Thiolation for Enhancing Photostability of Fluorophores at the Single‐Molecule Level

Fluorescent probes are essential for single‐molecule imaging. However, their application in biological systems is often limited by the short photobleaching lifetime. To overcome this, we developed a novel thiolation strategy for squaraine dyes. By introducing thiolation of the central cyclobutene of squaraine (thio‐squaraine), we observed a ≈5‐fold increase in photobleaching lifetime. Our single‐molecule data analysis attributes this improvement to improved photostability resulting from thiolation. Interestingly, bulk measurements show rapid oxidation of thio‐squaraine to its oxo‐analogue under irradiation, giving the perception of inferior photostability. This discrepancy between bulk and single‐molecule environments can be ascribed to the factors in the latter, including larger intermolecular distances and restricted mobility, which reduce the interactions between a fluorophore and reactive oxygen species produced by other fluorophores, ultimately impacting photobleaching and photoconversion rate. We demonstrate the remarkable performance of thio‐squaraine probes in various imaging buffers, such as glucose oxidase with catalase (GLOX) and GLOX+trolox. We successfully employed these photostable probes for single‐molecule tracking of CD56 membrane protein and monitoring mitochondria movements in live neurons. CD56 tracking revealed distinct motion states and the corresponding protein fractions. This investigation is expected to propel the development of single‐molecule imaging probes, particularly in scenarios where bulk measurements show suboptimal performance. Single‐molecule imaging can elucidate the intricate biomolecular dynamics in vivo. We present a straightforward thiolation of squaraine that enhances its photobleaching lifetime by approximately fivefold at the single molecule level. This advancement allows for extended single‐molecule tracking of the membrane protein CD56 in live cells, effectively tripling the duration of our observations.