LASER-SCANNING-CONFOCAL-MICROSCOPY

LASERSCANNINGCONFOCALMICROSCOPYNathanS.Claxton,ThomasJ.Fellers,andMichaelW.DavidsonDepartmentofOpticalMicroscopyandDigitalImaging,NationalHighMagneticFieldLaboratory,TheFloridaStateUniversity,Tallahassee,Florida32310Keywords:confocal,laser,scanning,fluorescence,widefield,microscopy,opticalsections,resolution,AOTF,acousto-optictunablefilter,spinningdisk,volumerendering,photomultipliers,point-spreadfunction,Airydisks,fluorophores,AlexaFluor,cyanine,fluorescentproteins,quantumdots,photobleachingAbstractLaserscanningconfocalmicroscopyhasbecomeaninvaluabletoolforawiderangeofinvestigationsinthebiologicalandmedicalsciencesforimagingthinopticalsectionsinlivingandfixedspecimensranginginthicknessupto100micrometers.Moderninstrumentsareequippedwith3-5lasersystemscontrolledbyhigh-speedacousto-optictunablefilters(AOTFs),whichallowverypreciseregulationofwavelengthandexcitationintensity.Coupledwithphotomultipliersthathavehighquantumefficiencyinthenear-ultraviolet,visibleandnear-infraredspectralregions,thesemicroscopesarecapableofexaminingfluorescenceemissionrangingfrom400to750nanometers.Instrumentsequippedwithspectralimagingdetectionsystemsfurtherrefinethetechniquebyenablingtheexaminationandresolutionoffluorophoreswithoverlappingspectraaswellasprovidingtheabilitytocompensateforautofluorescence.Recentadvancesinfluorophoredesignhaveledtoimprovedsyntheticandnaturallyoccurringmolecularprobes,includingfluorescentproteinsandquantumdots,whichexhibitahighlevelofphotostabilityandtargetspecificity.IntroductionandHistoricalPerspectiveThetechniqueoflaserscanningandspinningdiskconfocalfluorescencemicroscopyhasbecomeanessentialtoolinbiologyandthebiomedicalsciences,aswellasinmaterialsscienceduetoattributesthatarenotreadilyavailableusingothercontrastmodeswithtraditionalopticalmicroscopy(1-12).Theapplicationofawidearrayofnewsyntheticandnaturallyoccurringfluorochromeshasmadeitpossibletoidentifycellsandsub-microscopiccellularcomponentswithahighdegreeofspecificityamidnon-fluorescingmaterial(13).Infact,theconfocalmicroscopeisoftencapableofrevealingthepresenceofasinglemolecule(14).Throughtheuseofmultiply-labeledspecimens,differentprobescansimultaneouslyidentifyseveraltargetmoleculessimultaneously,bothinfixedspecimensandlivingcellsandtissues(15).Althoughbothconventionalandconfocalmicroscopescannotprovidespatialresolutionbelowthediffractionlimitofspecificspecimenfeatures,thedetectionoffluorescingmoleculesbelowsuchlimitsisreadilyachieved.ThebasicconceptofconfocalmicroscopywasoriginallydevelopedbyMarvinMinskyinthemid-1950s(patentedin1961)whenhewasapostdoctoralstudentatHarvardUniversity(16,17).Minskywantedtoimageneuralnetworksinunstainedpreparationsofbraintissueandwasdrivenbythedesiretoimagebiologicaleventsattheyoccurinlivingsystems.Minsky’sinventionremainedlargelyunnoticed,duemostprobablytothelackofintenselightsourcesnecessaryforimagingandthecomputerhorsepowerrequiredtohandlelargeamountsofdata.FollowingMinsky’swork,M.DavidEggerandMojmirPetran(18)fabricatedamultiple-beamconfocalmicroscopeinthelate1960sthatutilizedaspinning(Nipkow)diskforexaminingunstainedbrainsectionsandganglioncells.Continuinginthisarena,Eggerwentontodevelopthefirstmechanicallyscannedconfocallasermicroscope,andpublishedthefirstrecognizableimagesofcellsin1973(19).Duringthelate1970sandthe1980s,advancesincomputerandlasertechnology,coupledtonewalgorithmsfordigitalmanipulationofimages,ledtoagrowinginterestinconfocalmicroscopy(20).Fortuitously,shortlyafterMinsky’spatenthadexpired,practicallaserscanningconfocalmicroscopedesignsweretranslatedintoworkinginstrumentsbyseveralinvestigators.DutchphysicistG.FredBrakenhoffdevelopedascanningconfocalmicroscopein1979(21),whilealmostsimultaneously,ColinSheppardcontributedtothetechniquewithatheoryofimageformation(22).TonyWilson,BradAmos,andJohnWhitenurturedtheconceptandlater(duringthelate1980s)demonstratedtheutilityofconfocalimagingintheexaminationoffluorescentbiologicalspecimens(20,23).Thefirstcommercialinstrumentsappearedin1987.Duringthe1990s,advancesinopticsandelectronicsaffordedmorestableandpowerfullasers,high-efficiencyscanningmirrorunits,high-throughputfiberoptics,betterthinfilmdielectriccoatings,anddetectorshavingreducednoisecharacteristics(1).Inaddition,fluorochromesthatweremorecarefullymatchedtolaserexcitationlineswerebeginningtobesynthesized(13).Coupledtotherapidlyadvancingcomputerprocessingspeeds,enhanceddisplays,andlarge-volumestoragetechnologyemerginginthelate1990s,thestagewassetforavirtualexplosioninthenumberofapplicationsthatcouldbetargetedwithlaserscanningconfocalmicroscopy.Modernconfocalmicroscopescanbeconsideredascompletelyintegratedelectronicsystemswheretheopticalmicroscopeplaysacentralroleinaconfigurationthatconsistsofoneormoreelectronicdetectors,acomputer(forimagedisplay,processing,output,andstorage),andseverallasersystemscombinedwithwavelengthselectiondevicesandabeamscanningassembly.Inmostcases,integrationbetweenthevariouscomponentsissothoroughthattheentireconfocalmicroscopeisoftencollectivelyreferredtoasadigitalorvideoimagingsys