Fitting diffusion coefficients and dissociation time constants to Gaussian bleach profiles. (A) Rationale behind the experiment: illustrations show a GFP-tagged protein targeted to the plasma membrane; the membrane-bound pool of protein is assumed to be at equilibrium with a smaller, cytosolic pool. After bleaching with a pulse of light from a laser beam with a Gaussian intensity profile (i), the fluorescence intensity profile along the membrane will reflect this profile and have a defined Gaussian radius r and depth B. Lateral diffusion of bleached molecules along the plane of the membrane causes a widening of the profile radius r, while maintaining area under the curve (ii); conversely, exchange with the cytoplasmic pool maintains the radius but leads to a decrease in the area under the curve (iii). The model described in Eq. 2 accounts for both processes. (B) Fluorescence micrographs before bleaching of HEK cells expressing either single or tandem PH domains from PLCδ1 fused to GFP, or PM-YFP. The enlarged images show the bleached area at indicated times after bleaching. Bar, 10 µm. (C) Membrane fluorescence intensity profiles at the indicated postbleach times for the PH-GFP cell shown in B; the lines represent fits produced from Eq. 2 (see Materials and methods). (D and E) Data points show the changes in gaussian radius r (D) or the area under the curve (E) from membrane intensity profiles fitted with Eq. 1; the lines show the predicted diffusion coefficients D and membrane dissociation times τ established by fits of these curves with Eq. 1.