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.
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.
Periodically patterned photobleaching followed by spatial Fourier transform analysis of the recovery is shown to enable mapping of molecular diffusivity within spatially heterogeneous media.
Both pixel by pixel analysis method and segmented analysis method of Fourier transform fluorescence recovery after photobleaching (FT-FRAP) measurements and implemented for quantitatively evaluating diffusion of macromolecules in spatially and chemically complex media. With the development of FT-FRAP, the time-dependent recovery in fluorescence due to diffusion is measured in the spatial Fourier domain, with substantial improvements in the signal-to-noise ratio, the mathematical simplicity, and the compatibility with multi-photon excitation.
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