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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 20, Iss. 25 — Dec. 3, 2012
  • pp: 27337–27347

Three dimensional HiLo-based structured illumination for a digital scanned laser sheet microscopy (DSLM) in thick tissue imaging

Dipanjan Bhattacharya, Vijay Raj Singh, Chen Zhi, Peter T. C. So, Paul Matsudaira, and George Barbastathis  »View Author Affiliations

Optics Express, Vol. 20, Issue 25, pp. 27337-27347 (2012)

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Laser sheet based microscopy has become widely accepted as an effective active illumination method for real time three-dimensional (3D) imaging of biological tissue samples. The light sheet geometry, where the camera is oriented perpendicular to the sheet itself, provides an effective method of eliminating some of the scattered light and minimizing the sample exposure to radiation. However, residual background noise still remains, limiting the contrast and visibility of potentially interesting features in the samples. In this article, we investigate additional structuring of the illumination for improved background rejection, and propose a new technique, “3D HiLo” where we combine two HiLo images processed from orthogonal directions to improve the condition of the 3D reconstruction. We present a comparative study of conventional structured illumination based demodulation methods, namely 3Phase and HiLo with a newly implemented 3D HiLo approach and demonstrate that the latter yields superior signal-to-background ratio in both lateral and axial dimensions, while simultaneously suppressing image processing artifacts.

© 2012 OSA

OCIS Codes
(100.3020) Image processing : Image reconstruction-restoration
(110.6880) Imaging systems : Three-dimensional image acquisition
(180.2520) Microscopy : Fluorescence microscopy
(180.6900) Microscopy : Three-dimensional microscopy

ToC Category:

Original Manuscript: September 14, 2012
Revised Manuscript: October 25, 2012
Manuscript Accepted: October 26, 2012
Published: November 20, 2012

Virtual Issues
Vol. 8, Iss. 1 Virtual Journal for Biomedical Optics

Dipanjan Bhattacharya, Vijay Raj Singh, Chen Zhi, Peter T. C. So, Paul Matsudaira, and George Barbastathis, "Three dimensional HiLo-based structured illumination for a digital scanned laser sheet microscopy (DSLM) in thick tissue imaging," Opt. Express 20, 27337-27347 (2012)

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