Pixel-Wise Diffusion Imaging by Structured Illumination Microscopy Fluorescence Recovery after Photobleaching (SIM-FRAP)

Image reconstruction algorithms developed for structured illumination microscopy were integrated with spatial Fourier transform fluorescence recovery after photobleaching (FT-FRAP) to recover pixel-wise diffusion maps of molecular mobility across the dissolution front of model pharmaceutical compacts. The optics and acquisitions are unchanged; the novelty lies entirely in a SIM-based reconstruction that enables segmentation-free, pixel-wise diffusion mapping. The ability to quantify molecular diffusivity in amorphous solid dispersions (ASDs) is critical for rationally designing formulations containing biologically active molecules. Notably, drug loadings exceeding critical threshold values in ASDs can result in dramatic reductions in dissolution rates, which are hypothesized to arise from phase-separated drug-rich barrier layers. Molecular mobility is the defining property dictating these dissolution trends, varying spatially across the glassy core of the ASD compact, the hydrated compact, the gel-phase, and phase-separated domains. In this work, SIM-FRAP directly accesses the spatial diffusivity across this entire dissolution front in a single experiment. SIM-FRAP operates by using periodically structured illumination for photobleaching followed by spatial Fourier transformation, recentering peaks from 2D diffraction patterns, and inverse Fourier transformation to produce modulation-dependent amplitude maps. Pixel-wise fits to exponential decay functions recovered a unique diffusion coefficient at each pixel across the entire field of view (up to ∼1 million diffusion coefficients per experiment). Application of SIM-FRAP revealed both smoothly varying diffusivity across the compact and the gel phases and a discontinuous change to domains exhibiting uniform diffusivity, consistent with phase separation. Furthermore, the experimental simplicity of the SIM-FRAP approach supports its adoption in a broader scope of measurements.