流式细胞仪基本原理及应用

©byDrBaiqingLI免疫学教研室流式细胞仪在临床检测和临床科研中的应用李柏青蚌埠医学院免疫学教研室AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-2-流式细胞仪和流式细胞术－概述流式细胞仪的工作原理和测量参数流式细胞仪在临床检测和科研中的应用免疫学检测和科研应用血液病检测和科研应用肿瘤学检测和科研应用流式细胞仪的基本工作原理以及在基础和临床科研中的应用AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-3-一、流式细胞仪和流式细胞术的基本概念流式细胞仪(FlowCytometer，FCM):是集合光电子物理，光电测量技术，液流技术，计算机技术等现代高科技技术为一体的细胞测量和分析仪器。AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-4-一、流式细胞仪和流式细胞术的基本概念流式细胞术(FlowCytometry，FCM):是应用流式细胞仪，对处于快速流动液体中的细胞或生物颗粒进行多参数、快速测量和定量分析，以及分选的技术。AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-5-一、流式细胞仪和流式细胞术－特点多参数测量和分析（散射光和荧光）；快速测量和分析每秒可达1000-10000个细胞分选纯度可达99%以上。获取的数据分析结果更具有统计学意义；通常测量的对象如各种细胞细胞必须处在单个细胞悬液中。此外，也可测量微生物等生物颗粒，或者工合成微球非生物颗粒，AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-6-台式机——FACSCalibur双激光488nm635nm四荧光参数分选、浓缩系统7AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-8-大型机——FACSVantageSE科研型八荧光参数高速分选多路分选AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-9-流式细胞仪与荧光显微镜比较流式细胞仪荧光显微镜流动的细胞静止的细胞样本数量大数量少高速分选样本难以再利用细胞群体特征量分布可揭示细胞内部结构信息AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-10-二、流式细胞仪的工作原理和测量参数光路系统：激光光源，光路系统（透镜、滤光片）电子系统：光电倍增管（PMT），补偿电路液流系统：鞘液系统，样品流动系统数据系统：计算机，数据测量和分析软件。细胞分选系统（一）流式细胞仪的结构AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-11-流式细胞仪－光路系统激光(Laser)是一种相干光源，它能提供单一波长、单一方向、同步的稳定光照激光波长:488nm,635nm光收集系统是由若干组透镜,滤波片,小孔组成，将产生的光信号引导至检测器AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-12-光路系统AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-13-InjectorTipAllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-14-InjectorTipFluorescencesignalsFocusedlaserbeamSheathfluidAllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-15-流式细胞仪－光学系统激发光源与荧光标记物AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-16-光学检测信号AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-17-流式细胞仪的光学系统FACSCaliburAllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-18-流式细胞仪－液流系统AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-19-液流系统：鞘液系统＋样品流动系统AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-20-流式细胞仪的电子系统信号检测和分析当细胞携带荧光素,通过激光照射区,受到激发产生不同波长的、代表细胞内不同物质的荧光信号荧光信号由光电接收器接收，转变为电脉冲信号，电脉冲信号经A/D转换成数字信号数字信号传送到计算机，进行储存、作图和统计分析AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-21-数据系统AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-22-（二）流式细胞仪可检测的细胞参数细胞结构细胞大小细胞颗粒性程度细胞表面面积核浆比例DNA含量与细胞周期RNA含量细胞功能细胞表面蛋白抗原细胞胞浆内蛋白性抗原细胞内细胞因子细胞增殖细胞内钙离子浓度二、流式细胞仪的工作原理和测量参数AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-23-（二）流式细胞仪可检测的细胞的参数：散射光信号前向角散射光（FSC,ForwardScatter）入射光的前向(5)散射光信号,可反映：细胞相对大小及其表面积侧向角散射光（SSC,SideScatter）入射光90角的散射光信号，可反映：细胞颗粒性程度（胞内成分和构造和表面结构的复杂性）AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-24-散射光信号前向角散射光－FSCFALSSensorLaserAllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-25-散射光信号侧向角散射光－SSCFALSSensor90LSSensorLaserAllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-26-流式细胞的基本信息:“形态”AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-27-AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-28-外周全血细胞（红细胞溶解后）以散射光表示的双参数点图AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-29-外周全血细胞（红细胞溶解后）以散射光表示的双参数点图中性粒细胞单核细胞淋巴细胞AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-30-（二）流式细胞检测细胞参数:特征信息－荧光（Fluorescence）信号荧光素吸收激光（激发光波长）能量荧光素将吸收能量释放，转换为释放较入射光波长更长的光量子（发射光波长）荧光素与特异抗体结合（荧光抗体）荧光抗体与细胞抗原结合越多，产生荧光信号越强荧光素与细胞核酸结合，荧光强度反映核酸含量AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-31-（二）流式细胞检测细胞参数:特征信息－荧光（Fluorescence）信号Forwardscatter(FSC)Sidescatter(SSC)FL1（第一荧光）-FITC(异硫氰酸荧光素）FL2（第二荧光）-PE（藻红蛋白）FL3（第三荧光）-PerCP,PeCy5等PL4（第四荧光）-APC（藻青蛋白）AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-32-荧光信号FreqFluorescenceFALSSensorFluorescencedetector(PMT3,PMT4etc.)AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-33-AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-34-荧光信号双色直接染色AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-35-（三）流式细胞仪分析数据常见图形：（二维）点陈图(DotPlot)适用于：双参数分析AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-36-（三）流式细胞仪分析数据常见图形：直方图(HistogramPlot)适用于：单参数分析仅需要观察某个荧光强度的变化AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-37-流式细胞术检测细胞的荧光标记方法单标：一种单抗:FL1,FSC,SSC（三参数）双标：两种单抗：FL1,FL1,FSC,SSC（四参数）三标：三种单抗：FL1,FL2,FL3,FSC,SSC（五参数）四标－四种荧光标记抗体：四种单抗：FL1,FL2,FL3,FL4,FSC,SSC（六参数）AllrightsreservedbyDrBaiqingLIBengbuMedicalCollegeDepartmentofImmunology-38-淋巴细胞亚群分析细胞凋亡肿瘤学移植免疫三流式细胞仪在临床检测和科研中的应用血小板其它