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.