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Keywords:

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