In Situ Monitoring of Membrane Protein Dynamics Using High-Throughput Red-Light-Activated Single-Molecule Tracking

Single-molecule tracking offers nanometer resolution for studying individual molecule dynamics but is often limited by sparse labeling to avoid signal overlap. We present Red-Light-Activated Single-molecule Tracking (RE-LAST) strategy to address this challenge utilizing a photoactivatable probe, SiR670. SiR670 combines traditional silicon rhodamine with a photocage called SO, quenching fluorescence via photoinduced electron transfer (PET). Red light triggers SiR670 excitation, generating singlet oxygen that oxidizes the SO cage, halting PET and restoring fluorescence. RE-LAST used red light for both activation and imaging, eliminating harmful UV exposure. This method enables high-throughput single-molecule tracking, achieving approximately 9 times more tracks than conventional methods and allowing detailed classification of CD56 membrane protein motion. Furthermore, in situ imaging of single live cells revealed the effects of triplet quencher and oxygen scavenging system (OSS) on membrane protein dynamics. While triplet quenchers like Trolox had minimal impact on protein movement patterns, OSS significantly accelerated protein movement and increased the proportion of mobile proteins. This approach provides a comprehensive method for investigating membrane protein dynamics in living cells, contributing to further developments in cellular and molecular biology.