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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 22, Iss. 14 — Jul. 14, 2014
  • pp: 16706–16721

Designing the focal plane spacing for multifocal plane microscopy

Amir Tahmasbi, Sripad Ram, Jerry Chao, Anish V. Abraham, Felix W. Tang, E. Sally Ward, and Raimund J. Ober  »View Author Affiliations

Optics Express, Vol. 22, Issue 14, pp. 16706-16721 (2014)

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Multifocal plane microscopy (MUM) has made it possible to study subcellular dynamics in 3D at high temporal and spatial resolution by simultaneously imaging distinct planes within the specimen. MUM allows high accuracy localization of a point source along the z-axis since it overcomes the depth discrimination problem of conventional single plane microscopy. An important question in MUM experiments is how the number of focal planes and their spacings should be chosen to achieve the best possible localization accuracy along the z-axis. Here, we propose approaches based on the Fisher information matrix and report spacing scenarios called strong coupling and weak coupling which yield an appropriate 3D localization accuracy. We examine the effect of numerical aperture, magnification, photon count, emission wavelength and extraneous noise on the spacing scenarios. In addition, we investigate the effect of changing the number of focal planes on the 3D localization accuracy. We also introduce a new software package that provides a user-friendly framework to find appropriate plane spacings for a MUM setup. These developments should assist in optimizing MUM experiments.

© 2014 Optical Society of America

OCIS Codes
(100.2000) Image processing : Digital image processing
(180.2520) Microscopy : Fluorescence microscopy
(180.6900) Microscopy : Three-dimensional microscopy
(110.3055) Imaging systems : Information theoretical analysis

ToC Category:

Original Manuscript: May 2, 2014
Revised Manuscript: June 12, 2014
Manuscript Accepted: June 20, 2014
Published: June 30, 2014

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

Amir Tahmasbi, Sripad Ram, Jerry Chao, Anish V. Abraham, Felix W. Tang, E. Sally Ward, and Raimund J. Ober, "Designing the focal plane spacing for multifocal plane microscopy," Opt. Express 22, 16706-16721 (2014)

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