Towards scanning nanostructure X-ray microscopy.

This article demonstrates spatial mapping of the local and nanoscale structure of thin film objects using spatially resolved pair distribution function (PDF) analysis of synchrotron X-ray diffraction data. This is exemplified in a lab-on-chip combinatorial array of sample spots containing catalytically interesting nanoparticles deposited from liquid precursors using an ink-jet liquid-handling system. A software implementation is presented of the whole protocol, including an approach for automated data acquisition and analysis using the atomic PDF method. The protocol software can handle semi-automated data reduction, normalization and modeling, with user-defined recipes generating a comprehensive collection of metadata and analysis results. By slicing the collection using included functions, it is possible to build images of different contrast features chosen by the user, giving insights into different aspects of the local structure.

3D synchrotron imaging of muscle tissues at different atrophic stages in stroke and spinal cord injury: a proof-of-concept study.

Synchrotron X-ray computed tomography (SXCT) allows 3D imaging of tissue with a very large field of view and an excellent micron resolution and enables the investigation of muscle fiber atrophy in 3D. The study aimed to explore the 3D micro-architecture of healthy skeletal muscle fibers and muscle fibers at different stages of atrophy (stroke sample = muscle atrophy; spinal cord injury (SCI) sample = severe muscle atrophy). Three muscle samples: a healthy control sample; a stroke sample (atrophic sample), and an SCI sample (severe atrophic sample) were imaged using SXCT, and muscle fiber populations were segmented and quantified for microarchitecture and morphology differences. The volume fraction of muscle fibers was 74.7%, 70.2%, and 35.3% in the healthy, stroke (atrophic), and SCI (severe atrophic) muscle fiber population samples respectively. In the SCI (severe atrophic sample), 3D image analysis revealed fiber splitting and fiber swelling. In the stroke sample (atrophic sample) muscle fiber buckling was observed but was only visible in the 3D analysis. 3D muscle fiber population analysis revealed new insights into the different stages of muscle fiber atrophy not to be observed nor quantified with a 2D histological analysis including fiber buckling, loss of fibers and fiber splitting.

Single-shot, omni-directional x-ray scattering imaging with a laboratory source and single-photon localization.

Omni-directional, ultra-small-angle x-ray scattering imaging provides a method to measure the orientation of micro-structures without having to resolve them. In this letter, we use single-photon localization with the Timepix3 chip to demonstrate, to the best of our knowledge, the first laboratory-based implementation of single-shot, omni-directional x-ray scattering imaging using the beam-tracking technique. The setup allows a fast and accurate retrieval of the scattering signal using a simple absorption mask. We suggest that our new approach may enable faster laboratory-based tensor tomography and could be used for energy-resolved x-ray scattering imaging.

Revealing misfit dislocations in InAs x P1-x -InP core-shell nanowires by x-ray diffraction.

InAs x P1-x nanowires are promising building blocks for future optoelectronic devices and nanoelectronics. Their structure may vary from nanowire to nanowire, which may influence their average optoelectronic properties. Therefore, it is highly important for their applications to know the average properties of an ensemble of the nanowires. Structural properties of the InAs x P1-x -InP core-shell nanowires were investigated using the coplanar x-ray diffraction performed at a synchrotron facility. Studies of series of symmetric and asymmetric x-ray Bragg reflections allowed us to determine the 26% ± 3% of As chemical composition in the InAs x P1-x core, core-shell relaxation, and the average tilt of the nanowires with respect to the substrate normal. Based on the x-ray diffraction, scanning, and transmission electron microscopy measurements, a model of the core-shell relaxation was proposed. Partial relaxation of the core was attributed to misfit dislocations formed at the core-shell interface and their linear density was estimated to be 3.3 ± 0.3 × 104 cm-1.

Muscle fibre morphology and microarchitecture in cerebral palsy patients obtained by 3D synchrotron X-ray computed tomography.

BACKGROUND: Synchrotron X-ray computed tomography (SXCT) allows for three-dimensional imaging of objects at a very high resolution and in large field-of-view. PURPOSE: The aim of this study was to use SXCT imaging for morphological analysis of muscle tissue, in order to investigate whether the analysis reveals complementary information to two-dimensional microscopy. METHODS: Three-dimensional SXCT images of muscle biopsies were taken from participants with cerebral palsy and from healthy controls. We designed morphological measures from the two-dimensional slices and three-dimensional volumes of the images and measured the muscle fibre organization, which we term orientation consistency. RESULTS: The muscle fibre cross-sectional areas were significantly larger in healthy participants than in participants with cerebral palsy when carrying out the analysis in three dimensions. However, a similar analysis carried out in two dimensions revealed no patient group difference. The present study also showed that three-dimensional orientation consistency was significantly larger for healthy participants than for participants with cerebral palsy. CONCLUSION: Individuals with CP have smaller muscle fibres than healthy control individuals. We argue that morphometric measures of muscle fibres in two dimensions are generally trustworthy only if the fibres extend perpendicularly to the slice plane, and otherwise three-dimensional aspects should be considered. In addition, the muscle tissue of individuals with CP showed a decreased level of orientation consistency when compared to healthy control tissue. We suggest that the observed disorganization of the tissue may be induced by atrophy caused by physical inactivity and insufficient neural activation.

Tuning and Tracking of Coherent Shear Waves in Molecular Films.

We have determined the time-dependent displacement fields in molecular sub-micrometer thin films as response to femtosecond and picosecond laser pulse heating by time-resolved X-ray diffraction. This method allows a direct absolute determination of the molecular displacements induced by electron-phonon interactions, which are crucial for, for example, charge transport in organic electronic devices. We demonstrate that two different modes of coherent shear motion can be photoexcited in a thin film of organic molecules by careful tuning of the laser penetration depth relative to the thickness of the film. The measured response of the organic film to impulse heating is explained by a thermoelastic model and reveals the spatially resolved displacement in the film. Thereby, information about the profile of the energy deposition in the film as well as about the mechanical interaction with the substrate material is obtained.

X-ray Bragg Ptychography on a Single InGaN/GaN Core-Shell Nanowire.

The future of solid-state lighting can be potentially driven by applications of InGaN/GaN core-shell nanowires. These heterostructures provide the possibility for fine-tuning of functional properties by controlling a strain state between mismatched layers. We present a nondestructive study of a single 400 nm-thick InGaN/GaN core-shell nanowire using two-dimensional (2D) X-ray Bragg ptychography (XBP) with a nanofocused X-ray beam. The XBP reconstruction enabled the determination of a detailed three-dimensional (3D) distribution of the strain in the particular nanowire using a model based on finite element method. We observed the strain induced by the lattice mismatch between the GaN core and InGaN shell to be in the range from -0.1% to 0.15% for an In concentration of 30%. The maximum value of the strain component normal to the facets was concentrated at the transition region between the main part of the nanowire and the GaN tip. In addition, a variation in misfit strain relaxation between the axial growth and in-plane directions was revealed.

Spatial distribution of soluble insulin in pig subcutaneous tissue: Effect of needle length, injection speed and injected volume.

The spatial distribution of a soluble insulin formulation was visualized and quantified in 3-dimensions using X-ray computed tomography. The drug distribution was visualized for ex vivo injections in pig subcutaneous tissue. Pig subcutaneous tissue has very distinct layers, which could be separated in the tomographic reconstructions and the amount of drug in each tissue class was quantified. With a scan time of about 45min per sample, and a robust segmentation it was possible to analyze differences in the spatial drug distribution between several similar injections. It was studied how the drug distribution was effected by needle length, injection speed and injected volume. For an injected volume of 0.1ml and injection depth of 8mm about 50% of the injections were partly intramuscular. Using a 5mm needle resulted in purely subcutaneous injections with minor differences in the spatial drug distribution between injections. Increasing the injected volume from 0.1ml to 1ml did not increase the intramuscular volume fraction, but gave a significantly higher volume fraction placed in the fascia separating the deep and superficial subcutaneous fat layers. Varying the injection speed from 25l/s up to 300l/s gave no changes in the drug concentration distribution. The method presented gives novel insight into subcutaneous injections of soluble insulin drugs and can be used to optimize the injection technique for subcutaneous drug administration in preclinical studies of rodents.

Fast Strain Mapping of Nanowire Light-Emitting Diodes Using Nanofocused X-ray Beams.

X-ray nanobeams are unique nondestructive probes that allow direct measurements of the nanoscale strain distribution and composition inside the micrometer thick layered structures that are found in most electronic device architectures. However, the method is usually extremely time-consuming, and as a result, data sets are often constrained to a few or even single objects. Here we demonstrate that by special design of a nanofocused X-ray beam diffraction experiment we can (in a single 2D scan with no sample rotation) measure the individual strain and composition profiles of many structures in an array of upright standing nanowires. We make use of the observation that in the generic nanowire device configuration, which is found in high-speed transistors, solar cells, and light-emitting diodes, each wire exhibits very small degrees of random tilts and twists toward the substrate. Although the tilt and twist are very small, they give a new contrast mechanism between different wires. In the present case, we image complex nanowires for nanoLED fabrication and compare to theoretical simulations, demonstrating that this fast method is suitable for real nanostructured devices.

Application of high resolution synchrotron micro-CT radiation in dental implant osseointegration.

The purpose of this study was to describe a refined method using high-resolution synchrotron radiation microtomography (SRmicro-CT) to evaluate osseointegration and peri-implant bone volume fraction after titanium dental implant insertion. SRmicro-CT is considered gold standard evaluating bone microarchitecture. Its high resolution, high contrast, and excellent high signal-to-noise-ratio all contribute to the highest spatial resolutions achievable today. Using SRmicro-CT at a voxel size of 5 µm in an experimental goat mandible model, the peri-implant bone volume fraction was found to quickly increase to 50% as the radial distance from the implant surface increased, and levelled out to approximately 80% at a distance of 400 µm. This method has been successful in depicting the bone and cavities in three dimensions thereby enabling us to give a more precise answer to the fraction of the bone-to-implant contact compared to previous methods.

Structural Properties of wurtzite InP-InGaAs nanowire core-shell heterostructures.

We report on growth and characterization of wurtzite InP-In(1-x)Ga(x)As core-shell nanowire heterostructures. A range of nanowire structures with different Ga concentration in the shell was characterized with transmission electron microscopy and X-ray diffraction. We found that the main part of the nanowires has a pure wurtzite crystal structure, with occasional stacking faults occurring only at the top and bottom. This allowed us to determine the structural properties of wurtzite In(1-x)Ga(x)As. The InP-In(1-x)Ga(x)As core-shell nanowires show a triangular and hexagonal facet structure of {1100} and {101̅0} planes. X-ray diffraction measurements showed that the core and the shell are pseudomorphic along the c-axis, and the strained axial lattice constant is closer to the relaxed In(1-x)Ga(x)As shell. Microphotoluminescence measurements of the nanowires show emission in the infrared regime, which makes them suitable for applications in optical communication.

Novel X-ray phase-contrast tomography method for quantitative studies of heat induced structural changes in meat.

The objective of this study was to evaluate the use of X-ray phase-contrast tomography combined with 3D image segmentation to investigate the heat induced structural changes in meat. The measurements were performed at the Swiss synchrotron radiation light source using a grating interferometric setup. The non-destructive method allowed the same sample to be measured before and after cooking. Heat denaturation resulted in a 36% decrease in the volume of the muscle fibers, while solubilization of the connective tissues increased the volume from 8.4%to 24.9%. The cooking loss was quantified and separated into a water phase and a gel phase formed by the sarcoplasmic proteins in the exudate. The results show that X-ray phase contrast tomography offers unique possibilities in studies both the meat structure and the different meat component such as water, fat, connective tissue and myofibrils in a qualitative and quantitative manner without prior sample preparation as isolation of single muscle components, calibration or histology.

Visualisation by high resolution synchrotron X-ray phase contrast micro-tomography of gas films on submerged superhydrophobic leaves.

Floods can completely submerge terrestrial plants but some wetland species can sustain O2 and CO2 exchange with the environment via gas films forming on superhydrophobic leaf surfaces. We used high resolution synchrotron X-ray phase contrast micro-tomography in a novel approach to visualise gas films on submerged leaves of common cordgrass (Spartina anglica). 3D tomograms enabled a hitherto unmatched level of detail regarding the micro-topography of leaf gas films. Gas films formed only on the superhydrophobic adaxial leaf side (water droplet contact angle, Φ=162°) but not on the abaxial side (Φ=135°). The adaxial side of the leaves of common cordgrass is plicate with a longitudinal system of parallel grooves and ridges and the vast majority of the gas film volume was found in large ∼180µm deep elongated triangular volumes in the grooves and these volumes were connected to each neighbouring groove via a fine network of gas tubules (∼1.7µm diameter) across the ridges. In addition to the gas film retained on the leaf exterior, the X-ray phase contrast micro-tomography also successfully distinguished gas spaces internally in the leaf tissues, and the tissue porosity (gas volume per unit tissue volume) ranged from 6.3% to 20.3% in tip and base leaf segments, respectively. We conclude that X-ray phase contrast micro-tomography is a powerful tool to obtain quantitative data of exterior gas features on biological samples because of the significant difference in electron density between air, biological tissues and water.

Model study of the pressure build-up during subcutaneous injection.

In this study we estimate the subcutaneous tissue counter pressure during drug infusion from a series of injections of insulin in type 2 diabetic patients using a non-invasive method. We construct a model for the pressure evolution in subcutaneous tissue based on mass continuity and the flow laws of a porous medium. For equivalent injection forces we measure the change in the infusion rate between injections in air at atmospheric pressure and in tissue. From a best fit with our model, we then determine the flow permeability as well as the bulk modulus of the tissue, estimated to be of the order 10-11-10-10 m2 and 105 Pa, respectively. The permeability is in good agreement with reported values for adipose porcine tissue. We suggest our model as a general way to estimate the pressure build-up in tissue during subcutaneous injection.

Effect of phospholipid composition and phase on nanodisc films at the solid-liquid interface as studied by neutron reflectivity.

Nanodiscs are disc-like self-assembled structures formed by phospholipids and amphipatic proteins. The proteins wrap like a belt around the hydrophobic part of the lipids, basically producing nanometer-sized patches of lipid bilayers. The bilayer in the nanodisc constitutes a native-like model of the cell membrane and can act as a nanometer-sized container for functional single membrane proteins. In this study, we present a general nanodisc-based system, intended for structural and functional studies of membrane proteins. In this method, the nanodiscs are aligned at a solid surface, providing the ability to determine the average structure of the film along an axis perpendicular to the interface as measured by neutron reflectivity. The nanodisc film was optimized in terms of nanodisc coverage, reduced film roughness, and stability for time-consuming studies. This was achieved by a systematic variation of the lipid phase, charge, and length of lipid tails. Herein, we show that, although all studied nanodiscs align with their lipid bilayer parallel to the interface, gel-phase DMPC nanodiscs form the most suitable film for future membrane protein studies since they yield a dense irreversibly adsorbed film with low roughness and high stability over time. This may be explained by the appropriate matching between the thickness of the hydrophobic lipid core of gel phase DMPC and the height of the belt protein. Moreover, once formed the gel-phase DMPC nanodiscs film can be heated up to melt the lipid bilayer, thus providing a more biologically friendly environment for membrane proteins.

Imaging of metastatic lymph nodes by X-ray phase-contrast micro-tomography.

Invasive cancer causes a change in density in the affected tissue, which can be visualized by x-ray phase-contrast tomography. However, the diagnostic value of this method has so far not been investigated in detail. Therefore, the purpose of this study was, in a blinded manner, to investigate whether malignancy could be revealed by non-invasive x-ray phase-contrast tomography in lymph nodes from breast cancer patients. Seventeen formalin-fixed paraffin-embedded lymph nodes from 10 female patients (age range 37-83 years) diagnosed with invasive ductal carcinomas were analyzed by X-ray phase-contrast tomography. Ten lymph nodes had metastatic deposits and 7 were benign. The phase-contrast images were analyzed according to standards for conventional CT images looking for characteristics usually only visible by pathological examinations. Histopathology was used as reference. The result of this study was that the diagnostic sensitivity of the image analysis for detecting malignancy was 100% and the specificity was 87%. The positive predictive value was 91% for detecting malignancy and the negative predictive value was 100%. We conclude that x-ray phase-contrast imaging can accurately detect density variations to obtain information regarding lymph node involvement previously inaccessible with standard absorption x-ray imaging.

Monitoring shifts in the conformation equilibrium of the membrane protein cytochrome P450 reductase (POR) in nanodiscs.

Nanodiscs are self-assembled ∼50-nm(2) patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.

Graphene oxide as a monoatomic blocking layer.

Monolayer graphene oxide (mGO) is shown to effectively protect molecular thin films from reorganization and function as an atomically thin barrier for vapor-deposited Ti/Al metal top electrodes. Fragile organic Langmuir-Blodgett (LB) films of C(22) fatty acid cadmium salts (cadmium(II) behenate) were covered by a compressed mosaic LB film of mGO flakes. These hybrid LB films were examined with atomic force microscopy (AFM) and X-ray reflectivity, both with and without the metal top electrodes. While the AFM enabled surface and morphology analysis, the X-ray reflectivity allowed for a detailed structural depth profiling of the organic film and mGO layer below the metal top layers. The structure of the mGO-protected LB films was found to be perfectly preserved; in contrast, it has previously been shown that metal deposition completely destroys the first two LB layers of unprotected films. This study provides clear evidence of the efficient protection offered by a single atomic layer of GO.

X-ray vector radiography for bone micro-architecture diagnostics.

The understanding of large biophysical systems at the systems level often depends on a precise knowledge of their microstructure. This is difficult to obtain, especially in vivo, because most imaging methods are either limited in terms of achievable field of view, or make use of penetrating ionizing radiations such as x-rays, in which case the resolution is severely limited by the deposited dose. Here, we describe a new method, x-ray vector radiography (XVR), which yields various types of information about the local orientation, anisotropy and average size of the sample microstructures. We demonstrate the feasibility by showing first experimental XVRs of human vertebra bone samples, giving information on the trabecular structures even with a pixel resolution of half a millimetre, much larger than the structures themselves. This last point is critical for the development of low-dose measurement methods which will allow for in vivo studies and potentially in the future for new medical diagnostics methods of bone metabolic disorder diseases such as osteoporosis.

Aligning nanodiscs at the air-water interface, a neutron reflectivity study.

Nanodiscs are self-assembled nanostructures composed of a belt protein and a small patch of lipid bilayer, which can solubilize membrane proteins in a lipid bilayer environment. We present a method for the alignment of a well-defined two-dimensional layer of nanodiscs at the air-water interface by careful design of an insoluble surfactant monolayer at the surface. We used neutron reflectivity to demonstrate the feasibility of this approach and to elucidate the structure of the nanodisc layer. The proof of concept is hereby presented with the use of nanodiscs composed of a mixture of two different lipid (DMPC and DMPG) types to obtain a net overall negative charge of the nanodiscs. We find that the nanodisc layer has a thickness or 40.9 ± 2.6 Å with a surface coverage of 66 ± 4%. This layer is located about 15 Å below a cationic surfactant layer at the air-water interface. The high level of organization within the nanodiscs layer is reflected by a low interfacial roughness (~4.5 Å) found. The use of the nanodisc as a biomimetic model of the cell membrane allows for studies of single membrane proteins isolated in a confined lipid environment. The 2D alignment of nanodiscs could therefore enable studies of high-density layers containing membrane proteins that, in contrast to membrane proteins reconstituted in a continuous lipid bilayer, remain isolated from influences of neighboring membrane proteins within the layer.