The quantitative analysis of fluorescence perturbation experiments such as fluorescence recovery after photobleaching (FRAP) requires suitable analytical models to be developed. When diffusion in 3D environments is considered, the description of the initial condition produced by the perturbation (i.e., the photobleaching of a selected region in FRAP) represents a crucial aspect. Though it is widely known that bleaching profiles approximations can lead to errors in quantitative FRAP measurements, a detailed analysis of the sources and the effects of these approximations has never been conducted until now. In this study, we measured the experimental 3D bleaching distributions obtained in conventional and two-photon excitation schemes and analyzed the deviations from the ideal cases usually adopted in FRAP experiments. In addition, we considered the non-first-order effects generated by the high energy pulses usually delivered in FRAP experiments. These data have been used for finite-element simulations mimicking FRAP experiments on free diffusing molecules and compared with FRAP model curves based on the ideal bleach distributions. The results show that two-photon excitation more closely fits ideal bleaching patterns even in the event of fluorescence saturation, achieving estimations of diffusion coefficients within accuracy of the correct value.
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