This conference presentation was prepared for the Advanced Chemical Microscopy for Life Science and Translational Medicine 2023 conference at SPIE BiOS, 2023.
AF-PTIR microscopy is shown to enable chemically-specific imaging of an active pharmaceutical ingredient (API) and discrimination between two crystal forms of the API with parts per million (ppm) detection limit. AF-PTIR is based on detecting highly-localized temperature-induced variations in native autofluorescence intensity following the absorption of mid-IR radiation generated by an array of 32 independent narrowband quantum cascade lasers (QCLs). AF-PTIR was demonstrated to improve the detection limit in trace crystal form impurities analysis by two orders of magnitude as compared to commercial powder X-ray diffraction instrumentation, suggesting potential applications in pharmaceutical formulations analysis.
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.
Conventional three-dimensional (3D) images of biological samples are typically assembled from a stack of twodimensional images acquired sequentially at different focal planes. This time-consuming manner hinders the application of 3D imaging techniques to the investigation of fast biochemical dynamics and light-sensitive biological events. The concept of multifocus imaging, which enables simultaneous acquisition of images from multiple focal planes, was introduced to achieve rapid 3D imaging. In the present study, we achieved multifocus imaging through polarization wavefront shaping via a micro-retarder array which splits the incident linearly polarized light into three beamlets that are focused to three axially-offset focal planes with ~100 μm separation. Append to an existing beam-scanning microscope, this multifocus system enables rapid 3D imaging compatible with a variety of optical microscopic approaches including laser transmittance, two-photon excited fluorescence, and second harmonic generation microscopy.
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