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Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble Using a Polarized Vortex Point Spread Function

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Download Hi-Res ImageDownload to MS-PowerPointCite This:J. Phys. Chem. B 2021, 125, 46, 12718-12729
    B: Biophysical and Biochemical Systems and Processes

    Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble Using a Polarized Vortex Point Spread Function
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    • Tianben Ding
      Tianben Ding
      Department of Electrical and Systems Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, United States
      Center for Science and Engineering of Living Systems, Washington University in St. Louis, St. Louis, Missouri 63130, United States
      More by Tianben Ding
      Orcidhttps://orcid.org/0000-0003-0710-2344
    • Matthew D. Lew*
      Matthew D. Lew
      Department of Electrical and Systems Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, United States
      Center for Science and Engineering of Living Systems, Washington University in St. Louis, St. Louis, Missouri 63130, United States
      Institute of Materials Science and Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, United States
      *Email: [email protected]. Phone: +1 (314)935 6790.
      More by Matthew D. Lew
      Orcidhttps://orcid.org/0000-0002-5614-3292
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    The Journal of Physical Chemistry B

    Cite this: J. Phys. Chem. B 2021, 125, 46, 12718–12729
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    https://doi.org/10.1021/acs.jpcb.1c08073
    Published November 12, 2021
    Copyright © 2021 American Chemical Society
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    Abstract

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    Within condensed matter, single fluorophores are sensitive probes of their chemical environments, but it is difficult to use their limited photon budget to image precisely their positions, 3D orientations, and rotational diffusion simultaneously. We demonstrate the polarized vortex point spread function (PSF) for measuring these parameters, including characterizing the anisotropy of a molecule’s wobble, simultaneously from a single image. Even when imaging dim emitters (∼500 photons detected), the polarized vortex PSF can obtain 12 nm localization precision, 4°–8° orientation precision, and 26° wobble precision. We use the vortex PSF to measure the emission anisotropy of fluorescent beads, the wobble dynamics of Nile red (NR) within supported lipid bilayers, and the distinct orientation signatures of NR in contact with amyloid-beta fibrils, oligomers, and tangles. The unparalleled sensitivity of the vortex PSF transforms single-molecule microscopes into nanoscale orientation imaging spectrometers, where the orientations and wobbles of individual probes reveal structures and organization of soft matter that are nearly impossible to perceive by using molecular positions alone.

    Copyright © 2021 American Chemical Society

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.1c08073.

    • Additional experimental notes on the optical instrumentation, procedure of optical aberration retrieval, analysis and processing of SMOLM images, and generation of synthetic data; supporting figures providing characterization of the polarized vortex PSF and comparison of the Cramér–Rao bound of different PSFs; Monte Carlo simulations evaluating the performance of the polarized vortex PSF with the RoSE-O estimator; retrieved optical aberrations in the polarized imaging system; quantitative characterization of NR blinking in SLBs and additional orientation measurements of NR molecules on amyloid oligomers and fibrils; and table comparing localization, orientation, and rotational diffusion statistics of SMOLM data estimated by the polarized vortex PSF (PDF)

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    This article is cited by 35 publications.

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    2. Liam A. Koch, Megan K. Dunlap, Duncan P. Ryan, James H. Werner, Peter M. Goodwin, Christopher M. Green, Sebastián A. Díaz, Igor L. Medintz, Kimihiro Susumu, Michael H. Stewart, Martin P. Gelfand, Alan Van Orden. Super-Resolved Fluorescence Lifetime Imaging of Single Cy3 Molecules and Quantum Dots Using Time-Correlated Single Photon Counting with a Four-Pixel Fiber Optic Array Camera. The Journal of Physical Chemistry A 2025, 129 (1) , 3-13. https://doi.org/10.1021/acs.jpca.4c05143
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    9. Aranyak Sarkar, Vinu Namboodiri, Manoj Kumbhakar. Single-Molecule Orientation Imaging Reveals Two Distinct Binding Configurations on Amyloid Fibrils. The Journal of Physical Chemistry Letters 2023, 14 (21) , 4990-4996. https://doi.org/10.1021/acs.jpclett.3c00823
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    21. Tingting Wu, Weiyan Zhou, Jai S. Rudra, Rohit V. Pappu, Matthew D. Lew, , . 6D single-fluorogen orientation-localization microscopy for elucidating the architecture of beta-sheet assemblies and biomolecular condensates. 2024, 2. https://doi.org/10.1117/12.3005992
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    Cite this: J. Phys. Chem. B 2021, 125, 46, 12718–12729
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.jpcb.1c08073
    Published November 12, 2021
    Copyright © 2021 American Chemical Society
    Request reuse permissions

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