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Flow Cytometry and Sorting

Flow cytometry (also known as FACS/ fluorescence activated cell sorting) is the science of examining physical and chemical properties of cells (or bacteria, particles etc.), usually labelled with fluorescent probes, as they pass in a fluid stream through a measuring apparatus. The technology uses laser excitation and signal detection for analysis applications such as fluorochrome-labelled antibody binding to surface receptors, intracellular proteins, cytokines, activation markers etc., apoptosis, cell cycle and ploidy analyses, cell tracking and proliferation studies, kinetics/calcium flux, membrane potential and pH analyses, along with many new and emerging applications. Size, morphology, granularity, and structural complexity can also be deduced based on laser light scatter. Flow cytometry offers a more objective, quantifiable analysis of cells than a fluorescence microscope, with more parameters on many more cells in a very short time.

Flow cytometry is an extremely powerful and versatile technology and has important applications in virtually all life sciences, research, clinical and diagnosis, being vital in such areas as immunology, biochemistry, cell biology, cancer and HIV research, drug discovery, as well as other disciplines such as marine and microbiology etc.

Flow cytometry operates through a system whereby cells (or particles etc.) in suspension enter into a slow moving sample stream which is then injected into a faster moving sheath stream. Laminar flow and surface tension cause this sample stream to be whisked into the sheath stream where it is focused in the centre and the cells flow at high speeds in single file. This phenomenon is known as hydrodynamic focusing, and occurs in the flow cell. Here, one or more laser beams transect the stream and when cells pass this interrogation point, emitted photons are detected by a detector and this signal is converted to an electronic, and then digital, signal which can be acquired and analysed.

As a cell passes through the laser, it refracts or scatters light in every direction. Forward Scatter is the light scattered in the forward direction as laser light strikes the cell. The intensity of forward scatter signal is roughly proportional to the size of the cell. This scattered light is quantified by a photodiode detector that converts intensity into voltage. An obscuration bar in front of the forward scatter detector prevents any of the intense laser light from reaching the detector directly. As a cell passes the laser, light is scattered around the obscuration bar and is collected by the detector.

Light scattering at larger angles, and usually focussed through a lens system and collected by a detector at 90^o from the laser path, is caused by granularity and structural complexity inside the cell, and is commonly known as Side Scatter. Fluorescent molecules/ dyes can be added to the cell sample and if the molecule or dye binds to the cell, the cell will fluoresce. When laser light of the appropriate wavelength strikes the fluorochrome on the cell, a fluorescent signal is emitted and directed through a series of filters and mirrors, and detected by the appropriate detector (usually a photomultiplier tube). The more fluorescence bound to the cell, the more intense the signal generated, and hence the greater the output voltage. As each fluorochrome has a characteristic spectrum, more than one fluorochrome can be used at a time, and additional lasers may be added to the system.

Some flow cytometers are equipped to physically isolate cells from a heterogenous population, based on scatter and fluorescence characteristics. In this system, the stream will form droplets as the sample and sheath fluid are injected into air from the nozzle. By vibrating the nozzle at a defined frequency, the size and position of these droplets along the stream can be controlled. The sheath fluid is charged and these droplets can be charged and deflected downstream of laser excitation whereby a magnetic charge is added to the cell-containing droplet and the population of interest is diverted with magnetic-charge plates, and collected into a receptacle (5, 15, 50 ml tubes/ micro titre plates/ slides etc.). Up to four different populations can be sorted at once at ultra high speeds, and the population can be enriched or purified for down-stream applications, as well as single cell clone generation etc.