In addition to light scattering, detecting fluorochromes provides information about cell surface markers on cells passing through the laser beam. The monoclonal antibodies (MoAbs) included in the stains used for flow cytometry are labeled with fluorescent markers, or "tags," called fluorochromes. These MoAbs will bind specifically to corresponding cell surface antigens and can emit light at measurable wavelengths due to their fluorescent "tags."
Since blood cells may have multiple surface antigens, a single cell can bind to more than one type of labeled MoAb, depending upon the MoAbs used in the staining reagents. Different combinations of antibodies can be used within the same staining tube as long as the fluorochromes will emit signals that can be separated from one another. Remember that compensation for spectral overlap may be necessary. For example, if two separate monoclonal antibodies are labeled with a red and orange fluorochrome, respectively. In that case, it is important to compensate for the closeness of the two colors on the color spectrum and to ensure that the red-tagged MoAb signal is captured as red and the orange-tagged MoAb signal is captured as orange (and red isn't registered as orange and vice versa).
In the image shown on the right, the cells were stained with MoAb-green fluorochrome and MoAb-orange fluorochrome. The cell shown expresses both antigens and antibodies bound to the respective antigens. In the clinical setting, flow cytometers can handle upwards of 4-8 fluorochromes or more, depending on the instrument used.
The laser beam excites the fluorochromes, emitting fluorescent signals. Filters and mirrors capture these signals and separate them from other fluorescent signals.
