Shot-noise limited single-molecule FRET histograms: Comparison between theory and experiments

Eyal Nir, Xavier Michalet, Kambiz M. Hamadani, Ted A. Laurence, Daniel Neuhauser, Yevgeniy Kovchegov, Shimon Weiss

Research output: Contribution to journalArticlepeer-review

265 Scopus citations


We describe a simple approach and present a straightforward numerical algorithm to compute the best fit shot-noise limited proximity ratio histogram (PRH) in single-molecule fluorescence resonant energy transfer diffusion experiments. The key ingredient is the use of the experimental burst size distribution, as obtained after burst search through the photon data streams. We show how the use of an alternated laser excitation scheme and a correspondingly optimized burst search algorithm eliminates several potential artifacts affecting the calculation of the best fit shot-noise limited PRH. This algorithm is tested extensively on simulations and simple experimental systems. We find that dsDNA data exhibit a wider PRH than expected from shot noise only and hypothetically account for it by assuming a small Gaussian distribution of distances with an average standard deviation of 1.6 Å. Finally, we briefly mention the results of a future publication and illustrate them with a simple two-state model system (DNA hairpin), for which the kinetic transition rates between the open and closed conformations are extracted.

Original languageEnglish
Pages (from-to)22103-22124
Number of pages22
JournalJournal of Physical Chemistry B
Issue number44
StatePublished - 9 Nov 2006
Externally publishedYes

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry


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