Publikationen Rasterionenmikroskopie (Zellbestrahlung)

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2018

Physics at the Munich Tandem Accelerator Laboratory
G. Dollinger and T. Faestermann; Nuclear Physics News 28 (1) (2018) 5-12.
Abstract: The Tandem accelerator situated in Garching, just 20 km north of Munich, is of the “Emperor” (MP) series manufactured by High Voltage Engineering Corporation (HVEC). It delivered the first beams for experiments in 1970 and came close to its design voltage of 10 MV. In 1991 the tubes were exchanged to the extended version of HVEC. Routine operation at a terminal voltage of 14 MV was then possible.
BibTeX:
	@article{Dollinger2018,
	  author = {Günther Dollinger and Thomas Faestermann},
	  title = {Physics at the Munich Tandem Accelerator Laboratory},
	  journal = {Nuclear Physics News},
	  publisher = {Taylor & Francis},
	  year = {2018},
	  volume = {28},
	  number = {1},
	  pages = {5-12},
	  url = {https://www.tandfonline.com/doi/full/10.1080/10619127.2018.1427405},
	  doi = {https://doi.org/10.1080/10619127.2018.1427405}
	}
	
Physics at the Munich Tandem Accelerator Laboratory
G. Dollinger and T. Faestermann; ArXiv (v2) (2018) .
Abstract: This review reports on the science performed in various fields at the Munich tandem accelerator during the past decade. It covers nuclear structure studies, also with respect to astro- and particle physics as well as for the understanding of fundamental symmetries, the extremely sensitive detection of long-lived radionuclides from Supernova or r-process production with accelerator mass spectrometry and studies of the elemental composition of thin films with extreme depth resolution and sensitivity by elastic recoil detection (ERD). The ion microbeam is used for 3D hydrogen microscopy as well as in radiobiology to study the response of living cells on well-defined irradiations. In medical research new therapeutic methods of tumour irradiation are tested using proton minibeams as well as the determination of ion ranges in tissue with iono-acoustics. Primary and secondary beams from the accelerator are also used for development and testing of detector components in large setups, e.g. at the LHC, and for testing new kinds of fuel materials of high uranium density to use them as medium enriched fuels at the Munich research reactor FRM II in the future.
BibTeX:
	@openaccess{Dollinger2018a,
	  author = {Günther Dollinger and Thomas Faestermann},
	  title = {Physics at the Munich Tandem Accelerator Laboratory},
	  journal = {ArXiv},
	  type = {OpenAccess},
	  year = {2018},
	  number = {v2},
	  url = {https://arxiv.org/abs/1802.07057}
	}
	

2017

Low LET proton microbeam to understand high-LET RBE by shaping spatial dose distribution
C. Greubel, K. Ilicic, T. Rösch, J. Reindl, C. Siebenwirth, M. Moser, S. Girst, D.W. Walsh, T.E. Schmid and G. Dollinger; Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms 404 (Supplement C) (2017) 155 - 161.
Abstract: High LET radiation, like heavy ions, are known to have a higher biological effectiveness (RBE) compared to low LET radiation, like X- or γ-rays. Theories and models attribute these higher effectiveness mostly to their extremely inhomogeneous dose deposition, which is concentrated in only a few micron sized spots. At the ion microprobe SNAKE, low LET 20 MeV protons (LET in water of 2.6 keV/μm) can be applied to cells either randomly distributed or focused to submicron spots, approximating heavy ion dose deposition. Thus, the transition between low and high LET energy deposition is experimentally accessible and the effect of different spatial dose distributions can be analysed. Here, we report on the technical setup to cultivate and irradiate 104 cells with submicron spots of low LET protons to measure cell survival in unstained cells. In addition we have taken special care to characterise the beam spot of the 20 MeV proton microbeam with fluorescent nuclear track detectors.
BibTeX:
	@article{Greubel2017,
	  author = {Christoph Greubel and Katarina Ilicic and Thomas Rösch and Judith Reindl and Christian Siebenwirth and Marcus Moser and Stefanie Girst and Dietrich W.M. Walsh and Thomas E. Schmid and Günther Dollinger},
	  title = {Low LET proton microbeam to understand high-LET RBE by shaping spatial dose distribution},
	  journal = {Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms},
	  year = {2017},
	  volume = {404},
	  number = {Supplement C},
	  pages = {155 - 161},
	  note = {Proceedings of the 15th International Conference on Nuclear Microprobe Technology and Applications},
	  url = {http://www.sciencedirect.com/science/article/pii/S0168583X16305109},
	  doi = {https://doi.org/10.1016/j.nimb.2016.11.032}
	}
	
Chromatin organization revealed by nanostructure of irradiation induced γH2AX, 53BP1 and Rad51 foci
J. Reindl, S. Girst, D.W.M. Walsh, C. Greubel, B. Schwarz, C. Siebenwirth, G.A. Drexler, A.A. Friedl and G. Dollinger; Scientific Reports 7 (2017) 40616.
Abstract: The spatial distribution of DSB repair factors γH2AX, 53BP1 and Rad51 in ionizing radiation induced foci (IRIF) in HeLa cells using super resolution STED nanoscopy after low and high linear energy transfer (LET) irradiation was investigated. 53BP1 and γH2AX form IRIF with same mean size of (540 ± 40) nm after high LET irradiation while the size after low LET irradiation is significantly smaller. The IRIF of both repair factors show nanostructures with partial anti-correlation. These structures are related to domains formed within the chromatin territories marked by γH2AX while 53BP1 is mainly situated in the perichromatin region. The nanostructures have a mean size of (129 ± 6) nm and are found to be irrespective of the applied LET and the labelled damage marker. In contrast, Rad51 shows no nanostructure and a mean size of (143 ± 13) nm independent of LET. Although Rad51 is surrounded by 53BP1 it strongly anti-correlates meaning an exclusion of 53BP1 next to DSB when decision for homologous DSB repair happened.
BibTeX:
	@article{Reindl2017,
	  author = {Reindl, Judith and Girst, Stefanie and Walsh, Dietrich W. M. and Greubel, Christoph and Schwarz, Benjamin and Siebenwirth, Christian and Drexler, Guido A. and Friedl, Anna A. and Dollinger, Günther},
	  title = {Chromatin organization revealed by nanostructure of irradiation induced γH2AX, 53BP1 and Rad51 foci},
	  journal = {Scientific Reports},
	  year = {2017},
	  volume = {7},
	  pages = {40616},
	  url = {http://www.nature.com/articles/srep40616},
	  doi = {https://doi.org/10.1038/srep40616}
	}
	
Nanoskopische Analyse von DNA Doppelstrangbrüchen in menschlichen Krebszellen nach Ionenbestrahlung
Judith Reindl; Dissertation, Universität der Bundeswehr München, 2017.
Abstract: Ionisierende Strahlung induziert beim Durchgang durch menschliche Zellen Doppelstrangbrüche mit unterschiedlicher Dichte und Komplexität abhängig vom Linearen Energietransfer (LET) der Teilchen. Diese Arbeit beschreibt die quantitative, höchstauflösende STED (engl.: stimulated emission depletion) Mikroskopie an menschlichen Zellen mit einer lateralen Auflösung von  100 nm. Die Zellen wurden hierzu am Rasterionenmikroskop SNAKE am 14 MV Tandembeschleuniger in Garching oder der alpha-Bestrahlungsquelle an der Universität der Bundeswehr München bestrahlt.

Damit konnten strukturelle und funktionale Domänen der Anlagerung von Proteinen, welche für das Auffinden und die Reparatur von Doppelstrangbrüchen verantwortlich sind, detailliert untersucht werden.Weitergehend konnten diese Domänen mit der Chromatinstruktur höherer Ordnung, also der Lage der DNA innerhalb des Zellkerns, verknüpft werden. Hierzu wurde die Korrelation der wichtigen Reparaturproteine 53BP1,γH2AX, Rad51 sowie Brca1 nach hoch- und niedrig-LET Bestrahlungen untersucht. Hierbei zeigen γH2AX und 53BP1, obwohl sie der gleichen Reparaturdomäne, dem sogenannten ''flanking chromatin'' zugeordnet werden nur teilweise Korrelation, welche unabhängig vom linearen Energietransfer der Teilchen ist. 53BP1 und Rad51 schließen sich ebenso LET unabhängig gegenseitig aus, was deren unterschiedliche Rolle während der Reparatur und die Zugehörigkeit zu verschiedenen Domänen widerspiegelt. Als Mediator zwischen den beiden Proteinen und somit Domänen wurde Brca1 identifiziert, welches ähnliche Zeitverläufe, wie Rad51 zeigt, jedoch nur teilweise räumlich mit Rad51 korreliert. Weitergehend wurden bei den Proteinen 53BP1 und γ-H2AX, welche sich nach Kohlenstoffbestrahlung in (540 ± 60) nm großen IRIF (engl.: ionizing radiation induced foci) und nach Protonenbestrahlung in (410 ± 30) nm großen IRIF anlagern, Nanostrukturen innerhalb der IRIF identifiziert. Diese Strukturen haben unabhängig vom LET eine Größe von 120 nm - 140 nm und entsprechen der ebenso LET unabhängigen IRIF Größe von Rad51 von (142 ± 13) nm, welche selbst keine Nanostruktur zeigen. Diese Strukturen in Kombination mit den Korrelationsmessungen lassen sich mit der Chromatinstruktur höherer Ordnung verbinden. So konnte Rad51 als direkte Markierung des Schadensorts identifiziert werden, welche in einer Region von dekondensierter DNA liegt, dem sogenannten Perichromatin. Dies hat eine Breite von 100 nm - 200 nm und wird um den Schaden in einem größeren Bereich durch das Protein 53BP1 stabilisiert. Das phosphorylierte Histon H2AX (γ-H2AX) markiert hingegen direkt die Chromatin Territorien und somit die DNA. Als zweites wesentliches Ergebnis wurde die initiale Anzahl an IRIF des Schadensmarkers DNA-PKcs wenige Minuten nach Bestrahlung mit Hilfe höchstauflösender STED Mikroskopie bestimmt. Die IRIF haben eine mittlere Größe von  190 nm, was die Trennung von DSB ermöglicht, welche nur durch solch kleine Abstände getrennt sind. Für 27 MeV Kohlenstoffbestrahlung (LET = 500 keV/μm) am Rasterionenmikroskop SNAKE in Garching wurden (4,5 ± 0,9) IRIF/μm und für 20 MeV Lithiumbestrahlung (LET = 116 keV/μm) wurden (2,8 ± 0,5) IRIF/μm . Beides verbessert nicht nur bisherige Messungen um einen Faktor  3 sondern übersteigt auch die Anzahl von durch Monte- Carlo basierten Simulationen mit PARTRAC vorhergesagten Schadenszahlen um einen Faktor 2 - 2,5. Diese Messungen stellen damit eine gegenüber bisherigen Messungen essentiell verbesserte Datenbasis dar, um die erhöhte relative biologische Wirksamkeit von hoch-LET Strahlung besser modellieren und damit verstehen zu können.

BibTeX:
	@phdthesis{Reindl2017diss,
	  author = {Judith Reindl},
	  title = {Nanoskopische Analyse von DNA Doppelstrangbrüchen in menschlichen Krebszellen nach Ionenbestrahlung},
	  school = {Universität der Bundeswehr München},
	  year = {2017},
	  url = {https://athene-forschung.rz.unibw-muenchen.de/node?id=120718}
	}
	
Optimization of beam arrangements in proton minibeam radiotherapy by cell survival simulations
M. Sammer, C. Greubel, S. Girst and G. Dollinger; Medical Physics 44 (11) (2017) 6096-6104.
Abstract: Purpose: Proton minibeam radiotherapy using submillimeter beam dimensions allows to enhance tissue sparing in the entrance channel by spatial fractionation additionally to advantageous proton depth dose distribution. In the entrance channel, spatial fractionation leads to reduced side effects compared to conventional proton therapy. The submillimeter sized beams widen with depth due to small angle scattering and enable therefore, in contrary to x-ray microbeam radiation therapy (MRT), the homogeneous irradiation of a tumor. Proton minibeams can either be applied as planar minibeams or pencil shaped with an additional possibility to vary between a quadratic and a hexagonal arrangement for pencil minibeams. The purpose of this work is to deduce interbeam distances to achieve a homogeneous dose distribution for different tumor depths and tumor thicknesses. Furthermore, we aim for a better understanding of the sparing effect on the basis of surviving cells calculated by the linear-quadratic model.

Methods: Two-dimensional dose distributions are calculated for proton minibeams of different shapes and arrangements. For a tumor in 10-15 cm depth, treatment plans are calculated with initial beam size of σ0 = 0.2 mm in a water phantom. Proton minibeam depth dose distributions are finally converted into cell survival using a linear-quadratic model.

Results: Inter proton beam distances are maximized under the constraint of dose homogeneity in the tumor for tumor depths ranging from 4 to 15 cm and thickness ranging from 0.5 to 10 cm. Cell survival calculations for a 5 cm thick tumor covered by 10 cm healthy tissue show less cell death by up to 85%, especially in the superficial layers, while keeping the cell death in the tumor as in conventional therapy. In the entrance channel, the pencil minibeams result in higher cell survival in comparison to the planar minibeams while all proton minibeam irradiations show higher cell survival than conventional broadbeam irradiation.

Conclusion: The deduced constraints for interbeam distances simplify treatment planning for proton minibeam radiotherapy applications in future studies. The cell survival results indicate that proton minibeam radiotherapy reduces side effects but keeps tumor control as in conventional proton therapy. It makes proton minibeam, especially pencil minibeam radiotherapy a potentially attractive new approach for radiation therapy.

BibTeX:
	@article{Sammer2017,
	  author = {Sammer, M. and Greubel, C. and Girst, S. and Dollinger, G.},
	  title = {Optimization of beam arrangements in proton minibeam radiotherapy by cell survival simulations},
	  journal = {Medical Physics},
	  year = {2017},
	  volume = {44},
	  number = {11},
	  pages = {6096-6104},
	  note = {cited By 0},
	  url = {http://onlinelibrary.wiley.com/doi/10.1002/mp.12566/abstract;jsessionid=36AD12B1DEFB0CD51C862B75F5B5EF5D.f02t01},
	  doi = {https://doi.org/10.1002/mp.12566}
	}
	
The influence of reference radiation photon energy on high-LET RBE: comparison of human peripheral lymphocytes and human--hamster hybrid AL cells
T.E. Schmid, C. Greubel, G. Dollinger and E. Schmid; Radiation and Environmental Biophysics 56 (2017) 79-87.
Abstract: The relative biological effectiveness (RBE) based on the induction of dicentrics in any cell type is principally an important information for the increasing application of high-LET radiation in cancer therapy. Since the standard system of human lymphocytes for measuring dicentrics are not compatible with our microbeam irradiation setup where attaching cells are essential, we used human--hamster hybrid AL cells which do attach on foils and fulfil the special experimental requirement for microbeam irradiations. In this work, the dose--response of AL cells to photons of different energy, 70 and 200 kV X-rays and 60Co γ-rays, is characterized and compared to human lymphocytes. The total number of induced dicentrics in AL cells is approximately one order of magnitude smaller. Despite the smaller α and β parameters of the measured linear--quadratic dose--response relationship, the α/β-ratio versus photon energy dependence is identical within the accuracy of measurement for AL cells and human lymphocytes. Thus, the influence of the reference radiation used for RBE determination is the same. For therapy relevant doses of 2 Gy (60Co equivalent), the difference in RBE is around 20% only. These findings indicate that the biological effectiveness in AL cells can give important information for human cells, especially for studies where attaching cells are essential.
BibTeX:
	@article{Schmid2017,
	  author = {Schmid, T. E. and Greubel, C. and Dollinger, G. and Schmid, E.},
	  title = {The influence of reference radiation photon energy on high-LET RBE: comparison of human peripheral lymphocytes and human--hamster hybrid AL cells},
	  journal = {Radiation and Environmental Biophysics},
	  year = {2017},
	  volume = {56},
	  pages = {79--87},
	  url = {http://link.springer.com/article/10.1007%2Fs00411-016-0680-3},
	  doi = {https://doi.org/10.1007/s00411-016-0680-3}
	}
	
Gezielte Bestrahlung zellulärer und nukleärer Substrukturen am Ionenmikrostrahl SNAKE
Christian Siebenwirth; Dissertation, Universität der Bundeswehr München, 2017.
Abstract: In dieser Arbeit wurde ein Aufbau zur gezielten Bestrahlung von zellulären und nukleären Substrukturen am Rasterionenmikroskop SNAKE entwickelt, erfolgreich installiert und charakterisiert. Diese Entwicklung bildete die methodische Grundlage für die Untersuchung der Sensitivität des Nucleolus im Zellkern auf ionisierende Strahlung.

Das präsentierte neue Zielbestrahlungskonzept ermöglicht es, Substrukturen in Zellen mit einer Genauigkeit von besser als (0,4 ± 0,7) μm in X-Richtung und (-0,2 ± 0,8) μm in Y-Richtung mit einzelnen, abgezählten Ionen zu bestrahlen. So wird ein Nucleolus mit 3 μm Durchmesser von einem einzeln applizierten Ion zu mehr als 80% Wahrscheinlichkeit getroffen. Die Bestrahlung von 15-20 Zellen eines Kamerablickfeldes dauert dabei etwa 1 min, wodurch auf einer Probe mehr als 1000 Zellen pro Stunde gezielt bestrahlt werden können.

Diese methodische Entwicklung macht die Behandlung neuer Fragestellungen in der Strahlenbiologie möglich und wird schon erfolgreich für Projekte, wie der Untersuchung der Strahlensensitivität von Mitochondrien oder dem Vergleich von UV-Mikrobestrahlungen mit Ionenbestrahlungen angewendet. Als treibende Idee dieser Entwicklung wurde in dieser Arbeit erstmals mit einer gezielten Nucleolusbestrahlung mit 55 MeV Kohlenstoffionen die Hypothese getestet, ob der Zellkern homogen auf Strahlung sensitiv ist. Dazu wurde gezielt der Nucleolus oder der Zellkern mit ausgesparten Nucleoli mit 3 Ionen auf einen Punkt bestrahlt, was auf den Zellkern gemittelt etwa 1,1 Gy entspricht, und ein Zytokineseblock-Mikrokerntest durchgeführt. Es ergaben sich mit (0,34 ± 0,04) nach Nucleolusbestrahlung (NB) und (0,35 ± 0,04) nach „Zellkern ohne Nucleolus“-Bestrahlung (ZB) die gleichen Mikrokernraten pro doppelkerniger Zelle, die aber deutlich erhöht zu den Kontrollpositionen mit (0,073 ± 0,019) und (0,073 ± 0,022) sind. Nach NB wurde eine signifikant höhere Doppelkernrate von (0,60 ± 0,04) pro bestrahlter Zelle beobachtet als nach ZB mit (0,508 ± 0,023). Bei unbestrahlten Zellen lag die Doppelkernrate bei (0,600 ± 0,024). Offensichtlich wird der Zellzyklus nach NB etwas weniger verzögert als nach ZB. Bei beiden Endpunkten ist der Unterschied jedoch deutlich geringer als man anhand des DNADichteunterschieds annehmen würde (DNA-Dichte im Nucleolus ≈ 5% DNA-Dichte im Zellkern). Damit wirken sich im Nucleolus erzeugte DNA-Schäden scheinbar schwerer aus als im restlichen Zellkern.

Zusätzlich wurde überprüft, ob eine gezielte Bestrahlung des Nucleolus mit 1, 10, 50 und 100 Kohlenstoffionen eine Stressantwort der Nucleoli in der Zelle hervorruft. Eine auftretende nucleoläre Segregation mittels Färbung des UBF-Proteins, wie nach UV-Bestrahlung beobachtet wird, wurde weder in allen Nucleoli eines Zellkerns noch an dem bestrahlten Nucleolus in Folge der Ionenbestrahlung beobachtet. Jedoch ergab die Analyse der Transkription, dass an der Stelle eines Nucleolustreffer zu mehr als (90 ± 20)% das Signal des 5EU-Einbaus in die rRNA des Nucleolus verringert ist. Während-auch keine generelle Umverteilung des Parp1-Proteins über den kompletten bestrahlten Nucleolus beobachtet wurde, kam es jedoch zu (57 ± 15)% lokal an der Stelle-des reduzierten 5EU-Signals zu einer lokalen Verringerung des Parp1-Signals. Dies lässt auf eine von der Ionenzahl unabhängige lokale Hemmung der rRNA-Transkription -im Nucleolus schließen.

Abstract:

In this thesis, a setup for targeted irradiation of cellular and nuclear substructures at the ion microbeam SNAKE was developed, successfully installed and characterized. This development builds the methodical basis for investigations into the sensitivity of the nucleolus, which is in the nucleus, to ionizing radiation.

The presented new targeted irradiation concept enables the irradiation of substructures in the nucleus with an accuracy of less than (0.4 ± 0.7) μm in X-direction and (-0.2 ± 0.8) μm in Y-direction with single counted ions. Thus a nucleolus of 3 μm diameter is hit by a single applied ion with a probability of more than 80%. Irradiation of 15-20 cells in one field of view of the microscope camera takes about 1 min, whereby in one sample more than 1000 cells per hour can be irradiated.

This methodical development enables the investigation of new questions in radiobiologyand is successfully used in projects like the investigation of the radiation sensitivity of mitochondria or the comparison of UV microirradiations with ion irradiations. For the first time, as the main motivation for this development, in this
thesis the hypothesis was tested by a targeted irradiation of the nucleolus with 55 MeV carbon ion, if the nucleus is homogenously sensitive towards radiation. For
this purpose, the nucleolus or the nucleus without the nucleoli were irradiated with 3 ions in one spot, which equates to an average dose of 1.1 Gy to the nucleus. Following the irradiation a cytokinesis-block micronucleus assay done. The micronuclei yield per binucleated cell were similar with (0.34 ± 0.04) after nucleolus irradiation (NB) and (0.35 ± 0.4) after ''nucleus without nucleolus'' irradiation (ZB), but clearly higher than at the control positions with (0.073 ± 0.019) and (0.073 ± 0.022). After NB a significantly higher yield in binucleated cells of (0.60 ± 0.04) was investigated than after ZB with (0.508 ± 0.023). In unirradiated cells the yield of binucleated cells was (0.600 ± 0.024). Obviously, after NB the cell cycle is less delayed than after ZB. However, in both endpoints the difference is clearly smaller than expected using the DNA density difference (DNA density in the nucleolus ≈ 5% DNA density in the nucleus). So DNA damages caused in the nucleolus seem to have more impact than in the residual nucleus.

Additionally, it was tested, if targeted irradiation of the nucleolus with 1, 10, 50 and100 carbon ions induces a stress response in the nucleoli. An occurring nucleolar segregationusing a staining of the protein UBF, as observed after UV irradiation, couldnot be observed in all nucleoli of the nucleus nor in the irradiated nucleolus using ions. However, analysis of the transcription showed, that at the spot of a nucleolus hit with more than (90 ± 20)% probability the signal of the incorporation of 5EU in the rRNA of the nucleolus is decreased. While no general reorganization of Parp1 in the complete irradiated nucleolus was observed, (57 ± 15)% of the spots with reduced 5EU signal showed a local reduction of the Parp1 signal. Thus, independent of the ion number the rRNA transcription in the nucleolus was locally inhibited.

BibTeX:
	@phdthesis{Siebenwirth2017diss,
	  author = {Christian Siebenwirth},
	  title = {Gezielte Bestrahlung zellulärer und nukleärer Substrukturen am Ionenmikrostrahl SNAKE},
	  school = {Universität der Bundeswehr München},
	  year = {2017},
	  url = {http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:706-5214}
	}
	
Live cell imaging of mitochondria following targeted irradiation in situ reveals rapid and highly localized loss of membrane potential
D.W.M. Walsh, C. Siebenwirth, C. Greubel, K. Ilicic, J. Reindl, S. Girst, G. Muggiolu, M. Simon, P. Barberet, H. Seznec, H. Zischka, G. Multhoff, T.E. Schmid and G. Dollinger; Scientific Reports 7 (2017) 46684.
Abstract: The reliance of all cell types on the mitochondrial function for survival makes mitochondria an interesting target when trying to understand their role in the cellular response to ionizing radiation. By harnessing highly focused carbon ions and protons using microbeams, we have performed in situ live cell imaging of the targeted irradiation of individual mitochondria stained with Tetramethyl rhodamine ethyl ester (TMRE), a cationic fluorophore which accumulates electrophoretically in polarized mitochondria. Targeted irradiation with both carbon ions and protons down to beam spots of <1 μm induced a near instant loss of mitochondrial TMRE fluorescence signal in the targeted area. The loss of TMRE after targeted irradiation represents a radiation induced change in mitochondrial membrane potential. This is the first time such mitochondrial responses have been documented in situ after targeted microbeam irradiation. The methods developed and the results obtained have the ability to shed new light on not just mitochondria’s response to radiation but to further elucidate a putative mechanism of radiation induced depolarization and mitochondrial response.
BibTeX:
	@article{Walsh2017,
	  author = {Walsh, Dietrich W. M. and Siebenwirth, Christian and Greubel, Christoph and Ilicic, Katarina and Reindl, Judith and Girst, Stefanie and Muggiolu, Giovanna and Simon, Marina and Barberet, Philippe and Seznec, Hervé and Zischka, Hans and Multhoff, Gabriele and Schmid, Thomas E. and Dollinger, Guenther},
	  title = {Live cell imaging of mitochondria following targeted irradiation in situ reveals rapid and highly localized loss of membrane potential},
	  journal = {Scientific Reports},
	  year = {2017},
	  volume = {7},
	  pages = {46684},
	  url = {https://www.nature.com/articles/srep46684},
	  doi = {https://doi.org/10.1038/srep46684}
	}
	

2016

A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells
A. Buchfellner, L. Yurlova, S. Nüske, A.M. Scholz, J. Bogner, B. Ruf, K. Zolghadr, S.E. Drexler, G.A. Drexler, S. Girst, C. Greubel, J. Reindl, C. Siebenwirth, T. Romer, A.A. Friedl and U. Rothbauer; PLOS ONE 11 (3) (2016) 1-23.
Abstract: Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (  15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.
BibTeX:
	@article{Buchfellner2016,
	  author = {Buchfellner, Andrea AND Yurlova, Larisa AND Nüske, Stefan AND Scholz, Armin M. AND Bogner, Jacqueline AND Ruf, Benjamin AND Zolghadr, Kourosh AND Drexler, Sophie E. AND Drexler, Guido A. AND Girst, Stefanie AND Greubel, Christoph AND Reindl, Judith AND Siebenwirth, Christian AND Romer, Tina AND Friedl, Anna A. AND Rothbauer, Ulrich},
	  title = {A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells},
	  journal = {PLOS ONE},
	  year = {2016},
	  volume = {11},
	  number = {3},
	  pages = {1-23},
	  url = {http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151041},
	  doi = {https://doi.org/10.1371/journal.pone.0151041}
	}
	
Proton Minibeam Radiation Therapy Reduces Side Effects in an In Vivo Mouse Ear Model
S. Girst, C. Greubel, J. Reindl, C. Siebenwirth, O. Zlobinskaya, D. Walsh, K. Ilicic, M. Aichler, A. Walch, J. Wilkens, G. Multhoff, G. Dollinger and T. Schmid; International Journal of Radiation Oncology * Biology * Physics 95 (2016) 234-241.
Abstract: Purpose: Proton minibeam radiation therapy is a novel approach to minimize normal tissue damage in the entrance channel by spatial fractionation while keeping tumor control through a homogeneous tumor dose using beam widening with an increasing track length. In the present study, the dose distributions for homogeneous broad beam and minibeam irradiation sessions were simulated. Also, in an animal study, acute normal tissue side effects of proton minibeam irradiation were compared with homogeneous irradiation in a tumor-free mouse ear model to account for the complex effects on the immune system and vasculature in an in vivo normal tissue model.

Methods and Materials: At the ion microprobe SNAKE, 20-MeV protons were administered to the central part (7.2 × 7.2 mm2) of the ear of BALB/c mice, using either a homogeneous field with a dose of 60 Gy or 16 minibeams with a nominal 6000 Gy (4 × 4 minibeams, size 0.18 × 0.18 mm2, with a distance of 1.8 mm). The same average dose was used over the irradiated area.

Results: No ear swelling or other skin reactions were observed at any point after minibeam irradiation. In contrast, significant ear swelling (up to fourfold), erythema, and desquamation developed in homogeneously irradiated ears 3 to 4 weeks after irradiation. Hair loss and the disappearance of sebaceous glands were only detected in the homogeneously irradiated fields.

Conclusions: These results show that proton minibeam radiation therapy results in reduced adverse effects compared with conventional homogeneous broad-beam irradiation and, therefore, might have the potential to decrease the incidence of side effects resulting from clinical proton and/or heavy ion therapy.

BibTeX:
	@article{Girst2016,
	  author = {Girst, S. and Greubel, C. and Reindl, J. and Siebenwirth, C. and Zlobinskaya, O. and Walsh, D.W.M. and Ilicic, K. and Aichler, M. and Walch, A. and Wilkens, J.J. and Multhoff, G. and Dollinger, G. and Schmid, T.E.},
	  title = {Proton Minibeam Radiation Therapy Reduces Side Effects in an In Vivo Mouse Ear Model},
	  journal = {International Journal of Radiation Oncology * Biology * Physics},
	  year = {2016},
	  volume = {95},
	  pages = {234--241},
	  url = {http://www.sciencedirect.com/science/article/pii/S0360301615265856},
	  doi = {https://doi.org/10.1016/j.ijrobp.2015.10.020}
	}
	
Proton Minibeam Radiotherapy
Stefanie Girst; Dissertation, Universität der Bundeswehr München, 2016.
Abstract: The risk of developing adverse side effects in the normal tissue after radiotherapy is often limiting for the dose that can be applied to the tumor. Proton minibeam radiotherapy, a spatially fractionated radiotherapy method using sub-millimeter proton beams, similar to grid therapy or microbeam radiation radiotherapy (MRT) using X-rays, has recently been invented at the ion microprobe SNAKE in Munich. The aim of this new concept is to minimize normal tissue injuries in the entrance channel and especially in the skin by irradiating only a small percentage of the cells in the total irradiation field, while maintaining tumor control via a homogeneous dose in the tumor, just like in conventional broad beam radiotherapy. This can be achieved by optimizing minibeam sizes and distances according to the prevailing tumor size and depth such that after widening of the minibeams due to proton interactions in the tissue, the overlapping minibeams produce a homogeneous dose distribution throughout the tumor.

The aim of this work was to elucidate the prospects of minibeam radiation therapy compared to conventional homogeneous broad beam radiotherapy in theory and in experimental studies at the ion microprobe SNAKE. Treatment plans for model tumors of different sizes and depths were created using the planning software LAP-CERR, to elaborate suitable minibeam sizes and distances for the individual tumors. Radiotherapy-relevant inter-beam distances required to obtain a homogeneous dose in the target volume were found to be in the millimeter range.

First experiments using proton minibeams of only 10 μm and 50 μm size (termed microchannels in the corresponding publication Zlobinskaya et al. 2013) and therapy-conform larger dimensions of 100 μm and 180 μm were performed in the artificial human in-vitro skin model EpiDermFTTM (MatTek). The corresponding inter-beam distances were 500 μm, 1 mm and 1.8 mm, respectively, leading to irradiation of only a few percent of the cells in the skin tissue, but with significantly increased doses (up to 5000 Gy) compared to the average dose of 2 Gy, which was applied homogeneously in further skin samples for comparison. Gaussian-shaped minibeams of even larger sizes (σ = 260 μm and 520 μm, inter-beam distance 1.8 mm) were analyzed in further experiments to evaluate the effect of increasing beam sizes as in deeper-lying tissues. Acute side effects were quantified via the MTT tissue viability test and the release of inflammatory proteins into the culture medium and showed improved results for minibeam compared to homogeneous irradiation. Genetic damage, an indicator for secondary tumor induction, was analyzed via the micronucleus test in the epidermal keratinocytes and was less than half for minibeams up to 180 μm size compared to homogeneous fields. Increasing minibeam sizes, i.e. increasing fractions of irradiated skin receiving a dose higher than the average dose of 2 Gy) increased the number of micronuclei per divided cell, but never exceeded the genetic damage induced by a homogeneous dose distribution.

A more authentic and representative in-vivo skin model, accounting for higher complexity with blood vessels, further cell types, follicles glands and especially a working immune system, was used in the next step to further examine the side effects of minibeam radiotherapy compared to homogeneous irradiation. The central part of the ear of adult BALB/c mice was irradiated with 20 MeV protons, using an average dose of 60 Gy in a field of 7.2×7.2 mm2. The 4×4 minibeams of nominal 6000 Gy had a size of 180×180 μm2 and inter-beam distances of 1.8 mm, as in previous in-vitro skin experiments. Minibeam irradiation induced no ear swelling or other visible skin reaction at any time, while significant ear swelling (up to 4-fold), skin reddening (erythema) and desquamation developed in homogeneously irradiated ears 3-4 weeks after irradiation. Loss of hair and sebaceous glands only occurred in the homogeneous irradiation fields and did not recover during the monitoring phase of 90 days.

Taken together all theoretical considerations and experimental findings, proton minibeam radiation therapy appears suitable for the implementation in clinical tumor therapy using protons and/or heavy ions, as it reduces side effects in the normal tissue compared to conventional broad beam irradiation. However, the upper limit of the minibeam size for tissue sparing and the technical feasibility are still to be elucidated as current technologies might have to be improved and adapted for the generation of sub-millimeter proton beams of energies up to 250 MeV at therapy plants.

BibTeX:
	@phdthesis{Girst2016diss,
	  author = {Stefanie Girst},
	  title = {Proton Minibeam Radiotherapy},
	  school = {Universität der Bundeswehr München},
	  year = {2016},
	  url = {http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:706-4569}
	}
	
Double-Strand Break Distributions along high-LET Particle Tracks in Human HeLa Cells
Josef Huber; Masters-Thesis, Universität der Bundeswehr München, 2016.
Abstract: Ionizing radiation finds widespread application in cancer treatment because it induces DNA double-strand breaks (DSB), which are causal to the killing of tumor cells and are ultimately required for a patient’s recovery. Based on its clinical relevance, it is of great importance to study the influence of radiation quality on the number of induced DSB. In previous experiments, the number of observed damage sites in the cell for low-LET X-rays matched well with the number of DSB predicted by simulations. However, for high-LET ionizing radiation, a saturation in the number of damage sites was observed that is less than the predicted number of DSB. The cause of this saturation is attributed to the method of visualizing DSB. It is performed by imaging the distribution of proteins like 53BP1 and γH2AX, which are involved in DSB-signalling and form 1 μm-sized ionizing radiation induced foci (IRIF) at these sites. Due to a decreased spacing between consecutive DSB with increasing LET, single DSB can no longer be resolved within the IRIF. The proteins KU70/80 and DNA-PKcs might be a promising alternative to these conventional damage markers, since one copy each binds to the end of double-stranded DNA immediately after damage induction. Thus, for these proteins significantly smaller IRIF are expected, which might allow the visual- ization of single DSB.

The aim of this work was to count every DSB that is induced by high-LET ionizing radiation in human HeLa cells. For this, the IRIF-formation of DNA-PKcs and KU70/80 was tested and examined. Induction of DSB was achieved by irradiation with α-particles and small angle irradiation at the ion microprobe SNAKE with lithium and carbon ions. Visualization of the target proteins’ distribution in the cell was accom- plished through the method of indirect secondary immunofluorescence staining and imaging was performed with the help of a super-resolution STED-microscope.

In the experiments, no IRIF-formation was detected for primary antibodies specifically targeting KU80 and DNA-PKcs after α-irradiation. The abundant presence of 400000 proteins of each type masked the signal of single proteins bound to double-stranded DNA, indicating that the proteins are not suitable for the counting of single DSB in their indistinguishable collective natural state. DNA-PKcs bound to DSB reportedly undergo phosphorylation at Thr2609, which leads to the dissociation from the DSB. Although this way the expected strong localisation at sites of DSB is lost, it allows for the discrimination of DNA-PKcs proteins that are not involved in damage response. Based on these findings in the relevant literature, the experiments were carried out and IRIF-formation for a primary antibody specific to phosphorylated DNA-PKcs was tested positive after α-particle irradiation. Furthermore, particle tracks were visible after lithium and carbon ion irradiation for samples fixed 2, 3 and 5 minutes post- irradiation. Evaluation of 30 particle tracks for each time point yielded an average number of 2.5±0.4 IRIF per micron after 2 minutes, which increased to 3.2±0.6 IRIF per micron 5 minutes after irradiation for lithium ions with LET=116±10 keV/μm. For carbon ions with LET=500±80 keV/μm , the number of observed IRIF increased from 4.1±0.6 per micron to 4.5±0.7 per micron from 2 to 5 minutes after irradiation. The increase for both ion types can be attributed to a delayed accumulation of protein to a fraction of DSB, which become accessible by changes in the conformation of hete- rochromatin at later times. PARTRAC simulations predict 2.7±0.4 DSB per micron for lithium and 10.2±2.2 DSB per micron for carbon ions. The number of observed IRIF for lithium ions exceeded the number from linear scaling of low-LET X-rays and matched well with the predicted number from PARTRAC. However, the observed number of IRIF for carbon ions was only half the number of the predicted DSB by PARTRAC. Thus, it is concluded that the goal of counting single DSB for high-LET irradiation can only be partly fulfilled: up to LET=116±10 keV/μm , the average spacing between DSB can be resolved by the IRIF-size with diameters from 188±36 nm to 205±49 nm. However, at LET=500±80 keV/μm , the decreased average spacing between consecutive DSB can no longer be resolved and is exceeded by the minimal observed IRIF-diameter of 178±40 nm. PARTRAC takes into account that DSB in close vicinity may not be resolvable and provides a reduced number of observable IRIF, which is derived by assigning all DSB within 150 nm to one observable cluster. This results in a predicted number of 3.3±0.3 observable IRIF per micron for carbon ions, which is matched and partly exceeded by the actual observed number of IRIF per micron. This indicates that the experimental results of this thesis are compatible with PARTRAC simulations.

BibTeX:
	@mastersthesis{Huber2016ma,
	  author = {Huber, Josef},
	  title = {Double-Strand Break Distributions along high-LET Particle Tracks in Human HeLa Cells},
	  school = {Universität der Bundeswehr München},
	  year = {2016}
	}
	
Depletion of Histone Demethylase Jarid1A Resulting in Histone Hyperacetylation and Radiation Sensitivity Does Not Affect DNA Double-Strand Break Repair
C. Penterling, G.A. Drexler, C. Böhland, R. Stamp, C. Wilke, H. Braselmann, R.B. Caldwell, J. Reindl, S. Girst, C. Greubel, C. Siebenwirth, W.Y. Mansour, K. Borgmann, G. Dollinger, K. Unger and A.A. Friedl; PLoS ONE 11 (6) (2016) e0156599.
Abstract: Histone demethylases have recently gained interest as potential targets in cancer treatment and several histone demethylases have been implicated in the DNA damage response. We investigated the effects of siRNA-mediated depletion of histone demethylase Jarid1A (KDM5A, RBP2), which demethylates transcription activating tri- and dimethylated lysine 4 at histone H3 (H3K4me3/me2), on growth characteristics and cellular response to radiation in several cancer cell lines. In unirradiated cells Jarid1A depletion lead to histone hyperacetylation while not affecting cell growth. In irradiated cells, depletion of Jarid1A significantly increased cellular radiosensitivity. Unexpectedly, the hyperacetylation phenotype did not lead to disturbed accumulation of DNA damage response and repair factors 53BP1, BRCA1, or Rad51 at damage sites, nor did it influence resolution of radiation-induced foci or rejoining of reporter constructs. We conclude that the radiation sensitivity observed following depletion of Jarid1A is not caused by a deficiency in repair of DNA double-strand breaks.
BibTeX:
	@article{Penterling2016,
	  author = {Penterling, Corina and Drexler, Guido A. and Böhland, Claudia and Stamp, Ramona and Wilke, Christina and Braselmann, Herbert and Caldwell, Randolph B. and Reindl, Judith and Girst, Stefanie and Greubel, Christoph and Siebenwirth, Christian and Mansour, Wael Y. and Borgmann, Kerstin and Dollinger, Günther and Unger, Kristian and Friedl, Anna A.},
	  title = {Depletion of Histone Demethylase Jarid1A Resulting in Histone Hyperacetylation and Radiation Sensitivity Does Not Affect DNA Double-Strand Break Repair},
	  journal = {PLoS ONE},
	  year = {2016},
	  volume = {11},
	  number = {6},
	  pages = {e0156599},
	  url = {http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156599},
	  doi = {https://doi.org/10.1371/journal.pone.0156599}
	}
	
Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci
R.L. Perez, G. Best, N.H. Nicolay, C. Greubel, S. Rossberger, J. Reindl, G. Dollinger, K.-J. Weber, C. Cremer and P.E. Huber; Faseb 30 (2016) 2767-2776.
Abstract: 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 (Ø700-1000 nm) were composed of elongated subfoci with a size of  100 nm consisting of even smaller subfoci elements (Ø40-60 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. Subfoci 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.
BibTeX:
	@article{Perez2016,
	  author = {Ramon Lopez Perez and Gerrit Best and Nils H. Nicolay and Christoph Greubel and Sabrina Rossberger and Judith Reindl and Günther Dollinger and Klaus-Josef Weber and Christoph Cremer and Peter E. Huber},
	  title = {Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci},
	  journal = {Faseb},
	  year = {2016},
	  volume = {30},
	  pages = {2767-2776},
	  url = {http://www.fasebj.org/content/30/8/2767},
	  doi = {https://doi.org/10.1096/fj.201500106R}
	}
	
Feinstruktur von BRCA1- und Rad51-Foci nach α-Bestrahlung
Benjamin Schwarz; Masters-Thesis, Ludwig-Maximilians-Universität München, 2016.
Abstract: DNA-Doppelstrangbrüche (DSB) sind kritische Schäden für das Überleben einer Zelle, wobei eine Vielzahl von extra- und intrazellulärer Faktoren zu einem DSB führen können. Aus diesem Grund hat die Natur besonders spezialisierte und dadurch gleichfalls komplexe Methoden zur DSB-Reparatur entwickelt. Einer dieser Prozesse ist die Homologe Rekombination (HR), bei welcher durch ein komplexes Zusammenspiel verschiedenster Proteine, akkumuliert in so genannten Foci, unter Verwendung des homologen Schwesterchromatids, die losen DNA-Enden wieder zusammengefügt werden. Wichtige Schlüsselfunktionen übernehmen dabei die Proteine Rad51 und BRCA1, wobei ihr gesamtes Aufgabenspektrum noch nicht bekannt ist. Die geringe Größe solcher Foci von wenigen 100 nm macht eine Abbildung ihrer inneren Strukturen für herkömmliche Lichtmikroskopie aufgrund der beugungsbegrenzten Auflösung von 250 nm unmöglich. In dieser Arbeit wurden immunhistochemische Methoden in Kombination mit STED Mikroskopie (STimulated Emission Depletion) verwendet, um die Feinstruktur der Reparaturfoci α-strahlungsinduzierter DSB an Hand von BRCA1 und Rad51 unterhalb der Beugungsgrenze abzubilden und über die Zeit von 24h zu charakterisieren. Dabei deuten die Ergebnisse der Korrelationsanalyse auf drei Phasen gemeinsamer Aktion am Doppelstrangbruch hin, welche in Early Stage, Processing Stage und Late Stage aufgeteilt werden können. Des Weiteren weisen Intensitätsplots quer durch einzelne Foci gemessen, auf eine lokale Exklusion der beiden Proteine im Zentrum des DSB hin und stärken Hypothesen, welche keinen direkten Protein-Protein-Kontakt beschreiben.

DNA Double-Strand Breaks (DSB) are critical damages for a living cell. A variety of extra- and intracellular factors are able to induce DSB. Therefore, nature developed several specific and complex DSB-Repair mechanisms. One of them is homologous recombination (HR). It depends on a complex interaction between different proteins, accumulating in so called foci. These proteins use the homologous sister chromatid as a template to rejoin the loose DNA ends. The proteins BRCA1 and Rad51 have a key function in HR, but their specific responsibilities are not completely understood yet. The small size of the foci (few 100 nm) rules out a resolution via light microscopy because of the diffraction barrier of 250 nm. During this thesis, immunohistochemistry and STED microscopy (STimulated Emission Depletion) was used to image foci of BRCA1 and Rad51 during the repair of α-radiation induced DSB with a resolution below the diffraction limit. The resulting data of the correlation analysis of BRCA1 and Rad51 imply a subdivision of BRCA1 and Rad51 interaction in 3 phases during HR (early stage, processing stage, late stage). Intensity plots of the localization of BRCA1 and Rad51 show local exclusion within the foci and therefore support predictions of non-existing direct protein interaction.

BibTeX:
	@mastersthesis{Schwarz2016ma,
	  author = {Schwarz, Benjamin},
	  title = {Feinstruktur von BRCA1- und Rad51-Foci nach α-Bestrahlung},
	  school = {Ludwig-Maximilians-Universität München},
	  year = {2016}
	}
	

2015

Live cell imaging at the Munich ion microbeam SNAKE - a status report
G.A. Drexler, C. Siebenwirth, S.E. Drexler, S. Girst, C. Greubel, G. Dollinger and A.A. Friedl; Radiation Oncology 10 (2015) 42.
Abstract: Ion microbeams are important tools in radiobiological research. Still, the worldwide number of ion microbeam facilities where biological experiments can be performed is limited. Even fewer facilities combine ion microirradiation with live-cell imaging to allow microscopic observation of cellular response reactions starting very fast after irradiation and continuing for many hours. At SNAKE, the ion microbeam facility at the Munich 14 MV tandem accelerator, a large variety of biological experiments are performed on a regular basis. Here, recent developments and ongoing research projects at the ion microbeam SNAKE are presented with specific emphasis on live-cell imaging experiments. An overview of the technical details of the setup is given, including examples of suitable biological samples. By ion beam focusing to submicrometer beam spot size and single ion detection it is possible to target subcellular structures with defined numbers of ions. Focusing of high numbers of ions to single spots allows studying the influence of high local damage density on recruitment of damage response proteins.
BibTeX:
	@article{Drexler2015,
	  author = {Drexler, Guido A. and Siebenwirth, Christian and Drexler, Sophie E. and Girst, Stefanie and Greubel, Christoph and Dollinger, Günther and Friedl, Anna A.},
	  title = {Live cell imaging at the Munich ion microbeam SNAKE - a status report},
	  journal = {Radiation Oncology},
	  year = {2015},
	  volume = {10},
	  pages = {42},
	  url = {http://ro-journal.biomedcentral.com/articles/10.1186/s13014-015-0350-7},
	  doi = {https://doi.org/10.1186/s13014-015-0350-7}
	}
	
Improved normal tissue protection by proton and X-ray microchannels compared to homogeneous field irradiation
S. Girst, C. Marx, E. Bräuer-Krisch, A. Bravin, S. Bartzsch, U. Oelfke, C. Greubel, J. Reindl, C. Siebenwirth, O. Zlobinskaya, G. Multhoff, G. Dollinger, T. Schmid and J. Wilkens; Physica Medica 31 (0) (2015) 615-620.
Abstract: The risk of developing normal tissue injuries often limits the radiation dose that can be applied to the tumour in radiation therapy. Microbeam Radiation Therapy (MRT), a spatially fractionated photon radiotherapy is currently tested at the European Synchrotron Radiation Facility (ESRF) to improve normal tissue protection. MRT utilizes an array of microscopically thin and nearly parallel X-ray beams that are generated by a synchrotron. At the ion microprobe SNAKE in Munich focused proton microbeams (“proton microchannels”) are studied to improve normal tissue protection. Here, we comparatively investigate microbeam/microchannel irradiations with sub-millimetre X-ray versus proton beams to minimize the risk of normal tissue damage in a human skin model, in vitro. Skin tissues were irradiated with a mean dose of 2 Gy over the irradiated area either with parallel synchrotron-generated X-ray beams at the ESRF or with 20 MeV protons at SNAKE using four different irradiation modes: homogeneous field, parallel lines and microchannel applications using two different channel sizes. Normal tissue viability as determined in an MTT test was significantly higher after proton or X-ray microchannel irradiation compared to a homogeneous field irradiation. In line with these findings genetic damage, as determined by the measurement of micronuclei in keratinocytes, was significantly reduced after proton or X-ray microchannel compared to a homogeneous field irradiation. Our data show that skin irradiation using either X-ray or proton microchannels maintain a higher cell viability and DNA integrity compared to a homogeneous irradiation, and thus might improve normal tissue protection after radiation therapy.
BibTeX:
	@article{Girst2015,
	  author = {Girst, S. and Marx, C. and Bräuer-Krisch, E. and Bravin, A. and Bartzsch, S. and Oelfke, U. and Greubel, C. and Reindl, J. and Siebenwirth, C. and Zlobinskaya, O. and Multhoff, G. and Dollinger, G. and Schmid, T.E. and Wilkens, J.J.},
	  title = {Improved normal tissue protection by proton and X-ray microchannels compared to homogeneous field irradiation},
	  journal = {Physica Medica},
	  year = {2015},
	  volume = {31},
	  number = {0},
	  pages = {615--620},
	  url = {http://www.sciencedirect.com/science/article/pii/S1120179715000952},
	  doi = {https://doi.org/10.1016/j.ejmp.2015.04.004}
	}
	
The influence of the channel size on the reduction of side effects in microchannel proton therapy
S. Girst, C. Greubel, J. Reindl, C. Siebenwirth, O. Zlobinskaya, G. Dollinger and T.E. Schmid; Radiation and Environmental Biophysics 54 (3) (2015) 335-342.
Abstract: The potential of proton microchannel radiotherapy to reduce radiation effects in the healthy tissue but to keep tumor control the same as in conventional proton therapy is further elucidated. The microchannels spread on their way to the tumor tissue resulting in different fractions of the healthy tissue covered with doses larger than the tumor dose, while the tumor gets homogeneously irradiated. The aim of this study was to evaluate the effect of increasing channel width on potential side effects in the normal tissue. A rectangular 180 × 180 µm2 and two Gaussian-type dose distributions of σ = 260 µm and σ = 520 µm with an interchannel distance of 1.8 mm have been applied by 20-MeV protons to a 3D human skin model in order to simulate the widened channels and to compare the irradiation effects at different endpoints to those of a homogeneous proton irradiation. The number of protons applied was kept constant at all irradiation modes resulting in the same average dose of 2 Gy. All kinds of proton microchannel irradiation lead to higher cell viability and produce significantly less genetic damage than homogeneous proton irradiation, but the reduction is lower for the wider channel sizes. Our findings point toward the application of microchannel irradiation for clinical proton or heavy ion therapy to further reduce damage of normal tissues while maintaining tumor control via a homogeneous dose distribution inside the tumor.
BibTeX:
	@article{Girst2015a,
	  author = {Girst, Stefanie and Greubel, Christoph and Reindl, Judith and Siebenwirth, Christian and Zlobinskaya, Olga and Dollinger, Günther and Schmid, Thomas E.},
	  title = {The influence of the channel size on the reduction of side effects in microchannel proton therapy},
	  booktitle = {Radiation and Environmental Biophysics},
	  journal = {Radiation and Environmental Biophysics},
	  year = {2015},
	  volume = {54},
	  number = {3},
	  pages = {335--342},
	  url = {http://link.springer.com/article/10.1007%2Fs00411-015-0600-y},
	  doi = {https://doi.org/10.1007/s00411-015-0600-y}
	}
	
Nanoscopic exclusion between Rad51 and 53BP1 after ion irradiation in human HeLa cells
J. Reindl, G.A. Drexler, S. Girst, C. Greubel, C. Siebenwirth, S.E. Drexler, G. Dollinger and A.A. Friedl; Physical Biology 12 (6) (2015) 066005.
Abstract: Many proteins involved in detection, signalling and repair of DNA double-strand breaks (DSB) accumulate in large number in the vicinity of DSB sites, forming so called foci. Emerging evidence suggests that these foci are sub-divided in structural or functional domains. We use stimulated emission depletion (STED) microscopy to investigate localization of mediator protein 53BP1 and recombination factor Rad51 after irradiation of cells with low linear energy transfer (LET) protons or high LET carbon ions. With a resolution better than 100 nm, STED microscopy and image analysis using a newly developed analyzing algorithm, the reduced product of the differences from the mean, allowed us to demonstrate that with both irradiation types Rad51 occupies spherical regions of about 200 nm diameter. These foci locate within larger 53BP1 accumulations in regions of local 53BP1 depletion, similar to what has been described for the localization of Brca1, CtIP and RPA. Furthermore, localization relative to 53BP1 and size of Rad51 foci was not different after irradiation with low and high LET radiation. As expected, 53BP1 foci induced by low LET irradiation mostly contained one Rad51 focal structure, while after high LET irradiation, most foci contained >1 Rad51 accumulation.
BibTeX:
	@article{Reindl2015,
	  author = {Judith Reindl and Guido A Drexler and Stefanie Girst and Christoph Greubel and Christian Siebenwirth and Sophie E Drexler and Günther Dollinger and Anna A Friedl},
	  title = {Nanoscopic exclusion between Rad51 and 53BP1 after ion irradiation in human HeLa cells},
	  journal = {Physical Biology},
	  year = {2015},
	  volume = {12},
	  number = {6},
	  pages = {066005},
	  url = {http://stacks.iop.org/1478-3975/12/i=6/a=066005},
	  doi = {https://doi.org/10.1088/1478-3975/12/6/066005}
	}
	
Investigation of EBT2 and EBT3 films for proton dosimetry in the 4–20 MeV energy range
S. Reinhardt, M. Würl, C. Greubel, N. Humble, J. Wilkens, M. Hillbrand, A. Mairani, W. Assmann and K. Parodi; Radiation and Environmental Biophysics 54 (1) (2015) 71-79.
Abstract: Radiochromic films such as Gafchromic EBT2 or EBT3 films are widely used for dose determination in radiation therapy because they offer a superior spatial resolution compared to any other digital dosimetric 2D detector array. The possibility to detect steep dose gradients is not only attractive for intensity-modulated radiation therapy with photons but also for intensity-modulated proton therapy. Their characteristic dose rate-independent response makes radiochromic films also attractive for dose determination in cell irradiation experiments using laser-driven ion accelerators, which are currently being investigated as future medical ion accelerators. However, when using these films in ion beams, the energy-dependent dose response in the vicinity of the Bragg peak has to be considered. In this work, the response of these films for low-energy protons is investigated. To allow for reproducible and background-free irradiation conditions, the films were exposed to mono-energetic protons from an electrostatic accelerator, in the 4–20 MeV energy range. For comparison, irradiation with clinical photons was also performed. It turned out that in general, EBT2 and EBT3 films show a comparable performance. For example, dose–response curves for photons and protons with energies as low as 11 MeV show almost no differences. However, corrections are required for proton energies below 11 MeV. Care has to be taken when correction factors are related to an average LET from depth–dose measurements, because only the dose-averaged LET yields similar results as obtained in mono-energetic measurements.
BibTeX:
	@article{Reinhardt2015,
	  author = {Reinhardt, S. and Würl, M. and Greubel, C. and Humble, N. and Wilkens, J.J. and Hillbrand, M. and Mairani, A. and Assmann, W. and Parodi, K.},
	  title = {Investigation of EBT2 and EBT3 films for proton dosimetry in the 4–20 MeV energy range},
	  journal = {Radiation and Environmental Biophysics},
	  year = {2015},
	  volume = {54},
	  number = {1},
	  pages = {71--79},
	  url = {http://link.springer.com/article/10.1007%2Fs00411-014-0581-2},
	  doi = {https://doi.org/10.1007/s00411-014-0581-2}
	}
	
Sub-micrometer 20 MeV protons or 45 MeV lithium spot irradiation enhances yields of dicentric chromosomes due to clustering of DNA double-strand breaks
T. Schmid, W. Friedland, C. Greubel, S. Girst, J. Reindl, C. Siebenwirth, K. Ilicic, E. Schmid, G. Multhoff, E. Schmitt, P. Kundrát and G. Dollinger; Mutation Research/Genetic Toxicology and Environmental Mutagenesis 793 (2015) 30-40.
Abstract: Abstract In conventional experiments on biological effects of radiation types of diverse quality, micrometer-scale double-strand break (DSB) clustering is inherently interlinked with clustering of energy deposition events on nanometer scale relevant for DSB induction. Due to this limitation, the role of the micrometer and nanometer scales in diverse biological endpoints cannot be fully separated. To address this issue, hybrid human-hamster AL cells have been irradiated with 45 MeV (60 keV/μm) lithium ions or 20 MeV (2.6 keV/μm) protons quasi-homogeneously distributed or focused to 0.5 × 1 μm2 spots on regular matrix patterns (point distances up to 10.6 × 10.6 μm), with pre-defined particle numbers per spot to provide the same mean dose of 1.7 Gy. The yields of dicentrics and their distribution among cells have been scored. In parallel, track-structure based simulations of DSB induction and chromosome aberration formation with PARTRAC have been performed. The results show that the sub-micrometer beam focusing does not enhance DSB yields, but significantly affects the DSB distribution within the nucleus and increases the chance to form DSB pairs in close proximity, which may lead to increased yields of chromosome aberrations. Indeed, the experiments show that focusing 20 lithium ions or 451 protons per spot on a 10.6 μm grid induces two or three times more dicentrics, respectively, than a quasi-homogenous irradiation. The simulations reproduce the data in part, but in part suggest more complex behavior such as saturation or overkill not seen in the experiments. The direct experimental demonstration that sub-micrometer clustering of DSB plays a critical role in the induction of dicentrics improves the knowledge on the mechanisms by which these lethal lesions arise, and indicates how the assumptions of the biophysical model could be improved. It also provides a better understanding of the increased biological effectiveness of high-LET radiation.
BibTeX:
	@article{Schmid2015,
	  author = {Schmid, T.E. and Friedland, W. and Greubel, C. and Girst, S. and Reindl, J. and Siebenwirth, C. and Ilicic, K. and Schmid, E. and Multhoff, G. and Schmitt, E. and Kundrát, P. and Dollinger, G.},
	  title = {Sub-micrometer 20 MeV protons or 45 MeV lithium spot irradiation enhances yields of dicentric chromosomes due to clustering of DNA double-strand breaks},
	  booktitle = {Insights into formation and consequences of chromosome aberrations: Report on the 11th International Symposium on Chromosomal Aberrations (ISCA 11), Rhodes , Greece, September 12-14, 2014},
	  journal = {Mutation Research/Genetic Toxicology and Environmental Mutagenesis},
	  year = {2015},
	  volume = {793},
	  pages = {30--40},
	  url = {http://www.sciencedirect.com/science/article/pii/S1383571815002053},
	  doi = {https://doi.org/10.1016/j.mrgentox.2015.07.015}
	}
	
Determination of the accuracy for targeted irradiations of cellular substructures at SNAKE
C. Siebenwirth, C. Greubel, S. Drexler, S. Girst, J. Reindl, D. Walsh, G. Dollinger, A. Friedl, T. Schmid and G. Drexler; Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms 348 (0) (2015) 137-142.
Abstract: In the last 10 years the ion microbeam SNAKE, installed at the Munich 14 MV tandem accelerator, has been successfully used for radiobiological experiments by utilizing pattern irradiation without targeting single cells. Now for targeted irradiation of cellular substructures a precise irradiation device was added to the live cell irradiation setup at SNAKE. It combines a sub-micrometer single ion irradiation facility with a high resolution optical fluorescence microscope. Most systematic errors can be reduced or avoided by using the same light path in the microscope for beam spot verification as well as for and target recognition. In addition online observation of the induced cellular responses is possible. The optical microscope and the beam delivering system are controlled by an in-house developed software which integrates the open-source image analysis software, CellProfiler, for semi-automatic target recognition.
BibTeX:
	@article{Siebenwirth2015,
	  author = {Siebenwirth, C. and Greubel, C. and Drexler, S.E. and Girst, S. and Reindl, J. and Walsh, D.W.M. and Dollinger, G. and Friedl, A.A. and Schmid, T.E. and Drexler, G.A.},
	  title = {Determination of the accuracy for targeted irradiations of cellular substructures at SNAKE},
	  journal = {Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms},
	  year = {2015},
	  volume = {348},
	  number = {0},
	  pages = {137--142},
	  url = {http://www.sciencedirect.com/science/article/pii/S0168583X15000865},
	  doi = {https://doi.org/10.1016/j.nimb.2015.01.064}
	}
	

2014

The Effects of Ultra-High Dose Rate Proton Irradiation on Growth Delay in the Treatment of Human Tumor Xenografts in Nude Mice
O. Zlobinskaya, C. Siebenwirth, C. Greubel, V. Hable, R. Hertenberger, N. Humble, S. Reinhardt, D. Michalski, B. Röper, G. Multhoff, G. Dollinger, J. Wilkens and T. Schmid; Radiation Research 181 (2) (2014) 177-183.
Abstract: The new technology of laser-driven ion acceleration (LDA) has shown the potential for driving highly brilliant particle beams. Laser-driven ion acceleration differs from conventional proton sources by its ultra-high dose rate, whose radiobiological impact should be investigated thoroughly before adopting current clinical dose concepts. The growth of human FaDu tumors transplanted onto the hind leg of nude mice was measured sonographically. Tumors were irradiated with 20 Gy of 23 MeV protons at pulsed mode with single pulses of 1 ns duration or continuous mode (?100 ms) in comparison to controls and to a dose-response curve for 6 MV photons. Tumor growth delay and the relative biological effectiveness (RBE) were calculated for all irradiation modes. The mean target dose reconstructed from Gafchromic films was 17.4 ± 0.8 Gy for the pulsed and 19.7 ± 1.1 Gy for the continuous irradiation mode. The mean tumor growth delay was 34 ± 6 days for pulsed, 35 ± 6 days for continuous protons, and 31 ± 7 days for photons 20 ± 1.2 Gy, resulting in RBEs of 1.22 ± 0.19 for pulsed and 1.10 ± 0.18 for continuous protons, respectively. In summary, protons were found to be significantly more effective in reducing the tumor volume than photons (P < 0.05). Together with the results of previous in vitro experiments, the in vivo data reveal no evidence for a substantially different radiobiology that is associated with the ultra-high dose rate of protons that might be generated from advanced laser technology in the future.
The new technology of laser-driven ion acceleration (LDA) has shown the potential for driving highly brilliant particle beams. Laser-driven ion acceleration differs from conventional proton sources by its ultra-high dose rate, whose radiobiological impact should be investigated thoroughly before adopting current clinical dose concepts. The growth of human FaDu tumors transplanted onto the hind leg of nude mice was measured sonographically. Tumors were irradiated with 20 Gy of 23 MeV protons at pulsed mode with single pulses of 1 ns duration or continuous mode (?100 ms) in comparison to controls and to a dose-response curve for 6 MV photons. Tumor growth delay and the relative biological effectiveness (RBE) were calculated for all irradiation modes. The mean target dose reconstructed from Gafchromic films was 17.4 ± 0.8 Gy for the pulsed and 19.7 ± 1.1 Gy for the continuous irradiation mode. The mean tumor growth delay was 34 ± 6 days for pulsed, 35 ± 6 days for continuous protons, and 31 ± 7 days for photons 20 ± 1.2 Gy, resulting in RBEs of 1.22 ± 0.19 for pulsed and 1.10 ± 0.18 for continuous protons, respectively. In summary, protons were found to be significantly more effective in reducing the tumor volume than photons (P < 0.05). Together with the results of previous in vitro experiments, the in vivo data reveal no evidence for a substantially different radiobiology that is associated with the ultra-high dose rate of protons that might be generated from advanced laser technology in the future.
BibTeX:
	@article{Zlobinskaya2014,
	  author = {Zlobinskaya, O. and Siebenwirth, C. and Greubel, C. and Hable, V. and Hertenberger, R. and Humble, N. and Reinhardt, S. and Michalski, D. and Röper, B. and Multhoff, G. and Dollinger, G. and Wilkens, J.J. and Schmid, T.E.},
	  title = {The Effects of Ultra-High Dose Rate Proton Irradiation on Growth Delay in the Treatment of Human Tumor Xenografts in Nude Mice},
	  booktitle = {Radiation Research},
	  journal = {Radiation Research},
	  year = {2014},
	  volume = {181},
	  number = {2},
	  pages = {177--183},
	  url = {http://www.rrjournal.org/doi/abs/10.1667/RR13464.1},
	  doi = {https://doi.org/10.1667/RR13464.1}
	}
	

2013

Subdiffusion Supports Joining Of Correct Ends During Repair Of DNA Double-Strand Breaks
S. Girst, V. Hable, G.A. Drexler, C. Greubel, C. Siebenwirth, M. Haum, A.A. Friedl and G. Dollinger; Scientific Reports 3 (2013) 2511.
Abstract: The mobility of damaged chromatin regions in the nucleus may affect the probability of mis-repair. In this work, live-cell observation and distance tracking of GFP-tagged DNA damage response protein MDC1 was used to study the random-walk behaviour of chromatin domains containing radiation-induced DNA double-strand breaks (DSB). Our measurements indicate a subdiffusion-type random walk process with similar time dependence for isolated and clustered DSBs that were induced by 20 MeV proton or 43 MeV carbon ion micro-irradiation. As compared to normal diffusion, subdiffusion enhances the probability that both ends of a DSB meet, thus promoting high efficiency DNA repair. It also limits their probability of long-range movements and thus lowers the probability of mis-rejoining and chromosome aberrations.
BibTeX:
	@article{Girst2013,
	  author = {Girst, S. and Hable, V. and Drexler, G. A. and Greubel, C. and Siebenwirth, C. and Haum, M. and Friedl, A. A. and Dollinger, G.},
	  title = {Subdiffusion Supports Joining Of Correct Ends During Repair Of DNA Double-Strand Breaks},
	  journal = {Scientific Reports},
	  year = {2013},
	  volume = {3},
	  pages = {2511},
	  url = {http://www.nature.com/articles/srep02511},
	  doi = {https://doi.org/10.1038/srep02511}
	}
	
Einfluss der zeitlichen und räumlichen Fokussierung auf die strahlenbiologische Wirksamkeit von Protonen.
Christoph Greubel; Dissertation, Universität der Bundeswehr München, 2013.
Abstract: In dieser Arbeit wurde der Einfluss von auf Nanosekunden gepulster (zeitlich fokussierter) Dosisdeposition, im zweiten Teil von auf Submikrometer (räumlich) fokussierter Dosisdeposition auf die relative biologische Wirksamkeit, RBE, studiert. Die Effekte gepulster Bestrahlung auf Nanosekunden Zeitskala sind vor allem für eine mögliche Anwendung der Laserbeschleunigung von Ionen in der Tumortherapie, welche die Dosisdeposition auf einer Nanosekunden Zeitskala erwarten lässt, von Bedeutung. Zur Untersuchung wurde die Wachstumsverzögerung von zwei menschlichen Plattenepitelkarzinomen aus dem Mund- und Rachenraum, FaDu und XF354, im Mausmodell nach Bestrahlung mit einer Fraktion von nominell 20 Gy gemessen. In Ermangelung geeigneter lasergetriebener Ionenstrahlen wurde hierzu mittels konventioneller Technik am Rasterionenmikroskop SNAKE am Müchener Tandembeschleuniger ein auf 1,3 ns (volle Halbwertsbreite) gepulster 23 MeV Protonenstrahl mit einer Fluenz pro Einzelpuls von bis zu 109 cm-2 präpariert, sowie ein kontinuierlicher Protonenstrahl zur Dosisdeposition auf Millisekunden Zeitskala für direkte Vergleichsmessungen. Die Bestrahlung der maximal 4 mm tiefen und 7 mm im Durchmesser messenden Tumore erfolgt voxelweise, wobei die komplette Fluenz eines Voxels mit einem Nanosekunden Puls appliziert wird. An jedem Punkt im Tumor deponiert mindestens ein Puls eine Dosis zwischen 1,0 Gy und 2,7 Gy. Der RBE für die Wachstumsverzögerung von FaDu Tumoren bezüglich 6 MV Röntgenstrahlung wurde nach kontinuierlicher Dosisdeposition zu 1,10 ± 0,14, nach gepulster Dosisdeposition zu 1,22 ± 0,17 gemessen. Auch für die XF354 Tumore konnte kein signifikanter Unterschied in der Wachstumsverzögerung gemessen werden. Die Messungen zeigen keine Anzeichen für eine geänderte Wirksamkeit von Nanosekunden gepulster Dosisdeposition. Im zweiten Teil der Arbeit wurden die Auswirkungen von räumlich fokussierter Dosisdeposition am Endpunkt der Induktion von dizentrischen Chromosomen und Mikrokernen untersucht. Durch die Submikrometer Fokussierung von niedrig-LET 20 MeV Protonen kann eine räumliche Dosisverteilung generiert werden, welche qualitativ jener von Schwerionen mit hohem LET ähnelt, so dass die Wirkung von dichtionisierender hoch-LET Strahlung modelliert werden kann. Hierzu wurden AL-Zellen mit einer Dosis von jeweils 1,7 Gy in drei verschiedenen Modi bestrahlt: Die Bestrahlung mit Submikrometer fokussierten 20 MeV Protonen folgt einer 5,4 µm x 5,4 µm Matrix, wobei 117 Protonen pro Matrixpunkt appliziert werden. Die Bestrahlung mit 55 MeV Kohlenstoffionen erfolgt im selben Muster mit je einem Ion pro Matrixpunkt. Zufällig verteilte 20 MeV Protonen werden mit einer Fluenz von 4,01 µm-2 appliziert. Der RBE für die Induktion von Mikrokernen steigt durch die Fokussierung der Protonen von 1,28 ± 0,07 nach zufällig verteilter Protonenbestrahlung auf 1,48 ± 0,07 nach fokussierter Protonenapplikation, der RBE für die Induktion von dizentrischen Chromosomen steigt von 1,41 ± 0,14 auf 1,92 ± 0,15. Der von Kohlenstoffionen induzierte RBE ist mit 2,20 ± 0,09 für Mikrokerne und 3,21 ± 0,27 für dizentrische Chromosomen nochmal deutlich höher. Die signifikante Erhöhung der Induktion von Chromosomenaberrationen alleine durch die Fokussierung der Protonen und damit der räumlichen Dosisverteilung zeigt, dass die räumliche Dosisverteilung für den RBE maßgeblich ist. Die Experimente stellen somit die erste experimentelle Bestätigung der Grundannahme des Local Effect Models dar, welches in der Tumortherapie mit schweren Ionen zur Modellierung des RBE für die Dosisplanung verwendet wird. Rechnungen mit dem Local Effect Model III zeigen jedoch, dass dieses den RBE für die Endpunkte der Chromosomenaberrationen für die drei Bestrahlungsmodi zwar qualitativ, nicht aber quantitativ beschreiben kann.
BibTeX:
	@phdthesis{Greubel2013diss,
	  author = {Greubel, Christoph},
	  title = {Einfluss der zeitlichen und räumlichen Fokussierung auf die strahlenbiologische Wirksamkeit von Protonen.},
	  school = {Universität der Bundeswehr München},
	  year = {2013},
	  url = {http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:706-3415}
	}
	
Reduced side effects by proton microchannel radiotherapy: Study in a human skin model
O. Zlobinskaya, S. Girst, C. Greubel, V. Hable, C. Siebenwirth, D. Walsh, G. Multhoff, J. Wilkens, T. Schmid and G. Dollinger; Radiation and Environmental Biophysics 52 (1) (2013) 123-133.
Abstract: The application of a microchannel proton irradiation was compared to homogeneous irradiation in a three-dimensional human skin model. The goal is to minimize the risk of normal tissue damage by microchannel irradiation, while preserving local tumor control through a homogeneous irradiation of the tumor that is achieved because of beam widening with increasing track length. 20 MeV protons were administered to the skin models in 10- or 50-μm-wide irradiation channels on a quadratic raster with distances of 500 μm between each channel (center to center) applying an average dose of 2 Gy. For comparison, other samples were irradiated homogeneously at the same average dose. Normal tissue viability was significantly enhanced after microchannel proton irradiation compared to homogeneous irradiation. Levels of inflammatory parameters, such as Interleukin-6, TGF-Beta, and Pro-MMP1, were significantly lower in the supernatant of the human skin tissue after microchannel irradiation than after homogeneous irradiation. The genetic damage as determined by the measurement of micronuclei in keratinocytes also differed significantly. This difference was quantified via dose modification factors (DMF) describing the effect of each irradiation mode relative to homogeneous X-ray irradiation, so that the DMF of 1.21 ± 0.20 after homogeneous proton irradiation was reduced to 0.23 ± 0.11 and 0.40 ± 0.12 after microchannel irradiation using 10- and 50-μm-wide channels, respectively. Our data indicate that proton microchannel irradiation maintains cell viability while significantly reducing inflammatory responses and genetic damage compared to homogeneous irradiation, and thus might improve protection of normal tissue after irradiation.
BibTeX:
	@article{Zlobinskaya2013,
	  author = {Zlobinskaya, O. and Girst, S. and Greubel, C. and Hable, V. and Siebenwirth, C. and Walsh, D.W.M. and Multhoff, G. and Wilkens, J.J. and Schmid, T.E. and Dollinger, G.},
	  title = {Reduced side effects by proton microchannel radiotherapy: Study in a human skin model},
	  booktitle = {Radiation and Environmental Biophysics},
	  journal = {Radiation and Environmental Biophysics},
	  year = {2013},
	  volume = {52},
	  number = {1},
	  pages = {123--133},
	  url = {http://link.springer.com/article/10.1007%2Fs00411-012-0450-9},
	  doi = {https://doi.org/10.1007/s00411-012-0450-9}
	}
	

2012

A laser-driven nanosecond proton source for radiobiological studies
J. Bin, K. Allinger, W. Assmann, G. Dollinger, G.A. Drexler, A.A. Friedl, D. Habs, P. Hilz, R. Hoerlein, N. Humble, S. Karsch, K. Khrennikov, D. Kiefer, F. Krausz, W. Ma, D. Michalski, M. Molls, S. Raith, S. Reinhardt, B. Roeper, T.E. Schmid, T. Tajima, J. Wenz, O. Zlobinskaya, J. Schreiber and J.J. Wilkens; Applied Physics Letters 101 (24) (2012) 243701.
Abstract: Ion beams are relevant for radiobiological studies and for tumor therapy. In contrast to conventional accelerators, laser-driven ion acceleration offers a potentially more compact and cost-effective means of delivering ions for radiotherapy. Here, we show that by combining advanced acceleration using nanometer thin targets and beam transport, truly nanosecond quasi-monoenergetic proton bunches can be generated with a table-top laser system, delivering single shot doses up to 7Gy to living cells. Although in their infancy, laser-ion accelerators allow studying fast radiobiological processes as demonstrated here by measurements of the relative biological effectiveness of nanosecond proton bunches in human tumor cells.
BibTeX:
	@article{Bin2012,
	  author = {Bin, Jianhui and Allinger, Klaus and Assmann, Walter and Dollinger, Guenther and Drexler, Guido A. and Friedl, Anna A. and Habs, Dieter and Hilz, Peter and Hoerlein, Rainer and Humble, Nicole and Karsch, Stefan and Khrennikov, Konstantin and Kiefer, Daniel and Krausz, Ferenc and Ma, Wenjun and Michalski, Doerte and Molls, Michael and Raith, Sebastian and Reinhardt, Sabine and Roeper, Barbara and Schmid, Thomas E. and Tajima, Toshiki and Wenz, Johannes and Zlobinskaya, Olga and Schreiber, Joerg and Wilkens, Jan J.},
	  title = {A laser-driven nanosecond proton source for radiobiological studies},
	  journal = {Applied Physics Letters},
	  year = {2012},
	  volume = {101},
	  number = {24},
	  pages = {243701},
	  url = {http://link.aip.org/link/doi/10.1063/1.4769372},
	  doi = {https://doi.org/10.1063/1.4769372}
	}
	
Recruitment kinetics of DNA repair proteins Mdc1 and Rad52 but not 53BP1 depend on damage complexity
V. Hable, G.A. Drexler, T. Brüning, C. Burgdorf, C. Greubel, A. Derer, J. Seel, H. Strickfaden, T. Cremer, A.A. Friedl and G. Dollinger; PLoS One 7 (7) (2012) e41943.
Abstract: The recruitment kinetics of double-strand break (DSB) signaling and repair proteins Mdc1, 53BP1 and Rad52 into radiation-induced foci was studied by live-cell fluorescence microscopy after ion microirradiation. To investigate the influence of damage density and complexity on recruitment kinetics, which cannot be done by UV laser irradiation used in former studies, we utilized 43 MeV carbon ions with high linear energy transfer per ion (LET = 370 keV/μm) to create a large fraction of clustered DSBs, thus forming complex DNA damage, and 20 MeV protons with low LET (LET = 2.6 keV/μm) to create mainly isolated DSBs. Kinetics for all three proteins was characterized by a time lag period T0 after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times τ1. Mdc1 accumulates faster (T0 = 17±2 s, τ1 = 98±11 s) than 53BP1 (T0 = 77±7 s, τ1 = 310±60 s) after high LET irradiation. However, recruitment of Mdc1 slows down (T0 = 73±16 s, τ1 = 1050±270 s) after low LET irradiation. The recruitment kinetics of Rad52 is slower than that of Mdc1, but exhibits the same dependence on LET. In contrast, the mean recruitment time τ1 of 53BP1 remains almost constant when varying LET. Comparison to literature data on Mdc1 recruitment after UV laser irradiation shows that this rather resembles recruitment after high than low LET ionizing radiation. So this work shows that damage quality has a large influence on repair processes and has to be considered when comparing different studies.
BibTeX:
	@article{Hable2012,
	  author = {Hable, Volker and Drexler, Guido A. and Brüning, Tino and Burgdorf, Christian and Greubel, Christoph and Derer, Anja and Seel, Judith and Strickfaden, Hilmar and Cremer, Thomas and Friedl, Anna A. and Dollinger, Günther},
	  title = {Recruitment kinetics of DNA repair proteins Mdc1 and Rad52 but not 53BP1 depend on damage complexity},
	  journal = {PLoS One},
	  year = {2012},
	  volume = {7},
	  number = {7},
	  pages = {e41943},
	  url = {http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0041943},
	  doi = {https://doi.org/10.1371/journal.pone.0041943}
	}
	
Super resolution microscopy of repair foci after ion irradiation of human HeLa cells
Judith (Seel) Reindl; Masters-Thesis, Ludwigs-Maximilians-Universität München, 2012.
Abstract: High LET (linear energy transfer) irradiation of living cells using heavy ions generates a high amount of DNA double-strand breaks (DSB) in close vicinity to each other along the ion track. Various repair proteins cluster to the damage sites, such as gH2AX and 53BP1, forming so-called repair foci of a gross size of about 1 µm. Due to the fact that one focus covers more than one DSB, a fine-structure within the focus can be expected. First indications for such a fine-structure were found in wide field images of
cells taken one hour after irradiation with 55MeV carbon ions in a 5x5 µm matrix performed at the ion microprobe SNAKE. While a typical focus with the diameter of about 1 µm can be easily resolved using a conventional fluorescence microscope, its substructures cannot be resolved due to the diffraction limit of about 250 nm in conventional fluorescence microscopy. Therefore, for analyzing foci fine-structures systematically, super-resolution microscopy techniques like structured illumination microscopy (SIM), stimulation emission depletion microscopy (STED) or localization microscopy (SPDM) which provide a lateral resolution of about 130 nm (SIM) to 50 nm (SPDM) fwhm are utilized. Since with these techniques the lateral resolution is even better than the z-resolution we used an irradiation configuration, where the cells are irradiated at a small angle to the image plane. Thus, the complete ion track appears as a line within one layer of a 3D microscope image. Due to these improvements the super resolution images clearly indicate a fine-structure when e. g. 53BP1 is stained with two colors.
For quantification of the results the Pearson correlation coefficient is calculated for a pixel wise shift in x-direction as well as in y-direction of one color channel with respect to the other (Van Steensel approach). This proves the existence of a fine-structure of a scale of about 200-230 nm, which becomes obvious by an extra correlation peak with a fwhm of this size. Using the same Van Steensel approach with images where one color marks 53BP1 and the other gH2AX, it can be shown that there is no total correlation of the fine-structure between 53BP1 and gH2AX on the small scale.
Using the product of the difference of the mean (PDM) for 2D profiles the images where one protein is labeled with two colors show large regions with total correlation of the to color channels and only small regions at the rim of the focus with no total correlation. In addition, in the PDM approach two different damage markers each labeled in one color show colocalisation in small regions inside the focus but anticorrelation in the outer regions of the focus. These analysis lead to different results:
first of all a single repair marker seems to cluster systematically to the damage site and not in a random way. Secondly 53BP1 and gH2AX cluster in a different way and therefore no full colocalisation can be reached.
With this experimental and analytical methods it is possible to determine the way of clustering to DSB of one single DNA damage marker to clarify the structure of a DSB and the structure of the chromatin architecture as well as the comparison of two
damage markers to get deeper understanding to the interaction of repair markers and repair proteins and at the end decode the way of DNA repair.
BibTeX:
	@mastersthesis{Reindl2012ma,
	  author = {Reindl, Judith (Seel)},
	  title = {Super resolution microscopy of repair foci after ion irradiation of human HeLa cells},
	  school = {Ludwigs-Maximilians-Universität München},
	  year = {2012}
	}
	
Low LET protons focused to submicrometer shows enhanced radiobiological effectiveness
T.E. Schmid, C. Greubel, V. Hable, O. Zlobinskaya, D. Michalski, S. Girst, C. Siebenwirth, E. Schmid, M. Molls, G. Multhoff and G. Dollinger; Physics in Medicine and Biology 57 (19) (2012) 5889-5907.
Abstract: This study shows that enhanced radiobiological effectiveness (RBE) values can be generated focusing low linear energy transfer (LET) radiation and thus changing the microdose distribution. 20 MeV protons (LET = 2.65 keV µm −1 ) are focused to submicrometer diameter at the ion microprobe superconducting nanoprobe for applied nuclear (Kern) physics experiments of the Munich tandem accelerator. The RBE values, as determined by measuring micronuclei (RBE MN = 1.48 ± 0.07) and dicentrics (RBE D = 1.92 ± 0.15), in human–hamster hybrid (A L ) cells are significantly higher when 117 protons were focused to a submicrometer irradiation field within a 5.4 × 5.4 µm 2 matrix compared to quasi homogeneous in a 1 × 1 µm 2 matrix applied protons (RBE MN = 1.28 ± 0.07; RBE D = 1.41 ± 0.14) at the same average dose of 1.7 Gy. The RBE values are normalized to standard 70 kV (dicentrics) or 200 kV (micronuclei) x-ray irradiation. The 117 protons applied per point deposit the same amount of energy like a 12 C ion with 55 MeV total energy (4.48 MeV u −1 ). The enhancements are about half of that obtained for 12 C ions (RBE MN = 2.20 ± 0.06 and RBE D = 3.21 ± 0.10) and they are attributed to intertrack interactions of the induced damages. The measured RBE values show differences from predictions of the local effect model (LEM III) that is used to calculate RBE values for irradiation plans to treat tumors with high LET particles.
BibTeX:
	@article{Schmid2012,
	  author = {Schmid, T. E. and Greubel, C. and Hable, V. and Zlobinskaya, O. and Michalski, D. and Girst, S. and Siebenwirth, C. and Schmid, E. and Molls, M. and Multhoff, G. and Dollinger, G.},
	  title = {Low LET protons focused to submicrometer shows enhanced radiobiological effectiveness},
	  journal = {Physics in Medicine and Biology},
	  year = {2012},
	  volume = {57},
	  number = {19},
	  pages = {5889-5907},
	  url = {http://stacks.iop.org/0031-9155/57/i=19/a=5889},
	  doi = {https://doi.org/10.1088/0031-9155/57/19/5889}
	}
	
Induction and repair of DNA double-strand breaks assessed by gamma-H2AX foci after irradiation with pulsed or continuous proton beams
O. Zlobinskaya, G. Dollinger, D. Michalski, V. Hable, C. Greubel, G. Du, G. Multhoff, B. Röper, M. Molls and T.E. Schmid; Radiation and Environmental Biophysics 51 (1) (2012) 23-32.
Abstract: In particle tumor therapy including beam scanning at accelerators, the dose per voxel is delivered within about 100 ms. In contrast, the new technology of laser plasma acceleration will produce ultimately shorter particle packages that deliver the dose within a nanosecond. Here, possible differences for relative biological effectiveness in creating DNA double-strand breaks in pulsed or continuous irradiation mode are studied. HeLa cells were irradiated with 1 or 5 Gy of 20-MeV protons at the Munich tandem accelerator, either at continuous mode (100 ms), or applying a single pulse of 1-ns duration. Cells were fixed 1 h after 1-Gy irradiation and 24 h after 5-Gy irradiation, respectively. A dose–effect curve based on five doses of X-rays was taken as reference. The total number of phosphorylated histone H2AX (gamma-H2AX) foci per cell was determined using a custom-made software macro for gamma-H2AX foci counting. For 1 h after 1-Gy 20-MeV proton exposures, values for the relative biological effectiveness (RBE) of 0.97 ± 0.19 for pulsed and 1.13 ± 0.21 for continuous irradiations were obtained in the first experiment 1.13 ± 0.09 and 1.16 ± 0.09 in the second experiment. After 5 Gy and 24 h, RBE values of 0.99 ± 0.29 and 0.91 ± 0.23 were calculated, respectively. Based on the gamma-H2AX foci numbers obtained, no significant differences in RBE between pulsed and continuous proton irradiation in HeLa cells were detected. These results are well in line with our data on micronucleus induction in HeLa cells.
BibTeX:
	@article{Zlobinskaya2012,
	  author = {Zlobinskaya, O. and Dollinger, G. and Michalski, D. and Hable, V. and Greubel, C. and Du, G. and Multhoff, G. and Röper, B. and Molls, M. and Schmid, T. E.},
	  title = {Induction and repair of DNA double-strand breaks assessed by gamma-H2AX foci after irradiation with pulsed or continuous proton beams},
	  journal = {Radiation and Environmental Biophysics},
	  year = {2012},
	  volume = {51},
	  number = {1},
	  pages = {23--32},
	  url = {http://link.springer.com/article/10.1007%2Fs00411-011-0398-1},
	  doi = {https://doi.org/10.1007/s00411-011-0398-1}
	}
	

2011

Survival of tumor cells after proton irradiation with ultra-high dose rates
S. Auer, V. Hable, C. Greubel, G.A. Drexler, T.E. Schmid, C. Belka, G. Dollinger and A.A. Friedl; Radiation Oncology 6 (1) (2011) 139.
Abstract: Background Laser acceleration of protons and heavy ions may in the future be used in radiation therapy. Laser-driven particle beams are pulsed and ultra high dose rates of >109 Gy s-1may be achieved. Here we compare the radiobiological effects of pulsed and continuous proton beams. Methods The ion microbeam SNAKE at the Munich tandem accelerator was used to directly compare a pulsed and a continuous 20 MeV proton beam, which delivered a dose of 3 Gy to a HeLa cell monolayer within < 1 ns or 100 ms, respectively. Investigated endpoints were G2 phase cell cycle arrest, apoptosis, and colony formation. Results At 10 h after pulsed irradiation, the fraction of G2 cells was significantly lower than after irradiation with the continuous beam, while all other endpoints including colony formation were not significantly different. We determined the relative biological effectiveness (RBE) for pulsed and continuous proton beams relative to x-irradiation as 0.91 ± 0.26 and 0.86 ± 0.33 (mean and SD), respectively. Conclusions At the dose rates investigated here, which are expected to correspond to those in radiation therapy using laser-driven particles, the RBE of the pulsed and the (conventional) continuous irradiation mode do not differ significantly.
BibTeX:
	@article{Auer2011,
	  author = {Auer, Susanne and Hable, Volker and Greubel, Christoph and Drexler, Guido A. and Schmid, Thomas E. and Belka, Claus and Dollinger, Günther and Friedl, Anna A.},
	  title = {Survival of tumor cells after proton irradiation with ultra-high dose rates},
	  journal = {Radiation Oncology},
	  year = {2011},
	  volume = {6},
	  number = {1},
	  pages = {139},
	  url = {http://ro-journal.biomedcentral.com/articles/10.1186/1748-717X-6-139},
	  doi = {https://doi.org/10.1186/1748-717X-6-139}
	}
	
Spatial Dynamics of DNA Damage Response Protein Foci along the Ion Trajectory of High-LET Particles
G. Du, G.A. Drexler, W. Friedland, C. Greubel, V. Hable, R. Krücken, A. Kugler, L. Tonelli, A.A. Friedl and G. Dollinger; Radiation Research 176 (6) (2011) 706-715.
Abstract: High-linear energy transfer (LET) ion irradiation of cell nuclei induces complex and severe DNA lesions, and foci of repair proteins are formed densely along the ion trajectory. To efficiently discriminate the densely distributed/overlapping foci along the ion trajectory, a focus recognition algorithm called FociPicker3D based on a local fraction thresholding technique was developed. We analyzed high-resolution 3D immunofluorescence microscopic focus images and obtained the kinetics and spatial development of γ-H2AX, 53BP1 and phospho-NBS1 foci in BJ1-hTERT cells irradiated with 55 MeV carbon ions and compared the results with the dynamics of double-strand break (DSB) distributions simulated using the PARTRAC model. Clusters consisting of several foci were observed along the ion trajectory after irradiation. The spatial dynamics of the protein foci supports that the foci clusters are not formed by neighboring foci but instead originate from the DSB cluster damage induced by high-LET radiations.
BibTeX:
	@article{Du2011,
	  author = {Du, Guanghua and Drexler, Guido A. and Friedland, Werner and Greubel, Christoph and Hable, Volker and Krücken, Reiner and Kugler, Alexandra and Tonelli, Laura and Friedl, Anna A. and Dollinger, Günther},
	  title = {Spatial Dynamics of DNA Damage Response Protein Foci along the Ion Trajectory of High-LET Particles},
	  booktitle = {Radiation Research},
	  journal = {Radiation Research},
	  year = {2011},
	  volume = {176},
	  number = {6},
	  pages = {706--715},
	  url = {http://www.bioone.org/doi/10.1667/RR2592.1},
	  doi = {https://doi.org/10.1667/RR2592.1}
	}
	
Anomalous subdiffusion of DNA repair protein foci after ion microirradiation.
Stefanie Girst; Diplomarbeit, Technische Universität München, 2011.
Abstract: DNA repair processes, starting after the irradiation of cell nuclei, can be made visible by tagging DNA repair proteins (here MDC1) with the green fluorescent protein GFP, so that microscopic accumulations of the repair proteins ( at the ion-induced
damages (mostly DNA double-strand breaks) can be observed and analyzed "live" under a fluorescence microscope.
The aim of this work is to determine the dynamics of the MDC1-foci in the nucleus. Living U2OS osteosarcoma cells were irradiated in a 5x5 µm^2 matrix pattern with one carbon ion (43MeV) per point or 32 protons (20 MeV) respectively at the ion microprobe SNAKE at the Munich 14MV Tandem accelerator. The relative movement (i.e. the distance) of neighboring foci within the living cells was monitored over several hours "online" at the irradiation site at SNAKE. This relative measure is more robust against cell movement than absolute position determination. The distribution of the change of distance dl between two foci in a time interval dt is a measure for the underlying diffusion. The square of its standard deviation sigma^2(dt) is in general described by
sigma^2(dt) = G*dt^a, with a = 1 for normal, a < 1 for anomalous subdiffusion.
The diffusion data gathered in the performed experiments are in agreement with an anomalous subdiffusion. The anomalous diffusion exponent found is a = 0.50 +/- 0.04 for both proton and carbon irradiation on a time scale of dt =10 s till 10 000 s,
indicating that the degree of anomality does not depend on the density of double-strand breaks. The transport coefficient G and thus the apparent and the instantaneous diffusion coefficient, however, were clearly bigger in proton-irradiated cell nuclei
(G = (7+/-2)x10^(-3) µm^2/s^0.5) than in those irradiated with the higher-LET carbon ions (G = (3 +/- 1) x 10^(-3) µm^2/s^0.5). This probably arises from the fact that protons produce isolated double-strand breaks (DSBs) which move faster than the larger number of DSBs that form the foci in a carbon ion track.
BibTeX:
	@mastersthesis{Girst2011da,
	  author = {Girst, Stefanie},
	  title = {Anomalous subdiffusion of DNA repair protein foci after ion microirradiation.},
	  school = {Technische Universität München},
	  year = {2011}
	}
	
Scanning irradiation device for mice in vivo with pulsed and continuous proton beams
C. Greubel, W. Assmann, C. Burgdorf, G. Dollinger, G. Du, V. Hable, A. Hapfelmeier, R. Hertenberger, P. Kneschaurek, D. Michalski, M. Molls, S. Reinhardt, B. Röper, S. Schell, T.E. Schmid, C. Siebenwirth, T. Wenzl, O. Zlobinskaya and J.J. Wilkens; Radiation and Environmental Biophysics 50 (3) (2011) 339-344.
Abstract: A technical set-up for irradiation of subcutaneous tumours in mice with nanosecond-pulsed proton beams or continuous proton beams is described and was successfully used in a first experiment to explore future potential of laser-driven particle beams, which are pulsed due to the acceleration process, for radiation therapy. The chosen concept uses a microbeam approach. By focusing the beam to approximately 100 × 100 μm2, the necessary fluence of 109 protons per cm2 to deliver a dose of 20 Gy with one-nanosecond shot in the Bragg peak of 23 MeV protons is achieved. Electrical and mechanical beam scanning combines rapid dose delivery with large scan ranges. Aluminium sheets one millimetre in front of the target are used as beam energy degrader, necessary for adjusting the depth–dose profile. The required procedures for treatment planning and dose verification are presented. In a first experiment, 24 tumours in mice were successfully irradiated with 23 MeV protons and a single dose of 20 Gy in pulsed or continuous mode with dose differences between both modes of 10%. So far, no significant difference in tumour growth delay was observed.
BibTeX:
	@article{Greubel2011,
	  author = {Greubel, Christoph and Assmann, Walter and Burgdorf, Christian and Dollinger, Günther and Du, Guanghua and Hable, Volker and Hapfelmeier, Alexander and Hertenberger, Ralf and Kneschaurek, Peter and Michalski, Dörte and Molls, Michael and Reinhardt, Sabine and Röper, Barbara and Schell, Stefan and Schmid, Thomas E. and Siebenwirth, Christian and Wenzl, Tatiana and Zlobinskaya, Olga and Wilkens, Jan J.},
	  title = {Scanning irradiation device for mice in vivo with pulsed and continuous proton beams},
	  journal = {Radiation and Environmental Biophysics},
	  year = {2011},
	  volume = {50},
	  number = {3},
	  pages = {339--344},
	  url = {http://link.springer.com/article/10.1007%2Fs00411-011-0365-x},
	  doi = {https://doi.org/10.1007/s00411-011-0365-x}
	}
	
Echtzeitbeobachtung schneller Reaktionskinetiken in lebenden Zellen nach Ionenmikrobestrahlung
Volker Hable; Dissertation, Universität der Bundeswehr München, 2011.
Abstract: Diese Arbeit beschreibt den Aufbau einer Lebendzellmikroskopieumgebung am Rasterionenmikroskop SNAKE, welches am Münchner 14 MV Tandembeschleuniger installiert ist. An dessen Zellbestrahlungsplatz können lebende Zellen mit Protonen und Schwerionen unter Lebendbedingungen mit einer Genauigkeit von ca. 0,5 µm und mit genau definierter Dosis bestrahlt werden. Die nach der Bestrahlung im Zellkern ablaufenden Reparaturvorgänge können durch eine mikroskopische Betrachtung der an der Reparatur beteiligten Proteine analysiert werden. Hierfür ist die Markierung dieser Proteine mittels Fluoreszenzfarbstoffen nötig. Dazu werden die Zellen auf gentechnischem Wege so verändert, dass an Proteine, die an der Reparatur der ioneninduzierten Schäden beteiligt sind, Fluoreszenzproteine (z. B. GFP, green fluorescent protein) angehängt werden. Mikroskopische Proteinanlagerungen an die Schadensorte, sogenannte Foci, können mit dem im Rahmen dieser Arbeit realisierten Aufbau unmittelbar nach und sogar während der Bestrahlung "online“ analysiert werden. Ein kommerziell erhältliches Fluoreszenzmikroskop (Zeiss Axiovert 200M) wurde hierzu am Bestrahlungsplatz angebracht. An dessen Probentisch befinden sich die Zellen während der Bestrahlung und der nachfolgenden Mikroskopie unter optimalen Umgebungsbedingungen in neu entwickelten Zellkulturgefäßen. Erste Experimente an dem neuen Aufbau dienten der Untersuchung von Kinetiken (= zeitlicher Ablauf der Focibildung) der Proteine Mdc1, 53BP1 und Rad52. Nach Applizierung einer mittleren Dosis von 4,4 Gy mit 55 MeV Kohlenstoffionen mit einem linearen Energietransfer LET = 310 keV/µm beginnt Mdc1 nach T0 = 17 ± 2 s mit der Anlagerung an die Schadensorte. Dies geschieht mit einer Zeitkonstante t = 98 ± 11 s. Wird dieselbe Dosis mit 20 MeV Protonen appliziert (LET = 2,65 keV/µm), läuft die Focibildung langsamer ab (T0 = 73 ± 16 s, t = 1050 ± 270 s). Eine höhere Bestrahlungsdosis durch Erhöhung der pro Punkt applizierten Protonen beschleunigt die Kinetik. Die Zeitkonstanten des Proteins 53BP1 weisen keine solch ausgeprägte Abhängigkeit von der Bestrahlungsart auf. Für alle Bestrahlungsbedingungen liegt hier T0 in der Größenordnung von 100 s und t in der Größenordnung von 300 s. Das nur qualitativ betrachtete Reparaturprotein Rad52 zeigt eine deutlich langsamere Kinetik, die allerdings wieder stark von der Dosis und vom LET der Strahlung abhängt. Während bereits ca. zehn Minuten nach Bestrahlung mit 4,7 Gy mit 55 MeV Kohlenstoffionen erste Foci sichtbar werden, dauert deren Erscheinen nach Applizierung von 5,7 Gy durch 20 MeV Protonen (117 Protonen pro Punkt) ca. drei Stunden. Eine Erhöhung der pro Punkt applizierten Protonenzahl auf 256 (und somit der Dosis auf 12 Gy) verkürzt diese Zeit auf ca. eine Stunde. Eine weitere Verdopplung von Protonenzahl und Dosis führt zu einem Sichtbarwerden der Foci nach weniger als zehn Minuten.
BibTeX:
	@phdthesis{Hable2011diss,
	  author = {Hable, Volker},
	  title = {Echtzeitbeobachtung schneller Reaktionskinetiken in lebenden Zellen nach Ionenmikrobestrahlung},
	  school = {Universität der Bundeswehr München},
	  year = {2011},
	  url = {http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:706-2487}
	}
	
Subdiffusion von DNS-Doppelstrangbrüchen unter dem Einfluss von Zellkernverformungen
Michael Haum; Bachelors-Thesis, Universität der Bundeswehr München, 2011.
Abstract: Die Untersuchung von Schäden an biologischem Material durch ionisierende Strahlung stellt immer noch ein großes Forschungsgebiet von Medizin und Biologie dar. Insbesondere die Reparaturvorgänge nach der Schädigung der DNS im Zellkern werfen noch viele offene Fragen auf, dabei vor allem die der gefährlichsten Doppelstrangbrüche (DSB). Für ein besseres Verständnis der raumzeitlichen Dynamik der DSB wurden lebende Zellen am Münchner 14 MV Tandembeschleuniger mit 43 MeV Kohlenstoff-Ionen beschossen, um so die DNS gezielt zu schädigen und die erzeugten DSB über die sich dort gebildeten fluoreszenzmarkierten Reparaturproteincluster („Foci“) zu beobachten.
Für die Analyse der Dynamik wurde die zeitliche Änderung der Abstände benachbarter Foci ( l≈5μm ) herangezogen. Die Standardabweichung der Abstandsänderung über ein Zeitintervall dt kann mit der Gleichung sigma^2= G * dt^a beschrieben werden, die eine Aussage über die Art der Diffusion macht. Es zeigte sich, dass der Diffusionsexponent mit a=0,49±0,05 deutlich kleiner ist als der einer normalen Diffusion ( a=1 ) und der Transportkoeffizient bei G=(1,7± 0,6) x 10^(−3) μm^2/s^0,49 liegt, sodass der Bewegung eine anomale Subdiffusion zugrunde liegt [S. Girst, 2011]. Durch die Betrachtung der Abstände anstelle von absoluten Positionen soll ausgeschlossen werden, dass eine Bewegung oder Deformation der gesamten Zelle unbeabsichtigt
in die Auswertung mit einfließt.
Ziel dieser Arbeit war es zu untersuchen, ob auch bei der Auswertung von größeren Foci-Abständen eine anomale Subdiffusion vorliegt. Hierfür wurden die Abstandsänderungen eines Foci zu seinem übernächsten Nachbarn ( l≈10μm ) herangezogen. Es ergab sich, dass auch hier eine anomale Subdiffusion vorliegt, mit dem Diffusionsexponenten a=0,58± 0,03 und dem Transportkoeffizienten G=(1,6± 0,3) x 10^(−3) μm^2 /s^0,58 . Trotz des größeren Diffusionsexponenten liegt auch nach dieser Auswertung eine anomale Subdiffusion vor, sodass das für kleine Abstände gefundene Ergebnis bestätigt wird. Der größere Diffusionsexponent ist allerdings ein Hinweis darauf, dass sich bei großen Foci-Abständen eine Verformung der Zelle in der Auswertung stärker
bemerkbar macht.
BibTeX:
	@mastersthesis{Haum2011ba,
	  author = {Haum, Michael},
	  title = {Subdiffusion von DNS-Doppelstrangbrüchen unter dem Einfluss von Zellkernverformungen},
	  school = {Universität der Bundeswehr München},
	  year = {2011}
	}
	
The effectiveness of 20 MeV protons at nanosecond pulse lengths in producing chromosome aberrations in human-hamster hybrid cells
T.E. Schmid, G. Dollinger, V. Hable, C. Greubel, O. Zlobinskaya, D. Michalski, S. Auer, A.A. Friedl, E. Schmid, M. Molls and B. Röper; Radiation Research 175 (6) (2011) 719-727.
Abstract: Laser accelerated radiotherapy is a potential cancer treatment with proton and carbon-ion beams that is currently under development. Ultra-fast high-energy laser pulses will accelerate ion beams that deliver their dose to a patient in a “pulsed mode” that is expected to differ from conventional irradiation by increasing the dose delivery rate to a tissue voxel by approximately 8 orders of magnitude. In two independently performed experiments at the ion microprobe SNAKE of the 14 MV Munich tandem accelerator, AL cells were exposed either to protons with 1-ns pulse durations or to protons applied over 150 ms in continuous irradiation mode. A slightly but consistently lower aberration yield was observed for the pulsed compared to the continuous mode of proton irradiation. This difference was not statistically significant when each aberration type was analyzed separately (P values between 0.61 and 0.85 in experiment I and P values between 0.32 and 0.64 in experiment II). However, excluding the total aberrations, which were not analyzed as independent radiation-induced effects, the mean ratio of the yields of dicentrics, centric rings and excess acentrics scored together showed (with 95% CI) a significant difference of 0.90 (0.81; 0.98) between the pulsed and the continuous irradiation modes. A similar tendency was also determined for the corresponding RBE values relative to 70 kV X rays. Since the different findings for the comparisons of individual chromosome aberration types and combined comparisons could be explained by different sample sizes with the consequence that the individual comparisons had less statistical power to identify a difference, it can be concluded that 20 MeV protons may be slightly less effective in the pulsed mode.
BibTeX:
	@article{Schmid2011,
	  author = {Schmid, T. E. and Dollinger, G. and Hable, V. and Greubel, C. and Zlobinskaya, O. and Michalski, D. and Auer, S. and Friedl, A. A. and Schmid, E. and Molls, M. and Röper, B.},
	  title = {The effectiveness of 20 MeV protons at nanosecond pulse lengths in producing chromosome aberrations in human-hamster hybrid cells},
	  booktitle = {Radiation Research},
	  journal = {Radiation Research},
	  year = {2011},
	  volume = {175},
	  number = {6},
	  pages = {719--727},
	  url = {http://www.bioone.org/doi/10.1667/RR2465.1},
	  doi = {https://doi.org/10.1667/RR2465.1}
	}
	
Double-strand break-induced transcriptional silencing is associated with loss of tri-methylation at H3K4
D. Seiler, J. Rouquette, V. Schmid, H. Strickfaden, C. Ottmann, G. Drexler, B. Mazurek, C. Greubel, V. Hable, G. Dollinger, T. Cremer and A. Friedl; Chromosome Research 19 (7) (2011) 883-899.
Abstract: Epigenetic alterations induced by ionizing radiation may contribute to radiation carcinogenesis. To detect relative accumulations or losses of constitutive post-translational histone modifications in chromatin regions surrounding DNA double-strand breaks (DSB), we developed a method based on ion microirradiation and correlation of the signal intensities after immunofluorescence detection of the histone modification in question and the DSB marker γ-H2AX. We observed after ionizing irradiation markers for transcriptional silencing, such as accumulation of H3K27me3 and loss of active RNA polymerase II, at chromatin regions labeled by γ-H2AX. Confocal microscopy of whole nuclei and of ultrathin nuclear sections revealed that the histone modification H3K4me3, which labels transcriptionally active regions, is underrepresented in γ-H2AX foci. While some exclusion of H3K4me3 is already evident at the earliest time amenable to this kind of analysis, the anti-correlation apparently increases with time after irradiation, suggesting an active removal process. Focal accumulation of the H3K4me3 demethylase, JARID1A, was observed at damaged regions inflicted by laser irradiation, suggesting involvement of this enzyme in the DNA damage response. Since no accumulation of the repressive mark H3K9me2 was found at damaged sites, we suggest that DSB-induced transcriptional silencing resembles polycomb-mediated silencing rather than heterochromatic silencing.
BibTeX:
	@article{Seiler2011,
	  author = {Seiler, D.M. and Rouquette, J. and Schmid, V.J. and Strickfaden, H. and Ottmann, C. and Drexler, G.A. and Mazurek, B. and Greubel, C. and Hable, V. and Dollinger, G. and Cremer, T. and Friedl, A.A.},
	  title = {Double-strand break-induced transcriptional silencing is associated with loss of tri-methylation at H3K4},
	  journal = {Chromosome Research},
	  year = {2011},
	  volume = {19},
	  number = {7},
	  pages = {883--899},
	  url = {http://link.springer.com/article/10.1007%2Fs10577-011-9244-1},
	  doi = {https://doi.org/10.1007/s10577-011-9244-1}
	}
	

2010

Quantitative Analyse der LET- und Strahlungsdosisabhängigkeit von Proteinkinetiken nach Ionenmikrobestrahlung
Christian Burgdorf; Diplomarbeit, Universität der Bundeswehr München, 2010.
Abstract: In dieser Arbeit wurde eine quantitative Analyse von Proteinkinetiken nach Ionenmikrobestrahlung hinsichtlich einer LET- und Strahlungsdosisabhängigkeit durchgeführt.
Zur Auswertung der ablaufenden Reparaturprozesse wurden die mit dem Rasterionenmikroskop SNAKE fluoreszenzmikroskopisch aufgenommenen Zeitserien analysiert. In diesen Zeitserien bildeten sich in bestrahlten Bereichen innerhalb von verschiedenen Zeitintervallen Foci aus. Diese Foci beschreiben Orte, in denen sich die Konzentration von Proteinen erhöht, was mit der Anlagerung von Reparaturproteinen an beschädigten DNA-Sequenzen gleich zusetzen ist. Bei Beobachtung dieser Focibildung wurde des Weiteren deutlich, dass die Foci mit den Bestrahlungsorten kolokalisieren.
In dieser Arbeit wurden die Kinetiken der Proteine MDC1 und 53BP1 mit Hilfe von Helligkeitsmessungen ihrer Foci ausgewertet. Eine entwickelte Modellfunktion wurde an die gemessenen Helligkeitsverläufe angepasst. Die Proteinanlagerung und der Proteinabbau wurden mit Hilfe von zwei Zeitkonstanten Tau_1 und Tau_2 charakterisiert. Eine mögliche zeitliche Verzögerung beim Start des Reparaturvorganges konnte mit einem Zeitoffset T0 modelliert werden.
Zum Abschluss der Helligkeitsmessungen wurden probenübergeifend einzelne Bestrahlungsexperimente zusammengefasst, die unter gleichen biophysikalischen Bedingungen durchgeführt wurden. Die Klassifizierung erfolgte nach der verwendeten Strahlungsart und -dosis sowie nach dem untersuchten Reparaturprotein.
Hinsichtlich einer LET- und Strahlungsdosisabhängigkeit konnten für das Reparaturprotein MDC1 nach 20MeV H+, wie auch bei einer 55MeV C+ Bestrahlung, Abhängigkeiten festgestellt werden. Dabei zeigte sich für die Bestrahlung mit H+, dass die Erhöhung der Strahlungsdosis von 4,8 Gy auf 12,05 Gy (Faktor 2,5) eine Beschleunigung der Anlagerungszeit Tau_1,( 4,8 Gy) = 1052 ± 272 s zu Tau_1,(12,05 Gy) = 522 ± 148 s zur Folge hatte. Das Starten der Reparaturprozesse hingegen war nahezu konstant nach einem Zeitoffset von
T0,(4,8 Gy) = 73 ± 16 s und T0,(12,05 Gy) = 80 ± 11 s.
Für die Zeitkonstanten nach 55MeV C+ Bestrahlung zeigte sich ein ähnliches Bild, wobei deutlich wurde, dass weitaus geringere Strahlungsdosen nötig waren, um vergleichsweise schnelle Reaktionen für die Proteinanlagerung zu erreichen. Die Zeit für den Anlagerungsprozess wurde mit steigender Strahlungsdosis weiter verringert. Bei einer Dosis von 3,1Gy betrug Tau_1,(3,1 Gy) = 218 ± 55 s, die sich bei der Dosis von 4,4 Gy auf Tau_1,(4,4 Gy) = 98 ± 11 s verringerte. Eine signifikante Dosisabhängigkeit für die Offset-Zeiten
T0 konnte nicht bestimmt werden (T0,(3,1 Gy) = 14 ± 4 s, T0,(4,4 Gy) = 17 ± 2 s).
Die Auswertung des zweiten Reparaturproteins 53BP1 erbrachte für die Bestrahlung mit 20MeV H+ keine linearen Dosisabhängigkeiten. Die Werte für die Zeitkonstanten Tau_1 liegen in niedrigen (3,4 Gy) und hohen Strahlungsdosisbereichen (13,7 Gy) nahezu konstant
bei Tau_1,(3,4 Gy) = 237 ± 33 s und Tau_1,(13,7 Gy) = 226 ± 60 s. Für den mittleren Dosisbereich ist mit Tau_1,(6,9 Gy) = 460 ± 100 s die benötigte Zeit für die Proteinanlagerung doppelt so groß. Derselbe Effekt ist auch bei der Offset-Zeit T0 zu erkennen (T0, 3,4 Gy = 118 ± 14 s, T0, 6,9 Gy = 160 ± 12 s, T0, 13,7 Gy = 120 ± 22 s).
Die Auswertung des 53BP1 nach 55 MeVC+ Bestrahlung erbrachte ein Tau_1,(6,3 Gy) = 375 ± 58 s und einen Zeitoffset T0,(6,3 Gy) = 89 ± 8 s. Dabei wurde deutlich, dass sich die Zeitkonstanten für unterschiedliche Strahlungsarten trotz einer ähnlichen applizierten Strahlungsdosis stark unterschieden. Dennoch zeigte sich, wie bei der Untersuchung von MDC1, dass 55MeV C+ bestrahlte 53BP1 Proben eine schnellere Reaktion zeigten.
BibTeX:
	@mastersthesis{Burgdorf2010da,
	  author = {Burgdorf, Christian},
	  title = {Quantitative Analyse der LET- und Strahlungsdosisabhängigkeit von Proteinkinetiken nach Ionenmikrobestrahlung},
	  school = {Universität der Bundeswehr München},
	  year = {2010}
	}
	
Life cell micro-irradiation
G. Dollinger; Nuclear Physics News 20 (3) (2010) 27-32.
Abstract: A main subject of modern experiments in radiobiology is the detailed investigation of the biological response on a microscopic scale when a living organism is irradiated by ionizing radiation. As known for long, a DNA double strand break (DSB) is one of the most harmful threats that can be induced by ionizing radiation (Figure 1a). Thus, the response of cells to DSBs on a microscopic scale interests in view of cell surveillance strategies. There are already a lot of proteins known that are omnipresent in cell nuclei and that are involved in the repair of DSBs. Some of them cluster around a DSB forming a “repair focus” (Figure 1b). The spatio-temporal development of the repair processes and the interaction of the different proteins within repair pathways are to a large extent still unknown. A precise irradiation of cells by means of a nuclear microprobe, for example, using SNAKE ( S uperconducting N anoscope for A pplied nuclear ( K ern-) physics E xperiments) at the Munich tandem accelerator, is an ideal tool to perform accurate radiobiological experiments and to investigate cell surveillance strategies in general [1].
BibTeX:
	@article{Dollinger2010,
	  author = {Dollinger, G.},
	  title = {Life cell micro-irradiation},
	  journal = {Nuclear Physics News},
	  year = {2010},
	  volume = {20},
	  number = {3},
	  pages = {27--32},
	  note = {cited By (since 1996)0},
	  url = {http://www.nupecc.org/index.php?display=npn/issues},
	  doi = {https://doi.org/10.1080/10619127.2010.506125}
	}
	
Differences in the kinetics of γ-H2AX fluorescence decay after exposure to low and high LET radiation
T.E. Schmid, G. Dollinger, W. Beisker, V. Hable, C. Greubel, S. Auer, A. Mittag, A. Tarnok, A.A. Friedl, M. Molls and B. Röper; International Journal of Radiation Biology 86 (8) (2010) 682-691.
Abstract: Purpose:
In order to obtain more insight into heavy ion tumour therapy, some features of the underlying molecular mechanisms controlling the cellular response to high linear energy transfer (LET) radiation are currently analysed.

Materials and methods:
We analysed the decay of the integrated fluorescence intensity of γ-H2AX (phosphorylated histone H2AX) which is thought to reflect the repair kinetics of radiation-induced DNA double-strand breaks (DSB) using Laser-Scanning-Cytometry. Asynchronous human HeLa cells were irradiated with a single dose of either 1.89 Gy of 55 MeV carbon ions or 5 Gy of 70 kV X-rays.

Results:
Measurements of the γ-H2AX-intensities from 15–60 min resulted in a 16 % decrease for carbon ions and in a 43 % decrease for X-rays. After 21 h, the decrease was 77 % for carbon ions and 85 % for X-rays. The corresponding time-effect relationship was fitted by a bi-exponential function showing a fast and a slow component with identical half-life values for both radiation qualities being 24 ± 4 min and 13.9 ± 0.7 h, respectively. Apparent differences in the kinetics following high and low LET irradiation could completely be attributed to quantitative differences in their contributions, with the slow component being responsible for 47 % of the repair after exposure to X-rays as compared to 80 % after carbon ion irradiation.

Conclusion:
γ-H2AX loss kinetics follows a bi-exponential decline with two definite decay times independent of LET. The higher contribution of the slow component determined for carbon ion exposure is thought to reflect the increased amount of complex DSB induced by high LET radiation.

BibTeX:
	@article{Schmid2010,
	  author = {Schmid, Thomas E. and Dollinger, Günther and Beisker, Wolfgang and Hable, Volker and Greubel, Christoph and Auer, Susanne and Mittag, Anja and Tarnok, Attila and Friedl, Anna A. and Molls, Michael and Röper, Barbara},
	  title = {Differences in the kinetics of γ-H2AX fluorescence decay after exposure to low and high LET radiation},
	  journal = {International Journal of Radiation Biology},
	  year = {2010},
	  volume = {86},
	  number = {8},
	  pages = {682--691},
	  note = {PMID: 20569192},
	  url = {http://informahealthcare.com/doi/abs/10.3109/09553001003734543},
	  doi = {https://doi.org/10.3109/09553001003734543}
	}
	
Relative biological effectiveness of pulsed and continuous 20 MeV protons for micronucleus induction in 3D human reconstructed skin tissue
T.E. Schmid, G. Dollinger, V. Hable, C. Greubel, O. Zlobinskaya, D. Michalski, M. Molls and B. Röper; Radiotherapy and Oncology 95 (1) (2010) 66-72.
Abstract: Background and purpose: Laser accelerated radiotherapy is a prospect for cancer treatment with proton and/or carbon ion beams that is currently under fast development. In principal, ultra fast, high-energy laser pulses will lead to a "pulsed" delivery of the induced ion beam with pulse durations of 1 ns and below, whereas conventional proton beams deriving from a cyclotron or synchrotron apply the dose within 100 ms ("continuous"). Materials and methods: A simulation of both irradiation modes could be established at the Munich tandem accelerator with a 20 MeV proton beam, and a wide-field fast scanning system was implemented that allowed for application of up to 5 Gy per tissue voxel in a single pulse. The relative biological effectiveness (RBE) of pulsed and continuous modes of irradiation with 20 MeV protons relative to the reference radiation 70 kV X-rays was examined in a human tissue model (3D human reconstructed skin, EpiDermFT™) which preserves the three-dimensional geometric arrangement and communication of cells present in tissues in vivo. Using the induction of micronuclei (MN) in keratinocytes as the biological endpoint, the RBE was calculated as the ratio between the dose of 70 kV X-rays and 3 Gy of 20 MeV protons (pulsed or continuous) which produced equal response. Results: For pulsed and continuous 20 MV proton exposures of the human skin model, RBE values of 1.08 ± 0.20 and 1.22 ± 0.15 versus 70 kV X-rays were obtained in a first experiment and 1.00 ± 0.14 and 1.13 ± 0.14 in a second experiment during distinct beam access times, respectively. The ∼10% difference in RBE between the respective irradiation modes in both experiments was associated with large uncertainties which were not statistically significant (p ≈ 0.5). Conclusion: These findings represent an important step on the way towards application of laser-accelerated protons for clinical radiotherapy. Further clinically relevant endpoints in normal and tumor tissue have to be evaluated.
BibTeX:
	@article{Schmid2010a,
	  author = {Schmid, Thomas E. and Dollinger, Günther and Hable, Volker and Greubel, Christoph and Zlobinskaya, Olga and Michalski, Dörte and Molls, Michael and Röper, Barbara},
	  title = {Relative biological effectiveness of pulsed and continuous 20 MeV protons for micronucleus induction in 3D human reconstructed skin tissue},
	  journal = {Radiotherapy and Oncology},
	  year = {2010},
	  volume = {95},
	  number = {1},
	  pages = {66--72},
	  url = {http://www.sciencedirect.com/science/article/pii/S0167814010001623},
	  doi = {https://doi.org/10.1016/j.radonc.2010.03.010}
	}
	
Tumorbestrahlung mit gepulsten und kontinuierlichen Protonen am Mausmodell.
Christian Siebenwirth; Diplomarbeit, Technische Universität München, 2010.
Abstract: Zur Qualifizierung der Tumortherapie mit gepulsten Protonenstrahlen mit Pulsbreiten von 1 ns, wie sie bei der Laserbeschleunigung erzeugt werden, wurden am Münchner 14 MV Tandembeschleuniger menschliche Tumore am Mausmodell mit 20 Gy bestrahlt. Anhand des Parameters der Tumorwachstumsverzögerung wurde überprüft, ob ein Unterschied in der relativen biologischen Wirksamkeit (RBW) zwischen Protonenstrahlung, die ihre Dosis in Pulsen der Breite von 1 ns applizieren, und kontinuierlicher Protonenbestrahlung auftritt.
Da es noch keine laserbeschleunigten Ionenstrahlen in hinreichender Qualität gibt, um eine Tumorbestrahlung durchzuführen, wurde am Rasterionenmikroskop SNAKE ein laserbeschleunigter Protonenstrahl simuliert. Dazu wurde das 5 MHz Pulsungssystem des Tandembeschleunigers verwendet, das ein 23 MeV Protonenstrahl mit einer Pulsbreite von 1 ns erzeugt. Durch die Fokussierung des Strahls an SNAKE auf einen Durchmesser von 100 μm konnte in einem einzelnen Puls eine Ionenstrahldichte von 10^9 Protonen/cm² erreicht werden und so eine Dosis von 20 Gy mit einem Puls im Target deponiert werden. Die Strahlflecke wurden in lateraler Richtung durch Strahlablenkung und Bewegen des Tumors inklusive Maus zu einem homogenen Feld von ca. 1 cm² zusammengesetzt. Die homogene Tiefendosis wurde mittels Aluminiumplättchen als diskrete Energieabsorber kurz vor dem Target verwirklicht. So besaß das homogen bestrahlte Gesamtvolumen eine Tiefe von 4,8 mm und einen Durchmesser von 9 mm. Durch die Realisierung der kontinuierlichen Protonenbestrahlung am selben Gerät, wurden systematische Fehler im Vergleich der beiden Bestrahlungsarten minimiert.
Zur Kontrolle der Protonenfluenz diente ein vor dem Tumor platzierter Gafchromic EBT2 Film, der in Abhängigkeit von der durch die Protonen deponierten Dosis verdunkelt. Damit konnte die Dosis der gepulsten und kontinuierlichen Bestrahlung mit einer relativen Genauigkeit von 3 % rekonstruiert werden.
Es wurden insgesamt 11 XF354 und 12 FaDu Tumore bestrahlt, davon 12 im gepulsten und 11 im kontinuierlichen Modus. Die sich aus der Dosisrekonstruktion ergebende mittlere Tiefendosis lag für die gepulsten Bestrahlungen durchschnittlich bei 17,6 Gy mit einer Breite von 0,2 Gy bzw. für die kontinuierliche Bestrahlung bei 19,6 Gy mit einer Breite von 0,3 Gy. Annähernd die Hälfte des 10 % Dosisunterschieds zwischen gepulst und kontinuierlicher Bestrahlung konnten auf systematische Fehler der Bestrahlungsdurchführung und der Dosisrekonstruktion zurückgeführt werden. Diese sind
in zukünftigen Experimenten einfach zu korrigieren. Die andere Hälfte liegt vermutlich in der Strahlstrommessung begründet und sollte nach näheren Untersuchungen ebenfalls reduziert werden können.
Bei den XF354 Tumoren erreichte ein Tumor je Bestrahlungsmodus das dreifache Bestrahlungsvolumen, das für die Wachstumsverzögerung als Bezugspunkt dient, wobei die Wachstumsverzögerung 103 d für die gepulste und 35 d für die kontinuierliche Bestrahlung ergab. Die übrigen Tumore wurden kontrolliert, wodurch sich wegen der geringen Statistik keine Aussage über eine unterschiedliche RBW treffen lässt. Für die FaDu Tumore konnte eine mittlere Wachstumsverzögerung von (34 ± 4) d aus fünf gepulst bestrahlten und (36 ± 4) d aus vier kontinuierlich bestrahlten nicht kontrollierten Tumoren bestimmt werden.
Die gewonnenen Ergebnisse zeigen keinen signifikanten Unterschied bezüglich der Tumorwachstumsverzögerung von gepulster und kontinuierlicher Protonenbestrahlung.
BibTeX:
	@mastersthesis{Siebenwirth2010da,
	  author = {Siebenwirth, Christian},
	  title = {Tumorbestrahlung mit gepulsten und kontinuierlichen Protonen am Mausmodell.},
	  school = {Technische Universität München},
	  year = {2010}
	}
	

2009

Nanosecond pulsed proton microbeam
G. Dollinger, A. Bergmaier, V. Hable, R. Hertenberger, C. Greubel, A. Hauptner and P. Reichart; Nuclear Instruments and Methods in Physics Research Section B 267 (12-13) (2009) 2008-2012.
Abstract: We show the preparation of a pulsed 20 MeV proton beam at the Munich tandem accelerator which offers a fluence of more than 1 × 10e9 protons/cm2 being deposited in a beam spot smaller than 100 μm in diameter and within a time span of 0.9 ns fwhm. Such a beam is produced by an ECR type proton source using charge exchange in cesium vapor to obtain a beam of negative hydrogen of high brightness that is bunched, chopped, accelerated and then focused by the superconducting multipole lens of the microprobe SNAKE. Single beam pulses are generated in order to irradiate cell samples or tissue and to measure their biological effect in comparison to continuous proton or X-ray irradiation.
BibTeX:
	@article{Dollinger2009,
	  author = {Dollinger, G. and Bergmaier, A. and Hable, V. and Hertenberger, R. and Greubel, C. and Hauptner, A. and Reichart, P.},
	  title = {Nanosecond pulsed proton microbeam},
	  booktitle = {Proceedings of the 11th International Conference on Nuclear Microprobe Technology and Applications and the 3rd International Workshop on Proton Beam Writing},
	  journal = {Nuclear Instruments and Methods in Physics Research Section B},
	  year = {2009},
	  volume = {267},
	  number = {12-13},
	  pages = {2008--2012},
	  url = {http://www.sciencedirect.com/science/article/pii/S0168583X09003310},
	  doi = {https://doi.org/10.1016/j.nimb.2009.03.006}
	}
	
The live cell irradiation and observation setup at SNAKE
V. Hable, C. Greubel, A. Bergmaier, P. Reichart, A. Hauptner, R. Krücken, H. Strickfaden, S. Dietzel, T. Cremer, G. Drexler, A. Friedl and G. Dollinger; Nuclear Instruments and Methods in Physics Research Section B 267 (12-13) (2009) 2090-2097.
Abstract: We describe a new setup at the ion microprobe SNAKE (Superconducting Nanoscope for Applied nuclear (Kern-) physics Experiments) at the Munich 14 MV Tandem accelerator that facilitates both living cell irradiation with sub micrometer resolution and online optical imaging of the cells before and after irradiation by state of the art phase contrast and fluorescence microscopy. The cells are kept at standard cell growth conditions at 37 °C in cell culture medium. After irradiation it is possible to switch from single ion irradiation conditions to cell observation within 0.5 s. First experiments were performed targeting substructures of a cell nucleus that were tagged by TexasRed labeled nucleotides incorporated in the cellular DNA by 55 MeV single carbon ion irradiation. In addition we show first online sequences of short time kinetics of Mdc1 protein accumulation in the vicinity of double strand breaks after carbon ion irradiation.
BibTeX:
	@article{Hable2009,
	  author = {Hable, V. and Greubel, C. and Bergmaier, A. and Reichart, P. and Hauptner, A. and Krücken, R. and Strickfaden, H. and Dietzel, S. and Cremer, T. and Drexler, G.A. and Friedl, A.A. and Dollinger, G.},
	  title = {The live cell irradiation and observation setup at SNAKE},
	  booktitle = {Proceedings of the 11th International Conference on Nuclear Microprobe Technology and Applications and the 3rd International Workshop on Proton Beam Writing},
	  journal = {Nuclear Instruments and Methods in Physics Research Section B},
	  year = {2009},
	  volume = {267},
	  number = {12-13},
	  pages = {2090--2097},
	  url = {http://www.sciencedirect.com/science/article/pii/S0168583X09003504},
	  doi = {https://doi.org/10.1016/j.nimb.2009.03.071}
	}
	
No Evidence for a Different RBE between Pulsed and Continuous 20 MeV Protons
T.E. Schmid, G. Dollinger, A. Hauptner, V. Hable, C. Greubel, S. Auer, A.A. Friedl, M. Molls and B. Röper; Radiation Research 172 (5) (2009) 567-574.
Abstract: To obtain greater insight into the future potential of tumor radiotherapy using proton beams generated from high-intensity lasers, it is important to characterize the ionization quality of the new beams by measuring the relative biological effectiveness (RBE) under conditions where the full dose at one irradiation site will be deposited by a few proton pulses less than 1 ns in duration. HeLa cells attached to a Mylar foil were irradiated with 70 kV X rays to obtain a reference dose–response curve or with 3 Gy of 20 MeV protons at the Munich tandem accelerator (Garching), either using a continuous mode where a cell sample was irradiated within a 100-ms time span or using a pulsed mode where radiation was given in a single proton pulse of about 1 ns. After irradiation cytochalasin B was added; 24 h later cells were fixed and stained with acridine orange and micronuclei were counted. The X-ray dose–response curve for the production of micronuclei in HeLa cells followed a linear-quadratic model. The corresponding RBE values for 20 MeV protons in pulsed and continuous irradiation modes were 1.07 ± 0.08 and 1.06 ± 0.10 in the first proton experiment and 1.09 ± 0.08 and 1.05 ± 0.11 in the second, respectively. There was no evidence for a difference in the RBE for pulsed and continuous irradiation of HeLa cells with 20 MeV protons.
BibTeX:
	@article{Schmid2009,
	  author = {Schmid, T. E. and Dollinger, G. and Hauptner, A. and Hable, V. and Greubel, C. and Auer, S. and Friedl, A. A. and Molls, M. and Röper, B.},
	  title = {No Evidence for a Different RBE between Pulsed and Continuous 20 MeV Protons},
	  booktitle = {Radiation Research},
	  journal = {Radiation Research},
	  year = {2009},
	  volume = {172},
	  number = {5},
	  pages = {567--574},
	  note = {cited By (since 1996)18},
	  url = {http://www.bioone.org/doi/10.1667/RR1539.1},
	  doi = {https://doi.org/10.1667/RR1539.1}
	}
	
Differences in gamma-H2AX foci formation after irradiation with continuous and pulsed proton beams
O. Zlobinskaya, T. Schmid, G. Dollinger, V. Hable, C. Greubel, D. Michalski, J. Wilkens, G. Du, M. Molls and B. Röper; In: , O. Dössel and W.C. Schlegel (Eds.), IFMBE Proceedings 25 (2009) 142-145 , Springer International Publishing AG.
Abstract: Introduction: Classical particle accelerators offer proton pulses of some milliseconds duration. In contrast, the new technology of the high-intensity laser acceleration will produce ultimately shorter particle packages (up to one nanosecond) with substantially lower pulse frequency and higher pulse-dose achievement. Very little is known about the relative biological effectiveness (RBE) of this new beam quality, which could be a possible future application in radiation oncology. In our present study we investigate possible differences based on quantitative analysis of γ-H2AX fluorescence - a known marker of DNA double strand breaks (DSBs). Methods: HeLa cells were irradiated with 1 Gy of 20 MeV protons at the Munich tandem accelerator, either at continuous mode (100 ms), or at pulsed mode with a single pulse of 1 ns duration. A dose-effect-curve based on five doses of 75 kV x-rays served for reference. The total number of γ-H2AX foci per cell was determined using a self-developed macro (ImageJ, NIH, USA). Results: Quantitative analysis of γ-H2AX fluorescence revealed no significant difference (p=0.16) in yield of foci formation after irradiation with pulsed or continuous proton beams. γ-H2AX data for cell samples exposed to 1 Gy of 20 MeV protons at pulsed or continuous irradiation modes were 23.29 ± 2.04 and 26.54 ± 2.54 foci per cell, respectively. The corresponding RBE values for 20 MeV protons were 0.96 ± 0.18 and 1.13 ± 0.21 (p=0.21) for pulsed and continuous irradiation modes. However, the percentage of foci smaller than 5-10 pixels was slightly decreased and foci tended to cluster after irradiation with pulsed protons. Conclusions: Based on γ-H2AX foci formation no significant difference in the RBE between pulsed and continuous proton irradiation beams in HeLa cells has been detected so far. These results are well in line with our data on micronucleus induction in HeLa cells.
BibTeX:
	@inproceedings{Zlobinskaya2009,
	  author = {Zlobinskaya, O. and Schmid, T.E. and Dollinger, G. and Hable, V. and Greubel, C. and Michalski, D. and Wilkens, J. and Du, G. and Molls, M. and Röper, B.},
	  title = {Differences in gamma-H2AX foci formation after irradiation with continuous and pulsed proton beams},
	  booktitle = {IFMBE Proceedings},
	  publisher = {Springer International Publishing AG},
	  year = {2009},
	  volume = {25},
	  number = {3},
	  pages = {142--145},
	  editor = {Olaf Dössel and Wolfgang C. Schlegel},
	  note = {World Congress on Medical Physics and Biomedical Engineering: Radiation Protection and Dosimetry, Biological Effects of Radiation; Munich; Germany; 7 September 2009 through 12 September 2009;},
	  url = {http://link.springer.com/book/10.1007/978-3-642-03902-7},
	  doi = {https://doi.org/10.1007/978-3-642-03902-7}
	}
	

2008

Quantitative Analyse von Proteinkinetiken nach Bestrahlung lebender Zellen mit energetischen Schwerionen am Rasterionenmikroskop SNAKE
Tino Brüning; Diplomarbeit, Universität der Bundeswehr München, 2008.
BibTeX:
	@mastersthesis{Bruening2008da,
	  author = {Brüning, Tino},
	  title = {Quantitative Analyse von Proteinkinetiken nach Bestrahlung lebender Zellen mit energetischen Schwerionen am Rasterionenmikroskop SNAKE},
	  school = {Universität der Bundeswehr München},
	  year = {2008}
	}
	
Realisierung einer Schnittstelle für die externe Steuerung der Software AxioVision 4.6.3 in Verbindung mit dem Rasterionenmikroskop SNAKE
Christian Burgdorf; Studienarbeit, Universität der Bundeswehr München, 2008.
BibTeX:
	@thesis{Burgdorf2008sa,
	  author = {Burgdorf, Christian},
	  title = {Realisierung einer Schnittstelle für die externe Steuerung der Software AxioVision 4.6.3 in Verbindung mit dem Rasterionenmikroskop SNAKE},
	  school = {Universität der Bundeswehr München},
	  year = {2008}
	}
	
Quantitative analysis of DNA-damage response factors after sequential ion microirradiation
C. Greubel, V. Hable, G.A. Drexler, A. Hauptner, S. Dietzel, H. Strickfaden, I. Baur, R. Krücken, T. Cremer, A.A. Friedl and G. Dollinger; Radiation and Environmental Biophysics 47 (4) (2008) 415-422.
Abstract: Several proteins are known to form foci at DNA sites damaged by ionizing radiation. We study DNA damage response by immunofluorescence microscopy after microirradiation of cells with energetic ions. By using microirradiation, it is possible to irradiate different regions on a single dish at different time-points and to differentiate between cells irradiated earlier and later. This allows to directly compare immunofluorescence intensities in both subsets of cells with little systematic error because both subsets are cultivated and stained under identical conditions. In addition, by using irradiation patterns such as crossing lines, it is possible to irradiate individual cells twice and to differentiate between immunofluorescence signals resulting from the cellular response to the earlier and to the later irradiation event. Here, we describe the quantitative evaluation of immunofluorescence intensities after sequential irradiation.
BibTeX:
	@article{Greubel2008,
	  author = {Greubel, Christoph and Hable, Volker and Drexler, Guido A. and Hauptner, Andreas and Dietzel, Steffen and Strickfaden, Hilmar and Baur, Iris and Krücken, Reiner and Cremer, Thomas and Friedl, Anna A. and Dollinger, Günther},
	  title = {Quantitative analysis of DNA-damage response factors after sequential ion microirradiation},
	  journal = {Radiation and Environmental Biophysics},
	  year = {2008},
	  volume = {47},
	  number = {4},
	  pages = {415--422},
	  url = {http://link.springer.com/article/10.1007%2Fs00411-008-0181-0},
	  doi = {https://doi.org/10.1007/s00411-008-0181-0}
	}
	
Competition effect in DNA damage response
C. Greubel, V. Hable, G. Drexler, A. Hauptner, S. Dietzel, H. Strickfaden, I. Baur, R. Krücken, T. Cremer, G. Dollinger and A. Friedl; Radiation and Environmental Biophysics 47 (4) (2008) 423-429.
Abstract: We have built an ion-microbeam for studies of the nuclear topography and kinetics of double-strand break repair at the single cell level. Here, we show that a first and a second, delayed single ion exposure at different nuclear sites led to comparable accumulations of phospho-ATM, γ-H2AX and Mdc1 at both earlier (e) and later (l) microirradiated sites. In contrast, accumulations of 53BP1 and the recombination protein Rad51 were strongly reduced at l-sites. This apparent competition effect is accompanied by a reduced amount of 53BP1 in undamaged areas of the irradiated nuclei. We suggest that a critically limited pool size combined with strong binding at irradiated sites leads to the exhaustion of unbound factors freely roaming the nuclear space. The undersupply of these factors at l-sites requires in addition a long-lasting binding at e-sites or a weaker binding at l-sites. The observed effects suggest that DNA damage response at individual nuclear sites depends on the time course of damage load. This may have implications for therapeutic radiation treatments.
BibTeX:
	@article{Greubel2008a,
	  author = {Greubel, C. and Hable, V. and Drexler, G.A. and Hauptner, A. and Dietzel, S. and Strickfaden, H. and Baur, I. and Krücken, R. and Cremer, T. and Dollinger, G. and Friedl, A.A.},
	  title = {Competition effect in DNA damage response},
	  journal = {Radiation and Environmental Biophysics},
	  year = {2008},
	  volume = {47},
	  number = {4},
	  pages = {423--429},
	  url = {http://link.springer.com/article/10.1007%2Fs00411-008-0182-z},
	  doi = {https://doi.org/10.1007/s00411-008-0182-z}
	}
	
Relative biological effectiveness (RBE) of 20 MeV protons for induction of micronuclei in HeLa cells at continuous and pulsed irradiation modes
T.E. Schmid, G. Dollinger, A. Hauptner, V. Hable, C. Greubel, A.A. Friedl, M. Molls and B. Röper; In: , M. Baumann (Ed.), Proceedings des 17. Symposiums Experimentelle Strahlentherapie und Klinische Strahlenbiologie : Dresden, 28. Februar - 01. März 2008 17 (2008) 105-108 , Inst. für Biophysik u. Strahlenbiologie.
BibTeX:
	@inproceedings{Schmid2008,
	  author = {Schmid, T. E. and Dollinger, G. and Hauptner, A. and Hable, V. and Greubel, C. and Friedl, A. A. and Molls, M. and Röper, B.},
	  title = {Relative biological effectiveness (RBE) of 20 MeV protons for induction of micronuclei in HeLa cells at continuous and pulsed irradiation modes},
	  booktitle = {Proceedings des 17. Symposiums Experimentelle Strahlentherapie und Klinische Strahlenbiologie : Dresden, 28. Februar - 01. März 2008},
	  publisher = {Inst. für Biophysik u. Strahlenbiologie},
	  year = {2008},
	  volume = {17},
	  number = {17},
	  pages = {105--108},
	  editor = {Baumann, Michael}
	}
	

2007

Entwicklung von Auswertemethoden zur Bestimmung von Proteinkinetiken nach Zellbestrahlungen am Rasterionenmikroskop SNAKE
Tino Brüning; Studienarbeit, Universität der Bundeswehr München, 2007.
BibTeX:
	@thesis{Bruening2007sa,
	  author = {Brüning, Tino},
	  title = {Entwicklung von Auswertemethoden zur Bestimmung von Proteinkinetiken nach Zellbestrahlungen am Rasterionenmikroskop SNAKE},
	  school = {Universität der Bundeswehr München},
	  year = {2007}
	}
	

2006

Hydrogen microscopy and analysis of DNA repair using focused high energy ion beams
G. Dollinger, A. Bergmaier, A. Hauptner, S. Dietzel, G. Drexler, C. Greubel, V. Hable, P. Reichart, R. Krücken, T. Cremer and A. Friedl; Nuclear Instruments and Methods in Physics Research Section B 249 (1-2) (2006) 270-277.
Abstract: The ion microprobe SNAKE (Supraleitendes Nanoskop für Angewandte Kernphysikalische Experimente) at the Munich 14 MV tandem accelerator achieves beam focussing by a superconducting quadrupole doublet and can make use of a broad range of ions and ion energies, i.e. 4-28 MeV protons or up to 250 MeV gold ions. Due to these ion beams, SNAKE is particularly attractive for ion beam analyses in various fields. Here we describe two main applications of SNAKE. One is the unique possibility to perform three-dimensional hydrogen microscopy by elastic proton-proton scattering utilizing high energy proton beams. The high proton energies allow the analysis of samples with a thickness in the 100 μm range with micrometer resolution and a sensitivity better than 1 ppm. In a second application, SNAKE is used to analyse protein dynamics in cells by irradiating live cells with single focussed ions. Fluorescence from immunostained protein 53BP1 is used as biological track detector after irradiation of HeLa cells. It is used to examine the irradiated region in comparison with the targeted region. Observed patterns of fluorescence foci agree reasonably well with irradiation patterns, indicating an overall targeting accuracy of about 2 μm while the beam spot size is less than 0.5 μm in diameter. This performance shows successful adaptation of SNAKE for biological experiments where cells are targeted on a sub-cellular level by energetic ions.
BibTeX:
	@article{Dollinger2006,
	  author = {Dollinger, G. and Bergmaier, A. and Hauptner, A. and Dietzel, S. and Drexler, G.A. and Greubel, C. and Hable, V. and Reichart, P. and Krücken, R. and Cremer, T. and Friedl, A.A.},
	  title = {Hydrogen microscopy and analysis of DNA repair using focused high energy ion beams},
	  booktitle = {Proceedings of the Seventeenth International Conference on Ion Beam Analysis},
	  journal = {Nuclear Instruments and Methods in Physics Research Section B},
	  year = {2006},
	  volume = {249},
	  number = {1-2},
	  pages = {270--277},
	  url = {http://www.sciencedirect.com/science/article/pii/S0168583X06004587},
	  doi = {https://doi.org/10.1016/j.nimb.2006.04.012}
	}
	
Radiobiological Experiments at the Munich Microprobe SNAKE
A.A. Friedl, G.A. Drexler, M. Deutsch, H. Strickfaden, S. Dietzel, T. Cremer, A. Hauptner, R. Krücken, C. Greubel, V. Hable and G. Dollinger; In: Proceedings of the 7th International Workshop: Microbeam Probes of Cellular Radiation Response , Radiation Research 166 (2006) 668 , Radiation Research Society.
BibTeX:
	@inproceedings{Friedl2006,
	  author = {Friedl, A. A. and Drexler, G. A. and Deutsch, M. and Strickfaden, H. and Dietzel, S. and Cremer, T. and Hauptner, A. and Krücken, R. and Greubel, C. and Hable, V. and Dollinger, G.},
	  title = {Radiobiological Experiments at the Munich Microprobe SNAKE},
	  booktitle = {Proceedings of the 7th International Workshop: Microbeam Probes of Cellular Radiation Response},
	  journal = {Radiation Research},
	  publisher = {Radiation Research Society},
	  year = {2006},
	  volume = {166},
	  number = {4},
	  pages = {668},
	  url = {http://www.bioone.org/doi/abs/10.1667/RR0683.1},
	  doi = {https://doi.org/10.1667/RR0683.1}
	}
	
The Munich Microprobe SNAKE, a Single-Ion Cell Irradiation Facility
C. Greubel, V. Hable, G. Dollinger, A. Hauptner, R. Krücken, H. Strickfaden, S. Dietzel, T. Cremer, G.A. Drexler, M. Deutsch and A.A. Friedl; In: Proceedings of the 7th International Workshop: Microbeam Probes of Cellular Radiation Response , Radiation Research 166 (2006) 654 , Radiation Research Society.
BibTeX:
	@inproceedings{Greubel2006,
	  author = {Greubel, C. and Hable, V. and Dollinger, G. and Hauptner, A. and Krücken, R. and Strickfaden, H. and Dietzel, S. and Cremer, T. and Drexler, G. A. and Deutsch, M. and Friedl, A. A.},
	  title = {The Munich Microprobe SNAKE, a Single-Ion Cell Irradiation Facility},
	  booktitle = {Proceedings of the 7th International Workshop: Microbeam Probes of Cellular Radiation Response},
	  journal = {Radiation Research},
	  publisher = {Radiation Research Society},
	  year = {2006},
	  volume = {166},
	  number = {4},
	  pages = {654},
	  url = {http://www.bioone.org/doi/abs/10.1667/RR0683.1},
	  doi = {https://doi.org/10.1667/RR0683.1}
	}
	
Dynamics of DNA Repair Proteins after Directed Heavy-Ion Cell Irradiation.
V. Hable, G. Dollinger, C. Greubel, A. Hauptner, R. Krücken, S. Dietzel, T. Cremer, G.A. Drexler and A.A. Fried; In: Proceedings of the 7th International Workshop: Microbeam Probes of Cellular Radiation Response , Radiation Research 166 (2006) 676 , Radiation Research Society.
BibTeX:
	@inproceedings{Hable2006,
	  author = {Hable, V. and Dollinger, G. and Greubel, C. and Hauptner, A. and Krücken, R. and Dietzel, S. and Cremer, T. and Drexler, G. A. and Fried, A. A.},
	  title = {Dynamics of DNA Repair Proteins after Directed Heavy-Ion Cell Irradiation.},
	  booktitle = {Proceedings of the 7th International Workshop: Microbeam Probes of Cellular Radiation Response},
	  journal = {Radiation Research},
	  publisher = {Radiation Research Society},
	  year = {2006},
	  volume = {166},
	  number = {4},
	  pages = {676},
	  url = {http://www.bioone.org/doi/abs/10.1667/RR0683.1},
	  doi = {https://doi.org/10.1667/RR0683.1}
	}
	
Methods for quantitative evaluation of dynamics of repair proteins within irradiated cells
V. Hable, G. Dollinger, C. Greubel, A. Hauptner, R. Krücken, S. Dietzel, T. Cremer, G. Drexler, A. Friedl and R. Löwe; Nuclear Instruments and Methods in Physics Research Section B 245 (1) (2006) 298-301.
Abstract: Living HeLa cells are irradiated well directed with single 100 MeV oxygen ions by the superconducting ion microprobe SNAKE, the Superconducting Nanoscope for Applied Nuclear (=Kern-) Physics Experiments, at the Munich 14 MV tandem accelerator. Various proteins, which are involved directly or indirectly in repair processes, accumulate as clusters (so called foci) at DNA-double strand breaks (DSBs) induced by the ions. The spatiotemporal dynamics of these foci built by the phosphorylated histone γ-H2AX are studied. For this purpose cells are irradiated in line patterns. The γ-H2AX is made visible under the fluorescence microscope using immunofluorescence techniques. Quantitative analysis methods are developed to evaluate the data of the microscopic images in order to analyze movement of the foci and their changing size.
BibTeX:
	@article{Hable2006a,
	  author = {Hable, V. and Dollinger, G. and Greubel, C. and Hauptner, A. and Krücken, R. and Dietzel, S. and Cremer, T. and Drexler, G.A. and Friedl, A.A. and Löwe, R.},
	  title = {Methods for quantitative evaluation of dynamics of repair proteins within irradiated cells},
	  booktitle = {Proceedings of the Sixth International Symposium on Swift Heavy Ions in Matter (SHIM 2005)},
	  journal = {Nuclear Instruments and Methods in Physics Research Section B},
	  year = {2006},
	  volume = {245},
	  number = {1},
	  pages = {298--301},
	  url = {http://www.sciencedirect.com/science/article/pii/S0168583X05020628},
	  doi = {https://doi.org/10.1016/j.nimb.2005.11.118}
	}
	
Spatial Distribution of DNA Double-Strand Breaks from Ion Tracks
A. Hauptner, W. Friedland, S. Dietzel, G.A. Drexler, C. Greubel, V. Hable, H. Strickfaden, T. Cremer, A.A. Friedl, R. Krücken, H.G. Paretzke and G. Dollinger; In: P. Sigmund (Ed.), Ion Beam Science: Solved and Unsolved Problems , Vol. 52 , p. 59-85 , Royal Danish Academy of Sciences and Letter , 2006.
Abstract: Theoretical and experimental approaches are developed to investigate the spatial distribution of DNA damage induced by energetic ions in cell nuclei, with a special emphasis on DNA double-strand breaks (DSB). Using a phenomenological description for the relationship between energy dose and DSB induction, the total number of DSBs and their average number per unit pathlength can be calculated analytically for single ion tracks in cell nuclei. A simple approach to microscopic DNA damage description is offered by analytical representations which give the average energy dose in dependence of the radial distance from the ion track. However, the extreme fluctuations in the DNA damage per volume, which is due to the inhomogeneous ionisation events of the individual secondary electron paths and the structure of chromatin in the nucleus, make a true follow-up of the ionisation and excitation events desirable, e.g. by using Monte Carlo methods. The visualisation of DSBs by staining proteins which accumulate in large amounts at DSB repair sites, thus forming so-called foci, allows to analyse the spatial distribution of DSB sites under the fluorescence microscope. With this method, generally a much lower number of DSB sites along an ion track is observed than expected on basis of calculations. This observation hints at insufficient consideration of gross structures in the organisation of nuclear DNA or at a fast clustering of DSBs, possibly to form repair factories.
BibTeX:
	@incollection{Hauptner2006,
	  author = {Hauptner, A. and Friedland, W. and Dietzel, S. and Drexler, G. A. and Greubel, C. and Hable, V. and Strickfaden, H. and Cremer, T. and Friedl, A. A. and Krücken, R. and Paretzke, H. G. and Dollinger, G.},
	  title = {Spatial Distribution of DNA Double-Strand Breaks from Ion Tracks},
	  booktitle = {Ion Beam Science: Solved and Unsolved Problems},
	  publisher = {Royal Danish Academy of Sciences and Letter},
	  year = {2006},
	  volume = {52},
	  pages = {59--85},
	  editor = {P. Sigmund},
	  url = {http://www.sdu.dk/Bibliotek/matfys}
	}
	
DNA-repair protein distribution along the tracks of energetic ions
A. Hauptner, R. Krücken, C. Greubel, V. Hable, G. Dollinger, G. Drexler, M. Deutsch, R. Löwe, A. Friedl, S. Dietzel, H. Strickfaden and T. Cremer; Radiation Protection Dosimetry 122 (1-4) (2006) 147-149.
Abstract: A simple model of homogenous chromatin distribution in HeLa-cell nuclei suggests that the track of an energetic ion hits 30 nm chromatin fibers with a mean distance of 0.55 μm. To test this assumption, living HeLa-cells were irradiated at the irradiation setup of the ion microprobe SNAKE using the ion beams provided by the Munich 14 MV tandem accelerator. After irradiation, the distribution of 53BP1 protein foci was studied by immunofluorescence. The observed 53BP1 distribution along the tracks of 29 MeV 7Li ions and 24 MeV 12C ions differed significantly from the expectations resulting from the simple chromatin model, suggesting that the biological track structure is determined by cell nuclear architecture with higher order organisation of chromatin.
BibTeX:
	@article{Hauptner2006a,
	  author = {Hauptner, A. and Krücken, R. and Greubel, C. and Hable, V. and Dollinger, G. and Drexler, G.A. and Deutsch, M. and Löwe, R. and Friedl, A.A. and Dietzel, S. and Strickfaden, H. and Cremer, T.},
	  title = {DNA-repair protein distribution along the tracks of energetic ions},
	  journal = {Radiation Protection Dosimetry},
	  year = {2006},
	  volume = {122},
	  number = {1-4},
	  pages = {147--149},
	  url = {http://rpd.oxfordjournals.org/content/122/1-4/147.abstract},
	  doi = {https://doi.org/10.1093/rpd/ncl420}
	}
	
Irradiation of living cells with single ions at the ion microprobe SNAKE
A. Hauptner, T. Cremer, M. Deutsch, S. Dietzel, G. Drexler, C. Greubel, V. Hable, R. Krücken, R. Löwe, H. Strickfaden, G. Dollinger and A. Friedl; Acta Physica Polonica A 109 (3) (2006) 273-278.
Abstract: The irradiation setup at the ion microprobe SNAKE is used to irradiate living cells with single energetic ions. The irradiation accuracy of 0.55 µm and respectively 0.40 µm allows to irradiate substructures of the cell nucleus. By the choice of ion atomic number and energy the irradiation can be performed with a damage density adjustable over more than three orders of magnitude. Immunofluorescence detection techniques show the distribution of proteins involved in the repair of DNA double-strand breaks. In one of the first experiments the kinetics of appearance of irradiation-induced foci in living HeLa cells was examined. In other experiments a new effect was detected which concerned the interaction between irradiation events performed at different time points within the same cell nucleus.
BibTeX:
	@article{Hauptner2006b,
	  author = {Hauptner, A. and Cremer, T. and Deutsch, M. and Dietzel, S. and Drexler, G.A. and Greubel, C. and Hable, V. and Krücken, R. and Löwe, R. and Strickfaden, H. and Dollinger, G. and Friedl, A.A.},
	  title = {Irradiation of living cells with single ions at the ion microprobe SNAKE},
	  journal = {Acta Physica Polonica A},
	  year = {2006},
	  volume = {109},
	  number = {3},
	  pages = {273--278},
	  note = {Proceedings of the XL Zakopane School of Physics, Zakopane 2005},
	  url = {http://przyrbwn.icm.edu.pl/APP/SPIS/a109-3.html},
	  doi = {0.12693/APhysPolA.109.273}
	}
	
Mikroskopisch genaue Zellbestrahlung mit hochenergetischen Ionen.
Andreas Hauptner; Dissertation, Technische Universität München, 2006.
Abstract: Im Rahmen der Arbeit wurde die physikalisch-biologische Schädigungswirkung von hochenergetischer Ionenstrahlung in Modell-Zellkernen auf mikroskopischer Ebene abgeschätzt. Zur Durchführung von Bestrahlungsexperimenten wurde am Rasterionenmikroskop SNAKE des Münchener 14 MV Tandembeschleunigers ein Einzel-Ionen-Bestrahlungsaufbau für lebende Zellen realisiert. An HeLa-Zellen konnten damit Bestrahlungen mit einer räumlichen Auflösung von 0,5 µm durchgeführt und mittels Immunofluoreszenz-Methoden Proteine nachgewiesen werden, die an der Reparatur von DNA-Doppelstrangbrüchen beteiligt sind. Dies ermöglichte das Studium der Chromatin-Dynamik an geschädigten Zellkernbereichen sowie die Charakterisierung eines neu entdeckten "Konkurrenzeffekts" der DNA-Reparatur nach fraktionierter Bestrahlung. Durch Änderung der Bestrahlungsgeometrie konnten Schädigungsereignisse in Form sogenannter Foci entlang von Ionenspuren mit hoher Auflösung untersucht und mit Modellrechnungen verglichen werden.
BibTeX:
	@phdthesis{Hauptner2006diss,
	  author = {Hauptner, Andreas},
	  title = {Mikroskopisch genaue Zellbestrahlung mit hochenergetischen Ionen.},
	  school = {Technische Universität München},
	  year = {2006},
	  url = {http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:91-diss20060915-1726116123}
	}
	

2005

Microirradiation of cells with energetic heavy ions
G. Dollinger, V. Hable, A. Hauptner, R. Krücken, P. Reichart, A. Friedl, G. Drexler, T. Cremer and S. Dietzel; Nuclear Instruments and Methods in Physics Research Section B 231 (1-4) (2005) 195-201.
Abstract: The ion microprobe SNAKE (superconducting nanoscope for applied nuclear (Kern) physics experiments) at the Munich 14 MV tandem accelerator achieves beam focusing by a superconducting quadrupole doublet and can make use of a broad range of ions and ion energies, from 20 MeV protons to 200 MeV gold ions. This allows to adjust the number of DNA single strand breaks (SSBs) and double strand breaks (DSBs) per ion and per cell nucleus from about 0.1 DSBs per ion to several 100 DSBs per ion. When irradiating with single 100 MeV 16O ions, the adapted setup permits a fwhm irradiation accuracy of 0.55 μm in x-direction and 0.4 μm in y-direction, as demonstrated by retrospective track etching of polycarbonate foils. The experiments point to investigate protein dynamics after targeted irradiation. As an example for such experiments we show a kind of three dimensional representation of foci of γ-H2AX which are visible 0.5 h after the irradiation with 100 MeV 16O ions took place. It shows the gross correlation with the irradiation pattern but also distinct deviations which are attributed to protein dynamics in the cell.
BibTeX:
	@article{Dollinger2005,
	  author = {Dollinger, G. and Hable, V. and Hauptner, A. and Krücken, R. and Reichart, P. and Friedl, A.A. and Drexler, G. and Cremer, T. and Dietzel, S.},
	  title = {Microirradiation of cells with energetic heavy ions},
	  booktitle = {Proceedings of the 9th International Conference on Nuclear Microprobe Technology and Applications},
	  journal = {Nuclear Instruments and Methods in Physics Research Section B},
	  year = {2005},
	  volume = {231},
	  number = {1-4},
	  pages = {195--201},
	  url = {http://www.sciencedirect.com/science/article/pii/S0168583X05000765},
	  doi = {https://doi.org/10.1016/j.nimb.2005.01.056}
	}
	
Dynamik der Verteilung von DNA-Reparaturfaktoren in lebenden Zellen nach fraktionierter Bestrahlung am Rasterionenmikroskop SNAKE
Christoph Greubel; Diplomarbeit, Technische Universität München, 2005.
BibTeX:
	@mastersthesis{Greubel2005da,
	  author = {Greubel, Christoph},
	  title = {Dynamik der Verteilung von DNA-Reparaturfaktoren in lebenden Zellen nach fraktionierter Bestrahlung am Rasterionenmikroskop SNAKE},
	  school = {Technische Universität München},
	  year = {2005}
	}
	

2004

Untersuchung der Dynamik von DNA-Reparaturproteinen nach Bestrahlung lebender Zellen am Rasterionenmikroskop SNAKE.
Volker Hable; Diplomarbeit, Technische Universität München, 2004.
Abstract: In dieser Arbeit wurde die Dynamik der Reparaturvorgänge von DNA-Schäden in biologischen Zellen nach Schwerionenbestrahlung untersucht. Dazu wurden lebende HeLa-Zellen am Rasterionenmikroskop SNAKE mit 100MeV Sauerstoff-Ionen des Münchner 14MV Tandembeschleuniger bestrahlt. Die dort installierte Bestrahlungseinrichtung ermöglicht es, Zellkernen eine definierte Anzahl von Ionen und somit eine definierte Dosis mikroskopisch genau zu applizieren. Die erreichbare Strahlauflösung konnte im Rahmen dieser Arbeit mittels einer 50 Hz-Pulsung in x auf 0.55μm fwhm und in y auf 0.40μm verbessert werden. Durch die Entwicklung mikrostrukturierter Zellträgerfolien wurde das Auffinden der bestrahlten Zellen deutlich erleichtert und somit erstmals Experimente zur gezielten Bestrahlung einzelner Zellkerne ermöglicht.
Die schwersten Schäden, die hochenergetische Ionen in Zellkernen bewirken, sind Doppelstrangbrüche der DNA. Zu deren Reparatur stehen der Zelle verschiedene Mechanismen zur Verfügung. An der Reparatur direkt oder indirekt beteiligte Proteine
wie gH2AX, 53BP1, Rad51 und Mdc1 werden an Doppelstrangbrüchen angehängt und bilden sogenannte Foci aus. Ihre Funktion und Dynamik wurde in dieser Arbeit untersucht. Mittels biochemischer Prozesse wurden diese Proteine nach der Bestrahlung
angefärbt und unter dem Fluoreszenzmikroskop in einer Fokusserie abgebildet. So gewonnene und rechnergestützt entfaltete, dreidimensionale Bilder lieferten die Grundlage für eine quantitative Auswertung der Proteinverteilungen, um so die Dynamik der
Reparaturproteine zu studieren.
In Zeitreihenstudien wurde in der Zellkernmitte innerhalb der ersten 2 – 4 Stunden nach Bestrahlung ein Anwachsen der Focigröße (fwhm) von 1.2μm auf 1.5μm bei gH2AX und von 0.8μm auf 1.1μm bei 53BP1 beobachtet. In den folgenden zwei Stunden
fällt sie wieder in etwa auf den Anfangswert ab, und bleibt über 24 Stunden nahezu konstant. Am Zellkernrand wächst die Größe der gH2AX-Foci von ebenfalls 1.2μm innerhalb der ersten Stunde um knapp 0.1μm an und fällt daraufhin auf ca. 0.8μm
ab.
Des weiteren wurde die Bewegung der geschädigten DNA im Zellkern untersucht. Die hierbei gewonnenen Ergebnisse sind mit dem Modell einer Diffusion verträglich. Die Diffusionskonstante ließ sich zu (7 · 10^(−7) ± 4 · 10^(−7))μm^2/s bestimmen. Dabei waren keine signifikanten Unterschiede zwischen Zellkernmitte und -rand erkennbar.
Darüber hinaus wurde durch markiertes Bestrahlen zu zwei verschiedenen Zeitpunkten festgestellt, dass in Zellkernen, die gerade Doppelstrangbrüche reparieren, neu hinzukommende Strahlenschäden eine Unterversorgung von Protein 53BP1 erleiden.
Dieser Effekt tritt auf, wenn die Zeitdauer zwischen den beiden Bestrahlungen unter einer Stunde liegt.
BibTeX:
	@mastersthesis{Hable2004da,
	  author = {Hable, Volker},
	  title = {Untersuchung der Dynamik von DNA-Reparaturproteinen nach Bestrahlung lebender Zellen am Rasterionenmikroskop SNAKE.},
	  school = {Technische Universität München},
	  year = {2004}
	}
	
Microirradiation of cells with energetic heavy ions
A. Hauptner, S. Dietzel, G.A. Drexler, P. Reichart, R. Krücken, T. Cremer, A.A. Friedl and G. Dollinger; Radiation and Environmental Biophysics 42 (4) (2004) 237-245.
Abstract: The ion microprobe SNAKE at the Munich 14 MV tandem accelerator achieves beam focussing by a superconducting quadrupole doublet and can make use of a broad range of ions and ion energies, from 20 MeV protons to 200 MeV gold ions. Because of these properties, SNAKE is particularly attractive for biological microbeam experiments. Here we describe the adaptation of SNAKE for microirradiation of cell samples. This includes enlarging of the focal distance in order to adjust the focal plane to the specimen stage of a microscope, construction of a beam exit window in a flexible nozzle and of a suitable cell containment, as well as development of procedures for on-line focussing of the beam, preparation of single ions and scanning by electrostatic deflection of the beam. When irradiating with single 100 MeV 16O ions, the adapted set-up permits an irradiation accuracy of 0.91 µm (full width at half maximum) in the x-direction and 1.60 µm in the y-direction, as demonstrated by retrospective track etching of polycarbonate foils. Accumulation of the repair protein Rad51, as detected by immunofluorescence, was used as a biological track detector after irradiation of HeLa cells with geometric patterns of counted ions. Observed patterns of fluorescence foci agreed reasonably well with irradiation patterns, indicating successful adaptation of SNAKE. In spite of single ion irradiation, we frequently observed split fluorescence foci which might be explained by small-scale chromatin movements.
BibTeX:
	@article{Hauptner2004,
	  author = {Hauptner, A. and Dietzel, S. and Drexler, G. A. and Reichart, P. and Krücken, R. and Cremer, T. and Friedl, A. A. and Dollinger, G.},
	  title = {Microirradiation of cells with energetic heavy ions},
	  journal = {Radiation and Environmental Biophysics},
	  year = {2004},
	  volume = {42},
	  number = {4},
	  pages = {237--245},
	  url = {http://link.springer.com/article/10.1007%2Fs00411-003-0222-7},
	  doi = {https://doi.org/10.1007/s00411-003-0222-7}
	}
	
The Munich Microprobe Setup for Single-Ion Irradiation of Cells
A. Hauptner, G. Dollinger, G. Datzmann, H.-J. Körner, R. Krücken and P. Reichart; In: Proceedings of the 6th International Workshop/12th L. H. Gray Workshop: Microbeam Probes of Cellular Radiation ResponseMarch 29–31, 2003 , Radiation Research 161 (2004) 98 , Radiation Research Society.
BibTeX:
	@inproceedings{Hauptner2004a,
	  author = {Hauptner, A. and Dollinger, G. and Datzmann, G. and Körner, H.-J. and Krücken, R. and Reichart, P.},
	  title = {The Munich Microprobe Setup for Single-Ion Irradiation of Cells},
	  booktitle = {Proceedings of the 6th International Workshop/12th L. H. Gray Workshop: Microbeam Probes of Cellular Radiation ResponseMarch 29–31, 2003},
	  journal = {Radiation Research},
	  publisher = {Radiation Research Society},
	  year = {2004},
	  volume = {161},
	  number = {1},
	  pages = {98},
	  url = {http://www.rrjournal.org/toc/rare/161/1},
	  doi = {https://doi.org/10.1667/RR3091}
	}
	

2002

Design of the munich microprobe facility for single-ion irradiation of cells
A. Hauptner, G. Datzmann, G. Dollinger, H.-J. Körner, P. Reichart and O. Schmelmer; In: Proceedings of the 5th International Workshop: Microbeam Probes of Cellular Radiation Response , Radiation Research 158 (2002) 367 , Radiation Research Society.
BibTeX:
	@inproceedings{Hauptner2002,
	  author = {Hauptner, A. and Datzmann, G. and Dollinger, G. and Körner, H.-J. and Reichart, P. and Schmelmer, O.},
	  title = {Design of the munich microprobe facility for single-ion irradiation of cells},
	  booktitle = {Proceedings of the 5th International Workshop: Microbeam Probes of Cellular Radiation Response},
	  journal = {Radiation Research},
	  publisher = {Radiation Research Society},
	  year = {2002},
	  volume = {158},
	  number = {3},
	  pages = {367},
	  note = {Stresa, Lago Maggiore, Italy,May 26–27, 2001},
	  url = {http://www.rrjournal.org/toc/rare/158/3},
	  doi = {https://doi.org/10.1667/0033-7587(2002)158%5B0365:POTIWM%5D2.0.CO;2}
	}
	

1999

Der 0°-Spektrograph am Raster-Ionenmikroskop SNAKE.
Andreas Hauptner; Diplomarbeit, Technische Universität München, 1999.
Abstract: Das Ziel dieser Arbeit bestand im Aufbau und der Inbetriebnahme des 0°-Spektrographen am Raster-Ionenmikroskop SNAKE. Dieses Instrument erweitert die vielfältigen Anwendungsmöglichkeiten des Ionenmikroskops um Transmissionsmessungen mit einer Energieauflösung im Bereich von dE/E   1 x 10^(-5). Dadurch werden sowohl Dickenmessungen mit Auflösungen bis zu einatomaren Schichten als auch ganz grundlegende Experimente möglich, die sich mit der Wechselwirkung zwischen hochenergetischen Ionen und Materie beschäftigen.
Die ionenoptischen Grundlagen des Spektrographen werden ausführlich behandelt. Der vertikale 90°-Magnet als zentrales Element erlaubt dabei eine ionenoptische Abbildung in die Fokalebene mit hoher Qualität. Um die projektierte Energieauflösung
zu erreichen, ist jedoch eine weitergehende, flexible Fokussierung des Ionenstrahls notwendig.
Daher wurde der Spektrograph durch zwei Quadrupol-Linsen vervollständigt.
Um den Spektrographen betreiben zu können, wurde ein CCD (charge coupled device) Zeilensensor als Fokalebenendetektor gewählt. Dieser bietet eine Ortsauflösung von 14µm. Seine prinzipielle Eignung für die Detektion sowohl von leichten wie auch
von schweren Ionen wurde experimentell mit 20 MeV Protonen und 90 MeV Schwefelionen nachgewiesen. Bei 20 MeV Protonen konnten dabei effektive Zählraten von ca. 100kHz erreicht werden. Es zeigte sich, dass die Strahlenbeständigkeit des CCD-
Detektors ausreicht, um auf einem Pixel des Detektors zwischen 10^7 und 10^8 Protonen nachzuweisen.
In ersten Experimenten konnte die Einsetzbarkeit des Spektrographen einschließlich des Fokalebenendetektors demonstriert werden.
Mit 20 MeV Protonen wurde eine relative Energieauflösung von dE_(FWHM)/E = 1.3 x 10^(-4) erreicht und Energieverlustmessungen an Goldfolien durchgeführt. Die Auflösung war hier noch durch den Strahl beschränkt.
In einer Strahlzeit mit 90 MeV Schwefelionen wurde eine relative Energieauflösung von dE_(FWHM)/E = 3.8 x 10^(-5) erreicht. Dadurch scheint die projektierte Energieauflösung und damit auch der Einsatz des 0°-Spektrographen für die geplanten Experimente in absehbarer Zeit möglich.
BibTeX:
	@mastersthesis{Hauptner1999da,
	  author = {Hauptner, Andreas},
	  title = {Der 0°-Spektrograph am Raster-Ionenmikroskop SNAKE.},
	  school = {Technische Universität München},
	  year = {1999}
	}