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Journal Article

Quantitative localization microscopy: Effects of photophysics and labeling stoichiometry.

MPS-Authors
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Bates,  M.
Department of NanoBiophotonics, MPI for biophysical chemistry, Max Planck Society;

Fulltext (public)

2166607.pdf
(Publisher version), 2MB

Supplementary Material (public)

2166607_Suppl.zip
(Supplementary material), 5MB

Citation

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.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0027-A688-A
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.