Biological low voltage field emission scanning electron microscopy PDF
Preview Biological low voltage field emission scanning electron microscopy
Biological Low-Voltage Scanning Electron Microscopy Edited by Heide Schatten Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, Missouri, USA James B. Pawley Department of Zoology, University of Wisconsin, Madison, Wisconsin, USA HeideSchatten JamesB.Pawley DepartmentofVeterinaryPathobiology DepartmentofZoology UniversityofMissouri-Columbia UniversityofWisconsin 1600E.RollinsStreet 250N.MillsStreet Columbia,MO65211 Madison,WI53706 USA USA [email protected] [email protected] LibraryofCongressControlNumber:2007931613 ISBN978-0-387-72970-1 e-ISBN978-0-387-72972-5 (cid:2)c 2008SpringerScience+BusinessMedia,LLC Allrightsreserved.Thisworkmaynotbetranslatedorcopiedinwholeorinpartwithoutthewritten permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY10013,USA),exceptforbriefexcerptsinconnectionwithreviewsorscholarlyanalysis.Usein connectionwithanyformofinformationstorageandretrieval,electronicadaptation,computersoftware, orbysimilarordissimilarmethodologynowknownorhereafterdevelopedisforbidden. Theuseinthispublicationoftradenames,trademarks,servicemarksandsimilarterms,eveniftheyare notidentifiedassuch,isnottobetakenasanexpressionofopinionastowhetherornottheyaresubject toproprietaryrights. Printedonacid-freepaper. 9 8 7 6 5 4 3 2 1 springer.com Preface Eversinceitsadventinthe1950s,thescanningelectronmicroscopehsofferedan imageofsurfacestructuresthatwasbothuniquelydetailedandeasilyinterpretable. Although initially, the resolution of the images it produced did not match that of thosemadeusingthetransmissionelectronmicroscope,thisdeficitnolongerrele- vantonbiologicalspecimenswhere,forbothinstruments,imagequalityofisnow limitedfundamentallybythefragilityofthespecimen. High-resolution, low-voltage scanning electron microscopy (LVSEM) is now a powerful tool to study biological structure. Particularly when coupled with novel specimen preparation techniques, it has allowed us to understand in three dimen- sions objects that previously could only be imaged from serial-sectioned material analyzedwithtransmissionelectronmicroscopy. Because LVSEM provides images with clear topographic contrast from spec- imens coated with only an extremely thin metal coating, it can provide high- resolutionimagesofmacromolecularcomplexesandofstructuralinteractionsthat are free from the confusion of structural overlap. What is more, it provides this analysis in the context of being able to view the large specimen areas needed to providecontext. These capabilities are particularly useful for applications in cellular biology. In addition,specificmolecularcomponentsonsurfacesandinternalcellstructurescan be identified by using colloidal-gold labeling techniques. Particular internal struc- tures can be viewed either by isolating them or by using novel fracturing and sec- tioningtechniquesthatcausetheinternalcomponentstooccurontheoutersurface of the specimen. There, the LVSEM can image them with a degree of topological precisionthatisoftennotpossiblewithconventionalTEM. Giventhesetremendouscapabilities,itseemedtousbothsurprisingandunfor- tunate that that LVSEM was not used more often to study biological structure. In a time when interest is extending from the genome to the proteome and when we increasingly want to know not only the existence but also the localization and interactions of specific molecules and molecular complexes, it seemed to us that LVSEM was the ideal modality for answering a myriad of important questions in cellular biology and development. What seemed to be needed was a way to make potentialusersmoreawareofLVSEM’suniqueandpowerfulcapabilitiesandalso toprovidethereaderwithbothmeaningfulexamplesfromavarietyofapplications andsuitableprotocolsforpreparingspecimens. v vi Preface Weapproachedanumberofleadersinthefieldwiththisideaandreceivedamost enthusiastic response. The topics chosen were selected to be of interest to scien- tists,technicians,students,teachers,andtoallwhoareinterestedinexpandingtheir knowledgerelatedtoLVSEM.Thespecifictopicscoveredinthisbookincludehigh- resolution LVSEM applications to cellular biology and detailed specimen prepa- ration techniques for molecular labeling and correlative microscopy, cryoSEM of biological samples, and new developments in LVFESEM instrumentation in x-ray microanalysisatlowbeamvoltage. Biological Low Voltage Scanning Electron Microscopy covers many aspects of specimenpreparationandprovidesspecificprotocolsforpracticalapplicationsthat are commonly not available in research papers. It also gives general as well as detailed insights into the theoretical aspects of LVSEM. The book is intended for alargeaudience asareference bookonthesubject.Byproviding boththeoryand practicalapplications relatedtoimaging biological structureswithLVFESEM, we hopethatitwillfillagapintheliterature. During the editing process of this book, two of our most treasured colleagues, who have advanced the field immensely, passed away. Both the late Dr. Hans Ris andthelateDr.StanleyErlandsenwerepassionateabouttheusefulnessofLVSEM to enhance their own research, and as such, they left a wealth of new knowledge, noveltechniques,andideasfornewapplicationsforthescientificcommunity.Their contributionsareofgreatvaluetofuturescientist,students,technicalstaff,andmany otherusingLVSEM. Theeditorsaremostgratefultoallauthorswhohavecontributedtheirsuperband uniqueexpertisetothisprojectandsharedtheirinsightswiththepresentcommunity interestedinmicroscopyandthosewhowillenterthefieldinthefuture. We would like to thank Kathy Lyons, our ever-so-patient editor at Springer. In addition, one of the editors (JP) would also like to thank Bill Feeny, the Zoology Departmentalartist,andKandisElliot,theBotanyDepartmentartist,fortheirhelp inpreparingthefigures. HeideSchatten,Columbia,MissouriUSA JimPawley,Madison,WisconsinUSA Contents Preface............................................................ v ListofContributors ................................................ ix 1 TheEarlyDevelopmentoftheScanningElectronMicroscope DennisMcMullan............................................. 1 2 LVSEMforBiology JamesB.Pawley.............................................. 27 3 TheAberration-CorrectedSEM DavidC.Joy................................................. 107 4 NoiseandItsEffectsontheLow-VoltageSEM DavidC.Joy................................................. 129 5 High-Resolution,LowVoltage,Field-EmissionScanningElectron Microscopy (HRLVFESEM) Applications for Cell Biology and SpecimenPreparationProtocols HeideSchatten ............................................... 145 6 Molecular Labeling for Correlative Microscopy: LM, LVSEM, TEM,EF-TEMandHVEM RalphAlbrechtandDarylMeyer................................. 171 7 LowkVandVideo-Rate,Beam-TiltStereoforViewingLive-Time ExperimentsintheSEM AlanBoyde.................................................. 197 8 Cryo-SEMofChemicallyFixedAnimalCells StanleyL.Erlandsen .......................................... 215 9 High-ResolutionandLow-VoltageSEMofPlantCells GuyCox,PeterVesk,TeresaDibbayawan,TobiasI.Baskin,andMaret Vesk....................................................... 229 vii viii Contents 10 High-ResolutionCryoscanningElectronMicroscopyofBiological Samples PaulWalther ................................................. 245 11 DevelopmentsinInstrumentationforMicroanalysisinLow-Voltage ScanningElectronMicroscopy DaleE.Newbury ............................................. 263 Index .............................................................305 List of Contributors RalphAlbrecht DavidC.Joy DepartmentofAnimalSciences Science and Engineering Research DepartmentofPediatrics Facility DivisionofPharmaceuticalSciences UniversityofTennessee UniversityofWisconsin-Madison Knoxville,Tennessee,USA Madison,Wisconsin,USA DennisMcMullan 59CourtfieldGardens TobiasI.Baskin LondonSW50NF,UK BiologyDepartment UniversityofMassachusetts DarylMeyer Amherst,Massachusetts,USA DepartmentofAnimalSciences UniversityofWisconsin-Madison AlanBoyde Madison,Wisconsin,USA BiophysicsOGD,QMUL DaleE.Newbury DentalInstitute Surface and Microanalysis Science London,UK Division National Institute of Standards and GuyCox Technology ElectronMicroscopeUnit Gaithersburg,Maryland,USA UniversityofSydney NSW,Australia JamesB.Pawley DepartmentofZoology TeresaDibbayawan UniversityofWisconsin ElectronMicroscopeUnit Madison,Wisconsin, UniversityofSydney USA NSW,Australia HeideSchatten DepartmentofVeterinaryPathobiology StanleyL.Erlandsen UniversityofMissouri-Columbia (Deceased) Columbia,Missouri,USA DepartmentofGenetics CellBiologyandDevelopment PeterVesk University of Minnesota Medical LecturerintheSchoolofBotany School UniversityofMelbourne Minneapolis,Minnesota,USA Melbourne,AU ix x ListofContributors MaretVesk PaulWalther ElectronMicroscopeUnit CentralElectronMicroscopyUnit UniversityofSydney UniversityofUlm NSW,Australia Ulm,Germany Chapter1 The Early Development of the Scanning Electron Microscope DennisMcMullan It has been forty years since the scanning electron microscope (SEM) became a significant instrument in the scientific community. In 1965, the Cambridge Instru- ment Company in the United Kingdom marketed their Stereoscan 1 SEM, which wasfollowedabout6monthslaterbyJEOLofJapanwiththeJSM-1.Before1965, there were about thirty years of intermittent SEM development in Germany, the UnitedStates,England,andJapan,althoughJapanesedevelopmentwasapparently notcoveredinthepublishedliterature.Developmentbeganinthe1930sinGermany, andthenbegantowardstheendofthatdecadeintheUnitedStates. During these early years, there were two different approaches: the first, which had some specific relevance to the low voltage scanning transmission microscope (LVSEM);andthelateronethatwaslinkedtothetransmissionelectronmicroscope (TEM) and led to the scanning transmission electron microscope (STEM) and the futureformofthecurrentSEM.Butfirst,wemustshareafewwordsabouttheearly historyofscanninganditsapplicationinmicroscopy. InventionofScanning Inthe1840s,AlexanderBain,aScottishclockmaker,inventedtheprincipleofdis- sectinganimagebyscanning,andhewasgrantedaBritishpatent(Bain1843)for the first fax machine (McMullan 1990). At the transmitter, a stylus mounted on a pendulum contacts the surface of metal type forming the message, thus closing an electrical circuit. At the receiver, a similar stylus, also on a pendulum, records electrochemically on dampened paper. Following each swing of the pendulums, the type and the recording paper are lowered by one line. The means for starting the pendulums swinging simultaneously and synchronising them magnetically are describedinthepatent. ScanningOpticalMicroscopy The first proposal in print for applying scanning to microscopy was made in Dublin by Edward Synge (1928). The proposal was for a scanning optical microscope, and his goal was to overcome the Abbe limit on resolution by H.Schatten,J.B.Pawley(eds.),BiologicalLow-VoltageScanning 1 ElectronMicroscopy.(cid:2)C Springer2008 2 D.McMullan what is now called “near-field microscopy”—that is, the production of a very smalllightprobebycollimationthroughanaperturesmallerthanthewavelengthof thelight. Synge was a scientific dilettante who had original ideas in several scientific fields, but did not attempt to put them into practice (McMullan 1990). However, he considered some of the problems that would be encountered with a scanning microscopeandheproposedtheuseofpiezo-electricactuators(Synge1932),which are now used with great success in the scanning tunneling microscope and other probe instruments—including, of course, the near-field optical microscope itself. Heenvisagedfastscanningofthesamplesothatavisibleimagecouldbedisplayed onaphosphorscreen,andhealsopointedoutthepossibilityofcontrastexpansion to enhance the image from a low contrast sample—probably the first mention of imageprocessingbyelectronicmeans(asdistinctfromphotographic). ChargedParticleBeams A proposal for using an electron beam in a scanning instrument was described in German patents by Hugo Stintzing of Giessen University (1929). These patents wereconcernedwiththeautomaticdetection,sizing,andcountingofparticlesusing a light beam, or—for those of below-light microscopic size—a beam of electrons. The focusing of electrons was (at that time) unknown to him, as to most others, andheproposedobtainingasmalldiameterprobeusingcrossedslits.Thesamples wouldbeeithermechanicallyscannedinthecaseofalightbeam,oronecoulduse electricormagneticfieldstodeflectanelectronbeam.Suitabledetectorswouldbe usedtodetectthetransmittedbeamthatwasattenuatedbyabsorptionorscattering. The output was to be recorded on a chart recorder so that the linear dimension of a particle could be given by the width of a deflection, and the thickness by the amplitude—theproductionofatwo-dimensionalimagewasnotsuggested.Stintz- ingdidnotapparentlyattempttheconstructionofthisinstrumentandthereareno drawingsaccompanyingthepatentspecification.ThomasMulvey(1962),however, publishedablock-schematicdiagramofStintzing’sproposalmuchlater. TheTransmissionElectronMicroscope Intheearly1930s,themaincenterforthedevelopmentoftheelectronmicroscope wastheBerlinTechnischeHochschuleinthelaboratoryofProfessorMatthiaswhere Max Knoll was a research assistant supervising students—including Ernst Ruska, whose subject area was electron optics. Arising from this work, Knoll and Ruska demonstratedthefirsttransmissionelectronmicroscopewithamagnificationofx16. From the very beginning of electron microscopy, the imaging of solid samples wasanimportantgoal,particularlyasthemethodsforproducingthinsampleswere not developed until later. The first attempt was by Ruska (1933), with the sample
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