The Effects of ex vivo Handling Procedures on the Near-Infrared Raman Spectra of Normal Mammalian Tissues

Abstract

Recent studies in the literature have investigated the feasibility of tissue diagnostics based on Raman spectroscopy. The majority of these compare the ex vivo spectra of normal and diseased tissue. Due to the time lapse between tissue excision and spectroscopic examination, samples must be frozen or otherwise preserved to maintain their native biochemical states. In order to establish optimum procedures for ex vivo Raman spectroscopy of tissue, the effects of tissue drying, formalin fixing, snap freezing, tissue freezing in optimal cutting temperature (OCT) medium and extended post-thaw durations were studied to determine if any of these handling procedures introduced spectral artifacts. Experiments on representative tissues indicated that tissue heating due to the excitation light did not change the spectra significantly. With minor exceptions, OCT and formalin did not contaminate tissue spectra, so that samples stored for histological examination could also be studied with Raman spectroscopy. Tissue dehydration caused disruption of the protein vibrational modes, which caused spectral artifacts. It is concluded that ex vivo tissue samples should be frozen in OCT. Prior to spectral analysis, the tissue should then be acclimatized at room temperature in phosphate-buffered saline (PBS) and immersed in PBS during spectroscopic examination.