Periodically structured illumination coupled with spatial Fourier-transform fluorescence recovery after photobleaching (FT-FRAP) was used to support diffusivity mapping for segmented domains of arbitrary shape, for example, a phase-separated domains of amorphous solid dispersion spin-cast thin films. Periodic “comb-bleach” patterns of the excitation beam for photobleaching encoded maps of diffusion onto harmonic peaks in the spatial Fourier transform. Diffusion manifests as simple exponential decays of the spatial harmonics in the FT-domain, improving signal to noise and simplifying mathematical analysis. Image segmentation prior to Fourier transformation was performed to support pooling for signal to noise enhancement for regions of arbitrary shape expected to exhibit constant diffusivity within a domain. Following simulations with known ground-truth maps, spatially resolved diffusion differences within phase-separated domains of model amorphous solid dispersion spin-cast thin films were mapped via diffusion imaging by FT-FRAP. Multi-harmonic analysis by FT-FRAP was able to discriminate and quantify the roles of internal diffusion and exchange to higher mobility interfacial layers in modeling the recovery kinetics within thin amorphous/amorphous phase separated domains, with interfacial diffusion playing a critical role in recovery. These results have direct implications for the design of amorphous systems for stable storage and efficacious delivery of therapeutic molecules.

Periodic photobleaching with structured illumination as a method of diffusion imaging

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