FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy
Abstract Background Phosphorylated histone H2AX, also known as γH2AX, forms μm-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, γH2AX immunoassays have become the gold standard for studying DS...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2020-01-01
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| Series: | BMC Bioinformatics |
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| Online Access: | https://doi.org/10.1186/s12859-020-3370-8 |
| _version_ | 1818615891318800384 |
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| author | Simon Memmel Dmitri Sisario Heiko Zimmermann Markus Sauer Vladimir L. Sukhorukov Cholpon S. Djuzenova Michael Flentje |
| author_facet | Simon Memmel Dmitri Sisario Heiko Zimmermann Markus Sauer Vladimir L. Sukhorukov Cholpon S. Djuzenova Michael Flentje |
| author_sort | Simon Memmel |
| collection | DOAJ |
| description | Abstract Background Phosphorylated histone H2AX, also known as γH2AX, forms μm-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, γH2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of γH2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of γH2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected. Results To overcome the limitations of 2D foci counting, we present a freely available ImageJ-based plugin (FocAn) for automated 3D analysis of γH2AX foci in z-image stacks acquired by confocal fluorescence microscopy. The image-stack processing algorithm implemented in FocAn is capable of automatic 3D recognition of individual cell nuclei and γH2AX foci, as well as evaluation of the total foci number per cell nucleus. The FocAn algorithm consists of two parts: nucleus identification and foci detection, each employing specific sequences of auto local thresholding in combination with watershed segmentation techniques. We validated the FocAn algorithm using fluorescence-labeled γH2AX in two glioblastoma cell lines, irradiated with 2 Gy and given up to 24 h post-irradiation for repair. We found that the data obtained with FocAn agreed well with those obtained with an already available software (FoCo) and manual counting. Moreover, FocAn was capable of identifying overlapping foci in 3D space, which ensured accurate foci counting even at high DSB density of up to ~ 200 DSB/nucleus. Conclusions FocAn is freely available an open-source 3D foci analyzer. The user-friendly algorithm FocAn requires little supervision and can automatically count the amount of DNA-DSBs, i.e. fluorescence-labeled γH2AX foci, in 3D image stacks acquired by laser-scanning microscopes without additional nuclei staining. |
| first_indexed | 2024-12-16T16:41:06Z |
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| institution | Directory Open Access Journal |
| issn | 1471-2105 |
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| spelling | doaj.art-b3edee96807d41059ae6d519f6d520322022-12-21T22:24:19ZengBMCBMC Bioinformatics1471-21052020-01-0121111110.1186/s12859-020-3370-8FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopySimon Memmel0Dmitri Sisario1Heiko Zimmermann2Markus Sauer3Vladimir L. Sukhorukov4Cholpon S. Djuzenova5Michael Flentje6Department of Radiation Oncology, University Hospital WürzburgLehrstuhl für Biotechnologie und Biophysik, Biozentrum, Universität WürzburgFraunhofer Institute for Biomedical Engineering (IBMT)Lehrstuhl für Biotechnologie und Biophysik, Biozentrum, Universität WürzburgLehrstuhl für Biotechnologie und Biophysik, Biozentrum, Universität WürzburgDepartment of Radiation Oncology, University Hospital WürzburgDepartment of Radiation Oncology, University Hospital WürzburgAbstract Background Phosphorylated histone H2AX, also known as γH2AX, forms μm-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, γH2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of γH2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of γH2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected. Results To overcome the limitations of 2D foci counting, we present a freely available ImageJ-based plugin (FocAn) for automated 3D analysis of γH2AX foci in z-image stacks acquired by confocal fluorescence microscopy. The image-stack processing algorithm implemented in FocAn is capable of automatic 3D recognition of individual cell nuclei and γH2AX foci, as well as evaluation of the total foci number per cell nucleus. The FocAn algorithm consists of two parts: nucleus identification and foci detection, each employing specific sequences of auto local thresholding in combination with watershed segmentation techniques. We validated the FocAn algorithm using fluorescence-labeled γH2AX in two glioblastoma cell lines, irradiated with 2 Gy and given up to 24 h post-irradiation for repair. We found that the data obtained with FocAn agreed well with those obtained with an already available software (FoCo) and manual counting. Moreover, FocAn was capable of identifying overlapping foci in 3D space, which ensured accurate foci counting even at high DSB density of up to ~ 200 DSB/nucleus. Conclusions FocAn is freely available an open-source 3D foci analyzer. The user-friendly algorithm FocAn requires little supervision and can automatically count the amount of DNA-DSBs, i.e. fluorescence-labeled γH2AX foci, in 3D image stacks acquired by laser-scanning microscopes without additional nuclei staining.https://doi.org/10.1186/s12859-020-3370-8DNA double-strand breaksImageJ pluginγH2AX-fociAutomated analysisIonizing radiationOpen-source tool |
| spellingShingle | Simon Memmel Dmitri Sisario Heiko Zimmermann Markus Sauer Vladimir L. Sukhorukov Cholpon S. Djuzenova Michael Flentje FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy BMC Bioinformatics DNA double-strand breaks ImageJ plugin γH2AX-foci Automated analysis Ionizing radiation Open-source tool |
| title | FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy |
| title_full | FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy |
| title_fullStr | FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy |
| title_full_unstemmed | FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy |
| title_short | FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy |
| title_sort | focan automated 3d analysis of dna repair foci in image stacks acquired by confocal fluorescence microscopy |
| topic | DNA double-strand breaks ImageJ plugin γH2AX-foci Automated analysis Ionizing radiation Open-source tool |
| url | https://doi.org/10.1186/s12859-020-3370-8 |
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