High-density volumetric super-resolution microscopy

Abstract

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce biological throughput and applicability by requiring lower labeling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We demonstrate this optical robustness experimentally with high accuracy ( > 99.2 ± 0.1%, 0.1 locs μm−2) and sensitivity ( > 86.6 ± 0.9%, 0.1 locs μm−2) through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high-density volumetric imaging (0.15 locs μm−2) in dense cytosolic tubulin datasets.

Metadata

Item Type:	Article
Authors/Creators:	Daly, S. https://orcid.org/0000-0002-8559-3161 Ferreira Fernandes, J. https://orcid.org/0000-0002-8161-6709 Bruggeman, E. https://orcid.org/0000-0001-6100-1443 Handa, A. Peters, R. Benaissa, S. Zhang, B. Beckwith, J.S. https://orcid.org/0000-0003-4726-230X Sanders, E.W. https://orcid.org/0000-0002-8972-7702 Sims, R.R. https://orcid.org/0000-0001-9864-6405 Klenerman, D. https://orcid.org/0000-0001-7116-6954 Davis, S.J. O’Holleran, K. Lee, S.F. https://orcid.org/0000-0003-4492-5139
Copyright, Publisher and Additional Information:	© Crown 2024. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Keywords:	Microscopy; Imaging, Three-Dimensional; Single Molecule Imaging; Nanotechnology
Dates:	Published: 2 March 2024 Published (online): 2 March 2024 Accepted: 1 February 2024 Submitted: 1 May 2023
Institution:	The University of Sheffield
Academic Units:	The University of Sheffield > Faculty of Science (Sheffield) > School of Mathematical and Physical Sciences
Depositing User:	Symplectic Sheffield
Date Deposited:	30 Jan 2025 09:38
Last Modified:	30 Jan 2025 09:38
Status:	Published
Publisher:	Springer Science and Business Media LLC
Refereed:	Yes
Identification Number:	10.1038/s41467-024-45828-5
Related URLs:	PubMed URL Dataset Software or Code Software or Code
Open Archives Initiative ID (OAI ID):	oai:eprints.whiterose.ac.uk:222403

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High-density volumetric super-resolution microscopy

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High-density volumetric super-resolution microscopy

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