Co-expression of BRC3 and RFP in live Rad51GFP/WT ES cells affecting RAD51-GFP diffusion. Several cells in this field were imaged by oblique illumination using a TIRF setup (Eclipse Ti-E microscope; Nikon). RAD51-GFP and RFP fluorescence is indicated in green and red, respectively. Cells that solely display RAD51-GFP fluorescence are apparently nontransfected and, thus, display spontaneous foci and slowly diffusing RAD51-GFP particles. The red signal observed in one cell indicates sufficient RFP and BRC3 expression so that BRCA2–RAD51 binding is interfered with. This results in uniform RAD51-GFP fluorescence originating from the loss of (transient) binding interactions and concomitant faster diffusion. The laser beam passing into the sample had a 75° angle with respect to the objective optical axis. Frames were continuously collected for 10 s using a frame acquisition time of 50 ms. The display rate is 7 frames per second. Compare Fig. 4 B. Bar, 5 µm.
