Fig. 1.

Fluorescence photobleaching and recovery, FPR. The top panels diagram a portion of the cell surface labeled with a fluorescence probe. A focused laser beam defines a spot, typically 1 µm². The attenuated beam measures the molecules (gray dots) in the spot in terms of their fluorescence, F_pre. At t = 0, the attenuator is removed for a few msec and high-intensity laser light bleaches some of the molecules in the spot (white dots) so fluorescence is decreased to F₀. The attenuator is then returned, and the weak laser beam follows recovery of fluorescence in the spot in terms of increasing fluorescence as bleached molecules diffuse out of the spot and unbleached, fluorescent, molecules diffuse in. The graph of an FPR experiment is shown in the lower panel. Fluorescence recovers to some asymptote, F_∞, which is usually less than F_pre. Diffusion coefficients are calculated from the spot area and the time, t½, taken for fluorescence to recover to half of F_∞. Mobile fractions, R, are calculated as R = F_∞ − F₀/F_pre − F₀. A review of FPR is in [33]