Subnuclear foci quantification using high-throughput 3D image cytometry
Name
Engelward_Subnuclear foci.pdf
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Author(s)
Wadduwage, Dushan N.
Matsudaira, Paul
So, Peter T. C.
Parrish, Marcus Curtis
Choi, Heejin
Engelward, Bevin P
Date Issued
July 2015
Journal
Proceedings of SPIE--the International Society for Optical Engineering
Publisher
SPIE
Citation
Wadduwage, Dushan N. et al. “Subnuclear Foci Quantification Using High-Throughput 3D Image Cytometry.” Ed. Emmanuel Beaurepaire et al. N.p., 2015. 953607. CrossRef. Web. 20 Mar. 2017.
Version
Final published version
Abstract
Ionising radiation causes various types of DNA damages including double strand breaks (DSBs). DSBs are often recognized by DNA repair protein ATM which forms gamma-H2AX foci at the site of the DSBs that can be visualized using immunohistochemistry. However most of such experiments are of low throughput in terms of imaging and image analysis techniques. Most of the studies still use manual counting or classification. Hence they are limited to counting a low number of foci per cell (5 foci per nucleus) as the quantification process is extremely labour intensive. Therefore we have developed a high throughput instrumentation and computational pipeline specialized for gamma-H2AX foci quantification. A population of cells with highly clustered foci inside nuclei were imaged, in 3D with submicron resolution, using an in-house developed high throughput image cytometer. Imaging speeds as high as 800 cells/second in 3D were achieved by using HiLo wide-field depth resolved imaging and a remote z-scanning technique. Then the number of foci per cell nucleus were quantified using a 3D extended maxima transform based algorithm. Our results suggests that while most of the other 2D imaging and manual quantification studies can count only up to about 5 foci per nucleus our method is capable of counting more than 100. Moreover we show that 3D analysis is significantly superior compared to the 2D techniques.
MIT Department
Massachusetts Institute of Technology. Institute for Medical Engineering & Science
Massachusetts Institute of Technology. Department of Biological Engineering
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Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1117/12.2185005
