Correlation between Steady-State and Time-Resolved Fluorescence Anisotropy Data

Abstract

The most common fluorescence depolarization measurement is a steady-state experiment. Continuous illumination with monochromatic polarized light is used to excite fluorescent probes embedded in the lipid regions of the membrane sample. One measures the fluorescence intensities parallel (I‖) and perpendicular (I⊥) to the polarization direction of the excitation light. The relevant parameter is the steady-state fluorescence anisotropy (FA), defined as $${r_s} = \left( {{I_\parallel } - {I_ \bot }} \right)/\left( {{I_\parallel } + 2{I_ \bot }} \right)$$ (1) Here we are concerned with the FA of the probe 1,6-diphenyl-1,3,5hexatriene (DPH) in a membrane system which is macroscopically isotropic. In that case we have 0 ≤rs ≤ro; theoretically ro equals 0.4, experimental values lie between 0.362 [1] and 0.395 [2]. The steady-state FA, rs, reflects the hindrance of the probe rotation; rs = ro means that the probes do not rotate within the fluorescence lifetime and rs = 0 means that the probe molecules rotate rapidly and without restriction within that time.