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Flow Cytometry
Figure 1. Forward Angle Light Scatter (FSC) is the scattered light produced in the same axis as the laser light.
Flow cytometry is a powerful technique to simultaneously analyze multiple characteristics of thousands of individual cells in a relatively short period of time. Unlike other biochemical techniques, flow cytometry makes these multiparametric measurements on single cells as opposed to population measurements. By definition, cytometry means cell measurement (Cyto = Cell; Metry = measurement). Flow cytometry measures many different characteristics of cells as they move in a stream and are excited by a light source (laser). As cells are exposed to the laser, they scatter light in different directions and emit fluorescence. 
Figure 2. Side Light Scatter (SSC) is the light scattered to the side or perpendicular to the axis the laser light is traveling.
When scattered light is along the same axis as the laser light, it is detected in the forward scatter channel. Forward scatter tends to be more sensitive to surface properties of the cell, giving information about the cell size. Light scattered to the side or perpendicular to the axis the laser light is traveling is detected in the side scatter channel. Side scatter tends to be more sensitive to inclusions within the cell, giving information about the cell granularity. In addition to scattered light, fluorescence signals are created when cells that have been exposed to a fluorescent molecule intercept the laser beam. The excitation and emission spectrum is different for each flourophore, and it is important to know which laser and what filter the cytometer has in order to decide which flourochrome to use. 
Figure 3. List of the most common laser lines used in flow Cytometry and some commonly used fluorescent probes.
There are many different types of fluorescent probes commercially available. There are fluorescent probes that interact directly with cellular components, such as fluorochromes that bind directly to DNA, RNA, or intracellular ions. There are also molecules that are attached to other probes, which provide specificity, such as fluorochromes attached to monoclonal antibodies or biological ligands. In some cases, a fluorescent molecule can be produced by the cell as a reporter, exemplified by the green fluorescent protein. Multiple probes with different excitation (requiring more than one laser wavelength) and/or different emission wavelengths can be combined to provide multiparameter resolution for an individual sample. The molecules measured may be on the cell surface, in the cytoplasm, nucleus, or other intracellular organelle, depending on the specificity of the probe. Flow cytometry also permits the physical isolation of various subpopulations of cells based on specific cellular characteristics by electrostatic separation. Four distinct populations of cells can be simultaneously sorted for a given sample. Cell sorting can be performed under sterile conditions, and the cells can be collected into various types of containers, including eppendorf tubes or microscope slides. |
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