Scanning Transmission X-ray Microscopy Data Research Articles

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.

Wet–dry cycles impact DOM retention in subsurface soils

Abstract. Transport and reactivity of carbon in the critical zone are highly controlled by reactions of dissolved organic matter (DOM) with subsurface soils, including adsorption, transformation and exchange. These reactions are dependent on frequent wet–dry cycles common to the unsaturated zone, particularly in semi-arid regions. To test for an effect of wet–dry cycles on DOM interaction and stabilization in subsoils, samples were collected from subsurface (Bw) horizons of an Entisol and an Alfisol from the Catalina-Jemez Critical Zone Observatory and sequentially reacted (four batch steps) with DOM extracted from the corresponding soil litter layers. Between each reaction step, soils either were allowed to air dry (wet–dry treatment) before introduction of the following DOM solution or were maintained under constant wetness (continually wet treatment). Microbial degradation was the dominant mechanism of DOM loss from solution for the Entisol subsoil, which had higher initial organic C content, whereas sorptive retention predominated in the lower C Alfisol subsoil. For a given soil, bulk dissolved organic C losses from solution were similar across treatments. However, a combination of Fourier transform infrared (FTIR) and near-edge X-ray absorption fine structure (NEXAFS) spectroscopic analyses revealed that wet–dry treatments enhanced the interactions between carboxyl functional groups and soil particle surfaces. Scanning transmission X-ray microscopy (STXM) data suggested that cation bridging by Ca2+ was the primary mechanism for carboxyl association with soil surfaces. STXM data also showed that spatial fractionation of adsorbed OM on soil organo-mineral surfaces was diminished relative to what might be inferred from previously published observations pertaining to DOM fractionation on reaction with specimen mineral phases. This study provides direct evidence of the role of wet–dry cycles in affecting sorption reactions of DOM to a complex soil matrix. In the soil environment, where wet–dry cycles occur at different frequencies from site to site and along the soil profile, different interactions between DOM and soil surfaces are expected and need to be considered for the overall assessment of carbon dynamics.

The effect of mesomorphology upon the performance of nanoparticulate organic photovoltaic devices

Scanning transmission X-ray microscopy (STXM) compositional mapping has been used to probe the mesomorphology of nanoparticles (NPs) synthesized from two very different polymer:fullerene blends: poly(3-hexylthiophene) (P3HT): phenyl-C61-butyric acid methyl ester (PCBM) and poly[4,8-bis(2-ethylhexyloxy)benzo(1,2-b:4,5-b′)dithiophene-alt-5, 6-bis(octyloxy)-4,7-di(thiophen-2-yl)(2,1,3-benzothiadiazole)-5,5′-diyl] (PSBTBT): PCBM. The STXM data shows that both blends form core–shell NP structures with similar shell compositions, but with different polymer:fullerene ratios in the core regions. P3HT:PCBM and PSBTBT:PCBM NP organic photovoltaic (OPV) devices have been fabricated and exhibit similar device efficiencies, despite the PSBTBT being a much higher performing low band gap material. By comparing the measured NP shell and core compositions with the optimized bulk hetero-junction (BHJ) compositions, we show that the relatively higher performance of the P3HT:PCBM NP device arises from the fact that its shell composition is much closer to the optimal BHJ value than that of the PSBTBT:PCBM NP device.

Ordering in bio-inorganic hybrid nanomaterials probed by in situ scanning transmission X-ray microscopy.

Phospholipid bilayer coated Si nanowires are one-dimensional (1D) composites that provide versatile bio-nanoelectronic functionality via incorporation of a wide variety of biomolecules into the phospholipid matrix. The physiochemical behaviour of the phospholipid bilayer is strongly dependent on its structure and, as a consequence, substantial modelling and experimental efforts have been directed at the structural characterization of supported bilayers and unsupported phospholipid vesicles; nonetheless, the experimental studies conducted to date have exclusively involved volume-averaged techniques, which do not allow for the assignment of spatially resolved structural variations that could critically impact the performance of the 1D phospholipid-Si NW composites. In this manuscript, we use scanning transmission X-ray microscopy (STXM) to probe bond orientation and bilayer thickness as a function of position with a spatial resolution of ∼30 nm for Δ9-cis 1,2-dioleoyl-sn-glycero-3-phosphocholine layers prepared Si NWs. When coupled with small angle X-ray scattering measurements, the STXM data reveal structural motifs of the Si NWs that give rise to multi-bilayer formation and enable assignment of the orientation of specific bonds known to affect the order and rigidity of phospholipid bilayers.

Inside and Outside: X-ray Absorption Spectroscopy Mapping of Chemical Domains in Graphene Oxide

The oxidative chemistry of graphite has been investigated for over 150 years and has attracted renewed interest given the importance of exfoliated graphene oxide as a precursor to chemically derived graphene. However, the bond connectivities, steric orientations, and spatial distribution of functional groups remain to be unequivocally determined for this highly inhomogeneous nonstoichiometric material. Here, we demonstrate the application of principal component analysis to scanning transmission X-ray microscopy data for the construction of detailed real space chemical maps of graphene oxide. These chemical maps indicate very distinct functionalization motifs at the edges and interiors and, in conjunction with angle-resolved near-edge X-ray absorption fine structure spectroscopy, enable determination of the spatial location and orientations of functional groups. Chemical imaging of graphene oxide provides experimental validation of the modified Lerf-Klinowski structural model. Specifically, we note increased contributions from carboxylic acid moieties at edge sites with epoxide and hydroxyl species dominant within the interior domains.

Nano-domain behaviour in P3HT:PCBM nanoparticles, relating material properties to morphological changes

In this article we report a morphological study of the poly(3-hexylthiophene) (P3HT): phenyl-C61-butyric acid methyl ester (PCBM) material system in nanoparticle organic thin films. The morphology and chemical composition of unannealed and annealed P3HT:PCBM nanoparticles has been investigated using scanning transmission X-ray microscopy (STXM) for a range of P3HT molecular weights (Mw=5–72kgmol−1). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) have been used to support the STXM data. We find that unannealed P3HT:PCBM nanoparticles (NPs) exhibit a common core–shell morphology, with a PCBM-rich core and P3HT-rich shell. The morphology of the thermally annealed NP films is highly dependent upon the molecular weight of the P3HT and is determined by PCBM diffusion through the P3HT matrix. Two PCBM diffusion mechanisms operate within this system: (1) at high molecular weights diffusion of molecular PCBM dominates whilst, (2) at low molecular weights diffusion of the PCBM cores is significant. The Stokes–Einstein continuum model for diffusion has been used to determine a threshold molecular weight at which the diffusion of PCBM cores is activated in these films. The calculated value (Mw~38–25kgmol−1) is shown to agree very well with experimental observations. Finally, a model for the morphological evolution of annealed P3HT:PCBM NP films is developed.

Experimental and Theoretical Comparison of the O K-Edge Nonresonant Inelastic X-ray Scattering and X-ray Absorption Spectra of NaReO4

Accurate X-ray absorption spectra (XAS) of first row atoms, e.g., O, are notoriously difficult to obtain due to the extreme sensitivity of the measurement to surface contamination, self-absorption, and saturation affects. Herein, we describe a comprehensive approach for determining reliable O K-edge XAS data for ReO(4)(1-) and provide methodology for obtaining trustworthy and quantitative data on nonconducting molecular systems, even in the presence of surface contamination. This involves comparing spectra measured by nonresonant inelastic X-ray scattering (NRIXS), a bulk-sensitive technique that is not prone to X-ray self-absorption and provides exact peak intensities, with XAS spectra obtained by three different detection modes, namely total electron yield (TEY), fluorescence yield (FY), and scanning transmission X-ray microscopy (STXM). For ReO(4)(1-), TEY measurements were heavily influenced by surface contamination, while the FY and STXM data agree well with the bulk NRIXS analysis. These spectra all showed two intense pre-edge features indicative of the covalent interaction between the Re 5d and O 2p orbitals. Density functional theory calculations were used to assign these two peaks as O 1s excitations to the e and t(2) molecular orbitals that result from Re 5d and O 2p covalent mixing in T(d) symmetry. Electronic structure calculations were used to determine the amount of O 2p character (%) in these molecular orbitals. Time dependent-density functional theory (TD-DFT) was also used to calculate the energies and intensities of the pre-edge transitions. Overall, under these experimental conditions, this analysis suggests that NRIXS, STXM, and FY operate cooperatively, providing a sound basis for validation of bulk-like excitation spectra and, in combination with electronic structure calculations, suggest that NaReO(4) may serve as a well-defined O K-edge energy and intensity standard for future O K-edge XAS studies.

Ultrastructural and chemical study of modern and fossil sporoderms by Scanning Transmission X-ray Microscopy (STXM)

Diverse microscopy techniques have been extensively used in the past to study the ultrastructure of modern and fossil sporoderms for various purposes such as taxonomic identification or study of sporoderm formation and its evolution in different groups of plants. Optical microscopy and Scanning and Transmission Electron Microscopies (SEM and TEM) have been particularly useful in providing structural information at the μm- and nm-scales. In parallel, chemical analyses have been conducted on bulk samples to document variations in sporopollenin composition between major groups and compositional evolution/preservation during coalification processes . Here, we describe a microscopy tool, Scanning Transmission X-ray Microscopy (STXM), which offers valuable capabilities for palynological studies as demonstrated by applications of this technique to fossil spores over the past decade. This microscopy technique, which uses soft X-rays produced by synchrotron radiation sources, provides 25-nm spatial resolution element-based image contrast, and K or L near-edge X-ray absorption fine structure (NEXAFS) spectra at each 25-nm x 25-nm pixel in a STXM image for a number of key elements (e.g., K-edges of C, N, O, F, Na, Mg, Al, and Si and L-edges of K, Ca, Ti-Zn, Ga, Ge, As, Se, Rb, and Sr). STXM can be performed in situ on thin samples with no need for prior chemical extraction or staining. Comparison with conventional imaging techniques such as optical microscopy and TEM shows that STXM is an ideal complement to these microscopies. We present new STXM data obtained on modern Lilium longiflorum Thunb. pollen grains and on well-preserved lycopsid spores, identified as Annalepis zeilleri (Fliche) Grauvogel-Stamm and Duringer, collected from the Lettenkohle Formation of the Wasselonne Quarry (NE France). Using this technique, different biochemical compounds such as sporopollenin or cellulose can be specifically imaged at the 25-nm scale. Additionally, NEXAFS spectra can provide information on elemental speciation, i.e. the types of C-containing functional groups (e.g., aromatic, aliphatic, carboxylic, alcoholic…) and the redox state of iron and manganese. Based on these analyses, it is possible to document the chemical preservation of fossil spores that experienced diagenesis and possibly metamorphism.

Calibration of High-Resolution X-Ray Tomography With Atomic Force Microscopy.

For two-dimensional x-ray imaging of thin films, the technique of scanning transmission x-ray microscopy (STXM) has achieved images with feature sizes as small as 40 nm in recent years. However, calibration of three-dimensional tomographic images that are produced with STXM data at this scale has not yet been described in the scientific literature, and the calibration procedure has novel problems that have not been encountered by x-ray tomography carried out at a larger scale. In x-ray microtomography, for example, one always has the option of using optical imaging on a section of the object to verify the x-ray projection measurements; with STXM, on the other hand, the sample features are too small to be resolved by light at optical wavelengths. This fact implies that one must rely on procedures with higher resolution, such as atomic force microscopy (AFM), for the calibration. Such procedures, however, generally depend on a highly destructive sectioning of the sample, and are difficult to interpret because they give surface information rather than depth information. In this article, a procedure for calibration is described that overcomes these limitations and achieves a calibration of an STXM tomography image with an AFM image and a scanning electron microscopy image of the same object.A Ge star-shaped pattern was imaged at a synchrotron with a scanning transmission x-ray microscope. Nineteen high-resolution projection images of 200 × 200 pixels were tomographically reconstructed into a three-dimensional image. Features in two-dimensional images as small as 40 nm and features as small as 80 nm in the three-dimensional reconstruction were resolved. Transverse length scales based on atomic force microscopy, scanning electron microscopy, x-ray transmission and tomographic reconstruction agreed to within 10 nm. Toward the center of the sample, the pattern thickness calculated from projection images was (51 ± 15) nm vs (80 ± 52) nm for tomographic reconstruction, where the uncertainties are evaluated at the level of two standard deviations.