This lecture answers the question, What is flow cytometry? It describes the principles underlying the technique, explains its advantages and disadvantages and outlines the major applications. The last part of the lecture introduces some of the concepts used in data analysis.
As its name implies, flow cytometry is the measurement of single cells in a flow system. The flow system delivers particles singly past a point at which a measurement is made.
The particles may be:
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Cells (mammalian, plant, algae, yeast, bacteria)
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Small beads (possibly fluorescent)
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Light is focused at point of measurement. The light source can be a:
A laser emits an intense beam of monochromatic light, that is, light at a single wavelength. The large majority of flow cytometers use an argon-ion laser emitting monochromatic blue light at 488 nm. The commonest laser is air-cooled and produces 15 mW of light. One or two additional lasers may be fitted; these are usually either a helium-neon laser producing red light at 630 nm or a helium-cadmium laser producing UV at 325 nm. Some cytometers are now being fitted with diode lasers – red laser emitting at 630 nm, a green laser emitting at 535 nm or a violet laser emitting at 405 nm.
If an arc lamp is used, the desired wavelength of light must be selected using an optical filter.
As the cells pass through the focused beam of light, we measure:
The cells (or other particles) may be autofluorescent or they may be labelled with one or more of a variety of fluorescent compounds.
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Light scattered by the cells
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The important feature is that measurements are made on single cells, individually.