Far‐Red Aggregation‐Induced Emission Hydrogel‐Reinforced Tissue Clearing for 3D Vasculature Imaging of Whole Lung and Whole Tumor

Abstract Understanding the vascular formation and distribution in metastatic lung tumors is a significant challenge due to autofluorescence, antibody/dye diffusion in dense tumor, and fluorophore stability when exposed to solvent‐based clearing agents. Here, an approach is presented that redefines 3D vasculature imaging within metastatic tumor, peritumoral lung tissue, and normal lung. Specifically, a far‐red aggregation‐induced emission nanoparticle with surface amino groups (termed as TSCN nanoparticle, TSCNNP) is designed for in situ formation of hydrogel (TSCNNP@Gel) inside vasculatures to provide structural support and enhance the fluorescence in solvent‐based tissue clearing method. Using this TSCNNP@Gel‐reinforced tissue clearing imaging approach, the critical challenges are successfully overcome and comprehensive visualization of the whole pulmonary vasculature up to 2 µm resolution is enabled, including its detailed examination in metastatic tumors. Importantly, features of tumor‐associated vasculature in 3D panoramic views are unveiled, providing the potential to determine tumor stages, predict tumor progression, and facilitate the histopathological diagnosis of various tumor types.