Label-free imaging for T staging of gastric carcinoma by multiphoton microscopy

Gastric cancer is one of the most common malignancies worldwide. The accurate diagnosis of tumor invasion depth is critical for therapeutic strategy and prognosis. Without fluorescent labelling, multiphoton microscopy (MPM) imaging could directly reveal tissue architecture based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). In this study, we aimed to explore the feasibility of MPM imaging to assess the gastric tumor morphology and infiltration. Unstained slides of 18 fresh gastric tissues with different T staging were examined by multiphoton microscopy. Morphological and quantitative analyses were both conducted. The nuclear area was defined as the area of nuclear boundary. Collagen content was defined as the ratio of SHG pixels to all pixels. Gastric normal and tumor tissues under different T stages visually presented with cellular and subcellular features on fluorescent imaging. The nuclear areas of normal and cancerous cells were 32.01 ± 2.89 and 58.41 ± 6.06 μm² (P < 0.001), respectively. Collagen content was quantified as 0.087 ± 0.012 in normal mucosa but 0.020 ± 0.007 in cancerous mucosa (P < 0.001). All results were in accord with the paired H&E-stained slides. Our findings suggested the convincing potential of MPM for judging T staging of gastric cancer. Without staining intervention, TPEF and SHG of MPM imaging could objectively and quantitatively indicate the subcellular and molecular changes during carcinogenesis. With the advancement of deep penetration, self-focus imaging and three-dimensional (3D) visualization, label-free MPM imaging compacted with endoscopy could be further introduced to realize the real-time in vivo assessment of tumor invasion clinically.