What is a loss cytometry flow cytometry and what is a flow cytometer

How a flow cytometer is constructed and works.

The flow cytometer is mainly composed of the following five parts: Flow chamber and flow drive system Laser light source and beam forming system Optical system Signal detection and storage, display and analysis system Cell sorting system.

Flow chamber and fluidic drive system.

The flow chamber is the heart of the flow cytometer and is made of quartz glass. The single-cell suspension is surrounded by sheath fluid in the cell flow chamber through a hole of a certain pore size in the flow chamber, and the detection area is in the center of the hole, where the cells intersect perpendicularly with the laser, and the cells are arranged in a single row through the laser detection area under the constraint of the sheath fluid.

Laser light source and beam forming system.

The commonly used laser tube is the argon-ion gas laser tube, which emits light at a wavelength of 488 m, and can also be equipped with a helium-neon ion gas laser tube (wavelength 633 m) or an ultraviolet laser tube. There are two cylindrical lenses between the laser light source and the flow chamber, which focus the laser beam with a circular cross-section emitted by the laser light source into an elliptical laser beam (22 m 66 m) with a small cross-section, so that the irradiation intensity of the cells passing through the laser detection area is consistent.

Signal storage, display, analysis system.

The optical system of the flow cytometer is composed 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 back of the flow chamber. The optical system in front of the flow chamber is composed of a lens and a small hole, and the lens and the small hole focus the laser beam with a circular cross-section from the laser light source into an elliptical laser beam with a small cross-section, so that the irradiation intensity of the cells passing through the laser detection area is consistent and the interference of stray light is minimized

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A flow cytometer is a device that automatically analyzes and sorts cells. It can quickly measure, store, and display a range of important biophysical and biochemical characteristic parameters of dispersed cells suspended in liquids, and can sort out specific cell subsets from them according to a pre-selected range of parameters. Most flow cytometers are zero-resolution instruments, which can only measure the amount of total nucleic acids and total proteins of a cell, but cannot identify and measure the amount of nucleic acids or proteins in a specific site.

That is, it has a detail resolution of zero. The flow chamber is composed of a sample tube, a sheath fluid tube and a nozzle, which is commonly made of transparent and stable materials such as optical glass and quartz. Meticulously designed and manufactured, it is the heart of the fluidic system.

The sample tube stores the sample, and a single cell suspension is ejected from the sample tube under the action of liquid flow pressure; The sheath fluid flows from all sides to the nozzle from the sheath tube, encloses the periphery of the sample and exits the nozzle. In order to ensure that the liquid flow is stable, the liquid flow speed is generally limited to <10m s. Due to the action of the sheath fluid, the cells being tested are confined to the axis of the fluid flow.

The flow chamber is equipped with a piezoelectric crystal that vibrates when exposed to an oscillating signal.

Flow cytometry is based on flow cytometry, which can quickly and accurately quantify and sort the physical and chemical properties of individual cells. The analysis process is as follows: prepare a single-cell suspension, stain with antibodies labeled with specific fluorescent dyes, the cells to be tested after staining are pressed into the flow chamber under a certain gas pressure, and the cells are arranged in a single row under the wrapping of the sheath fluid, and pass through the detection area in turn, and the cells will produce scattered light and excitation fluorescence under the irradiation of the laser beam.

Both signals are received by a photomultiplier tube (PMT) at the same time. After receiving, it can be converted into an electrical signal, and through an analog-to-digital converter, the continuous electrical signal is converted into a digital signal that can be recognized by a computer for analysis.

As mentioned above, the flow cytometer is composed of three parts: the fluidics system, the optical path system, and the detection and analysis system, and some instruments also have a sorting function, which can charge and deflect the particles to achieve cell sorting.

Fluidics system: By generating pressure to make the sample containing the cells of interest quickly enter the instrument, the pressure difference between the sample and the sheath fluid is used in a closed sample tube to collect the sample into the optical analysis system;

Optical path system: The laser emitted by different lasers is irradiated to the cell surface to produce light signals, and then received by different optical path systems. At the analysis point in the flow cytometry irradiation chamber, the laser is scattered and refracted on the cell surface, and scattered light including forward scattered light (FSC) and side scattered light (SSC) is emitted, and the fluorescein on the cell surface is excited to emit fluorescence.

Forward-scattered light and side-scattered light detectors collect scattered light and convert it into an electrical signal; The fluorescence is collected by the concentrator, and the fluorescence of different colors is turned to different photomultiplication detectors by a two-color reflector, which also converts the fluorescence signal into an electrical signal. FSC and SSC are two very basic parameters in flow cytometry. The value of FSC can represent the size of the cell, and the larger the volume of the cell, the larger the FSC.

SSC represents the granularity of the cell, and the larger the intracellular organelles and granularity, the larger the SSC.

Detection and analysis system: The intensity of the fluorescent signal represents the intensity of the antigen on the surface of the cell membrane or the concentration of the material inside the cell, and the light signal is converted into an electrical signal that can be recognized by a computer. The computer processes and analyzes the various signals measured.

Flow cytometry sorting is performed on the basis of flow cytometry. An ultra-high frequency piezoelectric crystal is placed above the nozzle in the flow chamber, which generates vibrations that cause the ejected liquid stream to form uniform droplets in which the cells to be tested are dispersed. These droplets are charged with positive and negative charges (cell droplets labeled with fluorescein have a negative charge; Cell droplets that are not labeled with fluorescein are positively charged; Droplets that do not contain cells are not charged), as the droplets flow through the deflection plate, they are deflected by a high-voltage electric field and fall into their respective collection containers, and the uncharged droplets fall into the waste containers, thus achieving cell separation.

I'll put it simply.

Bai a little:

1.First, the machine sucks up the du

Cell suspension, injected into zhi

It is composed of a sheath fluid (generally phosphorus.

DAO Acid Buffer).

Due to the large flow rate of the sheath fluid, it belongs to the form of cell suspension through axial flow, which is generally in the form of a single cell queue.

2.The cells then pass through the detection window. There is a laser beam irradiation. Generally, cells are fluorescently stained, and after being irradiated by a laser, different wavelengths of excitation light are generated. The machine is equipped with a detector to detect the intensity of the excitation light.

3.According to the intensity of the excitation light, the binding of cells and chromatic substances is determined, so as to obtain the results to be detected (such as antigen amount, DNA amount, etc.).

Nanoflow bai

The detector can realize the particle size and distribution of individual DU nanoparticles (7-1000 nm), particle DAO concentration, and biochemistry.

High sensitivity and high selectivity of internal traits.

Capacitive and high-throughput assays will be a powerful characterization tool for life science, nanoscience, and nanotechnology research. The nanoflow detector is an advanced single-particle, multi-parameter, high-throughput nanoparticle quantitative characterization platform developed based on sheath flow single-molecule fluorescence detection technology, which has an unprecedented sensitivity of 24 nm for the scattering detection of a single low refractive index nanoparticle, and has the advantages of high sensitivity, fast speed, simultaneous detection of physical and biochemical properties, and suspension detection.

Advantages: Detection sensitivity: scattering detection of 24 nm low refractive index nanoparticles, fluorescence detection of individual phycoerythrin.

Nanoparticle (<100 nm) multiparametric quantitative characterization flow cytometry equipment. The detection range covers the complete particle size of exosomes (30-200 nm).

Generally, there is a user guide, please follow the instructions to use it.

This is the setting for the column chart when analyzing the data.

Max scaling means comparing the peaks of multiple column charts, comparing the proportional relationship between the short peaks and the highest one. The y-axis is now no longer a cell count, but a percentage.