Measuring the Distribution of Taurine Molecule Inside Biological Tissue via Intrinsic Molecular Vibrations using Nonlinear Raman Spectroscopy

Abstract

Distributions of small molecular weight (less than 300 Da) organic compounds inside biological tissue have been obscure because of the lack of appropriate methods to measure them. Although fluorescence techniques are widely used to characterize the localization of large molecules such as proteins and nucleic acids, they cannot be easily applied to the cases with small molecule compounds: Fluorescent labels are relatively large compared to the target compounds and can interfere with the chemical properties of them. Raman spectroscopy is a technique to study vibrational information intrinsic to and characteristic of the chemical species of compounds. We used coherent anti-Stokes Raman scattering (CARS) spectroscopy to detect and identify a small molecule compound, taurine, in aqueous environment without labeling. Molecular species could be uniquely identified from the spectral shape of the broadband vibrational spectra of target compound. The local distribution of the compound could be determined from the spectral intensity. We have developed a phase-sensitive CARS spectroscopy capable of measuring the broadband spectrum simultaneously without losing high frequency resolution. We also utilized a time-resolved technique to remove non-resonant noise signals over a wide spectral range produced by water molecules. We combined these techniques to selectively detect resonant vibrational CARS signals from a target compound. We measured taurine inside mouse cornea tissue soaked in solution as an initial model experiment. We detected a Raman peak of taurine near 1000 wavenumber / cm inside cornea, and successfully characterized its depth profile in the tissue. Our CARS spectra measurement can be a promising method to measure and visualize the distribution of small bio-related compounds in biological background without using any labeling, paving the way for new cell biological analysis in various disciplines.