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
Introduction: Fourier domain Optical coherence tomography (OCT) is a widely used high-resolution optical imaging technique. It is useful for various applications in medical imaging, such as ophthalmology (e.g. retinal imaging for diagnosing complications like glaucoma or macular degeneration), dermatology, oncology, and cardiology. The ability to noninvasively measure both the refractive index and thickness of biological tissues could have various medical applications and enable earlier disease detection. For example, observing changes in the refractive index can help distinguish between tissues with normal or abnormal function. Methods: In this study, the theoretical framework for simultaneous measurement of the refractive index and physical thickness of multilayer systems is proposed and tested for two different samples, each having three layers, a glass/NaCl solution/glass sample and a glass/sugar solution/glass sample. The whole signal processing procedure and the experimental setup are described. Results: The refractive index and thickness of salt water and sugar water samples in the Fourier-domain OCT (FD-OCT) system were obtained. The resulting data were compared with reference measurements and showed a deviation of about 1% for the samples. Conclusion: We tested the proposed framework for the simultaneous extraction of the refractive index and thickness of multilayer systems of salt water and sugar water from its FD-OCT data. We showed that the measured parameters were in agreement with reference amounts.
