Measurement of restricted rotational diffusion of fluorescent lipids in supported planar phospholipid monolayers...

A theory describing the shapes of polarized fluorescence photobleaching recovery (PFPR) curves for a population of fluorophores undergoing restricted rotational diffusion in two-dimensional systems such as planar membranes has been developed. In this model, restricted rotational diffusion of the fluorophores is described by using reflective boundary conditions, in which the fluorophores are assumed to diffuse freely but only within an angular space of width 2 omega. The magnitude and apparent rate of the PFPR postbleach fluorescence curves are a function of both omega and the angle between the bleaching and observation beam polarizations psi. It is shown that estimates of the degree of rotational restriction omega may be obtained from changes in the psi-dependent postbleach fluorescence intensities. Using angle-dependent PFPR, slow rotational reorientations of the fluorescent lipid analogue 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine in distearoylphosphatidylcholine Langmuir-Blodgett monolayers deposited on octadecyltrichlorosilane-treated fused quartz were measured. As theoretically predicted for a rotationally restricted fluorophore population, both the initial F psi (0) and final F psi (infinity) postbleach fluorescence intensities varied as a function of psi, and no measurable change in the postbleach fluorescence intensities was observed for psi = 45 degrees. Using the theory for restricted rotational motion, the psi-dependent variations of the final fluorescence intensities F psi (infinity) obtained at two bleaching intensities gave an average apparent omega approximately 52 degrees. However, to adequately fit the F psi (0) data, inclusion of the theoretical effects of rapid (faster than the duration of the photobleaching pulse) fluorophore dynamics was also required. Best fits of the F psi (0) and F psi (infinity) data were obtained when the fluorophores were assumed to rapidly wobble within a cone of semiangle delta approximately 30 degrees-50 degrees while slowly rotating within an angular space defined by semiangle omega approximately 35 degrees-60 degrees. Subsequent analysis of the time- and psi-dependent changes in the post-bleach fluorescence curves F psi (t) gave apparent diffusion coefficients ranging from D approximately 10(-3) s-1 to 4 x 10(-2) s-1.