Download Sample preparation for Transmission electron microscopy (TEM

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
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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‐160C.
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:
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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)
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