Shortwave infrared fluorofluorophores for multicolor in vivo imaging

Developing chemical tools to detect and influence biological processes in complex systems is a cornerstone of the field of chemical biology. Here we combine two tools which rely on orthogonality– perfluorocarbons and multiplexed shortwave infrared fluorescence imaging– to track the biodistribution of nanoemulsions in real time in living mice. Drawing inspiration from fluorous and shortwave infrared (SWIR) fluorophore development, we prepared two SWIR-emissive chromenylium polymethine dyes that are soluble in perfluorocarbons. These are the most red-shifted fluorous fluorophores– also known as “fluorofluorophores”– to date. After characterizing the fluorofluorophores, their in vivo utility was demonstrated through tracking perfluorocarbon nanoemulsion biodistribution in mice. We used an excitation-multiplexed approach to visualize two variables simultaneously, which halves the number of animals necessary per experimental group. These experiments provided insight into the importance of size and identity of the surfactant in biodistribution profiles in addition to demonstrating the success of fluorofluorophores for imaging perfluorocarbons in vivo.