Periodic Photobleaching with Structured Illumination for Diffusion Imaging

Abstract

The use of periodically structured illumination coupled with spatial Fourier-transform fluorescence recovery after photobleaching (FT-FRAP) was shown to support diffusivity mapping within segmented domains of arbitrary shape. Periodic "comb-bleach" patterning of the excitation beam during photobleaching encoded spatial maps of diffusion onto harmonic peaks in the spatial Fourier transform. Diffusion manifests as a simple exponential decay of a given harmonic, improving the signal to noise ratio and simplifying mathematical analysis. Image segmentation prior to Fourier transformation was shown to support pooling for signal to noise enhancement for regions of arbitrary shape expected to exhibit similar diffusivity within a domain. Following proof-ofconcept analyses based on simulations with known ground-truth maps, diffusion imaging by FT-FRAP was used to map spatiallyresolved diffusion differences within phase-separated domains of model amorphous solid dispersion spin-cast thin films. 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. These results have direct implications for the design of amorphous systems for stable storage and efficacious delivery of therapeutic molecules.

Diffusion Mapping with Diffractive Optical Elements for Periodically Patterned Photobleaching

“…These observations are in contrast to prior FT-FRAP studies of ritonavir in PVPVA, following water vapor exposure, in which substantial contributions from interfacial exchange were recorded. 23 The differences are tentatively attributed to differences in glass transition temperatures, T g , for the two active pharmaceutical ingredients. Whereas T g is well below ambient temperatures of 20 °C for ibuprofen (−42 °C), it is substantially above for ritonavir (50 °C).…”

“…In beam-scanning confocal microscopes, we have shown previously that integration of FT-FRAP with comb-bleach illumination simply requires software/firmware updates to the slow-scan mirror used to direct the beam across the FoV during the imaging and photobleaching stages, with no required changes to the physical hardware at all. 23 In this work, relatively minor retrofitting of a commercial wide-field microscope with a focal plane array detector was performed by a relatively straightforward addition of the DOE to the beam path and an upgrade to the photobleach laser source. These two microscope designs (confocal beam-scanning and wide field detection with a focal plane array) collectively comprise the overwhelming bulk of systems routinely used for fluorescence microscopy, and correspondingly, for FRAP analysis.…”

Spatially Resolving Size Effects on Diffusivity in Nanoporous Extracellular Matrix-like Materials with Fluorescence Correlation Spectroscopy Super-Resolution Optical Fluctuation Imaging

“…Single particle tracking (SPT) has been extensively applied to extract high-spatiotemporal information of molecular diffusion. − However, SPT measurements can be limited by the capabilities of tracking software analyses used to analyze the data that can include challenges of emitters that cross paths, photoblink, or have low signal. These challenges can result in a limited capability to detect and track particles moving at very high speeds for multiple consecutive frames or in complex matrices. , Finally, other optical techniques designed to measure the diffusion coefficients, such as fluorescence recovery after photobleaching (FRAP), require much larger length scales (∼10–100 μm 2 ) for accurate imaging. − Moreover, FRAP extracts an average diffusion coefficient over the entire area, but provides no local diffusion information.…”

Fast Diffusion Characterization by Multiphoton Excited Fluorescence Recovery while Photobleaching

“…22 Following the initial demonstration, FT-FRAP has recently been applied to characterize the mobility and chemical composition of phase-separated microdomains in amorphous pharmaceutical formulations. 23 In FT-FRAP, a periodic line bleach is achieved by controlling fast-scanning mirrors (either resonant or galvanometer scan mirrors) that rapidly translate the excitation beam across the FoV. In this implementation, a significant reduction in local heating is achieved by distributing the incident power along multiple equally separated rapidly scanned lines.…”

“…Several previous studies have demonstrated the use of line and patterned illumination profiles to circumvent some of the limitations of conventional FRAP analysis. − Recently, Geiger et al have demonstrated the theoretical framework and proof-of-concept experiments for Fourier-transform FRAP (FT-FRAP) with a comb-bleaching pattern across a large (∼1 × 1 mm) field of view (FoV) . Following the initial demonstration, FT-FRAP has recently been applied to characterize the mobility and chemical composition of phase-separated microdomains in amorphous pharmaceutical formulations . In FT-FRAP, a periodic line bleach is achieved by controlling fast-scanning mirrors (either resonant or galvanometer scan mirrors) that rapidly translate the excitation beam across the FoV.…”