**6. Conclusion and perspectives**

*DNA - Damages and Repair Mechanisms*

*cited figure legend under CC BY license in [147].*

**60**

**Figure 10.**

**Figure 9.**

*(A) Single molecule localization microscopy analysis of Alu clustering and dose dependent effects of numbers of Alu labelling points after exposure to ionizing photon radiation. (B) Density distribution of heterochromatin labelling in concentric rings around the center of ALU clusters. The reduction of the density peak corresponding to heterochromatin relaxation around the Alu clusters was independent of the dose. (C) Linear quadratic dose response observed by SMLM of specific oligonucleotide nanoprobe labeling of Alu elements in SkBr3 cells after exposure to different doses of* γ*-radiation. (D) Linear dose response observed by SMLM of specific oligonucleotide nanoprobe labeling of Alu elements in SkBr3 cells after exposure to low doses of* γ*-radiation.* 

*"Normalized histograms of the frequencies of similarity values of barcodes (Jaccard indices) of 53BP1 clusters in NHDF and U87 cells irradiated under 10° or 90° irradiation angle and fixed 2h post irradiation. The distributions of the average similarity of dimension 0 and 1 barcodes of 53BP1 clusters in NHDF and U87 cells are shown. The similarity distributions of clusters in cells irradiated under an angle of 10° are shown in blue, the similarity distributions of clusters in cells irradiated under 90° are shown in orange, and the similarity distributions obtained when comparing clusters in cells irradiated with 10° to clusters in cells irradiated with 90° are depicted in green". Note: These figures are modified and were originally published together with the* 

*Note: These figures are modified and were originally published under CC BY license in [16, 167].*

With this article, we have addressed scientists, researchers, and clinicians working in interdisciplinary fields, which are searching for a brief introduction to current radiobiology, its fundamental principles and methodologies. We would further like to have caught the attention of radiation biologists in laboratories, clinics, and industry by demonstrating novel super-resolution microscopy techniques that have the potential to drive radiobiology to a next generation. Single molecule localization allows geometrical and topological analyses on the meso- and nano-scale at the single-cell level in situ with the advantages of easy practice and the applicability to already existing experimental methods (e.g. immunostaining, FISH). As superresolution microscopy techniques are still not a wide-spread routine in molecular biology laboratories, the long history of fluorescence microscopy data from radiobiological studies provides a solid basis for validation. We have shown that radiobiology can be an application of SMLM based nanoscopy and its versatile data analysis method which allow the investigation of new perspectives of DNA damage induction and repair. It can even help to discover novel markers of biological dosimetry as demonstrated by our recent studies assessing dose dependent effects on retrotransposon Alu availability. Nano-scaled analysis of repair foci architecture and dynamics by assessing foci like 53BP1, Mre11, etc. will give further insight into the molecular mechanisms of DNA damage response and fate of repair pathway of individual damage sites in single cells. Indeed, evidence grows that nanostructure and function of chromatin are highly interdependent aspects that govern the fundamentals of molecular genetics, such as cell type differentiation, gene expression, DNA damage repair and reproduction. Thus, super-resolution radiobiology could serve as a general proof of principle for many other molecular biology applications in future. Finally, we believe that single-molecule localization microscopy will develop to a standard application of radiation biology and might even add to the repertoire of diagnostic technologies in clinical facilities in the future.

#### **Acknowledgements**

The successful collaborations with Felix Bestvater, German Cancer Research Center (DKFZ), Heidelberg, Christoph Cremer, Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Dieter W. Heermann, Institute for Theoretical Physics, Heidelberg University, Harry Scherthan, Institute for Radiobiology of the Bundeswehr, Munich, and Martin Falk, Institute of Biophysics, Czech Academy of Sciences, Brno, are gratefully acknowledged. The work was supported by the project grant ("Einflüsse strahleninduzierter, multipler und einzelner spezifisch-targetierter DNA-Strangschäden auf die übergeordnete mesound nanoskalige Chromatinarchitektur und die Topologie von Reparaturfoci" (NANOSTRANG)) of the Federal Ministry of Education and Research.
