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flow-cytometer

Flow Cytometer

Flow cytometer used in laboratory

What is flow cytometer?

cytometry
Flow cytometer 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.

Working principle of flow cytometer

FACS flow cytometry

A. Principle of parameter measurement

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.

B. Principle of sample separation

The sorting function of the flow cytometer is performed by a cell sorter. The overall process is: the liquid column from the nozzle is divided into a series of small water droplets, according to a certain parameter selected by the logic circuit to determine whether will be sorted, and then the charging circuit charges the selected cell droplets, the charged droplets carry cells through the electrostatic field and deflect, fall into the collector; Other liquids are aspirated as waste, and some types of instruments also use capture tubes for sorting.

The stable droplet is formed by the vibration of the piezoelectric crystal on the flow chamber under the action of tens of kHz electrical signal, which forces the liquid flow to break evenly. Generally, the droplet spacing is about hundreds of μm. The experimental empirical formula f=v/4.5d gives the oscillation signal frequency of stable water droplets. Where v is the liquid flow velocity and d is the diameter of the nozzle hole. It can be seen that using different orifices and changing the flow velocity may change the sorting effect. The deflection of the sorted cell-containing droplets in the electrostatic field is accomplished by the charging circuit and the deflection plate. The charging voltage is usually +150V or -150V; The potential difference between the deflection plates is thousands of volts. The charging pulse generator in the charging circuit is controlled by the logic circuit, so it takes a period of delay from parameter determination through logic selection to pulse charging, which is generally tens of ms. Accurate determination of the delay time is the key to determining the quality of sorting. The digital circuit of the shift register is used to generate the delay. It can be adjusted appropriately according to specific requirements.

Use of flow cytometer

cytoflex

Cytometry

A. Test and calibration

Flow cytometry should be carefully adjusted before and even during use to ensure the reliability and optimality of work. The main debugging items are laser intensity, liquid flow velocity, and the optical path of the measurement area.

Laser intensity: In addition to adjusting the Angle of the mirror to adjust the laser light to the desired wavelength, it is necessary to combine the spectrum curve on the display screen to maximize the laser intensity output.

Liquid flow speed: It can be supervised by a digital display on the operating table, and the gas pressure can be adjusted to obtain a stable liquid flow speed.

Measurement area light path adjustment: this is the key to debugging work. It is necessary to ensure that the liquid flow, laser beam, 90 scattering measurement photoelectric system in the measurement area is perpendicular orthogonal, and the intersection point is small. In general, it can be done in calibration with standard fluorescent microspheres.

The quantity measured in flow cytometry is relative value, so it is necessary to calibrate or calibrate the system before or during use, so that absolute significance can be obtained through relative measurement. Therefore, the calibration in FCM has a dual function: instrument alignment adjustment and quantitative scaling. The STANDARD sample should be stable, the shape of the tangible component should be relatively uniform and spherical in size, the sample dispersion performance is good, and the economy is easy to obtain. Standard fluorescent microspheres are commonly used as non-biological standard samples and chicken red blood cells as biological standard samples. The microspheres are made of resin material and either labeled with or without fluorescein. The preparation process of the standard sample of chicken blood red blood cells used was as follows: chicken blood anticoagulant with 3.8% citrate or heparin (anticoagulant: chicken blood = 1:4) was washed three times with PBS, then mixed with 5 ~ 10ml of 1.0% glutaraldehyde and washed with cleaned chicken red blood cells after shaking at room temperature for 24h. Finally, it was washed again with PBS and stored in a refrigerator at 4℃ for later use. It should be pointed out that without fluorescence staining, the measured light signal is the spontaneous fluorescence of chicken haemoglobin.

B. Operation and use of instruments

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.

C. Precautions in operation and use

a. The photomultiplier tube requires stable working conditions. After exposure to strong light, it needs a long time of "dark adaptation" to eliminate or reduce part of the dark current background before it can work. Also pay attention to magnetic shielding.

b. The light source shall not be turned off and on in a short time (usually about 1h); The use of light source must be preheated and pay attention to the cooling system is working normally.

c. The liquid flow system must be kept smooth at any time to avoid bubble embolization, and the used sheath fluid should be filtered and disinfected before use.

d. Pay attention to the selection of appropriate filter system and type of amplifier according to the transformation of the measurement object.

e. Special emphasis is placed on the need for a control group for each measurement.

How to buy flow cytometer?

ANTITECK provide lab equipment, lab consumable, manufacturing equipment in life sciences sector.
If you are interested in our flow cytometer or have any questions, please write an e-mail to info@antiteck.com, we will reply to you as soon as possible.


    AntiTeck Life Sciences Limited

    A1-519, XingGang GuoJi, Yingbin Road, Huadu, Guangzhou, China, 510810
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