Flow Cytometry involves the use of a beam of laser light projected through a liquid stream that contains cells, or other particles, which when struck by the focussed light give out signals which are picked up by detectors. These signals are then converted for computer storage and data analysis, and can provide information about various cellular properties.
The term "flow cytometry" derives from the measurement (meter) of single cells (cyto) as they flow past a series of detectors. The fundamental concept is that cells flow one at a time through a region of interogation where multiple biophysical properties of each cell can be measured at rates of over 1000 cells per second. These biophysical properties are then correlated with biological and biochemical properties of interest. The high through-put of cells allows for rare cells, which may have inherent or inducible differences, to be easily detected and identified from the remainder of the cell population.
In order to make the measurement of biological/biochemical properties of interest easier, the cells are usually stained with fluorescent dyes which bind specifically to cellular constituents. The dyes are excited by the laser beam, and emit light at a longer wavelengths. This emitted light is picked up by detectors, and these analogue signals are converted to digital so that they may be stored, for later display and analysis.
Many larger flow cytometers are also "cell sorters", instruments which have the ability to selectively deposit cells from particular populations into tubes, or other collection vessels. These selected cells can then be used for further experiments, cultured, or stained with another dye/antibody and reanalysed.
In order to sort cells, the instruments electronics interprets the signals collected for a each cell as it is interogated by the laser beam, compares the signal with sorting criteria set on the computer. If the cell meets the required criteria, an electrical charge is applied to the liquid stream which is being accurately broken into droplets containing the cells. This charge is applied to the stream at the precise moment the cell of interest is about to break off from the stream, then removed when the charged droplet has broken from the stream. As the droplets fall, they pass between two metal plates, which are strongly positively or negatively charged. Charged droplets get drawn towards the metal plate of the opposite polarity, and deposited in the collection vessel, or onto a microscope slide, for further examination. Computer Analysis: Following aquisition of data on the flow cytometer, analysis is undertaken to find out how many cells from the sampled population meet a criteria of interest. For example, does a population of cells express one molecule, but not another. The data can be displayed in a number of different formats, each having advantages and disadvantages. The common methods are histogram, or dotplots.
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