Fast Diffusion Characterization by Multiphoton Excited Fluorescence Recovery while Photobleaching.

Multiphoton-excited fluorescence recovery while photobleaching (FRWP) is demonstrated as a method for quantitative measurements of rapid molecular diffusion over microsecond to millisecond timescales. Diffusion measurements are crucial in assessing molecular mobility in cell biology, materials science, and pharmacology. Optical and fluorescence microscopy techniques enable non-invasive rapid analysis of molecular diffusion but can be challenging for systems with diffusion coefficients exceeding ∼100 µm2/s. As an example, fluorescence recovery after photobleaching (FRAP) operates on the implicit assumption of a comparatively fast photobleaching step prior to a relatively slow recovery and is not generally applicable for systems exhibiting substantial recovery during photobleaching. These challenges are exacerbated in multiphoton excitation by the lower excitation efficiency and competing effects from local heating. Herein, beam-scanning FRWP with patterned line-bleach illumination is introduced as a technique that addresses FRAP limitations and further extends its application range by measuring faster diffusion events. In FRWP, the recovery of fluorescence is continuously probed after each pass of a fast-scanning mirror, and the upper bound of measurable diffusion rates is, therefore, only limited by the mirror scanning frequency. A theoretical model describing transient fluctuations in fluorescence intensity arising as a result of combined contributions from photobleaching and localized photothermal effect is introduced along with a mathematical framework for quantifying fluorescence intensity temporal curves and recovering room-temperature diffusion coefficients. FRWP is then tested by characterization of normal diffusion of rhodamine-labeled bovine serum albumin, green fluorescence protein, and immunoglobulin G molecules in aqueous solutions of varying viscosity.

Periodic Photobleaching with Structured Illumination for Diffusion Imaging.

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-of-concept analyses based on simulations with known ground-truth maps, diffusion imaging by FT-FRAP was used to map spatially-resolved 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.

Parts-per-Million Detection of Trace Crystal Forms Using AF-PTIR Microscopy.

Autofluorescence-detected photothermal mid-infrared (AF-PTIR) microscopy was shown to enable parts-per-million detection of α-indomethacin impurity in γ-indomethacin samples. Subtle differences in the photothermal response of the UV-autofluorescence of two indomethacin crystal polymorphs were used for sub-micron chemical discrimination based on fingerprint region mid-IR spectroscopy. The AF-PTIR assignment was independently confirmed by second harmonic generation (SHG) microscopy, which was shown to reduce the total analysis time by rapidly identifying the suitable fields of view. AF-PTIR microscopy has the potential to assist in the early identification of crystal form impurities in the solid dosage forms development pipeline.

Label-Free Autofluorescence-Detected Mid-Infrared Photothermal Microscopy of Pharmaceutical Materials.

Label-free autofluorescence-detected photothermal mid-IR (AF-PTIR) microscopy is demonstrated experimentally and applied to test the distribution of active pharmaceutical ingredients (APIs) in a mixture containing representative pharmaceutical excipients. Two-photon excited UV-fluorescence (TPE-UVF) supports autofluorescence of native aromatic moieties using visible-light optics. Thermal modulation of the fluorescence quantum yield serves to report on infrared absorption, enabling infrared spectroscopy in the fingerprint region with a spatial resolution dictated by fluorescence. AF-PTIR provides high selectivity and sensitivity in image contrast for aromatic APIs, complementing broadly applicable optical photothermal IR (O-PTIR) microscopy based on photothermal modulation of refractive index/scattering. Mapping the API distribution is critical in designing processes for powdered dosage form manufacturing, with high spatial variance potentially producing variability in both delivered dosage and product efficacy. The ubiquity of aromatic moieties within API candidates suggests the viability of AF-PTIR in combination with O-PTIR to improve the confidence of chemical classification in spatially heterogeneous dosage forms.

Fluorescence-Detected Mid-Infrared Photothermal Microscopy.

We demonstrate instrumentation and methods to enable fluorescence-detected photothermal infrared (F-PTIR) microscopy and then demonstrate the utility of F-PTIR to characterize the composition within phase-separated domains of model amorphous solid dispersions (ASDs) induced by water sorption. In F-PTIR, temperature-dependent changes in fluorescence quantum efficiency are shown to sensitively report on highly localized absorption of mid-infrared radiation. The spatial resolution with which infrared spectroscopy can be performed is dictated by fluorescence microscopy, rather than the infrared wavelength. Intrinsic ultraviolet autofluorescence of tryptophan and protein microparticles enabled label-free F-PTIR microscopy. Following proof of concept F-PTIR demonstration on model systems of polyethylene glycol (PEG) and silica gel, F-PTIR enabled the characterization of chemical composition within inhomogeneous ritonavir/polyvinylpyrrolidone-vinyl acetate (PVPVA) amorphous dispersions. Phase separation is implicated in the observation of critical behaviors in ASD dissolution kinetics, with the results of F-PTIR supporting the formation of phase-separated drug-rich domains upon water sorption in spin-cast films.