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Robust and bias-free localization of individual fixed dipole emitters achieving the Cramér Rao bound for applications in cryo-single molecule localization microscopy

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  • Fabian Hinterer
  • Magdalena C Schneider
  • Simon Hubmer
  • Montserrat López-Martinez
  • Philipp Zelger
  • Alexander Jesacher
  • Ronny Ramlau
  • Gerhard J Schütz

Abstract

Single molecule localization microscopy (SMLM) has the potential to resolve structural details of biological samples at the nanometer length scale. Compared to room temperature experiments, SMLM performed under cryogenic temperature achieves higher photon yields and, hence, higher localization precision. However, to fully exploit the resolution it is crucial to account for the anisotropic emission characteristics of fluorescence dipole emitters with fixed orientation. In case of slight residual defocus, localization estimates may well be biased by tens of nanometers. We show here that astigmatic imaging in combination with information about the dipole orientation allows to extract the position of the dipole emitters without localization bias and down to a precision of 1 nm, thereby reaching the corresponding Cramér Rao bound. The approach is showcased with simulated data for various dipole orientations, and parameter settings realistic for real life experiments.

Suggested Citation

  • Fabian Hinterer & Magdalena C Schneider & Simon Hubmer & Montserrat López-Martinez & Philipp Zelger & Alexander Jesacher & Ronny Ramlau & Gerhard J Schütz, 2022. "Robust and bias-free localization of individual fixed dipole emitters achieving the Cramér Rao bound for applications in cryo-single molecule localization microscopy," PLOS ONE, Public Library of Science, vol. 17(2), pages 1-15, February.
    • Handle: RePEc:plo:pone00:0263500
    DOI: 10.1371/journal.pone.0263500
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    1. Christiaan N. Hulleman & Rasmus Ø. Thorsen & Eugene Kim & Cees Dekker & Sjoerd Stallinga & Bernd Rieger, 2021. "Simultaneous orientation and 3D localization microscopy with a Vortex point spread function," Nature Communications, Nature, vol. 12(1), pages 1-14, December.
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