Flow cytometer (FCM) is an instrument for flow cytometry. It is a laboratory that combines electronic technology, computer technology, laser technology, fluid theory, cell fluorescence chemistry technology and monoclonal antibody technology. "CT".
Flow cytometry is a multi-parameter, rapid quantitative analysis and sorting technique for functionally analyzing individual cells or other biological particles. It can analyze tens of thousands of cells at high speed and simultaneously from one cell. It has many advantages, such as high speed, high precision and good accuracy. It is the most advanced cell quantitative analysis technology in the world. Since the advent of the first flow cytometer in 1969, flow cytometry has evolved into an increasingly sophisticated cell sorting and analysis technique.
FCM instruments are divided into two categories: one is a desktop computer, which is characterized by fixed optical path adjustment system and high degree of automation; the other is a mainframe, which is characterized by rapid sorting of cells of interest and Individual or designated cells can be sorted into specific culture wells or culture plates, and a wide range of wavelengths and types of laser heads can be selected for a wider range of scientific research.
The structure of FCM can be generally divided into five parts: 1 flow chamber and liquid flow drive system; 2 laser light source and light speed forming system; 3 optical system; 4 signal detection and storage, display and analysis system; 5 cell sorting system.
The working principle of the flow cytometer is to stain the cells to be tested and prepare a single cell suspension. The sample to be tested is pressed into the flow chamber with a certain pressure, and the phosphate buffer containing no cells is ejected from the sheath tube under high pressure, and the direction of the sheath tube population is at an angle to the flow of the sample to be tested, so that the sheath liquid is It can wrap around the sample at a high speed to form a circular stream, and the cells to be tested are arranged in a single row under the coating of the sheath fluid, and sequentially pass through the detection area.
Flow cytometry usually uses a laser as a source of illumination. The focused and shaped beam is vertically irradiated on the sample stream, and the fluorescently stained cells generate a scattered light signal and a fluorescent signal under the irradiation of the laser beam.
1. The scattered light signal is divided into a forward angular scattered light signal (FSC) and a lateral angle-scattered light signal (SSC) collected at 90[deg.] with the laser beam. FSC basically reflects the size of the cell volume, usually used as a threshold in the FCM detection to exclude various fragments in the sample and small particles in the sheath fluid; SSC mainly reflects the particle characteristics of the cells, can distinguish lymphocytes, monocytes and granulocytes .
2. The fluorescence signal mainly represents the intensity of the surface antigen of the cell membrane measured or the concentration of the substance in the nucleus. The fluorescent signal is separated by a series of dichroic mirrors and band-pass filters before being detected, forming a plurality of different Fluorescent signal of wavelength.
The above signals are simultaneously received by the forward photodiode and the photomultiplier tube in the 90° direction, and are received by the photomultiplier tube and converted into an electrical signal, and then converted into a computer-recognizable electrical signal by an analog-to-digital converter. Digital signal. The computer performs computer processing on the measured signals and displays the analysis results on a computer screen.
The display of FCM test data can be selected in various forms depending on the measurement parameters. Single parameter data is expressed in the form of a histogram, with the X axis being the measured intensity and the Y axis being the number of cells. In general, the resolution of a flow cytometer axis has a resolution of 512 or 1024 channels, depending on the resolution of the analog-to-digital converter. For two-parameter or multi-parameter data, you can display the histogram of each parameter separately, or you can select a two-dimensional three-point map, a contour map, a grayscale map, or a three-dimensional stereo view.
Fluorescence signal scattered light signal detection mode
The cell sorting function of flow cytometry is achieved by isolating droplets containing individual cells. An ultra-high frequency transistor is arranged on the nozzle of the flow chamber, and vibrates after charging to break the discharged liquid stream into uniform droplets, and the cells to be measured are dispersed in the droplets. The droplets are charged with positive and negative charges. When the droplets flow through a deflection plate with several thousand volts, they are deflected by the high voltage electric field and fall into the respective collection containers. The uncharged droplets fall. A waste container in the middle of the person to achieve cell separation.
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