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Carbon ion radiation is a promising new form of radiotherapy for cancer, but the central question about the biologic effects of charged particle radiation is yet incompletely understood. Key to this question is the understanding of the interaction of ions with DNA in the cell's nucleus. Induction and repair of DNA lesions including double-strand breaks (DSBs) are decisive for the cell. Several DSB repair markers have been used to investigate these processes microscopically, but the limited resolution of conventional microscopy is insufficient to provide structural insights. We have applied superresolution microscopy to overcome these limitations and analyze the fine structure of DSB repair foci. We found that the conventionally detected foci of the widely used DSB marker γH2AX (Ø 700-1000 nm) were composed of elongated subfoci with a size of ∼100 nm consisting of even smaller subfocus elements (Ø 40-60 nm). The structural organization of the subfoci suggests that they could represent the local chromatin structure of elementary DSB repair units at the DSB damage sites. Subfocus clusters may indicate induction of densely spaced DSBs, which are thought to be associated with the high biologic effectiveness of carbon ions. Superresolution microscopy might emerge as a powerful tool to improve our knowledge of interactions of ionizing radiation with cells.-Lopez Perez, R., Best, G., Nicolay, N. H., Greubel, C., Rossberger, S., Reindl, J., Dollinger, G., Weber, K.-J., Cremer, C., Huber, P. E. Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci.

In many scientific fields such as biology, medicine, and material sciences, microscopy has become a major analytical tool. Electron microscopy (EM) with nanometer resolution, on the one hand, and light microscopy with a broad applicability, on the other hand, allowed for groundbreaking scientific achievements. Even though EM delivers unmatched resolution, light microscopy has never lost its relevance. Because of the vast propagation of fluorescent labeling techniques in recent years, the method of fluorescence microscopy has actually become one of the most important imaging techniques in the life sciences. However, the intrinsically limited resolution of standard fluorescencemicroscopy compared to nonlight-optical methods is still a major drawback as many biological specimens are of a size in the nanometer to micrometer range. Therefore, the standard light-microscopic resolution defined by Rayleigh (1896) (Chapter 2) is often not sufficient to resolve the objects of interest. Over last years, different techniques denoted as super-resolution fluorescence microscopy have been established to compensate for the deficiency of low spatial resolution. These approaches use fluorescence excitation because this circumstance allows – in combination with other techniques – access to high-resolution object information. These super-resolution methods (i.e., 4Pi (Cremer and Cremer, 1978; Hell and Stelzer, 1992; Hell et al., 1994; Hanninen et al., 1995 stimulated emission depletion (STED) (Chapter 10), SIM/PEM (Gustafsson, 2000; Heintzmann and Cremer, 1999), and localization methods (Chapter 8)) seemingly break the conventional resolution limit. However, it should be noted that the fundamental resolution limit is not broken directly. The super-resolution methods are based on conditions differing from the assumptions of Rayleigh. Rayleigh’s conclusions for self-luminous (i.e., fluorescent) objects do not consider spatial or temporal variations of the light intensity. Basically, all super-resolution techniques depend on juggling with the fluorescence excitation or emission.