Measurement of restricted rotational diffusion of fluorescent lipids in supported planar phospholipid monolayers using angle‐dependent polarized fluorescence photobleaching recovery

Abstract

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 2ω. The magnitude and apparent rate of the PFPR postbleach fluorescence curves are a function of both ω and the angle between the bleaching and observation beam polarizations ψ. It is shown that estimates of the degree of rotational restriction ω may be obtained from changes in the ψ‐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_ψ(0) and final F_ψ(∞) postbleach fluorescence intensities varied as a function of ψ, and no measurable change in the postbleach fluorescence intensities was observed for ψ = 45°. Using the theory for restricted rotational motion, the ψ‐dependent variations of the final fluorescence intensities F_ψ(∞) obtained at two bleaching intensities gave an average apparent ω ≈︁ 52°. However, to adequately fit the F_ψ(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_ψ(0) and F_ψ(∞) data were obtained when the fluorophores were assumed to rapidly wobble within a cone of semiangle δ ≈︁ 30°–50° while slowly rotating within an angular space defined by semiangle ω ≈︁ 35°–60°. Subsequent analysis of the time‐ and ψ‐dependent changes in the postbleach fluorescence curves F_ψ(t) gave apparent diffusion coefficients ranging from D ≈︁ 10⁻³ s⁻¹ to 4 × 10⁻² s⁻¹. © 1993 John Wiley & Sons, Inc.