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To improve surgical precision, we employed multiphoton microscopy (MPM) to characterize the microstructural and spectral features of both normal tissue and tumor boundary. The spectral differences between normal choroid and tumor boundary were systematically analyzed using lambda mode MPM. Collagen parameters were quantified by combining with image processing techniques to elucidate the structural alterations of normal tissues and choroidal melanomas. The results of the study demonstrated that the MPM enables label\u2010free identification of tumor boundary, with NADH molecular changes serving as a critical basis for qualitative differentiation. Furthermore, utilizing self\u2010developed image processing algorithms, the alteration of collagen fiber provides a potential quantitative index for identifying the tumor boundary. This study confirms the capability of MPM in identifying tumor boundary. With the advancement of multiphoton endoscopes, it holds promise for providing precise intraoperative guidance during choroidal melanoma resection.<\/jats:p>","DOI":"10.1002\/jbio.70241","type":"journal-article","created":{"date-parts":[[2026,3,11]],"date-time":"2026-03-11T05:07:59Z","timestamp":1773205679000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Label\u2010Free Identification of Choroidal Melanoma Infiltration Boundaries Using Multiphoton Microscopy Combined With Image Analysis"],"prefix":"10.1002","volume":"19","author":[{"given":"Linjing","family":"Shi","sequence":"first","affiliation":[{"name":"School of Medical Technology and Engineering Fujian Medical University  Fuzhou China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liwen","family":"Hu","sequence":"additional","affiliation":[{"name":"Department of Pathology The First Affiliated Hospital of Fujian Medical University  Fuzhou 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