Can DyeCycling break the photobleaching limit in single-molecule FRET?

Benjamin Vermeer, Sonja Schmid*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

8 Citations (Scopus)


Biomolecular systems, such as proteins, crucially rely on dynamic processes at the nanoscale. Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that control biomolecular systems. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to observe in real-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states. Currently, this technique is fundamentally limited by irreversible photobleaching, causing the untimely end of the experiment and thus, a narrow temporal bandwidth of ≤ 3 orders of magnitude. Here, we introduce “DyeCycling”, a measurement scheme with which we aim to break the photobleaching limit in smFRET. We introduce the concept of spontaneous dye replacement by simulations, and as an experimental proof-of-concept, we demonstrate the intermittent observation of a single biomolecule for one hour with a time resolution of milliseconds. Theoretically, DyeCycling can provide > 100-fold more information per single molecule than conventional smFRET. We discuss the experimental implementation of DyeCycling, its current and fundamental limitations, and specific biological use cases. Given its general simplicity and versatility, DyeCycling has the potential to revolutionize the field of time-resolved smFRET, where it may serve to unravel a wealth of biomolecular dynamics by bridging from milliseconds to the hour range. [Figure not available: see fulltext.]

Original languageEnglish
Pages (from-to)9818-9830
JournalNano Research
Issue number11
Early online date13 May 2022
Publication statusPublished - Nov 2022


  • biomolecular dynamics
  • conformational changes
  • photobleaching
  • single-molecule fluorescence resonance energy transfer (smFRET)
  • single-molecule kinetics


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