An approach is described and evaluated for spectrally parallel hyperspectral mid-infrared imaging with optical spatial resolution. Dual-comb mid-infrared spectroscopy using a commercial QCL DCS system (IRis-core, IRsweep) enables acquisition of infrared spectra at high speed (<1 millisecond) through generation of optical beat patterns and radio-frequency detection. The high-speed nature of the spectral acquisition is shown to support spectral mapping in microscopy measurements. Direct detection of the transmitted infrared beam yields excellent spectral information, but the long infrared wavelength imposes low diffraction-limited spatial resolution. Use of fluorescence-detected photothermal infrared (FPTIR) imaging provides high spatial resolution tied directly to the integrated IR absorption. Computational imaging using a multi-agent consensus equilibrium (MACE) approach combines the high spatial resolution of FPTIR and the high spectral information of dual-comb infrared transmission in a single optimized equilibrium hyperspectral data cube.
Broadband photothermal imaging using synchrotron radiation of the Lawrence Berkeley National Laboratory Advanced Light Source is demonstrated. Synchrotron-based photothermal spectroscopy simultaneously provides a significantly improved bandwidth over commercial laser-based approaches and enables sub-micron chemical imaging with at least a tenfold resolution improvement over FTIR microscopy. Synchrotron PTIR is shown to enable studying low-frequency aromatic bending modes of polymer samples. Furthermore, fluorescence detection of the photothermal effect was employed to demonstrate cell-specific broadband infrared imaging in brain tissues using nucleus-specific fluorescence dyes.
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.
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