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Integrating engineered point spread functions into the phasor-based single-molecule localization microscopy framework.

Authors
  • Martens, Koen J A1
  • Jabermoradi, Abbas2
  • Yang, Suyeon2
  • Hohlbein, Johannes3
  • 1 Laboratory of Biophysics, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands; Laboratory of Bionanotechnology, Wageningen University and Research, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands. , (Netherlands)
  • 2 Laboratory of Biophysics, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands. , (Netherlands)
  • 3 Laboratory of Biophysics, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands; Microspectroscopy Research Facility, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands. Electronic address: [email protected] , (Netherlands)
Type
Published Article
Journal
Methods
Publisher
Elsevier
Publication Date
Sep 01, 2021
Volume
193
Pages
107–115
Identifiers
DOI: 10.1016/j.ymeth.2020.07.010
PMID: 32745620
Source
Medline
Language
English
License
Unknown

Abstract

In single-molecule localization microscopy (SMLM), the use of engineered point spread functions (PSFs) provides access to three-dimensional localization information. The conventional approach of fitting PSFs with a single 2-dimensional Gaussian profile, however, often falls short in analyzing complex PSFs created by placing phase masks, deformable mirrors or spatial light modulators in the optical detection pathway. Here, we describe the integration of PSF modalities known as double-helix, saddle-point or tetra-pod into the phasor-based SMLM (pSMLM) framework enabling fast CPU based localization of single-molecule emitters with sub-pixel accuracy in three dimensions. For the double-helix PSF, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations. For the analysis of saddle-point or tetra-pod PSFs, we present a novel phasor-based deconvolution approach entitled circular-tangent pSMLM. Saddle-point PSFs were experimentally realized by placing a deformable mirror in the Fourier plane and modulating the incoming wavefront with specific Zernike modes. Our pSMLM software package delivers similar precision and recall rates to the best-in-class software package (SMAP) at signal-to-noise ratios typical for organic fluorophores and achieves localization rates of up to 15 kHz (double-helix) and 250 kHz (saddle-point/tetra-pod) on a standard CPU. We further integrated pSMLM into an existing software package (SMALL-LABS) suitable for single-particle imaging and tracking in environments with obscuring backgrounds. Taken together, we provide a powerful hardware and software environment for advanced single-molecule studies. Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

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