Simulation of heterogenous diffusion and fitting with the StrExp function. A 2-dimensional bleaching experiment was simulated on a disk with a circular bleached area of radius r1=0.5 μm using FeniCS, an automated computational modelling suite (http://www.fenicsproject.org). The bleaching rate is set to k=10 sec-1 and the diffusion coefficient is d1=0.2 μm2/sec and d2=0.8 μm2/sec on the left and right half disk, respectively (A). Fluorescence loss inside and outside the bleached region was fitted at every pixel position with the StrExp function. For this purpose the PixBleach plugin to ImageJ was used . The regression recovers a map of the stretching parameter, h, color-coded between 0.4 to 2.0 (B), the time constant distribution color-coded between 1.0 to 15.0 sec (C) and the χ2-map color-coded between 0 to 50 (E). A FIRE-LUT was used for color-coding, where dark blue and yellow indicate lowest and highest values, respectively. The reconstructed stack exactly resembles the simulated data set, as seen in D (upper row, simulated data (‘sim‘), lower row, regression (‘fit‘)). F, profile of time constants, as estimated from fitting the StrExp function to the simulated FLIP data along the line shown in panel C. G-L, rate coefficients were calculated on a pixel-by-pixel basis according to with self-programmed macros to ImageJ. Rate coefficients are color-coded using a FIRE-LUT in the range from 0.017 (dark blue) to 0.97 (yellow). G, shows montage of all time points; H, first frame of montage with some regions of interest (boxes 1 to 4) used for analysis in panel I-L. Rate coefficient as function of time averaged for box 1 (I), box 2 (J), box 3 (K) and box 4 (L) from the whole sequence.