A Calcium imaging setup at the single ion hit facility at GSI and its application to the investigation of Calcium response to ion irradiation in mammalian cells

This thesis reports the construction of a Calcium imaging system at the single ion hit facility at GSI and its application to the investigation of Calcium responses to ion irradiation in mammalian cells. It has been recently reported that not only Calcium ions regulate the cellular responses to ionizing radiation but also that cells show intracellular Calcium responses to ionizing radiation. All previous investigations on this matter were so far done with ionizing radiations with a low linear-energy-transfer (LET). Here we are interested in the Calcium response of cells exposed to heavy ion irradiation, which has a much higher LET and has not been investigated before. To accomplish this, a Calcium imaging system based on ratiometric fluorescence imaging was built at the single ion hit facility for cell irradiation at GSI. The signal-to-noise ratio of the measurements based on fluorescence images reached an average of 96:1 using established image analysis methods. The setup successfully detected the expected Calcium responses in cells treated with Hydrogen Peroxide and spontaneous Ca2+ oscillations in AG1522 cells. These oscillations occurred in standard cell dishes which were sealed air-tight, and could be prevented by using gas permeable cell dishes for experiments. In parallel, the targeting stability of the single ion hit facility was improved and a technique, namely an online hit verification method, was developed to check the targeting accuracy before the cell irradiation. Using the combination of the Calcium imaging system and the single ion hit facility, the intracellular Calcium concentration was examined in HeLa cells and human foreskin fibroblast AG1522-D cells before and immediately after irradiation with 4.8 MeV/nucleon Carbon or Argon ions. No changes of intracellular Calcium level was observed in these two cell lines, both in cell dishes with hypoxic stress or in gas permeable cell dishes without stress. The irradiated AG1522-D samples showed the same behavior of Calcium signaling as control samples under hypoxia stress, under the aspects of the percentage of responding cells, the onset of the oscillations, the frequency and amplitude of the oscillations. It is concluded from these experiments that ion irradiation, and the subsequent cellular responses such as DNA double strand break and phosphorylation of H2A histone protein, do neither induce Calcium signaling nor influence Calcium signaling cascades stimulated by hypoxia stress.