Enhanced Accuracy of Single-Molecule Diffusion Measurements with a Photocleavable Energy-Transfer Dyad

A photocleavable energy‐transfer dyad was synthesized, characterized, and applied to single‐molecule fluorescence microscopy. After photocleavage, a combination of independent two‐color single‐molecule tracking and analysis of single‐molecule energy‐transfer efficiencies allows the determination of the temporal evolution of the relative distances between both fragments from the nm to the μm scale. This gives access to a broad range of diffusion coefficients.