Afewcelltypesarethinenoughtobevieweddirectlyinamicroscope(algae,protozoa,blood,tissuecultures),butmosttissues(kidney,liver,brain)aretoothicktoallowlighttobetransmittedthroughthem.Thetissuescanbeslicedintoverythinsectionsprovidedtheyarefirstprocessedtopreventcelldamage.Theprocessinginvolvesaseriesofsteps;fixation,dehydration,embedmentandsubsequentsectioningwithamicrotome.Thesesteps,explainedindetaillaterinthischapter,aretimeconsumingandoftenalterthecellstructureinsubtleways.Fixingcellswithformaldehyde,forexample,willpreservethegeneralorganellestructureofthecell,butmaydestroyenzymesandantigenswhicharelocatedinthecell.

Pathologistsroutinelyexaminetissueswhichhavebeenfixedinformaldehydeandembeddedinparaffinwaxpriortosectioning.Theprocessrequiresaminimumof24hours,andusuallymoreifautomatedequipmentisnotavailable.Thistimedelaycanbecrucialwhenadiagnosisofbenignormalignantcancerisatstake.Valuabletimecanbesavedbyskippingthefixationanddehydrationstepsrequiredforparaffinembedding,andfreezingthetissueinamodifiedmicrotome,thecryostat.Sectionscanbepreparedwithinminutesanddiagnosesmadewhilethepatientremainsontheoperatingtable.Additionally,frozensectionswillmoreoftenretaintheirenzymeandantigenfunctions.Theuseoffrozensectionscanreducetheprocessingtime,butitisnotapanacea.Freezingisnotadequateforlongtermpreservationofthetissuesandtheformationoficecrystalswithinthecellsdestroyssubcellulardetail.Frozensectionsarealsothickersinceicedoesnotsectionasthinasparaffin.Thisresultsinpoormicroscopicresolutionandpoorimagesofwhatsubcellularstructuresremain.Iftimeorenzymefunctioniscriticalfrozensectionsarethepreferredprocess.Ifsubcellulardetailisimportant,otherproceduresmustbeused.SelectionofthecorrectproceduredependsonwhatthecellBIOLOGistislookingforandtoapoint,becomesanartform.Thehistologistmustchooseamonghundredsofprocedurestopreparetissuesinamannerthatismostappropriatetothetaskathand.

Fixation

Sincecellulardecompositionbeginsimmediatelyafterthedeathofanorganism,biologistsmustfixthecellstopreventalterationsintheirstructurethroughdecomposition.Routinefixationinvolvesthechemicalcross-linkingofproteins(topreventenzymeactionanddigestion)andtheremovalofwatertofurtherdenaturetheproteinsofthecell.Heavymetalsmayalsobeusedfortheirdenaturingeffect.

Atypicallaboratoryprocedureinvolvestheuseofanaldehydeastheprimaryfixative.Glutaraldehydeisusedfortransmissionelectronmicroscopy(TEM),andformaldehydeisusedforroutinelightmicroscopy.TheformaldehydesolutionmostoftenemployedwasoriginallyformulatedbyBakerin1944.

Baker"sFormalinFixativecontains:

calciumchloride	1.0g
cadmiumchloride	1.0g
formalin,concentrated	10.0ml
distilledwater	100.0ml

Blocksoftissue(liver,kidney,pancreas,etc.)ofapproximately1cmarerapidlyremovedfromafreshlykilledorganismandplacedinthefixative.Theyareallowedtoremaininthefixativeforaminimumoffourhoursbutusuallyovernight.Thelongertheblocksremaininthefixative,thedeeperthefixativepenetratesintotheblockandthemoreproteincross--linkingoccurs.Thefixativeisthereforetermedprogressive.Blocksmayremaininthisfixativeindefinitely,althoughthetissueswillbecomeincreasinglybrittlewithlongexposuresandwillbemoredifficulttosection.Whileitisnotrecommended,sectionshavebeencutfromblocksleftforyearsinformalin.

Formalinhaslatelybeenimplicatedasacausativeagentforstrongallergyreactions(contactdermatitiswithprolongedexposure)andmaybeacarcinogen----itshouldbeusedwithcareandalwaysinawellventilatedenvironment.Formalinisa39%solutionofformaldehydegas.Thefixativeisgenerallyusedasa10%formalinortheequivalent4%formaldehydesolution.Thekeyoperativetermhereisgas--formaldehydeshouldbehandledinahood,ifpossIBLe.Asagas,itisquitecapableoffixingnasalpassages,lungsandcorneas.

Dehydration

Fixatives,suchasformaldehyde,havethepotentialtofurtherreactwithanystainingprocedurewhichmaybeusedlaterintheprocess.Consequently,anyremainingfixativeiswashedoutbyplacingtheblocksinrunningwaterovernightorbysuccessivechangesofwaterand/orabuffer.Therearemyriadmeansofwashingthetissues(usingtemperature,pHandosmoticallycontrolledbuffers),butusuallysimplewashingintapwaterissufficient.

Ifthetissuesaretobeembeddedinparaffinorplastic,alltracesofwatermustberemoved:waterandparaffinareimmiscible.Theremovalofwaterisdehydration.Thedehydrationprocessisaccomplishedbypassingthetissuethroughaseriesofincreasingalcoholconcentrations.Theblocksoftissuearetransferredsequentiallyto30%,50%,70%,80%,90%,95%,and100%alcoholsforabouttwohourseach.Theblocksarethenplacedinasecond100%ethanolsolutiontoensurethatallwaterisremoved.Notethatethanolishydroscopicandabsorbswatervaporfromtheair.Absoluteethanolisonlyabsoluteifstepsaretakentoensurethatnowaterhasbeenabsorbed.

Itisimportanttodistinguishbetweendehydrationanddrying.TissuesshouldNEVERbeallowedtoairdry.Dehydrationinvolvesslowsubstitutionofthewaterinthetissuewithanorganicsolvent.Forcomparativepurposes,considerthegrape.Aproperlydehydratedgrapewouldstilllooklikeagrape.Adriedgrapeisaraisin.Itisvirtuallyimpossibletomakearaisinlooklikeagrapeagain,anditisequallyimpossibletomakeacelllooknormalafteryouallowittodry.

Embedding

Afterdehydration,thetissuescanbeembeddedinparaffin,nitrocelluloseorvariousformulationsofplastics.Paraffinistheleastexpensiveandthereforethemostcommonlyusedmaterial.Morerecently,plasticshavecomeintoincreaseduse,primarilybecausetheyallowthinnersections(about1.5micronscomparedto5--7micronsforparaffin).

Paraffin

Forparaffinembedding,firstclearthetissues.Clearingreferstotheuseofanintermediatefluidthatismisciblewithethanolandparaffin,sincethesetwocompoundsareimmiscible.Benzene,chloroform,tolueneorxylolarethemostcommonlyusedclearingagents,althoughsomehistologistsprefermixturesofvariousoils(cedarwoodoil,methylsalicylate,creosote,cloveoil,amylacetateorCellosolve).Dioxaneisfrequentlyusedandhastheadvantageofshortpreparationtimes.Ithasthedistinctdisadvantageofinducingliverandkidneydamagetotheuserandshouldonlybeusedwithadequateventilationandprotection.

Bewaryofallorganicsolvents.Mostareimplicatedascarcinogenicagents.Heedallprecautionsfortheproperuseofthesecompounds.

Themostoftenusedclearingagentistoluene.Itisusedbymovingtheblocksintoa50:50mixtureofabsoluteethanol:toluenefortwohours.Theblocksarethenplacedintopuretolueneandthenintoamixtureoftolueneandparaffin(also50:50).Theyarethenplacedinanovenat56-58°C(themeltingtemperatureofparaffin).

Theblocksaretransferredtopureparaffinintheovenfor1hourandthenintoasecondpotofmeltedparaffinforanadditional2--3hours.Duringthistimethetissueblockiscompletelyinfiltratedwithmeltedparaffin.

Subsequenttoinfiltration,thetissueisplacedintoanembeddingmoldandmeltedparaffinispouredintothemoldtoformablock.Theblocksareallowedtocoolandarethenreadyforsectioning.

Plastic

Morerecentdevelopmentsintheformulationofplasticresinshavebeguntoalterthewaysectionsareembedded.Forelectronmicroscopythatrequiresultrathinsections,paraffinissimplynotsuitable.Paraffinandnitrocellulosearetoosofttoyieldthinenoughsections.

Instead,specialformulationsofhardplasticsareused,andthebasicprocessissimilartothatforparaffin.Thealterationsinvolveplacingadehydratedtissuesampleofabout1mmintoaliquidplasticwhichisthenpolymerizedtoformahardblock.TheplasticblockistrimmedandsectionedwithanultramicrotometoobtainsectionsofafewhundredAngstroms.Table2.1presentsacomparisonofparaffinembeddingwiththetypicalEponembedmentforTEM.

Softerplasticsarealsobeingusedforroutinelightmicroscopy.Theaveragethicknessofaparaffin-sectionedtissueisbetween7and10microns.Oftenthiswillconsistoftwocelllayersand,consequentlylackdefinitionforcytoplasmicstructures.WithaplasticsuchaspolysciencesJB--4itispossibletosectiontissuesinthe1--3micronrangewithincreasedsharpness.Thisisparticularlyhelpfulifphotomicrographsaretobetaken.Withthedecreaseinsectionthickness,however,comesalossofcontrast,andthinsections(1micron)usuallyrequiretheuseofaphasecontrastmicroscopeaswellasspecialstainingprocedures.Thesharpimagemakestheeffortworthwhile.

Table	Light	Electron
SampleSize	1cm	1mm
Fixative	Formaldehyde	Glutaradehyde
Post-Fixation	None	OsmiumTetroxide
Dehydration	GradedAlcohol	AlcoholorAcetone
ClearingAgent	Xylol/Toluene	PropyleneOxide
EmbeddingMaterial	Pfaffin	VariousPlastics
SectionThickness	5-10µ	60-90nm
Stains	Coloreddyes	HeavyMetals

Table2.1Lightandelectronmicroscopypreparations.

Thesesoftplasticscanbesectionedwithastandardsteelmicrotomebladeanddonotrequireglassordiamondknives,aswiththeharderplasticsusedforEMwork.

Sectioning

Figure2.1AOmicrotomeforparaffinsectioning

Figure2.1presentsaphotographoftheAOstandardmicrotome.Thisdeviceisfounduniversallyincellbiologylaboratoriesandremainsafundamentalinstrumentforhistology.

Thisrathersimpledeviceconsistsofastationaryknifeholder/bladeandaspecimenholderwhichadvancesbypre-setintervalswitheachrotationoftheflywheelmountedontherighthandside.Inoperation,itissimilartothemeatandcheeseslicersfoundwithindelicatessans.Acontrolknobadjustsinternalcamswhichadvancetheparaffinblockwitheachstroke.Itisrelativelyeasytosectionparaffinat10micronsbutrequiresalotofskillandpracticetocutat5microns.Sinceeachsectioncomesoffoftheblockserially,itispossibletoalignallofthesectionsonamicroscopeslideandproduceaserialsectionfromoneendofatissuetotheother.

Whilevirtuallyanyonecancutasectionwithinminutesofbeingintroducedtothemicrotome,properuseofthemicrotomeisanartformandrequirespracticeandinventiveness.Manyacellbiologyresearchprojecthasdependedontheskillsinherentintheuseofthisinstrument.Amicroscopeisnearlyuselesswithoutagoodthin,flat,andundistortedsectionfromproperlyfixed,dehydratedandembeddedtissue.

TheUltramicrotome

Figure2.2Sorvallultramicrome

Figure2.2presentsaviewofanultramicrotome.Inprinciple,itistheoffspringofthestandardmicrotome,inthatitalsoisamechanicaldevicethatinvolvesastationaryknife(glassordiamond)andamovingspecimen.Thespecimen,orblock,isaplasticembeddedtissuethatadvancesinnanometersratherthanmicrons.

Operationally,theonlydifferenceisthatsmallersamplesarehandled,whichinturnrequiresabinoculardissectingmicroscopemountedovertheblade.Thetissuesectionsaretoothintoseetheirthicknesswiththenakedeye,oneusuallyestimatesthicknessbythecolorofthediffractionpatternonthesectionasitfloatsofftheknifeontothesurfaceofawaterbath.Thesectionsarealsotoothintobehandleddirectly,andtheyarethereforetransferredwithwireloops,orpickedoffthewaterdirectlyontoanEMgrid.Thisprocessrequiresagoodlightsourcemountedtocastthelightatjustthecorrectangletoseethecolorpattern.

Sincetheplasticsarehardenoughtobreaksteelknives,freshlypreparedglassknivesorcommerciallyavailablediamondknivesareused.Aglassknifecostsseveraldollarseach,whileagooddiamondknifewillcostinexcessof$3,000.Eithercanbepermanentlydamagedwithasinglecarelessstrokebytheoperator.Diamondknivesareusedinresearchlaboratoriesbytrainedtechniciansbecausetheyhavetheadvantageofaconsistentknifeedge(unlikeglasswhichvarieswitheachuse)andcanlastforyearsiftreatedproperly.Theycanusuallyberesharpenedseveraltimesbeforediscarding.

Tominimizevibrations(whichleadtounevensections)ultramicrotomesarecastinheavymetal,aremountedonshockabsorbenttablesand,preferrably,keptindraftfreeenvironmentsofrelativelyconstanttemperature.Tofurtherminimizevibrations,somemanufacturershavereplacedtheblock"smechanicaladvancemechanismwithathermalbar,whichadvancesthetissuebyheatingametalrod.Thiscanbeexquisitelypreciseandistheultimateinthinsectioning.Ofcoursewiththisadvancementcomesincreasedcostandmaintenance,anddecreasedABIlitytowithstandroughtreatment.Themechanicallyadvancedultramicrotomeremainsastheworkhorseofthecellbiologylaboratory.

TheCryostat

Figure2.3Moderncryostat

Whetherthesectioningisperformedwithamicrotomeoranultramicrotome,oneofthemajordelaysinpreparingatissuesectionisthetimerequiredtodehydrateandembedthetissue.Thiscanbeovercomebydirectsectioningofafrozentissue.Typicallyapieceoftissuecanbequickfrozentoabout-15to-20°C(forlightmicroscopicwork)andsectionedimmediatelyinadevicetermedacryostat.Thecryostatismerelyamicrotomemountedwithinafreezerbox.Figure2.3presentsamoderncryostat.

Apieceoftissueisremovedfromanorganism,placedontoametalstubandcoveredwithaviscousembeddingcompoundtokeepitinaformconvenientforsectioning.Thestudandtissueareplacedwithinthecryostatandquickfrozen.

Thismethodhastheadvantageofspeed,maintenanceofmostenzymeandimmunologicalfunctions(fixationisunnecessary)andrelativeeaseofhandling(farfewerstepstomanipulate).Ithasthedisadvantagethaticecrystalsformedduringthefreezingprocesswilldistorttheimageofthecell(burstingvacuolesandmembranesforexample)andtheblockstendtofreeze-dryorsublimate.Thus,theblocksmustbeusedimmediatelyandgreatcaremustbetakentoguardagainstinducedartifactfromthefreezingprocess.

Whentemperature-sensitive(orlipid-soluble)moleculesaretobestudied,orwherespeedisoftheessence(suchaspathologicalexaminationduringanoperation)thisisthepreferredmethod.Sectioningoperationwiththecryostatissimilartothatofthemicrotome,withtheexceptionthatonehandlessinglefrozensectionsandthusalloperationsmustbehandledatreducedtemperatures.

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