Counterion-Enhanced Brightness of Fluorous-Soluble Heptamethine Cyanine Dyes for Near- and Shortwave Infrared Fluorescence Imaging

Fluorescence imaging across the near-infrared (NIR, 700–1000 nm) and shortwave infrared (SWIR, 1000–2000 nm) regions offers significant advantages for biomedical applications. Over the past decade, we have advanced a unique class of fluorophores–deemed fluorofluorophores–that are soluble in the fluorous phase. The unique properties of the fluorous phase, including low polarizability and high oxygen content, render this medium challenging for fluorophore brightness and photostability. However, the low dielectric constant of perfluorocarbon solvents causes the counterion to play a significant role in the resulting photophysical properties, offering a nontraditional avenue for fluorophore optimization. Here, we demonstrate that counterion exchange provides a straightforward strategy to enhance the brightness of two fluorous-soluble heptamethine cyanine dyes for NIR and SWIR imaging. Exchanging a chloride counterion with bulkier fluorinated aryl borate counterions boosts the brightness up to 10-fold and photostability up to 60-fold in perfluorooctyl bromide. We showcase the utility of these bright, NIR fluorofluorophores for imaging perfluorocarbon nanoemulsion uptake in macrophage cells, visualizing droplet actuation for mechanobiology studies in zebrafish, and mapping vasculature using high-resolution SWIR detection in mice. Overall, this simple modification provides a practical approach to optimize fluorofluorophores for in vivo imaging without the need to redesign the entire fluorophore scaffold.