Figure 2.

Analysis of synthetic fluorescence images with illumination shading using PixBleach. Synthetic images were simulated using the Java-based Macro language of ImageJ, using the photobleaching model on a pixel-by-pixel, Equation (17), as described in the text. Spatially varying illumination intensity simulated by a ramp-like q-map (A) was used as input for the model at each pixel position with additional random variation of the intrinsic bleach rate constant, k₂, according to Equation (18). The first frame of this simulation with M₁ = 5000 and M₂ = 7000 being constant throughout the cytoplasm and nuclear compartment, respectively, is shown in panel (B); (C) selected frames of the simulated bleach stack (C, upper two rows) and the reconstructed data from pixel-wise fitting of a mono-exponential decay to the synthetic image set (C, lower two rows); Panel (D) shows fitting results also with a FIRE LUT, from left to right (‘amplitude’, background’, ‘time constant’ and ‘RMSE’); (E) various forms of the time-integrated emission (TiEM); from left to right, calculated as summed pixel intensity in PixBleach, either without including background into the calculation (‘TiEm-background’) or with the estimated background included (‘TiEm + background’) and as product of the regression-derived amplitude and time constant (‘TiEm from formula’). The most right panel in E show the ration of ‘TiEm-background’ and ‘TiEm from formula’ in the indicated range (1.03 in dark blue to 1.05 in yellow/white). G, surface plots of the indicated parameter maps. See text for further explanations.