Flow Cytometry Flow cytometry is a system for sensing individual cells in a physiologic saline solution as they move in a focused liquid stream through a fixed laser beam scattering light and emitting fluorescence that is measured and converted into digitized data.
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When a cell suspension is run through the cytometer, sheath fluid is used to hydrodynamically focus the cell suspension through a small nozzle. The tiny stream of fluid takes the cells past the laser light one cell at a time. Essentially, the fluidics system consists of a central channel/core through which the sample is injected, enclosed by an outer sheath that contains faster flowing fluid. As the sheath fluid moves, it creates a massive drag effect on the narrowing central chamber. This alters the velocity of the central fluid whose flow front becomes parabolic with greatest velocity at its center and zero velocity at the wall (see Figure). The effect creates a single file of particles and is called hydrodynamic focusing. Under optimal conditions (laminar flow) the fluid in the central chamber will not mix with the sheath fluid. Light scattered from the cells or particles is detected as they go through the laser beam. A detector in front of the light beam measures forward scatter (FS) and several detectors to the side measure side scatter (SS). Fluorescence detectors measure the fluorescence emitted from positively stained cells or particles.
Cells or particles passing through the beam scatter light, which is detected as FS and SS. FS correlates with cell size and SS is proportional to the granularity of the cells. In this manner, cell populations can often be distinguished based on differences in their size and granularity alone.
Figure left: Light scatter as the blue laser interrogates the cell. The direction of light scattered by the cell correlates to cell size and granularity.
When light hits a photodetector a small current (a few microamperes) is generated. Its associated voltage has an amplitude proportional to the total number of light photons received by the detector. This voltage is then amplified by a series of linear or logarithmic amplifiers, and by analog to digital convertors (ADCs), into electrical signals large enough (5–10 volts) to be plotted graphically.
Log amplification is normally used for fluorescence studies because it expands weak signals and compresses strong signals, resulting in a distribution that is easy to display on a histogram. Linear scaling is preferable where there is not such a broad range of signals e.g. in DNA analysis.
The measurement from each detector is referred to as a 'parameter' e.g. forward scatter, side scatter or fluorescence. The data acquired in each parameter are known as the 'events' and refer to the number of cells displaying the physical feature or marker of interest.
See Principle of "Fluorescence Measurement"