Biological Experiments Related Radiation-Induced Responses with Synchrotron X-Ray Microbeam at the Photon Factory

An X-ray microbeam irradiation system using synchrotron radiation has been developed at the Photon Factory, KEK, Japan, and now fully operated for various biological experiments. The energy of microbeam X-rays is 5.35 keV which is suitable for experiments using mammalian cells, because the X-rays can penetrate the thickness of cells and the range of secondary electrons are within few micrometers. Detail of the system and future developments are presented elsewhere in these abstracts.The synchrotron radiation has extremely low divergence, so it is easy to get X-ray beam with micrometer-dimension. Thus, the high-precision slit installed in the system can provide a simplest way to get microbeam without focusing. The minimum beamsize by the slit is 5-um square, and the size of the microbeam can be changed quickly. This spec might be ideal to investigate the damage localization in the cell or cell nuclei irradiated with subcellular-sized beam. We tried to visualize the DNA damage produced in the microbeam-irradiated human cells with an immunostaining of phosphorylated histone H2AX (gamma-H2AX). Human fibroblast cells (NB1RGB) were grown in alfa-MEM containing 10% FBS and inoculated to the specially-designed dishes, the bottom of which was made of a thin polymer film (Mylar or polypropylene). The cells were incubated for 12 hours to attach the surface of the polymer. Before irradiation, the cell nuclei were stained with the medium containing 1uM Hoechst 33258 for 30 min. The dish was set on the microbeam system and the positions of cell nuclei were recorded with the specially-developed software and then irradiated with X-ray microbeam. The cells were irradiated with two different beam-size of microbeam, either 5- or 10-um square. The fluence of microbeam were 1.4 x 10^4 photons/(10 um)^2 per sec, which is equivalent to the X-rays of 0.4 Gy per sec. After irradiation, cells were incubated at 37 degree in C for 15 min and fixed in 100% methanol for 20 min and 100% acetone for 10 sec at 0 degree in C. Then cells were blocked with 1% FBS for 10 min, incubated with the gamma-H2AX antibody (provided by Sawady Technology), washed, and incubated with FITC-conjugated donkey anti-rabbit IgG (Santa Cruz Biotechnology, Inc). The nuclei of the cells were also stained with 1 ug/ml of propidium iodide after the immunostaining process. The dish was set on the microbeam system again, and the system automatically revisited the position of the targeted cells. The targeted cells were viewed with the epi-fluorescent microscope installed in the system. We also observed the cells by a confocal laser scanning microscope installed recently as an "off-line detection system". All the irradiated cells could be found at the revisited position, and could be distinguished from surrounding unirradiated cells by their high yield of fluorescence of gamma-H2AX. Most fluorescent foci were observed in localized area in cell nuclei, the sizes of which were almost the same as the beam size. Difference in size of the stained area can be easily recognized between cells irradiated 5-um beam and those irradiated with 10-um beam. This result also demonstrates that the system has enough accuracy and repeatability to target a part of the individual cell nuclei. Dose dependence of gamma-H2AX induction was also clearly observed, within the dose range equivalent to 2 - 6 Gy X-rays. Recently, we have started the investigation to visualize the accumulation of DNA-repair-related proteins by using Chinese hamster cells with GFP-Rad51. Within 2 hours after irradiation, there are significant number of foci of GFP-Rad51 were clearly observed in the nuclei of the microbeam-irradiated cells. Experiments for the time-course of the protein accumulation is now in progress.7th International Workshop Microbeam probes of cellular radiation respons