Fig. 2. Paxillin proximity to PY sites, as detected by FRET measurements via acceptor photobleaching. (A) Cells expressing paxillin-CFP and dSH2-YFP were placed on an inverted microscope. For FRET measurements CFP and YFP images were captured before and after YFP photobleach. FRET efficiency (E%) was calculated pixel by pixel: 1-CFP before photobleaching and CFP after photobleaching. Note high FI values are seen in `hotspots' of the total focal adhesion area. Bar, 4 µm. (B) Left, focal adhesion donor (CFP) image before (red) and after (green) acceptor photobleaching. Right, a line scan through the focal adhesion images showing the increase in donor fluorescence as a result of the acceptor bleaching.