Figure 2.

(A) High-resolution spatio-temporal image of the FRAP recovery for GFP-tagged glucocorticoid receptor (GFP-GR) stably expressed in the nuclei of mouse adenocarcinoma cells (McNally et al, 2000) generated by repeated line scans (yellow double-arrow) that bisect the bleach spot (in dashed black circle), normalized to their pre-bleach value. (B) Instead of fitting the full spatio-temporal image in (A), we fit a subset of the scanned lines that were exponentially distributed in time (more lines earlier in the recovery and less later). This ensured the fast early part of the FRAP recovery was sufficiently sampled to generate an accurate fit. The best-fit surface (shaded green) to the data (blue dots) is shown in 3D, with r representing the radial distance from the centre of the bleach spot. The difference (orange lines) between each data point and the fitting surface is also shown. This yielded D=2.9 μm²/s, t_a=7.7 s, and t_r=3.1 s, from which we calculated the fraction of bound to free molecules bound/free ∼0.4 using Supplementary equations (S.1) and (S.2). (C) For clarity, the fit from (B) is displayed at select times, with increasing times colour coded from 0 to 40 s, as shown in the bar to the right.