流式细胞仪器参数设置与追踪(cst)PPT演示课件

2,CytometerPerformanceCS&T,2,2,KnowYourCytometer,CytometerCharacterizationForeachfluorescenceparameter:DetectorEfficiencyQrBackgroundSignalBrResolution(highsignal)%rCVforabrightparticleLinearRangeLinearityMinandMaxLimitsStandardDeviationofElectronicNoiseSDEN,2,3,DetectionEfficiencyQr,QrisproportionaltoQ=,#fluorochromemolecules,#photoelectrons,Cytometer1APCdetectorphotoelectrons,Cytometer2APCdetectorphotoelectrons,WhichcytometerhashigherQrforAPCdetection?,2,4,WhyisQrImportant?,HigherQrmoresensitivity.Whenotherfactorsareheldconstant,sensitivity(asquantifiedbystainindex)isproportionalto.,LowQr,HighQr,2,5,FactorsAffectingDetectorEfficiency,LaserpowerandalignmentOpticalefficiencyOpticscleanlinessordamagePMTredspectrumsensitivityPoorPMTperformance(age)DirtyflowcellDirtyordegradedfilter,2,6,Brisameasureofopticalbackground.,Unboundantibodyorfluorochrome,Spectraloverlapfromanothermarkeronacell,Autofluorescence,Ramanscatter,Scatterfromtheflowcellandambientlight,BackgroundSignalBr,Bristhenumberoffluorochromemoleculesthatwouldproduceasignalequivalenttothebackgroundsignal.,2,7,WhyisBrImportant?,HigherBrlesssensitivity.Whenotherfactorsareheldconstant,sensitivity(asquantifiedbystainindex)isproportionalto.,LowBr,HighBr,2,8,FactorsAffectingBr,Dirtyflowcell.Damagedopticalcomponent.BluelaserparametersFITCandPEexhibithighBrduetoRamanscatter.VioletlaserparametersexhibithighBrduetoautofluorescence.,2,9,QrandBr:ALookatSomeRealData,HighQrLowBr,HighQrHighBr,LowQrHighBr,QrandBrcharacterizationsareforbothFITCandPerCP-Cy5.5parameters.PlotsgatedonCD3+lymphocytes.,2,10,Resolutionistheabilitytodistinguishbetweenpopulationsbasedonfluorescense.Resolutionisakeyfactorinsensitivity.,Resolution,GoodResolution,PoorResolution,FluorescenceIntensity,Apracticalmeasureofresolutionisthe%rCVofareferenceparticleinthemidtoupperendofthescale.,2,11,CytometerFactorsAffectingResolution,UniformexcitationofparticlesLaserpowerLaserbeamshapeandalignmentFlowrateWindowextension,2,12,Linearityistheproportionalityofoutputtoinputforfluorescencemeasurements.,XAb,2XAbs,0,1000,2000,3000,WhatisLinearity?,XAbs,2XAbs,=,1000,2000,2,13,WhyisLinearityImportant?,AccuratefluorescencecompensationSmalldeviationsinlinearityinthelastdecadecanresultinsignificantcompensationerrors.QuantitativemeasurementstudiesDNAAntigen-antibodybinding,2,14,EffectofNon-LinearityonCompensation,FITCMFIPEMFI,179675,6880,592180,73000365,D,B,A,C,SamplesareBDCompBeadparticlesstainedwithvaryinglevelsofFITC-Ab.CompensationiscalculatedusingsamplesAandC.Thiscytometerhasapoorlinearrange,with2%deviationfromlinearityabovetheMaxLinearityChannelat50,000.,2,15,CytometerFactorsAffectingLinearity,QualityofPMTtubeanditssocketPreampandelectronicsLaserbeamshapeandalignment,2,16,SDENisthesignalvariationduetolowlevelbackgroundnoisecontributedbytheelectronicssystem.LowSDENhighersensitivityandlargerdynamicrange.Mostcriticalforparameterswithlowcellularautofluorescence:APC-H7PE-Cy7PerCP-Cy5.5APC,StandardDeviationofElectronicNoiseSDEN,2,17,PMTconnectorsPMTnoiseCablesnearpowersourcesAnalogtodigitalconversionerrorWindowextension,FactorsAffectingSDEN,2,18,WindowExtension,intensity,processedevent,time,electronicabort,windowextensionsoverlap,threshold,windowextensions,windowgate,belowthreshold,2,19,SmallerwindowextensionscanlowertheSDENcontributiontovariationrelativetotheintegratedpulsesignal.,WindowExtensionandSDEN,time,intensity,threshold,windowextensions,Caution:smallerwindowextensionscanamplifyothermeasurementerrors.LaserdelayLinearity,SensitivityExercise,2,20,CS&T,CS&Tisafullyautomatedsoftwareandresearchreagentsystem,uniquetoBDdigitalcytometers,providing:CharacterizationSetupTracking,2,21,CS&TBeads,Twosizesandthreeintensitiesofuniformbeads:2mdimfluorescenceintensity3mmidfluorescenceintensity3mbrightfluorescenceintensityExcitedanddetectedinallBDpreconfiguredcytometers.,Dim,Bright&Mid,3m,2m,Bright,Mid,Dim,FSC-A,FITC-A,(x1,000),2,22,CS&TWorkflow,AdministratorAccess,AllUsersAccess,Performinitiallyforeachconfiguration.Rerunbaselineatothertimesincludingaftermajorserviceandwhenexistingbaselineexpires.,Performinitiallyforeachapplication,andalsoafteranewbaseline.,Performdailybeforerunningexperiments.,Performwitheachexperiment.,2,23,CreateCytometerConfiguration,CS&TWorkflow,Definetheopticallayoutofthecytometerincludingfilters,mirrors,andparameterlabels.,CheckPerformance,CreateApplicationsSettings,DefineBaseline,CheckPerformance,ApplyApplicationSettings,2,24,CS&TWorkflow,Determinethebaselineperformanceofthecytometerbymeasuringkeyfactorssuchaslinearity,detectorefficiency,opticalbackground,andelectronicnoise.DetermineatargetMFIforeachparameter.Determinealinearrangeforeachparameter.,CreateCytometerConfiguration,CheckPerformance,CreateApplicationsSettings,DefineBaseline,CheckPerformance,ApplyApplicationSettings,2,25,BaselinePMTVsandMFITargetValues,Foreachparameter,CS&TdeterminesabaselinePMTVthatplacesthedimbeadatanMFIofabout10 xSDEN.,SDEN=18.11,MFI=10 xSDEN=181.1,PMTV=451,TheMFItargetvalueistheMFIofthebrightbeadatthebaselinePMTV.,2,26,IfaPMTVissettoolow,electronicnoisewilldecreasesensitivity.,PMTV=360,WhyareApplicationPMTVsImportant?,PMTV=560,IfaPMTVissettoohigh,positivepopulationsmaybeoutofthelinearrangeoroffscale.,PMTV=460,OptimalPMTVprovidesgoodsensitivity,positivepopulationsinlinearrange.,2,27,TheMFItargetvalueistheMFIofthebrightbeadatthebaselinePMTV.,WhatareTargetValues?,2,28,Day1,Day2,Day2,550V,550V,560V,WhyareTargetValuesImportant?,Bydeterminingthevoltageneededtoplacethebrightbeadsatthetargetvalue,CS&Tsupportstrackingandreproducibilityofresultsovertime.,OnDay2,theCS&TPerformanceCheckdeterminesthatthePMTvoltagemustberaisedfrom550to560toremainatthetargetvalue.,2,29,HowisLinearityCalculated?,MFIRatio=,MidbeadMFI,BrightbeadMFI,Linearrange,Non-linear,Non-linear,MFIRatioMIDistheconstantratiothroughthemiddleofthescale,%Deviation=100 x,MFIRatioMID,(MFIRatioMIDMFIRatio),LinearityMinandMaxChannelboundariessetsothatlinearrangehas%Deviation2%,BrightbeadMFI,2,30,BaselineLaserSettings,Characteristicsofthelasersaremeasuredandreported.Thelaserdelay,areascalingfactors,andthewindowextensionareset.FortheBDFACSAriacytometer,thelaserdelayissetforaspecificsheathpressure.Areascalingmayneedtobeoptimizedfordifferentcelltypes.,2,31,RobustStandardDeviationrSD,rSDisarobustestimateofstandarddeviation(SD)thatislessaffectedbythebehaviorofoutliers.,outliers,population2,TheSDofpopulation1issignificantlylowerthantheSDofpopulation2.TherSDsofthetwopopulationsareidentical.,2,32,RobustCoefficientofVariation%rCV,%rCVisarobustestimateof%CVthatislessaffectedbythebehaviorofoutliers.%CV=100 xSD/Mean%rCV=100 xrSD/Median,outliers,population1,outliers,population2,The%CVofpopulation1issignificantlylowerthanthe%CVofpopulation2.The%rCVsofthetwopopulationsareidentical.,2,33,LinearityMinChannelandLinearityMaxChannel:linearrangeElectronicNoise(EN)RobustSDQr:detectorefficiencyBr:background,PMTV:optimizedPMTvoltagesNewTargetValue:MFItargetforbrightbeadBrightBead%RobustCV,CytometerConfiguration,2,34,CS&TWorkflow,Mustberunbeforecreatingapplicationsettings.,CreateCytometerConfiguration,CheckPerformance,CreateApplicationsSettings,DefineBaseline,CheckPerformance,ApplyApplicationSettings,2,35,CS&TWorkflow,DetermineoptimalMFItargetsforyourapplicationpopulations.,CreateCytometerConfiguration,CheckPerformance,CreateApplicationsSettings,DefineBaseline,CheckPerformance,ApplyApplicationSettings,2,36,CS&TWorkflow,CreateCytometerConfiguration,CheckPerformance,CreateApplicationsSettings,DefineBaseline,CheckPerformance,ApplyApplicationSettings,Measurethevariationfromthebaselineandrecordsdatafortrackingday-to-daycytome