Subnuclear foci quantification using high-throughput 3D image cytometry
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 through...
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SPIE
2017
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| Sarrera elektronikoa: | http://hdl.handle.net/1721.1/107492 https://orcid.org/0000-0002-5472-3621 https://orcid.org/0000-0003-3681-7410 |
| _version_ | 1826215080828600320 |
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| author | Wadduwage, Dushan N. Matsudaira, Paul So, Peter T. C. Parrish, Marcus Curtis Choi, Heejin Engelward, Bevin P |
| author2 | Massachusetts Institute of Technology. Institute for Medical Engineering & Science |
| author_facet | Massachusetts Institute of Technology. Institute for Medical Engineering & Science Wadduwage, Dushan N. Matsudaira, Paul So, Peter T. C. Parrish, Marcus Curtis Choi, Heejin Engelward, Bevin P |
| author_sort | Wadduwage, Dushan N. |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T16:16:14Z |
| format | Article |
| id | mit-1721.1/107492 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T16:16:14Z |
| publishDate | 2017 |
| publisher | SPIE |
| record_format | dspace |
| spelling | mit-1721.1/1074922022-10-02T07:28:19Z Subnuclear foci quantification using high-throughput 3D image cytometry Wadduwage, Dushan N. Matsudaira, Paul So, Peter T. C. Parrish, Marcus Curtis Choi, Heejin Engelward, Bevin P Massachusetts Institute of Technology. Institute for Medical Engineering & Science Massachusetts Institute of Technology. Department of Biological Engineering Parrish, Marcus Curtis Choi, Heejin Engelward, Bevin P 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. 2017-03-20T14:55:10Z 2017-03-20T14:55:10Z 2015-07 Article http://purl.org/eprint/type/ConferencePaper 0277-786X 1996-756x http://hdl.handle.net/1721.1/107492 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. https://orcid.org/0000-0002-5472-3621 https://orcid.org/0000-0003-3681-7410 en_US http://dx.doi.org/10.1117/12.2185005 Proceedings of SPIE--the International Society for Optical Engineering 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. application/pdf SPIE SPIE |
| spellingShingle | Wadduwage, Dushan N. Matsudaira, Paul So, Peter T. C. Parrish, Marcus Curtis Choi, Heejin Engelward, Bevin P Subnuclear foci quantification using high-throughput 3D image cytometry |
| title | Subnuclear foci quantification using high-throughput 3D image cytometry |
| title_full | Subnuclear foci quantification using high-throughput 3D image cytometry |
| title_fullStr | Subnuclear foci quantification using high-throughput 3D image cytometry |
| title_full_unstemmed | Subnuclear foci quantification using high-throughput 3D image cytometry |
| title_short | Subnuclear foci quantification using high-throughput 3D image cytometry |
| title_sort | subnuclear foci quantification using high throughput 3d image cytometry |
| url | http://hdl.handle.net/1721.1/107492 https://orcid.org/0000-0002-5472-3621 https://orcid.org/0000-0003-3681-7410 |
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