RAD54-GFP diffusion in a live Rad54GFP/− ES cell. An individual Rad54GFP/− nucleus was imaged by oblique illumination using a TIRF setup (Eclipse Ti-E microscope; Nikon). Two pools of RAD54 protein in live cells are apparent: fast diffusing RAD54-GFP contributes to uniform nuclear fluorescence, whereas partially bound RAD54 is accumulated in spontaneous foci. By comparison, we can estimate that each of these accumulations contains tens to hundreds of RAD54-GFP molecules. 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.
