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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 μ m  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.

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