激光共聚焦显微镜原理与应用详细资料.docx

激光共聚焦显微镜原理与应用详细资料.docx

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激光共聚焦显微镜原理与应用详细资料

ConfocalMicroscopy

ConfocalMicroscopy:

BasicConcepts

Confocalmicroscopyoffersseveraladvantagesoverconventionalopticalmicroscopy,includingshallowdepthoffield,eliminationofout-of-focusglare,andtheabilitytocollectserialopticalsectionsfromthickspecimens.Inthebiomedicalsciences,amajorapplicationofconfocalmicroscopyinvolvesimagingeitherfixedorlivingcellsandtissuesthathaveusuallybeenlabeledwithoneormorefluorescentprobes.

Whenfluorescentspecimensareimagedusingaconventionalwidefieldopticalmicroscope,secondaryfluorescenceemittedbythespecimenthatappearsawayfromtheregionofinterestofteninterfereswiththeresolutionofthosefeaturesthatareinfocus.Thissituationisespeciallyproblematicforspecimenshavingathicknessgreaterthanabout2micrometers.Theconfocalimagingapproachprovidesamarginalimprovementinbothaxialandlateralresolution,butitistheabilityoftheinstrumenttoexcludefromtheimagethe"out-offocus"flarethatoccursinthickfluorescentlylabeledspecimens,whichhascausedtherecentexplosioninpopularityofthetechnique.Mostcurrentconfocalmicroscopesarerelativelyeasytooperateandhavebecomepartofthebasicinstrumentationofmanymulti-userimagingfacilities.Becausetheresolutionpossibleinthelaserscanningconfocalmicroscope（LSCM）issomewhatbetterthanintheconventionalwidefieldopticalmicroscope,butstillconsiderablylessthanthatofthetransmissionelectronmicroscope,ithasinsomewaysbridgedthegapbetweenthetwomorecommonlyusedtechniques.Figure1illustratestheprincipallightpathwaysinabasicconfocalmicroscopeconfiguration.

Inaconventionalwidefieldmicroscope,theentirespecimenisbathedinlightfromamercuryorxenonsource,andtheimagecanbevieweddirectlybyeyeorprojecteddirectlyontoanimagecapturedeviceorphotographicfilm.Incontrast,themethodofimageformationinaconfocalmicroscopeisfundamentallydifferent.Theilluminationisachievedbyscanningoneormorefocusedbeamsoflight,usuallyfromalaser,acrossthespecimen（Figure2）.Theimagesproducedbyscanningthespecimeninthiswayarecalledopticalsections.Thisterminologyreferstothenoninvasivemethodbywhichtheinstrumentcollectsimages,usingfocusedlightratherthanphysicalmeanstosectionthespecimen.

Theconfocalapproachhasfacilitatedmuchmoreusefulimagingoflivingspecimens,enabledtheautomatedcollectionofthree-dimensional（z-series）data,andimprovedtheimagesobtainedofspecimensusingmultiplelabeling.Figure3presentsacomparisonofaconventionalepifluorescenceimagewithaconfocalimageofsimilarregionsofawholemountofabutterflypupalwingepitheliumstainedwithpropidiumiodide.ThereisastrikingimprovementofresolutionofnucleiintheLSCMimageduetoeliminationofout-of-focusfluorescenceflare.

Thelaserscanningconfocalmicroscope（LSCM）iscurrentlythemostwidelyusedconfocalvariationforbiomedicalresearchapplications.EmphasisisplacedontheLSCMinthisintroduction,sinceitisthedesignmostlikelytobeencounteredbythenoviceuser.Otheralternativedesignsoftheinstrumentsarefavoredinspecificnicheswithinthefieldofbiologicalimaging.Mostoftheprotocolsforspecimenpreparationcanbeused,withminormodification,foranyoftheconfocalinstrumentvariants,aswellasforothermethodologiesforproducingopticalsectionssuchasdeconvolutiontechniquesandmultiple-photonimaging.

EvolutionofConfocalMicroscopy

TheinventionoftheconfocalmicroscopeisusuallyattributedtoMarvinMinsky,whoproducedaworkingmicroscopein1955.Thedevelopmentoftheconfocalapproachwaslargelydrivenbythedesiretoimagebiologicaleventsastheyoccurinlivingtissue（invivo）,andMinskyhadthegoalofimagingneuralnetworksinunstainedpreparationsoflivingbrains.TheprincipleofconfocalimagingadvancedbyMinsky,andpatentedin1957,isemployedinallmodernconfocalmicroscopes.Figure1illustratestheconfocalprinciple,asappliedinepifluorescencemicroscopy,whichhasbecomethebasicconfigurationofmostmodernconfocalsystemsusedforfluorescenceimaging.Minsky'soriginalconfigurationusedapinholeplacedinfrontofazirconiumarcsourceasthepointsourceoflight.

Thepointoflightwasfocusedbyanobjectivelensatthedesiredfocalplaneinthespecimen,andlightthatpassedthroughitwasfocusedbyasecondobjectivelensatasecondpinholehavingthesamefocusasthefirstpinhole（thetwowereconfocal）.Anylightthatpassedthesecondpinholestruckalow-noisephotomultiplier,whichgeneratedasignalthatwasrelatedtothebrightnessofthelightfromthespecimen.Thesecondpinholepreventedlightoriginatingfromaboveorbelowtheplaneoffocusinthespecimenfromreachingthephotomultiplier.Theuseofspatialfilteringtoeliminateout-of-focuslightorflare,inspecimensthatarethickerthantheplaneoffocus,isthekeytotheconfocalapproach.InMinsky'swritingshealsodescribedareflectedlightversionofthemicroscopethatusedasingleobjectivelensandadichromaticmirrorarrangementthatbecamethebasisforthesystemscurrentlyinuse.

Inordertobuildanimageusingtheconfocalprinciple,thefocusedspotoflightmustbescannedacrossthespecimeninsomeway.IntheoriginalinstrumentbuiltbyMinskythebeamwaskeptstationaryandthespecimenitselfwasmovedonavibratingstage.Thisarrangementhastheadvantagethatthescanningbeamisheldstationaryontheopticalaxisofthemicroscope,whichcaneliminatemostlensdefectsthatwouldaffecttheimage.Forbiologicalspecimens,however,movementofthespecimencancausewobbleanddistortion,resultinginalossofresolutionintheimage.Furthermore,itisimpossibletoperformvariousmanipulationsonthespecimensuchasmicroinjectionoffluorescentlylabeledprobeswhenthestageandspecimenaremoving.

Regardlessofthemeansbywhichtheilluminatingbeamisscannedacrossthespecimen,animageofthespecimenmustbeproduced.ArealimagewasnotformedinMinsky'soriginaldesign,butinsteadtheoutputfromthephotomultiplierwastranslatedintoanimageonthescreenofamilitarysurpluslongpersistenceoscilloscopethathadnoprovisionforrecording.Followingthedebutofhisinvention,Minskylaterwrotethattheimagequalityinhismicroscopewasnotveryimpressivebecauseofthequalityoftheoscilloscopedisplayandnotbecauseofpoorresolutionachievedbythemicroscopeitself.ItisnowclearthatthetechnologywasnotavailabletoMinskyin1955tofullydemonstratethepotentialoftheconfocalapproach,especiallyforimagingbiologicalstructures.Hestatedthatthisispossiblyareasonthatconfocalmicroscopywasnotimmediatelyembracedbythebiologicalcommunity,whowere,andstillare,ahighlydemandinggroupconcerningthequalityoftheirimages.Atthetime,theyhadavailablelightmicroscopeswithexcellentoptics,andcouldeasilyviewandphotographtheirbrightlystainedandcolorfulhistologicaltissuesectionsontohigh-resolutioncolorfilm.Intoday'sconfocalmicroscopes,theimageisseriallybuiltupfromtheoutputofaphotomultipliertubeorcapturedusingadigitalcameraincorporatingacharge-coupleddevice,directlyprocessedinacomputerimagingsystem,displayedonahigh-resolutionvideomonitor,andoutputonahardcopydevice,withoutstandingresults.TheinformationflowinamodernlaserscanningconfocalmicroscopeisdiagramedinFigure4.

Thebasicopticsoftheopticalmicroscopehaveremainedfundamentallyunchangedfordecadesbecausethefinalresolutionachievedbytheinstrumentisgovernedbythewavelengthoflight,theobjectivelens,andthepropertiesofthespecimenitself.Thedyesusedtoaddcontrasttospecimens,andothertechnologyassociatedwiththemethodsofopticalmicroscopy,haveimprovedsignificantlyoverthepast20years.Thegrowthandrefinementoftheconfocalapproachisadirectresultofarenaissanceinopticalmicroscopythathasbeenfueledlargelybyadvancementsinmoderntechnology.AnumberofmajortechnologicaladvancesthatwouldhavebeenabenefittoMinsky'sconfocaldesignhavegraduallybecomeavailable（ormoreaffordable）tobiologistsandothermicroscopists.Amongthesearestablemultiwavelengthlasersforimprovedpointlightsources,improveddichromaticmirrors,sensitivelow-noisephotodetectors,fastmicrocomputerswithimageprocessingcapabilitiesenhancedbyavailabilityofaffordablelarge-capacitymemorychips,sophisticatedimageanalysissoftwarepackages,andhigh-resolutionvideodisplaysanddigitalimageprinters.

Thesetechnologiesweredevelopedindependently,andsince1955,havebeengraduallyincorporatedintoconfocalimagingsystems.Asoneexample,digitalimageprocessingtechniqueswerefirstappliedeffectivelyintheearly1980sbyresearchersattheWoodsHoleOceanographicInstitute.Usingwhattheytermed"video-enhancedmicroscopes"theywereabletoimagecellularstructuressuchasmicrotubules,whicharejustbeyondthetheoreticalresolutionoftheopticalmicroscope.Theapparentincreaseinresolutionwasenabledbydigitalenhancementofimagesthatwerecapturedusingalowlightlevelsiliconintensifiedtarget（SIT）videocameraconnectedtoadigitalimageprocessor.Thecellularstructureswereimagedusingdifferentialinterferencecontrast（DIC）optics,andtheimageswerefurtherenhancedusingdigitalprocessingmethods.

Theclassificationofconfocalmicroscopedesignsisusuallydoneonthebasisofthemethodbywhichthespecimensarescanned.Thetwofundamentalmeanso