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Proceedings Paper

Issues in the benchmarking of image analysis algorithms for superresolution microscopy
Author(s): Shane P. Stahlheber; Alexander R. Small
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Paper Abstract

Superresolution localization microscopy requires accurate and precise localization algorithms. We have developed a plugin for ImageJ, called M2LE, which can localize molecules quickly and distinguish between single-molecule and multiple-molecule images using a shape test that requires only a single iteration. Localization is accomplished via a fast maximum-likelihood algorithm that uses the separable property of the Gaussian to independently fit two 1-D Gaussians along the x- and y-directions. To assess the performance of M2LE, we tested the plugin with realistic simulated images of single and multiple molecule images. We first found the optimal shape test parameters that accept most single-molecule images, and then the optimal signal-to-noise cutoff parameter for identifying potential molecules from noise. These two parameters have the greatest impact on what parts of the image go on to be analyzed. Using these optimal parameters, we then assessed (1) the tendency of the algorithm to find molecules from the tail of a point-spread function in high signal-to-noise cases, (2) the effects of regions-of-interest size and overlap tolerances, (3) the ability of shape tests to identify multi-molecule images as a function of molecular separation and ratio of photon counts from two molecules, and (4) the performance of the entire process{the number of molecules identified and their corresponding localization precision and accuracy. These methods and results can be used to identify the optimal M2LE parameters to use for experiments, as well as to compare the performance with other localization microscopy software.

Paper Details

Date Published: 22 February 2013
PDF: 6 pages
Proc. SPIE 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, 859013 (22 February 2013); doi: 10.1117/12.2002872
Show Author Affiliations
Shane P. Stahlheber, California State Polytechnic Univ., Pomona (United States)
Alexander R. Small, California State Polytechnic Univ., Pomona (United States)

Published in SPIE Proceedings Vol. 8590:
Single Molecule Spectroscopy and Superresolution Imaging VI
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

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