RAD51-GFP diffusion in a live Rad51GFP/WT ES cell. An individual Rad51GFP/WT nucleus was imaged by oblique illumination using a TIRF setup (Eclipse Ti-E microscope; Nikon). The nucleus contains both slowly diffusing RAD51-GFP particles and larger (apparently) immobile RAD51 accumulations, often referred to as spontaneous foci. Because of the elevated protein concentration of 50 nM, compared with BRCA2-GFP, not all oligomeric RAD51 entities with few individual proteins can be clearly detected. Note that, in this context, true single molecules will only be detected at concentrations well below 10 nM. 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 with Fig. 4 A. Bar, 5 µm.
