Detector Parameter
FL1 FL2 FL3 FL4
Filter
530/30 585/42 670LP 661/16
23
Electronics
—Converts analog signals to proportional digital signals
—Computes Height for each pulse —Calculates width and area —Interfaces with the computer for data transfer
60 mL/min
Laminar Flow
Sheath
Sheath
Sample
18
Sheath
Sheath
Sample
Review Question
Which of the following would cause disturbance in the laminar flow of the optical cuvette?
24
Creation of a Voltage Pulse - Analog Signal
Voltage
Laser Laser Laser
25
Voltage
Voltage
Time Time Time
Quantification of a Voltage Pulse
— Height is a measurement for all parameters. — Width = Area/Height
fluidic stream
• Collection optics consist of:
Filters that direct the signals to the appropriate optical detectors
20
Optical Filters
Longpass
460 500 540
Pulse Area
0 Pulse
Width Time
26
Volts
Pulse Height
Effect of the Instrument
Controls on the Data
Detecto r
Instrument Controls
FSC detector 250 gain
13
Freq
Two-Color Cell Analysis
Which of the three populations has the most Ab A binding sites?
Ab A
14
Ab B
双色荧光散Leabharlann 图15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
Shortpass
460 500 540
Bandpass
460 500 540
LP 500
SP 500
BP500/50
21
FACSCalibur光路图
22
Optics
Laser 488 nm
635 nm
Fluorochromes
FITC PE PerCP APC
GFP PI PerCP-CY5.5
A. bubbles B. cellular concentration C. sample flow rate
19
Optics
• Excitation optics consist of:
Lasers Lenses and mirrors that route the laser light to the
9
散点图——Dot Plot
lysed whole blood
10
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?
研究的微粒特性包括多种物理及生物学特征，并加以 定量
2
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
3
Fluorescence signals
Focused laser beam
4
Injector Tip
6
前向角散射光 ——FSC
—Forward Angle Light Scatter
Laser
FALS Sensor
7
侧向角散射光——SSC
Laser
FALS Sensor
90LS Sensor
8
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
将光信号转化为电信号，并使其数字化以供 计算机分析
16
液流系统
— 液流系统将样本悬液聚焦在光源的中心处
17
Sample Flow in Optical Cuvette
Low Sample Flow Rate
12 mL/min
Optical Cuvette
Laminar Flow
High Sample Flow Rate
11
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
12
荧光检测器
FALS Sensor
Fluorescence
Fluorescence detector (PMT3, PMT4 etc.)
Sheath fluid
流式细胞仪的光信号
—散射光信号 —荧光信号
5
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。
流式细胞术原理及应用
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