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Proceedings Paper

A comparison of methods for optical sectioning using structured illumination microscopy
Author(s): Benjamin Thomas; Michelle Momany; Peter Kner
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Paper Abstract

Structured Illumination Microscopy is a simple and effective method to remove out-of-focus light in widefield fluorescence microscopy. Neil et al. originally proposed a simple square-law method for calculating the optically sectioned image from the three raw images with the structured illumination pattern super-imposed. However, the Neil method does not make the most efficient use of the three raw images. The three structured illumination images can also be used to separate three copies of the image covering shifted regions of frequency space in a similar manner to that developed by Gustafsson et al. These can then be combined using a generalized Wiener filter to create an image with a well-behaved optical transfer function in which the missing cone has been filled in, providing optical sectioning. Here, we compare the Neil and Gustafsson methods and show that the Gustafsson method provides an image with higher fidelity and a better Signal to Noise Ratio (SNR) at low photon counts. We apply the two methods to images of fluorescent beads and GFP labeled septins in Aspergillus nidulans.

Paper Details

Date Published: 22 February 2013
PDF: 8 pages
Proc. SPIE 8589, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XX, 85890I (22 February 2013); doi: 10.1117/12.2002567
Show Author Affiliations
Benjamin Thomas, The Univ. of Georgia (United States)
Michelle Momany, The Univ. of Georgia (United States)
Peter Kner, The Univ. of Georgia (United States)

Published in SPIE Proceedings Vol. 8589:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XX
Carol J. Cogswell; Thomas G. Brown; Jose-Angel Conchello; Tony Wilson, Editor(s)

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