is a device for the automatic analysis and sorting of cells. It can rapidly measure, store and display a series of important biophysical AND biochemical characteristic parameters of dispersed cells suspended in liquid, and can classify the specified cell subsets according to the pre-selected parameter range. Most flow cytometers are zero-resolution instruments, which can only measure the total amount of nucleic acid, total protein, and other indicators of a cell, but cannot identify and detect the amount of nucleic acid or protein in a particular part. In other words, it has zero detail resolution.
Multi-parameter measurement can be performed simultaneously by flow cytometry
, and the information mainly comes from specific fluorescence signals and non-fluorescence scattering signals. The measurement is carried out in the measurement area, the so-called measurement area is the vertical intersection point of the laser beam and the liquid flow beam ejecting the nozzle. When a single cell in the center of the liquid flow passes through the measurement area, it is irradiated by the laser and scatters light to the whole space with a solid Angle of 2π. The wavelength of the scattered light is the same as the wavelength of the incident light. The intensity and spatial distribution of scattered light are closely related to the size, morphology, plasma membrane, and internal structure of the cell because these biological parameters are related to the optical properties of the cell, such as the reflection and refraction of light. Cells that have not suffered any damage have characteristic light scattering, so different scattered light signals can be used to analyze and sort living cells without staining. The scattered light signal of fixed and stained cells is certainly different from that of living cells due to the altered optical properties. The scattered light is not only related to the parameters of the cell as the scattering center, but also to abiotic factors such as the scattering Angle and the solid Angle at which the scattered light is collected.
In flow cytometry
measurement, two kinds of scattered light are commonly used: a. Forward Angle (0 Angle) scattering (FSC); b. Side scattering (SSC), also known as 90 Angle scattering. The Angle referred to here refers to the Angle roughly formed between the direction of the laser beam and the axial direction of the photomultiplier tube that collects the scattered light signal. Generally speaking, the intensity of forward Angle scattered light is related to the size of cells and increases with the increase of cell cross-sectional area for the same cell population. The experiments on living spherical cells show that it is basically linear with the size of the cross-sectional area in the range of a small solid Angle. The complex shape and orientation of the cell may be very different, especially needing to pay attention. The measurement of side scattered light is mainly used to obtain information about the granular properties of the fine internal structure of the cell. Although the side-scattered light is also related to the size and shape of the cell, it is more sensitive to the refractive index of the cell membrane, cytoplasm, and nuclear membrane, and can also give a sensitive response to the larger particles in the cytoplasm.
In practice, the instrument must first measure the light-scattering signal. When used in combination with fluorescent probes, light scattering analysis can distinguish stained and unstained cells in a sample. The most effective use of light scattering measurements is to identify certain subgroups from heterogeneous populations. Fluorescence signal mainly includes two parts: a. Spontaneous fluorescence, that is, without fluorescence staining, the fluorescence of fluorescent molecules in the cell after light irradiation; b. Characteristic fluorescence, that is, the fluorescence emitted by the cells after staining combined with the fluorescent dye on the light, its fluorescence intensity is weak, and the wavelength is different from that of the irradiation laser. The autofluorescence signal is a noise signal, which can interfere with the discrimination and measurement of specific fluorescence signals in most cases. In the measurement of immunocytochemistry, how to improve the signal-to-noise ratio is the key for fluorescent antibodies with low binding levels.
In general, the higher the content of molecules (e.g., riboflavin, cytochrome, etc.) that can produce autofluorescence in the cellular components, the stronger the autofluorescence.
In general, the higher the content of molecules (e.g., riboflavin, cytochrome, etc.) that can produce autofluorescence in the cellular components, the stronger the autofluorescence. The higher the ratio of dead cells to live cells, the stronger the autofluorescence. The higher the proportion of bright cells in the cell sample, the stronger the autofluorescence. The main measures to reduce autofluorescence interference and improve signal-to-noise ratio are as follows: a. Choose bright fluorescent dyes as far as possible; b. Select the appropriate laser and filter optical system; c. The background contribution of autofluorescence is compensated by an electronic compensation circuit.
a. Turn on the power and preheat the system.
b. Open the gas threshold, adjust the pressure, and obtain the appropriate liquid flow velocity; Turn on the light source cooling system.
c. Add deionized water to the sample tube, rinse nozzle system.
d. Using the calibration standard sample, adjust the instrument so that the fluorescence intensity of 0 and 90 scattering is the highest, and the coefficient of variation is required to be the minimum, on the basis of the laser power, the voltage of the photomultiplier tube and the gain of the amplifier circuit.
e. Selected flow rate, cell number measurement, measurement parameters, etc., in the same working conditions to measure the sample and control sample; At the same time, select the display mode of the data on the computer screen, so as to intuitively grasp the measurement process.
f. After the sample is measured, deionized water is used to flush the fluid flow system.
g. Because the experimental data has been stored in the computer hard disk (some machines also have a CD-ROM system, so storage capacity is greater), so you can close the gas, and measuring device, and use a computer alone for data processing.
h. Print out the required results.