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An approach is described for spectrally parallel hyperspectral mid-infrared imaging with spatial resolution dictated by fluorescence imaging. Quantum cascade laser (QCL)-based dual-comb mid-infrared spectroscopy enables the acquisition of infrared spectra at high speed (<1 millisecond) through the 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 high signal-to-noise spectral information, but long infrared wavelengths impose low diffraction-limited spatial resolution. The use of fluorescence detected photothermal infrared (F-PTIR) 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 F-PTIR and the high spectral information of dual-comb infrared transmission in a single optimized equilibrium hyperspectral data cube. 

Fourier transform-fluorescence recovery after photobleaching (FT-FRAP) using a diffractive optical element (DOE) is shown to support distance-dependent diffusion analysis in biologically relevant media. Integration of DOEs enables patterning of a dot array for parallel acquisition of point-bleach FRAP measurements at multiple locations across the field of view. In homogeneous media, the spatial harmonics of the dot array analyzed in the spatial Fourier transform domain yield diffusion recovery curves evaluated over specific well-defined distances. Relative distances for diffusive recovery in the spatial Fourier transform domain are directly connected to the 2D (h,k) Miller indices of the corresponding lattice lines. The distribution of the photobleach power across the entire field of view using a multidot array pattern greatly increases the overall signal power in the spatial FT-domain for signal-to-noise improvements. Derivations are presented for the mathematical underpinnings of FT-FRAP performed with 2D periodicity in the photobleach patterns. Retrofitting of FT-FRAP into instrumentation for high-throughput FRAP analysis (Formulatrix) supports automated analysis of robotically prepared 96-well plates for precise quantification of molecular mobility. Figures of merit are evaluated for FT-FRAP in analysis for both slow diffusion of fluorescent dyes in glassy polymer matrices spanning several days and model proteins and monoclonal antibodies within aqueous solutions recovering in matters of seconds. ■ INTRODUCTION