Flow cytometry consists of three basic structures: flow system, optical and signal conversion test system, and digital signal processing and amplification computer system. In the multi-parameter rapid automatic analysis of single cells or ultrastructures in cell suspensions, thousands to tens of thousands of cells can be measured per second, and specific cells can be designed according to experimental design requirements during the analysis. Analysis, flow cytometry with cell sorting system can also sort out the same type of cells with the same characteristics according to the experimental design requirements for culture or further research.
I. Working principle The working principle of flow cytometry draws on the fluorescence microscope technology, and the excitation light source of fluorescent microscope is changed into laser, which has better monochromaticity and excitation efficiency, and utilizes fluorescent dye and monoclonal antibody technology. The development has improved the sensitivity and specificity of the detection, and changed the fixed specimen table into a flowing single cell suspension, and processed the data processing of the optical signal with a computer to improve the accuracy of the detection speed and statistical analysis. Can obtain a variety of parameter data from a single cell at the same time.
(1) Basic composition
1. The flow system consists of a sample and a sheath. The cells to be tested are prepared as a single cell suspension, stained with a fluorescent dye-labeled monoclonal antibody, placed in a sample tube, and introduced into a flow chamber under a pressure of a cleaning gas to form a sample stream; the sheath fluid is a substrate for which the auxiliary sample stream is normally detected. The main function of the liquid is to wrap around the sample flow so that it remains at the center of the nozzle to ensure detection accuracy, while preventing cells in the sample flow from approaching the orifice wall to block the orifice. The pore size of the cell suspension into the sheath fluid is usually 50 to 300 μm.
2. The optical system consists of a laser source, a beam splitter, a beam shaper, a lens group, and a photomultiplier tube.
(1) Laser light source: Most of the modern flow cytometers use air-cooled argon ion lasers;
(2) Dichroic mirror: The function is to reflect longer wavelength light and pass shorter wavelength light.
(3) Beam shaper: consists of two cylindrical lenses placed in a crisscross shape, which is used to focus the laser beam emitted by the laser into an elliptical spot with a height of 15 μm and a width of 57 μm.
(4) Lens group: There are three lenses, which function to convert laser light and fluorescence into parallel light while removing discrete indoor light.
(5) Filter: The long-pass filter allows light to pass longer than the set wavelength; the short-pass filter allows light to pass shorter than the set wavelength; the band-pass filter allows a certain wavelength to pass, other wavelengths Light cannot pass, there are 4 525 nmBP, 575 nmBP, 620 nmBP, and 675 nmBP.
(6) Photomultiplier tube (PMT): composed of FS, SS, FL1, FL2, FL3, FIA, the main function is to detect fluorescence and scattered light, and at the same time convert optical signals into electrical pulse (digital data) signals. When the PMT voltage is adjusted, the pulse signal also changes.
3. The data processing system is mainly composed of a computer and its software. It is more intelligent and automated to store, display and analyze experimental analysis data, and is an important part of the components of flow cytometry.
(II) Basic working principle According to the detection requirements, the single cell suspension and sheath liquid of specific fluorescent dye are labeled, and respectively enter the flow chamber through the silicidation tube to form a steady-state single-cell liquid column of the sheath liquid-enclosed cell suspension, the liquid column High-speed shot through a nozzle in a stable laminar flow, the liquid column perpendicularly intersects the highly focused laser beam in the horizontal direction, and the fluorescent dye labeled on a single cell is excited to generate specific fluorescence when passing through the laser spot, and at the same time, due to mixing The size of the cells in the cell population and the number of intracellular particles are excited to produce different scattered light. In the direction perpendicular to the liquid column of the incident beam, there are a fluorescence detecting system and a scattered light sensing system for collecting the fluorescent signal and the side scattered light signal, and the forward scattered light sensor detects the forward light signal at the forward small angle. The received photoelectric signal is converted into a voltage pulse and an integrated pulse by a photomultiplier tube to amplify the signal, and the signal enters a computer system for data conversion, storage, analysis, processing, and comprehensive analysis of the results according to different detection designs using corresponding software programs. And displayed on the screen with images and data, including single parameter and 2D or 3D image data, percentage of positive cells, x±s, slope, peak, peak area and other multi-parameter data. A good flow cytometer can measure 15 000 cells per second and measure the particles of 1 000 fluorescent dye molecules, which is currently not possible with other instruments.
I. Working principle The working principle of flow cytometry draws on the fluorescence microscope technology, and the excitation light source of fluorescent microscope is changed into laser, which has better monochromaticity and excitation efficiency, and utilizes fluorescent dye and monoclonal antibody technology. The development has improved the sensitivity and specificity of the detection, and changed the fixed specimen table into a flowing single cell suspension, and processed the data processing of the optical signal with a computer to improve the accuracy of the detection speed and statistical analysis. Can obtain a variety of parameter data from a single cell at the same time.
(1) Basic composition
1. The flow system consists of a sample and a sheath. The cells to be tested are prepared as a single cell suspension, stained with a fluorescent dye-labeled monoclonal antibody, placed in a sample tube, and introduced into a flow chamber under a pressure of a cleaning gas to form a sample stream; the sheath fluid is a substrate for which the auxiliary sample stream is normally detected. The main function of the liquid is to wrap around the sample flow so that it remains at the center of the nozzle to ensure detection accuracy, while preventing cells in the sample flow from approaching the orifice wall to block the orifice. The pore size of the cell suspension into the sheath fluid is usually 50 to 300 μm.
2. The optical system consists of a laser source, a beam splitter, a beam shaper, a lens group, and a photomultiplier tube.
(1) Laser light source: Most of the modern flow cytometers use air-cooled argon ion lasers;
(2) Dichroic mirror: The function is to reflect longer wavelength light and pass shorter wavelength light.
(3) Beam shaper: consists of two cylindrical lenses placed in a crisscross shape, which is used to focus the laser beam emitted by the laser into an elliptical spot with a height of 15 μm and a width of 57 μm.
(4) Lens group: There are three lenses, which function to convert laser light and fluorescence into parallel light while removing discrete indoor light.
(5) Filter: The long-pass filter allows light to pass longer than the set wavelength; the short-pass filter allows light to pass shorter than the set wavelength; the band-pass filter allows a certain wavelength to pass, other wavelengths Light cannot pass, there are 4 525 nmBP, 575 nmBP, 620 nmBP, and 675 nmBP.
(6) Photomultiplier tube (PMT): composed of FS, SS, FL1, FL2, FL3, FIA, the main function is to detect fluorescence and scattered light, and at the same time convert optical signals into electrical pulse (digital data) signals. When the PMT voltage is adjusted, the pulse signal also changes.
3. The data processing system is mainly composed of a computer and its software. It is more intelligent and automated to store, display and analyze experimental analysis data, and is an important part of the components of flow cytometry.
(II) Basic working principle According to the detection requirements, the single cell suspension and sheath liquid of specific fluorescent dye are labeled, and respectively enter the flow chamber through the silicidation tube to form a steady-state single-cell liquid column of the sheath liquid-enclosed cell suspension, the liquid column High-speed shot through a nozzle in a stable laminar flow, the liquid column perpendicularly intersects the highly focused laser beam in the horizontal direction, and the fluorescent dye labeled on a single cell is excited to generate specific fluorescence when passing through the laser spot, and at the same time, due to mixing The size of the cells in the cell population and the number of intracellular particles are excited to produce different scattered light. In the direction perpendicular to the liquid column of the incident beam, there are a fluorescence detecting system and a scattered light sensing system for collecting the fluorescent signal and the side scattered light signal, and the forward scattered light sensor detects the forward light signal at the forward small angle. The received photoelectric signal is converted into a voltage pulse and an integrated pulse by a photomultiplier tube to amplify the signal, and the signal enters a computer system for data conversion, storage, analysis, processing, and comprehensive analysis of the results according to different detection designs using corresponding software programs. And displayed on the screen with images and data, including single parameter and 2D or 3D image data, percentage of positive cells, x±s, slope, peak, peak area and other multi-parameter data. A good flow cytometer can measure 15 000 cells per second and measure the particles of 1 000 fluorescent dye molecules, which is currently not possible with other instruments.
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