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.

Drop Coating Deposition Raman Spectroscopy of Fluorescein Isothiocyanate Labeled Protein

“…This result is consistent with the previous reports that the photobleaching rate increases with laser power density. 40 The increased fluorescence intensity observed in the DCDR spectra of 10FTIC-BSA after brief and/or low laser power illumination is very similar to the FITC fluorescence photobleaching profile reported by Hirschfeld. 41 The fact that enhanced fluorescence intensity was only seen in the 10FITC-BSA DCDR spectra, but not in its solution Raman spectra or in the DCDR spectra of 1FITC-BSA, is consistent with the concentration quenching effect proposed by Hirschfeld.…”

“…This result is consistent with the previous reports that the photobleaching rate increases with laser power density. 40 …”

“…The FITC Raman photobleaching is believed to be primarily due to the photochemical reaction of the FITC molecules in an electronically excited state. Given the fact that fluorescence background and Raman photobleaching are most commonly observed in biological and biochemical Raman applications, 40,[42][43][44] understanding their photobleaching mechanisms can be of great importance for correct interpretation of Raman spectral data obtained with such samples.…”

Near‐infrared Raman spectroscopy for the classification of epithelial pre‐cancers and cancers

“…A ð 80 ultra-long working distance lens was used to focus the laser beam (power at the sample of 32 š 1.1 mW) to a spot size of 2-3 µm on the tissue surface and collect the scattered photons in non-confocal mode. At least five spectra were acquired from each larynx sample (mean number D 7.96, range 5-12) and a minimum of 10 spectra were acquired from each oesophagus sample (mean number D 14, range [10][11][12][13][14][15][16][17][18][19][20]. The scattered Raman signal was integrated for 30 s and measured over a spectral range of 400-1800 cm 1 with respect to the excitation frequency.…”

Real‐time in vivo cancer diagnosis using raman spectroscopy

“…Raman cross section has a wavelength dependence of 1/λ 4 , where λ represents the excitation wavelength. Excitation with UV light is not suitable for in vivo applications due to possible photochemical tissue damage . Visible excitation generates strong auto‐fluorescence in biological samples.…”