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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 17, Iss. 8 — Apr. 13, 2009
  • pp: 6881–6898

Improved single particle localization accuracy with dual objective multifocal plane microscopy

Sripad Ram, Prashant Prabhat, E. Sally Ward, and Raimund J. Ober  »View Author Affiliations

Optics Express, Vol. 17, Issue 8, pp. 6881-6898 (2009)

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In single particle imaging applications, the number of photons detected from the fluorescent label plays a crucial role in the quantitative analysis of the acquired data. For example, in tracking experiments the localization accuracy of the labeled entity can be improved by collecting more photons from the labeled entity. Here, we report the development of dual objective multifocal plane microscopy (dMUM) for single particle studies. The new microscope configuration uses two opposing objective lenses, where one of the objectives is in an inverted position and the other objective is in an upright position. We show that dMUM has a higher photon collection efficiency when compared to standard microscopes. We demonstrate that fluorescent labels can be localized with better accuracy in 2D and 3D when imaged through dMUM than when imaged through a standard microscope. Analytical tools are introduced to estimate the nanoprobe location from dMUM images and to characterize the accuracy with which they can be determined.

© 2009 Optical Society of America

OCIS Codes
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:

Original Manuscript: January 15, 2009
Revised Manuscript: March 19, 2009
Manuscript Accepted: April 3, 2009
Published: April 10, 2009

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

Sripad Ram, Prashant Prabhat, E. Sally Ward, and Raimund J. Ober, "Improved single particle localization accuracy with dual objective multifocal plane microscopy," Opt. Express 17, 6881-6898 (2009)

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