Everything You Never Wanted to Know About Polarization -But Were Afraid You Might Find Out
Most practitioners of cytometry, whether "flow jocks" or more casual users ("Joe and/or Josie Three-Color"), measure the intensity of scattered light and fluorescence emission in one or more selected wavelength regions, and pay no attention to the degree or direction of polarization of that light. Most of the time, we have gotten away with it. However, the article by Asbury, Uy, and van den Engh in this issue of Cytometry provides incontrovertible evidence that the time has come to pay more attention to what may be the biggest skeleton in our cytometric closet.
Because the light emitted by most lasers used as light sources in cytometry is polarized, the light scattered by, and the fluorescence emitted by, cells are also polarized. Scattering and emission occur in many directions, over a large range of angles to the incident beam; the polarized nature of the scattered and emitted light makes the intensity of detected signals more dependent on the angle and direction at which they are detected than would otherwise be the case. Differences from instrument to instrument in optical geometry, and in the polarization response of optical elements such as lenses, dichroics, and filters, may therefore lead to otherwise inexplicable differences in the intensity of signals measured from supposedly identical cells or particles. Further complications may be introduced by the fact that different fluorescent probes exhibit differing degrees of fluorescence polarization, some intrinsic to the molecular structure of the probes,