Fluorescence Photobleaching Recovery Experiments Research Articles

Fast diffusion along defects and corrugations in phospholipid P beta, liquid crystals

The diffusion of a fluorescent lipid analogue in liquid crystals of the anisotropic P beta, phase of dimyristoylphosphatidylcholine (DMPC) had been found to be highly variable, suggesting structural defect pathways. Fluorescence photobleaching recovery (FPR) experiments imply two effective diffusion pathways with coefficients differing by at least 100. This is consistent with fast diffusion along submicroscopic bands of disordered material ("defects") in the bilayer corrugations characteristic of this phase. Due to strains during transformation from the L alpha phase, the axis of the corrugations is ordinarily disrupted by mosaic patches rotationally disoriented within the mean plane of the molecular bilayers, although larger oriented domains are sometimes adventitiously aligned into microscopically visible striped textures. The corrugations are also systematically aligned along positive disclinations pairs or "oily streaks." Thus, fast diffusion occurs parallel to the disclination lines and along the textured stripes. FPR results yield an upper limit on the effective diffusion in the ordered material of D less than or equal to 2 X 10(-16) cm2/s at 22 degrees C, D less than or equal to 3 X 10(-17) cm2/s at 13 degrees C. In contrast the diffusion coefficient along defect pathways where disordered ribbons are aligned is D approximately 4 X 10(-11) cm2/s at 16 degrees C.

Theoretical analysis of fluorescence photobleaching recovery experiments

We derive an exact closed formula for the fluorescence recovery curve measured in fluorescence photobleaching recovery experiments employing uniform circular laser beams. In contrast to the expression used currently, this result is very simple and free of mathematical drawbacks, thus facilitating the quantitative analysis of experimental data.

Polymerization-induced changes in the fluorescence of actin labeled with iodoacetamidotetramethylrhodamine

Rabbit skeletal muscle actin has been labeled with the fluorescent sulfhydryl reagent iodoacetamidotetramethylrhodamine (rhodamine). The label is probably located on Cys-373 because prior treatment of the actin with N-ethylmaleimide prevents incorporation of rhodamine. When the rhodamine-actin is polymerized with MgSO 4 or KCl, there is approximately a 1.5-fold increase in fluorescence. The change in fluorescence is correlated with incorporation of monomeric globular actin into polymer and can therefore be used as a quantitative measure of polymerization. Trace quantities of rhodamine-actin can be used to follow the kinetics of polymerization of unlabeled actin without disturbing the sample, and it is shown that the use of a capillary viscometer accelerates the rate of polymerization. Fluorescence photobleaching recovery experiments, which measure the diffusion of rhodamine-labeled actin, show that nondiffusible (apparent diffusion coefficient < 10 −10 cm 2/s) filaments appear during the polymerization process but that immediately after shearing these filaments are readily diffusible. These results demonstrate the destructive nature of hydrodynamic shear stress on polymerized actin.

Effect of bleaching light on measurements of lateral diffusion in cell membranes by the fluorescence photobleaching recovery method.

The Fc receptors for IgE on rat basophil leukemia 2H3 cells were labeled with either rhodamine- or fluorescein-conjugated IgE and then with Fab fragments of anti-IgE conjugated with the other dye. Fluorescence photobleaching recovery measurements of lateral diffusion were performed with one of the dyes before and after extensive bleaching of the other dye over an entire cell. Bleaching one dye did not affect subsequent measurements made with the other dye. We thus detect no evidence for photoinduced artifacts in fluorescence photobleaching recovery experiments.

Mobility measurement by analysis of fluorescence photobleaching recovery kinetics

Fluorescence photobleaching recovery (FPR) denotes a method for measuring two-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled molecules in approximately 10 mum2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. We present the theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments. Information obtainable from FPR experiments includes: (a) identification of transport process type, i.e. the admixture of random diffusion and uniform directed flow; (b) determination of the absolute mobility coefficient, i.e. the diffusion constant and/or flow velocity; and (c) the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, we describe some model experiments on aqueous solutions of rhodamine 6G.