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Flow Cytometry

Flow Cytometry


Flow cytometry uses laser technology to analyse a heterogeneous population of different cell types, for example, blood. Using this method it is possible to gather a large amount of data about the population such as cell size, internal complexity and phenotype.

A flow cytometer is made up of a fluidic system which delivers the sample to the laser beam, lasers which direct a beam into the flowing sample, and a range of optics, which can guide and detect scattered and refracted light from the flow of particles. There is also a powerful computer system which analyses the results and presents them as a series of histograms for easy analysis.

Flow cytometers use the principle of hydrodynamic focusing to present one cell at a time from a sample into the laser beam. This is accomplished in most instruments by injecting the sample into a flowing stream of carrier fluid which then passes through a narrow aperture. This presents cells to the interrogation point.

The interrogation point is at the heart of the system. It is the point at which the cells cross the laser beam. Typically cells of between 1 and fifteen microns are easily detected. However, filters can also be applied which can analyse cells which lie on either side of this range.

Each cell is analysed by the way that light is scattered from it. An obscuration board is placed in front of the detectors to omit light which enters directly from the laser beam itself. All other light which is incident on the detectors is scattered from the cells. Forward scattered light is proportional to the cell size. The light is converted into a voltage pulse by the detectors, which in turn is plotted as a histogram. A small cell emits a small amount of forward scatter, which in turn creates a low voltage pulse. Similarly larger cells are responsible for a larger voltage pulse, hence a larger histogram. 
The internal complexity of the cell creates side scatter. If a cell is simple with very little internal granularity, there is little side scatter. If a cell is very granular in nature, then more of the focussed laser beam is scattered to the sides. Once again, this can be plotted on a histogram or more usefully, on a scatter plot representing the whole sample.

In this way, cells of the whole sample can be analysed. Other parameters can also be measured. Commonly, fluorophores attached to antibodies are used to define populations of cells within a sample. This is a very elegant and sophisticated method of cell analysis and can be used to rapidly define many different characteristics of a substance.