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SamplepreparationforTransmissionelectronmicroscopy(TEM) TEMisamicroscopytechniquewherebyabeamofelectronsistransmittedthroughan ultrathinspecimen,interactingwiththespecimenasitpassesthroughit.Animageis formedfromtheelectronstransmittedthroughthespecimen,magnifiedandfocusedby anobjectivelensandappearsonanimagingscreen,afluorescentscreeninmostTEMs, plusamonitor,oronalayerofimagingplate,ortobedetectedbyasensorsuchasaCCD camera. Biologicalmaterialscontainlargequantitiesofwater.Tobeabletoviewitinthe electronmicroscopy,thefirststageinpreparingisthefixation,oneofthemost importantandmostcriticalstages.Weneedtofixeditinawaythattheultrastructureof thecellsortissuesremainasclosetothelivingmaterialaspossible.Thesampleisthen dehydratedthroughanacetoneorethanolseries,passedthrougha“transitionsolvent” suchaspropyleneoxideandtheninfiltratedandembeddedinaliquidresinsuchas epoxyandLRWhiteresin.Afterembeddingtheresinblockisthenthinsectionedbya processknownasultramicrotomy,sectionsof50‐70nmthicknessarecollectedon metalmesh'grids'andstainedwithelectrondensestainsbeforeobservationinthe TEM.Sectioningthesampleallowsustolookatcross‐sectionsthroughsamplestoview internal(ultra)structure.Manymodificationstothebasicprotocolcanbeappliedto achievemanydifferentgoals,immunogoldlabelingforexample;theinsitulocalization ofspecifictissueconstituentsusingantibodyandcolloidalgoldmarkersystems. Everysampleisdifferent.PleaseconsultwiththeEMStaffbeforestartingaproject. SupportfilmonTEMgrids Formvarfilmisusefulforthesupportofultrathinsectionsonthefinermeshgrids.Using ofsupportfilmareidealforthoseapplicationsrequiringlargeviewingareaswithout gridbarinterference.Thesefilmsmustbestrong,cleanandremainattachedtothe specimengridsduringspecimenpreparation. AFormvarfilmcoveredwitha"light"layerofcarbonwillhelptostabilizethefilmwhen thefilmisexposedtotheelectronbeam. Sectioningwithultramicrotome MaterialsforTEMmustbespeciallypreparedtothicknesseswhichallowelectronsto transmitthroughthesample,muchlikelightistransmittedthroughmaterialsin conventionalopticalmicroscopy.Becausethewavelengthofelectronsismuchsmaller thanthatoflight,theoptimalresolutionattainableforTEMimagesismanyordersof magnitudebetterthanthatfromalightmicroscope. Theblockiscutintosemithinsections(1µm)withaglassknife,usingan ultramicrotome.ThesectionsarethenstainedwithToluidineBlueforLMfor orientation,andforselectingofasmallareaforultrathinsectioning.Ultrathinsections aremadeat50‐70nmusingadiamondknifeandplaced/collectedonagridofmetal. Positivestaining Side1 Detailsinlightmicroscopesamplescanbeenhancedbystainsthatabsorblight; similarlyTEMsamplesofbiologicaltissuescanutilizehighatomicnumberstainsto enhancecontrast.Thestainabsorbselectronsorscatterspartoftheelectronbeam whichotherwiseisprojectedontotheimagingsystem.Usesheavymetalssuchaslead anduraniumtoscatterimagingelectronsandthusgivecontrastbetweendifferent structures,sincemany(especiallybiological)materialsarenearly"transparent"to electrons(weakphaseobjects). Heavymetalsaltsattachtovariousorganellesormacromoleculeswithinthesectionsto increasetheirelectrondensityandtheyappeardarkagainstalighterbackground. Uranylionsreactstronglywithphosphateandaminogroupssothatnucleicacidsand certainproteinsarehighlystained.Leadionsbindtonegativelychargedcomponents andosmium‐reactedareas(membranes). Gridsarestainedwithheavymetals,suchasuranylacetateandleadcitrate.Thegrids, withthespecimensidedown,remainin4%uranylacetatefor25minutesandarethen rinsedinaseriesoffourbeakersofpurewater.Afterrinsing,thegridsarethenstained with1%leadcitratefor5minutes,rinsedagaininpurewater,andstoredinagridbox. PC‐3cell,aprostatecancer cellline. Columnarcellsfromsmall intestine,showingciliaand organelles. Cilia,transversesection. Glomerulusinkidney. Skeletalmuscle. Nervusvagus,rat Side2 Immunogoldlabelling Thistechniqueusesantibodiestodetecttheintracellularlocationofstructuresof particularproteinsbyelectronmicroscopy.Ultrathinsectionsarelabelledwith antibodiesagainsttherequiredantigenandthenlabelledwithgoldparticles.Gold particlesofdifferentdiametersenabletwoormoreproteinstobestudied. EMLabcanofferpost‐embeddingimmunogoldlabellingofsamplesinresin(Epoxy,LR WhiteandLowicryl)andonfrozenhydratedultrathinsections(Tokuyasu‐method). Theinvestigatormustsupplytheprimaryandsecondaryantibodies.Theinvestigator shoulddoimmunolabellingatthefluorescentlightmicroscopylevelbeforeattemptingit attheEMlevel. Stomaccancertissue,conventionallyfixed andembeddedinepoxyresin.Immun‐ olabelledwithanti‐CgA,enhancedgold labelling. Bar:2µm. Visualizingofcell‐surfacelocated epidermalgrowthfactor(EGF)receptorsin culturedA431cells(epidermoid carcinoma)usingTokuyasu‐method. Bar:0,5µm. Immunogoldlabellingofmyrosincellin Arabidopsisthalianawithantibodyagainst TGG1.Theplantwasfreezesubstitutedand embeddedinlowicrylHM20resin. Stomaccancertissue,poorlydifferentiated, conventionallyfixedandembeddedin epoxyresin.Immunolabelledwithanti‐CgA. Side3 Cryotechniques/Lowtemperature Cryo‐ultramicrotomyistheultrathinsectioningofunfixed/fixed,cryo‐protectedand/or rapidlyfrozensamplesatverylowtemperatures.TheLeicaEMFC6cryochamberis designedforlowtemperaturesectioningofsamplesattemperaturesfrom‐15to‐160C. Freezesubstitutionisaprocesswherethewatermoleculeswithinthesamplesare exchangedwithasolvent(usuallymethanoloracetone),then,thesolventwitharesin (Lowicryl,LRWhiteorEpoxyresins).Thismethod,workingattemperaturesbelow0ºC, reducesthelossofcomponentsfromthesampleandminimizesthedenaturizationof theproteins.Intheend,thesampleisfullyinfiltratedwithpureresin.Polymerizationof theresinisperformedoutside(Epoxyresin)orinsidethemachinewhenusingLowicryl resin.ThislatterresinispolymerizedunderaUVlamp,startingat‐45ºC,thengradually movinguptoroomtemperature(LowicrylHM20).Attheendoftheprocesshardplastic blocksaregeneratedreadytobecutbyultramicrotomy.TheLeicaEMAFSiscapableof freezesubstitution,progressiveloweringoftemperaturetechniques,andlow temperatureembeddingandpolymerizationofresinsusingUVlightinsteadofheatfor improvedpreservationofultrastructureandantigenicity. TEMservicesinclude: Conventionalspecimenfixation,dehydrationandembedding. Specimensectioning: Semithinsectioning(1µm)withToluidinebluestain Ultrathinsectioning(50–70nm)ofresinembeddedmaterial(Epoxy, LRWhite,Lowicryletc) Cryo‐ultrathinsectioningforTokuyasu‐method(sucrose‐infiltrated) Cryo‐ultrathinsectioningforX‐raymicroanalysis(unfixed) Freezesubstitutionfollowedbyresinembedding(Epoxy,LRWhite,Lowicryletc) Immunolabellingofsections(resinandfrozen) Positivestaining PreparationofsamplesforX‐raymicroanalysis Coatingofgrids(formvar) Imageprocessing(softwareiTEMandTIA) Side4