**Abstract**

Past efforts in radiobiology, radio-biophysics, epidemiology and clinical research strongly contributed to the current understanding of ionizing radiation effects on biological materials like cells and tissues. It is well accepted that the most dangerous, radiation induced damages of DNA in the cell nucleus are double strand breaks, as their false rearrangements cause dysfunction and tumor cell proliferation. Therefore, cells have developed highly efficient and adapted ways to repair lesions of the DNA double strand. To better understand the mechanisms behind DNA strand repair, a variety of fluorescence microscopy based approaches are routinely used to study radiation responses at the organ, tissue and cellular level. Meanwhile, novel super-resolution fluorescence microscopy techniques have rapidly evolved and become powerful tools to study biological structures and bio-molecular (re-) arrangements at the nano-scale. In fact, recent investigations have increasingly demonstrated how super-resolution microscopy can be applied to the analysis of radiation damage induced chromatin arrangements and DNA repair protein recruitment in order to elucidate how spatial organization of damage sites and repair proteins contribute to the control of repair processes. In this chapter, we would like to start with some fundamental aspects of ionizing radiation, their impact on biological materials, and some standard radiobiology assays. We conclude by introducing the concept behind super-resolution radiobiology using single molecule localization microscopy (SMLM) and present promising results from recent studies that show an organized architecture of damage sites and their environment. Persistent homologies of repair clusters indicate a correlation between repair cluster topology and repair pathway at a given damage locus. This overview over recent investigations may motivate radiobiologists to consider chromatin architecture and spatial repair protein organization for the understanding of DNA repair processes.

**Keywords:** ionizing radiation, DNA damage, DNA repair, super-resolution localization microscopy, chromatin nano-architecture, spatial repair protein organization, molecular cluster analysis, molecular topologies

### **1. Introduction**

Past efforts in epidemiological (nuclear power industry, atomic bomb explosions, nuclear reactor accidents, etc.) and clinical (diagnostic imaging, radiation oncology, radiation therapy planning etc.) research strongly contributed to the current understanding of ionizing radiation effects on human organs, tissues, and cells [1, 2]. In principle radiation biology is based on effects of instantaneous (10−18 s) [3], stochastic damaging interactions of ionizing radiation with cells, a main target being the genetic material, i.e. chromatin in the cell nucleus [4]. In this context, radio-sensitivity and radio-resistance as opposing terms describe the extent of individual cellular susceptibility or 'response' upon radiation exposure which are highly dependent on physical (e.g., radiation type, dose, dose rate, etc.), chemical (e.g., hydroxyl radicals, etc.) and biological (e.g., developmental and proliferative state of the affected cell type) factors. As the overall organismal radiation response results from the entirety of all individual radiation responses on the single cell level, deeper understanding of the underlying, complex molecular mechanisms and dynamics of radiation induced DNA damaging and repair on the cellular level is highly relevant for fundamental and applied radiation biology (for review see [1, 2, 5]).

Hence, cytometric analyses based on fluorescence microscopy have become the method of choice to study damaging effects of ionizing radiation and DNA repair. This has contributed a lot to today's knowledge. However, conventional fluorescence microscopy is limited to average lateral resolutions around 200 nm laterally and 600 nm axially [6] and thus is limited to the bulk analysis of molecular cellular processes and structures. In parallel, super-resolution fluorescence microscopy techniques have rapidly evolved during the last few decades and turned out to be powerful tools to study cellular structures and molecular architectures on the nanoscale (for review see [6–8]). Methods based on stochastic reversible photobleaching [9–15] of single molecules called Single Molecule Localization Microscopy (SMLM) [16] reach effective resolutions down to 10 nm and have become popular among modern super-resolution imaging techniques as their realization is highly practical and straightforward using established specimen preparation methods of standard fluorescence microcopy [17]. As such resolutions allow the detection of single molecules, such as nucleosomes [18], proteins [19, 20], receptors and junction proteins [21, 22], or even single chromatin loops [23] etc., super-resolution microscopy opens new avenues for the research of radiation induced damaging and repair processes [5, 24].

With this article, we attempt to introduce the novel SMLM approach to radiation biophysics and radiation biology. We start with a brief summary on the basics of ionizing radiation, induction of DNA damage and damage repair mechanisms, to follow up with some standard radiobiology analysis methods. We further provide an overview of the working principles of selected sub-diffraction microscopy techniques with a focus on SMLM. Finally, the successful application of localization microscopy in radiation biology research is demonstrated along examples of current works.
