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 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.

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy

“…Fit parameters were constrained (Eq. 14) by using the steady-state anisotropy of 0.19 (data not shown). Considering the uncertainties about a randomly oriented fraction (Axelrod, 1979) and a collective lipid tilt and/or wobbling motion (discussed in Timbs and Thompson, 1993), there is clearly reasonable agreement with the previous studies.…”

Single-Molecule Anisotropy Imaging

“…The molecular rotation about a central axis was described by a Gaussian distribution of rotation angles of mean 0 and width, 2 ϭ 2D rot t (Chandrasekhar, 1943), characterized by a rotational diffusion constant, D rot , and the time between absorption and emission of a photon, t. It was further assumed that the rotation could take place only about the membrane normal, i.e., in one dimension. This assumption is rationalized by former findings, which showed that Ͼ88% of such fluorophores align their transition dipole moments perpendicular to the membrane (Axelrod, 1979;Lieberherr et al, 1987;Timbs and Thompson, 1993). An initial decrease in the anisotropy due to a differ- ence in the direction of the transition dipole moments in absorption and emission, respectively, was neglected because of the high measured anisotropy here in DPPC membranes and that found in other references reporting the anisotropy of the fluorophores in viscous media (Timbs and Thompson, 1990;Schaffer et al, 1999).…”

“…From the latter values, a two-dimensional anisotropy, r ϭ (I p Ϫ I s )/(I p ϩ I s ), of 0.37 Ϯ 0.07 was determined. The twodimensional anisotropy has been taken here because it is known for fluorophores like rhodamines that they orient with their molecular plane parallel to the surface of a membrane (Axelrod, 1979;Lieberherr et al, 1987;Timbs and Thompson, 1993). From the definition of the anisotropy given above, the mean two-dimensional anisotropy on linear excitation, for a molecule rotating slowly with respect to its excited-state lifetime (a few nanoseconds) but fast with respect to the total time of observation (10 ms here), is r ϭ 0.5.…”