Principles of fluorescence polarization measurements.

Fluorescence polarization measurements using high-speed single-cell flow cytometry have found increasing use in cellular biology. In most flow systems, the detection axis generally is aligned orthogonally to the direction of flow and the excitation axis, and asymmetric apertures along the excitation and emission axes by cylindrical lenses and/or nonplanar transition between optical indices complicate the numerical correction of systematic depolarization effects. In addition, recent studies on fluorescence emission of structured particles have shown remarkable anisotropies of polarized fluorescence emission dependent on the direction of the detection axis. To deal with these problems, we proposed the detection of fluorescence emission in a backward direction, known as epi-illumination, together with an optical design of axial symmetry. The flow chamber maintains axial symmetry by an optical index match to the plane window. Internal corrections for equal sensitivity of both detector channels is achieved by paired runs with orthogonally changed polarization of the exciting beam. Asymmetries are numerically corrected using a minicomputer. This also allows a check on the intensity dependence of fluorescence anisotropy. For high-resolution measurements of fluorescence polarization, an on-line calculation may be necessary. Since light scattering on the excitation side as well as on the emission side in a structured particle or cell is a major contribution to depolarization, system performance should also be probed by using structured particles. We thus introduced green algae (Chlorella vulgaris) as test objects for highly depolarized fluorescence emission and fluorescent polystyrene microspheres as test objects to probe polarized emission