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A quantitative analytical method to discriminate among the various types of cancerous esophagus tissue is essential for accurate cancer staging. This paper reports on the use of ratiometric nonlinear optical microscopy to reveal the ratio of two-photon excited fluorescence (TPEF) to second harmonic generation (SHG) and forward to backward (F/B) SHG from single collagen fibers only in submucosa of esophageal squamous cell carcinoma. This makes it possible to accurately differentiate among the four stages of esophageal cancer, providing results that are in good agreement with histopathology. Furthermore, it is confirmed by polarization-dependent SHG that the varied SHG response in esophageal cancer tissues is mainly from the shrinkage in diameter of collagen fibers instead of the collagen triple helixes altered by cancer cells. Based on the results of TPEF/SHG and F/B SHG ratio, they can cooperatively improve the precision of diagnostics on esophageal cancer and could be transferred to other types of cancer diseases with changed collagen fibers. Impact statement The issue of classifying esophageal cancer at various developmental stages is crucial for determining the optimized treatment protocol for the patients, as well as the prognosis. Precision improvement in staging esophageal cancer keeps seeking quantitative and analytical imaging methods that could augment histopathological techniques. In this work, we used nonlinear optical microscopy for ratiometric analysis on the intrinsic signal of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) from single collagen fibers only in submucosa of esophageal squamous cell carcinoma (ESCC). The blind tests of TPEF/SHG and forward (F)/backward (B) SHG were demonstrated to compare with the histology conclusion. The discussion of sensitivity and specificity was provided via statistical comparison between the four stages of esophageal cancer. To the best of our knowledge, this is the first study of using these two ratios in combination for staging ESCC.

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