Fourier transform fluorescence recovery after photobleaching (FT-FRAP) is shown to support spatially resolved diffusion analysis in amorphous thin-films and heterogeneous polymer matrices. Characterization of diffusion is routine for the assessment of mobility in cell biology and lends insight into in vivo bioavailability of various small molecule drug products and biotherapeutics. Conventional point-bleach fluorescence recovery after photobleaching (FRAP) measures diffusion over a micrometer scale and is noninvasive with low sample volume requirements, however conventional FRAP is complicated by sample heterogeneity, deviations from Gaussian bleach profile, and limitations in bleach-depth. Periodically structured illumination allows for bleaching patterns across the full field of view and allows pooling of signal to discrete points in the spatial frequency domain increasing signal to noise and simplifying analysis. The architecture for FT-FRAP was extended to 2D analysis of the diffusion tensor through simple modification of a commercial high-throughput FRAP system by integration of diffractive optical elements (DOEs) for dot array patterned photobleaching.

FT-FRAP for diffusion mapping with diffractive optical elements for periodically patterned photobleaching

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