Abstract
We already reported our microbeam irradiation system using synchrotron monochromatic X-rays. Our system adopted a precise slit system to make X-ray microbeam, which enables us to change the beam size arbitrarily larger than 5 micron square. Using this advantage of our system, we have measured dose-survival relationships of V79 cell in two irradiation conditions with clonogenic assay method. One is to irradiate with 10-micron square beam aiming at nucleus only, the other with 50 micron square aiming at whole cell. This work revealed that hypersensitivity in low dose region is more enhanced in nucleus-irradiated cells than in whole-cell irradiated cells. We have observed also in bystander cells that bystander effect depends upon the energy-deposited area in the irradiated cells. These results suggest that intracellular communication between nucleus and cytoplasm plays an important role in determining the cell death in low dose region. For further investigation, we have developed a method to irradiate cytoplasm only without irradiating cell nucleus.
In order to shield the nucleus in the uniform irradiation field, we made a gold mask, 15 micron in diameter and 20 micron thick, on a very thin (200 nm thick) SiN film. The thickness of the gold was determined to decrease the intensity of 5.35 keV X-rays to less than one-thousandth. It was mounted on a small X-Y stage and set in the system between the slit system and the sample stage. Using a scintillater dish, we adjusted the position of the mask and the size of the beam. Finally, we got a 50 micron by 50 micron beam with uniform intensity, at the center of which located was the gold mask of 15 micron diameter. When we want to irradiate only cytoplasm of the cells, center of the mask was recognized as the beam position and cytoplasm of cells, nuclei of which were stained with Hoechst dye and recognized by its fluorescence, were irradiated with X-rays, leaving nucleus unirradiated. Survival curve of cytoplasm-irradiated cells will be presented.
