Water soluble chromenylium dyes for shortwave infrared imaging in mice

In vivo imaging using shortwave infrared light (SWIR, 1000-2000 nm) benefits from deeper penetration depths, decreased background autofluorescence, and high resolution. However, the development of biocompatible contrast agents for these low energy wavelengths has significant challenges. While there have been significant advances in SWIR chromophore scaffolds over the past 5 years, a major barrier for widespread utility of SWIR small molecule fluorophores is their hydrophobicity and tendency to form non-emissive aggregates. Here, we report a platform for generating a panel of soluble and functional dyes for SWIR imaging by late-stage functionalization of a fluorophore intermediate via click chemistry. The resulting fluorophores with sulfonate, ammonium or zwitterion functionalities are all water soluble with bright SWIR fluorescence in serum, allowing for fast imaging in mice. Specifically, the sulfonate-carrying derivative enables clear video-rate imaging of vasculature with as little as 0.05 nmol injected dye, and the ammonium-modified dye shows strong retention in cells that enables tracking of xenograft tumor growth. We further showcase the versatility of this design by incorporating phosphonate functionalities for imaging of bone in awake and moving mice. This modular design of functional SWIR fluorophores in water provides insights for facile derivatization of existing fluorophores to introduce solubility and bioactivity towards bioimaging applications.