Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry

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 nu...

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Main Authors:	Nieuwenhuizen, Robert P. J., Bates, Mark, Szymborska, Anna, Lidke, Keith A., Rieger, Bernd, Stallinga, Sjoerd
Format:	Online
Language:	English
Published:	Public Library of Science 2015
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4439177/

id	pubmed-4439177
recordtype	oai_dc
spelling	pubmed-44391772015-05-29 Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry Nieuwenhuizen, Robert P. J. Bates, Mark Szymborska, Anna Lidke, Keith A. Rieger, Bernd Stallinga, Sjoerd Research Article 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. Public Library of Science 2015-05-20 /pmc/articles/PMC4439177/ /pubmed/25992915 http://dx.doi.org/10.1371/journal.pone.0127989 Text en © 2015 Nieuwenhuizen et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Nieuwenhuizen, Robert P. J. Bates, Mark Szymborska, Anna Lidke, Keith A. Rieger, Bernd Stallinga, Sjoerd
spellingShingle	Nieuwenhuizen, Robert P. J. Bates, Mark Szymborska, Anna Lidke, Keith A. Rieger, Bernd Stallinga, Sjoerd Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry
author_facet	Nieuwenhuizen, Robert P. J. Bates, Mark Szymborska, Anna Lidke, Keith A. Rieger, Bernd Stallinga, Sjoerd
author_sort	Nieuwenhuizen, Robert P. J.
title	Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry
title_short	Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry
title_full	Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry
title_fullStr	Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry
title_full_unstemmed	Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry
title_sort	quantitative localization microscopy: effects of photophysics and labeling stoichiometry
description	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.
publisher	Public Library of Science
publishDate	2015
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4439177/
_version_	1613226196147896320

Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry

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