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Open AccessArticle

Systematic Assessment of Burst Impurity in Confocal-Based Single-Molecule Fluorescence Detection Using Brownian Motion Simulations

Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Faculty of Mathematics & Science, The Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel
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Author to whom correspondence should be addressed.
Molecules 2019, 24(14), 2557; https://doi.org/10.3390/molecules24142557
Received: 26 May 2019 / Revised: 5 July 2019 / Accepted: 12 July 2019 / Published: 13 July 2019
(This article belongs to the Special Issue Single-Molecule Fluorescence Spectroscopy)
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. View Full-Text
Keywords: single-molecule; fluorescence; burst; photon rate; effective detection volume; point-spread function; Brownian; diffusion; simulation; threshold single-molecule; fluorescence; burst; photon rate; effective detection volume; point-spread function; Brownian; diffusion; simulation; threshold
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Hagai, D.; Lerner, E. Systematic Assessment of Burst Impurity in Confocal-Based Single-Molecule Fluorescence Detection Using Brownian Motion Simulations. Molecules 2019, 24, 2557.

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  • Supplementary File 1:

    ZIP-Document (ZIP, 3887 KB)

  • Externally hosted supplementary file 1
    Doi: 10.5281/zenodo.3268780
    Link: https://doi.org/10.5281/zenodo.3268780
    Description: Systematic assessment of burst impurity in confocal-based single-molecule fluorescence detection using Brownian motion simulations - photon timetag simulation files
  • Externally hosted supplementary file 2
    Doi: 10.5281/zenodo.3268785
    Link: https://doi.org/10.5281/zenodo.3268785
    Description: Systematic assessment of burst impurity in confocal-based single-molecule fluorescence detection using Brownian motion simulations - Jupyter notebooks that document the photon timestamp simulations as well as their analysis by FRETbursts
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