Effects of multiple membranes on measurements of cell surface dynamics by fluorescence photobleaching.

Fluorescence photobleaching recovery curves on a pair of membranes at various separations were calculated from a detailed knowledge of the variation in relative illumination areas and intensities as well as in relative contributions to the collected intensity with membrane separation. The observed diffusion coefficients were found to be relatively insensitive to membrane separation in all cases. Only for membranes with very different mobilities are there significant differences in fractional recoveries. Systematic variations in fractional recoveries with cell thickness may be indicative of differential mobility in the apical and basal membranes.