Flow cytometry

Flow cytometry

Flow cytometry (flow cytometry) is a device for automatic analysis and sorting of cells. It can quickly measure, store and display a series of important biophysical and biochemical characteristic parameters of dispersed cells suspended in liquid, and sort out the specified cell subsets according to the preselected parameter range. Most flow cytometry is a zero resolution instrument , it can only measure the indexes of a cell, such as total nuclear acid and total protein, but can not identify and measure the number of nucleic acids or proteins in a specific part. That is, its detail resolution is zero.

form

Flow cytometry is mainly composed of four parts. They are: flow chamber and liquid flow system; Laser source and optical system; Photocell and detection system; Computer and analysis system. The figure above shows its structure.

Flow chamber and liquid flow system

The flow chamber is composed of sample tube, sheath tube and nozzle. It is usually made of transparent and stable materials such as optical glass and quartz. The design and manufacture are very fine, which is the heart of the liquid flow system. The sample tube stores the sample, and the single cell suspension is ejected from the sample tube under the action of liquid flow pressure; The sheath fluid flows from the sheath tube to the orifice, surrounds the periphery of the sample and then shoots out from the nozzle. In order to ensure that the liquid flow is stable, the liquid flow speed is generally limited ν< 10m / s due to the action of sheath fluid, the detected cells are limited to the axis of fluid flow. The flow chamber is equipped with a piezoelectric crystal, which can vibrate under the oscillation signal.

Laser source and optical system

Cells stained with specific fluorescence need a suitable light source to emit fluorescence for collection and detection. Common light sources include arc lamp and laser; Argon ion lasers are also popular, and krypton ion lasers or dye lasers are also matched. The selection of light source is mainly based on the excitation spectrum of the excited material. Mercury lamp is the most commonly used arc lamp, and its emission spectrum is mostly concentrated in 300 ~ 400 nm, which is very suitable for occasions requiring ultraviolet excitation. In the emission spectrum of argon ion laser, 514nm green light and 488nm blue light are the strongest, accounting for about 80% of the total light intensity; The spectrum of krypton ion laser is mostly concentrated in the visible part, with a strong wavelength of 647 nm. The excitation wavelength of some fluorescent dyes used in immunology is more than 550 nm, and dye lasers can be used. Using organic dye as a component of laser pump can change the spectrum of the original laser to meet the needs, that is, to form a dye laser. For example, by pumping a dye laser containing rhodamine 6G aqueous solution with the green light of an argon ion laser, a continuously adjustable laser of 550 ~ 650nm can be obtained, especially at 590nm, the conversion efficiency is the highest, accounting for about half. In order to evenly irradiate the cells and improve the resolution, the diameter of the laser spot irradiated on the cells should be close to the cell diameter. Therefore, it is necessary to converge the laser beam through the lens. The spot diameter D can be determined by the following formula: D = 4 λ f/πd. λ Is the laser wavelength; F is the focal length of the lens; D is the laser beam diameter. The dispersion prism is used to select the wavelength of the laser and adjust the angle of the mirror to tune to the required wavelength λ。 In order to make the detected emission fluorescence stronger and improve the signal-to-noise ratio of the fluorescence signal, a variety of filters are also used in the optical path. The band stop or band-pass filter selectively filters or passes the light in a certain filter length section. For example, only FITC is allowed when 525nm bandpass filter is used The 525nm green light emitted by (fluorescein isothiocyanate, fluorescein isothiocyanate) passes through. The long wave passes through the dichroic mirror, which only allows the light of more than one wavelength to pass through, and reflects the light of another specific wavelength below this wavelength. In immunoassay, it is often necessary to detect the fluorescence signals of more than two wavelengths at the same time, so the dichroic mirror or dichroic spectrometer is used, To effectively separate various fluorescence.

Photocell and detection system

The fluorescence generated by fluorescent stained cells excited by appropriate light is measured by converting it into electrical signal through photoelectric converter. Photomultiplier tube (PMT) is the most commonly used. The response time of payment is short, only in the order of NS; the spectral response characteristics are good. In the spectral region of 200 ~ 900nm, the light quantum yield is relatively high. The gain of photomultiplier tube can be continuously adjusted from 10 to 10, so it is very beneficial to weak light measurement. When the photomultiplier tube is running, pay special attention to the stability, the working voltage should be very stable, the working current and power should be very stable The rate should not be too high. The general power consumption is less than 0.5W, and the maximum anode current is several ma. In addition, pay attention to the dark adaptation of the photocell and good magnetic shielding. In use, pay attention to PMTs with different installation positions. Because of different spectral response characteristics, they should not be interchanged. There are also silicon photodiodes, which are more stable in strong light than payment.

The electrical signal output from the payment is still weak and needs to be amplified before it can be input into the analytical instrument. There are two types of amplifiers in flow cytometry. One is the linear relationship between the output signal radiance and the input signal, which is called linear amplifier. Linear amplifiers are suitable for signals that vary in a small range and signals representing biological linear processes, such as deoxyribonucleic acid measurement. The other is logarithmic amplifier. There is a common logarithmic relationship between output signal and input signal. Logarithmic amplifiers are often used in immunological measurements. Because immunoassay often shows three subpopulations of negative, positive and strong positive at the same time, their fluorescence intensity differs by 1 ~ 2 orders of magnitude; Moreover, in multicolor immunofluorescence measurement, the data collected by logarithmic amplifier is easy to explain. In addition, it has the advantages of convenient regulation and the distribution shape of cell population is not easy to be affected by external working conditions.

Computer and analysis system

The amplified electrical signal is sent to the computer analyzer. The number of multichannel channels corresponds to the pulse height of electrical signal and is also related to the strength of optical signal. The corresponding channel number year ordinate usually represents the relative number of cells that emit the signal. The signals from the multi-channel analyzer are then transmitted to the microcomputer processor through the analog-to-digital converter, compiled into data files, or stored on the hard disk and floppy disk of the computer, or stored in the instrument for call. The computer has a large storage capacity and can store 6 ~ 8 parameters of the same cell. The data stored in the computer can be reproduced offline after actual measurement, processed and analyzed, and finally the results are given. In addition to the above four main parts, it is also equipped with additional devices such as power supply and compressed gas
 

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