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: Blocksoftissue(liver,kidney,pancreas,etc.)ofapproximately1cm 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.Thealterationsinvolveplacingadehydratedtissuesampleofabout1mm Softerplasticsarealsobeingusedforroutinelightmicroscopy.Theaveragethicknessofaparaffin-sectionedtissueisbetween7and10microns.Oftenthiswillconsistoftwocelllayersand,consequentlylackdefinitionforcytoplasmicstructures.WithaplasticsuchaspolysciencesJB--4itispossibletosectiontissuesinthe1--3micronrangewithincreasedsharpness.Thisisparticularlyhelpfulifphotomicrographsaretobetaken.Withthedecreaseinsectionthickness,however,comesalossofcontrast,andthinsections(1micron)usuallyrequiretheuseofaphasecontrastmicroscopeaswellasspecialstainingprocedures.Thesharpimagemakestheeffortworthwhile. Thesesoftplasticscanbesectionedwithastandardsteelmicrotomebladeanddonotrequireglassordiamondknives,aswiththeharderplasticsusedforEMwork. Sectioning Figure2.1presentsaphotographoftheAOstandardmicrotome.Thisdeviceisfounduniversallyincellbiologylaboratoriesandremainsafundamentalinstrumentforhistology. Thisrathersimpledeviceconsistsofastationaryknifeholder/bladeandaspecimenholderwhichadvancesbypre-setintervalswitheachrotationoftheflywheelmountedontherighthandside.Inoperation,itissimilartothemeatandcheeseslicersfoundwithindelicatessans.Acontrolknobadjustsinternalcamswhichadvancetheparaffinblockwitheachstroke.Itisrelativelyeasytosectionparaffinat10micronsbutrequiresalotofskillandpracticetocutat5microns.Sinceeachsectioncomesoffoftheblockserially,itispossibletoalignallofthesectionsonamicroscopeslideandproduceaserialsectionfromoneendofatissuetotheother. Whilevirtuallyanyonecancutasectionwithinminutesofbeingintroducedtothemicrotome,properuseofthemicrotomeisanartformandrequirespracticeandinventiveness.Manyacellbiologyresearchprojecthasdependedontheskillsinherentintheuseofthisinstrument.Amicroscopeisnearlyuselesswithoutagoodthin,flat,andundistortedsectionfromproperlyfixed,dehydratedandembeddedtissue. TheUltramicrotome 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 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.calciumchloride 1.0g cadmiumchloride 1.0g formalin,concentrated 10.0ml distilledwater 100.0ml arerapidlyremovedfromafreshlykilledorganismandplacedinthefixative.Theyareallowedtoremaininthefixativeforaminimumoffourhoursbutusuallyovernight.Thelongertheblocksremaininthefixative,thedeeperthefixativepenetratesintotheblockandthemoreproteincross--linkingoccurs.Thefixativeisthereforetermedprogressive.Blocksmayremaininthisfixativeindefinitely,althoughthetissueswillbecomeincreasinglybrittlewithlongexposuresandwillbemoredifficulttosection.Whileitisnotrecommended,sectionshavebeencutfromblocksleftforyearsinformalin.
intoaliquidplasticwhichisthenpolymerizedtoformahardblock.TheplasticblockistrimmedandsectionedwithanultramicrotometoobtainsectionsofafewhundredAngstroms.Table2.1presentsacomparisonofparaffinembeddingwiththetypicalEponembedmentforTEM.
Table2.1Lightandelectronmicroscopypreparations.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 Figure2.1AOmicrotomeforparaffinsectioning
Figure2.2Sorvallultramicrome
Figure2.3Moderncryostat