In Vivo Two‐Photon 3D Fluorescence Angiography of Uterus by Silica Enhanced Aggregation‐Induced Emission Nanoparticles

Abstract The uterine vascular system is essential for female reproductive function, but its structural complexity challenges conventional two‐dimensional (2D) analysis. Herein, a three‐dimensional (3D) fluorescence angiography strategy is developed using aggregation‐induced emission (AIE) based nanoparticles (NPs) combined with two‐photon microscopy. An AIE fluorophore (AIEgen AF ) is developed, exhibiting near‐infrared emission at 715 nm. AF@SiO 2 NPs is engineered by encapsulating AIEgen AF within a hydrophobic silica core. Compared to micellar counterpart ( AF@F127 NPs ), AF@SiO 2 NPs exhibits enhanced fluorescence performance: a 2.99‐fold higher quantum yield (48.76%) and a 4.37‐fold greater two‐photon absorption cross‐section (266.56 GM). The silica surface of AF@SiO 2 NPs is further coated with dextran to improve biocompatibility and “stealth properties” in blood circulation. The obtained AF@SiO 2 ‐Dex NPs show minimal cytotoxicity and systemic toxicity. For in vivo imaging, a minimally invasive device is developed to expose the murine uterus. Following intravenous administration of AF@SiO 2 ‐Dex NPs , the uterine vascular network and collagen fibers are visualized using two‐photon excitation fluorescence (TPEF) and second harmonic generation (SHG), respectively. This approach achieves an effective imaging depth exceeding 450 µm in tissue (signal‐to‐noise ratio >3). Key vascular parameters, such as vessel morphology, density, blood flow, and permeability, are systematacially analyzed.