Innovative Strategies for Enhancing Signal-to-Noise Ratio in Single-molecule Imaging: The Influence of Molecular Motion

Single-molecule fluorescence imaging has extensively revealed the dynamic and structural characteristics of biomolecules. However, its application is limited by the upper concentration of fluorophore-tagged biomolecules, which is in the sub-ten nanomolar range. We found that the signal-to-noise ratio (SNR) in single-molecule fluorescence imaging is strongly influenced by the size of fluorophore-labeled molecules in solution. Our computational simulations suggest that the faster diffusion of background fluorophores can enhance the SNR of target molecules. Moreover, we identified that the molecular motion through fluid flow can improve SNR. This study provides a novel perspective by emphasizing the importance of molecular motion in SNR and propose a rapid barrier-free method to increase the upper concentration limit in single-molecule imaging.