Single-molecule orientation localisation microscopy using a polarisation camera Ezra Bruggeman 1 , Oumeng Zhang 2 , Lisa-Maria Needham 1,3 , Markus Körbel 1 , Sam Daly 1 , Matthew Cheetham 1 , Ruby Peters 1 , Tingting Wu 2 , Andrey S. Klymchenko 4 , Simon J. Davis 5 , Ewa K. Paluch 1 , David Klenerman 1 , Matthew D. Lew 2 , Kevin O’Holleran 1 and Steven F. Lee 1 1 University of Cambridge, UK, 2 Washington University, USA, 3 University of Wisconsin-Madison, USA, 4 Université de Strasbourg, France, 5 University of Oxford, UK Existing methods for measuring the orientation of single fluorescent molecules require optical setups and algorithms that can be prohibitively complex and slow. This limits the widespread adoption of molecular orientation estimation for biological applications.Here, we present POLCAM, an experimentally simplified single-molecule orientation estimation method based on polarised detection using a polarisation camera and a fast Stokes parameter estimation-based algorithm. POLCAM can be easily implemented by replacing the camera on any wide-field fluorescence microscope (Fig 1a). We evaluate the performance of polarisation cameras for SMOLM using simulations and dyes immobilised in polymer (Fig 1b), and apply the developed methodology to the study of alpha-synuclein fibrils (Fig 1c) in vitro and the actin cytoskeleton of mammalian cells.
Figure 1 a) Optical setup. b) Polarisation camera image of the emission of three SYTOX Orange molecules and corresponding polarisation colourmap (hue = angle of linear polarisation, saturation = degree of linear polarisation, value = intensity). c) Polarisation camera SMOLM reconstruction of alpha-synuclein fibrils. Rods specify the orientation of the emission dipole moments of Nile Red molecules.
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© The Author(s), 2023
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