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  Quantitative localization microscopy: Effects of photophysics and labeling stoichiometry.

Nieuwenhuizen, R. P. J., Bates, M., Szymborska, A., Lidke, K. A., Rieger, B., & Stallinga, S. (2015). Quantitative localization microscopy: Effects of photophysics and labeling stoichiometry. PLoS One, 10(5): e0127989. doi:10.1371/journal.pone.0127989.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0027-A688-A Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-6F0B-3
Genre: Journal Article

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Nieuwenhuizen, R. P. J., Author
Bates, M.1, Author              
Szymborska, A., Author
Lidke, K. A., Author
Rieger, B., Author
Stallinga, S., Author
Affiliations:
1Department of NanoBiophotonics, MPI for biophysical chemistry, Max Planck Society, ou_578627              

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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.

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Language(s): eng - English
 Dates: 2015-05-20
 Publication Status: Published online
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 Table of Contents: -
 Rev. Method: Peer
 Identifiers: DOI: 10.1371/journal.pone.0127989
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Title: PLoS One
Source Genre: Journal
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Pages: 18 Volume / Issue: 10 (5) Sequence Number: e0127989 Start / End Page: - Identifier: -