Claudie Mory

Electron energy loss spectrometry mapping

Among electron beam microanalytical techniques, electron energy loss spectrometry (EELS) offers unique advantages in terms of information content, sensitivity, limits of detection. This paper describes new methods and tools for acquiring families of spectra over many pixels on the specimen, i.e. spectrumimages, and for processing them. Applications in different fields of research, both in materials science and in life sciences, demonstrate the potential impact of the technique for characterizing nano-sized structures.

Unconventional modes for STEM imaging of biological structures.

In this paper recent developments are discussed in instrumentation and methodology associated with scanning transmission electron microscopes (STEM), which are of great potential interest for solving structural and chemical problems in biological specimens. After describing the main features of the instrument, an attempt is made to define which type of signal acquisition and processing is best suited to obtain a given type of information. Starting with a definition of cross sections of interest, a discussion follows of methods using angular selection, energy selection of the transmitted beam, and several ways of signal mixing. More specific attention is devoted to two main modes of processing signals: ratio contrast, which emphasizes slight changes in scattering factors, rather independent of thickness variations; and elemental mapping, which provides semi-quantitative information on the distribution of low Z elements of great significance in biological specimens. Data relevant to typical biological objects are presented and discussed; they allow for the definition of the capabilities and limitations of these methods. These unconventional imaging modes define a new attitude for improving the efficiency of this modern generation of electron microscopes.

Carbon nanotubes in macrophages: Imaging and chemical analysis by X-ray fluorescence microscopy

X-ray fluorescence microscopy (microXRF) is applied for the first time to study macrophages exposed to unpurified and purified single-walled (SW) and multiwalled (MW) carbon nanotubes (CNT). Investigating chemical elemental distributions allows one to (i) image nanotube localization within a cell and (ii) detect chemical modification of the cell after CNT internalization. An excess of calcium is detected for cells exposed to unpurified SWCNT and MWCNT and related toxicological assays are discussed.

Elemental analysis near the single-atom detection level by processing sequences of energy-filtered images

Abstract Chemical mapping on individual metal clusters in the nanometer range is achieved by processing sequences of energy-filtered images recorded at different energy losses before and after a characteristic edge. The experiments are performed with a dedicated FEG-STEM delivering a typical 0.5 nm probe on the surface of a thin carbon layer supporting the small aggregates of uranium and terbium. A quantitative analysis of the data in the annular dark field image and in the characteristic uranium map shows that the elemental identification of numbers of atoms below 10 can be performed with good signal-to-noise ratio. Consequently, detection limit values of the order of one atom are accessible within the presently used experimental conditions.

Energy filtered STEM imaging of thick biological sections

Energy filtered imaging of thick biological specimens was analysed using a dedicated STEM fitted with an energy loss spectrometer and interfaced with a sophisticated data collection setup. All images were digital, thus permitting a quantitative analysis of the data. We also present a mathematical explanation of the data, which is useful in pradicting the quality of thick specimen images formed with energy filtered electrons.

Experimental investigation of the ultimate EELS spatial resolution

Abstract The spatial resolution of an inelastic scattering process, such as a core excitation, governs the ultimate spatial resolution of an EELS (electron energy loss spectroscopy) measurement. An experimental study of the attainable resolution has been made through a detailed analysis of the structure of energy-filtered images on a characteristic edge (uranium O 45 ), as compared to the simultaneously recorded annular dark field images. When it has been possible to select specimen areas with a suitable random distribution of point objects (subnanometer uranium clusters), an upper limit of the degradation of the resolution is of the order of 0.3-0.4 nm for a 100 eV loss.

Multi-dimensional and multi-signal approaches in scanning transmission electron microscopes

Developments in instrumentation are essential to open new fields of science. This clearly applies to electron microscopy, where recent progress in all hardware components and in digitally assisted data acquisition and processing has radically extended the domains of application. The demonstrated breakthroughs in electron optics, such as the successful design and practical realization and the use of correctors, filters and monochromators, and the permanent progress in detector efficiency have pushed forward the performance limits, in terms of spatial resolution in imaging, as well as for energy resolution in electron energy-loss spectroscopy (EELS) and for sensitivity to the identification of single atoms. As a consequence, the objects of the nanoworld, of natural or artificial origin, can now be explored at the ultimate atomic level. The improved energy resolution in EELS, which now encompasses the near-IR/visible/UV spectral domain, also broadens the range of available information, thus providing a powerful tool for the development of nanometre-level photonics. Furthermore, spherical aberration correctors offer an enlarged gap in the objective lens to accommodate nanolaboratory-type devices, while maintaining angström-level resolution for general characterization of the nano-object under study.

Improved visualization of single- and double-stranded nucleic acids by STEM

Annular dark field STEM images have been used to visualize nucleic acid molecules positively stained with uranyl acetate. The regular distribution of uranium clusters makes clearly visible the existence of segments of double and single strands on partially denatured RNA molecules. Unpaired regions, as short as about 8 nm, are detectable by this combination of a highly efficient imaging mode and a well adapted preparation technique.

Scanning transmission electron microscopy of biological structures

The design of the scanning transmission electron microscope (STEM) has been conceived to optimize its detection efficiency of the different elastic and inelastic signals resulting from the interaction of the high energy primary electrons with the specimen. Its potential use to visualize and measure biological objects was recognized from the first studies by Crewe and coworkers in the seventies. Later the real applications have not followed the initial hopes. The purpose of the present paper is to describe how the instrument has practically evolved and recently begun to demonstrate all its potentialities for quantitative electron microscopy of a wide range of biological specimens, from freeze‐dried isolated macromolecules to unstained cryosections. Emphasis will be put on the mass‐mapping, multi‐signal and elemental mapping modes which are unique features of the STEM instruments.

Intracellular fate of carbon nanotubes inside murine macrophages: pH-dependent detachment of iron catalyst nanoparticles.

BackgroundCarbon nanotubes (CNT) are a family of materials featuring a large range of length, diameter, numbers of walls and, quite often metallic impurities coming from the catalyst used for their synthesis. They exhibit unique physical properties, which have already led to an extensive development of CNT for numerous applications. Because of this development and the resulting potential increase of human exposure, an important body of literature has been published with the aim to evaluate the health impact of CNT. However, despite evidences of uptake and long-term persistence of CNT within macrophages and the central role of those cells in the CNT-induced pulmonary inflammatory response, a limited amount of data is available so far on the CNT fate inside macrophages. Therefore, the overall aim of our study was to investigate the fate of pristine single walled CNT (SWCNT) after their internalization by macrophages.MethodsTo achieve our aim, we used a broad range of techniques that aimed at getting a comprehensive characterization of the SWCNT and their catalyst residues before and after exposure of murine macrophages: X-ray diffraction (XRD), High Resolution (HR) Transmission Electron Microscopy (TEM), High Angle Annular Dark Field-Scanning TEM (HAADF-STEM) coupled to Electron Energy Loss Spectroscopy (EELS), as well as micro-X-ray fluorescence mapping (μXRF), using synchrotron radiation.ResultsWe showed 1) the rapid detachment of part of the iron nanoparticles initially attached to SWCNT which appeared as free iron nanoparticles in the cytoplasm and nucleus of CNT-exposed murine macrophages, and 2) that blockade of intracellular lysosomal acidification prevented iron nanoparticles detachment from CNT bundles and protected cells from CNT downstream toxicity.ConclusionsThe present results, while obtained with pristine SWCNT, could likely be extended to other catalyst-containing nanomaterials and surely open new ways in the interpretation and understanding of CNT toxicity.