Yersinia pseudotuberculosis binding to host cells leads to local activation of Rac1 GTPase. COS1 cells, expressing mCFP-Rac1 and mYFP fused to the p21 binding domain of Pak1 (PBD) were incubated with an effector-deficient Y. pseudotuberculosis for 20 minutes, then fixed. GTP-loaded Rac1 interaction with the mYFP-PBD brings fused mCFP and mYFP into close proximity, allowing energy transfer. This energy transfer is recorded microscopically as a corrected FRET image. CFP, YFP, and FRET images were captured using appropriate filter cube sets and the FRET image was corrected for bleed-through and cross-excitation. Scale bar (applicable to all images except insets) = 10 μm. Activation of Rac1 in response to bacterial binding is depicted here. Images courtesy of Sima Mohammadi and Ralph Isberg.