Enhancing Photostability of Fluorescent Dye-Attached Molecular Machines at Air–Glass Interface Using Cyclooctatetraene

Single-molecule fluorescence microscopy at an air–solid interface severely suffers from photobleaching. In this study, we evaluated using a triplet-state quencher cyclooctatetraene (COT) group to enhance the photostability of cy5-attached molecular machines. For single-dye-modified nanocars, the photobleaching lifetime of the dye was extended by 2.1 times after the attachment of the COT group. For two-COT-two-dye-attached unimolecular submersible nanomachine (USN) molecules, both one-step and two-step photobleaching of the dyes were observed, similar to those cy5-USNs without COT protection. The fraction of one-step photobleaching events was nearly a constant under different laser powers, indicating that one-step photobleaching is a single-photon process and that the product of the first photobleaching event destroys the second dye. The COT-cy5-USNs showed a larger fraction of two-step photobleaching events as compared to cy5-USNs, indicating that the COT group provides further protection for the second dye from the oxidative product generated in the first dye photobleaching process. Overall, the protected, doubly labeled COT-cy5-USN molecules have a total photobleaching lifetime extended by 2.4 times over unprotected cy5-USN molecules or by 3.3 times over unprotected, single cy5-labeled molecules. This study shows the potentials and the limits of using triplet quencher COT to protect fluorescent dyes at the air–glass interface.