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Pointwise error estimates in localization microscopy

Pointwise localization of individual fluorophores is a critical step in super-resolution localization microscopy and single particle tracking. Although the methods are limited by the localization errors of individual fluorophores, the pointwise localization precision has so far been estimated using...

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Detalles Bibliográficos
Autores principales: Lindén, Martin, Ćurić, Vladimir, Amselem, Elias, Elf, Johan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418599/
https://www.ncbi.nlm.nih.gov/pubmed/28466844
http://dx.doi.org/10.1038/ncomms15115
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author Lindén, Martin
Ćurić, Vladimir
Amselem, Elias
Elf, Johan
author_facet Lindén, Martin
Ćurić, Vladimir
Amselem, Elias
Elf, Johan
author_sort Lindén, Martin
collection PubMed
description Pointwise localization of individual fluorophores is a critical step in super-resolution localization microscopy and single particle tracking. Although the methods are limited by the localization errors of individual fluorophores, the pointwise localization precision has so far been estimated using theoretical best case approximations that disregard, for example, motion blur, defocus effects and variations in fluorescence intensity. Here, we show that pointwise localization precision can be accurately estimated directly from imaging data using the Bayesian posterior density constrained by simple microscope properties. We further demonstrate that the estimated localization precision can be used to improve downstream quantitative analysis, such as estimation of diffusion constants and detection of changes in molecular motion patterns. Finally, the quality of actual point localizations in live cell super-resolution microscopy can be improved beyond the information theoretic lower bound for localization errors in individual images, by modelling the movement of fluorophores and accounting for their pointwise localization uncertainty.
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spelling pubmed-54185992017-07-06 Pointwise error estimates in localization microscopy Lindén, Martin Ćurić, Vladimir Amselem, Elias Elf, Johan Nat Commun Article Pointwise localization of individual fluorophores is a critical step in super-resolution localization microscopy and single particle tracking. Although the methods are limited by the localization errors of individual fluorophores, the pointwise localization precision has so far been estimated using theoretical best case approximations that disregard, for example, motion blur, defocus effects and variations in fluorescence intensity. Here, we show that pointwise localization precision can be accurately estimated directly from imaging data using the Bayesian posterior density constrained by simple microscope properties. We further demonstrate that the estimated localization precision can be used to improve downstream quantitative analysis, such as estimation of diffusion constants and detection of changes in molecular motion patterns. Finally, the quality of actual point localizations in live cell super-resolution microscopy can be improved beyond the information theoretic lower bound for localization errors in individual images, by modelling the movement of fluorophores and accounting for their pointwise localization uncertainty. Nature Publishing Group 2017-05-03 /pmc/articles/PMC5418599/ /pubmed/28466844 http://dx.doi.org/10.1038/ncomms15115 Text en Copyright © 2017, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Lindén, Martin
Ćurić, Vladimir
Amselem, Elias
Elf, Johan
Pointwise error estimates in localization microscopy
title Pointwise error estimates in localization microscopy
title_full Pointwise error estimates in localization microscopy
title_fullStr Pointwise error estimates in localization microscopy
title_full_unstemmed Pointwise error estimates in localization microscopy
title_short Pointwise error estimates in localization microscopy
title_sort pointwise error estimates in localization microscopy
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418599/
https://www.ncbi.nlm.nih.gov/pubmed/28466844
http://dx.doi.org/10.1038/ncomms15115
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