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REGULARS4Dates for the Diary23People & Places26Literature Highlights29What's New41Reader Enquiry formMICROSCOPY AND ANALYSIS NOVEMBER 20113CONTENTSwww.microscopy-analysis.comSee the Microscopy and Analysis online Diary for a full listing. Entries for the January issue are due 1 December. Email to: dates for the diaryDIARYMICROSCOPY 4AND ANALYSIS NOVEMBER 2011NOVEMBER 20118-11GatanEELS School, Austrian Centre for Electron Microscopy and Nanoanalysis, TU Graz, Microscopy in Materials Science, Eindhoven University of Technology, The Netherlands 12-16Neuroscience 2011: Washington DC, USAwww.sfn.org14-18NvVM European School on Nanobeams , Centre de Recherche Public - Gabriel Lippmann, Belvaux, Luxembourg 16Cryo Microscopy Group Annual Meeting, Boots Science Building, School of Pharmacy, University Park, Nottingham University, Scanning Probe Microscopy Conference and Users' Meeting, University of Manchester, drew.murray@bruker-nano.com28-2/122011 MRS Fall Meeting, Boston, Massachusetts, USAwww.mrs.orgDECEMBER 20113-4Functional Optical Imaging, Univeristy of Nottingham Ningbo Campus, Society for Cell Biology 51st Annual Meeting, Denver, Colorado, USAwww.ascb.orgJANUARY 20124-7Winter School on High-Resolution Electron Microscopy, Arizona State University, Tempe, AZ, FEBRUARY 20125-9APMC 10: 10th Asia-Pacific Microscopy Conference; ICONN 2012: International Conference on Nanoscience and Nanotechnology; ACMM 22: Australian Conference on Microscopy and Microanalysis, Perth, www.acmm-22.org25-2956th Biophysical Society Annual Meeting, San Diego, California, MARCH 201211-16Pittcon 2012, Orlando, FL, USAwww.pittcon.org18-23Course in Cryotechniques for Electron Microscopy, Rothamsted Research, Harpenden, 20121-4Focus on Microscopy 2012; 25th International Conference on 3D Image Processing in Microscopy; 24th International Conference on Confocal Microscopy, Singaporewww.focusonmicroscopy.org9-13MRS 2012 Spring Meeting and Exhibition, San Francisco, CA, 2012: 23rd International Trade Fair for Laboratory Technology, Analysis and Biotechnology and analytica Conference, Munich Trade Fair Centre, Munich, Germanywww.analytica.deMAY 20129-11AMTC3: 3rd International Symposium on Advanced Microscopy and Theoretical Calculations, Nagaragawa Convention Center, 53rd International Field Emission Symposium, University of Alabama, Tuscaloosa, AL, 2012: 39th Annual Meeting of Society for Cutaneous Ultrastructure Research, Lyon, France JUNE 20123-15Microscopy School, Lehigh University, Bethlehem, PA, USASharon Coe: Introduction to SEM and EDS for the New Operator4-8 SEM and X-Ray Microanalysis11-14 Focused Ion Beam: Instrumentation and Applications11-15 Problem Solving with SEM, X-Ray Microanalysis and Electron Backscatter Patterns11-15 Quantitative X-Ray Microanalysis: Problem Solving with EDS and WDS Techniques11-15 Scanning Transmission Electron Microscopy: From Fundamentals to Advanced Applications25-27Abercrombie Cell Biology Symposium, Oxford, UKBSCB: www.bscb.org27-30CARS 2012: Computer Assisted Radiology and Surgery, 26th Intl Congress and Exhibition, Joint Congress of CAR / ISCAS / CAD / CMI / EuroPACS, Congress Palace, Pisa, 20122-6 Electron Microscopy Summer School, University of Leeds, UKRMS: 2-6 Light Microscopy Summer School, University of York, UKRMS: 4-6Optics Within Life Sciences (OWLS), Genoa, ItalyOrganised by Italian Institute of Technology www.owls2012.org9-13Inter/Micro: 63rd Annual Applied Microscopy Conference, Chicago, IL, USAwww.mcri.org11-13CryoElectron Microscopy Short Course, University of Minnesota, MN, USANanostructural Materials and Processes Program of IPrime: 29-2/8Microscopy & Microanalysis 2012, Phoenix, Arizona, USAwww.microscopy.orgAUGUST 20126-10ULTRAPATH XVI: Conference on Diagnostic Electron Microscopy, Basic Research and Oncology, Regensburg, Germanywww.ultrapath.org26-3014th International Congress of Histochemistry and Cytochemistry, Kyoto, Japan 2012 16-21European Microscopy Congress, Central Convention Complex, Manchester, 201213-17Neuroscience 2012, New Orleans, Louisiana, NOVEMBER 201226-302012 MRS Fall Meeting & Exhibition, Boston, Massachusetts, 201215-19 ASCB 52nd Annual Meeting, San Francisco, CA, USAwww.ascb.orgMARCH 201317-21Pittcon 2013, Philadelphia, PA, USAwww.pittcon.orgAPRIL 20131-52013 MRS Spring Meeting & Exhibition, San Francisco, CA, 20134-8Microscopy & Microanalysis 2013, Indianapolis, IN, USAwww.microscopy.orgNOVEMBER 20139-13Neuroscience, San Diego, California, USAwww.sfn.orgLITERATUREHIGHLIGHTSliterature highlights?? "?? '!?? ??  "A large array of sub-10-nm sin-gle-grain gold nanodots for usein nanotechnology is describedby Nicolas Clément and col-leagues at the Institut d'Elec-tronique Microélectronique etNanotechnologie, CNRS, Univer-sity of Lille, France [Small7(18):2607-2613, 2011].A uniform array of single-grain gold nanodots, as small as5-8 nm, was be formed on siliconusing e-beam lithography. Theas-fabricated nanodots wereamorphous, and thermal annealing converted themto pure Au single crystals covered with a thin SiO2layer. These findings were based on physical mea-surements by AFM, atomic-resolution STEM, andchemical techniques using energy dispersive X-rayspectroscopy. The authors demonstrated the forma-tion by e-beam lithography of sub-10-nm Au dotswith small dispersion and perfect alignment. Suchprecise formation of small dots enabled them to iden-(a) STEM image showing the bulk silicon (Si), five annealed dots (Au), carbon layer (C), and platinum layers. (c) Coloured STEM image of a single annealed nanodot (260°C, 2 h). Reproduced with permission, Copyright ©2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. tify the critical size that determines whether a dot iscomposed of single or multiple crystal domains.Moreover, they showed that annealing at moderatetemperature can convert Au dots from amorphous tosingle-crystalline, and then they were covered with athin SiO2layer. After easy removal of the SiO2(diluteHF etching), these nanodots can be used as electrodesfor the characterization of organic self-assembledmonolayers (SAMs) with less than 200 molecules.??"?? ??  &# !?? 'A miniature stage device to overcome resolutionanisotropy in fluorescence light microscopy isdescribed by Florian Staier and colleagues at theKirchhoff Institute for Physics, University of Heidel-berg, Germany [Rev. Sci. Instrum. 82:093701, 2011].To overcome the limitation of fluorescence micro-scopes in anisotropic optical resolution or point local-ization precision micro-axial tomography was usedwhich allowed object tilting on the microscope stageand led to an improvement in localization precisionand spatial resolution. A glass fiber was placed in theobject space of the microscope lens and its rotationcontrolled by a miniaturized stepping motor. By Testparticles were fixed onto the glass fiber, opticallylocalized with high precision, and automaticallyrotated to obtain views from different perspectiveangles from which distances of corresponding pairs ofobjects were determined. From these angle depen-dent distance values, the real 3D distance was calcu-lated with a precision in the ten nanometer range(corresponding here to an optical resolution of 10-30nm) using standard microscopical equipment. As aproof of concept, the spindle apparatus of a maturemouse oocyte was imaged during metaphase II mei-otic arrest under different perspectives.??%# ???? $??!A technique for the use of two fluorophores in stim-ulated emission depletion (STED) microscopy of livingcells is reported by Patrina Pellett and co-workers atthe Department of Cell Biology, Yale School of Medi-cine, CT [Biomedical Optics Express2(8):2364-2371,2011]. Current applications of STED microscopy havebeen limited to single colour imaging of living cellsand multicolour imaging in fixed cells. However, tostudy active processes, such as protein interactions, atwo-colour STED imaging technique is needed in liv-ing cells. This was achieved for the first time by theauthors: the key to their success was in overcomingthe challenges in labeling target proteins in livingcells with dyes optimal for two-colour STEDmicroscopy. By incorporating fusion proteins, theresearchers were able to improve the targetingbetween the protein and the dye, effectively bridgingthe gap. This allowed the researchers to achieve reso-lutions of 78 nm and 82 nm for 22 sequential two-colour scans of epidermal growth factor and its recep-tor in living cells. ?? "?? ??#"????! "?? The three-dimensional point spread function of anaberration-corrected scanning transmission electronmicroscopy (STEM) has been simulated and experi-mentally tested by Andrew Lupini and Niels de Jongeat the Oak Ridge National Laboratory, TN [Microscopyand Microanalysis17:817-826, 2011].Aberration correction reduces the depth of field inSTEM and thus allows three-dimensional imaging bydepth sectioning. This imaging mode offers thepotential for sub-Ångstrom lateral resolution andnanometer-scale depth sensitivity. For biological sam-ples, which may be many ?m across and where highlateral resolution may not always be needed, opti-mizing the depth resolution even at the expense oflateral resolution may be desired, aiming to imagethrough thick specimens. Although there has beenextensive work examining and optimizing the probeformation in two dimensions, there is less knownabout the probe shape along the optical axis. The authors examined the probe shape in 3D in anattempt to better understand the depth resolution in ??'!!??????"??"??"!A fractal dimension analysis and mathematical mor-phology of structural changes in actin filamentsimaged by electron microscopy is reported by Yoshi-taka Kimori et al at the National Institutes of NaturalSciences, in Tokyo [Journal of Structural Biology176(1):1-8, Oct 2011].The authors examined structural changes of actinfilaments interacting with myosin visualized by quickfreeze deep-etch replica EM by using a new methodof image processing and analysis based on mathe-matical morphology. To quantify the degree of struc-tural changes, two characteristic patterns wereextracted from the EM images: the winding patternof the filament shape (WP) reflecting flexibility of the"?? ?? ??!??#"#??!??A. Beltrán and colleagues at the Department of Mate-rials Sciences, University of Cadiz, Spain, report thatthree dimensional atom tomography resolves thequantum ring morphology of self-assembled GaSbburied nanostructures [Ultramicroscopy111(8):1073-1076, July 2011].Unambiguous evidence of ring-shaped self-assem-bled GaSb nanostructures grown by molecular beamepitaxy is presented on the basis of atom-probetomography reconstructions and darkfield transmis-sion electron microscopy imaging. From atom-probetomography compositional distribution has beenobtained. The GaAs capping process causes a strongsegregation of Sb out of the center of GaSb quantumdots, leading to the self-assembled GaAsxSb1-x quan-tum rings of 20-30 nm in diameter with x~0.33.filament, and the surface pattern of the filament (SP)reflecting intramolecular domain mobility of the actinmonomers in the filament. EM images were processedby morphological filtering followed by box-countingto calculate the fractal dimensions for WP (DWP) andSP (DSP). The result indicated that DWP was largerthan DSP irrespective of the state of the filament(myosin-free or bound) and that both parameters formyosin-bound filaments were significantly largerthan those for myosin-free filaments. This work is thefirst quantitative insight into how conformational dis-order of actin monomers is correlated with themyosin-induced increase in flexibility of actin fila-ments along their length.this mode. They present examples of how aberrationschange the probe shape in three dimensions, and itwas found that off-axial aberrations may need to beconsidered for focal series of large areas. It was shownthat oversized or annular apertures theoreticallyimprove the vertical resolution for three-dimensionalimaging of nanoparticles. When imaging nanoparti-cles of several nanometers in size, regular scanningtransmission electron microscopy can thereby be opti-mized such that the vertical full-width at half-maxi-mum approaches that of the aberration-correctedSTEM with a standard aperture.26MICROSCOPY AND ANALYSIS NOVEMBER 2011CIRCLENO. 1 ORONLINEwww.microscopy-analysis.comThe cover shows a confocal image (top) of aDrosophilaembryo at stage 11, expressing the trachealmarker trh-LacZ (Cy3, red) and the cell membranemarker Dlg (Alexa 488, green). The enlarged view(below) shows invaginating tracheal placodes in X-Y(left) and Y-Z (right) projections. (Courtesy of DrTakefumi Kondo and Dr Shigeo Hayashi at theLaboratory for Morphogenetic Signalling, RIKEN Centrefor Developmental Biology, Japan.)The new UPLSAPO30xS and UPLSAPO60xS siliconoil-immersion objectives from Olympus are ideal for livecell experiments investigating thick samples or requiringlong-term imaging. Users can generate bright images athigher resolutions, since silicon oil significantly improvesoptical performance compared to water-based methodsand offers larger working distances than oil immersionobjectives. This maximises the effectiveness ofbrightfield, differential interference contrast,fluorescence, confocal laser scanning and multiphotonstudies. Silicon oil allows users to capture bright, highresolution images of living samples, even deep into cellsand tissues. This can be achieved because the refractiveindex of silicon oil (n = 1.40) is almost identical to thatof living biological samples (n =1.38 on average) sosilicon oil-immersion objectives minimise refractive indexmismatch and spherical aberration. This is a key factorfor improving the focal spot, which can be even furtheroptimised to correct for temperature changes byadjusting the available correction collar. The propertiesof silicon oil also make it ideal for long-term, time-lapsestudies since it is stable, viscous and of low volatility; itwill not dry out or absorb moisture over time. With its 30x magnification and high NA of 1.05, theUPLSAPO30xS delivers highly resolved images of anextensive sample area. The Olympus UPSLAPO60xSprovides increased magnification (60x) and NA (1.30),allowing researchers to produce highly detailed livesample images using fluorescence, confocal laserscanning and multiphoton excitation techniques. Thisobjective is also ideal for high resolution 3D imagingapplications by offering improved spherical aberrationcorrection and a shorter working distance of 0.3 mm.Please contact:OLYMPUS EUROPA HOLDING GMBHKatja AnsmannMarketing Communications ManagerTel: +49 40 2 37 73 - 5913Fax: +49 40 2 37 73 - 4784FEATURES9Bonding and TSV in 3D IC Integration: Physical Analysis witha Plasma FIB M M V Taklo, A Klumpp, P Ramm,L Kwakman and G Franz15Fracture Surface, Impact Energy and Hardness of Ni-FreeHigh-Mn Steels W Sha, H Haji Talib, E Wilson, R Rajendran,S Malinov, H Charlesworth, L Ibbitson21Variable Pressure Scanning Electron Microscopy of Viciafaba Stigmatic Papillae W Chen, F Stoddard and T BaldwinS5Atomic Resolution Secondary Electron Imaging in AberrationCorrected STEM H Inada, M Konno, K Tamura,Y Suzuki, K Nakamura, Y ZhuS11Development of a High Throughput Electron Microscope forNanoscale Analysis M Matsushita, S Kawai, T Iwama, K Tanaka,T Kuba, N Endo, T IsabellS19Low Beam-Energy Energy-Dispersive X-Ray Spectroscopy forNanotechnology P CamusS23Product Focus - Nanotechnology and Electron MicroscopyCOVER STORYMICROSCOPY & ANALYSISISSN 0958-1952 - UKISSN 2043-0655 - Europe© 2011 John Wiley & Sons, LtdIssued in: January, March, May, July, September, NovemberPublished by: John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, UK Tel: +44 (0)1243 770257 Fax: +44 (0)1243 770432Email: Website: www.microscopy-analysis.comAssociate Editorial Director: Dr Ray J. 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Taylor Assistant Editor: Samantha Moore samamoore@wiley.comEDITORIAL BOARDJim Bentley, Oak Ridge National Lab, TN, USAEd Boyes,University of York, UKRay Carpenter, Arizona State University, AZ, USAChristian Colliex,CNRS Physique des Solides, FranceAlby Diaspro, University of Genoa, ItalyPeter Hawkes,CNRS, Toulouse, FranceColin Humphreys,University of Cambridge, UKCornelis van Noorden,University of Amsterdam, The NetherlandsJim Pawley, University of Wisconsin, WI, USAJohn Spence,Arizona State University, AZ, USANestor Zaluzec, Argonne National Lab, IL, USANON-USAreturns should be sent to Microscopy and Analysis, Reader Services, JohnWiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ,United KingdomUS POSTMASTER:Send address corrections to Microscopy and Analysis, ReaderServices, c/o Mercury Airfreight International Ltd, 365 Blair Road, Avenel NJ 07001-9871 USA.Periodicals Postage Paid at Rahway NJ.©2011 John Wiley & Sons, Ltd. 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