Single‐Molecule Photoactivation FRET: A General and Easy‐To‐Implement Approach To Break the Concentration Barrier

Single-molecule fluorescence resonance energy transfer (sm-FRET) has become a widely used tool to reveal dynamic processes and molecule mechanisms hidden under ensemble measurements. However, the upper limit of fluorescent species used in sm-FRET is still orders of magnitude lower than the association affinity of many biological processes under physiological conditions. Herein, we introduce single-molecule photoactivation FRET (sm-PAFRET), a general approach to break the concentration barrier by using photoactivatable fluorophores as donors. We demonstrate sm-PAFRET by capturing transient FRET states and revealing new reaction pathways during translation using μm fluorophore labeled species, which is 2-3 orders of magnitude higher than commonly used in sm-FRET measurements. sm-PAFRET serves as an easy-to-implement tool to lift the concentration barrier and discover new molecular dynamic processes and mechanisms under physiological concentrations.