POLCAM: instant molecular orientation microscopy for the life sciences
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.
Authors
- Ezra Bruggeman (ORCID: https://orcid.org/0000-0001-6100-1443)
- Markus Körbel (ORCID: https://orcid.org/0000-0002-8344-8016)
- Ewa K. Paluch (ORCID: https://orcid.org/0000-0003-4691-2323)
- Oumeng Zhang (ORCID: https://orcid.org/0000-0001-7318-2130)
- Matthew D. Lew (ORCID: https://orcid.org/0000-0002-5614-3292)
- David A. Klenerman (ORCID: https://orcid.org/0000-0001-7116-6954)
- Ruby Peters (ORCID: https://orcid.org/0000-0003-2946-7504)
- Matthew R. Cheetham (ORCID: https://orcid.org/0000-0001-7412-2332)
- Steven F. Lee (ORCID: https://orcid.org/0000-0003-4492-5139)
- Simon J. Davis (ORCID: https://orcid.org/0000-0002-2280-1170)
- Lisa-Maria Needham (ORCID: https://orcid.org/0000-0002-8139-5862)
- Sam Daly (ORCID: https://orcid.org/0000-0002-8559-3161)
- Andrey S. Klymchenko (ORCID: https://orcid.org/0000-0002-2423-830X)
- Tingting Wu (ORCID: https://orcid.org/0000-0001-5736-4962)
- Kevin O’Holleran (ORCID: https://orcid.org/0000-0003-1039-2127)
Institutions
- University of Wisconsin–Madison (US)
- Washington University in St. Louis (US)
- University of Cambridge (GB)
- University of Oxford (GB)
- Laboratoire de Biophotonique et Pharmacologie (FR)
- MRC Weatherall Institute of Molecular Medicine (GB)
- Research Network (United States) (US)
- Université de Strasbourg (FR)
Publication Details
- Journal
- Nature Methods
- Published
- 2024-10-01
- DOI
- https://doi.org/10.1038/s41592-024-02382-8
- Citations
- 18
- Primary Topic
- Advanced Fluorescence Microscopy Techniques
- Type
- article
- Field-Weighted Citation Impact
- 11.92
Funders
- Michael J. Fox Foundation for Parkinson's Research