POLCAM: instant molecular orientation microscopy for the life sciences
Ezra Bruggeman, Oumeng Zhang, Lisa-Maria Needham, Markus Körbel, Sam Daly, Matthew R. Cheetham, Ruby Peters, Tingting Wu, Andrey S. Klymchenko, Simon J. Davis, Ewa K. Paluch, David Klenerman, Matthew D. Lew, Kevin O’Holleran, Steven F. Lee
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.