Pattern photobleaching of fluorescent lipid vesicles using polarized laser light

Abstract

A burst of linearly polarized laser radiation incident on a spherical lipid vesicle, liposome, or biological cell can produce a well-defined nonuniform distribution of membrane-bound fluorescent molecules, provided the absorption transition dipole moment of the fluorescent label has a nonrandom orientation relative to the membrane surface and can be photobleached by the laser radiation. The return (recovery) of fluorescent membrane-bound molecules to a uniform distribution can be monitored using the same polarized radiation source. Under appropriate conditions this recovery is characterized by a single exponential time constant tau. This time constant is related to the radius R of the vesicle and the lateral diffusion coefficient D of the fluorescent membrane-bound molecules by the equation R2 = 6D tau. In the case of vesicle membranes this result is not limited by diffraction and so should be applicable to vesicles whose radii are less than the wavelength of light. The above considerations are illustrated by the polarized light photobleaching-recovery of lipid vesicles containing a fluorescent lipid, N-4-nitro-benzo-2-oxa,1,3-diazole l-alpha-dimyristoylphosphatidylethanolamine (NBD-DMPE).