Quantitative localization microscopy: effects of photophysics and labeling stoichiometry.

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

Index: PLoS ONE 10 , e0127989, (2015)

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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.