Layered Analysis of Injury in the Rat Esophagus Induced by Photodynamic Therapy Using Two-Photon Microscopy

The esophagus is characterized by a multi-layered structure with compositional differences in each layer, resulting in layer-specific damage thresholds to photodynamic therapy (PDT). Accurate and efficient evaluation of the damages in each layer of healthy tissues is crucial for a successful PDT treatment when eradicating tumors in esophagus. However, conducting a comprehensive assessment of damage to each layer requires the integration of multiple traditional methods, which can be both time-consuming and labor-intensive. Here, we employed two-photon microscopy (TPM) to image rat esophageal sections and fresh samples with full-layer damage induced by PDT. We find that TPM can precisely identify injuries in each layer of the esophagus, including cellular hyperplasia, lamina propria detachment, loosened and tortuous collagen fibers, as well as vacuolated and atrophied muscle fibers, which are consistent with the detection results of traditional methods. Moreover, TPM possesses unique capabilities not present in traditional methods. For example, TPM successfully detected enhanced perinuclear fluorescence in necrotic esophageal epithelial cells, performed quantitative analysis of collagen fiber changes, and enabled three-dimensional (3D) visualization of structural and morphological alterations caused by the damage. Together, our findings demonstrate that TPM serves as an effective tool for evaluating layer-specific effects induced by PDT, and is expected to have a long-term influence for enhancing the targeting accuracy of PDT in tumor treatment of hollow organs.