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Virtual Journal for Biomedical Optics


  • Editor: Gregory W. Faris
  • Vol. 4, Iss. 6 — May. 26, 2009

Effect of aberration on height calibration in three-dimensional localization-based microscopy and particle tracking

Yi Deng and Joshua W. Shaevitz  »View Author Affiliations

Applied Optics, Vol. 48, Issue 10, pp. 1886-1890 (2009)

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Many single-particle tracking and localization-based superresolution imaging techniques use the width of a single lateral fluorescence image to estimate a molecule’s axial position. This determination is often done by use of a calibration data set derived from a source adhered to a glass–water interface. However, for sources deeper in solution, aberrations will change the relationship between the image width and the axial position. We analyzed the depth-varying point spread function of a high numerical aperture objective near an index of refraction mismatch at the water–glass interface using an optical trap. In addition to the well-known focal shift, spherical aberrations cause up to 30% relative systematic error in axial position estimation. This effect is nonuniform in depth, and we find that, although molecules below the focal plane are correctly localized, molecules deeper than the focal plane are found to be lower than their actual positions.

© 2009 Optical Society of America

OCIS Codes
(100.2960) Image processing : Image analysis
(100.6640) Image processing : Superresolution
(180.0180) Microscopy : Microscopy
(180.2520) Microscopy : Fluorescence microscopy
(180.6900) Microscopy : Three-dimensional microscopy
(150.1488) Machine vision : Calibration

ToC Category:

Original Manuscript: October 27, 2008
Revised Manuscript: February 3, 2009
Manuscript Accepted: February 13, 2009
Published: March 25, 2009

Virtual Issues
Vol. 4, Iss. 6 Virtual Journal for Biomedical Optics

Yi Deng and Joshua W. Shaevitz, "Effect of aberration on height calibration in three-dimensional localization-based microscopy and particle tracking," Appl. Opt. 48, 1886-1890 (2009)

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