X-ray microbeam measurement with radiophotoluminescent glass plate for single cell irradiation

A commercially available radiophotoluminescent (RPL) glass dosimeter was used for the profile measurement of an X-ray microbeam. The X-ray microbeam of 10μm diameter was produced by a microfocus X-ray tube and a glass capillary. An RPL glass plate of 1 mm thickness was irradiated with the X-ray microbeam. After the irradiation, the RPL image for the X-ray microbeam on the glass plate was observed with a conventional fluorescence microscope. In the RPL image analysis, the RPL photons around 600 nm wavelength were detected by exposure to UV photons in the wavelength range from 330 to 380 nm. The profile of the X-ray microbeam was well measured with the RPL glass dosimeter and the RPL intensity was proportional to the X-ray fluence. Normal human fibroblasts cells, AG01522B, were grown on the RPL glass plate for single cell irradiation. A pre-selected living cell was irradiated by the X-ray microbeam and the absorbed dose was estimated to be about 3 Gy. After the irradiation, the targeting cell was stained with anti-γ-H2AX antibody, Alexa Fluor 488 and propidium iodide, to visualize the effects on DNA double strand breaks in the nucleus of the cell. In fluorescence microscopy, foci in the nucleus corresponding to the phosphorylation of serine 139 of H2AX and the X-ray beam spot were simultaneously observed at the position of the targeting cell