Brief introduction to the technique of flow cytometry|同科生物-华强电子网

Flow Cytometry (FCM) is a powerful technique used for the rapid, quantitative analysis and sorting of cells or other biological particles—such as microspheres, bacteria, or small organisms—arranged in a single file within a liquid stream. Originating in the 1960s and 1970s, modern flow cytometry has evolved into a highly sophisticated tool that plays a crucial role in various scientific fields, from basic research to clinical applications. It is widely applied in cell biology, immunology, hematology, oncology, pharmacology, genetics, and clinical diagnostics. At its core, flow cytometry enables the rapid determination of biological properties of individual cells or organelles within a flow system. It allows for the classification of specific cells or structures based on multiple parameters such as size, granularity, fluorescence, light scattering, and absorption. These measurements help quantify key features like DNA content, cell volume, protein levels, enzyme activity, and surface markers. By analyzing these characteristics, researchers can isolate pure cell populations for further study, with sorting speeds reaching up to 30,000 cells per second. Modern flow cytometry integrates multiple technologies, including fluid mechanics, laser optics, electronics, photoelectric detection, computer systems, fluorescent chemistry, and monoclonal antibody techniques. This convergence of disciplines has led to continuous advancements in detection accuracy, sorting efficiency, and high-throughput analysis. As life sciences demand more precise and detailed cellular data, flow cytometry continues to evolve, offering groundbreaking tools for biological and medical research. The development of flow cytometry began in the early 20th century. In 1934, A. Mordawan introduced an automated method for counting red blood cells passing through a glass capillary. In 1956, WH Kurt developed a device using electrical resistance (Coulter principle) to measure cell volume. By 1965, LA Kamenzki created a multi-parameter flow cytometer, while MJ Furl Weller designed a cell sorter. In 1969, Van Dilla et al. introduced a flow cytometer with orthogonal optical alignment, marking a significant milestone in the field. Over time, improvements in cell sorting technology allowed for more accurate and efficient cell separation. However, early systems lacked the resolution to capture detailed cell morphology. Subsequent innovations, such as slit scanning techniques and mercury lamp-based excitation, enhanced the ability to analyze nuclear fluorescence and cell size. These developments laid the foundation for the advanced instruments we use today. The principle behind flow cytometry involves labeling cells with fluorescent dyes and suspending them in a fluid stream. The sample is then passed through a flow chamber where it intersects with a laser beam. The cells emit fluorescence, which is collected by detectors at right angles to the laser. Forward scatter measures cell size, while side scatter reflects internal complexity. The signals are processed using a multi-channel pulse height analyzer, generating histograms, scatter plots, and 3D visualizations for data interpretation. Cell sorting works by applying high-frequency vibrations to break the cell stream into droplets. Each droplet is electrically charged based on its measured properties. When the droplets pass through a charged deflector, they are separated into different collection channels, allowing for the isolation of specific cell types. In cell biology, flow cytometry is used to determine the cell cycle distribution by measuring DNA content. It also supports multi-parameter analysis, enabling simultaneous measurement of various cellular features. For example, staining cells with acridine orange allows differentiation between DNA (green) and RNA (red), providing insights into cellular metabolism and function. In genetic research, flow cytometry helps analyze chromosomal DNA content, creating a "flow karyotype" that reveals chromosome abundance and distribution. This technique is essential for genome mapping, cancer diagnosis, and understanding genetic disorders. Immunological studies benefit greatly from flow cytometry combined with immunofluorescence. It identifies and quantifies cells based on surface antigens, helping diagnose immune deficiencies, autoimmune diseases, and hematological malignancies. Additionally, it measures receptor density and ligand binding, offering valuable insights into cellular interactions. In oncology, flow cytometry detects abnormal DNA content in tumor cells, aiding in the diagnosis of cancers like leukemia and solid tumors. It also monitors the effectiveness of chemotherapy and radiation therapy by analyzing cell dynamics and response to treatment. Beyond these fields, flow cytometry is widely used in hematology, microbiology, and molecular biology. Ongoing advancements aim to improve sensitivity, speed, and the ability to capture morphological details, making it an indispensable tool in modern biomedical research.

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