Imaging of molecular mobility by spatial Fourier transform fluorescence recovery after photobleaching (FT-FRAP) with structured illumination

FT-FRAP with structured illumination is demonstrated for recovery of 2D molecular diffusion maps within heterogeneous systems. Distribution of the bleach power in comb illumination patterns maintained high integrated power, but avoids "hot spots" from local heating typical in point-bleach multi-photon excitation. Spatial Fourier transform approaches support recovery of diffusion coefficient imaging, in which 2D maps of spatially varying diffusion coefficients are recovered within a given field of view. Proof-of-concept simulations with known ground-truth behaviors enable algorithm assessment. Experiments are described to characterize molecular and macromolecular mobility within biological tissues and tissue-mimetic materials. Additional applications include mapping of small molecule diffusion within model heterogeneous pharmaceutical formulations to understand bioavailability and stability.