TEM Specimen Preparation：Preparative Techniques for the TEM

Forroutinetransmissionelectronmicroscopy(TEM),itisgenerallyacceptedthatspecimensshouldbethin,dryandcontainmoleculeswhichdiffractelectrons.BIOLOGicalspecimens,whicharelargeandconsistoflargeamountsofwater,alsodonotdefractelectronsandarethereforedifficulttoseeintheTEM.PreparingbiologicalspecimensfortheTEM,whilstretainingthestructuralmorphologyofthematerial,isachallenge.However,researchershavebeenlookingatbiologicalmaterialformanyyears,andmanyprotocolsexistwhichallowustolookatbiologicalmaterialinmanydifferentways.BelowisabriefoutlineofsomeofthemorecommonwaysoflookingatbiologicalsamplesintheTEM.

Smallorverythinobjectscanbeexamineddirectlybymountingthemontoasupportfilmandintroducingthemdirectlyintotheelectronbeam.Contrastisprovidedbyheavymetalprecipitationinoneofthreeways.

1. Positivestaining:Theobjectischemicallystainedwithasolutionofthemetalsaltandappearsdarkonabrightbackground.
2. Negativestaining:Theobjectremainsunstainedbutisembeddedinadriedfilmoftheheavymetalsalt.Thespecimenappearslightonadarkbackground.Thismethodofvisualizationhasbeenusedextensivelyinthestudyofvirusparticlesbutisalsousefulforcellfractions(e.g.coatedvesicles).Moredetailscanbefoundhere.
3. Shadowing:Athinlayerofheavymetalatomsisdepositedonthespecimenbyevaporationinavacuumchamber.Shadowingfromonedirectiononlyproducesapseudo-three-dimensionalimage.Rotaryshadowing,wherethespecimenisuniformlycoatedwithheavymetal,isusedtovisualizenucleicacidsandproteins.

ThemostpopulartechniqueforexaminingbiologicalmaterialsistoembedthematerialunderstudyinplasticandcutultrathinsectionsthatcanbeexaminedinaTEM.ThematerialisstABIlizedbychemicalfixation(usuallywithaldehydessuchasformaldehydeorgluteraldehyde),contrastedwithsolutionsofheavymetalsalts(osmiumtetroxideanduranylacetate),dehydratedinethanoloracetone,andembeddedinplastic(epoxyresin).Ultrathinsections(60nm)cutwithglassordiamondknivesusinganultramicrotomearefloatedonwater,transferredtospecimensupportgridsandexaminedintheTEM.Oftenthesectionsarefurthercontrastedwithuranylacetateandleadcitratepriortoexaminationinthemicroscope.

Insomecases,macromoleculescanbespecificallylabelledpriortoembeddingandsectioning.Forexample,thelocationofsomeenzymescanbevisualizedbyincubatingthetissuewithasubstratewhosereactionwiththeenzymeleadstothelocaldepositionofelectronopaquematerial.Alternately,antibodiescanbecoupledtosuchenzymes,andtheelectronopaquereactionproductisusedtolocalizetheantigensrecognizedbytheantibodies.Someembeddingresins(e.g.LowicrylresinsandLRWhiteresin)havebeendesignedtoenableantibodiesandelectronopaqueMarkers(suchascolloidalgoldparticles)tobeappliedtotheultrathinsections.Inthisway,subcellularantigensrecognizedbytheantibodiescanbelocalizedwiththeTEM.

Anothersectioningtechniquethatisincreasinginpopularityiscryosectioning(thesectioningofvitrified,frozenmaterial).Afterchemicalfixation,thetissueisimmersedincryo-protectant(usuallysucrose)andthenquicklyfrozeninliquidnitrogen.Thecryo-protectantallowsthebiologicalmaterialtobefrozenwithouttheformationoficecrystals,whichwoulddamageultrastructure.Thistypeoffreezing,orvitrification,ispossIBLeintheabsenceofcryo-protectantsbutistechnicallydemanding.Sectionscutfromthevitrifiedblockcanbethawedandincubatedwithantibodiesspecifictosubcellularantigens.ElectronopaquemarkersallowtheantibodiestobeseenintheTEM.

ColloidalgoldcoupledtoproteinA(aproteinfrombacterialcellwallswhichbindstotheFcportionofsomeimmunoglobulins)hasbeenusedextensivelyinrecentyearstolocalizeantibodiesonresinandfrozensectionsofbiologicalmaterials.Theabilitytoproducehomogeneouspopulationsofcolloidalgoldwithdifferentparticlesizeshasenabledresearcherstousetheseprobestocolocalizedifferentstructuresonthesamesection.

Itispossibletofreezebiologicalmaterialfastenoughtovitrifythewaterpresentinsidethecells.Vitrificationofwateroccurswhenthefreezinghasoccurredsofastthaticecrystalshavenotimetoform.Vitrifiedbiologicalmaterialcanbesectionedatlowtemperatures.Thinfilmsofvitrifiedwaterandsectionsofvitrifiedmaterialcanbeexaminedintransmissionelectronmicroscopesthatareequippedwithspecimenstagesthatcanbekeptcold.

RapidFreezingMethods

Therearesevenmainrapidfreezingmethodspresentlyavailable.Theyare

1. immersionfreezing-thespecimenisplungedintothecryogen.
2. slam(ormetalmirror)freezing-thespecimenisimpactedontoapolishedmetalsurfacecooledwithliquidnitrogenorhelium.
3. coldblockfreezing-twocold,polishedmetalblocksattachedtothejawsofapairofplierssqueeze-freezethespecimen.
4. sprayfreezing-afinesprayofsampleinliquidsUSPensionisshotintothecryogen(usuallyliquidpropane).
5. jetfreezing-ajetofliquidcryogenissprayedontothespecimen.
6. highpressurefreezing-freezingthespecimenathighpressuretosubcoolthewater.
7. excisionfreezing-acoldneedleisplungedintothespecimen,simultaneouslyfreezinganddissectingthesample.

Freeze-fracturefollowedbyfreezeetchandreplication

If,forsomereason,theobjecttobestudiedcannotbeexaminedintheTEM,thenathinreplicacanbemade.Thisisusuallymadebyevaporatingathinlayerofaheavymetal(usuallyplatinum)ontothespecimenandthencoatingthiswithathinlayerofcarbon.Theobjectandthereplicaareseparatedeitherbyfloatingoffthereplicaorbydigestingawaytheobject.Therearefourbasicstepstofollow

1. Thespecimenisfrozen(oftenwithoutregardtovitrification).
2. Thespecimenisfractured,whilestillfrozen,undervacuum.
3. Thefracturedspecimencanthenbeetchedbyleavingitfrozenandundervacuum.Dependingonthetimeofexposure,moreorlesswatersublimesfromthespecimen(freezedrying).
4. Areplicaofthefracturedsurfaceismadewhichisthenexaminedintheelectronmicroscope.

Arecentmodificationofthismethodemploysrapidfreezingachievedbyslammingcellsagainstacopperblockcooledto-269°Cwithliquidhelium.Ifthesefrozencellsarethenexposedtoextensivefreezedrying(deepetching),veryimpressiveimagesoftheinternalstructuresofcellsareuncovered.