In this work, multiphoton microscopy (MPM), based on the nonlinear optical processes two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), was extended to evaluate the feasibility of using MPM to distinguish layers of the bowel wall. It was found that MPM has the ability to identify the four-layer microstructures of colorectal tissues including mucosa, submucosa, muscularis propria, and serosa as there are many intrinsic signal sources in each layer. Our results also showed the capability of using the quantitative analyses of MPM images for quantifying some feature parameters including the nuclear area, nuclear-to-cytoplasmic ratio, and optical redox ratio. This work demonstrates that MPM has the potential in noninvasively monitoring the development and progression of colorectal diseases and then guiding effective treatment.
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