This study aimed to explore the potential of metal oxides such as Titanate Scrolled Nanosheets (TNs) in improving the radiosensitivity of sarcoma cell lines. Enhancing the response of cancer cells to radiation therapy is crucial, and one promising approach involves utilizing metal oxide nanoparticles. We focused on the impact of exposing two human sarcoma cell lines to both TNs and ionizing radiation (IR). Our research was prompted by previous in vitro toxicity assessments, revealing a correlation between TNs' toxicity and alterations in intracellular calcium homeostasis. A hydrothermal process using titanium dioxide powder in an alkaline solution produced the TNs. Our study quantified the intracellular content of TNs and analyzed their impact on radiation-induced responses. This assessment encompassed PIXE analysis, cell proliferation, and transcriptomic analysis. We observed that sarcoma cells internalized TNs, causing alterations in intracellular calcium homeostasis. We also found that irradiation influence intracellular calcium levels. Transcriptomic analysis revealed marked disparities in the gene expression patterns between the two sarcoma cell lines, suggesting a potential cell-line-dependent nano-sensitization to IR. These results significantly advance our comprehension of the interplay between TNs, IR, and cancer cells, promising potential enhancement of radiation therapy efficiency.

Exposure to ionizing radiation can induce genetic aberrations via unrepaired DNA strand breaks. To investigate quantitatively the dose–effect relationship at the molecular level, we irradiated dry pBR322 plasmid DNA with 3 MeV protons and assessed fragmentation yields at different radiation doses using long-read sequencing from Oxford Nanopore Technologies. This technology applied to a reference DNA model revealed dose-dependent fragmentation, as evidenced by read length distributions, showing no discernible radiation sensitivity in specific genetic sequences. In addition, we propose a method for directly measuring the single-strand break (SSB) yield. Furthermore, through a comparative study with a collection of previous works on dry DNA irradiation, we show that the irradiation protocol leads to biases in the definition of ionizing sources. We support this scenario by discussing the size distributions of nanopore sequencing reads in the light of Geant4 and Geant4-DNA simulation toolkit predictions. We show that integrating long-read sequencing technologies with advanced Monte Carlo simulations paves a promising path toward advancing our comprehension and prediction of radiation-induced DNA fragmentation.

We study the effects of irradiating water with 3 MeV protons at high doses by observing the motion of charged polystyrene beads outside the proton beam. By single-particle tracking, we measure a radial velocity of the order of microns per second. Combining electrokinetic theory with simulations of the beam-generated reaction products and their outward diffusion, we find that the bead motion is due to electrophoresis in the electric field induced by the mobility contrast of cations and anions. This work sheds light on the perturbation of biological systems by high-dose radiations and paves the way for the manipulation of colloid or macromolecular dispersions by radiation-induced diffusiophoresis.

Time-lapse fluorescence imaging coupled to micro-irradiation devices provides information on the kinetics of DNA repair protein accumulation, from a few seconds to several minutes after irradiation. Charged-particle microbeams are valuable tools for such studies since they provide a way to selectively irradiate micrometric areas within a cell nucleus, control the dose and the micro-dosimetric quantities by means of advanced detection systems and Monte Carlo simulations and monitor the early cell response by means of beamline microscopy. We used the charged-particle microbeam installed at the AIFIRA facility to perform micro-irradiation experiments and measure the recruitment kinetics of two proteins involved in DNA signaling and repair pathways following exposure to protons and α-particles. We developed and validated image acquisition and processing methods to enable a systematic study of the recruitment kinetics of GFP-XRCC1 and GFP-RNF8. We show that XRCC1 is recruited to DNA damage sites a few seconds after irradiation as a function of the total deposited energy and quite independently of the particle LET. RNF8 is recruited to DNA damage sites a few minutes after irradiation and its recruitment kinetics depends on the particle LET.

We describe a methodology to manipulate Caenorhabditis elegans (C. elegans) and irradiate the stem progenitor gonad region using three MeV protons at a specific developmental stage (L1). The consequences of the targeted irradiation were first investigated by considering the organogenesis of the vulva and gonad, two well-defined and characterized developmental systems in C. elegans. In addition, we adapted high-throughput analysis protocols, using cell-sorting assays (COPAS) and whole transcriptome analysis, to the limited number of worms (>300) imposed by the selective irradiation approach. Here, the presented status report validated protocols to (i) deliver a controlled dose in specific regions of the worms; (ii) immobilize synchronized worm populations (>300); (iii) specifically target dedicated cells; (iv) study the radiation-induced developmental alterations and gene induction involved in cellular stress (heat shock protein) and cuticle injury responses that were found.

Proton imaging can be carried out on microscopic samples by focusing the beam to a diameter ranging from a few micrometers down to a few tens of nanometers, depending on the required beam intensity and spatial resolution. Three-dimensional (3D) imaging by tomography is obtained from proton transmission (STIM: Scanning Transmission Ion Microscopy) and/or X-ray emission (PIXE: Particle Induced X-ray Emission). In these experiments, the samples are dehydrated for under vacuum analysis. In situ quantification of nanoparticles has been carried out at CENBG in the frame of nanotoxicology studies, on cells and small organisms used as biological models, especially on Caenorhabditis elegans (C. elegans) nematodes. Tomography experiments reveal the distribution of mass density and chemical content (in g.cm-3) within the analyzed volume. These density values are obtained using an inversion algorithm. To investigate the effect of this data reduction process, we defined different numerical phantoms, including a (dehydrated) C. elegans phantom whose geometry and density were derived from experimental data. A Monte Carlo simulation based on the Geant4 toolkit was developed. Using different simulation and reconstruction conditions, we compared the resulting tomographic images to the initial numerical reference phantom. A study of the relative error between the reconstructed and the reference images lead to the result that 20 protons per shot can be considered as an optimal number for 3D STIM imaging. Preliminary results for PIXE tomography are also presented, showing the interest of such numerical phantoms to produce reference data for future studies on X-ray signal attenuation in thick samples.

A compact low-energy and high-intensity x-ray source for radiation biology applications is presented. A laser-induced plasma moves inside a 30 kV diode and produces a beam of 1014 electrons at the anode location. An aluminum foil converts a part of the energy of these electrons into x-ray photons, which are characterized using filtered imaging plates. The dose that would be deposited by these x-ray photons in C. elegans larvae is calculated from Geant4 simulations. It can be set to a value ranging between 10 μGy and 10 mGy per laser shot by simply changing the aluminum foil thickness and the diode voltage. Therefore, this versatile and compact x-ray source opens a new path to explore the radiation effects induced by dose rates varying over several orders of magnitude.

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 µg.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

Charged-particle microbeams (CPMs) provide a unique opportunity to investigate the effects of ionizing radiation on living biological specimens with a precise control of the delivered dose, i.e. the number of particles per cell. We describe a methodology to manipulate and micro-irradiate early stage C. elegans embryos at a specific phase of the cell division and with a controlled dose using a CPM. To validate this approach, we observe the radiation-induced damage, such as reduced cell mobility, incomplete cell division and the appearance of chromatin bridges during embryo development, in different strains expressing GFP-tagged proteins in situ after irradiation. In addition, as the dosimetry of such experiments cannot be extrapolated from random irradiations of cell populations, realistic three-dimensional models of 2 cell-stage embryo were imported into the Geant4 Monte-Carlo simulation toolkit. Using this method, we investigate the energy deposit in various chromatin condensation states during the cell division phases. The experimental approach coupled to Monte-Carlo simulations provides a way to selectively irradiate a single cell in a rapidly dividing multicellular model with a reproducible dose. This method opens the way to dose-effect investigations following targeted irradiation.

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 µg.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

Hadron therapy is an innovative mode of radiotherapy (RT) for cancer treatment which enables tumor cells to be more effectively destroyed than conventional RT using photons. The precise knowledge of the lineal energy of particles is used in the field of microdosimetry (MKM model) as a fundamental parameter in the prediction of the relative biological efficiency (RBE) of clinical beams. Based on single‐crystal CVD (scCVD) super‐thin diamond membranes obtained using deep Ar/O2 plasma etching, prototypes of solid‐state microdosimeters for lineal energy measurements are produced at the Diamond Sensors Laboratory of CEA‐LIST. The response of a diamond membrane microdosimeter to single projectiles is investigated in ion microbeams. The microdosimeter is irradiated using a raster scanning method and the charge transport properties of the device are determined with sub‐micron precision by measuring the charge collection efficiency (CCE), the μSVs 3D spatial definition and the pulse‐height spectra. A prototype of this novel microdosimeter is then tested in a 100 MeV therapeutic proton beam at the Institute Curie − Proton Therapy Centre in Orsay. All results effectively demonstrate the great potential for this device to be used for studies of the RBE in clinical applications.

A new development of the TomoRebuild software package is presented, including “thick sample” correction for non linear X-ray production (NLXP) and X-ray absorption (XA). As in the previous versions, C++ programming with standard libraries was used for easier portability. Data reduction requires different steps which may be run either from a command line instruction or via a user friendly interface, developed as a portable Java plugin in ImageJ. All experimental and reconstruction parameters can be easily modified, either directly in the ASCII parameter files or via the ImageJ interface. A detailed user guide in English is provided. Sinograms and final reconstructed images are generated in usual binary formats that can be read by most public domain graphic softwares. New MLEM and OSEM methods are proposed, using optimized methods from the NiftyRec medical imaging library. An overview of the different medical imaging methods that have been used for ion beam microtomography applications is presented. In TomoRebuild, PIXET data reduction is performed for each chemical element independently and separately from STIMT, except for two steps where the fusion of STIMT and PIXET data is required: the calculation of the correction matrix and the normalization of PIXET data to obtain mass fraction distributions. Correction matrices for NLXP and XA are calculated using procedures extracted from the DISRA code, taking into account a large X-ray detection solid angle. For this, the 3D STIMT mass density distribution is used, considering a homogeneous global composition. A first example of PIXET experiment using two detectors is presented. Reconstruction results are compared and found in good agreement between different codes: FBP, NiftyRec MLEM and OSEM of the TomoRebuild software package, the original DISRA, its accelerated version provided in JPIXET and the accelerated MLEM version of JPIXET, with or without correction.

We report the development of thin single crystal diamond membranes suitable for dose control in targeted cell irradiation experiments with a proton microbeam. A specific design was achieved to deliver single protons with a hit detection efficiency approaching 100%. The membranes have thicknesses between 1.8 and 3 μm and are used as vacuum windows on the microbeam line. The impact of these transmission detectors on the microbeam spot size is estimated by Monte-Carlo simulations, indicating that a beam lateral resolution below 2 μm is achieved. This is confirmed by experiments showing the accumulation online of X-ray Repair Cross-Complementing protein 1 (XRCC1)-Green Fluorescent Protein (GFP) at DNA damaged sites in living cells.

As well as being a significant source of environmental radiation exposure, α-particles are increasingly considered for use in targeted radiation therapy. A better understanding of α-particle induced damage at the DNA scale can be achieved by following their tracks in real-time in targeted living cells. Focused α-particle microbeams can facilitate this but, due to their low energy (up to a few MeV) and limited range, α-particles detection, delivery, and follow-up observations of radiation-induced damage remain difficult. In this study, we developed a thin Boron-doped Nano-Crystalline Diamond membrane that allows reliable single α-particles detection and single cell irradiation with negligible beam scattering. The radiation-induced responses of single 3 MeV α-particles delivered with focused microbeam are visualized in situ over thirty minutes after irradiation by the accumulation of the GFP-tagged RNF8 protein at DNA damaged sites.

Although titanium dioxide nanoparticles (TiO2 NPs) have been extensively studied, their possible impact on health due to their specific properties supported by their size and geometry, remains to be fully characterized to support risk assessment. To further document NPs biological effects, we investigated the impact of TiO2 NPs morphology on biological outcomes. To this end, TiO2 NPs were synthesized as nanoneedles (NNs), titanate scrolled nanosheets (TNs), gel-sol-based isotropic nanoparticles (INPs) and tested for perturbation of cellular homeostasis (cellular ion content, cell proliferation, stress pathways) in three cell types and compared to the P25. We showed that TiO2 NPs were internalized at various degrees and their toxicity depended on both titanium content and NPs shape, which impacted on intracellular calcium homeostasis thereby leading to endoplasmic reticulum stress. Finally, we showed that a minimal intracellular content of TiO2 NPs was mandatory to induce toxicity enlightening once more the crucial notion of internalized dose threshold beside the well-recognized dose of exposure.

We present here a new development of the TomoRebuild software package, to perform quantitative Particle Induced X-ray Emission Tomography (PIXET) reconstruction. X-ray yields are obtained from the GUPIX code. The GUPIX data base is available for protons up to 5MeV and also in the 20–100MeV energy range, deuterons up to 6MeV, 3He and alphas up to 12MeV. In this version, X-ray yields are calculated for thin samples, i.e. without simulating X-ray attenuation. PIXET data reconstruction is kept as long as possible independent from Scanning Transmission Ion Microscopy Tomography (STIMT). In this way, the local mass distribution (in g/cm3) of each X-ray emitting element is reconstructed in all voxels of the analyzed volume, only from PIXET data, without the need of associated STIMT data. Only the very last step of data analysis requires STIMT data, in order to normalize PIXET data to obtain concentration distributions, in terms of normalized mass fractions (in μg/g). For this, a noise correction procedure has been designed in ImageJ. Moreover sinogram or image misalignment can be corrected, as well as the difference in beam size between the two experiments. The main features of the TomoRebuild code, user friendly design and modular C++ implementation, were kept. The software package is portable and can run on Windows and Linux operating systems. An optional user-friendly graphic interface was designed in Java, as a plugin for the ImageJ graphic software package. Reconstruction examples are presented from biological specimens of Caenorhabditis elegans – a small nematode constituting a reference model for biology studies. The reconstruction results are compared between the different codes TomoRebuild, DISRA and JPIXET, and different reconstruction methods: Filtered BackProjection (FBP) and Maximum Likelihood Expectation Maximization (MLEM).

Correlative microscopy is the application of two or more distinct microscopy techniques to the same region of a sample, generating complementary morphological and structural information that exceeds what is possible with any single technique to answer a biological question. We propose an approach based on a multimodal correlative microscopy, via two imaging and analytical techniques: fluorescence microscopy (FM) and ion beam analysis (IBA) to investigate in vitro nanoparticles (NPs) interactions. Indeed, the explosive growth in Nanotechnology has led to their utilization in a wide range of applications from therapeutics to multimodal imaging labeling. However, the risks for adverse health effects have not been clearly established. Detecting and tracking nanomaterials in biological systems are thus challenging and essential to understand the possible NPs-induced adverse effects. Indeed, assessing in situ NPs internalization at the single cell level is a difficult but critical task due to their potential use in nanomedicine. One of the main actual challenges is to control the number of NPs internalized per cell. The data obtained by both FM and IBA were strongly correlated in terms of detection, tracking, and colocalization of fluorescence and metal detection. IBA provides the in situ quantification not only of exogenous elements in a single cell but also of all the other endogenous elements and the subsequent variation in their cellular homeostasis. This unique property gives access to dose-dependent response analyses and therefore new perspectives for a better insight on the effect of metal oxide NPs on cellular homeostasis.

Quantification and imaging of chemical elements at the cellular level requires the use of a combination of techniques such as micro-PIXE, micro-RBS, STIM, secondary electron imaging associated with optical and fluorescence microscopy techniques employed prior to irradiation. Such a numerous set of methods generates an important amount of data per experiment. Typically for each acquisition the following data has to be processed: chemical map for each element present with a concentration above the detection limit, density and backscattered maps, mean and local spectra corresponding to relevant region of interest such as whole cell, intracellular compartment, or nanoparticles. These operations are time consuming, repetitive and as such could be source of errors in data manipulation.In order to optimize data processing, we have developed a new tool for batch data processing and imaging. This tool has been developed as a plugin for ImageJ, a versatile software for image processing that is suitable for the treatment of basic IBA data operations. Because ImageJ is written in Java, the plugin can be used under Linux, Mas OS X and Windows in both 32-bits and 64-bits modes, which may interest developers working on open-access ion beam facilities like AIFIRA. The main features of this plugin are presented here: listfile processing, spectroscopic imaging, local information extraction, quantitative density maps and database management using OMERO. (C) 2015 Elsevier B.V. All rights reserved.

The general purpose Geant4 Monte Carlo simulation toolkit is able to simulate radiative and non-radiative atomic de-excitation processes such as fluorescence and Auger electron emission, occurring after interaction of incident ionising radiation with target atomic electrons. In this paper, we evaluate the Geant4 modelling capability for the simulation of fluorescence spectra induced by 1.5MeV proton irradiation of thin high-Z foils (Fe, GdF3, Pt, Au) with potential interest for nanotechnologies and life sciences. Simulation results are compared to measurements performed at the Centre d’Etudes Nucléaires de Bordeaux-Gradignan AIFIRA nanobeam line irradiation facility in France. Simulation and experimental conditions are described and the influence of Geant4 electromagnetic physics models is discussed.