For complete removal of cancerous tissue in esophageal squamous cell carcinoma (ESCC), intramural metastasis (IM) should be identified preoperatively or intraoperatively. Here, multiphoton microscopy (MPM) was introduced for label-free identification of IM in the esophageal wall, by a combination of two-photon excited fluorescence (TPEF), second harmonic generation (SHG) imaging, and spectral analysis. Three-dimensional (3D) imaging of the IM region was also performed. Quantitative parameters, including 3D fiber orientation, were measured by 3D-weighted orientation vector summation. Overall, MPM showed the potential to identify IM. With the development of the advanced MPM endoscope, clinical identification of IM by MPM will be possible.
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