Flow cytometers are used to rapidly count and identify bacteria, cells, and other biological particles. Although flow cytometers measure only one cell at a time, they are capable of processing thousands of cells in just a few seconds. The cells may be living or fixed at the time of study, but in either case they must be in a monodisperse (single cell) suspension. Flow cytometers function by passing cells through a laser beam in a continuous single-file stream. Each cell scatters some of the laser light, and also emits fluorescent light excited by the laser. The cytometer measures a number of factors based on scattering and emission and uses this data to differentiate between and count the types of cells in the mixture. Some of the main aspects measured include low angle forward scatter intensity, approximately proportional to cell diameter; orthogonal (90 degree) scatter intensity, approximately proportional to the quantity of granular structures within the cell; and fluorescence intensities at several wavelengths. This data creates a “histogram” of each cell.
Some flow cytometers are able to sort cells after they have been identified. This involves physically separating particles of desired types from other entities, and depositing them into tubes or collection plates for further study. The two most common sorting methods include stream-in-air sorting and the closed flow cell method. In the stream-in-air method, particle-containing fluid is passed through a nozzle and broken up into individual droplets by a piezoelectric transducer. This technique requires aerosol containment for work with biohazardous substances. Stream-in-air sorting is usually employed by high performance, high-speed analysis instruments. Closed flow cell sorting is a mechanical sorting method that has a lower sorting rate than the stream-in-air sorting. This method does not produce aerosol.
Flow cytometers are widely used in biomedical research, as well as environmental testing and monitoring. Some typical applications include sorting and analyzing human and other biological lymphocytes, stem cells, dendritic cells, macrophages, monocytes, tumor cells, and bacteria; drug discovery and testing, genetics, marine biology and limnology, livestock sex selection, and fetal cell separation.