Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry

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

PLOS ONE, 2015, vol. 10, issue 5, 1-18

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.

Date: 2015
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Persistent link: https://EconPapers.repec.org/RePEc:plo:pone00:0127989

DOI: 10.1371/journal.pone.0127989

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