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

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@SiO₂ NPs is engineered by encapsulating AIEgen AF within a hydrophobic silica core. Compared to micellar counterpart (AF@F127 NPs), AF@SiO₂ 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@SiO₂ NPs is further coated with dextran to improve biocompatibility and "stealth properties" in blood circulation. The obtained AF@SiO₂-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@SiO₂-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.