光学显微与电子成像-激光扫描共焦显微镜

CONFOCALMICROSCOPY***2014.11.24Chapter12Outline1.PrincipleofconfocalmicroscopeScanmodesImagegenerationAdvantages2.ThefactorsaffectingthequalityoftheimagePinholesizeScanrateZoomPrinciplesofConfocalmicroscopyPrinciplesofconfocalmicroscopyScanmodesImagegenerationAdvantagesPrinciplesofConfocalmicroscopyInconfocalsystem:ApointlightsourceforilluminationApointlightfocuswithinthespecimenApinholeattheimagedetectingplaneWhatisconfocal?Thesethreepointsareopticallyconjugatedtogetherandalignedaccuratelytoeachotherinthelightpathofimageformationthisisconfocal.PrinciplesofConfocalmicroscopyPrinciplesofConfocalMicroscopy3213PMT,forTPEFPMT(transmission)PMT(4Channels)ForfluorescenceK-ScannerZ-axisscandeviceDichromaticMirrorCoolingdeviceforscannerPinholelensPrismUVPinholelensConfigurationofConfocalmicroscopes(Leica)PinholePrinciplesofConfocalmicroscopyPrinciplesofconfocalmicroscopyScanmodesImagegenerationAdvantagesScanmodesBeamscansystemsingle-beamscanning(currentlymostpopular,usedintheLSCM)multiple-beamscanningScanmodessingle-beamscanning(currentlymostpopular,usedintheLSCM)

ScanmodesScanningasinglemirrorontwoorthogonalaxestwo-mirrorconfigurationsingle-beamscanning00Scanmodesmultiple-beamscanningScanmodesEachdiskhasmorethan20,000pinholes.PrinciplesofConfocalmicroscopyPrinciplesofconfocalmicroscopyScanmodesImagegenerationAdvantagesImageGenerationOriginaldesign:ArealimagewasnotformedinMinsky'soriginaldesign,butinsteadtheoutputfromthephotomultiplierwastranslatedintoanimageonthescreenofamilitarysurpluslongpersistenceoscilloscopethathadnoprovisionforrecording.ImageGenerationPMT:detectslightandconvertsphotonhitsintoanalogueelectronflow.ADC:analogueelectronsignalisthensampledatseparatetimepoint,digitizedimageprocessor:dotbydotintoaline,andlinebylineintoaframeImageGenerationToday’sconfocalmicroscopePrinciplesofConfocalmicroscopyPrinciplesofconfocalmicroscopyScanmodesImagegenerationAdvantagesAdvantagesandfeaturesConfocalmicroscopyadvantages(comparedwithconventionalopticalmicroscopy):shallowdepthoffieldeliminationofout-of-focusglaretheabilitytocollectserialopticalsectionsfromthickspecimensAdvantagesandfeaturesConfocalimageshavefollowingfeatures:voidofinterferencefromlateralstraylight:highercontrast.voidofsuperimposeofout-of-focal-planesignal:lessblur,sharperimage.imagesderivedfromopticallysectionedslices.(depthdiscrimination,3D-view)X-,y-,z-,time-,colors-.(5D-view)ConfocaltraditionalAdvantagesandfeaturesThefactorsthataffectthequalityoftheimagesPinholesizeScanrateZoomPinholesizeResolution

thesizeoftheinnerdarkringofAirydisk.

theapertureofthepinholelensaperturePSFinnerringPinholesizeTheeffectofthepinholediameteronthethicknessoftheopticalsectionisshowninthefollowingpicture.PinholesizeAsmallpinholegivesthethinnestopticalsection.Italsoimproveshorizontalresolutionandcontrastintheimage.However,reducingthesizeofthepinholealsodecreasesthenumberofphotonsreachingthedetector.Ifwecompareimagesmadeatdifferentpinholesettingsbutthesameexposuretime,weseethattheimageacquiredusingawiderpinholeisbrighterandcontainslessnoise.PinholesizeTheoptimumaveragepinholesettingcorrespondstoanaperturegiving50%ofmaximumintensity.Atthisposition,75%ofthelightoftheAirydiskstillpassesthepinhole,andresolutionwillbe20%betterthanobtainedbyawide-fieldfluorescencesystem.ThefactorsthataffectthequalityoftheimagesPinholesizeScanrateZoom

Iftheamplitudeofdeflectionofthegalvanometermirrorsissmallandthescanrateishigh,asmallerimageareacanbepresentedonthemonitoratratesuptohundredsofframespersecond.ScanrateHighspeedisalsousefulformonitoringrapidkineticeventsinthespecimen.ScanrateThespeedforconfocalmicroscopeThespeedforconfocalmicroscopeislimitedbythelinefrequencyofthescanner.Generallyspeaking,intermsofspeed,confocalmicroscopyisslowerthanCCDcamerabasedsystemintheframebasedacquisition.Butthelinefrequencycanbeusedmore"economically"inconfocalinlinescanmodewherenomatrixof"mxn"isneeded.ThefactorsthataffectthequalityoftheimagesPinholesizeScanrateZoomZoomElectroniczoomisusedfortworeasons:1)