F F E C B D L D P O U S P M U P NB J O U B J O 9 : Q P T J U J P O ; G P D V T B U U I F O B O P NF U F S M F W F M 0 V S O F XF T U Q S P E V D U T G P S T V Q F S S F T P M V U J P O NJ D S P T D P Q Z Q V T I U I F F O W F M P Q F Z F U B H B J O * G Z P V O F F E V M U S B Q S F D J T F NP U J P O D P O U S P M NJ D S P T D P Q F B V U P NB U J P O P S B S P D L T P M J E T Z T U F N G P S E F NB O E J O H 0 & . B Q Q M J D B U J P O M P P L V T V Q " NF S J D B T

1 S P E V D U T B O E M P D B M T V Q Q P S U B W B J M B C M F G S P N & V S P Q F

& NB J M * O G P !B T J J NB H J O H D P N 8F C XXX B T J J NB H J O H D P N

& V S P Q F

NB J M * O G P !P Q U P Q I B T F D P N & NB J M * O G P !NJ D S B T Z T D P N & C XXX P Q U P Q I B T F D P N 8F C XXX NJ D S B T Z T D P N 8F

CIRCLE NO. 33 OR ONLINE: www.microscopy-analysis.com

CIRCLE NO. 32 OR ONLINE: www.microscopy-analysis.com

Hidenâ&#x20AC;&#x2122;s EQS and MAXIM SIMS analysers provide:

â&#x2014;?

chemical surface composition analysis for ion probe microscopy

â&#x2014;?

depth profiling and surface imaging at the nano scale

â&#x2014;?

EQ Sb olt -on

SIM Sp rob e

interface to existing systems

for further details of Hiden Analytical products contact:

info@hiden.co.uk www.HidenAnalytical.com Quadrupoles for advanced science

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

Whatsnew_Nov11_Euro_Page36_Whatsnew_May09_Page40.qxd 10/24/2011 2:42 PM Page 36

Backscatter Diffraction

Oxford Instruments NanoAnalysis, a world leader in microanalysis systems, launched a new EBSD detector at M&M 2011 in Nashville. The new NordlysNano addresses the growing requirements of nanoscale applications: it is 60% more sensitive than the previous generation of EBSD detector, the NordlysS. Increased sensitivity offers a number of benefits. Firstly, accurate EBSD data can be collected at lower beam energy, including low beam currents (<0.1 nA) and low kV. Using lower energy beams is essential for applications where spatial resolution is required, for example nanoscale applications. It is also important when looking at beam sensitive samples, or non-conducting samples. In addition, increased sensitivity enables faster data acquisition under comparable beam conditions. According to EBSD Business Manager, Dr Jenny Goulden, “As a business, Oxford Instruments aims to use innovation to turn smart science into world class products that delight our customers, and the NordlysNano is a classic example of achieving just that!” The new NordlysNano EBSD detector is 60% more sensitive than its predecessor, the NordlysS. It enables faster and higher resolution analysis of a crystal structure. Contact: Oxford Instruments www.oxford-instruments.com

Argon Ion Milling System

Hitachi High-Technologies has launched the IM4000 Ion Milling system. Used to prepare specimens for scanning electron microscope (SEM) imaging and analytical studies such as EDX and EBSP, the versatile IM4000 is capable of both pin-point cross-section and flat surface ion milling. Cross-section milling provides smooth cross-section specimens for high resolution imaging of subsurface structures, with the cross-section position accurately controllable by fine positioning of a beam-shielding mask edge. Flat milling provides uniform polishing of surfaces of 5 mm in diameter or more with variable angle milling, to either flatten surfaces or to selectively enhance specimen surface features (relief milling). The two IM4000 applications – cross-section and flat surface milling – are realised via two different removable sample stage units, allowing for convenient specimen setting and cutting edge definition using an external optical microscope. The IM4000 ion milling system features a new high-current argon ion gun that delivers cross-sectional milling rates of 300 um/hr in silicon for dramatically reduced cross-sectioning times. The wide Argon ion beam can define sharp cross sections even on samples of dissimilar materials with different hardness that can not be cut or broken without causing material deformations or dislocations. Sensitive materials like polymers or papers can be processed by freely selecting proper lower ion beam energies between 0 and 6 kV, without need for special sample cooling. Wide and smooth flat milled surfaces of approximately 5mm in diameter or more can be achieved within minutes by shifting the centre of the defocused ion beam from the sample rotation or swinging centre. The beam irradiation angle to the specimen surface is selectable from 0 to 90 degrees. Contact: Hitachi High-Technologies Corporation www.hht-eu.com

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

Environmental Cryo Chamber Boeckeler RMC Products recently celebrated the successful launch of its new environmental chamber for ultramicrotomy during a training demonstration at the Société Française Mu (SFmu) 2011 conference held in Strasbourg, CEMOVIS 50 nm serial sections of yeast cut on an RMC PT-PC system France last June. According to Boeckeler President Pat Brey, the demonstration included an impressive array of sectioning methods including sectioning for low voltage TEM (transmission electron microscopy), Tokuyasu immunolabeling and CEMOVIS (cryo-electron microscopy of vitreous sections) sectioning. The environmental chamber was critical to creating the parameters necessary for producing a continuous ribbon of 25 sections of a frozen hydrated sample 50 nm thick that had been flash frozen under high pressure. Such high-pressure freezing of biological specimens retains as much cell structure as possible for viewing under an electron microscope. The CEMOVIS method of sample preparation is experiencing resurgence in popularity among cell biologists since it was introduced in the 1980s, due in part to advances in overcoming some of the difficulties first encountered in the method. “These tiny ribbons are so thin, they could be free-floating,” Mr Brey says. “So, that means that the environment around the RMC ultramicrotome had to be especially free of air movement and static charges, which the chamber and ionizer successfully provided.” The work was performed on samples frozen in the lab of Daniele Spehner from the Institute of Genetics and Molecular and Cellular Biology. Colleague Caroline Kizilyaprak from Bruno Humbel’s lab at the University of Lausanne, and Greg Becker of RMC performed the sample trimming and sectioning, using a Diatome 35-degree diamond knife. Contact: Boeckeler Instruments Inc. www.rmcproducts.com

X-Ray Inspection Solution

The new phoenix x|aminer from GE’s Inspection Technologies business is a 5-axis, microfocus X-ray inspection system, which has been developed for quality control applications in the manufacture of electronics sub-assemblies. It is particularly suitable for the reliable and accurate inspection of soldered joints. The phoenix x|aminer features two megapixel high resolution and high magnification. In addition, its powerful imaging software permits intuitive programming and component manipulation is precise and easy using a computer mouse or joystick. An important design feature of the new system is its OVHM (oblique view high magnification) module, which allows an oblique angle view of up to 70° as well as a total magnification of up to >23,000⫻. This ensures best possible quality of defect information in the vertical direction. Ease of maintenance is also permitted with the system’s open, 160 kV microfocus tube design, whose easy cathode replacement ensures unlimited operating life, while its 20 W of tube power at target can penetrate even the most radiation-absorbing components. The new x|aminer is supplied with the recently launched phoenix x|act software, which is designed specially for electronics inspection. Contact: GE Energy Services www.ge-mcs.com

Prior_LumenLED_M&A_AD264x92_Layout 1 28/06/2011 09:24 Page 1

Quo rum

Tomorrowâ&#x20AC;&#x2122;s EM coating technology today . . .

Quorum Q series

> Fully automatic, touch screen control > Turbo or rotary pumped > Sputtering or carbon evaporation > Large specimen capability (wafers) > Dual head version (for sequential coating)

> Three year warranty

Q300

NEW Quorum Technologie s Quor um Technologies +44 (0)1233 646332

www.quorumtech.com

CIRCLE NO. 34 OR ONLINE: www.microscopy-analysis.com

CIRCLE NO. 40 OR ONLINE: www.microscopy-analysis.com

> Glow discharge, FTM and other options

Introducing LumenLEDâ&#x20AC;Ś Superior stability is just one of the advantages of LumenLED, an advanced illumination system for fluorescence microscopy. Thanks to a closed-loop optical feedback mechanism, LED intensity is continuously monitored to ensure constant illumination over hours, days and even longer! It also ensures reproducibility from one experiment to the next. For stability, fast switching times and ease of use choose LumenLED! For further information visit the web at www.prior.com or email a brochure request to uksales@prior.com

Focussed on microscopy Prior Scientific Instruments Cambridge CB21 5ET UK Telephone +44 (0)1223 881711

www.prior.com

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

200 kV Transmission Electron Microscope A new 200 kV transmission electron microscope from JEOL delivers high throughput nano-analysis for process and quality control of mass produced semiconductor and materials samples. The multi-function JEOL JEM-2800 features high resolution imaging in TEM, STEM, and SE modes; ultrasensitive elemental mapping with a large angle Energy Dispersive Spectrometer (EDS); Electron Energy Loss Spectroscopy (EELS) for chemical analysis; critical dimension analysis; tomography; and in situ observation of samples. The all-new TEM functions without use of the traditional fluorescent screen on the electron column. The JEM-2800 speeds specimen observation through fully automatic functions including adjustment of focus, astigmatism, contrast, brightness, crystal zone axis alignment, and height. Switching between analysis modes is seamless, and quick data collection shortens turnaround time between samples. An operator navigation system and on-screen operating guide make the JEM-2800 a high throughput, user-friendly TEM for any skill level. Additional features and key specifications of the JEM-2800 include a Schottky field-emission electron gun, highly stable eucentric side-entry goniometer stage, a magnification range of 100⫻ to

150,000,000⫻ using STEM, 0.1 nm TEM resolution and 0.20 nm BF/DF STEM resolution. The JEM-2800 is JEOL’s latest addition to its comprehensive lineup of 100-300kV TEMs and most recently the ARM200F atomic resolution TEM. The first US customer, a global semiconductor manufacturer, will take delivery of the new JEM-2800 with large solid angle EDS this summer. Contact: JEOL USA, Inc. www.jeolusa.com

CIRCLE NO. 35 OR ONLINE: www.microscopy-analysis.com

Widefield Superresolution Microscope With the new Leica SR GSD from Leica Microsystems, scientists can now achieve resolutions far below the limit of diffraction that have never been attained before in widefield fluorescence microscopy. The system is capable of resolving details as small as 20 nm. The SR GSD is based on GSDIM (ground state depletion followed by individual molecule return) technology exclusively licensed from MPI Goettingen. It has already delivered amazing results in scientific experiments during its test phase. One of the key advantages of the GSDIM method is that it can be used with conventional fluorescence labels routinely applied in fluorescence imaging applications. GSDIM provides the highest resolution possible with a light microscope today, almost equalling that of an electron microscope. The Leica SR GSD is based on a fully automated TIRF

system. Combining the benefits of super-resolution with TIRF microscopy is one of many options. The system can also be used for a wide range of applications in all areas of live cell microscopy and high-end fluorescence microscopy. As a flexible, multifunctional system, the Leica SR GSD gives researchers the freedom to tailor the system exactly to their needs. Contact: Leica Microsystems www.leica-microsystems.com

Web Microscopy 2.0 We live in the midst of Web 2.0 and Cloud computing revolution. Today you can store documents, images and music on the Cloud and access them from any web browser; you can share data instantly and collaborate in real time. If we can do so many things online, why not image analysis? With proliferation of high-speed internet and digital imaging, may be it is time to bring web 2.0 technology to microscopy? One aspiring technology startup thinks so: Smart Imaging Technologies, a VC-backed technology venture from Texas, pledges to bring Web 2.0 technology to microscopy with Simagis Live cloud software. “We want to bring power and convenience of web applications to microscopy and microanalysis, and make web technology available to millions of microscope users around the globe. If you have digital camera and Internet- you are ready for Web Microscopy,” says company founder Vitali Khvatkov. With Web Microscopy, a small software utility on your desktop will automatically upload your digital images to a cloud server so you can work with them online via a web interface. If you have automated microscope stage, you can stream individual image

tiles to the server for stitching and creating mosaic images or virtual slides, to view your entire specimen at full resolution online. Once your digital images or virtual slides are uploaded, they can be viewed, annotated and measured by any number of people at the same time from any computer or web tablet. You can upload composite images with several fluorescence channels and view them online with overlays. Web Viewers utilize zoom technology similar to Google Maps and allow panning and zooming on virtual slides of unlimited size. Contact: Smart Imaging Technologies Company www.live.simagis.com

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

W H AT ’ S N E W

Large Area EDS Detector for S/TEM Bruker has introduced the XFlash 5060 T, the latest addition to the XFlash family of detectors for X-ray micro- and nanoanalysis (EDS) in electron microscopy. The XFlash 5060 T is one of two detectors available for use on (scanning) transmission electron microscopes. Providing 60 mm² active area, it guarantees optimum solid angle for the analysis at low beam currents and of samples with low X-ray yield. The slender detector end cap and microscopespecific collimator design allow shortest detector-sample distances and provide a high take-off angle without requiring sample tilt. Compared to Si(Li) detectors, still commonly used for EDS on TEM, the XFlash 5060 T exhibits superior speed and drastically lower dead times, providing significant advantages in collection efficiency, even in low count rate situations. Additionally, the XFlash 5060 T can operate with good energy resolution at count rates far beyond what any Si(Li) or even competing SDD can handle on TEM. The detector is fully operational at input count rates of up to 750,000 cps, a big advantage for low-mag high count rate STEM mapping. Also, there is no danger of ‘locking up’ the spectrometer for minutes when hitting a support grid. The superb sensitivity of the XFlash 5060 T allows detection of high energy radiation. In combination with high-end signal processing electronics and sophisticated software, reliable quantitative analysis of element peaks at 40 kV and above is possible.

? 7 I R W S J E V A

'SRJSGEP ERH -RXIVJIVSQIXVMG 3TXMGEP 4VS´PIV Bruker‘s many years of experience in SDD and signal processing electronics design, have led to the outstanding energy resolution of the XFlash 5060 T at only moderate cooling temperatures, enabling reliable and efficient light element analysis of elements down to boron. The XFlash 5060 T, including electronics, is designed to cause minimum interference with all compatible transmission electron microscopes, conventional or aberration-corrected. Light-weight and with passive cooling, this detector causes minimal strain on the column and introduces no vibrations. The low temperature gradient provides stable measurement conditions and the completely non-magnetic detector head minimizes beam shift, when moving the detector in or out during TEM operation. Contact: Bruker Nano GmbH www.bruker-nano.com

Chemical Analysis in the S/TEM

Sensofar Plu neox for research and industry Confocal and Interferometer modes in the same instrument Color CCD camera White and blue LED light sources Vertical resolution down to 0.01 nm Lateral resolution down to 140 nm

FEI, a leading instrumentation company providing electron microscope systems for applications in research and industry, has announced the release of its Titan G2 80-200 with ChemiSTEM technology, a new member of the Titan G2 series of S/TEM (scanning / transmission electron microscopes). “By combining the Titan platform’s latest generation of electron optics with the revolutionary analytical sensitivity of ChemiSTEM Technology, we have created a microscope which can deliver atomic resolution elemental maps in minutes and adds new capabilities in addressing our customer’s applications in materials science, chemistry and nanotechnology,” said Trisha Rice, vice president and general manager of FEI’s Research Business Unit. Dr Paul Kotula of Sandia National Laboratories, said: “Our institute chose the FEI Titan G2 80-200 due to its innovative combination of the latest in probecorrection technology and large solid-angle, windowless silicon-drift X-ray detectors (SDDs). We esti-

mate that we will gain a factor of 50 to 100 in terms of analytical sensitivity, speed, and spatial resolution combined, over our existing FEG analytical electron microscope. It is already clear that atomic resolution Xray microanalysis is not only possible but practical with this new microscope. Once the domain only of electron energy loss spectrometry, atomic resolution microanalysis with x-rays gives us access to more of the periodic table and the possibility to use existing quantification methods to routinely analyze many materials at the highest resolution and sensitivity needed.” Contact: FEI Company www.fei.com/chemistem

We also supply

CIRCLE NO. 36 OR ONLINE: www.microscopy-analysis.com

GaAs Atomic EDX in the [110] crystal projection showing 1.4 Angstroms Ga-As dumbbells obtained with Titan G2 80-200 with ChemiSTEM technology. The GaAs [110] dumbbell splitting of 0.14 nm is clearly resolved by chemical mapping using energy dispersive X-ray analysis with a Titan G2 80-200 with ChemiSTEM technology and a probe Cs-corrector at 200 kV acceleration voltage, using a 200 pA probe current. On the right hand side the atomic structure of GaAs the [110] projection is shown along with the grayscale HAADF-STEM image. This represents the highest resolution ever obtained in atomic elemental mapping by any technique using an S/TEM.

AFMs for research, industry and teaching Nano-/Micro- Indenter and Tribology Tester, Nanoparticle size measurement, LEED/AES

Contact your nearest office Germany

info@schaefer-tec.com +49 6103 300 980

France

info@schaefer-tech.com +33 1 6449 6350

South-East Europe

see@schaefer-tec.com +39 0425 460 218

Switzerland

ch@schaefer-tec.com +41 34 423 7070

www.schaefer-tec.com

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

A N D

A N A L Y S I S

BUYER’S GUIDE For LM, SPM, EM, Bold entries are the standard types of instruments; other entries are the modes in which they are used. Use this also for web page cross referencing. C00 COMPOSITIONAL ANALYSIS L00 LIGHT MICROSCOPY C02 Calibration devices and standards L01 Transmitted light microscopes C03 Cathodoluminescence L02 Reflected light microscopes C04 Electron backscatter diffraction L03 Stereomicroscopes (EBSD) L04 Acoustic C05 Electron beam induced current L05 Confocal (EBIC) L06 Differential interference contrast C06 Electron diffraction, convergent L08 Field and mobile microscopes beam electron diffraction (ED,CBED) L10 Fluorescence lifetime C07 Electron energy loss spectroscopy, L11 Fluorescence resonance electron spectroscopic imaging energy transfer (EELS, ESI) L12 Fluorescence spectroscopy C08 Energy and wavelength L13 High resolution imaging (4Pi, STED) dispersive X-ray spectroscopy L14 Infrared (EDX, WDX) L15 Interference C09 Mass spectrometry (MS, SIMS) L16 Modulation contrast C10 X-ray diffraction, X-ray fluorescence, L17 Multiphoton X-ray photoelectron spectroscopy L18 Multispectral imaging (XRD, XRF, XPS) L19 Phase contrast L20 Polarization D00 DIGITAL IMAGING AND L21 Profilometry and metrology ANALYSIS L22 Raman D01 CCD cameras L23 Total internal reflection fluorescence D02 CMOS cameras L24 Ultraviolet D04 Frame grabbers L30 LM ACCESSORIES AND SUPPLIES D05 Image analysis hardware L31 Autofocusing devices D06 Image analysis software L32 Calibration devices and standards D07 Image archiving and reporting L33 Condensers D09 Image intensifiers L35 Environmental chambers D10 Machine vision systems L36 Eyepieces D11 Metrology L37 Filters, beamsplitters, polarizers D12 Particle counting L38 Lamps, Illuminators D13 Stereoscopic display L39 Lasers, LEDs D14 Video cameras L40 Micromanipulators and D15 Video processors microinjectors L41 Objectives P00 PHOTOGRAPHIC EQUIPMENT L42 Optics AND SUPPLIES L43 Scanning heads P01 Chemicals, film and paper L44 Stages L45 Stands F00 SPECIMEN PREPARATION EQUIPMENT AND SUPPLIES E00 ELECTRON, ION AND X-RAY F01 Coating units MICROSCOPES F02 Critical point dryers E01 Transmission electron microscopes F03 Cryofixation and cryosubstitution E02 Scanning electron microscopes devices E03 Ion beam microscopes F05 Cryostats E05 X-ray microscopes F07 Cutting, grinding, polishing and E07 Analytical TEM thinning devices E08 Atom probes F08 Cytochemical, immunochemical E09 Auger microscopes and in-situ probes E10 Cryoelectron microscopes F09 Electrolytic thinning E11 Dual beam microscopes F11 Fixatives, stains and chemicals E12 Energy filtering TEM F12 Freeze drying equipment E13 Environmental and variable F13 Freeze etch and fracture units pressure SEM F14 Glass/steel/diamond knives E14 Helium ion microscopes F15 Gold probes E15 High and intermediate voltage F16 Grids electron microscopes F17 Histology equipment and supplies E17 Low energy electron microscopes F18 Ion beam etching and thinning E18 Microprobes F19 Ion beam milling E19 Nanofabrication,nanolithography F20 Ion beam sputter coating E21 Secondary ion microscopes F21 Knifemakers E22 Tomography F22 Microtomes E23 X-ray microtomography F23 Microwave processing E30 EM ACCESSORIES AND SUPPLIES F24 Plasma cleaning E32 Anti-contamination systems F25 Plasma etching E33 Anti-vibration systems F26 Reactive ion beam etching E34 Apertures and filaments F27 Section stainers E35 Calibration standards F28 Tissue processors E36 Cryotransfer systems F29 Ultramicrotomes E37 Energy filters F30 Vibratomes E38 Magnetic field cancellation E39 Specimen holders for TEM G00 GENERAL MICROSCOPY EQUIPMENT, E40 Spectrometers (EDX, WDX, EELS) SUPPLIES AND SERVICES E41 Stages for SEM G01 Anti-vibration systems E42 Vacuum equipment G04 Image analysis service E44 Plasma cleaners G05 Maintenance contracts and servicing E45 Anti-contaminators G06 Microhardness and failure testing G07 Thermal analysis M00 SCANNING PROBE MICROSCOPES G08 Training M01 Scanning tunnelling microscopes G09 Used equipment M02 Atomic force microscopes G10 Workshops for microscopists M03 Nearfield scanning optical microscopes H00 SOCIETIES M04 Specialized SPMs H01 CONFERENCES, COURSES, AND M05 Dual mode (LM/SEM/TEM) SPMs EXHIBITIONS M06 SPM Nanolithography H03 RECRUITMENT SERVICES M10 SPM ACCESSORIES AND SUPPLIES M11 Calibration devices and standards M12 Cantilevers M13 Scanners M14 Stages M15 Tips

www. microscopy-analysis.com 40

M I C R O S C O P Y A N D A N A LY S I S N O V E M B E R 2 0 1 1

CIRCLE NO. 37 OR ONLINE: www.microscopy-analysis.com

ADVERTISERS INDEX Page

Company Name

S2 8 16 35 5 S15 31 32 30 S9 S1 28 43 6 25 35 14 S4 13 S10 S9 24 40 38 18 27 S22 S31 1 2 20 S16 34 33 37 37 39 44 S18 S22 S32

Agilent Andor APMC 2012 Applied Scientific Instrumentation Asylum Research Bruker GmbH Bruker GmbH Bruker Nano Cargille E2V FEI Company FEI Company FEI Company Gatan Herzan Hiden Hitachi Europe GmbH Hitachi Europe GmbH Jeol UK Jeol UK John Wiley & Sons Ltd. John Wiley & Sons Ltd. John Wiley & Sons Ltd. JPK Instruments Leica Leica Märzhäuser Wetzlar GmbH NT-MDT Olympus Europa Holding GmbH Olympus Europa Holding GmbH Olympus Soft Imaging Solutions GmbH Oxford Instruments Physik Instrumente Picoquant Prior Scientific Quorum Technologies Schaefer-Tec Skyscan Thermo Scientific XEI Scientific, Inc. Carl Zeiss

Reader Enq 12 5 8 32 3 17 28 29 27 14 11 26 38 4 40 33 7 6 16 16 15 24 37 35 9 25 20 22 1 2 10 18 31 30 34 40 36 39 19 21 23

NEW Reader card ALL_Layout 1 17/10/2011 12:42 Page 1

Please register me as a NEW READER

Please RE-REGISTER me My USER NO is (see address label)

Visit our website: www.microscopy-analysis.com

A N D

Title

A N A L Y S I S

Prof Dr Ing Mr Ms Mrs Other

TO REGISTER OR RE-REGISTER please fill in ALL sections on this page. TO SUBMIT PRODUCT ENQUIRIES please fill in your Name and Email details.

(please indicate)

First Names Last Names Job Title Organisation Name Department Name Organisation Address Please supply your full postal address remembering to include any Room Numbers, Post Box numbers or Mail Stop Codes.

We do not register home addresses.

City

State

Country

Postal Code

Telephone Number Fax Number

Email Address * * We regret that we are no longer able to accept paper registrations without an email address.

We periodically send registered readers e-newsletters about the content in the magazine as an aditional free information resource. If you do NOT wish us to email you please check this box. We may wish to send you emails from selected third-parties. If you would like to receive these emails please check this box

TECHNIQUE

INDUSTRIAL SECTOR Education: University Environmental Services Government Manufacturing Medical and Health Services Research and Development

MAIN INTERESTS Light Microscopy Electron Ion and XRay Microscopy Scanning Probe Microscopy

HOW DID YOU FIND US Colleague/Friend Conference/Exhibition Newswire Subscription Online advert Other website Press advert Search Engine Other

Light Microscopy

Compositional Analysis

11 12 14 15 16 18 60

26 EELS 33/34 EDX/WDX 35 ED/CBED 36 EBSD 38 XRD 39 XRF 41 AES 42 SIMS 44 FTIR 45 Raman 64 Cathodoluminescence

Transmitted/Reflection Stereo Confocal Multi Photon Fluorescence Polarised Digital/Slide Scanners

Electron Microscopy 20 21 22 24 25 61 62 63

TEM STEM SEM FIB Cryo EM EFTEM VPSEM Ion/Atom Probe/X-Ray

Scanning Probe Microscopy 28 STM 29 AFM 30 NSOM / SNOM

RESPONSIBILITY Approve equipment Purchase equipment

Specify equipment None of the above

JOB ROLE Professor/Lecturer PostDoc/Graduate Research Officer Scientist/Engineer Technician Forensics Military Sales/Corporate Role Librarian

SPECIALTY Earth Sciences Electronics Environment Food Technology Life Sciences Machine Vision Materials Science Medical Technology Quality Control Semiconductors

NEW Reader card ALL_Layout 1 17/10/2011 12:42 Page 4

www.microscopy-analysis.com

ONLY USE THIS CARD FOR ENQUIRIES FOR

UK/EUROPEAN MICROSCOPY AND ANALYSIS NOVEMBER/DECEMBER 2011 ENQUIRIES FOR MAGAZINE

Circle enquiry numbers for information

ENQUIRIES FOR BUYERâ&#x20AC;&#x2122;S GUIDE L00 L01 L02 L03 L04 L05 L06

Circle enquiry numbers for information

L07 L08 L09 L10 L11 L12 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L30 L31 L32 L33 L34 M00 M01 M02 M03 M04 M05 M10 M11 M12 M13 M14 M15

E00 E01 E02 E03

E04 E05 E06 E07 E08 E09 E10 E11 E12 E13 E14 E15 E16 E17 E18 E19 E20 E21 E22 E23 E30 E31 E32 E33 E34

E35 E36 E37 E38 E39

L35 L36 L37 L38 L39 L40 L41 L42 L43 L44 L45 L46 E40 E41 E42 E43 D13 D14 D15

C00 C01 C02 C03 C04 C05 C06 C07 C08 C09 C10 P00 P01 P02 P03 P04 P05

D00 D01 D02 D03 D04 D05 D06 D07 D08 D09 D10 D11 D12

F00 F01 F02 F03 F04 F05 F06 F07 F08 F09 F10

F19 F20 F21 F22 F23 F24 F25 F26 F27 F28 F29 F30

F11 F12 F13

G00 G01 G02 G03 G04 G05 G06 G07 G08 G09

F14 F15 F16 F17 F18

H00 H01 H02 H03

Please use this box to complete any comments for the magazine or additional enquiries

Fold here Fold here

Microscopy & Analysis Reader Services John Wiley & Sons, Ltd The Atrium Southern Gate Chichester West Sussex PO19 8SQ United Kingdom Affix Stamp Here DO NOT USE STAPLES

SEAL HERE

View, Analyze, & Create in 3D

View, Analyze, & Create in 3D with the most powerful FIB and SEM with the most powerful FIB and SEM Helios NanoLab™ 50 Series Helios NanoLab™ 50 Series

Surface of uncoated pollen, imaged using SEM at very Surface of uncoated pollen, low kV (50 V). imaged using SEM at very low kV (50The V). horizontal field width

is 51 field μm. Courtesy of The horizontal width FEI NanoPort. is 51 μm. Courtesy of FEI NanoPort.

Austenic-ferritic duplex

Ultra-stable,contamination contamination damage ••Ultra-stable, andand damage freeimaging imagingofofuncoated uncoated charging free charging or or beam-sensitive samples beam-sensitive samples ••Best preparation: precise Bestininclass classsample sample preparation: precise milling very lowlow kV kV millingofoflarge largevolume, volume, very polishing, process monitoring polishing, process monitoring ••Most integrated suite of of Mostcomplete completeand and integrated suite prototyping capabilities with SEM, FIB prototyping capabilities with SEM, FIB and andbeam beamchemistries chemistries

• Robust, reproducible and versatile • Robust, reproducible and versatile multi-signal 3D Slice and View automation

multi-signal 3D Slice and View automation

• Accurate and flexible sample positioning •and Accurate and flexible sample positioning handling

and handling

• Outstanding application and service support

Learnmore moreatatwww.fei.com/research www.fei.com/research Learn CIRCLE NO. 38

ONLINE: www.microscopy-analysis.com

Austenic-ferritic duplex steel, 16 x 12 x 18 μm3 steel, 16 x 12 x 18 μm3 volume acquired with the volume acquired with the AutoSlice and View™ AutoSlice and View™ application. A series of application. A series of top-down high energy, top-down high energy, angle SEM-BSE high anglehigh SEM-BSE images were collected images were collected automatically. The distance automatically. The distance between slice is 30 nm. between each slice iseach 30 nm. of FEI NanoPort. Courtesy ofCourtesy FEI NanoPort.

Platinum nanowire Platinum nanowire deposited and milled to

deposited and milled to

about 50 nm diameter for about 50 nm diameter for use as a gas sensor use as a gas sensor Courtesy of Peter Heard,

Courtesy of Peter Heard, Bristol University, Bristol University, United Kingdom. United Kingdom.

CIRCLE NO. 39

ONLINE: www.microscopy-analysis.com