Multistation multiparameter flow cytometry: Some influences of instrumental factors on system performance

In an effort aimed at design of a flow cytometer/sorter system in which light scattering, fluorescence, and extinction measurements could be made in as many as five spatially separated illuminating beams, the performance of different mechanical components, light sources, illumination and collection optics, and flow chambers was compared by changing individual components while maintaining the same basic instrument configuration. A protocol was developed, and is here described, allowing rapid setup of a flow cytometer using a mercury arc lamp and two laser beams for illumination. When two or more illuminating beams are derived from a single laser, it is generally necessary to incorporate automatic gain control in the detector electronics to compensate for source intensity fluctuations and so to achieve acceptable measurement precision. It is, however, possible to use a single laser beam as a source for several flow cytometers without the need for such compensation. Mercury arc lamps provide adequate excitation in the near ultraviolet (uv) for DNA fluorochromes, such as Hoechst 33342, and may be preferable to ion lasers for this purpose. Reported differences in precision and sensitivity between stream‐in‐air and enclosed flow systems appear owing more to differences in the illumination and collection optics used in different instruments; five flow chamber designs gave equivalent performance when tested in the same instrument. Relatively inexpensive achromatic microscope objectives of numerical aperture (NA) approximately 0.5 may offer the best combination of depth of focus and light gathering power when used as fluorescence collection lenses for flow cytometers; image plane apertures improve system performance in fluorescence measurement.