Effects of freeze-thaw and photobleaching on the ultraviolet resonance Raman spectra of human colon biopsies

We studied the effects of sample freezing and thawing, laser fluence, tempe rature, and oxygen on the ultraviolet resonance Raman (UVRR) spectra of hum an colon biopsies at 251 mn excitation. We show that the total adenylate co ntent of the tissue decreases as a result of freezing and subsequent thawin g to room temperature. We also show that photobleaching is the result of ox idative as well as thermal damage. Photobleaching consisted of a decrease i n the intensity of the purine spectral bands and broadening and intensity i ncrease of the aromatic amino acid bands. Both bio- and photodegradation of the tissue can be minimized by keeping the biopsy samples refrigerated dur ing testing and the laser fluence under 0.2 mJ/mum(2). Unlike amino acid ph otobleaching, purine photobleaching was also greatly reduced by placing the samples in an argon atmosphere. Sample storage could be achieved by refrig erating the fresh specimens and testing them within 30 h of surgical resect ion, without freezing. Alternatively, the fresh specimens could be snap-fro zen in liquid nitrogen, then thawed and tested for up to 30 min. Altogether , these studies can be utilized to guide future experimental design and dat a interpretation during ultraviolet spectroscopy of biological tissues.