Quantitative multiphoton imaging of cell metabolism, stromal fibers, and keratinization enables label-free discrimination of esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) features atypical clinical manifestations and a low 5-year survival rate (< 5% in many developing countries where most of the disease occurs). Precise ESCC detection and grading toward timely and effective intervention are therefore crucial. In this study, we propose a multidimensional, slicing-free, and label-free histopathological evaluation method based on multispectral multiphoton fluorescence lifetime imaging microscopy (MM-FLIM) for precise ESCC identification. To assess the feasibility of this method, comparative imaging on fresh human biopsy specimens of different ESCC grades is performed. By constructing fluorescence spectrum- and lifetime-coded images, ESCC-induced morphological variations are unveiled. Further quantification of cell metabolism and stromal fibers reveals potential indicators for ESCC detection and grading. The specific identification of keratin pearls provides additional support for the early detection of ESCC. These findings demonstrate the viability of using MM-FLIM and the series of derived indicators for histopathological evaluation of ESCC. As there is an increasing interest in developing multiphoton endoscopes and multiphoton FLIM systems for clinical use, the proposed method would probably allow noninvasive, label-free, and multidimensional histological detection and grading of ESCC in the future.