Polarization of scatter and fluorescence signals in flow cytometry.

BACKGROUND The pulses of light scatter and fluorescence measured in flow cytometers exhibit varying degrees of polarization. Flow cytometers are heterogeneously sensitive to this polarization, depending on the light source(s), the optical layout, and the types of mirrors and filters used. Therefore, fluorescence polarization can affect apparent intensity ratios between particles and interfere with schemes for interlaboratory standardization. METHODS We investigate the degree to which polarization affects common flow cytometry measurements. Our technique for determining polarization differs from previous methods because complete distributions of intensity versus polarization angle are measured, rather than intensities at just two orthogonal polarization angles. Theoretical models for scatter and fluorescence are presented and verified by making polarization measurements of calibration beads. RESULTS Measurements of cells stained with a variety of dyes illustrate that fluorescence polarization occurs frequently in flow cytometry. CONCLUSIONS Consequences for quantitative cytometry are discussed, and the use of the "magic angle" to make a flow cytometer insensitive to fluorescence polarization is proposed.