Systematic assessment of burst impurity in confocal-based single-molecule fluorescence detection using Brownian motion simulations

Dolev Hagai, Eitan Lerner

Research output: Contribution to journalArticlepeer-review


Single-molecule fluorescence detection (SMFD) experiments are useful in distinguishing sub-populations of molecular species when measuring heterogeneous samples. One experimental platform for SMFD is based on a confocal microscope, where molecules randomly traverse an effective detection volume. The non-uniformity of the excitation profile and the random nature of Brownian motion, produce fluctuating fluorescence signals. For these signals to be distinguished from the background, burst analysis is frequently used. Yet, the relation between the results of burst analyses and the underlying information of the diffusing molecules is still obscure and requires systematic assessment. In this work we performed three-dimensional Brownian motion simulations of SMFD, and tested the positions at which molecules emitted photons that passed the burst analysis criteria for different values of burst analysis parameters. The results of this work verify which of the burst analysis parameters and experimental conditions influence both the position of molecules in space when fluorescence is detected and taken into account, and whether these bursts of photons arise purely from single molecules, or not entirely. Finally, we show, as an example, the effect of bursts that are not purely from a single molecule on the accuracy in single-molecule Förster resonance energy transfer measurements.

Original languageAmerican English
Article number2557
Issue number14
StatePublished - 2019


  • Brownian
  • Burst
  • Diffusion
  • Effective detection volume
  • Fluorescence
  • Photon rate
  • Point-spread function
  • Simulation
  • Single-molecule
  • Threshold

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Chemistry (miscellaneous)
  • Molecular Medicine
  • Pharmaceutical Science
  • Drug Discovery
  • Physical and Theoretical Chemistry
  • Organic Chemistry


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