Bruggeman, E. orcid.org/0000-0001-6100-1443, Zhang, O. orcid.org/0000-0001-7318-2130, Needham, L.-M. et al. (12 more authors) (2024) POLCAM: instant molecular orientation microscopy for the life sciences. Nature Methods, 21. pp. 1873-1883. ISSN 1548-7091
Abstract
Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, which can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field-of-view errors, used simulations to optimize experimental design and developed a fast algorithm based on Stokes parameter estimation that can operate over 1,000-fold faster than the state of the art, enabling near-instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study α-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2024 The Authors. 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: | Humans; Software; Algorithms; Single Molecule Imaging; Microscopy, Fluorescence; Image Processing, Computer-Assisted; Animals; alpha-Synuclein; Actin Cytoskeleton; Cell Membrane; Biological Science Disciplines |
Dates: |
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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: | 31 Oct 2024 16:39 |
Last Modified: | 31 Oct 2024 16:39 |
Published Version: | http://dx.doi.org/10.1038/s41592-024-02382-8 |
Status: | Published |
Publisher: | Springer Science and Business Media LLC |
Refereed: | Yes |
Identification Number: | 10.1038/s41592-024-02382-8 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:219063 |