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Flow Cytometry is the quantitative analysis of cells and cell systems - measuring things like cell size, the number of cells, cell shape, and structure, types of proteins etc. Flow cytometry uses a fluid suspension, commonly known as sheath fluid, to analyze individual cells and simultaneously measure multiple parameters of each individual cell. Thousands of cells per second can be analyzed as they pass through the liquid suspension. This allows researchers to generate large datasets from samples can be small and limiting.
The purpose of the optical system is to illuminate the cells or particles present in the sheath fluid using lasers. Lasers are used in flow cytometers because of two main reasons - lasers are coherent and monochromatic sources of light. This means that a very narrow wavelength can be specified and the light output will always be in phase. These two properties of lasers are critical for flow cytometry. The sheath fluid flows at a high stream velocity, therefore it is critical that the laser illumination spot be very small and focused, to illuminate a cell or particle.
Another reason for using lasers is that their output power, which essentially measures of how many photons are output per unit time, is usually adjustable. Flow cytometer lasers typical range in power from 20mW to 100mW. plasma allowing for the smaller footprint of cytometers, low power requirements, and short warm-up periods.
The most common wavelengths of lasers used in flow cytometry are 355 nm, 375 nm, 405 nm, 488 nm, 532 nm, 561 nm, and 640 nm. The lasing medium in these lasers is solid, as opposed to a gas or plasma allowing for the smaller footprint of cytometers, low power requirements, and short warm-up periods.
When the laser of a specific wavelengths strikes a cell, the light is either reflected by the cell or it is absorbed. Depending on the response to the laser, various properties of the cell can be analyzed. Laser light reflected from a cell can either follow a forward scatter or a side scatter. Forward scattered (FSC) light is refracted by a cell in the same direction that the light was originally traveling. Side scattered (SSC) light is refracted by cells in a direction that is different from the original light path
If a cell has fluorescent properties or is "tagged" with fluorescent dyes, when laser light is shown on them, they absorb the energy of the light and "fluoresce" at a higher wavelength. E.g. fluorescein isothiocyanate (FITC) is a dye that efficiently absorbs light from a blue (488nm) laser and produces a green (525nm) emission while phycoerythrin (PE) also absorbs light from a blue (488nm) laser but produces an orange (575nm) emission.