Polarized fluorescence photobleaching recovery for measuring rotational diffusion in solutions and membranes.
A variation of fluorescence photobleaching recovery (FPR) suitable for measuring the rate of rotational molecular diffusion in solution and cell membranes is presented in theory and experimental practice for epi-illumination microscopy. In this technique, a brief flash of polarized laser light creates an anisotropic distribution of unbleached fluorophores which relaxes by rotational diffusion, leading to a time-dependent postbleach fluorescence. Polarized FPR (PFPR) is applicable to any time scales from seconds to microseconds. However, at fast (microsecond) time scales, a partial recovery independent of molecular orientation tends to obscure rotational effects. The theory here presents a method for overcoming this reversible photobleaching, and includes explicit results for practical geometries, fast wobble of fluorophores, and arbitrary bleaching depth. This variation of a polarized luminescence "pump-and-probe" technique is compared with prior ones and with "pump-only" time-resolved luminescence anisotropy decay methods. The technique is experimentally verified on small latex beads with a variety of diameters, common fluorophore labels, and solvent viscosities. Preliminary measurements on a protein (acetylcholine receptor) in the membrane of nondeoxygenated cells in live culture (rat myotubes) show a difference in rotational diffusion between clustered and nonclustered receptors. In most experiments, signal averaging, high laser power, and automated sample translation must be employed to achieve adequate statistical accuracy.
Velez
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Biophysics Research Division, University of Michigan, Ann Arbor 48109.
Axelrod
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D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D004058:Diffusion; D007700:Kinetics; D049449:Luminescence; D008433:Mathematics; D008567:Membranes, Artificial; D008962:Models, Theoretical; D009132:Muscles / Q000502:physiology; D010777:Photochemistry; D051381:Rats; D011950:Receptors, Cholinergic / Q000502:physiology; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*
D008567:Membranes, Artificial; D011950:Receptors, Cholinergic; D012996:Solutions