Multiphoton microscopy for label-free identification of intramural metastasis in human esophageal squamous cell carcinoma.

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.