Ph. Barberet

Development of a focused charged particle microbeam for the irradiation of individual cells

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, bas...

Monte-Carlo dosimetry on a realistic cell monolayer geometry exposed to alpha particles

The energy and specific energy absorbed in the main cell compartments (nucleus and cytoplasm) in typical radiobiology experiments are usually estimated by calculations as they are not accessible for a direct measurement. In most of the work, the cell geometry is modelled using the combination of simple mathematical volumes. We propose a method based on high resolution confocal imaging and ion beam analysis (IBA) in order to import realistic cell nuclei geometries in Monte-Carlo simulations and thus take into account the variety of different geometries encountered in a typical cell population. Seventy-six cell nuclei have been imaged using confocal microscopy and their chemical composition has been measured using IBA. A cellular phantom was created from these data using the ImageJ image analysis software and imported in the Geant4 Monte-Carlo simulation toolkit. Total energy and specific energy distributions in the 76 cell nuclei have been calculated for two types of irradiation protocols: a 3 MeV alpha particle microbeam used for targeted irradiation and a ²³⁹Pu alpha source used for large angle random irradiation. Qualitative images of the energy deposited along the particle tracks have been produced and show good agreement with images of DNA double strand break signalling proteins obtained experimentally. The methodology presented in this paper provides microdosimetric quantities calculated from realistic cellular volumes. It is based on open-source oriented software that is publicly available.

Development of a single ion irradiation system at CENBG for applications in radiation biology

Abstract A single event facility is currently under development at CENBG for applications in radiation biology. The aim is to induce the response of living cells by hitting selected individual specimens or specific sub-cellular compartments with an exact number of light ions. In order to avoid the construction of a complete beam line, it was decided to adapt the existing microprobe system. The line has been equipped with a removable final irradiation stage constituting a versatile system working on demand either in external beam mode or classical analysis under vacuum. The beam blanking is ensured by particle detectors which have been specifically designed to control MeV proton or alpha beams in a single event mode. This paper presents the overall experimental setup and first in-air experimental tests.

Simulation of ion propagation in the microbeam line of CENBG using GEANT4

For more than five years the use of microbeam setups for the irradiation of biological samples has opened a new field of investigation in the study of radiobiological effects at low doses. Since 1998, a single ion irradiation system has been developed on the microbeam line of CENBG. Compared to the already existing systems, based on collimated beams, the use of a focused microbeam gives the advantage of a faster irradiation procedure, since the beam can be positioned onto the targeted cells by means of a fast electrostatic deflection system. Single cells irradiation requires a precise control of the position of the incident ions on target, with a spatial resolution of a few microns. All the components of the microbeam line must be designed in order to minimize the spatial dispersion of the beam: single ion transmission detector, collimators used along the beam line, exit window... To understand and to control the different processes of ion diffusion which can occur all along the beam line is thus a crucial point. For this, a reliable beam transport simulation tool is required. For more than 20 years nuclear physicists have used the GEANT code to simulate particle–matter interaction. Its most recent version, GEANT4, with an object-oriented architecture, allows to simulate different components of a beam line, considered as objects that can be inserted into the code. The beam line and the detectors geometry can be easily implemented. Moreover, the simulation code can be interfaced with computer-assisted design systems and numerical analysis software packages. In this article, the first attempt to use GEANT4 to simulate components of a microbeam line with protons and alphas of a few MeV will be presented.

Measurement of lateral straggling using a microbeam

Abstract An original method has been developed to measure the lateral straggling of sub-micron proton and alpha beams after passing through polymer foils of different thickness. For this purpose, the microbeam line at CENBG has been used in scanning transmission ion microscopy (STIM) configuration to deliver the ions in normal incidence on foils. The lateral spreading of the beam was measured using a collimated charged particle detector centred on the beam axis and placed 6.3 mm behind the foil. When the beam was horizontally scanned in front of the collimator, its lateral distribution was measured step by step detecting particles passing trough the small aperture. Regular gaussian shapes were obtained and data were compared to the result of simulations carried out using the SRIM Monte-Carlo code for 2.5 MeV protons and alphas in Mylar and Formvar thin films.

ADVANCES IN MICROBEAM TECHNOLOGIES AND APPLICATIONS TO RADIATION BIOLOGY

Charged-particle microbeams (CPMs) allow the targeting of sub-cellular compartments with a counted number of energetic ions. While initially developed in the late 1990s to overcome the statistical fluctuation on the number of traversals per cell inevitably associated with broad beam irradiations, CPMs have generated a growing interest and are now used in a wide range of radiation biology studies. Besides the study of the low-dose cellular response that has prevailed in the applications of these facilities for many years, several new topics have appeared recently. By combining their ability to generate highly clustered damages in a micrometric volume with immunostaining or live-cell GFP labelling, a huge potential for monitoring radiation-induced DNA damage and repair has been introduced. This type of studies has pushed end-stations towards advanced fluorescence microscopy techniques, and several microbeam lines are currently equipped with the state-of-the-art time-lapse fluorescence imaging microscopes. In addition, CPMs are nowadays also used to irradiate multicellular models in a highly controlled way. This review presents the latest developments and applications of charged-particle microbeams to radiation biology.

Elemental maps in human allantochorial placental vessels cells: 1. High K+ and acetylcholine effects

Regulation of vascular tone in the fetal extracorporeal circulation most likely depends on circulating hormones, local paracrine mechanisms and changes in membrane potential of vascular smooth muscle cells (VSMCs) and of vascular endothelial cells (VECs). The membrane potential is a function of the physiological activities of ionic channels (particularly, K+ and Ca2+ channels in these cells). These channels regulate the ionic distribution into these cells. Micro-particle induced X-ray emission (PIXE) analysis was applied to determine the ionic composition of VSMC and of VEC in the placental human allantochorial vessels in a physiological survival medium (Hanks’ solution) modified by the addition of acetylcholine (ACh: which opens the calcium-sensitive K+ channels, KCa) and of high concentration of K+ (which blocks the voltage-sensitive K+ channels, Kdf). In VSMC (media layer), the addition of ACh induced no modification of the Na, K, Cl, P, S, Mg and Ca concentrations and high K+ medium increased significantly the Cl and K concentrations, the other ion concentrations remaining constant. In endothelium (VEC), ACh addition implicated a significant increase of Na and K concentration, and high K+ medium, a significant increase in Cl and K concentration. These results indicated the importance of Kdf, KCa and KATP channels in the regulation of K+ intracellular distribution in VSMC and VEC and the possible intervention of a Na–K–2Cl cotransport and corroborated the previous electrophysiological data.