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Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization

Alex von Diezmann1, Maurice Y Lee2, Matthew D Lew3

  • 1Department of Chemistry, Stanford University, Stanford, California 94305, USA.

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|March 15, 2016
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Summary

Accurate 3D super-resolution microscopy requires precise single-molecule localization. This study reveals and corrects field-dependent aberrations in 3D single-molecule imaging, improving nanoscale accuracy.

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Nanotechnology

Background:

  • Single-molecule localization microscopy (SMLM) offers subdiffraction imaging of molecular structures and dynamics.
  • Three-dimensional (3D) SMLM utilizes point spread function (PSF) engineering for depth information.
  • The field-dependent accuracy of 3D SMLM localization has not been thoroughly investigated.

Purpose of the Study:

  • To rigorously examine the nanoscale accuracy of localization across the field of view in 3D single-molecule microscopy.
  • To identify and quantify field-dependent aberrations affecting 3D localization precision.
  • To develop and validate a method for correcting these aberrations to enhance 3D imaging accuracy.

Main Methods:

  • Utilized regularly spaced subdiffraction apertures filled with fluorescent dyes as calibration standards.
  • Quantified field-dependent aberrations by measuring localization errors across the microscope's field of view.
  • Applied aberration correction strategies to engineered PSFs, including the double-helix and astigmatic PSFs.
  • Validated correction accuracy over an extended 3D focal volume.

Main Results:

  • Revealed significant field-dependent aberrations, ranging from 50-100 nm, across the 3D imaging volume.
  • Demonstrated successful correction of these aberrations to achieve localization accuracy below 25 nm.
  • Showcased the effectiveness of the correction method with both double-helix and astigmatic engineered PSFs.

Conclusions:

  • Field-dependent aberrations are a critical factor limiting nanoscale accuracy in 3D single-molecule microscopy.
  • The developed aberration correction method significantly improves localization precision over an extended 3D focal volume.
  • These findings are crucial for advancing 3D single-molecule tracking and super-resolution techniques requiring high spatial accuracy.