Flow cytometry

Flow cytometry is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid.

A beam of light (usually laser light) of a single frequency (colour) is directed onto a hydrodynamically focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors). Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals in the particle may be excited into emitting light at a lower frequency than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and by analysing fluctuations in brightness at each detector (one for each fluorescent emission peak) it is possible to deduce various facts about the physical and chemical structure of each individual particle.

Modern flow cytometers can analyse several thousand particles every second in "real time" and can actively separate out and isolate particles having specified properties.

The technology has applications in a number of fields, including molecular biology, pathology and marine biology. In the field of molecular biology it is especially useful when used with fluorescence tagged antibodies. These specific antibodies bind to antigens on the target cells and help to give information on specific characteristics of the cells being studied in the cytometer.

Modern instruments may have multiple lasers and fluorescent detectors (the current record for a commercial instrument is 4 lasers and 14 detectors)allowing multiple antibody labelling to be used to more precisely specify a target population.

Flow cytometers can also be configured as sorting instruments. As cells/particles pass through they can be selectively charged and on their exit can be deflected into separate paths of flow. It is therefore possible to separate up to 4 defined populations of cells from an original mix with a high degree of accuracy and speed (up to ~90,000cells per second in theory).

The data coming from flow-cytometers can be plotted in 1-D to produce histograms or seen in 2D as dot plots or in 3D with newer software. The regions on these plots can be sequentially separated by a series of subset extractions which are termed gates. Specific gating protocols exist for diagnostic and clinical purposes especially in relation to haematology. The plots are often made on logarithmic scales.

See also