Spatially selective multiphoton photothermolysis guided by reflectance confocal microscopy for precise vessel closure in the mouse eye limbus

Corneal neovascularization (CNV) is characterized by abnormal vessel growth into the cornea, often impairing vision. Conventional laser therapies for CNV lack precision and risk collateral damage. We developed a multiphoton photothermolysis (MPP) approach using femtosecond (fs) laser-based two-photon absorption guided by real-time reflectance confocal microscopy. Two MPP laser treatment configurations were implemented: dual lasers for imaging (785 nm CW) and treatment (830 nm fs), and a single fs laser for both functions. We demonstrated that in the mouse eye limbus, MPP achieved selective vessel closure without collateral tissue damage. Results also indicate therapeutic effects are mediated by two- rather than one-photon absorption, highlighting MPP's potential as a precise and safe CNV treatment.