Feasibility of non-imaging, random-sampling second harmonic generation measurements to distinguish colon cancer

Globally, colorectal cancer was the second leading cause of cancer death in 2020. Research suggests that collagen, a major structural protein, plays a pivotal role in cancer development and metastasis, and by extension, subject prognosis. Collagen surrounding tumor cells undergoes structural changes that can be quantitatively studied with second harmonic generation (SHG), a subset of multiphoton microscopy (MPM). MPM as an imaging modality is difficult to implement in an endoscope because of the complex and expensive miniaturized scanning components required. Endoscope complexity can be greatly reduced by implementing a simpler, non-synchronized scanning mechanism. This study investigates whether non-imaging, randomly sampled SHG intensity measurements are sufficient to distinguish normal tissue from tumor/tumor-adjacent tissue. Unstained tumor, normal, and adjacent formalin-fixed, paraffin-embedded thin sections from 12 colorectal cancer subjects were imaged using a multiphoton microscope with 850nm excitation and 400-430nm emission band, constant power, and consisting of 1024x1024 pixels over 425x425μm. SHG signal from collagen fibers was isolated by grayscale thresholding, and the grayscale mean of the thresholded image was calculated. Then, random supra-threshold pixels in the image were selected. The mean SHG signal from normal samples was significantly greater than adjacent samples (p = 0.014) and cancer samples (p = 0.007). For both tumor and adjacent comparisons to normal tissue, p value becomes reliable after randomly sampling only 1000 pixels. This study suggests that reliable diagnostic information may be obtained through simple non-imaging, random-sampling SHG intensity measurements. A simple endoscope with this capability could help identify suspicious masses or optimum surgical margins.