Multiphoton-microscopy-based imaging analysis of lung adenocarcinoma

Lung cancer has the highest incidence and mortality rates in China, with lung adenocarcinoma being the most prevalent histological subtype. Rapid and accurate intraoperative identification of tumor tissue is essential for achieving negative resection margins, reducing surgical time, and improving patient outcomes. However, conventional frozen section pathology is time-consuming and subject to interpretive variability, underscoring the need for more efficient diagnostic techniques. This study investigates the feasibility of using multiphoton microscopy (MPM) to detect microstructural alterations in lung adenocarcinoma. MPM, which leverages second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), enables label-free, subcellular-resolution imaging of tissue architecture—particularly effective for visualizing collagen remodeling in the tumor microenvironment. We applied MPM to normal lung tissue, adenocarcinoma in situ (AIS), and invasive adenocarcinoma (IAC), and compared the results with conventional hematoxylin and eosin (H&E) staining. MPM images revealed subcellular structures with high fidelity and strong concordance with H&E images. As disease progressed, tumor cell heterogeneity increased, and collagen fibers transitioned from a curled to an elongated morphology, eventually becoming fragmented and disorganized—changes often difficult to discern with H&E staining alone. These findings demonstrate that MPM provides high-resolution, label-free imaging and effectively detects collagen remodeling associated with tumor progression.