We studied the origin of the vibrational signatures in the sum-frequency generation (SFG) spectrum of fibrillar collagen type I in the carbon-hydrogen stretching regime. For this purpose, we developed an all-reflective, laser-scanning SFG microscope with minimum chromatic aberrations and excellent retention of the polarization state of the incident beams. We performed detailed SFG measurements of aligned collagen fibers obtained from rat tail tendon, enabling the characterization of the magnitude and polarization-orientation dependence of individual tensor elements Xijk2 of collagen's nonlinear susceptibility. Using the three-dimensional atomic positions derived from published crystallographic data of collagen type I, we simulated its Xijk2 elements for the methylene stretching vibration and compared the predicted response with the experimental results. Our analysis revealed that the carbon-hydrogen stretching range of the SFG spectrum is dominated by symmetric stretching modes of methylene bridge groups on the pyrrolidine rings of the proline and hydroxyproline residues, giving rise to a dominant peak near 2942 cm-1 and a shoulder at 2917 cm-1. Weak asymmetric stretches of the methylene bridge group of glycine are observed in the region near 2870 cm-1, whereas asymmetric CH2-stretching modes on the pyrrolidine rings are found in the 2980 to 3030 cm-1 range. These findings help predict the protein's nonlinear optical properties from its crystal structure, thus establishing a connection between the protein structure and SFG spectroscopic measurements.
Carbon , Collagen Type I , Hydrogen , Hydrogen/chemistry , Carbon/chemistry , Collagen Type I/chemistry , Rats , Animals , Spectrum Analysis/methods
Spectral imaging in the mid-infrared (MIR) range provides simultaneous morphological and chemical information of a wide variety of samples. However, current MIR technologies struggle to produce high-definition images over a broad spectral range at acquisition rates that are compatible with real-time processes. We present a novel spectral imaging technique based on nondegenerate two-photon absorption of temporally chirped optical MIR pulses. This approach avoids complex image processing or reconstruction and enables high-speed acquisition of spectral data cubes (xyω) at high-pixel density in under a second.
The emerging technique of mid-infrared optical coherence tomography (MIR-OCT) takes advantage of the reduced scattering of MIR light in various materials and devices, enabling tomographic imaging at deeper penetration depths. Because of challenges in MIR detection technology, the image acquisition time is, however, significantly longer than for tomographic imaging methods in the visible/near-infrared. Here we demonstrate an alternative approach to MIR tomography with high-speed imaging capabilities. Through femtosecond nondegenerate two-photon absorption of MIR light in a conventional Si-based CCD camera, we achieve wide-field, high-definition tomographic imaging with chemical selectivity of structured materials and biological samples in mere seconds.
Recent work on mid-infrared (MIR) detection through the process of non-degenerate two-photon absorption (NTA) in semiconducting materials has shown that wide-field MIR imaging can be achieved with standard Si cameras. While this approach enables MIR imaging at high pixel densities, the low nonlinear absorption coefficient of Si prevents fast NTA-based imaging at lower illumination doses. Here, we overcome this limitation by using InGaAs as the photosensor. Taking advantage of the much higher nonlinear absorption coefficient of this direct bandgap semiconductor, we demonstrate high-speed MIR imaging up to 500 fps with under 1 ms exposure per frame, enabling 2D or 3D mapping without pre- or post-processing of the image.
Chemical imaging based on mid-infrared (MIR) spectroscopic contrast is an important technique with a myriad of applications, including biomedical imaging and environmental monitoring. Current MIR cameras, however, lack performance and are much less affordable than mature Si-based devices, which operate in the visible and near-infrared regions. Here, we demonstrate fast MIR chemical imaging through non-degenerate two-photon absorption (NTA) in a standard Si-based charge-coupled device (CCD). We show that wide-field MIR images can be obtained at 100 ms exposure times using picosecond pulse energies of only a few femtojoules per pixel through NTA directly on the CCD chip. Because this on-chip approach does not rely on phase matching, it is alignment-free and does not necessitate complex postprocessing of the images. We emphasize the utility of this technique through chemically selective MIR imaging of polymers and biological samples, including MIR videos of moving targets, physical processes and live nematodes.
