Multimodal 2D and 3D microscopic mapping of growth cartilage by computational imaging techniques - a short review including new research.

Being able to image the microstructure of growth cartilage is important for understanding the onset and progression of diseases such as osteochondrosis and osteoarthritis, as well as for developing new treatments and implants. Studies of cartilage using conventional optical brightfield microscopy rely heavily on histological staining, where the added chemicals provide tissue-specific colours. Other microscopy contrast mechanisms include polarization, phase- and scattering contrast, enabling non-stained or 'label-free' imaging that significantly simplifies the sample preparation, thereby also reducing the risk of artefacts. Traditional high-performance microscopes tend to be both bulky and expensive.Computational imagingdenotes a range of techniques where computers with dedicated algorithms are used as an integral part of the image formation process. Computational imaging offers many advantages like 3D measurements, aberration correction and quantitative phase contrast, often combined with comparably cheap and compact hardware. X-ray microscopy is also progressing rapidly, in certain ways trailing the development of optical microscopy. In this study, we first briefly review the structures of growth cartilage and relevant microscopy characterization techniques, with an emphasis on Fourier ptychographic microscopy (FPM) and advanced x-ray microscopies. We next demonstrate with our own results computational imaging through FPM and compare the images with hematoxylin eosin and saffron (HES)-stained histology. Zernike phase contrast, and the nonlinear optical microscopy techniques of second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) are explored. Furthermore, X-ray attenuation-, phase- and diffraction-contrast computed tomography (CT) images of the very same sample are presented for comparisons. Future perspectives on the links to artificial intelligence, dynamic studies andin vivopossibilities conclude the article.

Orientational mapping of minerals in Pierre shale using X-ray diffraction tensor tomography.

Shales have a complex mineralogy with structural features spanning several length scales, making them notoriously difficult to fully understand. Conventional attenuation-based X-ray computed tomography (CT) measures density differences, which, owing to the heterogeneity and sub-resolution features in shales, makes reliable interpretation of shale images a challenging task. CT based on X-ray diffraction (XRD-CT), rather than intensity attenuation, is becoming a well established technique for non-destructive 3D imaging, and is especially suited for heterogeneous and hierarchical materials. XRD patterns contain information about the mineral crystal structure, and crucially also crystallite orientation. Here, we report on the use of orientational imaging using XRD-CT to study crystallite-orientation distributions in a sample of Pierre shale. Diffraction-contrast CT data for a shale sample measured with its bedding-plane normal aligned parallel to a single tomographic axis perpendicular to the incoming X-ray beam are discussed, and the spatial density and orientation distribution of clay minerals in the sample are described. Finally, the scattering properties of highly attenuating inclusions in the shale bulk are studied, which are identified to contain pyrite and clinochlore. A path forward is then outlined for systematically improving the structural description of shales.

Quantifying the hydroxyapatite orientation near the ossification front in a piglet femoral condyle using X-ray diffraction tensor tomography.

While a detailed knowledge of the hierarchical structure and morphology of the extracellular matrix is considered crucial for understanding the physiological and mechanical properties of bone and cartilage, the orientation of collagen fibres and carbonated hydroxyapatite (HA) crystallites remains a debated topic. Conventional microscopy techniques for orientational imaging require destructive sample sectioning, which both precludes further studies of the intact sample and potentially changes the microstructure. In this work, we use X-ray diffraction tensor tomography to image non-destructively in 3D the HA orientation in a medial femoral condyle of a piglet. By exploiting the anisotropic HA diffraction signal, 3D maps showing systematic local variations of the HA crystallite orientation in the growing subchondral bone and in the adjacent mineralized growth cartilage are obtained. Orientation maps of HA crystallites over a large field of view (~ 3 × 3 × 3 mm3) close to the ossification (bone-growth) front are compared with high-resolution X-ray propagation phase-contrast computed tomography images. The HA crystallites are found to predominantly orient with their crystallite c-axis directed towards the ossification front. Distinct patterns of HA preferred orientation are found in the vicinity of cartilage canals protruding from the subchondral bone. The demonstrated ability of retrieving 3D orientation maps of bone-cartilage structures is expected to give a better understanding of the physiological properties of bones, including their propensity for bone-cartilage diseases.

Nanoscale imaging of shale fragments with coherent X-ray diffraction.

Despite the abundance of shales in the Earth's crust and their industrial and environmental importance, their microscale physical properties are poorly understood, owing to the presence of many structurally related mineral phases and a porous network structure spanning several length scales. Here, the use of coherent X-ray diffraction imaging (CXDI) to study the internal structure of microscopic shale fragments is demonstrated. Simultaneous wide-angle X-ray diffraction (WAXD) measurement facilitated the study of the mineralogy of the shale microparticles. It was possible to identify pyrite nanocrystals as inclusions in the quartz-clay matrix and the volume of closed unconnected pores was estimated. The combined CXDI-WAXD analysis enabled the establishment of a correlation between sample morphology and crystallite shape and size. The results highlight the potential of the combined CXDI-WAXD approach as an upcoming imaging modality for 3D nanoscale studies of shales and other geological formations via serial measurements of microscopic fragments.

Real Time 3D Observations of Portland Cement Carbonation at CO2 Storage Conditions.

Depleted oil reservoirs are considered a viable solution to the global challenge of CO2 storage. A key concern is whether the wells can be suitably sealed with cement to hinder the escape of CO2. Under reservoir conditions, CO2 is in its supercritical state, and the high pressures and temperatures involved make real-time microscopic observations of cement degradation experimentally challenging. Here, we present an in situ 3D dynamic X-ray micro computed tomography (µ-CT) study of well cement carbonation at realistic reservoir stress, pore-pressure, and temperature conditions. The high-resolution time-lapse 3D images allow monitoring the progress of reaction fronts in Portland cement, including density changes, sample deformation, and mineral precipitation and dissolution. By switching between flow and nonflow conditions of CO2-saturated water through cement, we were able to delineate regimes dominated by calcium carbonate precipitation and dissolution. For the first time, we demonstrate experimentally the impact of the flow history on CO2 leakage risk for cement plugging. In-situ µ-CT experiments combined with geochemical modeling provide unique insight into the interactions between CO2 and cement, potentially helping in assessing the risks of CO2 storage in geological reservoirs.

3D Maps of Mineral Composition and Hydroxyapatite Orientation in Fossil Bone Samples Obtained by X-ray Diffraction Computed Tomography.

Whether hydroxyapatite (HA) orientation in fossilised bone samples can be non-destructively retrieved and used to determine the arrangement of the bone matrix and the location of muscle attachments (entheses), is a question of high relevance to palaeontology, as it facilitates a detailed understanding of the (micro-)anatomy of extinct species with no damage to the precious fossil specimens. Here, we report studies of two fossil bone samples, specifically the tibia of a 300-million-year-old tetrapod, Discosauriscus austriacus, and the humerus of a 370-million-year-old lobe-finned fish, Eusthenopteron foordi, using XRD-CT - a combination of X-ray diffraction (XRD) and computed tomography (CT). Reconstructed 3D images showing the spatial mineral distributions and the local orientation of HA were obtained. For Discosauriscus austriacus, details of the muscle attachments could be discerned. For Eusthenopteron foordi, the gross details of the preferred orientation of HA were deduced using three tomographic datasets obtained with orthogonally oriented rotation axes. For both samples, the HA in the bone matrix exhibited preferred orientation, with the unit cell c-axis of the HA crystallites tending to be parallel with the bone surface. In summary, we have demonstrated that XRD-CT combined with an intuitive reconstruction procedure is becoming a powerful tool for studying palaeontological samples.

High-resolution coherent x-ray diffraction imaging of metal-coated polymer microspheres.

Coherent x-ray diffraction imaging (CXDI) is becoming an important 3D quantitative microscopy technique, allowing structural investigation of a wide range of delicate mesoscale samples that cannot be imaged by other techniques like electron microscopy. Here we report high-resolution 3D CXDI performed on spherical microcomposites consisting of a polymer core coated with a triple layer of nickel-gold-silica. These composites are of high interest to the microelectronics industry, where they are applied in conducting adhesives as fine-pitch electrical contacts-which requires an exceptional degree of uniformity and reproducibility. Experimental techniques that can assess the state of the composites non-destructively, preferably also while embedded in electronic chips, are thus in high demand. We demonstrate that using CXDI, all four different material components of the composite could be identified, with radii matching well to the nominal specifications of the manufacturer. Moreover, CXDI provided detailed maps of layer thicknesses, roughnesses, and defects such as holes, thus also facilitating cross-layer correlations. The side length of the voxels in the reconstruction, given by the experimental geometry, was 16 nm. The effective resolution enabled resolving even the thinnest coating layer of ∼20 nm nominal width. We discuss critically the influence of the weak phase approximation and the projection approximation on the reconstructed electron density estimates, demonstrating that the latter has to be employed. We conclude that CXDI has excellent potential as a metrology tool for microscale composites.

Wavefront metrology for coherent hard X-rays by scanning a microsphere.

Characterization of the wavefront of an X-ray beam is of primary importance for all applications where coherence plays a major role. Imaging techniques based on numerically retrieving the phase from interference patterns are often relying on an a-priori assumption of the wavefront shape. In Coherent X-ray Diffraction Imaging (CXDI) a planar incoming wave field is often assumed for the inversion of the measured diffraction pattern, which allows retrieving the real space image via simple Fourier transformation. It is therefore important to know how reliable the plane wave approximation is to describe the real wavefront. Here, we demonstrate that the quantitative wavefront shape and flux distribution of an X-ray beam used for CXDI can be measured by using a micrometer size metal-coated polymer sphere serving in a similar way as the hole array in a Hartmann wavefront sensor. The method relies on monitoring the shape and center of the scattered intensity distribution in the far field using a 2D area detector while raster-scanning the microsphere with respect to the incoming beam. The reconstructed X-ray wavefront was found to have a well-defined central region of approximately 16 µm diameter and a weaker, asymmetric, intensity distribution extending 30 µm from the beam center. The phase front distortion was primarily spherical with an effective radius of 0.55 m which matches the distance to the last upstream beam-defining slit, and could be accurately represented by Zernike polynomials.

Using three-dimensional 3D grazing-incidence small-angle X-ray scattering (GISAXS) analysis to probe pore deformation in mesoporous silica films.

In the past decade, remarkable progress has been made in studying nanoscale objects deposited on surfaces by grazing-incidence small-angle X-ray scattering (GISAXS). However, unravelling the structural properties of mesostructured thin films containing highly organized internal three-dimensional (3D) structures remains a challenging issue, because of the lack of efficient algorithms that allow prediction of the GISAXS intensity patterns. Previous attempts to calculate intensities have mostly been limited to cases of two-dimensional (2D) assemblies of nanoparticles at surfaces, or have been adapted to specific 3D cases. Here, we demonstrate that highly organized 3D mesoscale structures (for example, porous networks) can be modeled by the combined use of established crystallography formalism and the Distorted Wave Born Approximation (DWBA). Taking advantage of the near-zero intensity of symmetry-allowed Bragg reflections, the casual extinction or existence of certain reflections related to the anisotropy of the form factor of the pores can be used as a highly sensitive method to extract structural information. We employ this generic method to probe the slightly compressed anisotropic shape and orientation of pores in a mesoporous silica thin film having P63/mmc symmetry.

Semi-metallic polymers.

Polymers are lightweight, flexible, solution-processable materials that are promising for low-cost printed electronics as well as for mass-produced and large-area applications. Previous studies demonstrated that they can possess insulating, semiconducting or metallic properties; here we report that polymers can also be semi-metallic. Semi-metals, exemplified by bismuth, graphite and telluride alloys, have no energy bandgap and a very low density of states at the Fermi level. Furthermore, they typically have a higher Seebeck coefficient and lower thermal conductivities compared with metals, thus being suitable for thermoelectric applications. We measure the thermoelectric properties of various poly(3,4-ethylenedioxythiophene) samples, and observe a marked increase in the Seebeck coefficient when the electrical conductivity is enhanced through molecular organization. This initiates the transition from a Fermi glass to a semi-metal. The high Seebeck value, the metallic conductivity at room temperature and the absence of unpaired electron spins makes polymer semi-metals attractive for thermoelectrics and spintronics.

Substrate-induced variations of molecular packing, dynamics, and intermolecular electronic couplings in pentacene monolayers on the amorphous silica dielectric.

Charge-carrier transport in thin-film organic field-effect transistors takes place within the first (few) molecular layer(s) of the active organic material in contact with the gate dielectric. Here, we use atomistic molecular dynamics simulations to evaluate how interactions with bare amorphous silica surfaces that vary in terms of surface potential influence the molecular packing and dynamics of a monolayer pentacene film. The results indicate that the long axis of the pentacene molecules has a non-negligible tilt angle away from the surface normal. Grazing-incidence X-ray diffraction patterns for these models are calculated, and we discuss notable differences in the shapes of the Bragg rods as a function of the molecular packing, also in relation to previously published experimental reports. Intermolecular electronic couplings (transfer integrals) evaluated for the monolayers show marked differences compared to bulk crystal calculations, a result that points to the importance of fully considering the molecular packing environment in charge-carrier mobility models for organic electronic materials.

Two-dimensional GIWAXS reveals a transient crystal phase in solution-processed thermally converted tetrabenzoporphyrin.

Thermally convertible organic materials are useful for the fabrication of multilayered thin film electronic devices such as solar cells. However, substantial changes in molecular ordering can occur during the conversion process that may lead to multiple polymorphs having differing electronic properties. In-situ grazing incidence wide-angle X-ray scattering with 2-D detection (2-D GIWAXS) was used to study the changes in the thin film crystal structure, texture, and crystallite size of a convertible small-molecule electron donor, tetrabenzoporphyrin (BP), during thermal conversion from the precursor bicycloporphyrin (CP) and the resulting crystal-crystal phase transition from a metastable phase (phase I) to a stable phase (phase II). The annealing temperature and the presence of an underlying BP layer both affect the phase-transition behavior. Phase II has a much weaker degree of crystalline texture than phase I, attributed to changes in molecular packing to achieve a herringbone arrangement. The unit cell for phase I was determined by electron diffraction and GIWAXS, and the thin film structure of phase II matched the previously determined bulk structure. The texture of crystallites in phase II was characterized by the simulation of the GIWAXS pattern. Transmission electron microscopy revealed differences in the morphology, grain size, and film coverage of the two polymorphs. Peak shape analysis with corrections for geometric smearing and paracrystalline disorder showed an increase in crystallite size from phase I to phase II. These results demonstrate the utility of in-situ 2-D GIWAXS in revealing polymorphic phases during the structural transition of thermally convertible organic semiconductors, the presence of which may impact the performance of solar cells.

Three-dimensional packing structure and electronic properties of biaxially oriented poly(2,5-bis(3-alkylthiophene-2-yl)thieno[3,2-b]thiophene) films.

We use a systematic approach that combines experimental X-ray diffraction (XRD) and computational modeling based on molecular mechanics and two-dimensional XRD simulations to develop a detailed model of the molecular-scale packing structure of poly(2,5-bis (3-tetradecylthiophene-2-yl)thieno[3,2-b]thiophene) (PBTTT-C(14)) films. Both uniaxially and biaxially aligned films are used in this comparison and lead to an improved understanding of the molecular-scale orientation and crystal structure. We then examine how individual polymer components (i.e., conjugated backbone and alkyl side chains) contribute to the complete diffraction pattern, and how modest changes to a particular component orientation (e.g., backbone or side-chain tilt) influence the diffraction pattern. The effects on the polymer crystal structure of varying the alkyl side-chain length from C(12) to C(14) and C(16) are also studied. The accurate determination of the three-dimensional polymer structure allows us to examine the PBTTT electronic band structure and intermolecular electronic couplings (transfer integrals) as a function of alkyl side-chain length. This combination of theoretical and experimental techniques proves to be an important tool to help establish the relationship between the structural and electronic properties of polymer thin films.

Columnar self-assembly and alignment of planar carbenium ions in Langmuir-Blodgett films.

Structural and optical properties of multilayer Langmuir-Blodgett (LB) films of two amphiphilic carbenium salts 2-didecylamino-6,10-bis(dimethylamino)-4,8,12-trioxatriangulenium hexafluorophosphate (ATOTA-1) and 2,6-bis(decylmethylamino)-10-dimethylamino-4,8,12-trioxatriangulenium hexafluorophosphate (ATOTA-2) are described. The LB films were prepared on lipophilic glass by standard vertical dipping. Grazing incidence X-ray diffraction (GIXD) measurements show that the planar organic cores, in spite of their positive charge, form closely packed columns with a repeating distance of ∼3.45 Å. Specular X-ray reflectivity (SXR) reveals the LB multilayers to consist of Y-type bilayers with thickness 31 Å for ATOTA-1 and 41 Å for ATOTA-2. This significant difference is ascribed to the different packing motifs of the alkyl chains in the two LB films. GIXD and polarized UV-vis absorption and emission spectroscopy show that the columnar aggregates in the LB films are oriented along the dipping direction. This alignment is attributed to shear effects during LB transfer. The main absorption band of the LB films is blue-shifted compared to that in solution, while the fluorescence is red-shifted by more than 100 nm. These findings suggest the presence of H-aggregates in agreement with the cofacial packing derived from the X-ray measurements. Polarized absorption spectroscopy with variable angle of incidence was used to resolve two perpendicular optical transitions in the visible range, one at 460 nm polarized perpendicular to the columnar direction, in the plane of the film, and one at 420 nm polarized along the film normal.

Understanding structure-mobility relations for perylene tetracarboxydiimide derivatives.

Discotic mesophases are known for their ability to self-assemble into columnar structures and can serve as semiconducting molecular wires. Charge carrier mobility along these wires strongly depends on molecular packing, which is controlled by intermolecular interactions. By combining wide-angle X-ray scattering experiments with molecular dynamics simulations, we elucidate packing motifs of a perylene tetracarboxdiimide derivative, a task which is hard to achieve by using a single experimental or theoretical technique. We then relate the charge mobility to the molecular arrangement, both by pulse-radiolysis time-resolved microwave conductivity experiments and simulations based on the non-adiabatic Marcus charge transfer theory. Our results indicate that the helical molecular arrangement with the 45 degrees twist angle between the neighboring molecules favors hole transport in a compound normally considered as an n-type semiconductor. Statistical analysis shows that the transport is strongly suppressed by structural defects. By linking molecular packing and mobility, we eventually provide a pathway to the rational design of perylenediimide derivatives with high charge mobilities.

Biaxially oriented CdSe nanorods.

The shape, structure, and orientation of rubbing-aligned cadmium selenide (CdSe) nanorods on polymer coated glass substrates have been studied using transmission electron microscopy (TEM) and grazing incidence X-ray scattering combined with computer simulations. The nanorods are found to be of wurtzite structure and highly monodisperse, and have an essentially ellipsoidal shape with short axes of approximately 8 nm and long axis of approximately 22 nm. The nanorods exhibit preferred biaxial orientation with the hexagonal a-c-plane parallel to the sample surface and the c-axis oriented along the rubbing direction of the sample. Some tendency of smectic-A ordering is observed. A quantitative model incorporating atomic structure, rod shape, and preferred orientation was developed for numerically simulating the diffraction peak positions, widths, and intensities, giving good correlation with the experimental observations.

Theoretical investigation of perylene dimers and excimers and their signatures in X-ray diffraction.

The structures of the ground and excimer states of perylene pairs are calculated [using density functional theory (DFT) and time-dependent DFT techniques] in a free as well as a crystal environment, and their spectroscopic properties are studied for the most stable configurations. The vertical transition energies for the absorption and emission bands are obtained, and they are in good agreement with experimental data. In these calculations, up to six excited states are considered. With the calculated structures of the ground and excimer states, the scattering factors are analyzed as a function of the concentration of excimers in a crystal. The intensity of the 110, 005, and 0 10 0 reflections are found to be fairly sensitive to the presence of excimers in the crystal. The finite (nanosecond) lifetime of the excimer may make it possible to observe this state using time-resolved X-ray diffraction techniques.

Synthesis of novel amphiphilic azobenzenes and X-ray scattering studies of their Langmuir monolayers.

We report a simple synthetic route to novel symmetrical alkylated and acylated amphiphilic 4,4'-diaminoazobenzene dyes, with their optical axis perpendicular to the amphiphilic direction of the molecule. Three different substitution patterns are reported, two of which are highly amphiphilic. At the air-water interface, the amphiphilic azobenzenes form noncrystalline but stable Langmuir films that display an unusual reversible monolayer collapse close to 35 mN/m. The structures and phase transitions were studied by X-ray reflectivity (XR) and grazing-incidence X-ray diffraction, both utilizing synchrotron radiation. Compression beyond the collapse point does not change the XR data, showing that the film is unchanged at the molecular level, even at areas less than half of that of the collapse. This leads to the conclusion that few macroscopic collapse sites are responsible for reversibly removing large amounts of material from the interface.

Multicomponent semiconducting polymer systems with low crystallization-induced percolation threshold.

Blends and other multicomponent systems are used in various polymer applications to meet multiple requirements that cannot be fulfilled by a single material. In polymer optoelectronic devices it is often desirable to combine the semiconducting properties of the conjugated species with the excellent mechanical properties of certain commodity polymers. Here we investigate bicomponent blends comprising semicrystalline regioregular poly(3-hexylthiophene) and selected semicrystalline commodity polymers, and show that, owing to a highly favourable, crystallization-induced phase segregation of the two components, during which the semiconductor is predominantly expelled to the surfaces of cast films, we can obtain vertically stratified structures in a one-step process. Incorporating these as active layers in polymer field-effect transistors, we find that the concentration of the semiconductor can be reduced to values as low as 3 wt% without any degradation in device performance. This is in stark contrast to blends containing an amorphous insulating polymer, for which significant reduction in electrical performance was reported. Crystalline-crystalline/semiconducting-insulating multicomponent systems offer expanded flexibility for realizing high-performance semiconducting architectures at drastically reduced materials cost with improved mechanical properties and environmental stability, without the need to design all performance requirements into the active semiconducting polymer itself.

Designing solution-processable air-stable liquid crystalline crosslinkable semiconductors.

Organic electronics technology, in which at least the semiconducting component of the integrated circuit is an organic material, offers the potential for fabrication of electronic products by low-cost printing technologies, such as ink jet, gravure offset lithography and flexography. The products will typically be of lower performance than those using the present state of the art single crystal or polysilicon transistors, but comparable to amorphous silicon. A range of prototypes are under development, including rollable electrophoretic displays, active matrix liquid crystal (LC) displays, flexible organic light emitting diode displays, low frequency radio frequency identification tag and other low performance electronics. Organic semiconductors that offer both electrical performance and stability with respect to storage and operation under ambient conditions are required. This work describes the development of reactive mesogen semiconductors, which form large crosslinked LC domains on polymerization within mesophases. These crosslinked domains offer mechanical stability and are inert to solvent exposure in further processing steps. Reactive mesogens containing conjugated aromatic cores, designed to facilitate charge transport and provide good oxidative stability, were prepared and their liquid crystalline properties evaluated. The organization and alignment of the mesogens, both before and after crosslinking, were probed by grazing incidence wide-angle X-ray scattering of thin films. Both time-of-flight and field effect transistor devices were prepared and their electrical characterization reported.