Simple SummaryX-ray microbeams are useful tools for elucidating the mechanisms underlying non-target effects, such as the radiation-induced bystander response (RIBR) that occurs under heterogeneous exposure conditions. The microbeam X-ray cell irradiation system at the Central Research Institute of Electric Power Industry (Tokyo, Japan) has been upgraded to enable irradiation with titanium characteristic X-rays (TiKX-rays), which have a longer penetration distance than aluminum characteristic X-rays. The beam size of the TiKX-rays was elliptical, with a long diameter of 7.5 μm and a short diameter of 6.9 μm. The dose rate at the sample position was approximately 0.8 Gy/min. Using this system, we irradiated the nuclei of HeLa cells with high precision and then analyzed RIBR. The percentage of bystander cells with pan-nuclear induction of phosphorylated histone H2AX on serine 139 (γ-H2AX) was significantly increased in one field of view, including microbeam-irradiated cells 180 and 360 min after TiKX-ray microbeam irradiation.AbstractRadiation-induced bystander response (RIBR) is a response induced in non-irradiated cells that receive bystander signals from directly irradiated cells. X-ray microbeams are useful tools for elucidating the mechanisms underlying RIBR. However, previous X-ray microbeams used low-energy soft X-rays with higher biological effects, such as aluminum characteristic X-rays, and the difference from conventional X-rays and γ-rays has often been discussed. The microbeam X-ray cell irradiation system at the Central Research Institute of Electric Power Industry has been upgraded to generate higher energy titanium characteristic X-rays (TiKX-rays), which have a longer penetration distance sufficient to irradiate 3D cultured tissues. Using this system, we irradiated the nuclei of HeLa cells with high precision and found that the pan-nuclear induction of phosphorylated histone H2AX on serine 139 (γ-H2AX) in the non-irradiated cells increased 180 and 360 min after irradiation. We established a new method to quantitatively evaluate bystander cells, using the fluorescence intensity of γ-H2AX as an indicator. The percentage of bystander cells increased significantly to 23.2% ± 3.2% and 29.3% ± 3.5% at 180 and 360 min after irradiation, respectively. Our irradiation system and the obtained results may be useful for studies of cell competition as well as non-targeted effects.