A study on multiphoton spectroscopy and imaging using multimodal nonlinear optical microspectroscopy

Abstract

Coherent anti-Stokes Raman scattering (CARS) is a technology developed from spontaneous Raman scattering and is a third-order nonlinear scattering phenomenon. When the energy difference between the two beams, 'pump' and 'Stokes', exactly matches the vibrational energy of the molecule, a photon with a new energy is generated. CARS is a label-free, molecularly selective and molecularly sensitive technique because it observes molecular vibrations. However, CARS signal intensity is proportional to the absolute square of the third-order nonlinear susceptibility, which consists of resonant and nonresonant terms. It is difficult to utilize the fingerprint region containing various information due to the CARS signal distortion that occurs in the mixture of Raman vibrational resonance and nonresonance. Therefore, it is necessary to reduce the nonresonant background (NRB) through the numerical methods based on maximum entropy method (MEM) and Kramers-Kronig (KK) relation or the experimental methods based on heterodyne detection and destructive interference. To make spectral information useful, we used phase retrieval via KK relation, which is one of the ways to reduce the constraints caused by NRB. CARS spectral phase retrieval for the 4 marine oils showed that the molecular bonding ratios of the natural oils were similar except for the ethyl ester form oil. Principal component analysis (PCA) was combined to classify different samples, which generated new axes that maximize variance within the data set. The construction of the scores plot showed that different samples were separated from each other and that the same samples were grouped. To investigate the chemical and biological properties of skin tissues ex vivo, we utilized a multimodal nonlinear optical system combining CARS and second harmonic generation (SHG). Cereblon (CRBN) is a substrate receptor for the cullin-4A/B RING E3 ubiquitin ligase complex that mediates various physiological and pathological functions in humans. To validate previous discoveries that CRBN plays a critical role in lipid metabolism and skin homeostasis, we utilized a label-free multimodal nonlinear optical system to analyze lipid and collagen profiles in CRBN deleted mouse skin. CARS measurements show that CRBN deletion can affect the degree of unsaturation of triacylglycerides in the mice. And we observed that the loss of CRBN decreased dermal collagen fibers in the mice, which was consistent with the result of a previous study.