Main structure and working principle of flow cytometry
A. Flow cytometry Overview
Flow Cytometry (FCM) is a high-scientific technology developed in the 1970s. It combines computer technology, laser technology, fluid mechanics, cytochemistry, and cellular immunology. It also has the function of analyzing and sorting cells. . It can not only measure cell size, internal particle traits, but also detect cell surface and cytoplasmic antigen, intracellular DNA, RNA content, etc., can analyze population cells at the single cell level, and analyze and analyze a large number of cells in a short time. And collect, store and process data for multi-parameter quantitative analysis; can collect (sort) a subset of cells, sorting purity >95%. Widely used in hematology, immunology, oncology, pharmacology, molecular biology and other disciplines.
The flow cytometer used in China is mainly produced by two manufacturers in the United States: BECKMAN-COULTER and Becton-Dickinson (BD). There are two main types of flow cytometry: clinical (also known as minicomputer, desktop) and integrated (also known as mainframe, analytical). BECKMAN-COULTER's latest products are EPICS ALTRA and EPICS XL/XL-MCL, and BD?'s latest products are FACS Vantage and FACS Calibur. EPICS XL/XL-MCL and FACS Calibur are clinical models; EPICS ALTRA and FACS Vantage are comprehensive types. In addition to detection and analysis functions, they also have cell sorting functions, which are used for scientific research.
II. Flow cytometry main technical indicators
1. Analysis speed of flow cytometry:
Generally, the flow cytometer detects 1000 to 5000 cells per second, and the mainframe can reach tens of thousands of cells per second.
2. Fluorescence detection sensitivity of flow cytometry: generally can detect <600 fluorescent molecules on a single cell, and the fluorescence difference between the two cells can be distinguished by >5%.
3. Forward Angle Scattering (FSC) Light Detection Sensitivity: Forward Angle Scattering (FSC) reflects the size of the cells being measured. Generally, flow cytometry can measure 0.2μm to 0.5μm.
4. The resolution of the flow cytometer: usually expressed by the coefficient of variation CV, the general flow cytometer can reach <2.0%, which is also required to adjust the instrument with fluorescent microspheres before measuring the specimen.
5. Sorting speed of flow cytometry: general flow cytometry sorting speed>1000/sec, sorting cell purity can reach more than 99%.
III. Flow cytometry and major construction works     Flow chamber and flow drive system
The flow cytometer is mainly composed of the following five parts: 1 flow chamber and liquid flow drive system 2 laser light source and beam forming system 3 optical system 4 signal detection and storage, display, analysis system 5 cell sorting system.
The flow cell (Flow Cell or Flow Chamber) is the core component of the flow cytometer. The flow cell is made of quartz glass. The single cell suspension is surrounded by the sheath fluid in the cell flow chamber through a certain aperture in the flow chamber. The detection zone is at the center of the well, where the cells intersect perpendicularly to the laser, and the cells are arranged in a single row through the laser detection zone under the constraint of the sheath fluid. The sheath flow in the flow chamber is a steady flow. The device for controlling the sheath flow is composed of a series of pressure systems and baroreceptors under the guidance of fluid mechanics theory. As long as the sheath fluid pressure and the specimen tube pressure are adjusted, the sheath fluid The flow wraps around the sample stream and maintains the sample flow in the axial direction of the flow, ensuring that each cell passes through the laser irradiation zone for the same amount of time, thereby making the laser-excited fluorescence information accurate. See Figure 12.1 for a flow cell diagram. The flow chamber pore size is 60μm, 100μm, 150μm, 250μm, etc., for researchers to choose. Small instruments typically have a flow chamber with a certain aperture.
Figure 12.1 Schematic diagram of the flow chamber (taken from the Coulter Training Guide)
 Laser source and beam forming system
The flow cytometer can be equipped with one or more laser tubes. The commonly used laser tube is an argon ion gas laser tube, which emits light at a wavelength of 488 ηm, and can be equipped with a helium ion gas laser tube (wavelength 633 ηm) and/or ultraviolet laser. tube.
The main measurement signal fluorescence of flow cytometry is excited by excitation light. The intensity of fluorescence signal is related to the intensity of excitation light and irradiation time. Laser is a kind of coherent light source, which can provide single wavelength, high intensity and high stability. The illumination is the ideal source of excitation light to meet this requirement.
There are two cylindrical lenses between the laser light source and the flow chamber, and the laser beam with a circular cross section emitted by the laser light source is focused into an elliptical laser beam (22 μm × 66 μm) having a small cross section, in this elliptical shape. The laser energy in the laser spot is normally distributed, so that the intensity of the cells passing through the laser detection zone is uniform.
Optical system
The optical system of the flow cytometer consists of several sets of lenses, small holes, and filters, which can be roughly divided into two groups: the front of the flow chamber and the latter of the flow chamber. The optical system in front of the flow chamber consists of a lens and a small hole. The main function of the lens and the small hole (generally 2 lenses, 1 small hole) is to focus the laser beam from the laser source into a circular cross section. The smaller elliptical laser beam causes the laser energy to be normally distributed, so that the intensity of the cells passing through the laser detection zone is uniform, and the interference of stray light is minimized; the optical system behind the flow chamber is mainly composed of multiple sets of filters. Composition, the main function of the filter is to send different wavelengths of fluorescent signals to different photomultiplier tubes. There are three main types of filters: long pass filters (LP) - only allow light above a certain wavelength to pass, short pass filters (SP) - allow only light below a certain wavelength to pass, band pass filter (BP) -- Only light of a specific wavelength is allowed to pass. Different combinations of filters can send different wavelengths of fluorescent signals to different photomultiplier tubes (PMT). For example, the filter configured in front of the PMT receiving green fluorescent (FITC) is LP550. With BP525, the filters configured in front of the PMT receiving color orange-red fluorescent (PE) are LP600 and BP575, and the filters configured in front of the PMT receiving red fluorescent (CY5) are LP650 and BP675. See Figure 12.2 for an optical system and signal detection system.
Signal detection system
The scattered light is divided into forward-scattering (Forward Scatter, FS) and lateral angular scattering or 900 scattering (Side) when the specimen is arranged in a single row in the order of the sheath fluid to sequentially pass through the laser detection zone. Scatter, SS), the scattered light is the physical parameters of the cell is not related to the preparation of the cell sample (such as staining); there are two kinds of fluorescent signals, one is the autofluorescence of the cell, it is generally weak, and the other is the cell sample is labeled with specific fluorescence. The fluorescence emitted by the laser after the monoclonal antibody is stained, it is the fluorescence we want to measure, and the fluorescence signal is strong. The simultaneous presence of these two kinds of fluorescent signals is the reason why we need to set a negative control when we measure. Fluorescence signals are subtracted from fluorescence produced by cell autofluorescence and non-specific binding of antibodies.
Forward scatter (FS) reflects the size of the cell being measured, which is received by the photodiode device facing the flow cell and converted into an electrical signal; lateral scatter or 900 scatter (SS) reflects the cell membrane and cytoplasm of the cell being tested The refractive index of the nuclear membrane and the properties of the intracellular particles, which are received by a photomultiplier tube (PMT) and converted into electrical signals, which are stored in the computer hard disk or floppy disk of the flow cytometer.
Flow cytometry has a variety of commonly used fluorescent dyes. Their molecular structures are different, and the excitation and emission spectra are also different. When selecting fluorescent dyes, the wavelength of the emitted light from the laser source (such as argon ions) must be selected according to the flow cytometer. A gas laser tube, which emits light 488 ηm, and a krypton ion gas laser tube emits light at a wavelength of 633 ηm). Fluorescent dyes commonly used in 488ηm laser sources are FITC (fluorescein isothiocyanate), PE (phycoerythrin), PI (propidium iodide), CY5 (green pigment), preCP (chlorophyll protein), ECD (algae) Protein - Texas Red) and so on. Their excitation and emission wavelengths are:
Excitation wavelength (ηm) emission peak (ηm)
FITC 488 525 (green)
PE 488 575 (orange red)
PI 488 630 (orange red)
ECD 488 610 (red)
CY5 488 675 (dark red)
PreCP 488 675 (dark red)
The various fluorescent signals are received by their respective photomultiplier tubes (PMT) and converted into electrical signals and stored in the computer hard disk or floppy disk of the flow cytometer. See Figure 12.2 for an optical system and signal detection system.
Signal storage, display, analysis system
(1) Signal storage The data stored in the computer hard disk or floppy disk of the flow cytometer is generally stored in the List mode. There are two advantages to using the List mode: 1 saving memory and disk space 2 Processing analysis.
(II) Signal display and analysis Due to the lack of intuitiveness of the data in the List mode method, the display and analysis of the data generally adopts a one-dimensional histogram (Fig. 12.3), a two-dimensional bitmap (Fig. 12.4, 12.5), and a contour map (Fig. 12.8) and density map (Figure 12.7).
1. Single parameter data display and analysis Each single parameter measurement data of the cell is displayed by a histogram. The abscissa indicates the relative intensity value of the scattered light or fluorescent signal. The unit is the number of tracks, which can be linear or logarithmic. The ordinate indicates the number of cells. See Figure 12.3 one-dimensional histogram, where the abscissa is the relative intensity value (logarithm) of the FITC fluorescence signal, and the ordinate indicates the number of cells; the appropriate “door†(linear door) has been set according to the negative control in the figure. The computer will give the measured values ​​(including positive cell % and mean fluorescence intensity).
2. Two-parameter data display and analysis The relationship between the two-parameter measurement data and the number of cells of the cells is displayed and analyzed using one-dimensional histograms, two-dimensional bitmaps, contour maps, and density maps. As shown in Figure 12.4, the two-dimensional dot matrix image is a dot matrix composed of forward scattered light (FS) and side scattered light (SS) of normal human peripheral blood leukocytes. The abscissa and ordinate are linear. Cells, monocytes, and granulocytes are clearly divided into 3 groups, which can be easily circled (Bitmap, amorphous gate) to analyze the data of each subpopulation; Figure 12.6 False 3D topographic map (X-axis: SSC, Y-axis: FSC Z-axis: cell number) shows this more clearly. Figure 12.5 Two-dimensional bitmap is a two-parameter data display of two fluorescences (FITC and RD1) of the cell. The abscissa and ordinate are logarithmic, the abscissa represents FITC, and the ordinate represents PE. The figure has been set appropriately. The "gate" (cross door), the D1, D2, D3, and D4 gates of the cross gate represent PE single positive cells, PE and FITC double positive cells, negative cells, and FITC single positive cells, respectively. The instrument's computer will give two fluorescence measurements (% of negative cells, % of positive cells for each of the two fluorescences, % of double positive cells for both fluorescence, and mean fluorescence intensity for each group of cells). Figure 12.7 and Figure 12.8 show the density and contour plots of the two fluorescence two-parameter data of the cells, respectively. The abscissa and the ordinate represent a kind of fluorescence parameter. Similarly, as long as the cross door is set, two kinds of fluorescence can be obtained. The various measured values, density maps and contour maps are more intuitive than bitmaps.
3. Three-parameter data display and analysis of the relationship between the three-parameter measurement data of the cells and the number of cells. Each pair of data consists of two pairs (three-parameter measurement data and cell number per two data can be composed of six pairs of data) with one-dimensional histogram, two-dimensional Dot matrix, contour plot and density map display and analysis. The relationship between the three fluorescence data is represented by a prism. The spectrogram can directly give 8 data (for example, ABC represents 3 kinds of fluorescence, and can have A+B+C+, A+B+C-, A+BC- , A-B+C+, A-B+C-, AB-C+, A+B-C+, ABC-). See Figure 12.9 prism, Figure 12.9 is a spectrogram of the results of human peripheral blood lymphocyte subsets, showing various results of CD3, CD4, CD8 combination, such as T helper cells (CD3 + CD4 + CD8-) is 42.0% For example, T suppressor cells (CD3+CD4+CD8-) were 17.4%.
Figure 12.5 Two-dimensional dot pattern of two fluorescences (taken from the Coulter Operaters Guide)
Figure 12.7. Density plot of two-parameter data (from the Coulter Operaters Guide)
Figure 12.6 Fake 3D topographic map and 2D bitmap (taken from the Coulter Operaters Guide)
Figure 12.8 Contour map of two-parameter data (taken from the Coulter Operaters Guide)
Figure 12.9 Spectrogram (prism)
Cell sorting system
For example, if the ultrasonic piezoelectric crystal is installed on the cell flow chamber, the high-frequency vibration of the ultrasonic piezoelectric crystal after the energization can drive the high-frequency vibration of the cell flow chamber, and the liquid flow from the nozzle of the cell flow chamber is broken into a series of uniform liquids. Drops, tens of thousands of droplets are formed every second. Each droplet contains a sample cell, and the cells in the droplet have been measured before the droplet is formed, can be charged if the predetermined requirement is met, and deflected to the left or right when passing through the high voltage electrostatic field of the deflector It is collected in a designated container, does not contain cell droplets or the cells do not meet the predetermined requirements. The droplets are not charged or deflected into the intermediate waste collector, thereby achieving sorting. ? Detailed principle and operation of sorting, please refer to relevant literature for those who are interested.
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