A beam of light (usually laser light) of a single wavelength is directed onto a hydrodynamically-focused stream of liquid. 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 or SSC).
Each suspended particle from 0.2 to 150
micrometers passing through the beam scatters the ray, and fluorescent
chemicals found in the particle or attached to the particle may be excited into
emitting light at a longer wavelength 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, it is then possible to derive various types of information
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 roughness). This is because the light is scattered off
of the internal components of the cell. Some flow cytometers on the market have
eliminated the need for fluorescence and use only light scatter for
measurement.(5.)
Fluorescent substances absorb light of an appropriate wavelength and re-emit light of a different wavelength. Fluorescein isothiocyanate (FITC), Texas red, and phycoerythrin (PE) are the most common fluorescent dyes used in the biomedical sciences. Light and/or fluorescence scatter signals are detected by a series of photodiodes and amplified. Optical filters are essential to block unwanted light and permit light of the desired wavelength to reach the photodetector. The resulting electrical pulses are digitized, and the data is stored, analyzed, and displayed through a computer system.
The end result is quantitative information about every cell analyzed. Since large numbers of cells are analyzed in a short period of time (>1,000/sec), statistically valid information about cell populations is quickly obtained.(6.)
Fluorescent substances absorb light of an appropriate wavelength and re-emit light of a different wavelength. Fluorescein isothiocyanate (FITC), Texas red, and phycoerythrin (PE) are the most common fluorescent dyes used in the biomedical sciences. Light and/or fluorescence scatter signals are detected by a series of photodiodes and amplified. Optical filters are essential to block unwanted light and permit light of the desired wavelength to reach the photodetector. The resulting electrical pulses are digitized, and the data is stored, analyzed, and displayed through a
computer system.
The end result is quantitative information about every cell analyzed. Since large numbers of cells are analyzed in a short period of time (>1,000/sec), statistically valid information about cell populations is quickly obtained.(6.)
A technology to COUNT & SORT cells (7.)
This technique can be best understood by visualizing.(8.) (The video gives a brief description on principle of flow cytometry.)
References :
5. Principle - http://en.wikipedia.org/wiki/Flow_cytometry#Principle
8. Video - Youtube - http://www.youtube.com/watch?v=3kkXzPaayAk&feature=related
