Effects of Freeze-Thaw and Photobleaching on the Ultraviolet Resonance Raman Spectra of Human Colon Biopsies

Abstract

We studied the effects of sample freezing and thawing, laser fluence, temperature, and oxygen on the ultraviolet resonance Raman (UVRR) spectra of human colon biopsies at 251 nm excitation. We show that the total adenylate content of the tissue decreases as a result of freezing and subsequent thawing to room temperature. We also show that photobleaching is the result of oxidative as well as thermal damage. Photobleaching consisted of a decrease in the intensity of the purine spectral bands and broadening and intensity increase of the aromatic amino acid bands. Both bio- and photodegradation of the tissue can be minimized by keeping the biopsy samples refrigerated during testing and the laser fluence under 0.2 mJ/μm2. Unlike amino acid photobleaching, purine photobleaching was also greatly reduced by placing the samples in an argon atmosphere. Sample storage could be achieved by refrigerating the fresh specimens and testing them within 30 h of surgical resection, without freezing. Alternatively, the fresh specimens could be snap-frozen in liquid nitrogen, then thawed and tested for up to 30 min. Altogether, these studies can be utilized to guide future experimental design and data interpretation during ultraviolet spectroscopy of biological tissues.