The geometric locations of ion traversals in mammalian cells constitute important information in the study of heavy ion-induced biological effects. We employed a contact microscopy technique, which was developed for boron imaging in boron neutron capture therapy to the irradiation mammalian cells by low-energy heavy ions. This method enables the simultaneous visualization of mammalian cells as a relief on a plastic track detector, CR-39, and the etch pits which indicate the positions of ion traversals. This technique provides visual geometric information about the cells and ion traversal, without any specially designed devices or microscopes. Only common laboratory equipment, such as a conventional optical microscope, a UV lamp, and commercially available CR-39 is required. To validate this method, CHO-K1 and HeLa cells were cultured on the CR-39 surface and then irradiated with low-energy Ar and Ne ions, respectively. The positions of induced DNA double strand breaks were detected as gamma-H2AX fluorescent spots, which coincided with the positions of the etch pits in the cell relief image.

African green monkey kidney cells, CV-1, were irradiated with Carbon ions (LET: 735 keV/µm Argon ions (LET: 3,000 keV/µm) to visualize ion tracks through the cell nucleus by labeling the 3'-OH termini result of DNA strand breaks. The 3'-OH termini of DNA were labeled with BrdU-triphosphate catalyzed by TdT. This method of TUNEL (TdT-mediated dUTP Nick End labeling) is based on the specific binding of TdT to 3'-OH termini of DNA. Subsequent immuno-fluorescent staining with the primary monoclonal antibody against BrdU, followed by a secondary antibody of Alexa Fluor 488, was performed to visualize the BrdU labeled DNA termini. Images of the cell nuclei were acquired by confocal laser microscopy. When cell monolayers were irradiated perpendicularly with argon ions, induced DSBs in cell nuclei were identifiable as fluorescent spots. In another irradiation setup, when cells were irradiated at a small angle with incident argon ions, DNA strand breaks were detected as fluorescent stripes across the cell nucleus. These results demonstrate the induction of 3'-OH termini at sites of DNA strand breaks along Argon ion tracks.

HeLa and CHO-K1 cells were irradiated with Fe ions (1.14 MeV/nucleon) near the Bragg peak to determine how many ion traversals through a cell nucleus are necessary to induce cell inactivation. The ion traversals through a cell nucleus were visualized by immunostaining the phosphorylated histone H2AX (gamma-H2AX), as an indicator of DNA double strand breaks (DSBs), to confirm that DSBs are actually induced along every Fe ion traversal through the nucleus. The survival curves after irradiation with Fe ions decreased exponentially with the ion fluence without a shoulder. The inactivation cross sections calculated from the slope of the survival curves and the standard errors were 96.9 +/- 1.8 and 57.9 +/- 5.4 microm2 for HeLa and CHO-K1 cells, respectively, corresponding to 0.442 and 0.456 of the mean value of each cell nucleus area. Taking the distribution of the cell nucleus area into consideration with an equation proposed by Goodhead et al. (1980), which calculates the average number of lesions per single ion track through the average area of a sensitive organelle (mainly nucleus), these two ratios were converted to 0.705 and 0.659 for HeLa and CHO-K1 cells, respectively. These ratios were less than one, suggesting that the average numbers of lethal hits per cell produced by a single ion traversal were less than one. We thus considered two possible explanations for ion traversals of more than one, necessary for cell inactivation.

We have developed a new system for irradiating biological samples in air with ions from H to Xe below 6.0 MeV/ nucleon near the Bragg peak. The irradiation system can provide ion beams with 20-mm diameter of which the central area of 100 mm 2 is uniform in fluence rate within a standard deviation of ±10%. For each ion, the linear energy transfer is selectable by irradiation positions in air, from the lowest at the surface of a vacuum window to the highest at the Bragg peak, for example, from 281 to 977 keV/µm for C ions. A wide range of fluence rates, 10 −3 -10 4 ions/ m 2 / s, can be provided by the system, which makes it possible to irradiate a variety of biological samples with different target sizes, from small plasmid DNA to living mammalian cells. The ion fluence irradiated to each sample is calculated from the output of the secondary electron monitor using the linear relationship between the output and ion fluence measured at the sample position by CR-39 track detectors. Survival curves and visualization of NBS1 foci for human cells are presented as examples of preliminary experiments using C ions near the Bragg peak.