Direct label-free measurement of the distribution of small molecular weight compound inside thick biological tissue using coherent Raman microspectroscopy.

Masahiko Kawagishi,Takayuki Suzuki,Yuki Obara,Sumio Terada,Kazuhiko Misawa,Masumi Hayashi

doi:10.1038/srep13868

Abstract

Distributions of small molecular weight (less than 300 Da) compounds inside biological tissue have been obscure because of the lack of appropriate methods to measure them. Although fluorescence techniques are widely used to characterise the localisation of large biomolecules, they cannot be easily applied to the cases with small molecule compounds. We used CARS spectroscopy to detect and identify a label-free small molecule compound. To facilitate detection in aqueous environment, we utilised time-resolved and phase-sensitive techniques to reduce non-resonant background generated from water. We applied this technique to detect small molecular weight compound, taurine, inside mouse cornea tissue immersed in taurine solution as an initial model experiment. We detected a Raman peak of taurine near wavenumber 1033 cm−1 inside cornea and successfully characterised its depth profile in the tissue. Our CARS spectra measurement can be a promising method to measure and visualise 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.

Highlights

IntroductionInfrared spectroscopy is used to get label-free information about small molecules
A visualisation technique that works without labeling is required
This feature is useful in cases of measuring other small molecular weight molecules where labeling interferes with the chemical properties of the target

Summary

Introduction

Infrared spectroscopy is used to get label-free information about small molecules It uses the spectral pattern of infrared absorption that is characteristic to each compound, to differentiate target chemical species and to perform spatial imaging[4]. Raman spectroscopy probes molecular vibrations of energy ranges similar to those probed in infrared spectroscopy It is less affected by water, but spontaneous Raman scattering is typically weak. It has been used for imaging cell chemical composition[8,9,10,11] and for label-free detection of histological structures[12,13]. We recently combined phase-sensitive CARS with time-resolved technique[39,40] This effectively removed NRB generated from water. We successfully identified label-free taurine inside cornea sample and measured its depth profile

Methods

Results

Conclusion