POLCAM: instant molecular orientation microscopy for the life sciences
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...
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| Veröffentlicht in: | Nature methods 2024-10, Vol.21 (10), p.1873-1883 |
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| creator | Bruggeman, Ezra Zhang, Oumeng Needham, Lisa-Maria Körbel, Markus Daly, Sam Cheetham, Matthew Peters, Ruby Wu, Tingting Klymchenko, Andrey S. Davis, Simon J. Paluch, Ewa K. Klenerman, David Lew, Matthew D. O’Holleran, Kevin Lee, Steven F. |
| description | 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.
Combining localization and polarization microscopy can yield detailed insights into subcellular structures. POLCAM uses a polarization camera and wide-field microscopy for rapid measurement of super-resolution orientation imaging in live cells. |
| doi_str_mv | 10.1038/s41592-024-02382-8 |
| format | Article |
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Combining localization and polarization microscopy can yield detailed insights into subcellular structures. 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| subjects | 631/1647/245/2225 631/1647/328 631/57/2265 Actin Algorithms Anisotropy Bioinformatics Biological Microscopy Biological Techniques Biomedical and Life Sciences Biomedical Engineering/Biotechnology Cameras Cytoskeleton Design of experiments Design optimization Experimental design Fibrils Image analysis Image processing Image resolution Life Sciences Localization Lymphocytes Lymphocytes T Mammalian cells Microscopy Orientation Parameter estimation Polarization Proteomics Software Stokes parameters Synuclein |
| title | POLCAM: instant molecular orientation microscopy for the life sciences |
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