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补充细节1：关于荧光素

荧光信号主要包括两部分：①自发荧光，即不经荧光染色细胞内部的荧光分子经光照射后所发出的荧光；②特征荧光，即由细胞经染色结合上的荧光染料受光照而发出的荧光，其荧光强度较弱，波长也与照射激光不同。
自发荧光信号为噪声信号，在多数情况下会干扰对特异荧光信号的分辨和测量。在免疫细胞化学等测量中，对于结合水平不高的荧光抗体来说，如何提高信噪比是个关键。一般说来，细胞成分中能够产生的自发荧光的分子(例核黄素、细胞色素等)的含量越高，自发荧光越强；培养细胞中死细胞/活细胞比例越高，自发荧光越强；细胞样品中所含亮细胞的比例越高，自发荧光越强。减少自发荧光干扰、提高信噪比的主要措施是：①尽量选用较亮的荧光染料；②选用适宜的激光和滤片光学系统；③采用电子补偿电路，将自发荧光的本底贡献予以补偿。

当应用流式细胞术对细胞表面或内部抗原进行检测时，除必要的抗体外，应用各种荧光素也是必不可少的，它包括单标记抗体荧光索、双标记抗体荧光素、三标记抗体荧光素等。荧光素发射荧光基本原理是：荧光素受到一定波长(激发波长)的激光激发后，其原子核外的电子由于吸收了激光的能量，由原本运动处于基态轨道跃迁到激发态轨道上运动，然后当电子由激发态重新回到基础态时，释放出能量并发射出一定波长(发射波长)的荧光。不同荧光素用不同的激发光波长的激发光来激发，所以要选择正确的激光器。例如，FITC和FE等的微发光波长均为488nm，所以可用产生可见光的氩离子激光器，而APC和PC5等的激发波长在红光范围，需使用发射630nm波长红光的氦—氖激光器。
此外，各种荧光素的发射波长也十分重要，可以据此确定其检测所需光电倍增管性质。如FITC，被激光激发后发射绿光，检测时要使用第一光电倍增管(即PMTl)；PE则发射橙包光，需用第二光电倍增管(即PMT2)；PC5和PcrCP等发射的是深红色光，这时需选择第二甚至第四光电倍增管(即PMT3或PMT4)等。
一定要注意激发波长和发射波长的区别！

常用荧光素
流式细胞仪测定常用的荧光染料有多种,他们分子结构不同,激发光谱和发射光谱也各异,选择荧光染料时必须依据流式细胞仪所配备的激光光源的发射光波长(如氩离子气体激光管,它的发射光波488ηm,氦氖离子气体激光管发射光波长633ηm）。488ηm激光光源常用的荧光染料有FITC（异硫氰酸荧光素）、PE（藻红蛋白）、PI（碘化丙啶）、CY5（化青素）、preCP(叶绿素蛋白)、ECD(藻红蛋白-得克萨斯红)等。他们的激发光和发射光波长分别是：
激发光波长（ηm） 发射光峰值（ηm）
FITC 488 525（绿）
PE 488 575（橙红）
PI 488 630（橙红）
ECD 488 610（红）
CY5 488 675（深红）
PreCP 488 675（深红）
（摘自《实用流式细胞术彩色图谱》）


常用荧光素分布图

FITC和PI
PI和EB 都具有嵌入到双链DNA和RNA的碱基对中并有与碱基对结合的特异性。为了获得特异的DNA分布，染色前必须用RNA酶处理细胞，排除双链RNA的干扰。PI和EB不能进入完整的细胞膜，因此又可以用于检测死细肥。PI和EB各种理化性质相似，但PI比EB的发射光光谱峰向长波方向移动，因而在作DNA和蛋白质双参数测量时，PI的红色荧光和FITC的绿色荧光更易于区分和测量。另外，Pl比EB测得的DNA分布的变异系数(CV值)低，所以PI得到更广泛的应用。
FITC 为一种小分子荧光素，其效率(即荧光强度)取决于溶液的pH值，因此在使用FITC时应注意溶液的酸碱度。

一些流式常用名字的概念和意义：
Amplifier 放大器 Electronic component of a flow cytometer that increases the signal by an adjustable factor.

Aneuploid 异倍体 Having an abnormal number of chromosomes. Aneuploid cells may also have an abnormal DNA content.

Average The mean value, which is the total amount divided by the number of contributors.

Channel 常说的荧光道数 The measured value of a parameter, representing the signal intensity of an event after amplification. To appear on a plot, data for an event must fall into one of either 256 channels (0-255) or one of 1024 channels (0-1023) depending on the resolution of the plot.

Compensation （荧光）补偿 An electronic calculation that removes signal overlap which the optical system cannot remove. Fluorescence compensation works for specific pairs of fluorescent parameters; for example, FL1 and FL2.

Cursor For FACScans: a highlight appearing in a data field that indicates you can modify this field. On a dot plot, a crosshair for drawing a polygon gate. On other flow cytometers: a line separating regions on single parameter histograms that are treated statistically separate. See marker.

%CV CV值！表示其集中分散趋势。Percent coefficient of variation is a measure of peak distribution. The percent coefficient of variation is the standard deviation of the peak divided by the mean channel number of the peak, times 100.

Diploid 二倍体Having the normal number of chromosome pairs for non-reproductive, mammalian cells. The amount of DNA in diploid cells defines the normal DNA content for the species.

DNA Index The ratio of GO/G1 peak channel in a DNA histogram of the experimental sample to the GO/G1 peak channel of the reference sample, when normal human diploid cells or nuclei are the reference. This is a measure of DNA aneuploidy, or abnormal DNA content.

Dot Plot A two parameter data graph used for acquisition and analysis. Each dot on the display represents one event that the flow cytometer analyzed.

Doublet 分选中常用的概念。 Two particles stuck together, which a flow cytometer records as one larger event. Particles may also occur in triplets and higher associations.

Event A unit of data representing one particle or cell. An event has a relative intensity value and channel number for each parameter.

Filter An optical device used to attenuate particular wavelengths or frequencies while passing others.

FL1, Green Fluorescence signal received by the photomultiplier tube (PMT), FL1. The range of the signal detected is dependent on the filters associated with the PMT. For FACScans and FACSCaliburs, FL1 measures light in the green range of the spectrum (515 to 545 nm).

FL2, Orange-Red Fluorescence signal received by the photomultiplier tube, FL2. For FACScans and FACSCaliburs, FL2 measures orange-red light (564-606 nm).

FL3, Red Fluorescence signal received by the photomultiplier tube, FL3. For FACScans, FL3 measures red light (above 650 nm). For FACSCaliburs, FL3 measures red light (above 670 nm).

FL4, Red-Orange Fluorescence signal received by the photomultiplier tube, FL4. For FACSCaliburs, FL4 measures red-orange light (653-669 nm).

Fluorescence A property of molecules to absorb light at one wavelength and emit light at a longer wavelength. Flow cytometers detect Fluorescence emission at a 90 degree angle to the exciting light beam.

Forward Scatter (FSC) 前向角 A parameter measuring light scattered less than 10 degrees as a cell passes through the laser beam. The FSC measurement is related to cell size.

G0 Phase of cell cycle designating cells that are quiescent and have not yet entered the growth cycle. Normal cells in this phase have exactly one set of chromosome pairs.

G1 Phase of cells cycle in which cells are committed to division. These cells have the same number of chromosomes and the same amount of DNA as GO phase cells. They are about to enter S phase where they synthesize DNA.

G2 Phase of cells cycle in which proliferating cells have duplicated their DNA and formed two sets of chromosome pairs, in preparation for division. G2 follows the S phase and precedes the M (mitosis) phase.

Gain 增益 An adjustment that modifies amplifier responses. Increasing the gain increases the electronic pulse (and the relative fluorescence intensity value and the channel value) in response to a light signal.

Gate 翻译过来后还是觉得称之为“gate”好！A boundary that defines a subset or sub-population of events. Gates are set by drawing boundaries around the subsets on data plots (dot plots or histograms). Use gates either for data acquisition or analysis. Inclusive gates select only the events that fall within (and on) the boundary. Exclusive gates select only the events that fall outside of the boundary.

Live gate 与上面的“gate”不同，是作为记录的条件的gate，不在gate之内的数据不被存入电脑！Gate through which any data from the flow cytometer/computer parallel interface must pass before acquisition. Any data outside the gate does not enter the computer.

Haploid 单倍体 Having one set of single chromosomes. Reproductive cells, like eggs and sperm, are haploid.

Histogram A data plot of a single parameter. This displays either the relative fluorescence intensity value or the channel number on the x axis and the number of events on the y axis.

Linear scale 线性The scale on which the output is directly proportional to the input. When the control is set at Linear, the voltage measured is directly proportional to the channel into which the event falls.

Logarithmic scale 对数 The scale on which the values increase logarithmically. This scale is used when the instrument is set to LOG. There are four decades across the 1024 channels on FACScan (256 channels per decade). The scale ranges from 1 to 10,000. For FACScan, the number of decades is fixed.

M Phase of the cell cycle, mitosis, during which a cell divides into two. This precedes GO/G1 and follows G2. M phase and G2 cells contain the same amount of DNA and the same number of chromosomes. In normal cells, this is the tetraploid number (4N) of chromosomes.

Marker 就是第五篇中的一张图上面的M1，M2等。作为统计时的分界区域限制。A line separating regions on single parameter histograms that are treated statistically separate. See cursor.

Parameter A measurement of light intensity, either scattered or emitted fluorescence, from a particle as it passes through a laser beam. For all five parameters (FSC, SSC, FL1, FL2, FL3) the height of the electronic pulse that is generated is used. In addition, for cell cycle analysis CellQuest software can measure either FL2 area and FL2 width (for staining with propidium iodide) or FL3 area and FL3 width (for staining with 7-amino-actinomycin D).

Photodiode 光电二极管，将光信号转化为电信号。A semiconductor device used to detect light and generate an electrical current. Typically used in forward scatter (FSC) detection.

Photomultiplier 光电倍增管 A photodetector, with adjustable voltage, that translates optical tube (PMT) signals into electrical current. PMT detectors are used for SSC and fluorescence parameters. Increasing the PMT voltage, increases the output signal for a given amount of light.

Ploidy 倍性 The number of chromosomes present in the cell. The amount of DNA in a cell gives an indication of the ploidy, but is not directly proportional. See Haploid, Diploid, and Aneuploid.

Pulse 不是脉搏，是脉冲！The electronic signal generated by a particle (cell or nucleus) in the flow cytometer.

Rate The number of events per second processed by the computer.

Reference Sample 就是作为一个内标的样本，通过它可以对一些物质进行定量检测！（这就是为什么在一些帖子里面，我们总是说流式是个定性，半定量工具，要做到半定量或定量，需要内标。不是所有的数据都可以定量的。） A sample of known DNA content mixed with an experimental sample to provide an internal standard.

S Phase of the cell cycle, DNA synthesis, in which the cell duplicates its DNA.

Scale The maximum number of events displayed for a channel in a histogram. A low scale number magnifies the data.

Side Scatter (SSC) Also called 90o scatter or right angle scatter. Light scattered at a 90 degree angle as a cell passes through the laser beam. This measurement is related to the internal granularity or complexity of a particle.

Singlet A single particle (cell or nucleus), which a flow cytometer records as one event.

Threshold 阈值，在流式中，这是个很重要的概念！ The lowest channel number for a selected parameter for which an event may be recorded. The flow cytometer sends to the computer only events above the threshold level.


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