MOBILITY MEASUREMENT BY ANALYSIS OF FLUORESCENCE PHOTOBLEACHING RECOVERY KINETICS

Fluorescence photobleaching recovery (FPR) denotes a method for measuring 2-dimensional lateral mobility of fluorescent particles, e.g., the motion of fluorescently labeled molecules in .apprx. 10 .mu.m2 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. The theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments are presented. Information obtainable from FPR experiments includes identification of transport process type, i.e., the admixture of random diffusion and uniform directed flow; determination of the absolute mobility coefficient, i.e., the diffusion constant and/or flow velocity; and the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, some model experiments on aqueous solutions of rhodamine 6G are described.