Genomic deoxyribonucleic acid (DNA) is continuously being damaged by endogenous processes such as metabolism or by exogenous events such as radiation. The specific phosphorylation of histone H2AX on serine residue 139, described as γ-H2AX, is an excellent indicator or marker of DNA double-strand breaks (DSBs). The yield of γ-H2AX (foci) is shown to have some correlation with the dose of radiation or other DSB-causing agents. However, there is some discrepancy in the DNA DSB foci yield among imaging and other methods such as gel electrophoresis. Super-resolution imaging techniques are now becoming widely used as essential tools in biology and medicine, after a slow uptake of their development almost two decades ago. Here we compare several super-resolution techniques used to image and determine the amount and spatial distribution of γ-H2AX foci formation after X-ray irradiation: stimulated emission depletion (STED), ground-state depletion microscopy followed by individual molecule return (GSDIM), structured illumination microscopy (SIM), as well as an improved confocal, Airyscan and HyVolution 2. We show that by using these super-resolution imaging techniques with as low as 30-nm resolution, each focus may be further resolved, thus increasing the number of foci per radiation dose compared to standard microscopy. Furthermore, the DNA repair proteins 53BP1 (after low-LET irradiations) and Ku70/Ku80 (from laser microbeam irradiation) do not always yield a significantly increased number of foci when imaged by the super-resolution techniques, suggesting that γ-H2AX, 53PB1 and Ku70/80 repair proteins do not fully co-localize on the units of higher order chromatin structure.
Bibliographical noteFunding Information:
We thank Professor Chris Hawes (Oxford Brookes University) for access to the Zeiss LSM 880 Airyscan confocal and Professors Tony Parker and Chris Stubbs (STFC, Rutherford Appleton Laboratory, CLF) for their review and discussion of the manuscript. We also thank Public Health England and Oxford University Radiation Oncology for time on the X-ray instruments where samples were irradiated. We thank Professor Dr. Dik van Gent (Department of Cell Biology and Genetics, Erasmus MC), who kindly provided the V15B cells. This work was supported by the STFC through the provision of access to laser laboratories at the Central Laser Facility, Octopus, Rutherford Appleton Laboratory. Support was also provided by Laserlab-Europe (no. EU-H2020 654148).
© 2018 by Radiation Research Society.