AdvancesinBioscienceEducationSummerWorkshopFluorescenceandElectronMicroscopyJune26-29,2007BiologicalElectronMicroscopeFacilityPacificBiosciencesResearchCenterUniversityofHawai’iatManoaWhatisaMicroscope?AtoolthatmagnifiesandimprovesresolutionofthecomponentsofastructureHasthreecomponents:sourcesofillumination,amagnifyingsystem,detectors.SourcesofIlluminationLightmicroscopesuseabeamoflightforilluminationandincludefluorescenceandconfocalmicroscopesElectronmicroscopesuseelectronsasasourceofilluminationandincludetransmissionandscanningelectronmicroscopes.LightandElectronMicroscopesLensesareusedtocontrolabeamofillumination,magnify,anddirectanimagetoadetectorImagesandpicturesareyourdata!EpifluorescenceMicroscopyCommonFluorescence

ApplicationsLocalize/identifyspecificorganellesDetectlivecellsvs.deadcells,necroticvs.apoptoticcellsDeterminecellmembranepermeabilityLocalizeantigen-specificmoleculesMultiplelabelingLaserScanningConfocalMicroscopeBetterresolutionSerialopticalsectionscanbecollectedfromthickspecimensLiveorfixedcellandtissueimagingLaserScanningConfocalMicroscopyPhotoscourtesyofGreggMeada&Dr.Gert

DeCouet,UHMAndDr.ChrisYuenandDr.DavidChristopherDrosophilaeyePlantProtoplastEpifluorescencevs.ConfocalSamplecourtesyGreggMeada&Dr.Gert

DeCouet,UHMScanningElectronMicroscopy(SEM)ViewoutersurfaceCoatspecimenwithgoldNosectioningHighMag(40xto300,000x)Highresolution(betterthan2nm)SEMImagesTransmissionElectronMicroscopy

(TEM)Viewinsidecellviasectionsmagnification120,000X50,000XConventionalTEMMicrographsSkinBacteriaincellApoptosisChloroplastCollagenVirusincellUltra-microtomyUltrathin(60-90nm)sectioningofresin-embeddedspecimensSeveralbrands/modelsavailableCryotechniquesUltrarapid

cryofixation

MetalmirrorimpactLiquidpropaneplungeFreezefracturewithBalzers400TCryosubstitution

Cryoultramicrotomy–Ultrathinfrozensections(primarilyforantibodylabeling)ImmunolocalizationLMFluor/confocalTEMSEMwithbackscatterdetectorApproachestoImmunolabelingDirectMethod:PrimaryantibodycontainslabelIndirectMethod:PrimaryantibodyfollowedbylabeledsecondaryantibodyAmplifiedMethod:MethodstoaddmorereportertolabeledsiteTwo-stepIndirectMethodforImmunolabelingFluorescent-conjugatedsecondaryantibodyattachestoprimaryantibodythatisboundtoantigenImmunolabelingforTransmissionElectronMicroscopyNormallydoTwo-StepMethodPrimaryantibodyappliedfollowedbycolloidalgold-labeledsecondaryantibodyMayalsobeenhancedwithsilverColloidalGoldImmunolabelingforTEMColloidalgoldofdefinedsizes,e.g.,5nm,10nm,20nm,easilyconjugatedtoantibodiesResultsinsmall,round,electron-denselabeleasilydetectedwithEMCanbeenhancedafterlabelingtoenlargesizeforLMorEMDouble-labelingMethodUseprimaryantibodiesderivedfromdifferentanimals(e.g.,onemouseantibodyandonerabbitantibody)ThenusetwodifferentsecondaryantibodiesconjugatedwithdifferentsizedgoldparticlesPreparationofBiologicalSpecimensforImmunolabelingPreservetissueascloselyaspossibletoitsnaturalstatewhileatthesametimemaintainingtheabilityoftheantigentoreactwiththeantibodyChemicalfixationORCryofixationChemicalFixationAntigenicsitesareeasilydenaturedormaskedduringchemicalfixationGlutaraldehydegivesgoodfixationbutmaymaskantigens,plusitisfluorescentParaformaldehydeoftenbetterchoice,butresultsinpoormorphology,especiallyforelectronmicroscopyMayusee.g.,4%paraformaldehydewith0.5%glutaraldehydeasagoodcompromiseEmbeddingDehydratedtissueisembeddedinaplasticresintomakeiteasiertocutthinsectionsStepsinLabelingofSectionsChemicalfixationDehydration,infiltration,embeddingandsectioningBlockingIncubationwithprimaryantibodyWashingIncubationwithsecondaryantibodycongugatedwithreporter(fluorescentprobe,colloidalgold)Washing,optionalcounterstainingMountandviewControls!Controls!Controls!OmitprimaryantibodyIrrelevantprimaryantibodyPre-immuneserumPerformpositivecontrolCheckforautofluorescenceCheckfornon-specificlabelingDilutionseriesLightMicroscopesLightPathinFluorescenceLightdeliveredthroughexcitationfilterandthenobjectivelenstospecimenwhereitisabsorbed;emittedlightgoesbackthroughobjectivelensthroughbarrierfilterandemissionfilterandthentodetector.FluorescenceLightbeamexcitesthefluorochrome,raisingittoahigherenergystate,Asitfallsbacktoit’soriginalstate,itreleasesenergyintheformofalightoflowerEandlongerwavelengththanoriginalbeamoflightPrimaryAb=PDI

secondaryAb=Alexafluor

Bluelight=excitingbeam

greenandredlightemittedKnowYourArtifacts

AutofluorescenceAndusethemtoyouradvantage!Greenislabel;orange-redisautofluorescenceActsascounterstainFluorescenceFluorochromesareexcitedbyspecificwavelengthsoflightandemitspecificwavelengthsofalowerenergy(longerwavelength)FilterCubesforFluorescenceFiltercubesgenerallyhaveanexcitationfilter,adichroicelement,andanemissionfilterTheelementsofacubeareselectedfortheexcitationandfluorescencedetectiondesiredChooseFluorochrome/FilterCombosLaserScanningConfocalMicroscopyFluorescencetechniqueUseslaserlightforexcitationImprovesimageresolutionoverconventionalfluorescencetechniquesOpticallyremovesout-of-focuslightanddetectsonlysignalfromfocalplaneCanconstructanin-focusimageofconsiderabledepthfromastackofimagestakenfromdifferentfocalplanesofathickspecimenCanthenmakea3-Dimagethatcanbetilted,rotated,andslicedPrincipalLightPathwayinConfocalMicroscopyLaserlightisscannedpixelbypixelacrossthesamplethroughtheobjectivelensFluorescentlightisreflectedbackthroughtheobjectiveandfilters(dichroicmirrors)AdjustablepinholeaperturesforPMTseliminateout-of-focusflareImageisdetectedbyphotomultiplier(s)anddigitizedoncomputerTEMTransmissionElectronMicroscopeIlluminationsourceisbeamofelectronsfromtungstenwireElectromagneticlensesperformsamefunctionasglasslensesinLMHigherresolutionandhighermagnificationofthinspecimensSpecimenPreparationforTEMChemicalfixationwithbufferedglutaraldehydeOr4%paraformaldehydewith>1%glutaraldehydePostfixationwithosmiumtetroxideOrnot,orwithsubsequentremovalfromsectionsDehydrationandinfiltrationwithliquidepoxyoracrylicresinPolymerizationofhardblocksbyheatorUVUltramicrotomy–60-80nmsectionsLabelingand/orstainingViewwithTEMHighpressurefreezing:Planttissueisflashfrozeninapressurebomb-197CWaterinthetissueisreplacedwithacetoneover5dayperiodAcetonesaturatedtissueisembeddedinresinResiniscutinthinsections,80nmthickAddantibodies-immunolabelingLookund