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, 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.