Optimal precision and accuracy in 4Pi-STORM using dynamic spline PSF models.
Journal
Nature methods
ISSN: 1548-7105
Titre abrégé: Nat Methods
Pays: United States
ID NLM: 101215604
Informations de publication
Date de publication:
05 2022
05 2022
Historique:
received:
28
05
2021
accepted:
23
03
2022
pubmed:
17
5
2022
medline:
24
5
2022
entrez:
16
5
2022
Statut:
ppublish
Résumé
Coherent fluorescence imaging with two objective lenses (4Pi detection) enables single-molecule localization microscopy with sub-10 nm spatial resolution in three dimensions. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation and the challenging nature of the data analysis. Here we report the development of a 4Pi-STORM microscope, which obtains optimal resolution and accuracy by modeling the 4Pi point spread function (PSF) dynamically while also using a simpler optical design. Dynamic spline PSF models incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal evolution of the optical system. Our method reaches the theoretical limits for precision and minimizes phase-wrapping artifacts by making full use of the information content of the data. 4Pi-STORM achieves a near-isotropic three-dimensional localization precision of 2-3 nm, and we demonstrate its capabilities by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.
Identifiants
pubmed: 35577958
doi: 10.1038/s41592-022-01465-8
pii: 10.1038/s41592-022-01465-8
pmc: PMC9119851
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
603-612Informations de copyright
© 2022. The Author(s).
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