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Optica, Optics Express, 20(30), p. 36761, 2022

DOI: 10.1364/oe.470146

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Deep-SMOLM: deep learning resolves the 3D orientations and 2D positions of overlapping single molecules with optimal nanoscale resolution

Journal article published in 2022 by Tingting Wu ORCID, Peng Lu ORCID, Md Ashequr Rahman, Xiao Li, Matthew D. Lew ORCID
This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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Data provided by SHERPA/RoMEO

Abstract

Dipole-spread function (DSF) engineering reshapes the images of a microscope to maximize the sensitivity of measuring the 3D orientations of dipole-like emitters. However, severe Poisson shot noise, overlapping images, and simultaneously fitting high-dimensional information–both orientation and position–greatly complicates image analysis in single-molecule orientation-localization microscopy (SMOLM). Here, we report a deep-learning based estimator, termed Deep-SMOLM, that achieves superior 3D orientation and 2D position measurement precision within 3% of the theoretical limit (3.8° orientation, 0.32 sr wobble angle, and 8.5 nm lateral position using 1000 detected photons). Deep-SMOLM also demonstrates state-of-art estimation performance on overlapping images of emitters, e.g., a 0.95 Jaccard index for emitters separated by 139 nm, corresponding to a 43% image overlap. Deep-SMOLM accurately and precisely reconstructs 5D information of both simulated biological fibers and experimental amyloid fibrils from images containing highly overlapped DSFs at a speed ~10 times faster than iterative estimators.