Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry

Title

Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry

 Author

Nieuwenhuizen, R.P.J.
Bates, M.
Szymborska, A.
Lidke, K.A.
Rieger, B.
Stallinga, S.

Faculty

Applied Sciences

 Department

ImPhys/Imaging Physics

 Date

2015-05-20

 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.

 Subject

stoichiometry
bleaching
fluorescence imaging
photons
absorption spectroscopy
fluorescence microscopy
oligomers
glass
OA-Fund TU Delft

 To reference this document use:

http://resolver.tudelft.nl/uuid:dc2964e9-2dea-4499-afa2-aae2f968c683

 Publisher

Public Library of Science

 ISSN

1932-6203

 Source

PLoS ONE, 10 (5), 2015

 Part of collection

Institutional Repository

 Document type

journal article

 Rights

© 2015 The Author(s)
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 credited.
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