Enhanced Single-Molecule Imaging via Intersystem Crossing Suppression

The red-emitting cyanine dye Cy5 is widely used in single-molecule imaging, but its utility is limited by triplet-state accumulation. This state facilitates energy transfer to molecular oxygen, producing reactive oxygen species (ROS) that lead to photobleaching. The triplet state also causes the dye's fluorescence intermittency and introduces temporal noise. Furthermore, Cy5 in this state is susceptible to nucleophilic attacks in cellular environments, resulting in an inactive off-state. To address these challenges, we engineered a series of phenyl-substituted meso-Cy5 derivatives to suppress intersystem crossing (ISC) and effectively reduce the triplet-state population. Theoretical calculations and transient absorption spectroscopy validated the suppression of ISC in these derivatives. Our lead compound, Cy5-N, featuring an electron-withdrawing nitro modification on the phenyl group, demonstrated a 790% increase in photostability and a 2.3-fold improvement in the signal-to-noise ratio (SNR) compared to conventional Cy5 in single-molecule imaging. Notably, Cy5-N maintained continuous emission even in the presence of the nucleophile β-mercaptoethanol (β-ME), indicating robust resistance to nucleophilic attacks. Using Cy5-N, we successfully performed long-term single-molecule tracking of membrane proteins in live U2OS cells, with enhanced SNR. This study introduces a novel molecular design strategy for enhancing cyanine dye performance in single-molecule imaging, offering substantial improvements in photostability and SNR with broad implications for bioimaging and sensing technologies.