A novel approach to double-strand DNA break analysis through γ-H2AX confocal image quantification and bio-dosimetry
Abstract DNA damage occurs in all living cells. γ-H2AX imaging by fluorescent microscopy is widely used across disciplines in the analysis of double-strand break (DSB) DNA damage. Here we demonstrate a method for the quantitative analysis of such DBSs. Ionising radiation, well known to induce DSBs,...
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Nature Portfolio
2024-11-01
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| Series: | Scientific Reports |
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| Online Access: | https://doi.org/10.1038/s41598-024-76683-5 |
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| author | Michael Valceski Elette Engels Sarah Vogel Jason Paino Dylan Potter Carolyn Hollis Abass Khochaiche Micah Barnes Matthew Cameron Alice O’Keefe Kiarn Roughley Anatoly Rosenfeld Michael Lerch Stéphanie Corde Moeava Tehei |
| author_facet | Michael Valceski Elette Engels Sarah Vogel Jason Paino Dylan Potter Carolyn Hollis Abass Khochaiche Micah Barnes Matthew Cameron Alice O’Keefe Kiarn Roughley Anatoly Rosenfeld Michael Lerch Stéphanie Corde Moeava Tehei |
| author_sort | Michael Valceski |
| collection | DOAJ |
| description | Abstract DNA damage occurs in all living cells. γ-H2AX imaging by fluorescent microscopy is widely used across disciplines in the analysis of double-strand break (DSB) DNA damage. Here we demonstrate a method for the quantitative analysis of such DBSs. Ionising radiation, well known to induce DSBs, is used in this demonstration, and additional DBSs are induced if high-Z nanoparticles are present during irradiation. As a deliberate test of the methodology, cells are exposed to a spatially fractionated ionising radiation field, characterised by regions of high and low absorbed radiation dose that are only ever qualitatively verified biologically via γ-H2AX imaging. Here we validate our bio-dosimetric quantification method using γ-H2AX assays in the assessment of DSB enhancement. Our method reliably quantifies DSB enhancement in cells when exposed to either a spatially contiguous or fractionated irradiation fields. Using the γ-H2AX assay, we deduce the biological dose response, and for the first time, demonstrate equivalence to the independently measured physical absorbed dose. Using our novel method, we are also able quantify the nanoparticle DSB enhancement at the cellular level, which is not possible using physical dose measurement techniques. Our method therefore provides a new paradigm in γ-H2AX image quantification of DSBs, as well as an independently validated bio-dosimetry technique. |
| format | Article |
| id | doaj-art-5f6c406cc4f841d6acda58190038600a |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-5f6c406cc4f841d6acda58190038600a2024-11-17T12:18:22ZengNature PortfolioScientific Reports2045-23222024-11-0114111710.1038/s41598-024-76683-5A novel approach to double-strand DNA break analysis through γ-H2AX confocal image quantification and bio-dosimetryMichael Valceski0Elette Engels1Sarah Vogel2Jason Paino3Dylan Potter4Carolyn Hollis5Abass Khochaiche6Micah Barnes7Matthew Cameron8Alice O’Keefe9Kiarn Roughley10Anatoly Rosenfeld11Michael Lerch12Stéphanie Corde13Moeava Tehei14Centre for Medical Radiation Physics, University of WollongongCentre for Medical Radiation Physics, University of WollongongCentre for Medical Radiation Physics, University of WollongongCentre for Medical Radiation Physics, University of WollongongCentre for Medical Radiation Physics, University of WollongongCentre for Medical Radiation Physics, University of WollongongCentre for Medical Radiation Physics, University of WollongongCentre for Medical Radiation Physics, University of WollongongAustralian Synchrotron - Australian Nuclear Science and Technology Organisation (ANSTO)Centre for Medical Radiation Physics, University of WollongongCentre for Medical Radiation Physics, University of WollongongCentre for Medical Radiation Physics, University of WollongongCentre for Medical Radiation Physics, University of WollongongCentre for Medical Radiation Physics, University of WollongongCentre for Medical Radiation Physics, University of WollongongAbstract DNA damage occurs in all living cells. γ-H2AX imaging by fluorescent microscopy is widely used across disciplines in the analysis of double-strand break (DSB) DNA damage. Here we demonstrate a method for the quantitative analysis of such DBSs. Ionising radiation, well known to induce DSBs, is used in this demonstration, and additional DBSs are induced if high-Z nanoparticles are present during irradiation. As a deliberate test of the methodology, cells are exposed to a spatially fractionated ionising radiation field, characterised by regions of high and low absorbed radiation dose that are only ever qualitatively verified biologically via γ-H2AX imaging. Here we validate our bio-dosimetric quantification method using γ-H2AX assays in the assessment of DSB enhancement. Our method reliably quantifies DSB enhancement in cells when exposed to either a spatially contiguous or fractionated irradiation fields. Using the γ-H2AX assay, we deduce the biological dose response, and for the first time, demonstrate equivalence to the independently measured physical absorbed dose. Using our novel method, we are also able quantify the nanoparticle DSB enhancement at the cellular level, which is not possible using physical dose measurement techniques. Our method therefore provides a new paradigm in γ-H2AX image quantification of DSBs, as well as an independently validated bio-dosimetry technique.https://doi.org/10.1038/s41598-024-76683-5Confocal microscopyImage analysisRadiotherapySynchrotronMicrobeam Radiation TherapyγH2AX |
| spellingShingle | Michael Valceski Elette Engels Sarah Vogel Jason Paino Dylan Potter Carolyn Hollis Abass Khochaiche Micah Barnes Matthew Cameron Alice O’Keefe Kiarn Roughley Anatoly Rosenfeld Michael Lerch Stéphanie Corde Moeava Tehei A novel approach to double-strand DNA break analysis through γ-H2AX confocal image quantification and bio-dosimetry Scientific Reports Confocal microscopy Image analysis Radiotherapy Synchrotron Microbeam Radiation Therapy γH2AX |
| title | A novel approach to double-strand DNA break analysis through γ-H2AX confocal image quantification and bio-dosimetry |
| title_full | A novel approach to double-strand DNA break analysis through γ-H2AX confocal image quantification and bio-dosimetry |
| title_fullStr | A novel approach to double-strand DNA break analysis through γ-H2AX confocal image quantification and bio-dosimetry |
| title_full_unstemmed | A novel approach to double-strand DNA break analysis through γ-H2AX confocal image quantification and bio-dosimetry |
| title_short | A novel approach to double-strand DNA break analysis through γ-H2AX confocal image quantification and bio-dosimetry |
| title_sort | novel approach to double strand dna break analysis through γ h2ax confocal image quantification and bio dosimetry |
| topic | Confocal microscopy Image analysis Radiotherapy Synchrotron Microbeam Radiation Therapy γH2AX |
| url | https://doi.org/10.1038/s41598-024-76683-5 |
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