Figure 5: Relationship between the GimRET ratio and protein diffusion.

(a) High-magnification fluorescence (left) and ratiometric (right) images of Cos7 cells transfected with GimRET. (b) Low-magnification ratiometric image of Cos7 cells transfected with GimRET. Colour bars indicate the ratio from 1.0 (black) to 3.0 (white) in (a) and (b). (c) Relationship of the ratios from the cytoplasm and nucleus (right lower) and histograms of the ratios from the cytoplasm (upper) and nucleus (left). The broken line indicates a 1:1 correlation. N = 91 cells. (d) Photorecovery curves of FRAP measurement inside the nuclei with ratios of 2.08 (blue), 1.54 (purple), and 1.15 (red). (e) Relationship between the ratio and diffusion coefficient from FRAP measurement in the cytoplasm (blue) and nucleus (red) N = 69 cells. Error bars indicate standard deviation. Light-coloured symbols indicate individual cells. (f) GimRET ratio map (left) and 15 × 15 RICS diffusion map (right). Coloured bars indicate the ratio from 0 (blue) to 0.5 (white) and 0 (red) to 30 (white), respectively. Broken lines indicate the boundary between the nucleus and cytoplasm. The image width/height is 22.5 μm (one pixel = 1.5 μm). (g) Relationship between the ratio and RICS diffusion coefficient in the nucleus (open) and cytoplasm (filled) of four cells. Each colour indicates a single cell. (h) Mean values of the intensity ratio (red) and diffusion coefficients obtained by FRAP (blue), RICS (green), and FCS (purple) in the nucleus (Nuc) and cytoplasm (Cyto). N = 71, 71, 71, 71, 11, 11, 18, and 14 cells, respectively. Error bars indicate standard deviation.