Cell Irradiation System Research Articles

Observation of Histone H2AX Phosphorylation by Radiation-Induced Bystander Response Using Titanium Characteristic X-ray Microbeam.

Radiation-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 (TiK X-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.

The effect of well-characterized, very low-dose x-ray radiation on fibroblasts.

The purpose of this study is to determine the effects of low-dose radiation on fibroblast cells irradiated by spectrally and dosimetrically well-characterized soft x-rays. To achieve this, a new cell culture x-ray irradiation system was designed. This system generates characteristic fluorescent x-rays to irradiate the cell culture with x-rays of well-defined energies and doses. 3T3 fibroblast cells were cultured in cups with Mylar® surfaces and were irradiated for one hour with characteristic iron (Fe) K x-ray radiation at a dose rate of approximately 550 μGy/hr. Cell proliferation, total protein analysis, flow cytometry, and cell staining were performed on fibroblast cells to determine the various effects caused by the radiation. Irradiated cells demonstrated increased proliferation and protein production compared to control samples. Flow cytometry revealed that a higher percentage of irradiated cells were in the G0/G1 phase of the cell cycle compared to control counterparts, which is consistent with other low-dose studies. Cell staining results suggest that irradiated cells maintained normal cell functions after radiation exposure, as there were no qualitative differences between the images of the control and irradiated samples. The result of this study suggest that low-dose soft x-ray radiation might cause an initial pause, followed by a significant increase, in proliferation. An initial “pause” in cell proliferation could be a protective mechanism of the cells to minimize DNA damage caused by radiation exposure. The new cell irradiation system developed here allows for unprecedented control over the properties of the x-rays given to the cell cultures. This will allow for further studies on various cell types with known spectral distribution and carefully measured doses of radiation, which may help to elucidate the mechanisms behind varied cell responses to low-dose x-rays reported in the literature.

A laser-plasma–produced soft X-ray laser at 89 eV generates DNA double-strand breaks in human cancer cells

While it has been expected that X-ray laser will be widely applied to biomedical studies, this has not been achieved to date and its biological effects such as DNA damage have not been evaluated. As a first step for its biological application, we developed a culture cell irradiation system, particularly designed for a plasma-driven soft X-ray laser pulse, to investigate whether the soft X-ray laser is able to induce DNA double strand breaks (DSBs) in living cells or not. The human adenocarcimona cell line A549 was irradiated with the soft X-ray laser at a photon energy of 89 eV and the repair focus formation of the DSBs was assessed by immunofluorescence staining with antiphosphorylated DNA-PKcs (p-DNA-PKcs), ATM (p-ATM) and γ-H2AX antibody. The p-DNA-PKcs, ATM, and γ-H2AX foci were clearly identified after soft X-ray laser irradiation. Furthermore, the increase in the X-ray laser shot number, even from a single shot, results in the increase in p-DNA-PKcs foci. These results are the first evidence that the 89 eV soft X-ray laser is able to induce DSB in living cells. Our study demonstrated that this irradiation system is a useful tool for investigating the radiobiological effect of soft X-ray laser.

A compact biological cell irradiation system with a Van de Graaff accelerator

This report describes the development of a compact microcell irradiation system in the 3MV KN Van de Graaff accelerator in the Accelerator Laboratory of National Tsing Hua University (NTHU). The main feature of this system is backscattering, a technique whereby a 100nm gold scattering foil is placed in the center of a scattering chamber, instead of a 90° bending magnet. The incident particles produced by the accelerator bombard the scattering foil and scatter isotropically. A set of micro pinholes was installed above the scattering foil and directed the 90° scattering particles to irradiate the cell target. This innovation simplifies the system design and massively reduces the cost and space requirements for system construction.

Microbeam Studies of Soft X-ray Induced Bystander Cell Killing Using Microbeam X-ray Cell Irradiation System at CRIEPI

The radiation induced bystander response is defined as a response in cells which have not been directly targeted by radiation, but which are in the neighborhood of cells which have been directly exposed. In many cases, the bystander response is saturated with increasing dose and is observed when only one cell in a population is targeted by high-LET particle radiations or ultrasoft X-rays (278 eV). However, in our studies using synchrotron X-ray microbeams (5.35 keV), the bystander cell killing effect in normal human fibroblast WI-38 cells had a parabolic relationship to the irradiating dose and was detected if 5 or more cell nuclei were irradiated. To evaluate the feature of the X-ray-induced bystander cell killing effect at a wider dose range and the existence of photon energy dependence, the effects were assessed by irradiating cell nuclei in confluent WI-38 cells with AlK X-ray microbeams (1.49 keV). The surviving fraction decreased when only a single cell nucleus was irradiated, suggesting the minimal number of targeted cells to induce the effect may depend on the energy of photons used. In this study, we found that the bystander cell killing effect showed a biphasic relationship to the irradiating dose. The decrease in bystander cell survival at the doses higher than 0.23 Gy was partially suppressed between 2.3 and 7.0 Gy, followed by level-off around 90% above 14 Gy, suggesting that the X-ray-induced bystander response is dose dependent. In addition, NO is one of chief initiators/mediators of the effect at least 0.47 Gy.

A dosimetric system for quantitative cell irradiation experiments with laser-accelerated protons

An integrated dosimetry and cell irradiation system (IDOCIS) with laser-accelerated proton beams was developed, characterized, calibrated and successfully used for systematic in vitro experiments. Due to the broad exponentially shaped energy spectrum, the low-energy range of the protons (<20 MeV) and the high pulse dose, the absolute dosimetry for this beam quality is challenging. Therefore, a dedicated Faraday cup is used as an energy and dose rate independent absolute dosimeter that has been calibrated consistently with three independent methods. A transmission ionization chamber providing online relative dose information is cross-calibrated against the Faraday cup. Providing both online and absolute dose information, the IDOCIS allows for quantitative dosimetric and radiobiological studies at current low-energy laser-accelerated proton beams. Finally, first dosimetric characterizations of a laser-accelerated proton beam with the IDOCIS are presented.

A Novel Cell Irradiation System Using 90$^{\circ}$-Scattering Technique

A novel cell irradiation system was developed for use with the HVEE Van de Graaff accelerator at the National Tsing Hua University. A 90°-scattering technique using an Au foil was designed, in which charged particles were directed from horizontal to vertical irradiation. Beam performance was tested with a silicon detector and a CR-39 nuclear track detector. The HeLa cells were irradiated with 2-MeV alpha particles and the formation of giant cells was observed. The result showed that this system provides a convenient way of generating a vertical particle beam and a cell-supporting chamber, thereby allowing a suitable cell growth condition during irradiation. Furthermore, establishment of such a system is more convenient and economical than the alternatives.

γ-H2AX and Phosphorylated ATM Focus Formation in Cancer Cells after Laser Plasma X Irradiation

The usefulness of laser plasma X-ray pulses for medical and radiation biological studies was investigated, and the effects of laser plasma X rays were compared with those of conventional sources such as a linear accelerator. A cell irradiation system was developed that used copper-Kα (8 keV) lines from an ultrashort high-intensity laser to produce plasma. The absorbed dose of the 8 keV laser plasma X-ray pulse was estimated accurately with Gafchromic® EBT film. When the cells were irradiated with approximately 2 Gy of laser plasma X rays, the circular regions on γ-H2AX-positive cells could be clearly identified. Moreover, the numbers of γ-H2AX and phosphorylated ataxia telangiectasia mutated (ATM) foci induced by 8 keV laser plasma X rays were comparable to those induced by 4 MV X rays. These results indicate that the laser plasma X ray source may be useful for radiation biology studies.

Cell irradiation setup and dosimetry for radiobiological studies at ELBE

The radiation source ELBE delivers different types of secondary radiation, which is used for cell irradiation studies in radiobiological research. Thereby an important issue is the determination of the biological effectiveness of photon radiation as a function of photon energy by using low-energetic, monochromatic channeling radiation (10–100 keV) and high-energetic bremsstrahlung (up to 40 MV). Radiobiological studies at the research facility ELBE demand special technical and dosimetric prerequisites. Therefore, a cell irradiation system (CIS) has been designed, constructed and installed at the beam line. The CIS allows automatic irradiation of a larger cell sample number and the compensation of spatial inhomogeneity of the dose distribution within the beam spot. The recently introduced GafChromic ® EBT radiochromic film model has been used to verify the cell irradiation dose deposition achieving a dose uncertainty of <5%. Both, the installed cell irradiation system and the developed dosimetric procedure based on the use of the EBT film have been experimentally tested at ELBE. The biological effectiveness of 34 MV bremsstrahlung with respect to 200 kV X-rays from a conventional X-ray tube has been determined. An RBE value of 0.75 has been measured in good agreement with literature.

Radiation-induced γ-H2AX in mammalian cells irradiated with a synchrotron X-ray microbeam

In order to study the radiobiological effects from low dose radiation, a cell irradiation system using synchrotron X-ray microbeam has been developed, by which cells can be recognised individually and irradiated one by one with the desired dose of monochromatic X rays. The minimum beam sizes obtained are 2 microm with the focusing optics and 5 microm square with the non-focused beam, and the beam size can be changed easily with a high-precision slit in the case of a non-focused beam. Human fibroblast cells were individually irradiated with this system, and immunostained by gamma-H2AX antibody to visualise the DNA damage. Most of the fluorescent foci were observed in a localised area in cell nuclei, the size of which was almost the same as the beam size.

An electron microbeam cell-irradiation system at KIRAMS: performance and preliminary experiments

An electron microbeam cell-irradiation (EMCI) system is now ready for routine operation in Korea. The system components include an electron gun operating at 1-100 keV, a beam transport chamber delivering a micron-sized beam, a cell image acquisition and positioning part and an automatic system control section. The present choice of source beam energy is 30 keV so that the radiation impact is conveyed to the targeted cells with a minimum spatial dispersion. The beam is available at 5 microm in diameter now, but can be changed in the range of 1-200 microm. The cellular dose is delivered with a standard deviation of 30% at 0.1 Gy, 10% at 1 Gy and 3% at 10 Gy. The cells are recognised by over 98% in a 1 mm x 1 mm area and the system is capable of irradiating up to 30,000 cells h(-1).

Development of a nanotechnology based low-LET multi-microbeam array single cell irradiation system

A novel single cell irradiation system using carbon nanotube (CNT) based field emission technology is proposed. The system can produce electron microbeam at a large range of pulsation frequencies and dose rates with energy between 20 and 60 keV. Different from any existing single beam microbeam device, the CNT-based system can have 10,000 microbeam pixels, each is approximately 10 microm in size and individually controlled. Microscope imaging will be used for targeting cell(s) and the coordinate(s) identification. A single cell or large number of individually selected cells can be simultaneously irradiated under real time microscope observation. This poster reports our preliminary results in the initial stage of the CNT multipixel microbeam array development-prototype single pixel CNT microbeam device development.

Beam characterisation of the KIRAMS electron microbeam system

An electron microbeam system has been installed at the Korea Institute of Radiological and Medical Sciences (KIRAMS) for use in radiation biology studies. The electron beam is produced from a commercial electron gun, and the beam size is defined by a 5 microm diameter pinhole. Beam energy can be varied in the range of 1-100 keV, covering a range of linear energy transfer from 0.4 to 12.1 keV microm-1. The micrometer-sized electron beam selectively irradiates cells cultured in a Mylar-bottomed dish. The positioning of target cells one by one onto the beam exit is automated, as is beam shooting. The electron beam entering the target cells has been calibrated using a Passivated Implanted Planar Silicon (PIPS) detector. This paper describes the KIRAMS microbeam cell irradiation system and its beam characteristics.

Improving of Irradiation Efficiency of the JAERI-Takasaki Heavy Ion Microbeam Cell Irradiation System (III)

Improving of Irradiation Efficiency of the JAERI-Takasaki Heavy Ion Microbeam Cell Irradiation System (III)

Microbeams of Heavy Charged Particles

We have established a single cell irradiation system, which allows selected cells to be individually hit with defined number of heavy charged particles, using a collimated heavy-ion microbeam apparatus at JAERI-Takasaki. This system has been developed to study radiobiological processes in hit cells and bystander cells exposed to low dose and low dose-rate high-LET radiations, in ways that cannot be achieved using conventional broad-field exposures. Individual cultured cells grown in special dishes were irradiated in the atmosphere with a single or defined numbers of 18.3 MeV/amu 12C, 13.0 MeV/amu 20Ne, and 11.5 MeV/amu 40Ar ions. Targeting and irradiation of the cells were performed automatically at the on-line microscope of the microbeam apparatus according to the positional data of the target cells obtained at the off-line microscope before irradiation. The actual number of particle tracks that pass through cell nuclei was detected with prompt etching of the bottom of the cell dish made of ion track detector TNF-1 (modified CR-39), with alkaline-ethanol solution at 37 degrees C for 15-30 minutes. Using this system, separately inoculated Chinese hamster ovary cells, confluent normal human fibroblasts, and single plant cells (tobacco protoplasts) have been irradiated. These are the first studies in which single-ion direct hit effect and the bystander effect have been investigated using a high-LET heavy particle microbeam.

Irradiation of single mammalian cells with a precise number of energetic heavy ions – Applications of microbeams for studying cellular radiation response

A single cell irradiation system has been developed for targeting cells individually with a precise number of high-LET heavy ions to elucidate radiobiological effects of single heavy ions and to investigate the interaction of damages produced by separate events. The system has been installed at a high-energy collimated heavy-ion microbeam apparatus under a vertical beam line of the JAERI-Takasaki AVF-cyclotron. Using the heavy ion microbeam apparatus, mammalian cells are irradiated in the atmosphere with a single or precise number of heavy ions, 13.0 MeV/u 20Ne or 11.5 MeV/u 40Ar. Positional data of the individual cells are obtained at an off-line microscope before irradiation, then the cells are targeted and irradiated semi-automatically using the on-line microscope of the microbeam apparatus according to the obtained data. The number of ions penetrating the cells attached on the ion track detector CR-39 were counted with a plastic scintillator-photomultiplier tube assembly and a constant fraction discriminator. Immediately after the irradiation, the position and the number of ion tracks traversed the cell was detected with etching of CR-39 from the opposite side of the cell with alkaline-ethanol solution at 37 °C. The growths of the cells were observed individually up to 60 h after irradiation. The continuous observation of the individual cell growth indicated that a single ion traversal of a cell nucleus resulted in complete growth inhibition of the irradiated cells.

Development of an automated single cell irradiation system combined with a high-energy heavy ion microbeam system

An automated single cell recognition and irradiation system has been developed at a high-energy heavy ion microbeam. The system has been installed on a vertical beam line of the JAERI Takasaki AVF-cyclotron accelerator to irradiate individual biological cells in an air environment with energies of more than 10 MeV/u by single ions. The system consists of high-precision stages, optical microscopes with CCD camera, a computer network and software packages to obtain positional data from a cluster of individual samples randomly placed on a holder before irradiation and to irradiate them with individual ions according to the obtained data. This paper will describe the new systems, and show results from irradiation experiments for CR-39.