FRAP by total internal reflection. (A) Epifluorescence and TIR fluorescence micrographs of the adhesion planes (“footprints”) of HEK cells expressing either the myristoylated and palmitoylated sequence of Lyn kinase fused to YFP (PM-YFP), GFP fused to the PH domain of PLCδ1, or GFP fused to full-length GAP1^IP4BP as indicated. The latter two are targeted to the plasma membrane via an interaction with PtdIns(4,5)P₂. (B) The rationale behind the FRAP experiment: after bleaching by TIR (1), entry of unbleached protein into the adhesion plane via lateral diffusion through the membrane should cause fluorescence recovery from the periphery (2), whereas exchange with a cytosolic pool would lead to uniform recovery across the footprint (3). (C) Example micrographs taken at the indicated times after an 8-s bleach; residual postbleach fluorescence was subtracted from the images (see Fig. S1 for details, available). Stills are taken from Videos 1–3. Bar, 10 µm.