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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. FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughtness). Measurable parameters are:
This list is very long and constantlty expanding.
Flow cytometers
Modern flow cytometers can analyse several thousand particles every second in "real time" and can actively separate out and isolate particles having specified properties. A flow cytometer is similar to a microscope, except high-throughput automated quantification of the set parameters for high number of single cells during each analysis session. Solid tissues have to be prepared to a single-cell suspension for analysis. An apparatus has 5 main components:
Earlier flow cytometers were more experimental devices, but nowdays there is considerable market of them as well as reagents for analysis (MAb's, chemicals). Each aparatus of different manufacture has it's specialities. Major manufacturers and their brands:
The technology has applications in a number of fields, including molecular biology, pathology, immunology 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. It is have broad application in medicine (especially in transplantation, heamatology, tumor immunology and chemotherapy, genetics).
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 by their phenotype.
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. Because of overlaping fluorescent dyes emission spectrum generated interfering signals by detectors signals have to be compensated electronicaly.