Shot-noise limited single-molecule FRET histograms: comparison between theory and experiments.
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 A. 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.
Nir
Eyal
E
Department of Chemistry and Biochemistry, and California NanoScience Institute, University of California at Los Angeles, Los Angeles, California 90095, USA.
Michalet
Xavier
X
Hamadani
Kambiz M
KM
Laurence
Ted A
TA
Neuhauser
Daniel
D
Kovchegov
Yevgeniy
Y
Weiss
Shimon
S
D000465:Algorithms; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D007834:Lasers; D009690:Nucleic Acid Conformation; D044367:Phase Transition
D004247:DNA