title: Quantitative localization microscopy: effects of photophysics and labeling stoichiometry.
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.
authors.affiliation: Quantitative Imaging Group, Delft University of Technology, Delft, The Netherlands.
authors.forename: Robert P J
authors.initials: RP
authors.lastname: Nieuwenhuizen
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Bates
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Szymborska
authors.affiliation: Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Lidke
authors.affiliation: Quantitative Imaging Group, Delft University of Technology, Delft, The Netherlands.
authors.forename: Bernd
authors.initials: B
authors.lastname: Rieger
authors.affiliation: Quantitative Imaging Group, Delft University of Technology, Delft, The Netherlands.
authors.forename: Sjoerd
authors.initials: S
authors.lastname: Stallinga
mesh_terms: D000818:Animals; D002138:Calibration; D002522:Chlorocebus aethiops; D016000:Cluster Analysis; D005456:Fluorescent Dyes / Q000737:chemistry; D008027:Light; D008856:Microscopy, Fluorescence / Q000379:methods*; D051381:Rats; D013194:Staining and Labeling; D014709:Vero Cells
chemical_list: D005456:Fluorescent Dyes