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流式细胞术简介

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. Injector Tip. Sheath

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流式细胞术简介

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  1. 流式细胞术简介 For FCM Training

  2. 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

  3. 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

  4. Injector Tip Sheath fluid Fluorescence signals Focused laser beam

  5. 流式细胞仪的光信号 • 散射光信号 • 荧光信号

  6. 1. 散射光信号 • 前向角散射光（FSC,Forward Scatter） 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, Side Scatter） 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。

  7. Laser FALS Sensor 前向角散射光 ——FSC • Forward Angle Light Scatter

  8. Laser FALS Sensor 90LS Sensor 侧向角散射光——SSC

  9. 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

  10. 散点图——Dot Plot lysed whole blood

  11. Review Question Dead cells are known to be smaller and to exhibit more internal complexity than live cells. Which of the populations on this plot would you expect to be dead? A B

  12. 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

  13. FALS Sensor Fluorescence Fluorescence detector (PMT3, PMT4 etc.) 荧光检测器

  14. Two-Color Cell Analysis Which of the three populations has the most Ab A binding sites? Ab B Ab A

  15. 双色荧光散点图

  16. 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

  17. 液流系统 • 液流系统将样本悬液聚焦在光源的中心处

  18. Sample Flow in Optical Cuvette Optical Cuvette High Sample Flow Rate 60 mL/min Low Sample Flow Rate 12 mL/min LaminarFlow LaminarFlow Sheath Sheath Sheath Sheath Sample Sample

  19. Review Question Which of the following would cause disturbance in the laminar flow of the optical cuvette? • bubbles • cellular concentration • sample flow rate

  20. 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

  21. Optical Filters Longpass Shortpass Bandpass 460 500 540 460 500 540 460 500 540 BP500/50 LP 500 SP 500

  22. FACSCalibur光路图

  23. Optics 488 nm 635 nm

  24. Electronics • Converts analog signals to proportional digital signals • Computes Heightfor each pulse • Calculates width and area • Interfaces with the computer for data transfer

  25. Laser Voltage Time Voltage Laser Time Voltage Laser Time Creation of a Voltage Pulse - Analog Signal

  26. Pulse Area Pulse Height Volts 0 Pulse Width Time Quantification of a Voltage Pulse • Height is a measurement for all parameters. • Width = Area/Height

  27. Effect of the Instrument Controls on the Data Detector Instrument Controls FSC detector 250 gain FSC detector 350 gain

  28. SSC FSC FL2 FL1 FL4 FL3 Review Data Processing

  29. 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.

  30. Review Questions • What would happen to the population below if you increased the Red parameter value in the Instrument controls?

  31. Review Questions • Which instrument components ensure that the fluorescence signal of a specific fluorochrome is only measured by a designated detector? For example, APC is only measured by the FL4 detector.

  32. 样本处理

  33. 细胞悬液的制备 • 细胞悬液： • 分离PBMC、PRP等：操作复杂，分离、离心步骤导致细胞特定群体丢失，并可能引入某些误差 • 直接使用外周血、骨髓：最接近生理状况，操作简便，样本用血量小 • 灌洗液、体腔积液 • 培养细胞、细胞系 • 实体组织： • 病理组织：新鲜样本/石蜡包埋样本 • 针吸组织：新鲜样本 • 鞘液、洗液等：清洁无颗粒杂质

  34. 步骤一：选择合适的荧光染料 • 必须能够被流式细胞仪上所配备的激光器所激发 • 激发的光谱必须在仪器上滤光片能够接受的合适范围内 • 荧光素光谱的重叠应当尽量减少

  35. 荧光产生过程

  36. Propidium Iodide Emisson Excitation 300 nm 400 nm 500 nm 600 nm 700 nm 400 nm 500 nm 600 nm 700 nm DNA PI

  37. Fluorescein (FITC) Emisson Excitation 300 nm 400 nm 500 nm 600 nm 700 nm Wavelength 400 nm 500 nm 600 nm 700 nm Protein

  38. Phycoerytherin (PE) Emisson Excitation 300 nm 400 nm 500 nm 600 nm 700 nm Protein

  39. Allophycocyanin (APC) Protein 632.5 nm (HeNe) 300 nm 400 nm 500 nm 600 nm 700 nm Excitation Emisson

  40. FITC和PE荧光光谱

  41. 补偿模型图

  42. 步骤二： 染色 • 体积 • 温度 • 孵育时间 • 对照

  43. 直接染色 • Fluorescent probe attached to antibody • Specific signal: weak • Nonspecific binding: low

  44. 间接染色 • Fluorescent probe attached to a 2nd antibody • Specific signal: strong, 5-6 2nd Ab/each 1st Ab; • Nonspecific binding: high

  45. Avidin-Biotin method I biotinylated primary Ab biotin avidin biotinylated dye

  46. 示意图

  47. 步骤三： 数据收集和分析 • 画图 • 寻找目标细胞 • 调整仪器设置到合适的状态 • 我们可以得到怎样的结果？它们意味着什么？

  48. 仪器设置调节 1. 用未染色细胞调整仪器PMT电压 2. 用单染色的细胞调节仪器补偿

  49. 关于同型对照 • 例：一个双色染色的实验 抗体A FITC，抗体B PE 对应的同型对照是IgG1 FITC,IgG1 PE 那么需要准备的是 阴性对照：细胞加上IgG1 FITC,IgG1 PE 单阳性对照： FITC: 细胞加上Ab A FITC, IgG1 PE PE: 细胞加上Ab B PE, IgG1 FITC

  50. 数据分析 • 设门 • 设定阴性与阳性群体的界限 • 确定阳性与阴性细胞群体 • 统计阳性或阴性细胞群体的百分率，平均荧光值，绝对数或抗体结合数

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