流式细胞术简介介绍.ppt

流式细胞术简介 For FCM Training 流式细胞术 流式细胞术（Flow Cytometry, 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 流式细胞仪的光信号 散射光信号 荧光信号 前向角散射光 ——FSC Forward Angle Light Scatter 侧向角散射光——SSC 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——Dot Plot Review Question 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 Two-Color Cell Analysis 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 Sample Flow in Optical Cuvette Review Question Optics Excitation optics consist of: Lasers Lenses and mirrors that route the laser light to the fluidic stream Collection optics consist of: Filters that direct the signals to the appropriate optical detectors Optical Filters FACSCalibur光路图 Optics Electronics Converts analog signals to proportional digital signals Computes Height for each pulse Calculates width and area Interfaces with the computer for data transfer Creation of a Voltage Pulse - Analog Signal Quantification of a Voltage Pulse Height is a measurement for all parameters. Width = Area/Height Effect of the Instrument Controls on the Data Review Review Questions Which of the following fluorochromes cannot be used with the FACSCalibur? DAPI (ex. 345 nm, emits 455 nm) Propidium Iodide (ex. 536 nm, emits 617 nm) Alexa Fluor 647 (ex. 650 nm, emits 668 nm) What are the three measurements of a particle that can be determined by FACSCalibur? Briefly describe the functions of the fluidics, optics, and electronics systems. Review Questions What would happen to the population below if you increased the Red parameter value in the Instrument controls? Review Questions Which instrument components ensure that the fluorescence signal of a specific fluorochrome is only measured by a designated d