Scanning Transmission X-ray Microscopy Images Research Articles

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.

In situ characterization of gas-filled microballoons using soft X-ray microspectroscopy.

In this study we describe the first direct, real-space characterization of a novel type of poly(vinyl alcohol) (PVA) based microballoons in aqueous environment using scanning transmission X-ray microscopy (STXM). From the oxygen K-edge near-edge X-ray absorption fine structure (NEXAFS) spectra taken from the microballoon interiors we could unambiguously distinguish between water- and air-filled particles. We also demonstrate that STXM imaging below and above the O K-edge (520 eV and 550 eV) can provide unique information on the composition of microballoons in water. It was found that the particular microballoon system investigated here has a remarkable high stability and is able to contain gases for more than 6 months, making it well suited for biomedical applications.

Chemical mapping of polymer photoresists by scanning transmission x-ray microscopy

Scanning transmission x-ray microscopy (STXM) is shown to be a powerful imaging technique that provides chemical selectivity and high spatial resolution (∼35nm) for studying chemically amplified photoresists. Samples of poly(4-t-butoxycarbonyloxystyrene) PTBOCST resist, imprinted by deep ultraviolet lithography with a line∕space pattern of 1.10μm∕0.87μm followed by a post-exposure bake, are used to demonstrate STXM imaging capabilities to extract photoresist latent images. Chemical contrast is obtained by measuring the x-ray absorption at an energy of 290.5 eV, corresponding to a carbon K shell electronic transition to the unoccupied π* molecular orbital of the PTBOCST carbonyl group. A quantitative analysis provides the spatial distribution of the fraction of the unexposed and deprotected polymers remaining after the post-exposure bake stage as well as the thickness of both regions. Both chemical and topographical contributions to the total contrast are estimated. Advantages and limitations of STXM in comparison with other imaging techniques with chemical specificity are discussed.

Spatially resolved characterization of biogenic manganese oxide production within a bacterial biofilm.

Pseudomonas putida strain MnB1, a biofilm-forming bacterial culture, was used as a model for the study of bacterial Mn oxidation in freshwater and soil environments. The oxidation of aqueous Mn+2 [Mn+2(aq)] by P. putida was characterized by spatially and temporally resolving the oxidation state of Mn in the presence of a bacterial biofilm, using scanning transmission X-ray microscopy (STXM) combined with near-edge X-ray absorption fine structure (NEXAFS) spectroscopy at the Mn L2,3 absorption edges. Subsamples were collected from growth flasks containing 0.1 and 1 mM total Mn at 16, 24, 36, and 48 h after inoculation. Immediately after collection, the unprocessed hydrated subsamples were imaged at a 40-nm resolution. Manganese NEXAFS spectra were extracted from X-ray energy sequences of STXM images (stacks) and fit with linear combinations of well-characterized reference spectra to obtain quantitative relative abundances of Mn(II), Mn(III), and Mn(IV). Careful consideration was given to uncertainty in the normalization of the reference spectra, choice of reference compounds, and chemical changes due to radiation damage. The STXM results confirm that Mn+2(aq) was removed from solution by P. putida and was concentrated as Mn(III) and Mn(IV) immediately adjacent to the bacterial cells. The Mn precipitates were completely enveloped by bacterial biofilm material. The distribution of Mn oxidation states was spatially heterogeneous within and between the clusters of bacterial cells. Scanning transmission X-ray microscopy is a promising tool for advancing the study of hydrated interfaces between minerals and bacteria, particularly in cases where the structure of bacterial biofilms needs to be maintained.

In Situ Characterization of Aluminum-Containing Mineral−Microorganism Aqueous Suspensions Using Scanning Transmission X-ray Microscopy

In situ characterization of colloidal particles under hydrous conditions is one of the key requirements for understanding their state of aggregation and impact on the transport of pollutants in aqueous environments. Scanning transmission X-ray microscopy (STXM) is one of the few techniques that can satisfy this need by providing element- and chemical-state-specific 2-D maps at a spatial resolution better than 50 nm using soft X-rays from synchrotron radiation wiggler or undulator sources tuned to the absorption edges of different elements. X-ray absorption near-edge structure (XANES) spectra can also be collected simultaneously at a similar spatial resolution and can provide phase identification in many cases. In this study, we report STXM images and XANES spectroscopy measurements at or above the Al K-edge (E = 1559.6 eV) of various Al-containing minerals and synthetic oxides [alpha-Al2O3 (corundum), gamma-Al2O3, gamma-AlOOH (boehmite), alpha-Al(OH)3 (bayerite), KAl2(AlSi3O10)(OH)2 (muscovite), (Al,Mg)8(Si4O10)4(OH)8.nH2O (montmorillonite), and Mg6Al2(OH)16CO3.4H2O (hydrotalcite)] and demonstrate the capability of this spectromicroscopic tool to identify different Al-containing mineral colloids in multiphase mixtures in aqueous solution. We also demonstrate that STXM imaging at or above the C K-edge (E = 284.2 eV) and Al K-edge can provide unique information on the interactions between bacteria and Al-containing nanoparticles in aqueous suspensions. STXM images of a mixture of Caulobacter crescentus and montmorillonite and corundum particles just above the C and Al K-edges show that the mineral particles and bacteria are closely associated in aggregates, which is likely due to the binding of bacteria to clay and corundum particles by extracellular polysaccharides.

Soft X-ray spectro-microscopy investigation of the formation and ageing of Eu(III)-induced humic acid aggregates

Aggregation of Aldrich humic acid at pH 4.3 and after loading with Eu(III)-cations at pH 5.3 is investigated by scanning transmission X-ray microscopy (STXM). C ls-NEXAFS spectra extracted from STXM image stacks for the low pH aggregates show no significant variation, whereas Eu(III)-humate formation is observed to be accompanied by segregation into zones with different optical densities. These zones exhibit distinctly different NEXAFS features, indicative of the presence of a separation of organic species or components. Increasing humate aggregate densification over a period of several months is observed. The spectrum of the unloaded HA cannot be reproduced by a superposition of spectra extracted from the segregated humate zones; there is a distinct metal bonding effect on the molecular states of the functional groups involved in complexation.

Optimization of scanning transmission X-ray microscopy for the identification and quantitation of reinforcing particles in polyurethanes

The morphology, size distributions, spatial distributions, and quantitative chemical compositions of co-polymer polyol-reinforcing particles in a polyurethane have been investigated with scanning transmission X-ray microscopy (STXM). A detailed discussion of microscope operating procedures is presented and ways to avoid potential artifacts are discussed. Images at selected photon energies in the C 1s, N 1s and O 1s regions allow unambiguous identification of styrene–acrylonitrile-based (SAN) copolymer and polyisocyanate polyaddition product-based (PIPA) reinforcing particles down to particle sizes at the limit of the spatial resolution (50 nm). Quantitative analysis of the chemical composition of individual reinforcing particles is achieved by fitting C 1s spectra to linear combinations of reference spectra. Regression analyses of sequences of images recorded through the chemically sensitive ranges of the C 1s, N 1s and O 1s spectra are used to generate quantitative compositional maps, which provide a fast and effective means of investigating compositional distributions over a large number of reinforcing particles. The size distribution of all particles determined by STXM is shown to be similar to that determined by TEM. The size distributions of each type of reinforcing particle, which differ considerably, were obtained by analysis of STXM images at chemically selective energies.

Effect of an Interactive Surface on the Equilibrium Contact Angles in Bilayer Polymer Films

Scanning transmission X-ray microscopy (STXM), atomic force microscopy (AFM), near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, and photoemission electron microscopy (PEEM) were used to obtain the three-dimensional concentration profiles and the late-stage morphology of liquid bilayer thin films of the two imiscible polymers polystyrene (PS) and (polybromostyrene) (PBrx=0.79S), where x = fraction of monomers brominated) as a function of the lower PS film thickness. The results showed that the apparent contact angle at the polymer/air interface decreases exponentially with increasing PS film thickness with a constant that scales with the PS radius of gyration (Rg). In contrast, the Neuman angle, as determined from the PBrS STXM images, increases with substrate thickness. NEXAFS and PEEM data show that the droplets consist of a PBrS core fully encapsulated by PS for substrate thicknesses greater than Rg. Only partial encapsulation is seen for substrates less than Rg. These results could not be explained with existing models developed for bulk viscoelastic fluids. We found that, for the case of entangled polymers, other factors such as the interfacial energy with the substrate, the film thickness, and the molecular weights must be considered. A model which includes a restoring force characterizing the change in stiffness of the substrate layer due to surface interactions provided good agreement with the experimental observations.