Improved identification of DNA double strand breaks: γ-H2AX-epitope visualization by confocal microscopy and 3D reconstructed images.

Ruprecht, Nico; Hungerbuehler, Martin; Böhm, Ingrid; Heverhagen, Johannes (2019). Improved identification of DNA double strand breaks: γ-H2AX-epitope visualization by confocal microscopy and 3D reconstructed images. Radiation and environmental biophysics, 58(2), pp. 295-302. Springer 10.1007/s00411-019-00778-1

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Currently, in the context of radiology, irradiation-induced and other genotoxic effects are determined by visualizing DSB-induced DNA repair through γ-H2AX immunofluorescence and direct counting of the foci by epifluorescence microscopy. This procedure, however, neglects the 3D nature of the nucleus. The aim of our study was to use confocal microscopy and 3D reconstructed images to improve documentation and analysis of γ-H2AX fluorescence signals after diagnostic examinations. Confluent, non-dividing MRC-5 lung fibroblasts were irradiated in vitro with a Cs-137 source and exposed to radiation doses up to 1000 mGy before fixation and staining with an antibody recognizing the phosphorylated histone variant γ-H2AX. The 3D distribution of γ-H2AX foci was visualized using confocal laser scanning microscopy. 3D reconstruction of the optical slices and γ-H2AX foci counting were performed using Imaris Image Analysis software. In parallel, γ-H2AX foci were counted visually by epifluorescence microscopy. In addition, whole blood was exposed ex vivo to the radiation doses from 200 to 1600 mGy. White blood cells (WBCs) were isolated and stained for γ-H2AX. In fibroblasts, epifluorescence microscopy alone visualized the entirety of fluorescence signals as integral, without correct demarcation of single foci, and at 1000 mGy yielded on average 11.1 foci by manual counting of 2D images in comparison to 36.1 foci with confocal microscopy and 3D reconstruction (p < 0.001). The procedure can also be applied for studies on WBCs. In contrast to epifluorescence microscopy, confocal microscopy and 3D reconstruction enables an improved identification of DSB-induced γ-H2AX foci, allowing for an unbiased, ameliorated quantification.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Department of Radiology, Neuroradiology and Nuclear Medicine (DRNN) > Institute of Diagnostic, Interventional and Paediatric Radiology
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > DBMR Forschung Mu35 > Forschungsgruppe Radiologie
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > DBMR Forschung Mu35 > Forschungsgruppe Radiologie

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UniBE Contributor:

Ruprecht, Nico; Hungerbuehler, Martin; Böhm, Ingrid and Heverhagen, Johannes

Subjects:

600 Technology > 610 Medicine & health

ISSN:

1432-2099

Publisher:

Springer

Language:

English

Submitter:

Maria de Fatima Henriques Bernardo

Date Deposited:

23 Apr 2019 07:50

Last Modified:

08 Aug 2019 10:01

Publisher DOI:

10.1007/s00411-019-00778-1

PubMed ID:

30799523

Uncontrolled Keywords:

3D reconstructed images Confocal microscopy DNA DSBs γ-H2AX foci

BORIS DOI:

10.7892/boris.127202

URI:

https://boris.unibe.ch/id/eprint/127202

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