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  Optimal precision and accuracy in 4Pi-STORM using dynamic spline PSF models

Bates, M., Keller-Findeisen, J., Przybylski, A., Hüper, A., Stephan, T., Ilgen, P., et al. (2022). Optimal precision and accuracy in 4Pi-STORM using dynamic spline PSF models. Nature Methods, 19(5), 603-612. doi:10.1038/s41592-022-01465-8.

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 Creators:
Bates, M.1, Author           
Keller-Findeisen, J.1, Author           
Przybylski, A.1, Author           
Hüper, A., Author
Stephan, T.2, Author           
Ilgen, P.2, Author           
Cereceda Delgado, A.R.1, Author           
D’Este, E., Author
Egner, A., Author
Jakobs, S.2, Author           
Sahl, S. J.1, Author           
Hell, S. W.1, Author                 
Affiliations:
1Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Max Planck Society, ou_3350048              
2Research Group of Mitochondrial Structure and Dynamics, Max Planck Institute for Multidisciplinary Sciences, Max Planck Society, ou_3350049              

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Free keywords: Fluorescence imaging; Single-molecule biophysics; Super-resolution microscopy
 Abstract: 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 capa-bilities by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.

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Language(s): eng - English
 Dates: 2022-05-162022-05
 Publication Status: Published in print
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 Rev. Type: Peer
 Identifiers: DOI: 10.1038/s41592-022-01465-8
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Title: Nature Methods
Source Genre: Journal
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Pages: - Volume / Issue: 19 (5) Sequence Number: - Start / End Page: 603 - 612 Identifier: ISSN: 1548-7091
ISSN: 1548-7105