In Vivo Detection of Rat Colorectal Cancers by using a Dual-Wavelength Excitation Method

Hypoxia is a characteristic feature of solid neoplasms, and insufficient oxygen supply increases cellular nicotinamide adenine dinucleotide (NADH) fluorescence, which is a main component of autofluorescence of the colorectal mucosa. We investigated whether a dual-wavelength excitation method which is optimized for sensing mucosal NADH fluorescence could be applicable to the detection of rat colorectal cancers in vivo. Rat colorectal adenocarcinomas were studied by using fluorescence stereomicroscopy. After autofluorescence images at 470 nm irradiated with dual-wavelength excitation at 365 nm (F365 ex) and 405 nm (F405 ex) were acquired, ratio images were produced by dividing F365 ex by F405 ex: The excitation-emission wavelength pairs in F365 ex and F405 ex were adjusted for acquisition of NADH fluorescence and reference fluorescence. Based on observations from the luminal surface in vivo, F365 ex/F405 ex ratio images indicated a 1.57-fold higher signal value in the cancers than in the surrounding normal mucosa. The signal values in F365 ex/F405 ex ratio images were less mutually related with the hemoglobin concentration index. Small adenocarcinomas (less than 4 mm) could be detected on F365 ex/F405 ex ratio images. The results showed that NADH fluorescence measurement with little interference from tissue hemoglobin is efficient for visualizing rat colorectal cancers in vivo, suggesting that the dual-wavelength excitation method has potential for label-free endoscopic detection of diminutive colorectal neoplasms.