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Journal of Biomedical Optics

Morphological spot counting from stacked images for automated analysis of gene copy numbers by fluorescence in situ hybridization
Author(s): Artyom M. Grigoryan; Edward R. Dougherty; Juha Kononen; Lukas Bubendorf; Galen Hosteter; Ollie Kallionienmi
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Paper Abstract

Fluorescence in situ hybridization (FISH) is a molecular diagnostic technique in which a fluorescent labeled probe hybridizes to a target nucleotide sequence of deoxyribose nucleic acid. Upon excitation, each chromosome containing the target sequence produces a fluorescent signal (spot). Because fluorescent spot counting is tedious and often subjective, automated digital algorithms to count spots are desirable. New technology provides a stack of images on multiple focal planes throughout a tissue sample. Multiple-focal-plane imaging helps overcome the biases and imprecision inherent in single-focalplane methods. This paper proposes an algorithm for global spot counting in stacked three-dimensional slice FISH images without the necessity of nuclei segmentation. It is designed to work in complex backgrounds, when there are agglomerated nuclei, and in the presence of illumination gradients. It is based on the morphological tophat transform, which locates intensity spikes on irregular backgrounds. After finding signals in the slice images, the algorithm groups these together to form three-dimensional spots. Filters are employed to separate legitimate spots from fluorescent noise. The algorithm is set in a comprehensive toolbox that provides visualization and analytic facilities. It includes simulation software that allows examination of algorithm performance for various image and algorithm parameter settings, including signal size, signal density, and the number of slices.

Paper Details

Date Published: 1 January 2002
PDF: 14 pages
J. Biomed. Opt. 7(1) doi: 10.1117/1.1428292
Published in: Journal of Biomedical Optics Volume 7, Issue 1
Show Author Affiliations
Artyom M. Grigoryan, Univ. of Texas/San Antonio (United States)
Edward R. Dougherty, Texas A&M Univ. (United States)
Juha Kononen, National Institutes of Health (United States)
Lukas Bubendorf, National Institutes of Health (United States)
Galen Hosteter, National Institutes of Health (United States)
Ollie Kallionienmi, National Institutes of Health (United States)

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