Polarization effects in flow cytometric DNA sizing

We have investigated the influence of the polarization direction of excitation Light on DNA sizing results obtained by a flow cytometric technique. We found strong fluorescence anisotropy of the fluorescent signal from lambda DNA stained with the bis-intercalating dye TOTO-1. Small fragments of DNA are less sensitive to polarization than larger pieces. This effect is more pronounced at faster flow speeds. These observations show a noticeable orientation of the DNA molecules introduced by the shear forces in the flow system. The data are consistent with an angle between the transition moment of fluorescence of TOTO-1, and the long axis of DNA is approximately 62 degrees. (C) 1999 Optical Society of America OCIS codes: 160.1190, 260.2510, 260.5430, 280.2490.