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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics


  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 7, Iss. 7 — Jun. 25, 2012

Using fixed fiduciary markers for stage drift correction

Sang Hak Lee, Murat Baday, Marco Tjioe, Paul D. Simonson, Ruobing Zhang, En Cai, and Paul R. Selvin  »View Author Affiliations

Optics Express, Vol. 20, Issue 11, pp. 12177-12183 (2012)

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To measure nanometric features with super-resolution requires that the stage, which holds the sample, be stable to nanometric precision. Herein we introduce a new method that uses conventional equipment, is low cost, and does not require intensive computation. Fiduciary markers of approximately 1 µm x 1 µm x 1 µm in x, y, and z dimensions are placed at regular intervals on the coverslip. These fiduciary markers are easy to put down, are completely stationary with respect to the coverslip, are bio-compatible, and do not interfere with fluorescence or intensity measurements. As the coverslip undergoes drift (or is purposely moved), the x-y center of the fiduciary markers can be readily tracked to 1 nanometer using a Gaussian fit. By focusing the light slightly out-of-focus, the z-axis can also be tracked to < 5 nm for dry samples and <17 nm for wet samples by looking at the diffraction rings. The process of tracking the fiduciary markers does not interfere with visible fluorescence because an infrared light emitting diode (IR-LED) (690 and 850 nm) is used, and the IR-light is separately detected using an inexpensive camera. The resulting motion of the coverslip can then be corrected for, either after-the-fact, or by using active stabilizers, to correct for the motion. We applied this method to watch kinesin walking with ≈8 nm steps.

© 2012 OSA

OCIS Codes
(110.0180) Imaging systems : Microscopy
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:

Original Manuscript: March 12, 2012
Revised Manuscript: May 7, 2012
Manuscript Accepted: May 7, 2012
Published: May 14, 2012

Virtual Issues
Vol. 7, Iss. 7 Virtual Journal for Biomedical Optics

Sang Hak Lee, Murat Baday, Marco Tjioe, Paul D. Simonson, Ruobing Zhang, En Cai, and Paul R. Selvin, "Using fixed fiduciary markers for stage drift correction," Opt. Express 20, 12177-12183 (2012)

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