EXPERIMENTAL FIGURE 4-23 Fluorescence recovery after photobleaching (FRAP) reveals the dynamics of molecules. In a live cell, following the distribution of a GFP-labeled protein provides a view of the overall distribution of the protein, but it doesn’t tell us how dynamic populations of individual molecules might be. (a) In FRAP, the GFP fluorochrome is bleached by a short burst of strong laser light focused on the region of interest (ROI). The light rapidly bleaches the molecules irreversibly, so they are not detected again. Restoration of fluorescence in the region tells us that unbleached molecules have moved into the ROI. (b) FRAP was used to determine the exchange rate of EBP50 and ezrin, two components of microvilli (seen as white lines), on the apical surface of epithelial cells. In cells expressing either GFP-EBP50 or ezrin-GFP, the GFP in a small region indicated by the green box was bleached, and recovery by exchange with unbleached protein was followed. The fluorescence of GFP-EBP50 returns very fast, indicating it has a fast exchange rate, whereas the fluorescence of ezrin-GFP returns slowly, indicating a slower exchange rate. (c) By quantifying the recovery, the dynamic properties of EBP50 and ezrin can be established.