Technical developments for computed tomography on the CENBG nanobeam line

The use of ion microbeams as probes for computedtomography has proven to be a powerful tool for the three-dimensional characterization of specimens a few tens of micrometers in size. Compared to other types of probes, the main advantage is that quantitative information about mass density and composition can be obtained directly, using specific reconstruction codes. At the Centre d’Etudes Nucléaires de Bordeaux Gradignan (CENBG), this technique was initially developed for applications in cellular biology. However, the observation of the cell ultrastructure requires a sub-micron resolution. The construction of the nanobeamline at the Applications Interdisciplinaires des Faisceaux d’Ions en Region Aquitaine (AIFIRA) irradiation facility has opened new perspectives for such applications. The implementation of computedtomography on the nanobeamline of CENBG has required a careful design of the analysis chamber, especially microscopes for precise sample visualization, and detectors for scanning transmission ion microscopy (STIM) and for particle induced X-ray emission (PIXE). The sample can be precisely positioned in the three directions X, Y, Z and a stepper motor coupled to a goniometer ensures the rotational motion. First images of 3D tomography were obtained on a reference sample containing microspheres of certified diameter, showing the good stability of the beam and the sample stage, and the precision of the motion

Simulation of cellular irradiation with the CENBG microbeam line using GEANT4

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.Comment: 6 pages, 8 figures, presented at the 2003 IEEE-NSS conference, Portland, OR, USA, October 20-24, 200

Paparamborde: a software dedicated to quantitative mapping of biological samples using scanning transmission ion microscopy

Paparamborde, Programme d'Analyse des PArticules RAlenties dans la Matière de BORDEaux, a software dedicated to the scanning transmission ion microscopy (STIM) analysis of biological samples was developed. The programme includes the reconstruction of the median and/or mean energy loss image of the sample, as well as accurate calculation of sample thickness performed over the whole or a part of the scanned surface. The calculation is based on the conversion of transmitted particle energy into areal density of matter ($\mu$g/cm$^2$) and uses SRIM code for the stopping power calculation. A new routine for the calculation of X-ray absorption correction factors (XCF), as needed for PIXE quantification of trace element, has been added in the latest version of Paparamborde, allowing the thickness heterogeneity of sample to be properly taken into account in the calculation of XCF

Beyond filtered backprojection: A reconstruction software package for ion beam microtomography data

A new version of the TomoRebuild data reduction software package is presented, for the reconstruction of scanning transmission ion microscopy tomography (STIMT) and particle induced X-ray emission tomography (PIXET) images. First, we present a state of the art of the reconstruction codes available for ion beam microtomography. The algorithm proposed here brings several advantages. It is a portable, multi-platform code, designed in C++ with well-separated classes for easier use and evolution. Data reduction is separated in different steps and the intermediate results may be checked if necessary. Although no additional graphic library or numerical tool is required to run the program as a command line, a user friendly interface was designed in Java, as an ImageJ plugin. All experimental and reconstruction parameters may be entered either through this plugin or directly in text format files. A simple standard format is proposed for the input of experimental data. Optional graphic applications using the ROOT interface may be used separately to display and fit energy spectra. Regarding the reconstruction process, the filtered backprojection (FBP) algorithm, already present in the previous version of the code, was optimized so that it is about 10 times as fast. In addition, Maximum Likelihood Expectation Maximization (MLEM) and its accelerated version Ordered Subsets Expectation Maximization (OSEM) algorithms were implemented. A detailed user guide in English is available. A reconstruction example of experimental data from a biological sample is given. It shows the capability of the code to reduce noise in the sinograms and to deal with incomplete data, which puts a new perspective on tomography using low number of projections or limited angle

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

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 1–3.5 MeV energy range with a spatial resolution down to about 1 µm 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 5 µm 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 ±2 µm. 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

Elemental maps in human allantochorial placental vessels cells: 2. MgCl2 and MgSO4 effects

Extracellular magnesium salts are known to interfere with ionic channels in the cellular membranes. The membrane potential, a regulator of vascular tone, 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-Particule, Induced X-ray Emission (PIXE) analysis was applied to determine the ionic composition of vascular smooth muscle cells (VSMC) and of vascular endothelial cells (VEC) in the placental human allantochorial vessels in a physiological medium (Hanks' solution) modified by the addition of 2 MM MgCl2 or 2 mM MgSO4 which block the calcium-sensitive K+ channels (K-Ca) the ATP-sensitive K+ channels (K-ATP) and the voltage-sensitive K+ (Kdf) and Ca2+ channels. In VSMC (media layer), the addition of MgCl2 induced no modification of the K, Cl, P, S and Ca concentrations but increased the Na and Mg concentrations and the addition of MgSO4 only significantly increased the Mg concentration, the other ion concentrations remaining constant. In endothelium (VEC), MgCl2 or MgSO4 addition implicated the same observations as in VSMC. These results confirmed the blockage of Kdf, K-Ca, K-ATP and Ca channels in VSMC and VEC by magnesium salts, the relationship between Mg2+ ions and internal Na and demonstrated the possible intervention of a Na+/Mg2+ exchanger

Elemental maps in human allantochorial placental vessels cells : 1. High K+K^{+} and acetycholine 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 Ca$^{2+}$ 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 K$_{df}$, K$_{Ca}$ 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