3D Elemental Distribution Research Articles

3D elemental distribution images by XRFμCT at LNLS—Brazil

An X-ray Transmission Microtomography (CT) system combined with an X-ray Fluorescence Microtomography (XRFμCT) system was implemented in the Brazilian Synchrotron Light Laboratory (LNLS), Campinas, Brazil. The main objective of this work is to determine the elemental distribution in biological samples (breast, prostate and lung samples) in order to verify the concentration of some elements correlated with characteristics and pathology of each tissue observed by the transmission CT. The experiments were performed at the X-Ray Fluorescence beamline (D09B-XRF) of the Brazilian Synchrotron Light Laboratory, Campinas, Brazil. A quasi-monochromatic beam produced by a multilayer monochromator was used as an incident beam. The sample was placed on a high precision goniometer and translation stages that allow its rotation as well as translation perpendicular to the beam. The fluorescence photons were collected with an energy dispersive HPGe detector placed at 90° to the incident beam, while transmitted photons were detected with a fast Na(Tl) scintillation counter placed behind the sample on the beam direction. The CT images were reconstructed using a filtered back-projection algorithm and the XRFμCT were reconstructed using a filtered back-projection algorithm with absorption corrections. The 3D images were reconstructed using the 3D-DOCTOR software.

New Approach To Quantitative Subcellular Imaging Of Phosphorus And Calcium Using Energy-filtered Transmission Electron Microscopy And Tomography

Analytical electron microscopy provides high-resolution distributions of chemical elements by measuring characteristic core-edge signals that originate from interaction of the incident electrons with a thin section of a cell. Such elemental images give information about the organization of specific biomolecules within cellular organelles, as well as the distribution of ions involved in regulation of cellular processes. For example, mapping phosphorus enables visualization of nucleic acid, and calcium provides distributions of a major intracellular second messenger. Elemental mapping at ∼10 nm spatial resolution is typically achieved using energy-dispersive x-ray spectroscopy (EDXS) or electron energy loss spectroscopy (EELS) in a scanning transmission electron microscope (STEM). We have developed a complementary approach based on energy-filtered transmission electron microscopy (EFTEM). It is demonstrated that quantitative 2D elemental distributions containing ∼106 pixels can be obtained from large regions of cells, and that 3D elemental distributions can be obtained when EFTEM is combined with electron tomography. It is found that an accurate elemental distribution can be derived from just two energy-selected images, above and below a core-edge. However, since the core-edge signals for elements like calcium and phosphorus are relatively weak, it is important to model the spectral background carefully by correcting for plural scattering. We have applied quantitative ETFEM imaging and tomography to determine the 3D distributions of DNA in the cell nucleus, and to measure calcium in mitochondria of neurons.

Analysis of 3D elemental mapping artefacts in biological specimens using Monte Carlo simulation

In this paper, we present Monte Carlo simulation results demonstrating the feasibility of using the focused ion beam based X-ray microanalysis technique (FIB-EDS) for the 3D elemental analysis of biological samples. In this study, we used a marine diatom Thalassiosira pseudonana as our model organism and NISTMonte for the Monte Carlo simulations. We explored several beam energies commonly used for the X-ray microanalysis to examine their effects on the resulting 3D elemental volume of the model organism. We also performed a preliminary study on the sensitivity of X-ray analysis for detecting nanoparticles in the model. For the conditions considered in this work, we show that the X-ray mapping performed using the 5 keV beam energy results in 3D elemental distributions that closely reflect the elemental distributions in the original model. At 5 keV, the depth resolution of the X-ray maps is about 250 nm for the model organism. We also show that the nanoparticles that are 50 nm in diameter or greater are easily located. Although much work is still needed in generating more accurate biological models and simulating experimental conditions relevant to these samples, our results indicate that FIB-EDS is a promising technique for the 3D elemental analysis of some biological specimens.

Imaging and 3D Elemental Characterization of Intact Bacterial Spores by High-Resolution Secondary Ion Mass Spectrometry

We present a quantitative, imaging technique based on nanometer-scale secondary ion mass spectrometry for mapping the 3D elemental distribution present in an individual micrometer-sized Bacillus spore. We use depth profile analysis to access the 3D compositional information of an intact spore without the additional sample preparation steps (fixation, embedding, and sectioning) typically used to access substructural information in biological samples. The method is designed to ensure sample integrity for forensic characterization of Bacillus spores. The minimal sample preparation/alteration required in this methodology helps to preserve sample integrity. Furthermore, the technique affords elemental distribution information at the individual spore level with nanometer-scale spatial resolution and high (microg/g) analytical sensitivity. We use the technique to map the 3D elemental distribution present within Bacillus thuringiensis israelensis spores.

Observation of three-dimensional elemental distributions of a Si device using a 360°-tilt FIB and the cold field-emission STEM system

A technique for preparation of a pillar-shaped specimen and its multidirectional observation using a combination of a scanning transmission electron microscope (STEM) and a focused ion beam (FIB) instrument has been developed. The system employs an FIB/STEM compatible holder with a specially designed tilt mechanism, which allows the specimen to be tilted through 360° [T. Yaguchi, M. Konno, T. Kamino, T. Hashimoto, T. Ohnishi, K. Umemura, K. Asayama, Microsc. Microanal. 9 (Suppl. 2) (2003) 118; T. Yaguchi, M. Konno, T. Kamino, T. Hashimoto, T. Ohnishi, M. Watanabe, Microsc. Microanal. 10 (Suppl. 2) (2004) 1030]. This technique was applied to obtain the three-dimensional (3D) elemental distributions around a contact plug of a Si device used in a 90-nm technology. A specimen containing only one contact plug was prepared in the shape of a pillar with a diameter of 200 nm and a length of 5 μm. Elemental maps were obtained from the pillar specimen using a 200-kV cold-field emission gun (FEG) STEM model HD-2300C equipped with the EDAX genesis X-ray energy-dispersive spectrometry (XEDS) system through a spectrum imaging technique. In this study, elemental distributions of minor elements with weak signals were enhanced by applying principal component analysis (PCA), which is a superior technique to extract weak signals from a large dataset. The distributions of elements, especially the metallization component Ti and minor dopant As in this particular device, were successfully extracted by PCA. Finally, the 3D elemental distributions around the contact plug could be visualized by reconstruction from the tilt series of maps.

3D Elemental Distributions in Biological Structures by EFTEM Tomography

3D Elemental Distributions in Biological Structures by EFTEM Tomography

A public software for energy filtering transmission electron tomography (EFTET-J): Application to the study of granular inclusions in bacteria from Riftia pachyptila

Energy-filtering transmission electron microscopy (EFTEM) allows the determination of elemental distributions out of a sequence of energy filtered images. Combined with electron tomography, EFTEM is a powerful tool to obtain three-dimensional chemical maps from sub-cellular structures. However, there is no existing software in the public-domain for the computation and analysis of 3D-chemical maps. Here, we present a Java-based program to compute 3D-elemental distribution. This program is available as a set of plug-ins for the public-domain Java image processing program ImageJ inspired by NIH Image. Its implemented algorithms have been successfully applied to the three-dimensional localization of iron granules in semi thin (200 nm) epon sections from the vent worm Riftia pachyptalia.

2D and 3D SIMS investigations on sintered steels

Powder metallurgy (PM) is a well-established method for manufacturing ferrous precision parts. Sintering is one of the important production steps and can be strongly enhanced (activated) by formation of a liquid phase during the sintering process. The liquid phase can be reached by the addition of alloying elements (e.g., copper) or sintering activators (e.g., phosphorus) and is formed by melting of eutectic phase mixtures or by incipient melting. The main investigations presented in this work are done by secondary ion mass spectrometry (SIMS): 2D and 3D elemental distribution. Additionally, impact energy and hardness measurements were performed in order to study the influence of phosphorus on mechanical properties. The concentration of P in different samples was varied between 0 and 1 weight percent (wt.%), the carbon content was consistently 0.5 wt.%. Nominal specimens were sintered at 1120 and 1250 °C in protective atmosphere of flowing nitrogen to determine the influence of sintering temperature.

TTPIXAN — the 4th generation

During the past six years the computer code TTPIXAN [I. Orlić et al., NIMB 49 (1990) 166] went through many changes and developments. From its infant stage when it was developed for a simple quantitative broad beam PIXE analysis, through a more sophisticated version [K.K. Loh et al., NIMB 77 (1993) 132] suited for applications in Nuclear Microscopy (NM). At present, the program is finally reaching its mature stage or its 4th metamorphosis [S.C. Liew et al., NIMB 104 (1995) 222]. It is now capable of not only simulating NM elemental images but also performing quantitative analysis and reconstruction of a complex 3D elemental composition encountered in NM applications. From a given initial 3D elemental distributions the program calculates exact elemental maps, compares them with the corresponding measured maps and by using a very efficient iterative Maximum-Likelihood Expectation-Maximisation (MLEM) algorithm, it calculates a new 3D elemental distributions. Energy loss of the incident particles and attenuation of the X-ray photons are incorporated into the reconstruction algorithm by using the most up-to date data base. The reconstruction algorithm has been successfully tested on several samples such as thin tungsten wire coated with paint, integrated circuits, and single aerosol particles. The computer code is briefly described and future plans outlined.

Volumetric rendering of 3D SIMS depth profiles

Dynamic secondary ion mass spectrometry (SIMS) can be used to uncover unique information about interfaces and whole structures which are “buried” within a solid. During a depth profile of a solid, a sequence of SIMS images is acquired for each element under study The sequence is correlated into a vertical “stack” which contains digitised three dimensional (3D) elemental distributions. Although such distributions are distorted by surface roughness, preferential sputtering and SIMS matrix effects, there is still considerable structural information contained in the volume and much potential for further information retrieval as the above-mentioned distorting effects are addressedOne of the major tools used to assess distributional information within the 3D volume has been visual rendering software. Using Sunvision software (Sun Microsystems Inc.) and a small workstation, images of the volume can be constructed which display pixels either in a “maximum value” perspective or in perspectives where the density of each phase can be adjusted to maximise structural detail.Several examples of the technique will be shown.