The occurrence of bystander effects has challenged the evaluation of risk for heavy ions, mainly in the context of space exploration and the increasing application of carbon ions in radiotherapy. In the present study, we addressed whether heavy-ion-induced DNA and cytogenetic damage is detectable in bystander cells. The formation of gamma-H2AX foci, sister chromatid exchanges and micronuclei were used as markers of damage to DNA. Normal human fibroblasts were exposed to low fluences of carbon and uranium ions, and alternatively single cells were targeted with heavy ions using the GSI microbeam. We did not observe a significant increase in the bystander formation of gamma-H2AX foci, sister chromatid exchanges or micronuclei. In addition, we performed for the first time parallel experiments at two microbeam facilities (GSI, JAEA) using the same cell line, culture conditions and irradiation protocols. No significant enhancement of the micronucleus frequencies in bystander cells was detected after targeted carbon-ion irradiation, confirming the results. Details regarding the history, culture conditions or support of the cells might be affecting the detection of bystander effects. On the other hand, the potential X-ray- and heavy-ion-induced bystander effects investigated herein clearly do not exceed the experimental error and thus are either lacking or are less pronounced than the effects reported in the literature for similar end points after a-particle and X-ray exposure. (c) 2009 by Radiation Research Society

Biological effects in unirradiated cells located near to irradiated cells are called bystander effects. Targeted irradiation of single cells by means of the heavy ion microprobe developed at GSI [1] was used to improve our understanding of these effects on a molecular and cellular basis. The protein of our interest was the cyclindependent kinase inhibitor CDKN1A (p21). Based on previous results obtained by broadbeam irradiation, we investigated the spatial distribution of the induction of p21 protein on the level of single bystander cells. We targeted single cells (3%) of a confluent monolayer of normal human fibroblasts (AG1522C) with 5 particles of carbon ions in 2 crossed lines per nucleus. Three hours after exposure we performed immunofluorescence (IF) staining for CDKN1A and 53BP1 protein. 53BP protein is recruited to sites of DNA damage immediately after irradiation by forming foci and is used to identify the targeted cells unambiguously (see figure 1a: the irradiated cell indicated with an arrow and enlarged in the box).

A single-ended HVEE® 3.5 MV Singletron electrostatic accelerator has been installed since October 2005 at the Centre d’Etudes Nucléaires de Bordeaux-Gradignan (CENBG) in France. This facility is equipped with a microbeam line dedicated to ion beam analysis (scanning transmission ion microscopy – STIM, particle induced X-ray emission – PIXE, Rutherford back scattering) and cellular irradiation in single event mode. A high demagnification nanobeam line will be installed on the same facility in the near future. This paper focuses on the simulation of the microbeam and nanobeam lines performances using the Geant4 Monte Carlo simulation toolkit. Comparisons with experimental data collected on the microbeam line are presented.

Since in nuclear power plants, risks of skin contact contamination by radiocobalt are significant, we focused on the impact of cobalt on a human cutaneous cell line, i.e. HaCaT keratinocytes. The present paper reports an interdisciplinary approach aimed at clarifying the biochemical mechanisms of metabolism and toxicity of cobalt in HaCaT cells. Firstly, a brief overview of the used instrumental techniques is reported. The following parts present description and discussion of results concerning: (i) toxicological studies concerning cobalt impact towards HaCaT cells (ii) structural and speciation fundamental studies of cobalt-bioligand systems, through X-ray absorption spectroscopy (XAS), ab initio and thermodynamic modelling (iii) preliminary results regarding intracellular cobalt speciation in HaCaT cells using size exclusion chromatrography/inductively coupled plasma-atomic emission spectroscopy (SEC/ICP-AES) and direct in situ analysis by ion beam micropobe analytical techniques.

A comparison of three cellular irradiation techniques using the Monte Carlo simulation toolkit Geant4 is presented in this paper. They involve electrodeposited source of alpha particle-emitting radionuclides, random classical alpha beam irradiation and single cell targeted irradiation using a focused alpha microbeam line. The simulation allows the calculation of hit distributions among the cellular population as well as the absorbed dose for two typical cellular geometries.

Scanning transmission ion microscopy tomography (STIM-T) was carried out on diatom cells with the aim of displaying their 3D structure and performing density measurements on their silica skeleton. Two software packages were compared for data reduction: TomoRebuild, based on a simple filtered backprojection algorithm, and DISRA, an iterative program. Silicon carbide microfibres of known density were also analysed as reference specimens. Similar results were obtained with both algorithms, demonstrating the ability of STIM-T to provide density measurements at the cell level without requiring any standard calibration samples. This unique feature stresses the interest of STIM-T to accurately normalise X ray emission micro-tomography data from synchrotron radiation (SXRF: synchrotron radiation X-ray fluorescence) or ion beam sources (PIXE: particle induced X-ray emission). Possible enhancements of the DISRA code are discussed in order to facilitate its use for the reconstruction of future PIXE/STIM tomography data. A "nanoprobe" coupled to a Singletron (R) accelerator, allowing a spatial resolution of a few tens of nanometers, is going to be built in the coming months at the Centre d'Etudes Nucleaires de Bordeaux Gradignan (CENBG). This new facility will bring promising applications in imaging and analysis at the sub-cellular level. (c) 2006 Elsevier B.V. All rights reserved.

A new data reduction software package has been developed at CENBG for scanning transmission ion microscopy tomography (STIM-T) data. This program, TomoRebuild, has been designed in Visual C++® in the Windows® environment. Fortran 77® routines from the Donner Library were translated to C++ for the reconstruction of 2D and 3D data using the filtered backprojection algorithm. Tomography images are stored as ASCII files to be displayed using a graphic interface. A graphic software based on the AMIRA® environment was developed to extract and display 3D structures according to their density. The whole program was designed to be interactive, simple to use and easily adjustable to different experimental conditions. 3D STIM-T was carried out on microcomposite materials, allowing determination of the density of a thin AlN layer deposited on a SiC microfibre.

The use of in vitro reconstructed human epidermis (RHE) by the cosmetic and pharmaceutical industries is increasing because of its similar physiological mechanisms to native human skin. With the advent of ethic laws on animal experimentation, RHE provides an helpful alternative for the test of formulations. The aim of this study is to check that the RHE mineral status is comparable to that of human native skin by investigating the elemental distributions in the epidermis strata. In addition, possible deleterious effects of the transport on the epidermis ionic content were studied by nuclear microscopy.

Because of the large natural inter-individual variability and of some difficulties in obtaining material from surgery or from healthy volunteers, the use of human native skin in experimentation comes up against fierce competition from experimental models, such as reconstituted epidermis or human skin grafted in animals. In this study, micro-PIXE was used in association with other ion beam microanalysis techniques to characterize different epidermis models on a microscopic scale. The ionic species were found to be highly compartmentalized in the different strata of human skin with a distribution that can be explained in the frame of the homeostatic barrier function. Reconstituted epidermis obtained from airlifted culture, epidermis of mice foot sole and human forehead skin grafts transplanted into mice are the models investigated and compared with human native skin. Very similar inorganic ion patterns were observed in all epidermis, suggesting comparable permeability barrier mechanisms and validating their use as alternative approaches to native skin experimentation. In the near future, we plan to use some of these models in penetration studies and for investigating effects of exposure to different environmental stresses. Copyright (c) 2005 John Wiley & Sons, Ltd.

The use of ion beams a few micrometers in diameter as a tomographic probe could constitute a powerful tool for displaying the 3D structure of samples a few tens or hundreds of micrometers thick in a non-destructive way. At CENBG, ion beam micro-tomography has been developed for biomedical applications at the cell level. The combination of STIM and computed tomography gives access to the 3D distribution of density (in g/cm(3)) within the analysed volume. The aim is to explore intracellular micro-structures, the sole preparation required being freeze-drying permitting analysis under vacuum.

Since the first experiments of Pontau et al. [1] and Fischer and Mühlbauer [2], applications of ion beam micro-tomography still remain scarce, although it could constitute a powerful tool for the 3D non-destructive study of small samples. At CENBG, this technique was initially developed for biomedical applications with the aim of exploring the internal structure of cells [3]. The combination of STIM (Scanning Transmission Ion Microscopy) and computed tomography gives access to the 3D distribution of density (in g/cm) with a high contrast, thanks to the high stopping power of protons in the energy range of a few MeV. The maximal profiling depth is limited by the range of protons in the material, typically a few tens to a few hundreds of micrometers.

Light-ion microbeams provide a unique opportunity to irradiate biological samples at the cellular level and to investigate radiobiological effects at low doses of high LET ionising radiation. Since 1998 a single-ion irradiation facility has been developed on the focused horizontal microbeam line of the CENBG 3.5 MV Van de Graaff accelerator. This setup delivers in air single protons and alpha particles of a few MeV onto cultured cells, with a spatial resolution of a few microns, allowing subcellular targeting. In this paper, we present results from the use of the GEANT4 toolkit to simulate cellular irradiation with the CENBG microbeam line, from the entrance to the microprobe up to the cellular medium.

An irradiation facility, able to expose cellular and subcellular targets to a precise number of particles, has been developed at CENBG for applications in radiobiology. The development of this facility was based on an existing horizontal focused microbeam developed in the early 90’s for material analysis. The focusing properties of the line allow the delivering of proton or alpha particle beams in the 1–3.5MeV energy range with a spatial resolution down to about 1μm under vacuum. For irradiation of living cells, a removable stage has been developed to extract the beam into air while preserving the analytical capabilities of the microbeam line under vacuum. This stage includes a high resolution epifluorescence microscope for online visualization of the cells and a motorized stage for cell positioning. Single particle control is ensured by a fast electrostatic deflector triggered by the signal induced by the particles through a transmission detector just before reaching the target. A dedicated software, based on an object-oriented architecture, has been designed to control the entire experiment. This includes semiautomatic calibration procedures (necessary to achieve the micron precision) and semiautomatic irradiation procedures used for targeting a large number of individual cells. In air irradiation of solid track detectors has permitted us to estimate that 99.5% of the particles are delivered on the target at a distance lower than 5μm from the beam center when an alpha particles beam is used. The targeting precision of the overall irradiation procedure, which reflects the alignment precision of the beam center with the target center, has been estimated to be within ±2μm. First experiments involving cells in culture have permitted to estimate an irradiation rate of 2000 cells per hour. This article presents the overall experimental facility and the tests performed for its validation for the irradiation of individual cells in their culture medium.

overall irradiated 50 µm 100 µm 150 µm 200 µm unirradiated b irradiated cell carbon (250 keV/µm -1 , 1.3 Gy nucleus -1 ) a 30 20 10 0 percentage of cells Biological effects in unirradiated cells located near to irradiated cells are called bystander effects. Targeted irradiation of single cells by means of the heavy ion microprobe developed at GSI (1) was used to improve our understanding of these effects on a molecular and cellular basis. The protein of our interest was the cyclin- dependent kinase inhibitor CDKN1A (p21). Based on previous results obtained by broadbeam irradiation, we investigated the spatial distribution of the induction of p21 protein on the level of single bystander cells. We targeted single cells (3%) of a confluent monolayer of normal human fibroblasts (AG1522C) with 5 particles of carbon ions in 2 crossed lines per nucleus. Three hours after exposure we performed immunofluorescence (IF) staining for CDKN1A and 53BP1 protein. 53BP protein is recruited to sites of DNA damage immediately after irradiation by forming foci and is used to identify the targeted cells unambiguously (see figure 1a: the irradiated cell indicated with an arrow and enlarged in the box).