Approaches to visualise the dynamics of DNA lesion processing substantially contribute to the understanding of the hierarchies of the DNA damage response pathways. Charged particle irradiation has recently emerged as a tool to generate discrete sites of subnuclear damage by means of its extremely localised dose deposition, thus facilitating the spatiotemporal analysis of repair events. In this regard low energy particles share similarity to the now widely used UV-laser microirradiation, with the advantage of a defined dose deposition and ionizing radiation properties similar to xor γ-rays. In addition, the special physical and biological properties of accelerated charged particles are of great interest for the application in advanced radiation therapy of solid tumours and, as a one of the major contributions of cosmic rays, for risk estimation of human space exploration (mars mission). A major challenge for the microscopy of early radiation effects with charged particles arises from the fact that the whole setup has to be controlled remotely during irradiation due to radiation safety regulations. The actual setup of the GSI beamline microscope consists of a modified Olympus IX71 frame, a piezo focusing device (pifoc, Physik-Instrumente), a PolyV monochromator (Till-photonics) and an Andor ixon EMCCD camera as key parts. The microscope frame has to be turned by 90° to be adapted to the horizontal beamline. The cells were cultivated on thin polymer foils and irradiated in medium filled chambers. Microscopic imaging was done with a 60x NA1.2 water immersion lens through 250 μm of cell medium. As a major application, the beamline microscopy allows determining the exact kinetics of fluorescently tagged repair proteins after irradiation with different charged particles inducing different lesion densities. The classification into fast recruited proteins like DNA-PK or XRCC1 or slower recruited ones like 53BP1 or MDC1 helps to establish the hierarchical organisation of damage recognition and subsequent repair events [1]. Additionally, motional analysis of DNA lesions induced by traversing particles provided information about the mobility of DSBs. Increased mobility might have direct consequences on the formation of chromosomal translocations and thus on the probability of cancer formation. Recently an 478 nm solid state laser for photobleaching experiments (FRAP) was coupled to the beamline microscope allowing to access the exchange and binding of the recruited repair proteins directly after irradiation with charged particles. In conclusion, the present version of the beamline microscope provides a tool to contribute to several open fields in radiation biology and DNA repair which will be discussed. Introducing small modifications, it was recently demonstrated that the device can also successfully be used at the high energy branch of the accelerator (1GeV/u) mimicking cosmic high energetic charged particles.

Since 2003, we use the GSI heavy-ion microprobe to irradiate living cells with ions from the UNILAC [1]. Two specific properties of the microbeam method make it stand out among other, more conventional irradiation procedures: First, we precisely control the dose by counting and switching individual particles that are to be delivered to the target, down to the low-dose limit of a single ion per irradiated object. Secondly, the microprobe delivers each ion into a predefined and deliberately chosen location of the target. The deviation of the actual ion impact from the point targeted defines the absolute targeting accuracy of a particle microbeam experiment. To allow for selective irradiation of tiny cellular sub-structures of interest, we strive for the best possible targeting accuracy. Here, we report on the first in-situ measurement of the absolute targeting accuracy of the GSI microbeam setup and the first sub-micron targeting accuracy measured at any biological particle microbeam.

In the cellular response to ionizing radiation a complex network of molecular pathways is activated leading to endpoints like repair, cell cycle arrest or apoptosis. Protein interactions constitute a fundamental process in these responses, but it is highly challenging to make them experimentally accessible. With the use of GFP and the production of numerous kinds of fluorescent derivates it became possible to measure Förster Resonance Energy Transfer (FRET) between fluorescently labelled proteins in living cells. Here we describe a setup to measure FRET by detecting fluorescence polarization anisotropy. The setup was then validated with live cell measurements of GFP and mCherry constructs.

Poly-ADP ribose polymerase 1 (PARP-1) is a protein well described as a sensor for single strand DNA breaks (SSB) [1]. After its activation a localized poly(ADPribosyl)ation of proteins near the DNA breaks is initiated, which is necessary for efficient binding of other repair proteins like XRCC1. The aprataxin protein has interaction domains for both SSB-repair proteins such as PARP1 and XRCC1 and double strand (DSB)-repair proteins like XRCC4 [2, 3]. This suggests a requirement for aprataxin in multiple repair pathways. Here the functional interaction of aprataxin and PARP-1 after heavy ion irradiation is analyzed using different imaging techniques [4].

In collaboration with GSI Biophysics, the microbeam, operated with ions from the UNILAC linear accelerator of GSI, is routinely employed for targeted irradiation of living cells in culture [1]. A custom-made fluorescence microscope is used both for beam targeting and for cell observation via fluorescent dyes that specifically highlight the biological agent of interest. Green Fluorescent Protein (GFP) is often used to study dynamics of repair proteins shortly after broad-beam ion irradiation in a beamline microscope [2, 3]. A combination of the high-quality in-situ GFP microscopy, currently available at the X6 beamline microscope, with the accurate targeting of single ions performed at the X0 microbeam would greatly widen the possibilities of the microbeam for radiobiological studies. Here, we report on technical improvements of the in-situ GFP microscopy setup at the microbeam facility.