Enhanced accuracy of single-molecule diffusion measurements with a photocleavable energy-transfer dyad

Abstract

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.

Keywords: Förster resonance energy transfer; diffusion; fluorescence microscopy; photolabile groups; single-molecule spectroscopy.