Flow cytometry is a technology that measures and analyzes the optical properties of mono-dispersed single particles, such as cells, bacteria, picoplankton, microbeads, yeast, platelets, nuclei and other similarly-sized particles, passing single file through a focused laser beam. The laser can excite fluorophores that have been used to mark various molecules or physiological functions of the particles. The use of fluorophores with different fluorescence characteristics, multiple lasers and multiple photo detectors allows flow cytometers to measure many characteristics of each particle simultaneously. An important feature of flow cytometry is that large numbers, for example thousands of particles per second, are analyzed and therefore provide a statistically significant picture of a specimen's physical and biochemical make-up. To perform these functions, cytometers requires a combined system of:
- Fluidics that introduces and restricts the particles for interrogation.
- An excitation source and collection optics to generate and collect the light signals.
- Electronics that convert the optical signals to proportionate electronic signals and digitize them for computer analysis.
A further strength of flow cytometry, unlike fluorescent microscopy, is that it is able to distinguish between multiple closely emitting fluorophores, such as eGFP and eYFP, in concurrently expressing cells if the appropriate optics and experimental controls exist. If sort criteria have been set up, some flow cytometers can physically sort selected sub-populations. Optical signals consisting of laser light scatter and fluorescence are sequentially generated by each single particle and consist of the following:
- Low angle forward laser light-scatter (FSC) intensity, approximately proportional to cell diameter
- Side (also known as orthogonal, 90 degree) laser light-scatter (SSC) intensity, approximately proportional to the quantity of granular structures within the cell
- Fluorescence intensities at many wavelengths (FL1, FL2, FL3, etc.)
Common applications of flow cytometry include the analysis and/or sorting of experiments studying:
- Apoptosis (intracellular homeostasis, free radical production, chromatin condensation, DNA fragmentation, plasma membrane integrity, mitochondrial function, caspase activation, phosphatidylserine translocation, etc.)
- Cell viability
- Cellular signal transduction (calcium flux, cytokine studies, intracellular pH and glutathione measurements, cell cycle analysis, cellular transport assays, drug uptake/efflux assays, fluorescent resonance energy transfer [FRET])
- Characterization of multi-drug resistance (MDR) of cancer cells
- DNA (cell cycle analysis, cell kinetics, proliferation, etc.)
- Immunophenotyping (cell surface antigens, intracellular antigens, etc.)
- Transfection efficiencies, and many more.
There is a nice overview article of flow cytometry in Nature Methods.
A valuable forum for the discussion of many aspects of flow cytometry is the Cytometry Electronic Mailing List. To subscribe, contact email@example.com.