Claire Michelet

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.

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.

STIM tomography at the cell level

Abstract STIM tomography has been recently implemented in the nuclear microbeam line of the CENBG. In addition to the design of the experimental device, the main development consisted in the elaboration of data reduction programs based on analytic reconstruction methods usually employed for X-ray tomography. This report describes the first results that we obtained in the analysis of biological samples. In a first step, plant specimens such as single pollen grains and stamens were investigated in order to assess the performances of the technique. Individual cells isolated from a human cancer cell line were then analysed. One of the main advantages pointed out by these experiments is the capability to reveal the cell ultrastructure using simply freeze-dried unsectioned specimens.

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.

Single Cell In Situ Detection and Quantification of Metal Oxide Nanoparticles Using Multimodal Correlative Microscopy

Assessing in situ nanoparticles (NPs) internalization at the level of a single cell 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 internalized NPs per cell. To in situ detect, track, and above all quantify NPs in a single cell, we propose an approach based on a multimodal correlative microscopy (MCM), via the complementarity of three imaging techniques: fluorescence microscopy (FM), scanning electron microscopy (SEM), and ion beam analysis (IBA). This MCM was performed on single targeted individual primary human foreskin keratinocytes (PHFK) cells cultured and maintained on a specifically designed sample holder, to probe either dye-modified or bare NPs. The data obtained by both FM and IBA on dye-modified NPs were strongly correlated in terms of detection, tracking, and colocalization of fluorescence and metal detection. IBA techniques should therefore open a new field concerning specific studies on bare NPs and their toxicological impact on cells. Complementarity of SEM and IBA analyses provides surface (SEM) and in depth (IBA) information on the cell morphology as well as on the exact localization of the NPs. Finally, IBA not only provides in a single cell the in situ quantification of exogenous elements (NPs) but also that all the other endogenous elements and the subsequent variation of their homeostasis. This unique feature opens further insights in dose-dependent response analyses and adds the perspective of a better understanding of NPs behavior in biological specimens for toxicology or nanomedicine purposes.

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.

The biological research programme of the nuclear microprobe at the National Accelerator Centre, Faure

Abstract The nuclear microprobe (NMP) unit of the National Accelerator Centre (NAC) has initiated a focused research programme on studies of biological material, ranging from applications in medicine to agriculture and botany. During this period a state-of-the-art cryo-preparation laboratory was also developed. This research programme has resulted in a wide range of projects, and has shown how well suited the NMP is for studies of biological material in general. This paper reports on some of the problems and demands in this field, as well as some of the results obtained using particle induced X-ray spectroscopy (PIXE) and Rutherford backscattering (RBS). True elemental imaging is routinely performed using the dynamic analysis (DA) method, which forms part of the GeoPIXE suite of programmes. A collaborative project, together with the CENBG group of Bordeaux–Gradignan in France, on the development of a facility with the aim of studying effects of single-events of radiation in living cells was recently established and is discussed.

In situ quantification of diverse titanium dioxide nanoparticles unveils selective endoplasmic reticulum stress-dependent toxicity

Abstract 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.

TRACE METAL CONTENT IN DISTINCT GENOTYPES OF HUMAN NEUROBLASTOMA CELLS : PRELIMINARY RESULTS

Abstract Some transition metals play important regulatory roles in gene expression. The disturbance of their cellular levels could be involved in oncogene expression and tumorigenesis. Nuclear Microprobe Analysis (NMPA) was used to measure cellular trace metal levels (Mn, Fe, Cu, Zn) in two human neuroblastoma cell lines characterized by distinct genotypes. In this paper, a specific protocol established for sample preparation of neuronal cultured cells is described. Trace metal concentrations in SK-N-SH and IGR-N-91 cells exhibiting respectively a single copy, and 60 copies, of the N-myc oncogene are reported. A brief discussion on experiment design for NMPA of trace metal functions in gene expression is also presented.

STIM tomography: A potential tool for the non-destructive characterisation of SiC microcomposite materials

Abstract Despite the fact that ion micro-tomography techniques were initiated several years ago, useful applications are still rare in material sciences. This work is a preliminary study on the non-destructive characterisation of a thin BN interphase in a ceramic matrix composite material by scanning transmission ion microscopy (STIM) tomography. The technique, initially developed at CENBG for applications in the biomedical field, was used here to investigate several model composites as processed and after oxidation tests at high temperature. The data reduction codes, based on analytic reconstruction methods, made it possible to determine the spatial extension of the BN interphase in transversal planes at different positions along the microcomposites (25 μm in diameter). This measurement was performed taking advantage of the density contrast between SiC and BN even for a layer thickness down to 3 μm. A first attempt was also made to separate the initial BN interphase from the B2O3 layer formed on its surface during oxidation.