Shortwave Infrared Fluorofluorophores for Multicolor In Vivo Imaging**

Developing chemical tools to detect and influence biological processes is a cornerstone of chemical biology. Here we combine two tools which rely on orthogonality– perfluorocarbons and multiplexed shortwave infrared (SWIR) fluorescence imaging– to visualize nanoemulsions in real time in living mice. Drawing inspiration from fluorous and SWIR fluorophore development, we prepared two SWIR-emissive, fluorous-soluble chromenylium polymethine dyes. These are the most red-shifted fluorous fluorophores– “fluorofluorophores”—to date. After characterizing the dyes, their utility was demonstrated by tracking perfluorocarbon nanoemulsion biodistribution in vivo. Using an excitation-multiplexed approach to image two variables simultaneously, we gained insight into the importance of size and surfactant identity on biodistribution.