Thursday, April 23, 2009

Flow cytometry

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Analysis of a marine sample of photosynthetic picoplankton by flow cytometry showing three different populations (Prochlorococcus, Synechococcus and picoeukaryotes)
Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus.
1 History
2 Principle of flow cytometry
3 Flow cytometers
4 Fluorescence-Activated Cell Sorting
5 Fluorescent labels
6 Measurable parameters
7 Applications
8 See also
9 Bibliography
10 References
11 External links
The first fluorescence-based flow cytometry device (ICP 11) was developed in the year 1968 by Wolfgang G?hde from the University of M榛眘ter, Germany (Patent No. DE1815352) and first commercialized in 1968/69 by German developer and manufacturer Partec through Phywe AG in G?ttingen. At that time absorption methods were still widely favored by other scientists over fluorescence methods [1]. The original name of the flow cytometry technology was pulse cytophotometry (German: Impulscytophotometrie). Only 10 years later in 1978, at the Conference of the American Engineering Foundation in Pensacola, Florida, the name was changed to flow cytometry, a term which quickly became popular. Subsequently introduced flow cytometry instruments have been the Cytofluorograph (1971) from Bio/Physics Systems Inc. (later: Ortho Diagnostics), the PAS 8000 (1973) from Partec, the first FACS instrument from Becton Dickinson (1974), the ICP 22 (1975) from Partec/Phywe and the Epics from Coulter (1977/79
Principle of flow cytometry
A beam of light (usually laser light) of a single wavelength is directed onto a hydro-dynamically 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 from 0.2 to 150 micrometers passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a higher 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 (one for each fluorescent emission peak) 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). Some flow cytometers on the market have eliminated the need for fluorescence and use only light scatter for measurement. Other flow cytometers form images of each cell's fluorescence, scattered light, and transmitted light.
Flow cytometers
Modern flow cytometers are able to analyse several thousand particles every second, in "real time", and can actively separate and isolate particles having specified properties. A flow cytometer is similar to a microscope, except that instead of producing an image of the cell, flow cytometry offers "high-throughput" (for a large number of cells) automated quantification of set parameters. To analyze solid tissues single-cell suspension must first be prepared.
A flow cytometer has 5 main components:
a flow cell - liquid stream (sheath fluid) carries and aligns the cells so that they pass single file through the light beam for sensing.
an optical system - commonly used are lamps (mercury, xenon); high power water-cooled lasers (argon, krypton, dye laser); low power air-cooled lasers (argon (488nm), red-HeNe (633nm), green-HeNe, HeCd (UV)); diode lasers (blue, green, red, violet) resulting in light signals.
a detector and Analogue-to-Digital Conversion (ADC) system - generating FSC and SSC as well as fluorescence signals from light into electrical signals that can be processed by a computer.
an amplification system - linear or logarithmic.
a computer for analysis of the signals.
Early flow cytometers were generally experimental devices, but recent technological advances have created a considerable market for the instrumentation, as well as the reagents used in analysis, such as fluorescently-labeled antibodies and analysis software.
Modern instruments usually have multiple lasers and fluorescence detectors (the current record for a commercial instrument is 4 lasers and 18 fluorescence detectors). Increasing the number of lasers and detectors allows for multiple antibody...(and so on)

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