Year: 2024
Type: article
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.
Source: Nature Methods
Institutions: University of Cambridge, Aligning Science Across Parkinson's, Washington University in St. Louis, University of Wisconsin–Madison, Laboratoire de Biophotonique et Pharmacologie, Université de Strasbourg, University of Oxford, MRC Weatherall Institute of Molecular Medicine, Cambridge Advanced Imaging Centre
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $12,290
OpenAlex ID: W4403195592