流式细胞术原理及应用ForFCMTraining流式细胞术流式细胞术(FlowCytometry,简称FCM)是一种可以快速、准确、客观,并且同时检测单个微粒(通常是细胞)的多项特性的技术,同时可以对特定群体加以分选研究对象为生物颗粒,如各种细胞、染色体、微生物、及人工合成微球等研究的微粒特性包括多种物理及生物学特征,并加以定量流式细胞仪系统简介—通过流式细胞仪我们可以得到以下信息-相对细胞大小-相对细胞颗粒密度和内部复杂度-染色过细胞的相对荧光强度InjectorTipFluorescenceFluorescencesignalssignalsFocusedlaserFocusedlaserbeambeamSheathfluid流式细胞仪的光信号—散射光信号—荧光信号1.散射光信号•前向角散射光(FSC,ForwardScatter)入射激光的同向散射光信号细胞相对大小及其表面积。•侧向角散射(SSC,SideScatter)入射激光90角的散射光信号细胞粒度及细胞内相对复杂性。前向角散射光——FSC—ForwardAngleLightScatterForwardAngleLightScatterFALSSensorLaser侧向角散射光——SSCFALSSensor90LSSensorLaser散射光—散射光能被用来区分不同细胞群体的基本形态上的差异-通常使用“散点图”来看散射光信号-散点图上的一个点就代表一个细胞颗粒的数据散点图——DotPlotlysedwholebloodReviewQuestionDeadcellsareknowntobesmallerandtoexhibitmoreinternalcomplexitythanlivecells.Whichofthepopulationsonthisplotwouldyouexpecttobedead?2.荧光信号2.荧光信号荧光素吸收激光能量荧光素将吸收能量释放,转换为振动能和热能释放较入射光波长更长的光量子荧光素与特异抗体结合荧光抗体与细胞抗原结合越多,产生的荧光信号越强荧光检测器FreqFluorescenceFALSSensorFluorescencedetector(PMT3,PMT4etc.)Two-ColorCellAnalysisWhichofthethreepopulationshasthemostAbAbindingsites?AbAAbB双色荧光散点图现代流式细胞仪包括—液流系统聚焦细胞以供检测—光学系统激发和收集光信号—电子系统将光信号转化为电信号,并使其数字化以供计算机分析液流系统—液流系统将样本悬液聚焦在光源的中心处SampleFlowinOpticalCuvetteSampleLaminarFlowLowSampleFlowRate12L/minSheathSheathSampleLaminarFlowSheathSheathOpticalCuvetteHighSampleFlowRate60L/minReviewQuestionWhichofthefollowingwouldcausedisturbanceinthelaminarflowoftheopticalcuvette?A.bubblesB.cellularconcentrationC.sampleflowrateOpticsExcitationopticsconsistof:LasersLensesandmirrorsthatroutethelaserlighttothefluidicstreamCollectionopticsconsistof:FiltersthatdirectthesignalstotheappropriateopticaldetectorsOpticalFilters460500540460500540460500540SP500SP500LP500LP500BP500/50BP500/50LongpassShortpassBandpassFACSCalibur光路图OpticsLaserFluorochromesDetectorParameterFilterFITCFITCGFPGFPFL1FL1530/30530/30PEPEPIPIFL2FL2585/42585/42PerCPPerCPPerCP-CY5.5PerCP-CY5.5FL3FL3670LP670LPAPCAPCFL4FL4661/16661/16488nm488nm635nm635nmElectronics—Convertsanalogsignalstoproportionaldigitalsignals—ComputesHeightforeachpulse—Calculateswidthandarea—InterfaceswiththecomputerfordatatransferCreationofaVoltagePulse-AnalogSignalLaserLaserLaserLaserLaserLaserTimeVoltageTimeVoltageTimeVoltageQuantificationofaVoltagePulseTimeVoltsPulseAreaPulseHeightPulseWidth0—Heightisameasurementforallparameters.—Width=Area/HeightEffectoftheInstrumentControlsontheDataInstrumentInstrumentControlsControlsDetecDetectortorFSCdetector250gainFSCdetector250gainFSCdetector350gainFSCdetector350gainReviewDataDataProcessinProcessinggFL2FL1SSCFSCFL4FL3ReviewQuestions1.WhichofthefollowingfluorochromescannotbeusedwiththeFACSCalibur?a.DAPI(ex.345nm,emits455nm)b.PropidiumIodide(ex.536nm,emits617nm)c.AlexaFluor647(ex.650nm,emits668nm)2.WhatarethethreemeasurementsofaparticlethatcanbedeterminedbyFACSC...