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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 9 — May. 6, 2013
  • pp: 10978–10988

Correcting chromatic offset in multicolor super-resolution localization microscopy

Miklos Erdelyi, Eric Rees, Daniel Metcalf, Gabriele S. Kaminski Schierle, Laszlo Dudas, Jozsef Sinko, Alex E. Knight, and Clemens F. Kaminski  »View Author Affiliations

Optics Express, Vol. 21, Issue 9, pp. 10978-10988 (2013)

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Localization based super-resolution microscopy techniques require precise drift correction methods because the achieved spatial resolution is close to both the mechanical and optical performance limits of modern light microscopes. Multi-color imaging methods require corrections in addition to those dealing with drift due to the static, but spatially-dependent, chromatic offset between images. We present computer simulations to quantify this effect, which is primarily caused by the high-NA objectives used in super-resolution microscopy. Although the chromatic offset in well corrected systems is only a fraction of an optical wavelength in magnitude (<50 nm) and thus negligible in traditional diffraction limited imaging, we show that object colocalization by multi-color super-resolution methods is impossible without appropriate image correction. The simulated data are in excellent agreement with experiments using fluorescent beads excited and localized at multiple wavelengths. Finally we present a rigorous and practical calibration protocol to correct for chromatic optical offset, and demonstrate its efficacy for the imaging of transferrin receptor protein colocalization in HeLa cells using two-color direct stochastic optical reconstruction microscopy (dSTORM).

© 2013 OSA

OCIS Codes
(100.6640) Image processing : Superresolution
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:

Original Manuscript: February 11, 2013
Revised Manuscript: April 8, 2013
Manuscript Accepted: April 18, 2013
Published: April 26, 2013

Virtual Issues
Vol. 8, Iss. 6 Virtual Journal for Biomedical Optics

Miklos Erdelyi, Eric Rees, Daniel Metcalf, Gabriele S. Kaminski Schierle, Laszlo Dudas, Jozsef Sinko, Alex E. Knight, and Clemens F. Kaminski, "Correcting chromatic offset in multicolor super-resolution localization microscopy," Opt. Express 21, 10978-10988 (2013)

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