Abstract
Fluorescence-based single-molecule techniques have become widely used tools to reveal dynamic processes of biomolecules and elucidate their molecular mechanisms. However, the concentration upper limit of labeled species that can be used in single-molecule fluorescence measurements is at the low nm range, which is below the Michaelis constants of many enzymatic reactions and physiological concentrations of many biomolecules. Such discrepancy limits the application of single-molecule fluorescence tools. Several techniques have been developed to break the concentration barrier. In this Concept, we focus on reviewing fundamental principles of these techniques and wish to inspire development of new and better tools to achieve this goal.
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