Flow cytometry is an outstanding technology that provides the means to investigate particles, such as prokaryotic or eukaryotic cells, organelles, chromosomes etc. as being a individual entities. Each particle or cell can be examined for multiple parameters in both quantitative and qualitative manners within only few microseconds. Subsequent data analysis provides exact statistics pertaining to the sample and subpopulations may be distinguished. In addition, these (sub) populations can be physically separated for further study by Fluorecence Activated Cell Sorting (FACS).
In most flow cytometers measurement of a particle’s characteristics is performed as they move in single file, hydrodynamically focussed in a liquid sheath stream through one or more excitation light sources past a sensitive detection area. While passing this illumination point light is diffracted, refracted and reflected in three dimensional spaces. This scattered light is then collected and transformed into a current by photon detectors (e.g. P
ubes or Photodiodes). Depending on the angle at which the scatter is measured, the resultant value is proportional to one of several different characteristics.
In general light is measured at low angles between 0.5–10° (Forward Scatter) and at right angles of about 90° (side scatter).
Light scattered in the forward direction is proportional to the square of the radius of a sphere and thus to the size of the cell or particle of similar refractive index.
Scattered light measured at 90° may be considered proportional to the granularity, surface topography and intracellular complexity of a spherical particle.
In addition to this initial discrimination by their different light scatter properties, each particle associated light emission can be measured and separated by wavelength using dichroic mirrors and/or interference filters.
Specific Antibodies coupled to dyes with fluorescent properties can be used to detect molecules bound to, expressed by or incorporated inside the target particle to further explore population diversity.
In addition to fluorescently tagged antibodies there are other reagents available to detect particle properties for certain applications. Some ommon Applications of flow cytometry are measurement of protein expression and localization, apoptosis, total DNA/RNA content, cell cycle analysis, cell kinetics, proliferation, enzymatic activity, pH, intracellular ionized calcium, membrane potential and many more.