George Kourousias

Recent developments at the TwinMic beamline at ELETTRA: an 8 SDD detector setup for low energy X-ray Fluorescence

The present manuscript reports the characteristics of a novel Low Energy X-ray Fluorescence (LEXRF) system installed at the TwinMic X-ray Microscopy station operated at Elettra synchrotron (Trieste, Italy). The setup has been recently upgraded to an 8 large area Silicon Drift Detectors (SDD) system able to collect the XRF fluorescence emitted by the specimen in the 180-2200 eV energy range. The LEXRF system in an X-ray microscope has allowed combining chemical specificity of the specimen with the morphological information acquired in transmission at submicron length scales. Distinct advances of the new set-up are related to software system that has introduced new algorithms and methods.

Fabrication of a Sealed Electrochemical Microcell for in Situ Soft X-ray Microspectroscopy and Testing with in Situ Co-Polypyrrole Composite Electrodeposition for Pt-Free Oxygen Electrocatalysis

In this paper we report on the fabrication and testing of a novel concept of sealed electrochemical microcell for in situ soft X-ray microspectroscopy in transmission, dedicated for nonvacuum compatible electrolytes. The microcell, fabricated using ultraviolet lithography, at variance with previous versions of electrochemical wet cells, that featured an optical window glued on top of the electrode system and a very limited electrolyte volume, the device presented here is a single solid block based around a microfabricated channel with fixed optical windows and apt for microfluidic work. Moreover, this cell allows to employ an advanced electrodic geometry developed in our group - so far used only in open electrochemical cells for work with vacuum-compatible electrolytes - also with low-vapor pressure liquids, possibly saturated with the required gases. The cell optimal electrode design allows three-electrode electrochemical control typical of traditional electrochemical experiments. The first electrochemical experiments with this new cell explore the electrochemical growth of a Co-polypyrrole, a composite electrocatalyst material with promising performance to replace the expensive Pt catalyst in fuel-cell oxygen electrodes. Morphological and chemical-state distributions of Co codeposited with polypyrrole has been followed as a function of time and position, yielding unprecedented information on the processes relevant to the synthesis of this catalyst.

Current status of the TwinMic beamline at Elettra: a soft X-ray transmission and emission microscopy station

The current status of the TwinMic beamline at Elettra synchrotron light source, that hosts the European twin X-ray microscopy station, is reported. The X-ray source, provided by a short hybrid undulator with source size and divergence intermediate between bending magnets and conventional undulators, is energy-tailored using a collimated plane-grating monochromator. The TwinMic spectromicroscopy experimental station combines scanning and full-field imaging in a single instrument, with contrast modes such as absorption, differential phase, interference and darkfield. The implementation of coherent diffractive imaging modalities and ptychography is ongoing. Typically, scanning transmission X-ray microscopy images are simultaneously collected in transmission and differential phase contrast and can be complemented by chemical and elemental analysis using across-absorption-edge imaging, X-ray absorption near-edge structure or low-energy X-ray fluorescence. The lateral resolutions depend on the particular imaging and contrast mode chosen. The TwinMic range of applications covers diverse research fields such as biology, biochemistry, medicine, pharmacology, environment, geochemistry, food, agriculture and materials science. They will be illustrated in the paper with representative results.

Soft X-Ray Microscopy Radiation Damage On Fixed Cells Investigated With Synchrotron Radiation FTIR Microscopy

Radiation damage of biological samples remains a limiting factor in high resolution X-ray microscopy (XRM). Several studies have attempted to evaluate the extent and the effects of radiation damage, proposing strategies to minimise or prevent it. The present work aims to assess the impact of soft X-rays on formalin fixed cells on a systematic manner. The novelty of this approach resides on investigating the radiation damage not only with XRM, as often reported in relevant literature on the topic, but by coupling it with two additional independent non-destructive microscopy methods: Atomic Force Microscopy (AFM) and FTIR Microscopy (FTIRM). Human Embryonic Kidney 293 cells were exposed to different radiation doses at 1 keV. In order to reveal possible morphological and biochemical changes, the irradiated cells were systematically analysed with AFM and FTIRM before and after. Results reveal that while cell morphology is not substantially affected, cellular biochemical profile changes significantly and progressively when increasing dose, resulting in a severe breakdown of the covalent bonding network. This information impacts most soft XRM studies on fixed cells and adds an in-depth understanding of the radiation damage for developing better prevention strategies.

In situ soft x-ray fluorescence and absorption microspectroscopy: A study of Mn-Co/polypyrrole electrodeposition

This paper reports the development and application of a novel thin-layer electrochemical microcell for in situ soft x-ray fluorescence and absorption microspectroscopy. The microcell, fabricated using ultraviolet lithography, is an improved version of concepts previously developed in our group, featuring a wide optical window that allows the extension of the range of accessible in situ microspectroscopy and imaging methods, including those requiring small emission take-off angles. The three-electrode design implemented in the cell enables optimal electrochemical control. The first in situ experiment employing this new cell explores the electrochemical growth of a novel Mn-Co/polypyrrole composite that is a prospective electrocatalyst for Pt replacement in air cathodes. Morphological, compositional, and chemical-state distributions of Mn and Co codeposited with polypyrrole are subsequently performed in situ as a function of time and position, yielding otherwise unachievable information regarding the electr...

Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice

Human exposure to asbestos can cause a wide variety of lung diseases that are still a current major health concern, even if asbestos has been banned in many countries. It has been shown in many studies that asbestos fibers, ingested by alveolar macrophages, disrupt lung iron homeostasis by sequestering iron. Calcium can also be deposited on the fibers. The pathways along which iron and above all calcium interact with fibers are still unknown. Our aim was that of investigating if the iron accumulation induced by the inhaled asbestos fibers also involves calcium ions accumulation. Lung sections of asbestos-exposed mice were analyzed using an extremely sensitive procedure available at the synchrotron facilities, that provides morphological and chemical information based on X-ray fluorescence microspectroscopy (μ-XRF). In this study we show that (1) where conventional histochemical procedures revealed only weak deposits of iron and calcium, μ-XRF analysis is able to detect significant deposits of both iron and calcium on the inhaled asbestos fibers; (2) the extent of the deposition of these ions is proportionally directly related and (3) iron and calcium deposition on inhaled asbestos fibers is concomitant with the appearance of inflammatory and hyperplastic reactions.

Feasibility of direct mapping of cerebral fluorodeoxy-D-glucose metabolism in situ at subcellular resolution using soft X-ray fluorescence

Glucose metabolism is difficult to image with cellular resolution in mammalian brain tissue, particularly with 18fluorodeoxy‐D‐glucose (FDG) positron emission tomography (PET). To this end, we explored the potential of synchrotron‐based low‐energy X‐ray fluorescence (LEXRF) to image the stable isotope of fluorine (F) in phosphorylated FDG (DG‐6P) at 1 μm2 spatial resolution in 3‐μm‐thick brain slices. The excitation‐dependent fluorescence F signal at 676 eV varied linearly with FDG concentration between 0.5 and 10 mM, whereas the endogenous background F signal was undetectable in brain. To validate LEXRF mapping of fluorine, FDG was administered in vitro and in vivo, and the fluorine LEXRF signal from intracellular trapped FDG‐6P over selected brain areas rich in radial glia was spectrally quantitated at 1 μm2 resolution. The subsequent generation of spatial LEXRF maps of F reproduced the expected localization and gradients of glucose metabolism in retinal Müller glia. In addition, FDG uptake was localized to periventricular hypothalamic tanycytes, whose morphological features were imaged simultaneously by X‐ray absorption. We conclude that the high specificity of photon emission from F and its spatial mapping at ≤1 μm resolution demonstrates the ability to identify glucose uptake at subcellular resolution and holds remarkable potential for imaging glucose metabolism in biological tissue.

Combining multiple imaging techniques at the TwinMic X-ray microscopy beamline

In synchrotron facilities, imaging techniques are on high demand from the scientific community. Those related to X-ray microscopy are among the most prominent ones. Such techniques include scanning transmission x-ray microscopy (STXM), full-field transmission x-ray microscopy (TXM), and coherent diffraction imaging (CDI) which have a wide spectrum of applications ranging from clinical and biomedical sciences to nanotechnology and cultural heritage. Their advancement is achieved through specialisation and focused studies, often requiring dedicated beamline end-stations. On the other hand, scientific applications benefit from the combination of techniques in a complementary manner. Beamlines suitably designed to offer multiple techniques, instead of a single one, can host efficiently such combinatorial studies. In this paper, we present the diverse Soft X-ray microscopy techniques in use at the TwinMic beamline at Elettra Sincrotrone Trieste, namely, STXM combined with XRF spectroscopy, full-field TXM and P...

Aluminum complexation with malate within the root apoplast differs between aluminum resistant and sensitive wheat lines

In wheat (Triticum aestivum), it is commonly assumed that Al is detoxified by the release of organic anions into the rhizosphere, but it is also possible that detoxification occurs within the apoplast and symplast of the root itself. Using Al-resistant (ET8) and Al-sensitive (ES8) near-isogenic lines of wheat, we utilized traditional and synchrotron-based approaches to provide in situ analyses of the distribution and speciation of Al within root tissues. Some Al appeared to be complexed external to the root, in agreement with the common assumption. However, root apical tissues of ET8 accumulated four to six times more Al than ES8 when exposed to Al concentrations that reduce root elongation rate by 50% (3.5 μM Al for ES8 and 50 μM for ET8). Furthermore, in situ analyses of ET8 root tissues indicated the likely presence of Al-malate and other forms of Al, predominantly within the apoplast. To our knowledge, this is the first time that X-ray absorption near edge structure analyses have been used to examine the speciation of Al within plant tissues. The information obtained in the present study is important in developing an understanding of the underlying physiological mode of action for improved root growth in systems with elevated soluble Al.

In Situ and Ex Situ X-Ray Microspectroelectrochemical Methods for the Study of Zinc–Air Batteries

Electrical energy storage based on Zn–air concepts is experiencing increasing interest for applications ranging from consumer electronics to automotive and grid storage, owing to their high energy density, intrinsic safety, environmental friendliness, and low cost. Their implementation is nevertheless daunted by several materials science riddles, affecting the actually available power density and durability. In this scenario, in operando dynamic physicochemical information at length scales between mesoscopic and nanometric is highly desirable for knowledge-based advancements. This overview summarizes recent contributions of in situ and quasi- in situ X-ray methods – absorption and fluorescence microspectroscopies and microtomography – to studies of cathodes, anodes, and model cells.