Quantitative, in situ visualization of intracellular insulin vesicles in pancreatic beta cells.

Characterizing relationships between Zn2+, insulin, and insulin vesicles is of vital importance to the study of pancreatic beta cells. However, the precise content of Zn2+ and the specific location of insulin inside insulin vesicles are not clear, which hinders a thorough understanding of the insulin secretion process and diseases caused by blood sugar dysregulation. Here, we demonstrated the colocalization of Zn2+ and insulin in both single extracellular insulin vesicles and pancreatic beta cells by using an X-ray scanning coherent diffraction imaging (ptychography) technique. We also analyzed the elemental Zn2+ and Ca2+ contents of insulin vesicles using electron microscopy and energy dispersive spectroscopy (EDS) mapping. We found that the presence of Zn2+ is an important characteristic that can be used to distinguish insulin vesicles from other types of vesicles in pancreatic beta cells and that the content of Zn2+ is proportional to the size of insulin vesicles. By using dual-energy contrast X-ray microscopy and scanning transmission X-ray microscopy (STXM) image stacks, we observed that insulin accumulates in the off-center position of extracellular insulin vesicles. Furthermore, the spatial distribution of insulin vesicles and their colocalization with other organelles inside pancreatic beta cells were demonstrated using three-dimensional (3D) imaging by combining X-ray ptychography and an equally sloped tomography (EST) algorithm. This study describes a powerful method to univocally describe the location and quantitative analysis of intracellular insulin, which will be of great significance to the study of diabetes and other blood sugar diseases.

Understanding the Growth of Electrodeposited PtNi Nanoparticle Films Using Correlated In Situ Liquid Cell Transmission Electron Microscopy and Synchrotron Radiation.

Electrodeposition is a versatile method for synthesizing nanostructured films, but controlling the morphology of films containing two or more elements requires a detailed understanding of the deposition process. We used liquid cell transmission electron microscopy to follow the electrodeposition of PtNi nanoparticle films on a carbon electrode during cyclic voltammetry. These in situ observations show that the film thickness increases with each cycle, and by the fourth cycle, branched and porous structures could be deposited. Synchrotron studies using in situ transmission X-ray microscopy further revealed that Ni was deposited in the oxide phase. Ex situ studies of bulk electrodeposited PtNi nanoparticle films indicated the number of cycles and the scanning rate were the most influential parameters, resulting in a different thickness, a different homogeneity, a different nanoparticle size, and a different surface structure, while the precursor concentration did not have a significant influence. By varying the potential range, we were able to obtain films with different elemental compositions.

3D imaging of magnetic domains in Nd2Fe14B using scanning hard X-ray nanotomography.

Nanoscale structural and electronic heterogeneities are prevalent in condensed matter physics. Investigating these heterogeneities in 3D has become an important task for understanding material properties. To provide a tool to unravel the connection between nanoscale heterogeneity and macroscopic emergent properties in magnetic materials, scanning transmission X-ray microscopy (STXM) is combined with X-ray magnetic circular dichroism. A vector tomography algorithm has been developed to reconstruct the full 3D magnetic vector field without any prior noise assumptions or knowledge about the sample. Two tomographic scans around the vertical axis are acquired on single-crystalline Nd2Fe14B pillars tilted at two different angles, with 2D STXM projections recorded using a focused 120 nm X-ray beam with left and right circular polarization. Image alignment and iterative registration have been implemented based on the 2D STXM projections for the two tilts. Dichroic projections obtained from difference images are used for the tomographic reconstruction to obtain the 3D magnetization distribution at the nanoscale.

Morphological, Microstructural, and In Situ Chemical Characteristics of Siderite Produced by Iron-Reducing Bacteria.

Dissimilatory iron-reducing bacteria (DIRB) oxidize organic matter or hydrogen and reduce ferric iron to form Fe(II)-bearing minerals, such as magnetite and siderite. However, compared with magnetite, which was extensively studied, the mineralization process and mechanisms of siderite remain unclear. Here, with the combination of advanced electron microscopy and synchrotron-based scanning transmission X-ray microscopy (STXM) approaches, we studied in detail the morphological, structural, and chemical features of biogenic siderite via a growth experiment with Shewanella oneidensis MR-4. Results showed that along with the growth of cells, Fe(II) ions were increasingly released into solution and reacted with CO32- to form micrometer-sized siderite minerals with spindle, rod, peanut, dumbbell, and sphere shapes. They are composed of many single-crystal siderite plates that are fanned out from the center of the particles. Additionally, STXM revealed Fh and organic molecules inside siderite. This suggests that the siderite crystals might assemble around a Fh-organic molecule core and then continue to grow radially. This study illustrates the biomineralization and assembly of siderite by a successive multistep growth process induced by DIRB, also provides evidences that the distinctive shapes and the presence of organic molecules inside might be morphological and chemical features for biogenic siderite.

Nitrogen-Rich Organic Matter Formation and Stabilization in Iron Ore Tailings: A Submicrometer Investigation.

Organic matter (OM) formation and stabilization are critical processes in the eco-engineered pedogenesis of Fe ore tailings, but the underlying mechanisms are unclear. The present 12 month microcosm study has adopted nanoscale secondary ion mass spectrometry (NanoSIMS) and synchrotron-based scanning transmission X-ray microscopy (STXM) techniques to investigate OM formation, molecular signature, and stabilization in tailings at micro- and nanometer scales. In this system, microbial processing of exogenous isotopically labeled OM demonstrated that 13C labeled glucose and 13C/15N labeled plant biomass were decomposed, regenerated, and associated with Fe-rich minerals in a heterogeneous pattern in tailings. Particularly, when tailings were amended with plant biomass, the 15N-rich microbially derived OM was generated and bound to minerals to form an internal organo-mineral association, facilitating further OM stabilization. The organo-mineral associations were primarily underpinned by interactions of carboxyl, amide, aromatic, and/or aliphatic groups with weathered mineral products derived from biotite-like minerals in fresh tailings (i.e., with Fe2+ and Fe3+) or with Fe3+ oxyhydroxides in aged tailings. The study revealed microbial OM generation and subsequent organo-mineral association in Fe ore tailings at the submicrometer scale during early stages of eco-engineered pedogenesis, providing a basis for the development of microbial based technologies toward tailings' ecological rehabilitation.

Arbuscular Mycorrhizal Fungi Drive Organo-Mineral Association in Iron Ore Tailings: Unravelling Microstructure at the Submicron Scale by Synchrotron-Based FTIR and STXM-NEXAFS.

Arbuscular mycorrhizal (AM) fungi play an important role in organic matter (OM) stabilization in Fe ore tailings for eco-engineered soil formation. However, little has been understood about the AM fungi-derived organic signature and organo-mineral interactions in situ at the submicron scale. In this study, a compartmentalized cultivation system was used to investigate the role of AM fungi in OM formation and stabilization in tailings. Particularly, microspectroscopic analyses including synchrotron-based transmission Fourier transform infrared (FTIR) and scanning transmission X-ray microspectroscopy combined with near-edge X-ray absorption fine structure spectroscopy (STXM-NEXAFS) were employed to characterize the chemical signatures at the AM fungal-mineral and mineral-OM interfaces at the submicron scale. The results indicated that AM fungal mycelia developed well in the tailings and entangled mineral particles for aggregation. AM fungal colonization enhanced N-rich OM stabilization through organo-mineral association. Bulk spectroscopic analysis together with FTIR mapping revealed that fungi-derived lipids, proteins, and carbohydrates were associated with Fe/Si minerals. Furthermore, STXM-NEXAFS analysis revealed that AM fungi-derived aromatic, aliphatic, and carboxylic/amide compounds were heterogeneously distributed and trapped by Fe(II)/Fe(III)-bearing minerals originating from biotite-like minerals weathering. These findings imply that AM fungi can stimulate mineral weathering and provide organic substances to associate with minerals, contributing to OM stabilization and aggregate formation as key processes for eco-engineered soil formation in tailings.

Quantifying signal quality in scanning transmission X-ray microscopy.

While the general effects of experimental conditions such as photon flux and sample thickness on the quality of data acquired by scanning transmission X-ray microscopy (STXM) are widely known at a basic level, the specific details are rarely discussed. This leaves the community open to forming misconceptions that can lead to poor decisions in the design and execution of STXM measurements. A formal treatment of the uncertainty and distortions of transmission signals (due to dark counts, higher-order photons and poor spatial or spectral resolution) is presented here to provide a rational basis for the pursuit of maximizing data quality in STXM experiments. While we find an optimum sample optical density of 2.2 in ideal conditions, the distortions considered tend to have a stronger effect for thicker samples and so ∼1 optical density at the analytical energy is recommended, or perhaps even thinner if significant distortion effects are expected (e.g. lots of higher-order light is present in the instrument). (Note that X-ray absorption calculations based on simple elemental composition do not include near-edge resonances and so cannot accurately represent the spectral resonances typically employed for contrast in STXM.) Further, we present a method for objectively assessing the merits of higher-order suppression in terms of its impact on the quality of transmission measurements that should be useful for the design of synchrotron beamlines.

Chemical and elemental mapping of spent nuclear fuel sections by soft X-ray spectromicroscopy.

Soft X-ray spectromicroscopy at the O K-edge, U N4,5-edges and Ce M4,5-edges has been performed on focused ion beam sections of spent nuclear fuel for the first time, yielding chemical information on the sub-micrometer scale. To analyze these data, a modification to non-negative matrix factorization (NMF) was developed, in which the data are no longer required to be non-negative, but the non-negativity of the spectral components and fit coefficients is largely preserved. The modified NMF method was utilized at the O K-edge to distinguish between two components, one present in the bulk of the sample similar to UO2 and one present at the interface of the sample which is a hyperstoichiometric UO2+x species. The species maps are consistent with a model of a thin layer of UO2+x over the entire sample, which is likely explained by oxidation after focused ion beam (FIB) sectioning. In addition to the uranium oxide bulk of the sample, Ce measurements were also performed to investigate the oxidation state of that fission product, which is the subject of considerable interest. Analysis of the Ce spectra shows that Ce is in a predominantly trivalent state, with a possible contribution from tetravalent Ce. Atom probe analysis was performed to provide confirmation of the presence and localization of Ce in the spent fuel.

Molecular Insights into Roles of Dissolved Organic Matter in Cr(III) Immobilization by Coprecipitation with Fe(III) Probed by STXM-Ptychography and XANES Spectroscopy.

The coprecipitation of heavy metals (HMs) with Fe(III) in the presence of dissolved organic matter (DOM) is a crucial process to control the mobility of HMs in the environment, but its underlying immobilization mechanisms are unclear. In this study, Cr(III) immobilization by coprecipitation with Fe(III) in the presence of straw-derived DOMs under different Fe/C molar ratios, pHs, and ionic strengths was investigated using scanning transmission X-ray microscopy (STXM) and ptychography and X-ray absorption near-edge structure (XANES) spectroscopy. The results showed that Cr(III) retention was enhanced in the presence of DOM, a maximum of which was achieved at an Fe/C molar ratio of 0.5. The increase of pH and ionic strength could also promote Cr(III) immobilization. Cr K-edge XANES results indicated that Fe (oxy)hydroxide fractions, instead of organics, provided the predominant binding sites for Cr(III), which was directly confirmed by high spatial resolution STXM-ptychography analysis at the sub-micron- and nanoscales. Moreover, organics could indirectly facilitate Cr immobilization by improving the aggregation and deposition of coprecipitate particles through DOM bridging or electrostatic interactions. Additionally, C K-edge XANES analysis further indicated that the carboxylic groups of DOM were complexed with Fe (oxy)hydroxides, which probably contributed to DOM bridging. This study provides a new insight into Cr(III) immobilization mechanisms in its coprecipitation with Fe(III) and DOM, which could have important implications on the management of Cr(III)-enriched soils, particularly with crop straw returning.

X-Ray Spectromicroscopy Investigation of Heterogeneous Sodiation in Hard Carbon Nanosheets with Vertically Oriented (002) Planes.

Hard carbon (HC) is a promising anode material for sodium-ion batteries, but the performance remains unsatisfactory and the sodiation mechanism in HC is one of the most debated topics. Here, from self-assembled cellulose nanocrystal sheets with crystallographic texture, unique HC nanosheets with vertically oriented (002) planes are fabricated and used as a model HC to investigate the sodiation mechanisms using synchrotron scanning transmission X-ray microscopy (STXM) coupled with analytical transmission electron microscopy (TEM). The model HC simplifies the 3D sodiation in a typical HC particle into a 2D sodiation, which facilitates the visualization of phase transformation at different states of charge. The results for the first time unveil that the sodiation in HC initiates heterogeneously, with multiple propagation fronts proceeding simultaneously, eventually merging into larger aggregates. The spatial correlation between the preferential adsorption and nucleation sites suggests that the heterogeneous nucleation is driven by the local Na-ion concentration, which is determined by defects or heteroatoms that have strong binding to Na ions. By identifying intercalation as the dominant sodium storage mechanism in the model HC, the findings highlight the importance of engineering the graphene layer orientation and the structural heterogeneity of edge sites to enhance the performances.

Soft X-ray nanospectroscopy for quantification of X-ray linear dichroism on powders.

X-ray linear dichroism (XLD) is a fundamental property of many ordered materials that can for instance provide information on the origin of magnetic properties and the existence of differently ordered domains. Conventionally, measurements of XLD are performed on single crystals, crystalline thin films, or highly ordered nanostructure arrays. Here, it is demonstrated how quantitative measurements of XLD can be performed on powders, relying on the random orientation of many particles instead of the controlled orientation of a single ordered structure. The technique is based on a scanning X-ray transmission microscope operated in the soft X-ray regime. The use of a Fresnel zone plate allows X-ray absorption features to be probed at ∼40 nm lateral resolution - a scale small enough to probe the individual crystallites in most powders. Quantitative XLD parameters were then retrieved by determining the intensity distributions of certain diagnostic dichroic absorption features, estimating the angle between their transition dipole moments, and fitting the distributions with four-parameter dichroic models. Analysis of several differently produced ZnO powders shows that the experimentally obtained distributions indeed follow the theoretical model for XLD. Making use of Monte Carlo simulations to estimate uncertainties in the calculated dichroic model parameters, it was established that longer X-ray exposure times lead to a decrease in the amplitude of the XLD effect of ZnO.

Why is my image noisy? A look into the terms contributing to a time-resolved X-ray microscopy image.

Through Monte Carlo simulations, we investigate how various experimental parameters can influence the quality of time-resolved scanning transmission X-ray microscopy images. In particular, the effect of the X-ray photon flux, of the thickness of the investigated samples, and of the frequency of the dynamical process under investigation on the resulting time-resolved image are investigated. The ideal sample and imaging conditions that allow for an optimal image quality are then identifed.

Combining X-ray excited optical luminescence and X-ray absorption spectroscopy for correlative imaging on the nanoscale.

X-ray absorption and optical luminescence can both provide valuable but very different information on the chemical and physical properties of materials. Although it is known that the spectral characteristics of many materials are highly heterogeneous on the micro- and/or nanoscale, no methodology has so far been shown to be capable of spatially resolving both full X-ray absorption and X-ray excited optical luminescence (XEOL) spectra on the nanoscale in a correlative manner. For this purpose, the scanning transmission X-ray microscope at the HERMES beamline of the SOLEIL synchrotron was equipped with an optical detection system capable of recording high-resolution XEOL spectra using a 40 nm soft X-ray probe. The functionality of the system was demonstrated by analyzing ZnO powder dispersions - showing simultaneously the X-ray linear dichroism and XEOL behavior of individual submicrometric ZnO crystallites.

A bidirectional scanning method for scanning transmission X-ray microscopy.

Scanning mode is a key factor for the comprehensive performance, including imaging efficiency, of scanning transmission X-ray microscopy (STXM). Herein is presented a bidirectional scanning method designed for STXM with an S-shaped moving track. In this method, artificially designed ramp waves are generated by a piezo-stage controller to control the two-dimensional scanning of the sample. The sample position information is measured using laser interferometric sensors and sent to a field-programmable gate array (FPGA) board which also acquires the X-ray signals simultaneously from the detector. Since the data recorded by the FPGA contain the real position of each scanned point, the influence of the backlash caused by the back-turning movement on the STXM image can be eliminated. By employing an adapted post-processing program, a re-meshed high-resolution STXM image can be obtained. This S-track bidirectional scanning method in fly-scan mode has been implemented on the STXM endstation at the Shanghai Synchrotron Radiation Facility (SSRF), and successfully resolved the ∼30 nm interval between the innermost strips of a Siemens star. This work removes the limitation on bidirectional scanning caused by motor backlash and vibration, and significantly improves the efficiency of STXM experiments.

Calculating absorption dose when X-ray irradiation modifies material quantity and chemistry.

X-ray absorption is a sensitive and versatile tool for chemical speciation. However, when high doses are used, the absorbed energy can change the composition, amount and structure of the native material, thereby changing the aspects of the absorption process on which speciation is based. How can one calculate the dose when X-ray irradiation affects the chemistry and changes the amount of the material? This paper presents an assumption-free approach which can retrieve from the experimental data all dose-sensitive parameters - absorption coefficients, composition (elemental molecular units), material densities - which can then be used to calculate accurate doses as a function of irradiation. This approach is illustrated using X-ray damage to a solid film of a perfluorosulfonic acid fluoropolymer in a scanning transmission soft X-ray microscope. This new approach is compared against existing dose models which calculate the dose by making simplifying assumptions regarding the material quantity, density and chemistry. While the detailed measurements used in this approach go beyond typical methods to experimental analytical X-ray absorption, they provide a more accurate quantitation of radiation dose, and help to understand mechanisms of radiation damage.

Three-dimensional fast elemental mapping by soft X-ray dual-energy focal stacks imaging.

The three-dimensional (3D) dual-energy focal stacks (FS) imaging method has been developed to quickly obtain the spatial distribution of an element of interest in a sample; it is a combination of the 3D FS imaging method and two-dimensional (2D) dual-energy contrast imaging based on scanning transmission soft X-ray microscopy (STXM). A simulation was firstly performed to verify the feasibility of the 3D elemental reconstruction method. Then, a sample of composite nanofibers, polystyrene doped with ferric acetylacetonate [Fe(acac)3], was further investigated to quickly reveal the spatial distribution of Fe(acac)3 in the sample. Furthermore, the data acquisition time was less than that for STXM nanotomography under similar resolution conditions and did not require any complicated sample preparation. The novel approach of 3D dual-energy FS imaging, which allows fast 3D elemental mapping, is expected to provide invaluable information for biomedicine and materials science.

Identification of Ca-rich dense granules in human platelets using scanning transmission X-ray microscopy.

Whole-mount (WM) platelet preparation followed by transmission electron microscopy (TEM) observation is the standard method currently used to assess dense granule (DG) deficiency (DGD). However, due to the electron-density-based contrast mechanism in TEM, other granules such as α-granules might cause false DG detection. Here, scanning transmission X-ray microscopy (STXM) was used to identify DGs and minimize false DG detection of human platelets. STXM image stacks of human platelets were collected at the calcium (Ca) L2,3 absorption edge and then converted to optical density maps. Ca distribution maps, obtained by subtracting the optical density maps at the pre-edge region from those at the post-edge region, were used to identify DGs based on the Ca richness. DGs were successfully detected using this STXM method without false detection, based on Ca maps for four human platelets. Spectral analysis of granules in human platelets confirmed that DGs contain a richer Ca content than other granules. The Ca distribution maps facilitated more effective DG identification than TEM which might falsely detect DGs. Correct identification of DGs would be important to assess the status of platelets and DG-related diseases. Therefore, this STXM method is proposed as a promising approach for better DG identification and diagnosis, as a complementary tool to the current WM TEM approach.

Time-of-arrival detection for time-resolved scanning transmission X-ray microscopy imaging.

A setup for time-resolved scanning transmission X-ray microscopy imaging is presented, which allows for an increase in the temporal resolution without the requirement of operating the synchrotron light source with low-α optics through the measurement of the time-of-arrival of the X-ray photons. Measurements of two filling patterns in hybrid mode of the Swiss Light Source are presented as a first proof-of-principle and benchmark for the performances of this new setup. From these measurements, a temporal resolution on the order of 20-30 ps could be determined.

Uranium(IV) adsorption by natural organic matter in anoxic sediments.

Uranium is an important carbon-free fuel source and environmental contaminant that accumulates in the tetravalent state, U(IV), in anoxic sediments, such as ore deposits, marine basins, and contaminated aquifers. However, little is known about the speciation of U(IV) in low-temperature geochemical environments, inhibiting the development of a conceptual model of U behavior. Until recently, U(IV) was assumed to exist predominantly as the sparingly soluble mineral uraninite (UO2+x) in anoxic sediments; however, studies now show that this is not often the case. Yet a model of U(IV) speciation in the absence of mineral formation under field-relevant conditions has not yet been developed. Uranium(IV) speciation controls its reactivity, particularly its susceptibility to oxidative mobilization, impacting its distribution and toxicity. Here we show adsorption to organic carbon and organic carbon-coated clays dominate U(IV) speciation in an organic-rich natural substrate under field-relevant conditions. Whereas previous research assumed that U(IV) speciation is dictated by the mode of reduction (i.e., whether reduction is mediated by microbes or by inorganic reductants), our results demonstrate that mineral formation can be diminished in favor of adsorption, regardless of reduction pathway. Projections of U transport and bioavailability, and thus its threat to human and ecosystem health, must consider U(IV) adsorption to organic matter within the sediment environment.

Lignans in Knotwood of Norway Spruce: Localisation with Soft X-ray Microscopy and Scanning Transmission Electron Microscopy with Energy Dispersive X-ray Spectroscopy.

Lignans are bioactive compounds that are especially abundant in the Norway spruce (Picea abies L. Karst.) knotwood. By combining a variety of chromatographic, spectroscopic and imaging techniques, we were able to quantify, qualify and localise the easily extractable lignans in the xylem tissue. The knotwood samples contained 15 different lignans according to the gas chromatography-mass spectrometry analysis. They comprised 16% of the knotwood dry weight and 82% of the acetone extract. The main lignans were found to be hydroxymatairesinols HMR1 and HMR2. Cryosectioned and resin-embedded ultrathin sections of the knotwood were analysed with scanning transmission X-ray microscopy (STXM). Cryosectioning was found to retain only lignan residues inside the cell lumina. In the resin-embedded samples, lignan was interpreted to be unevenly distributed inside the cell lumina, and partially confined in deposits which were either readily present in the lumina or formed when OsO4 used in staining reacted with the lignans. Furthermore, the multi-technique characterisation enabled us to obtain information on the chemical composition of the structural components of knotwood. A simple spectral analysis of the STXM data gave consistent results with the gas chromatographic methods about the relative amounts of cell wall components (lignin and polysaccharides). The STXM analysis also indicated that a torus of a bordered pit contained aromatic compounds, possibly lignin.