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Proceedings Paper

Subnuclear foci quantification using high-throughput 3D image cytometry
Author(s): Dushan N. Wadduwage; Marcus Parrish; Heejin Choi; Bevin P. Engelward; Paul Matsudaira; Peter T. C. So
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Paper 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.

Paper Details

Date Published: 14 July 2015
PDF: 5 pages
Proc. SPIE 9536, Advanced Microscopy Techniques IV; and Neurophotonics II, 953607 (14 July 2015); doi: 10.1117/12.2185005
Show Author Affiliations
Dushan N. Wadduwage, Singamore MIT Alliance for Research and Technology (Singapore)
National Univ. of Singapore (Singapore)
Marcus Parrish, Massachusetts Institute of Technology (United States)
Heejin Choi, Massachusetts Institute of Technology (United States)
Bevin P. Engelward, Massachusetts Institute of Technology (United States)
Paul Matsudaira, National Univ. of Singapore (Singapore)
Peter T. C. So, Singamore MIT Alliance for Research and Technology (Singapore)
Massachusetts Institute of Technology (United States)


Published in SPIE Proceedings Vol. 9536:
Advanced Microscopy Techniques IV; and Neurophotonics II
Emmanuel Beaurepaire; Francesco Pavone; Elizabeth M. Hillman; Peter T. C. So, Editor(s)

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