Measurement of scattered light and fluorescenceĪs well as measuring forward and side scattered light from all cells or particles in a sample, fluorescence detectors within the flow cytometer measure the fluorescence emitted from positively stained cells or particles. The type and order of dichroic filters allow the simultaneous detection of multiple signals. A dichroic filter acts as a mirror when placed at an angle, allowing specific wavelengths to pass through while others are reflected. Optical filters block certain wavelengths and let others pass.Detectors are known as photomultiplier tubes (PMTs) and will only detect fluorescence at a specific wavelength. For the instrument to detect the specific wavelength emitted by a fluorophore, the emitted light is passed through a series of mirrors and filters until it reaches the appropriate detector.If a cell is fluorescently labeled, the laser excites the fluorophore, and the emitted light is collected as fluorescence intensity. For each event, forward scatter and side scatter are subsequently recorded. As each cell passes the laser, the instrument records it as an event.Hydrodynamic focusing uses a controlled fluid flow to focus the sample into a narrow diameter, causing the cells to separate and align in a single file. When a sample is introduced into the multicolor flow cytometer flow chamber, it enters the fluidics system and separates into single cells in a process known as hydrodynamic focusing.Overview of basic multicolor flow cytometry technology Understanding the instrumentation basics The flow cytometerįigure 2. Measurement of scattered light and fluorescence.Measurement of forward and side scattered light.Understanding the instrumentation basics.If you are looking to get to grips with flow cytometry analysis, check out our free online flow cytometry training. Multicolor flow cytometry takes this further by analyzing multiple parameters on thousands of single cells or other particles in seconds 2,3. In multicolor flow cytometry, fluorescent markers are used to characterize and define different cell types of interest in heterogeneous cell populations, assess the purity of isolated subpopulations, and analyze cell size and shape. The cells are then incubated in tubes or microtiter plates with unlabeled or fluorophore-labeled antibodies and analyzed on the flow cytometer. The staining procedure involves making a single-cell suspension from cell culture or tissue samples. Flow cytometry assays can also assess the purity of isolated subpopulations, analyze cell size and volume, and sort different cell populations, known as fluorescence-activated cell sorting (FACS) 1.Ī flow cytometry test is usually based on measuring fluorescence intensity produced by fluorescently labeled antibodies specific to proteins on or in cells or ligands that bind to specific cell-associated molecules, such as propidium iodide binding to DNA. Recent trends in flow cytometry technology include single cell spectroscopy, single cell mass spectrometry, imaging of individual cells in flow, and the development of small inexpensive flow cytometers and sorters.There are many applications of flow cytometry in research and diagnostics, including simultaneous multi-parameter analysis of single cells and characterizing and defining different cell types in heterogeneous cell populations. Hallmarks of flow cytometry are analysis speed, detection sensitivity, the ability to measure many parameters simultaneously, and the ability to sort individual cells. Cells may be live or fixed, depending on the application, and individual cells can often be physically sorted.įlow cytometry is generally used for the multiparameter analysis of individual cells in suspension, and is widely used in immunology and haematopathology. Other optical signals can be measured, including light scatter. In addition, cytometry measures a host of other cellular features and behavior including cell viability, morphology, proliferation, secretion, endocytosis and phagocytosis, to name just a few. In general, cytometry measures optical properties of cells, and most often uses fluorescence to measure specific antigen molecules using antibodies, intracellular ions using indicator dyes, and DNA and RNA using nucleic acid-specific probes. Popular examples are flow cytometry and image cytometry, which are primarily optical methods, but the term can apply to any methodology used to extract quantitative information from individual cells. Deputy Director of National Reference CenterĬytometry is the quantitative analysis of cells and cell systems.
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