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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics


  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 9, Iss. 3 — Mar. 6, 2014

Correction of depth-dependent aberrations in 3D single-molecule localization and super-resolution microscopy

Ryan McGorty, Joerg Schnitzbauer, Wei Zhang, and Bo Huang  »View Author Affiliations

Optics Letters, Vol. 39, Issue 2, pp. 275-278 (2014)

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Single-molecule switching based super-resolution microscopy techniques have been extended into three dimensions through various 3D single-molecule localization methods. However, the localization accuracy in z can be severely degraded by the presence of aberrations, particularly the spherical aberration introduced by the refractive index mismatch when imaging into an aqueous sample with an oil immersion objective. This aberration confines the imaging depth in most experiments to regions close to the coverslip. Here we show a method to obtain accurate, depth-dependent z calibrations by measuring the point spread function (PSF) at the coverslip surface, calculating the microscope pupil function through phase retrieval, and then computing the depth-dependent PSF with the addition of spherical aberrations. We demonstrate experimentally that this method can maintain z localization accuracy over a large range of imaging depths. Our super-resolution images of a mammalian cell nucleus acquired between 0 and 2.5 μm past the coverslip show that this method produces accurate z localizations even in the deepest focal plane.

© 2014 Optical Society of America

OCIS Codes
(100.6640) Image processing : Superresolution
(180.6900) Microscopy : Three-dimensional microscopy
(220.1000) Optical design and fabrication : Aberration compensation

ToC Category:

Original Manuscript: October 10, 2013
Revised Manuscript: November 30, 2013
Manuscript Accepted: December 1, 2013
Published: January 8, 2014

Virtual Issues
Vol. 9, Iss. 3 Virtual Journal for Biomedical Optics

Ryan McGorty, Joerg Schnitzbauer, Wei Zhang, and Bo Huang, "Correction of depth-dependent aberrations in 3D single-molecule localization and super-resolution microscopy," Opt. Lett. 39, 275-278 (2014)

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