Flow cytometry is a laser-based technique that analyzes physical and fluorescent characteristics of cells or particles suspended in a fluid stream as they pass individually through a detection apparatus.
Principles and instrumentation
A flow cytometer transports a suspension of cells or other particles in a narrow stream so that they pass one at a time through one or more focused laser beams. Hydrodynamic focusing confines the sample within a sheath fluid, ensuring single-file passage. As each cell intersects the laser, detectors measure the light scattered in the forward direction, which correlates with cell size, and light scattered at 90 degrees, which relates to internal complexity or granularity. Additional detectors collect fluorescence emitted from fluorochromes bound to cell components. Optical filters and photomultiplier tubes separate and quantify signals from different fluorophores. The instrument’s electronics convert these signals into digital data representing multiple parameters for each event. Modern flow cytometers can analyze thousands of cells per second and measure dozens of fluorescent markers simultaneously. Software allows gating to define populations, compensation for spectral overlap, and statistical analysis of cell subsets. Proper calibration and quality control are essential to obtain accurate and reproducible results.
Applications in research and medicine
Flow cytometry is widely used to identify and quantify immune cell subsets based on surface markers, such as distinguishing CD4+ and CD8+ T cells in HIV monitoring. It aids in diagnosing leukemias and lymphomas by examining antigen expression patterns and DNA content. Researchers assess cell cycle distribution, proliferation and apoptosis using dyes like propidium iodide and annexin V. Flow cytometry can measure intracellular cytokines, detect phosphorylated signaling proteins and evaluate calcium flux. In microbiology, it is employed to assess bacterial viability, monitor yeast fermentation, and enumerate phytoplankton in environmental samples. High-throughput flow cytometry facilitates screening of drug responses in cancer cells and evaluation of vaccine-induced immunity. Sperm analysis and stem cell enumeration before transplantation are additional clinical applications. The ability to rapidly analyze large numbers of cells with multiparameter readouts makes flow cytometry an indispensable tool in biology and medicine.
By combining fluidics, optics and electronics, flow cytometry provides detailed single-cell information that informs diagnostics, basic research and bioprocess monitoring.
Related Terms: FACS, Immunophenotyping, Cytometry, Laser, Fluorescence