Flow Cytometry (FCM) is a technique for rapid quantitative analysis and sorting of cells or other biological particles (such as microspheres, bacteria, small model organisms, etc.) arranged in a single column in a liquid flow. As a technology platform for detection by flow cytometry, modern flow cytometry was produced in the 1960s and 1970s. After nearly 40 years of development and improvement, today’s flow cytometers are very mature and widely used in all aspects from basic research to clinical practice, covering cell biology, immunology, hematology, oncology , pharmacology, genetics and clinical testing and other fields, play an important role in various disciplines.
Flow Cytometry – Overview
A technique for rapidly determining the biological properties of individual cells or organelles and sorting and collecting specific cells or organelles from a population in a fluidic system. It is characterized by the rapid determination of Coulter resistance, fluorescence, light scattering and light absorption to quantify many important parameters such as cellular DNA content, cell volume, protein content, enzyme activity, cell membrane receptors and surface antigens. Cells of different properties are separated according to these parameters to obtain pure cell populations for biological and medical research. At present, the highest sorting speed has reached 30,000 cells per second.
Modern flow cytometry integrates fluid mechanics technology, laser technology, electronic physics technology, photoelectric measurement technology, computer technology, fluorescence chemistry technology and monoclonal antibody technology. It is the crystallization of technological progress in multiple disciplines and fields. With the rapid development of modern science and technology, in order to meet the higher-level requirements of life science for cell analysis, flow cytometry is still developing rapidly, and has made many breakthroughs in detection technology, sorting technology and high-throughput analysis. .
Flow Cytometry – A Brief History
In 1934, A. Mordavan first reported the automatic counting of cells by passing red blood cells in suspension through a glass capillary placed on a microscope stage. In 1956, W.H. Coulter introduced a device for counting cells and measuring cell volume by using the resistance change (called Coulter resistance) of cells in a conductive solution through a small hole (75-100 microns) between two chambers. . In 1965, L.A. Kamensky developed a multi-parameter flow cytometer that can measure cell size and nucleic acid content. In the same year, M.J. Fulweiler also made a cell sorter. In 1969, Van Diera et al. applied argon ion laser and layered shell flow technology to establish a flow cytometer in which the liquid flow, the illumination optical axis and the detector axis were orthogonal to each other.
Later, the improved cell sorter by H.R. Hewlett et al. can make the cells in the flowing liquid spray into the air for measurement. However, in the above systems, the laser beam used and the limiting diaphragm set in the detection direction of the collected fluorescence are larger than the cells in the liquid flow, so they cannot provide information about the cell morphology, so it is called zero resolution. system. Subsequently, L.L. Wellis and S.F. Patten developed a low-resolution slit-scanning technique to measure nuclear fluorescence, size of cells and nuclei. In 1969, W. Gerd and W. Dietrich described a flow cytometer using a mercury lamp as a light source to excite cells flowing parallel to the optical axis in a flow chamber under epi-light illumination.
Flow Cytometry – Principles
The sample to be tested (such as cells, chromosomes, sperm or bacteria, etc.) is dyed with fluorescent dyes to make a sample suspension, which enters the flow chamber through a sample injection tube surrounded by shell liquid under a certain pressure, and the cells are arranged in a single row. The nozzle of the chamber is ejected into a stream of cell fluid that intersects the incident laser beam. Cells are excited to produce fluorescence, which is collected by an optical system placed at 90° to the incident laser beam and cell fluid flow. Blocking filters in the optical system are used to block excitation light; dichroic beamsplitters and other blocking filters are used to select fluorescence wavelengths. The fluorescence detector is a photomultiplier tube. Scattered light detectors are photodiodes that collect forward scattered light. Small angle forward scatter is related to cell size.
The entire instrument processes fluorescence pulse signals and light scattering signals with a multi-channel pulse height analyzer. The results of the assay are represented by one-parameter histograms, two-parameter scatter plots, three-dimensional stereograms and contour (contour) maps.
The principle of sorting cells is that high-frequency oscillation is generated by an ultrasonic oscillator, which makes the flow chamber vibrate, and the cell liquid stream ejected from the nozzle is broken into a series of uniform droplets, some of which contain cells. These cells have their signals (representative of cell properties) measured by the optical system before forming droplets, if the measured signals are consistent with the properties of the cells selected to be sorted, or if found to be sorted When a selected cell is just forming a droplet, the instrument charges the entire stream with a brief positive or negative charge. When the droplet leaves the stream, the droplets of selected cells are charged, and the droplets of cells that are not selected are uncharged. When the droplets with positive or negative electricity pass through the high-voltage deflection plate, they are deflected to the cathode or to the anode, so as to achieve the purpose of sorting and collecting cells.
Flow Cytometry – Applications
Cell Biology Research
Flow cytometry can be used to determine the percentage of cells in each phase of the cell cycle. The DNA content distribution curve is obtained by measuring the DNA content of each cell in the cell population. For example, in the determination of the DNA content distribution curve of Hela cells, the first peak is G1/G0 (DNA synthesis prophase/quiescent phase) cells with 2CDNA content, and the second peak is G2+M (DNA synthesis late stage + mitosis) cells with 4CDNA content Phase) cells, the region from 2C to 4C is S phase (DNA synthesis phase) cells. The percentage of cells in each phase of the cell cycle in the entire cell population can be calculated by drawing or computer fitting.
Flow cytometry can be used for multiparametric analysis, that is, the simultaneous determination of multiple properties of a cell. Such as scattered light and fluorescence, or a variety of different colors of fluorescence. For example, after cells are stained with acridine orange, DNA fluoresces green and RNA fluoresces red. By measuring these two kinds of fluorescence, the DNA and RNA content in a cell can be known at the same time. The measurement results can be represented by a two-dimensional scatter diagram or a three-dimensional stereogram. In this way cells in G0 and G1, M and G2 phases can be identified based on DNA and RNA content. By combining flow cytometry with liquid scintillation technology, the time (T, Ts, T+M and its coefficient of variation) of cells passing through G1, S and G2+M phases can also be obtained. In recent years, the use of anti-bromodeoxyuridine (Brdurd ) monoclonal antibody can find bromodeoxyuridine that penetrates into cellular DNA. Combining this fluorescent antibody technology with the determination of cellular DNA content is a very useful technique for studying DNA synthesis and cell cycle. In addition, flow cytometry Cytometry can also determine the degree and stage of cell population synchronization, identify dead cells and live cells, use fluorescently labeled ligands, and quantitatively determine cell surface and internal receptors.
Using flow cytometry to determine the content of chromosomal DNA, the frequency distribution map of chromosomes can be obtained, which is called flow chromosomal karyotype analysis. A peak appears for the same type of chromosome, and the area of the peak represents the abundance of this type of chromosome. Flow chromosomal karyotyping technology can not only quickly analyze karyotypes, but also sort out different types of chromosomes and make a DNA library of each human chromosome, which can be used for human genome research, genetic disease and cancer diagnosis research.
Combined with immunofluorescence methods, flow cytometry can identify and count cells with different surface-specific antigens, such as using fluorescein-labeled immunoglobulin to identify T and B lymphocytes, and further distinguish between different cell surface antigens. T and B lymphocyte subsets, and the number, density and kinetic parameters of antigens carried by each cell. Flow cytometry can also be used to sort and collect cell populations with a specific antigen with “+” and without “-” for studying its functional properties.
Flow cytometry is an important feature for judging immunodeficiencies such as AIDS (acquired immunodeficiency syndrome), that is, changes in the proportion of T4 and T8 lymphocytes (large decrease in T4 cells), as well as judging autoimmune diseases and determining leukemia and lymphoma. Phenotypes, etc., are very useful. In addition, flow cytometry can be used to quantify fluorescein-labeled lectins bound to cells, measure cell surface area and relative density of fluorescein-binding sites, and measure the number, and study the competition of various lectins for binding to the cell surface.
Tumor cells generally contain abnormal amounts of DNA. Aneuploid cells are found in most solid tumors and acute leukemias, and flow cytometry can provide valuable diagnosis due to the simple method of sample preparation, accurate measurement results, and rapid access to DNA ploidy information data. The reliability of the diagnosis can be further improved if other parameters (such as different types of medium fibrin, protein content, cell size, nucleocytoplasmic ratio, etc.) are measured at the same time as the DNA content.
Flow cytometry can be used to evaluate the effects of tumor chemotherapy and radiotherapy both experimentally and clinically, and the monitoring of tumor treatment based on flow cytometry and cytokinetic data has been carried out in practice.
In addition, flow cytometry is also widely used in hematology, microbiology, molecular biology and other fields. Flow cytometry is developing towards high sensitivity, high speed, multi-parameter measurement, acquisition of morphological information, etc.