Comparison of Calculated and Simulation Extracted Diffusion Coefficient (a) Results of 13 single experiments using circular bleaching; the beam diameter is the value extracted from the post-bleach frame, using angular averaging. The resulting diffusion coefficient is clearly not correlated with the diameter of the bleached region. (b) Results of rectangular bleaching pattern. Box width (w) was left as a free fitting parameter in fitting Eq. (3) to the data. Error bars account for uncertainty in the determination of beam diameter (Gauss) or width (Box) (for analytical calculation) and iteration time (for simulation). (c, two leftmost columns) Comparison of mean (over all experiments: 13 for circular and 6 for rectangular) diffusion coefficient extracted with the analytical models, with the simulation approach. The mean differences between calculated (blue) and simulated (red) diffusion coefficients are statistically insignificant. (c, three rightmost columns) the mean diffusion coefficient of a large diameter Gaussian bleached spot, arbitrary shape and a third of a cell calculated using the simulation approach. Error bars represent SEM.