流式细胞术基本原理及应用VIP专享VIP免费

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SampleFlowinOpticalCuvetteSampleLaminarFlowLowSampleFlowRate12L/minSheathSheathSampleLaminarFlowSheathSheathOpticalCuvetteHighSampleFlowRate60L/minReviewQuestionWhichofthefollowingwouldcausedisturbanceinthelaminarflowoftheopticalcuvette?A.bubblesB.cellularconcentrationC.sampleflowrateOpticsExcitationopticsconsistof:LasersLensesandmirrorsthatroutethelaserlighttothefluidicstreamCollectionopticsconsistof: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...