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.