Label-free, multiphoton microscopy-based strategy for intraoperative grading of lung adenocarcinoma via quantitative extracellular matrix analysis.

Lung adenocarcinoma (LUAD) requires accurate subtyping for prognosis and treatment, yet conventional histopathology is time-consuming and subjective. This study applies label-free multiphoton microscopy (MPM) to quantify extracellular matrix remodeling, imaging collagen via SHG and elastin via TPEF in 88 specimens from 72 patients. We introduce a SHG/TPEF ratio (STR) system to assess tumor invasiveness. Results show progressive "fiber desertification," reduced fiber content, and decreased collagen alignment in high-grade LUAD subtypes. STR metrics strongly correlate with pathological grading and effectively distinguish aggressive patterns. This MPM-based framework enables rapid, objective intraoperative identification of high-grade LUAD, improving diagnostic precision and clinical decision-making.