Size‐Controlled Self‐Assembly for Bimodal In Vivo Imaging

Abstract Contrast agents (CAs) are essential in biomedical imaging to aid in the diagnosis and therapy monitoring of disease. However, they are typically restricted to one imaging modality and have fixed properties such as size, shape, toxicity profile, or photophysical characteristics, which hampers a comprehensive view of biological processes. Herein, rationally designed dye assemblies are introduced as a unique CA platform for simultaneous multimodal and multiscale biomedical imaging. To this end, a series of amphiphilic aza‐BODIPY dyes are synthesized with varying hydrophobic domains ( C 1 , C 8 , C 12, and C 16 ) that self‐assemble in aqueous media into nanostructures of tunable size (50 nm–1 µm) and photophysical properties. While C 1 exhibits oblique‐type exciton coupling and negligible emission, C 8 ‐ C 16 bearing longer alkyl chains undergo J ‐type aggregation with NIR absorption and emission and excellent photoacoustic properties. Given these advantageous features, aza‐BODIPY specific, semi‐quantitative fluorescence reflectance and photoacoustic imaging both in vitro and in vivo are established. Additionally, in vitro cell viability as well as murine in vivo biodistribution analysis with ex vivo validation showed excellent biocompatibility and a size‐dependent biodistribution of nanostructures to different organ beds. These results broaden the scope of aqueous self‐assembly to multimodal imaging and highlight its great potential for rationalizing numerous biomedical questions.