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

Koen J A Martens1, Abbas Jabermoradi2, Suyeon Yang2

  • 1Laboratory 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.

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|August 4, 2020
PubMed
Summary
This summary is machine-generated.

New phasor-based single-molecule localization microscopy (pSMLM) methods enable fast 3D localization using complex point spread functions (PSFs). This advanced technique offers high precision for single-molecule imaging and tracking, even in challenging conditions.

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Area of Science:

  • Microscopy
  • Biophysics
  • Optical Engineering

Background:

  • Single-molecule localization microscopy (SMLM) uses engineered point spread functions (PSFs) for 3D localization.
  • Conventional Gaussian fitting struggles with complex PSFs from phase masks or deformable mirrors.

Purpose of the Study:

  • Integrate novel PSFs (double-helix, saddle-point, tetra-pod) into the phasor-based SMLM (pSMLM) framework.
  • Enable fast, sub-pixel accurate 3D localization of single-molecule emitters using advanced PSFs.

Main Methods:

  • Developed pSMLM for double-helix PSFs, using lobe rotation for z-localization.
  • Introduced circular-tangent pSMLM for saddle-point/tetra-pod PSFs via phasor-based deconvolution.
  • Experimentally realized saddle-point PSFs using deformable mirrors and Zernike modes.

Main Results:

  • Achieved precision and recall comparable to SMAP at typical organic fluorophore signal-to-noise ratios.
  • Demonstrated high localization rates: up to 15 kHz (double-helix) and 250 kHz (saddle-point/tetra-pod) on a standard CPU.
  • Integrated pSMLM into SMALL-LABS for single-particle imaging and tracking in noisy backgrounds.

Conclusions:

  • pSMLM provides a powerful hardware and software solution for advanced single-molecule studies.
  • The developed methods significantly enhance 3D localization capabilities in SMLM.
  • Offers high-speed, high-accuracy 3D super-resolution imaging for various biological applications.