The principle of flow cytometry and its application and selection in plants
The principle of flow cytometry and its application and selection in plants Flow Cytometry 1 The concept of flow cytometry and its development history 2 Working principle and technical indicators of flow cytometry 3 Mainstream flow cytometry models and their characteristics The companies that currently have a large market share are BD (Becton-Dickinson) in the United States, Beckman-Coulter (formerly named Couter.ter) and Partec in Germany.
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Abstract: With the continuous deepening of the scientific and technological level, the continuous update of scientific research equipment, the country's investment in scientific research continues to increase, and plant detection technology has also been further developed. Flow cytometry plant detection technology is a new technology that has just emerged in China in recent years, and it is still relatively new to many plant science and technology workers. This article introduces the basic concepts, development history, working principles, well-known brands, common models and their application in plants that are necessary for flow cytometry analysis in more popular languages.
Keywords: flow cytometry; plant; detection technology
Zhao Hong (Beijing Vegetable Research Center, Bering 100089, China)
Abstract: With the development of science, with the updating of scientific equipments, and with the increasing investment by nation, the plant measurement technologies have been made great progress. Flow cytometry (FCM) is a new technology appeared recently in China. It is still strange to many plant scientific researchers. In this article we briefly introduce the bas—ic concept and history of FCM, simply explmn the principle, concretely present three famous companies and their model Tl models, and demonstrate some applications of FCM in plants science.
Key words: Flow cytometry; plant; measurement
1.1 Basic concepts of flow cytometry Flow cytometry (FCM) is an instrument for rapid quantitative measurement and analysis of high-speed straight-line flowing cells or biological particles, mainly including sample flow technology and cell counting And sorting technology, computer data collection and analysis technology, etc. Flow cytometry is based on flow cytometry. It is the crystallization of the comprehensive application of knowledge such as fluid mechanics, laser technology, electronic engineering, molecular immunology, cell fluorescence chemistry, and computer. Flow cytometry is a technique that automatically analyzes and sorts cells or subcells. Its characteristics are: fast measurement speed, large measured population, and multi-parameter measurement, that is, multi-parameter measurement of physical and biochemical characteristics of the same cell, and it is statistically effective.
1.2 A brief history of the development of flow cytometry The earliest prototype of flow cytometry was born in 1934. Moldavan proposed to make suspended single red blood cells flow through glass capillaries, count them with a microscope under a bright field of view, and measure them with a photoelectric recording device Assumptions. In 1953, Crosland-Taylor designed the flow chamber according to the law of Newtonian fluid flowing in a circular tube. After that, through continuous improvement of Coulter Parker Horst Kamentsky1 Gohde, Fulwyler Herzen-berg and others, they designed photoelectric detection equipment and cell sorting device, completed the physical connection between computer and flow cytometer, and recorded and analyzed multi-parameter data , Pioneered the cell's immunofluorescence staining and detection technology, popularized the application of flow cytometry in clinic? In the past 20 years, with the improvement of flow cytometry and its detection technology, people are increasingly devoted to the work of sample preparation, cell labeling, software development, etc., in order to expand the application field and use effect of FCM. Song Pinggen's "Principles and Applications of Flow Cytometry" is by far the most detailed and thorough Chinese work on flow cytometry and its techniques. This book introduces the history, structure, principles, and technical indicators of flow cytometry in great detail. It exemplifies its application in medicine and bioengineering, and is very suitable for those engaged in professional research in this field. Since this book was published 13 years ago, it basically did not involve plant flow cytometry detection technology. In addition, for those who only need a basic understanding of flow cytometry, this book is too complicated and esoteric. Xie Xiaomei mainly introduced the application of flow cytometry in bioengineering. Yang Rui summarized the working principle of flow cytometry and briefly mentioned the application of flow cytometry. After analyzing the advantages and disadvantages of these three works or articles, this article introduces some principles that must be understood to master the flow cytometry detection technology in a more popular language, and gives an objective performance summary of the mainstream models on the market.
2.1 Working principle of flow cytometer In addition to power supply, flow cytometer is mainly composed of four parts: flow chamber and liquid flow system: laser source and optical system; photoelectric tube and detection system; computer and analysis system, including flow chamber It is the core component of the instrument. These four components have completed the tasks of signal generation, conversion and transmission.
2.1.1 Signal generation, conversion and transmission Under the action of pressure, the sheath liquid in the sheath liquid tube is continuously pressed into the flow chamber, forming a stable continuous flow, ensuring that the sample liquid is stable The sheath fluid flows on the axis and passes through the laser irradiation area in the form of a single cell. The laser irradiation area, also known as the measurement area, refers to the point where the liquid flow intersects the laser beam perpendicularly. When the cell carries the fluorescein marker through the laser irradiation area, it generates fluorescent signals representing different substances and different wavelengths inside the cell, and these signals are centered on the cell and directed toward the space 360. Solid angle emission, generating scattered light and fluorescent signal. The scattered light does not depend on any cell sample preparation technology, so it is called the physical or intrinsic parameter of the cell. The scattered light includes forward angle scattering and direction finding scattering. The forward angle scattering is closely related to the square of the measured cell diameter. The measured angle scattered light is more sensitive to the refractive index of the cell membrane, cytoplasm, and nuclear membrane, and can provide information about the fine structure and particle properties in the cell. There are also two kinds of fluorescence signals; one is the weak fluorescence signal emitted by the cell itself under laser irradiation, and the other is the fluorescence signal obtained after the cells irradiated by the specific fluorescein label are excited and irradiated. In immunoassay, it is often necessary to detect fluorescence signals of more than two wavelengths at the same time, so a dichroic mirror or dichroic beam splitter is used to effectively separate various fluorescences. The fluorescence produced by fluorescently stained cells after being excited by suitable light is measured by converting the photoelectric converter into an electrical signal. The most commonly used photoelectric converter is a photomultiplier tube (PMT). The electrical signal output from the PMT needs to be amplified before it can be input into the analysis instrument. There are generally two types of amplifiers in flow cytometers. One type is a linear amplifier, whose output signal has a linear relationship with the input signal. Linear amplifiers are suitable for signals that vary within a relatively small range and signals that represent biological linear processes, such as DNA measurements. The other type is the logarithmic amplifier, which has a common logarithmic relationship between the output signal and the input signal. Logarithmic amplifiers are often used in immunological measurements. The amplified electrical signal is transmitted to the computer, and then transmitted to the microcomputer processor through the analog-to-digital converter to form a data file, which is saved on the computer. The data saved on the computer can be processed and analyzed after offline.
2.1.2 Sorting principle of flow cytometry Not all flow cytometers have the sorting function. The sorting function of the flow cytometer is performed by the cell sorter. The liquid column ejected from the nozzle vibrates under the action of an electric signal and breaks to form uniform small droplets. According to a selected parameter, the logic circuit determines whether it will be sorted, and then the charging circuit charges the selected cell droplets. The charged droplets carry the cells and deflect through the electrostatic field and fall into the collector. The use of nozzles with different apertures and changing the flow rate may change the sorting effect. It takes a delay time from parameter determination through logic selection to pulse charging. Accurate determination of the delay time is the key to determining the quality of sorting, and can be adjusted appropriately according to specific requirements. 2.1.3 Data display and analysis Data processing mainly includes data display and analysis. Single parameter histogram is the most used graphic display form, which can be used for both qualitative analysis and quantitative analysis. The single-parameter histogram is a two-dimensional plan composed of x and Y directions. The abscissa x is the intensity of the measured fluorescence or scattered light, which is represented by "Channel No." The selected amplifier type is different and the scale is different. The ordinate Y generally represents the absolute number of cells tested. Under normal circumstances, the graph obtained by data analysis is a graph with one or several peaks. The interpretation of the graph should be analyzed concretely. In addition to the histogram, the data display method also includes 2D dot plots, 2D contour plots, pseudo 3D plots, and list mode. Two-dimensional dot plots are also commonly used data display types. It shows the relationship between two independent parameters and the relative number of cells. It is also a two-dimensional plan. The horizontal and vertical coordinates can be determined by the measured parameters selected by themselves. The position of the points indicates the two measured parameters of the cells and particles Value. The amount of information provided by a two-dimensional dot plot is greater than a single parameter histogram. The methods of data analysis can be roughly divided into two categories: parametric method and non-parametric method. When the biological system being tested can use a certain mathematical model, parametric methods are often used. The non-parametric analysis method does not make any assumptions about the displayed image, nor does it use a mathematical model. The analysis procedure can be very simple. It can also be complicated. Non-parametric analysis is more commonly used in clinical medicine.
2.2 Technical indicators of flow cytometer performance The technical indicators of flow cytometer performance mainly include fluorescence resolution, fluorescence sensitivity, applicable sample concentration, and sorting purity. Fluorescence resolution refers to the minimum distance to resolve two adjacent peaks, usually expressed by the coefficient of variation (CV value). At present, the fluorescence resolution of mainstream models on the market should be less than 2.0%. Fluorescence sensitivity reflects the instrument's ability to detect the minimum fluorescence intensity. It is generally expressed by the minimum number of FITC molecules that can be measured on fluorescent microspheres. At present, the instrument can reach about 1000. When the instrument is working, the sample concentration is generally 105-107 cells / m1. Analysis speed / sorting speed refers to the number of particles that can be analyzed or sorted by the flow cytometer per second. The general analysis speed is 5000-10000, and the sorting speed is controlled below 1000. The data measured by the flow cytometer are relative values, so the system needs to be calibrated or calibrated before use. The calibration of the flow cytometer has two purposes, namely the alignment adjustment and quantitative scale of the instrument. Standard microspheres are usually used as non-biological standard samples, and chicken red blood cells are used as biological standard samples.
3.1 Introduction to BD Flow Cytometer The flow cytometer produced by BD is crowned with FACs (fluorescence activated cell sorter), that is, fluorescence activated cell sorter. Its models are relatively complete, such as the early FAC. Sort, FACS Canto, FACSean ,. There are five models currently on the market: FACSCount (small flow cytometer), FACSCAlibur (flow cytometer), FACSAfia (flow cytometer), FACSVantage SE (multicolor analysis and high-speed sorting flow cytometry Instrument), LSRII (digital analysis type flow cytometer). FACSCount is designed to accurately count the absolute number of lymphocytes CD3, CD4, CD8. FACSCalibur is a fully automatic multi-color flow system, focusing on the clinic. Its overall design helps clinicians to quickly achieve clinical analysis of conventional immunophenotypes, CD4T cell counts, DNA, reticulocytes, platelets, etc., and has a sorting function. Equipped with two laser tubes, it can detect 4 fluorescence parameters at the same time. Identify adherent cells.
The BD FACSAfia flow cytometer is a desktop high-speed cell sorter with an acquisition speed of 70,000 cells / s and an analysis speed of 50,000 fine s. Use quartz cup flow detection cell fixed optical path calibration technology. Using three lasers, multi-color analysis, analysis parameters up to 15 colors. Two-tube or four-tube sorting, you can use a variety of collection tubes. The liquid flow monitoring system monitors liquid flow breakpoints in vain, checks for blockages, and realizes unmanned operation of cell sorting. With the accessory BDACDU device, cells can be sorted quantitatively on microplates or carrier slides. FACSVantage SETM is a sorted enhanced flow cytometer based on the cell sorting function of FACSVantage. Six-color fluorescence analysis system, point-to-point sorting, equipped with FACS Diva digital system, providing comprehensive matching reagents. Speed ​​and function are better than FACSVantage. BD LSR II is a digitally upgraded version of LSR. Its performance is between FACSVantage SE and FACS Calibur. It is a desktop computer designed for life science research. Equipped with fixed and calibrated ultraviolet laser, four kinds of laser three-dimensional space excitation, simultaneous analysis of ten-color fluorescence, digitalization of electronic system, relatively easy to learn and use.
3.2 Introduction of Beckman-Coulter Flow Cytometer The flow cytometer produced by Beckman-Coulter is represented by Profile (early and now discontinued) and EPICS series. In recent years, Cytomics FC500 series has been launched. The EPICS series is a large-scale flow cytometer. At present, there are three models on the market: XL, XL-MCL and ALTRA. Among them, ALTRA has a sorting function and is suitable for immunology, cell physiology, molecular biology, genetics, microbiology, water quality Analysis and plant cell analysis. Cytomics FC500 series flow cytometer is small and can automatically analyze 5 colors. It is suitable for immunological detection, such as human HIV diagnosis. Especially the unique design of FC50OMPL can use 12 × 75mm centrifuge tubes and 24 or 96-well plates on the same system. Especially suitable for laboratories with large workload. These two companies are mainly designed and produced for medical research and clinical work, and their products can be applied to many fields of basic research in biomedicine and clinical testing, such as genetics, tumors, blood, immunity and many other research in red blood cells, T cells, lymph Cell subpopulation determination, detection of early apoptosis, tumor cell immunoassay, etc. The flow cytometers of these two companies are expensive, and the main units purchased and used in China are basically some medical institutions.
3.3 Introduction to the flow cytometer of Partee Company Compared with the products of BD and Becman-Coutler, the common characteristics of the flow cytometer produced by the German Partee company are small size, low cost, easy operation, easy to carry, suitable for botany Research is suitable for remote areas and developing countries. German Partec products are divided into three categories: CCA family, PAS family and CyFlow family (Galaxy is an early product, has been discontinued). The CCA family includes the cell counting analyzer CCA and the ploidy analyzer PA-I. It is a single- or dual-parameter desktop minicomputer, which can perform some routine analysis, such as nuclear DNA measurement (detect ploidy or cell cycle), cell count, and apoptosis. It is characterized by small size, easy operation, low price, wide detection range, and can detect the fluorescence emitted by various fluoresceins (such as PI, DAPI, Fluorescein). The PAS family includes particle analysis system PAS, particle analysis system III (PAS-III) and ploidy analyzer PA-II. Provide three combinations of three lasers, can detect more than ten kinds of fluorescent dyes. Up to eight independent fluorescence parameters can be detected and recorded. The ploidy analyzer PA can automatically measure the ploidy level of plants within 2 minutes and detect aneuploidy. Plant materials such as leaves, seedlings, seeds, peels, roots, flowers, etc. can be analyzed. In most plants, the detection resolution of aneuploid chromosomes is ± 1 chromosome. PA-I uses HBO-100 mercury lamp, which is an arc lamp, which can produce ultraviolet excitation light and blue light. A 488 am argon ion laser is added to PA-II, which can detect almost all fluorescent dyes, such as DAPI, Hoeehst, PI, EB, MMC, FITC, FDA, etc. Mercury lamp glow is caused by gas ionization when electric current passes through the gas. It can provide the best excitation wavelength. CyFlow (R) SL is equipped with three kinds of laser tubes, which can be used in researches such as human health, microbiology, industrial applications, process control, and ecology. Such as the count of immunolabeled cells in HIV scans, the count of microorganisms during food processing, and apoptosis. It uses l2 V direct current, especially suitable for remote areas and developing countries. Internationally, in the 1980s, flow cytometry was applied to the study of plants (Galbraith, 1983). Many scholars believe that flow cytometry is an accurate and rapid method for detecting DNA content (Michae | son, 1991). The scope of application is mainly to use flow cytometry to study the DNA content of various plants within and between genera (Baird), 1994; Jacob [, 1996; HALL, 2000) and ploidy level (Costich, 1993; Meng [101 , 2002), detection of DNA content of somatic cell hybrids (Pfosser ?, 1995; Kel-ler [", 1996) and free microspore culture regenerated strains (Kim¨3, 2003). In the past, plant research in China using this detection technology There are very few workers, and most of them are completed in foreign laboratories. The research content includes plant physiology III introduction, programmed death III, ploidy identification mI2, etc. The flow cytometer used in plant research is basically Partec ’s There are also a small number of flow cytometers produced by BD. BD's products are mainly aimed at medical research and application. Compared with Partec products, they are not suitable for the identification of plant chromosome ploidy. The main manifestation is that they cannot provide plant samples. Preparation techniques / reagents, data acquisition software can simultaneously detect a small number of ploidy.