Periodically patterned photobleaching followed by spatial Fourier transform analysis of the recovery is shown to enable mapping of molecular diffusivity within spatially heterogeneous media.
Image segmentation prior to Fourier transform fluorescence recovery after photobleaching (FT-FRAP) enabled quantitatively evaluating diffusion of macromolecules in spatially and chemically complex media. Notably,multi-harmonic analysis by FT-FRAP was able to definitively 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.
Fluorescence-detected photothermal mid-infrared (F-PTIR) microscopy is demonstrated experimentally and applied to characterize the chemical composition within micrometer-size phase-separated domains of ritonavir/copovidone amorphous solid dispersions formed upon water sorption. In F-PTIR, temperature-sensitive changes in fluorescence quantum efficiency report on highly localized absorption of mid-infrared radiation. Two-photon excited ultraviolet autofluorescence supported label-free F-PTIR microscopy of tryptophan microcrystals and lyophilized lysozyme particles. F-PTIR provides two degrees of chemical specificity, informing on infrared absorption selectively in the local environments immediately adjacent to fluorescent regions of interest.
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