Fig. 1. Example of specificity and sensitivity of FRET in a dorsal membrane ruffle. (A) GFP-Rac localization in a Swiss 3T3 fibroblast stimulated with serum. (B) GFP-Rac, Alexa-PBD and processed FRET images of the same ruffle. GFP-Rac localization illustrates the presence of Rac in the ruffle but does not provide information about its activation status. PBD localization cannot be used to quantify Rac activity because of the high background from unbound PBD in the cytoplasm, and because of lack of specificity. All images are color-coded with warmer colors representing higher concentrations or higher levels of FRET. Bar, 22 µm. N, nucleus. Figure reproduced with permission from the American Association for the Advancement of Science (Kraynov et al., 2000).