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...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2015-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4439177?pdf=render |
| _version_ | 1828803010846785536 |
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| author | Robert P J Nieuwenhuizen Mark Bates Anna Szymborska Keith A Lidke Bernd Rieger Sjoerd Stallinga |
| author_facet | Robert P J Nieuwenhuizen Mark Bates Anna Szymborska Keith A Lidke Bernd Rieger Sjoerd Stallinga |
| author_sort | Robert P J Nieuwenhuizen |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-12T07:14:28Z |
| format | Article |
| id | doaj.art-0a474da770af4da4ad950a15a1a13b7e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-12T07:14:28Z |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-0a474da770af4da4ad950a15a1a13b7e2022-12-22T00:33:32ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01105e012798910.1371/journal.pone.0127989Quantitative localization microscopy: effects of photophysics and labeling stoichiometry.Robert P J NieuwenhuizenMark BatesAnna SzymborskaKeith A LidkeBernd RiegerSjoerd StallingaQuantification 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.http://europepmc.org/articles/PMC4439177?pdf=render |
| spellingShingle | Robert P J Nieuwenhuizen Mark Bates Anna Szymborska Keith A Lidke Bernd Rieger Sjoerd Stallinga Quantitative localization microscopy: effects of photophysics and labeling stoichiometry. PLoS ONE |
| title | 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_short | Quantitative localization microscopy: effects of photophysics and labeling stoichiometry. |
| title_sort | quantitative localization microscopy effects of photophysics and labeling stoichiometry |
| url | http://europepmc.org/articles/PMC4439177?pdf=render |
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