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Fluorescence recovery after photobleaching (FRAP) measurements offer animportant tool for analyzing diffusion and binding processes. Confocal scanninglaser microscopes that are used in FRAP experiments bleach regions with aradially Gaussian distributed profile. Previous attempts to derive analyticalexpressions in the case of processes governed by fast diffusion have overlookedthe characteristics of the instruments used to perform FRAP measurements andtherefore led to approximating solutions. In the present paper, bleaching laserbeam characteristics are incorporated into an improved model to provide a morerigorous and accurate method. The proposed model simulates binding insidebounded regions, and it leads to FRAP curves that depend on the on and offrates that can be employed to determine the rate constants. It can be used inconjunction with experimental data acquired with confocal scanning lasermicroscopes to investigate the biophysical properties of proteins in livingcells. The model aims to improve the accuracy when determining rate constantsby taking into account amore realistic scenario of the light-matterinteraction.

Quantitative interpretation of binding reactions of rapidly diffusing species using fluorescence recovery after photobleaching