Targetable Conformationally Restricted Cyanines Enable Photon-Count Limited Applications

Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to parent species in a range of contexts including their use on a DNA-nano-antenna, in single-molecule Förster resonance energy transfer (FRET) applications, far-red fluorescence lifetime imaging microscopy (FLIM), and single-molecule localization microscopy. These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.