Ph. Moretto

Advantages of high speed scanning for microprobe analysis of biological samples

Abstract The damage caused by the impact of a high energy focused ion beam on biological targets can lead, in a number of cases, to erroneous analytical results. Some of the observed modifications result from cummulative radiolytic effects and cannot be avoided. However, those alterations, induced by a local increase of the sample temperature can be minimized by high speed scanning. We present here the first comparative results achieved by irradiation of organic samples by the CENBG Nuclear Microprobe for different scan frequencies.

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...

Simulation of RBS spectra for quantitative mapping of inhomogeneous biological tissue

Abstract A computational algorithm, capable to simulate RBS spectra recorded during scanning proton microprobe analysis of organic specimens with inhomogeneous thickness has been developed. This program is based on an enhancement of the RUMP source code. It allows us to normalize in terms of dry specimen mass the elemental concentrations obtained with simultaneous PIXE and RBS microanalysis. This code includes non-Rutherford cross sections for proton scattering from carbon, nitrogen and oxygen. It permits the computation of matrix correction factors for the analysis of low energy X-ray emitters. In this paper, the first version of the simulation algorithm is presented as also the application to the analysis of reference organic material.

3D mapping of individual cells using a proton microbeam

Abstract Various imaging techniques carried out with a nuclear microprobe make it possible to reveal by 2D mapping, the internal structure of isolated cells. An improvement of those techniques allows today 3D mapping of cells. STIM- and PIXE-Tomography have been recently implemented on the CENBG microbeam line. The performance offered by these methods, which are capable of resolving objects having diameters less then 100 μm, has been validated on reference specimens and on human cells from cultures. In addition to the fineness of the resolution, these techniques offer the advantage of performing volume analyses without prior cutting of the samples. The ultimate aim of this program of research is to perform 3D elemental chemical analysis of individual cells in the field of biomedicine.

PIXE microanalysis in human cells: physiology and pharmacology

Abstract The micro-PIXE technique has been regularly carried out for more than two years to provide elemental distributions in human cells. Using this technique in the framework of cellular biology, two research axes have been developed: the cellular pharmacology f chemotherapeutic agents and the physiology of ionic cellular exchanges. These studies are based upon in vitro experimental models of human cells, either under the form of isolated cultured cells or as part of well-structured tissues. The aim of this paper is to present the experimental procedures and methodological aspects of cellular and subcellular quantitative mapping. Cell processing, identification of intracellular structures, quantitatives results and beam damage will be discussed and illustrated by examples issuing from the above-mentioned studies.

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.

Imaging physicochemical reactions occurring at the pore surface in binary bioactive glass foams by micro ion beam analysis.

In this work, the physicochemical reactions occurring at the surface of bioactive sol-gel derived 3D glass scaffolds via a complete PIXE characterization were studied. 3D glass foams in the SiO(2)-CaO system were prepared by sol-gel route. Samples of glass scaffolds were soaked in biological fluids for periods up to 2 days. The surface changes were characterized using particle induced X-ray emission (PIXE) associated to Rutherford backscattering spectroscopy (RBS), which are efficient methods to perform quantitative chemical maps. Elemental maps of major and trace elements at the glass/biological fluids interface were obtained at the micrometer scale for every interaction time. Results revealed interconnected macropores and physicochemical reactions occurring at the surface of pores. The micro-PIXE-RBS characterization of the pores/biological fluids interface shows the glass dissolution and the rapid formation of a Ca rich layer with the presence of phosphorus that came from biological fluids. After 2 days, a calcium phosphate-rich layer containing magnesium is formed at the surface of the glass scaffolds. We demonstrate that quantities of phosphorus provided only by the biological medium have a significant impact on the development and the formation of the phosphocalcic layer.

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.

Microanalysis of the human skin structure: preliminary results

Abstract In this paper, we report micro-PIXE analysis of normal human skin sections. The aim of this preliminary study was to investigate the tissue morphology using the distribution pattern of mineral ions. We found that these minerals were compartmentalized enough to constitute useful markers of the tissue ultrastructure. The epidermis and its different skin strata, corneum, granulosum and spinosum, were actually identified on elemental maps. The present work also provided additional useful information about minerals in the intradermal part of hair tracts. Methodological points will be evoked on the basis of this experience, more especially those concerning the sampling procedure.

Light-element analysis with the CENBG nuclear microprobe

In the field of light-element detection (Z < 11), the capability of the nuclear muprobe to carry out nuclear reaction analysis offers significant advantages compared with other methods. But taking into account the low beam current available, reactions with large cross sections must be selected in order to achieve acquisition in a reasonable time. This restriction prevents the use of RBS analysis for detection of light elements in heavy matrices. Moreover, the 8 μm beryllium window of standard X-ray detectors does not permit the PIXE measurement of light elements. We present here some examples in the use of the resonant nuclear reactions 12C(p, p)12C and 16O(α, α)16O for carbon and oxygen determination in different materials. Results are discussed in terms of strength and limit of nuclear reactions compared with new possibilities of X-ray detectors following improvements in the transmission of low energy radiation through windows.