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Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry

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  • Robert P J Nieuwenhuizen
  • Mark Bates
  • Anna Szymborska
  • Keith A Lidke
  • Bernd Rieger
  • Sjoerd Stallinga

Abstract

Quantification in localization microscopy with reversibly switchable fluorophores is severely hampered by the unknown number of switching cycles a fluorophore undergoes and the unknown stoichiometry of fluorophores on a marker such as an antibody. We overcome this problem by measuring the average number of localizations per fluorophore, or generally per fluorescently labeled site from the build-up of spatial image correlation during acquisition. To this end we employ a model for the interplay between the statistics of activation, bleaching, and labeling stoichiometry. We validated our method using single fluorophore labeled DNA oligomers and multiple-labeled neutravidin tetramers where we find a counting error of less than 17% without any calibration of transition rates. Furthermore, we demonstrated our quantification method on nanobody- and antibody-labeled biological specimens.

Suggested Citation

  • Robert P J Nieuwenhuizen & Mark Bates & Anna Szymborska & Keith A Lidke & Bernd Rieger & Sjoerd Stallinga, 2015. "Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry," PLOS ONE, Public Library of Science, vol. 10(5), pages 1-18, May.
    • Handle: RePEc:plo:pone00:0127989
    DOI: 10.1371/journal.pone.0127989
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