Cell separations by flow cytometry

Flow cytometry is a means of measuring the physical and chemical characteristics of particles in a fluid stream as they pass one by one past a sensing point. The modern flow cytometer consists of a light source, collection optics and detectors, and a computer to translate signals into data. In effect, a flow cytometer can be described as a large and powerful fluorescence microscope in which the light source is of a highly specific wavelength, generally produced by a laser, and the human observer is replaced by a series of optical filters and detectors that aim to make the instrument more objective and more quantitative. As a cell passes through the laser beam, light is scattered in all directions, and also at this point any fluorochromes present on the cell are excited and emit light of a higher wavelength. Scattered and emitted light is collected by two lenses-one set in front of the light source and one set at right angles to it. By a series of beam splitters, optical filters, and detectors the wavelengths of light specific for particular fluorochromes can be isolated and quantitated-up to six fluorochromes can be measured in some flow cytometers. A simplified diagram of the optical setup for two-color analysis is shown in Fig. 1. The theory of operation of flow cytometers is well documented, and there are several good general books on the subject (1-3).