25E05
Radiation Biological Application of the laser plasma x-ray and soft x-ray laser
レーザー励起単色X線ならびに軟X線レーザーを用いた放射線生物学応用
Katsutoshi Sato1, Masaharu Nishikino2, Teshima Teruki3, and Hiroaki Nishimura4
佐藤 克俊, 錦野 将元，手島 昭樹，西村 博明
1

Advanced Radiation Biology Research Program, Research Center for Charged Particle Therapy,
National Institute of Radiological Science.
2
X-ray Laser Application Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency.
3
Department of Medical Physics and Engineering, Osaka University Graduate School of Medicine.
4
Institute of Laser Engineering, Osaka University.
Laser-plasma X-ray source has the attractive features such as monochromaticity, high energy, andultra-short pulse. Soft X-ray laser produced by laser-produced plasma especially possesses excellent spatial
coherence and large amount of photon number. When the laser-plasma X-ray source is applied to thebiomedical investigation, it is expected that the novel phenomenon and the dynamics of biologicalmolecules will be observed. For the first step of the biological application of the laser-plasma X-ray source,
we have developed the cell irradiation system using laser-plasma X-ray source and have assessed whether
DNA damage induced by the laser-plasma X-ray and soft X-ray laser is same as that of conventional X-ray
source. Here, we report the recent results of the in vitro cell irradiation experiment.

1. Introduction
Recently, intense and ultra-short pulse X-ray
from laser-produced plasma has opened a newhorizon of allocations including biomedical andoncological researches. The pulse width of the laser
plasma X-ray is several tens pico (10-12) second andthe large amount of X-ray photons are emitted
within the single pulse. Moreover, themonochromaticity is superior to the conventional
X-ray and therefore the X-ray can be effectively
focused with X-ray optics. In addition, the spatial
coherence of the X-ray laser is superior to that ofconventional X-ray source. When the X-ray laser isapplied to biomedical research, we can largely
contribute to the progression of biomedicine.
The purpose of this research is to apply thelaser plasma X-ray and soft X-ray laser to thebiomedical research.
2. Radiation biological effect of the laser-plasma
X-rayFirst, we have revealed whether radiation
biological effect of laser-plasma X-ray is same asthat of conventional X-ray. To generate thelaser-plasma X-ray, high intensity Ti;sapphire laser
was used. The laser intensity, pulse width andrepetition were 3.0 × 1017 W/cm2, 70 fs, and 10 Hz,respectively. The laser spot size was 30 µm on thetarget surface. In this system, the copper (Cu) slab
was used for the target and therefore the 8 KeV
X-ray, which is Cu Kα-line, was emitted from thetarget. The X-ray was focused with thepoly-capillary X-ray lens into the culture cells. Theabsorbed dose of the laser-plasma X-ray wasmeasured with Gafchromic EBT film.
In all experiments, the human lungadenocarcinoma cell line A549 was used. Thesilicon nitride (SiN) membrane was used for thecell culture substrate. A certain number of A549
were seeded onto the SiN membrane and incubatedwith 10 % fetal bovine serum (FBS) containing
Dulbecco’s modified Eagle’s medium (DMEM).
The cell culture dish we used in the irradiation
experiment has the 3 mm diameter hole at thecenter of bottom side. Immediately before theirradiation, the SiN membrane in which the cells
were adhered on the surface was attached to thehole of cell culture dish. The irradiation site waspositioned by the upright microscope and automaticstage that installed on the irradiation system. Afterthe positioning, the cells were irradiated withlaser-plasma X-ray at room temperature.
To measure the absorbed dose of thelaser-plasma X-ray, the changes in color density of
the Gafchromic EBT film was analyzed. After theirradiation, the Gafchromic EBT film was scannedwith the flatbed commercial scanner and the density
profile was acquired. The color density wascalibrated by the high energy X-ray generated withthe 4 MV medical LINAC. As the results, theabsorbed dose was 0.048 Gy/min. The X-ray spot
size was ranging from 785 to 954 and 796 to 976µm along the horizontal and vertical direction,
respectively.
The results of the film dosimetry indicatedthat the 40 to 50 min. of the irradiation time wasneeded to treat the cells with 2 Gy. To confirm
whether the DNA double strand break (DSB) is
induced by the laser-plasma X-ray, we irradiated theA549 with 2 Gy of laser-plasma X-ray and thenperformed the immunofluorescence staining withanti-γ-H2AX antibody. As the result, the region of
γ-H2AX positive cells was clearly identified as thecircular region. The diameter of this region is 922µm along the horizontal direction and the value
approximately corresponded with the X-ray spot
size measured by Gafchromic EBT film dosimetry.
To investigate whether the radiation biologicaleffect of laser-plasma X-ray was same as that of theconventional
X-ray,
weperformed
theimmunofluorescence staining with anti-γ-H2AXand anti-p-ATM antibody and quantified thenumber of γ-H2AX and p-ATM focus in the nucleus.
As the results, the number of γ-H2AX focus
induced by the laser-plasma X-ray were same asthat induced by high energy X-ray. Likewise, thenumber of the p-ATM focus showed same aspects
as the number of the γ-H2AX focus. These results
demonstrated that the radiation biological effect oflaser-plasma X-ray was same as that ofconventional X-ray such as high energy X-ray
generated from medical LINAC. It was confirmedthat the laser-plasma X-ray could be used for thebiomedical study [1, 2].
3. Soft X-ray laser-induced DNA double strandbreaks
We have applied the soft X-ray laser to theradiation biological study and investigated whether
the DSB was induced by the soft X-ray laser
irradiation. The soft X-ray laser used in this study
was generated by double-target method developingat JAEA. Briefly, Two silver (Ag) slab target wereirradiated with Nd:glass laser to generate thehighly-charged plasma (Nickel like Ag-ions) and 90
eV soft X-ray laser are emitted from the plasmagenerated in each target. The output energy, pulsewidth, photon number per laser shot and X-ray spot
size at the surface of incidence were approximately
300 nJ, 7 ps, 3.0×1010 photons and 90 μm in
diameter, respectively. The soft X-ray laser wasvertically reflected with molybdenum/silicon(Mo/Si) mirror to be incident to the cell culture
substrate.
The energy of soft X-ray laser used in thisstudy is 90 eV and therefore the X-ray photons areeasily absorbed by the air and water. This problem
is most critical for the radiobiological study because
the irradiation experiment must be performed at theair to keep cell living. For this reason, wedeveloped the custom-made irradiation dish toirradiate cells with soft X-ray laser. The dish ismade of Aluminum and a hole is opened at thecenter of the irradiation dish. By attaching the SiN
membrane to a hole, this dish can be used for thevacuum window of the target chamber. In addition,
the inside of the dish can be filled with the culture
medium and therefore the cells can be cultured on
the upper side of SiN membrane. The cells can beirradiated with the soft X-ray laser regardless of theX-ray attenuation by any air and water by using theirradiation dish we designed.
To investigate whether the DSB is induced by
the soft X-ray laser, we perform theimmunofluorescence staining with anti-p-ATM andp-DNA-PKcs antibody. As the result, p-ATM andp-DNA-PKcs were identified in the nucleus evenafter the single shot of soft X-ray laser although thephotons of the soft X-ray laser is almost absorbedby the cellular membrane and cytoplasm andtherefore is not reached nucleus. These data suggestthat the DNA damage response is induced by thethe large amount of energy transfer into the cellular
membrane and cytoplasm. The reproducibility ofthis result must be testified by the further irradiation
experiments as soon as possible.
4. Conclusion
In this research, We conformed that the effectof laser plasma X-ray on the DSB were same as thatof conventional X-ray and that 90 eV soft X-ray
laser could induce the DSB although the x-ray
energy was not enough to penetrate the cellular
membrane, cytoplasm, and nucleus.
Acknowledgments
We are indebted to Nariaki Matsuura, M.D.,
Professor of the Laboratory of Molecular Pathology,
Osaka University Graduate School of Medicine andHealth Science, Osaka, Japan. This work wasperformed in part under the auspices of the‘‘Mono-energetic quantum beamscience withpetawatt lasers’’ project supported by the Japan
Ministry of Education, Culture, Sports, Science andTechnology (MEXT).
References[1] K. Sato, M. Nishikino, Y. Okano, S. Ohshima, N.
Hasegawa, M. Ishino, T. Kawachi, H. Numasaki, T.
Teshima, and H. Nishimura: Radiat. Res. 174 (2010)436.
[2] M. Nishikino, K. Sato, N. Hasegawa, M. Ishino, S,Ohshima, Y. Okano, T. Kawachi, H. Numasaki, T.
Teshima, and H. Nishimura. Rev. Sci. Instrum. 81(2010) 026107.