Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (approximate to 200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy (dSTORM) with a lateral resolution of approximate to 20 nm to analyze the focal nanostructure of the DSB marker histone H2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained H2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that H2AX foci consisted of distinct circular subunits (nanofoci) with a diameter of approximate to 45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that H2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual H2AX-containing nucleosomes. dSTORM-based H2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of H2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between approximate to 0.6 and approximate to 1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.