On the Mechanisms of Cyanine Fluorophore Photostabilization

Cyanine fluorophores exhibit greatly improved photostability when covalently linked to stabilizers, such as cyclooctatetraene (COT), nitrobenzyl alcohol (NBA) or Trolox. However, the mechanism by which photostabilization is mediated has yet to be determined. Here we present spectroscopic evidence that COT, when covalently linked to Cy5, substantially reduces the lifetime of the Cy5 triplet state, and that the degree of triplet state quenching correlates with enhancements in photostability observed in single-molecule fluorescence measurements. By contrast, NBA and Trolox did not quench the Cy5 triplet state under our conditions suggesting that their mechanism of photostabilization is different from COT and does not target the fluorophore triplet state directly. These findings provide insights into the mechanisms of fluorophore photostabilization that may lead to improved fluorophore designs for biological imaging applications.