Synchrotron Small-angle X-ray Diffraction Research Articles

Using Multiscale Simulations as a Tool to Interpret Equatorial X-ray Fiber Diffraction Patterns from Skeletal Muscle.

Synchrotron small-angle X-ray diffraction is the method of choice for nm-scale structural studies of striated muscle under physiological conditions and on millisecond time scales. The lack of generally applicable computational tools for modeling X-ray diffraction patterns from intact muscles has been a significant barrier to exploiting the full potential of this technique. Here, we report a novel "forward problem" approach using the spatially explicit computational simulation platform MUSICO to predict equatorial small-angle X-ray diffraction patterns and the force output simultaneously from resting and isometrically contracting rat skeletal muscle that can be compared to experimental data. The simulation generates families of thick-thin filament repeating units, each with their individually predicted occupancies of different populations of active and inactive myosin heads that can be used to generate 2D-projected electron density models based on known Protein Data Bank structures. We show how, by adjusting only a few selected parameters, we can achieve a good correspondence between experimental and predicted X-ray intensities. The developments presented here demonstrate the feasibility of combining X-ray diffraction and spatially explicit modeling to form a powerful hypothesis-generating tool that can be used to motivate experiments that can reveal emergent properties of muscle.

Physical Models from Physical Templates Using Biocompatible Liquid Crystal Elastomers as Morphologically Programmable Inks For 3D Printing.

Advanced manufacturing has received considerable attention as a tool for the fabrication of cell scaffolds however, finding ideal biocompatible and biodegradable materials that fit the correct parameters for 3D printing and guide cells to align remain a challenge. Herein, a photocrosslinkable smectic-A (Sm-A) liquid crystal elastomer (LCE) designed for 3D printing is presented, that promotes cell proliferation but most importantly induces cell anisotropy. The LCE-based bio-ink allows the 3D duplication of a highly complex brain structure generated from an animal model. Vascular tissue models are generated from fluorescently stained mouse tissue spatially imaged using confocal microscopy and subsequently processed to create a digital 3D model suitable for printing. The 3D structure is reproduced using a Digital Light Processing (DLP) stereolithography (SLA) desktop 3D printer. Synchrotron Small-Angle X-ray Diffraction (SAXD) data reveal a strong alignment of the LCE layering within the struts of the printed 3D scaffold. The resultant anisotropy of the LCE struts is then shown to direct cell growth. This study offers a simple approach to produce model tissues built within hours that promote cellular alignment.

Janus Photochemical/Photothermal Azobenzene Inverse Opal Actuator with Shape Self-Recovery toward Sophisticated Motion.

Azobenzene actuators have aroused enormous research interest due to their excellent performance and promising applications in the fields of soft robots, artificial muscles, etc. However, there are still challenges for the fabrication of azobenzene actuators with a sophisticated actuation mode owing to the unitary actuation direction and slow thermal relaxation of cis- to trans-azobenzene mesogens. To solve these problems, this paper presents a facile fabrication method of a Janus azobenzene inverse opal actuator with one side made of the monodomain azobenzene polymer and the other side made of the polydomain azobenzene inverse opal structure. Gradient-layer spacing structure of the film in its cross section is proven by synchrotron small-angle X-ray diffraction. The introduction of the inverse opal structure mainly provides a polydomain mesogen alignment, large specific surface area, low elastic modulus, and structure color. The synergetic actuation of the photochemical/photothermal mode produces multiple actuation directions, a larger actuation force, and an alteration of the structure color. Shape self-recovery of this Janus azobenzene actuator contributes to some promising applications, such as crawling on a smooth surface, driving an engine axis, and logic electric circuit for the coding technique. This work is of great significance for the design and fabrication of novel-type photoactuators.

Cubic and Hexagonal Mesophases for Protein Encapsulation: Structural Effects of Insulin Confinement

Monoolein-based cubic and hexagonal mesophases were investigated as matrices for insulin loading, at low pH, as a function of temperature and in the presence of increasing amounts of oleic acid, as a structural stabilizer for the hexagonal phase. Synchrotron small angle X-ray diffraction, rheological measurements, and attenuated total reflection-Fourier transform infrared spectroscopy were used to study the effects of insulin loading on the lipid mesophases and of the effect of protein confinement in the 2D- and 3D-lipid matrix water channels on its stability and unfolding behavior. We found that insulin encapsulation has only little effects both on the mesophase structures and on the viscoelastic properties of lipid systems, whereas protein confinement affects the response of the secondary structure of insulin to thermal changes in a different manner according to the specific mesophase: in the cubic structure, the unfolding toward an unordered structure is favored, while the prevalence of parallel β-sheets, and nuclei for fibril formation, is observed in hexagonal structures.

Diffusionless transformation of soft cubic superstructure from amorphous to simple cubic and body-centered cubic phases

In a narrow temperature window in going from the isotropic to highly chiral orders, cholesteric liquid crystals exhibit so-called blue phases, consisting of different morphologies of long, space-filling double twisted cylinders. Those of cubic spatial symmetry have attracted considerable attention in recent years as templates for soft photonic materials. The latter often requires the creation of monodomains of predefined orientation and size, but their engineering is complicated by a lack of comprehensive understanding of how blue phases nucleate and transform into each other at a submicrometer length scale. In this work, we accomplish this by intercepting nucleation processes at intermediate stages with fast cross-linking of a stabilizing polymer matrix. We reveal using transmission electron microscopy, synchrotron small-angle X-ray diffraction, and angle-resolved microspectroscopy that the grid of double-twisted cylinders undergoes highly coordinated, diffusionless transformations. In light of our findings, the implementation of several applications is discussed, such as temperature-switchable QR codes, micro-area lasing, and fabrication of blue phase liquid crystals with large domain sizes.

Mechanical response of cortical bone in compression and tension at the mineralized fibrillar level in steroid induced osteoporosis

Nanocomposites like bone are used in vivo in a dynamic mechanical environment, and musculoskeletal fracture during traumatic events lead to pain and immobilisation, especially significant in elderly and patients with metabolic bone diseases. To mitigate against these occurrences, it is essential to understand the dynamic mechanical response of bone over short timescales, but the underlying matrix-level mechanisms are not well understood. Here, we studied the mechanical response of cortical bone at nano- and micro-scale in a mouse model with glucocorticoid induced osteoporosis and their wild type littermates using real-time synchrotron small-angle X-ray diffraction (SAXD) combined with in situ compression testing and micro-tensile testing under controlled strain rates. Under compression, the tissue modulus, yield stress, effective fibril modulus and fibrillar reorientation rate in osteoporotic bone is significantly lower than that in healthy bone. Under tension, when going from low to high strain rates, the effective fibril modulus of healthy bone increase by a factor of 4.8, but this tendency is suppressed in osteoporotic bone. Also, bone microstructure in osteoporotic showed large fraction of cavities with disrupted mineralization. Our results demonstrate how the nano- and microscale deformation mechanisms of bone ultrastructure change in osteoporotic bone under compression and tension. Our results suggest that material level changes of bone matrix contributed to the reduced mechanical competence of bone in metabolic bone diseases such as osteoporosis.

Myosin filament-based regulation of the dynamics of contraction in heart muscle

Myosin-based mechanisms are increasingly recognized as supplementing their better-known actin-based counterparts to control the strength and time course of contraction in both skeletal and heart muscle. Here we use synchrotron small-angle X-ray diffraction to determine the structural dynamics of local domains of the myosin filament during contraction of heart muscle. We show that, although myosin motors throughout the filament contribute to force development, only about 10% of the motors in each filament bear the peak force, and these are confined to the filament domain containing myosin binding protein-C, the "C-zone." Myosin motors in domains further from the filament midpoint are likely to be activated and inactivated first in each contraction. Inactivated myosin motors are folded against the filament core, and a subset of folded motors lie on the helical tracks described previously. These helically ordered motors are also likely to be confined to the C-zone, and the associated motor conformation reforms only slowly during relaxation. Myosin filament stress-sensing determines the strength and time course of contraction in conjunction with actin-based regulation. These results establish the fundamental roles of myosin filament domains and the associated motor conformations in controlling the strength and dynamics of contraction in heart muscle, enabling those structures to be targeted to develop new therapies for heart disease.

Bone matrix development in steroid-induced osteoporosis is associated with a consistently reduced fibrillar stiffness linked to altered bone mineral quality

Glucocorticoid-induced osteoporosis (GIOP) is a major secondary form of osteoporosis, with the fracture risk significantly elevated – at similar levels of bone mineral density – in patients taking glucocorticoids compared with non-users. The adverse bone structural changes at multiple hierarchical levels in GIOP, and their mechanistic consequences leading to reduced load-bearing capacity, are not clearly understood. Here we combine experimental X-ray nanoscale mechanical imaging with analytical modelling of the bone matrix mechanics to determine mechanisms causing bone material quality deterioration during development of GIOP. In situ synchrotron small-angle X-ray diffraction combined with tensile testing was used to measure nanoscale deformation mechanisms in a murine model of GIOP, due to a corticotrophin-releasing hormone promoter mutation, at multiple ages (8-, 12-, 24- and 36 weeks), complemented by quantitative micro-computed tomography and backscattered electron imaging to determine mineral concentrations. We develop a two-level hierarchical model of the bone matrix (mineralized fibril and lamella) to predict fibrillar mechanical response as a function of architectural parameters of the mineralized matrix. The fibrillar elastic modulus of GIOP-bone is lower than healthy bone throughout development, and nearly constant in time, in contrast to the progressively increasing stiffness in healthy bone. The lower mineral platelet aspect ratio value for GIOP compared to healthy bone in the multiscale model can explain the fibrillar deformation. Consistent with this result, independent measurement of mineral platelet lengths from wide-angle X-ray diffraction finds a shorter mineral platelet length in GIOP. Our results show how lowered mineralization combined with altered mineral nanostructure in GIOP leads to lowered mechanical competence. Significance StatementIncreased fragility in musculoskeletal disorders like osteoporosis are believed to arise due to alterations in bone structure at multiple length-scales from the organ down to the supramolecular-level, where collagen molecules and elongated mineral nanoparticles form stiff fibrils. However, the nature of these molecular-level alterations are not known. Here we used X-ray scattering to determine both how bone fibrils deform in secondary osteoporosis, as well as how the fibril orientation and mineral nanoparticle structure changes. We found that osteoporotic fibrils become less stiff both because the mineral nanoparticles became shorter and less efficient at transferring load from collagen, and because the fibrils are more randomly oriented. These results will help in the design of new composite musculoskeletal implants for bone repair.

DOPE–oleic acid–Ca2+ as DNA condensing agent

Abstract Phospholipid-based non-viral carriers composed of neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) and the binary mixture DOPE–oleic acid (OA) are examined as potential DNA delivery vectors. The process of DNA condensation in the presence of Ca2+ ions has been monitored through changes in emmision intensity of fluorescent probe ethidium bromide. The decline in fluorescence intensity with increasing Ca2+ concentration at two different time intervals was correlated with the binding capacity of complexes and possible release of DNA from the complex. The microstructure of DOPE–OA mixtures at different OA/DOPE molar ratios and that of DOPE–OA–DNA–Ca2+ complexes were determined using synchrotron small angle X-ray diffraction (SAXD). We identified inverted hexagonal phase HII as the dominant structure. OA affects the lattice parameter of HII formed by DOPE. With the increasing OA/DOPE molar ratio, the lattice parameter decreases, which results in significantly lower fraction of DNA bound to the OA-enriched complexes.

The lipolytic degradation of highly structured cubic micellar nanoparticles of soy phosphatidylcholine and glycerol dioleate by phospholipase A2 and triacylglycerol lipase

The effects of different lipolytic enzymes on the structure of lipid liquid crystalline nano-particles (LCNP) have been investigated by cryogenic transmission electron microscopy (cryo-TEM) and synchrotron small angle X-ray diffraction (SAXD). Here we used highly structured cubic micellar (Fd3m) nanoparticles of 50/50 (wt%/wt%) soy phosphatidyl choline (SPC)/glycerol dioleate (GDO) as substrate. Two types of lipolytic enzymes were used, phospholipase A2 (PLA2) that catalyses degradation of the phospholipid component, SPC, and porcine pancreatic triacylglycerol lipase (TGL) that facilitate the hydrolysis of the diglyceride, GDO. Evolution of the structure was found to be very different and linked to specificity of the two types of enzymes. PLA2, which hydrolyses the lamellar forming component, SPC, induces a reversed micellar lipid phase, while TGL which hydrolysis the reverse phase forming compound, GDO, induces a lamellar phase.

Body wall structure in the starfish Asterias rubens.

The body wall of starfish is composed of magnesium calcite ossicles connected by collagenous tissue and muscles and it exhibits remarkable variability in stiffness, which is attributed to the mechanical mutability of the collagenous component. Using the common European starfish Asterias rubens as an experimental animal, here we have employed a variety of techniques to gain new insights into the structure of the starfish body wall. The structure and organisation of muscular and collagenous components of the body wall were analysed using trichrome staining. The muscle system comprises interossicular muscles as well as muscle strands that connect ossicles with the circular muscle layer of the coelomic lining. The collagenous tissue surrounding the ossicle network contains collagen fibres that form loop‐shaped straps that wrap around calcite struts near to the surface of ossicles. The 3D architecture of the calcareous endoskeleton was visualised for the first time using X‐ray microtomography, revealing the shapes and interactions of different ossicle types. Furthermore, analysis of the anatomical organisation of the ossicles indicates how changes in body shape may be achieved by local contraction/relaxation of interossicular muscles. Scanning synchrotron small‐angle X‐ray diffraction (SAXD) scans of the starfish aboral body wall and ambulacrum were used to study the collagenous tissue component at the fibrillar level. Collagen fibrils in aboral body wall were found to exhibit variable degrees of alignment, with high levels of alignment probably corresponding to regions where collagenous tissue is under tension. Collagen fibrils in the ambulacrum had a uniformly low degree of orientation, attributed to macrocrimp of the fibrils and the presence of slanted as well as horizontal fibrils connecting antimeric ambulacral ossicles. Body wall collagen fibril D‐period lengths were similar to previously reported mammalian D‐periods, but were significantly different between the aboral and ambulacral samples. The overlap/D‐period length ratio within fibrils was higher than reported for mammalian tissues. Collectively, the data reported here provide new insights into the anatomy of the body wall in A. rubens and a foundation for further studies investigating the structural basis of the mechanical properties of echinoderm body wall tissue composites.

Structural properties of paeonol encapsulated liposomes at physiological temperature: Synchrotron small-angle and wide-angle X-ray diffraction studies

Structural properties of paeonol encapsulated liposomes at physiological temperature: Synchrotron small-angle and wide-angle X-ray diffraction studies

Interfibrillar stiffening of echinoderm mutable collagenous tissue demonstrated at the nanoscale

The mutable collagenous tissue (MCT) of echinoderms (e.g., sea cucumbers and starfish) is a remarkable example of a biological material that has the unique attribute, among collagenous tissues, of being able to rapidly change its stiffness and extensibility under neural control. However, the mechanisms of MCT have not been characterized at the nanoscale. Using synchrotron small-angle X-ray diffraction to probe time-dependent changes in fibrillar structure during in situ tensile testing of sea cucumber dermis, we investigate the ultrastructural mechanics of MCT by measuring fibril strain at different chemically induced mechanical states. By measuring a variable interfibrillar stiffness (EIF), the mechanism of mutability at the nanoscale can be demonstrated directly. A model of stiffness modulation via enhanced fibrillar recruitment is developed to explain the biophysical mechanisms of MCT. Understanding the mechanisms of MCT quantitatively may have applications in development of new types of mechanically tunable biomaterials.

A Dual Modulated Homochiral Helical Nanofilament Phase with Local Columnar Ordering Formed by Bent Core Liquid Crystals: Effects of Molecular Chirality.

Helical nanofilament (HNF) phases form as a result of an intralayer mismatch between top and bottom molecular halves in bent-core liquid crystals (BC-LCs) that is relieved by local saddle-splay geometry. HNFs are immensely attractive for photovoltaic and chiral separation applications and as templates for the chiral spatial assembly of guest molecules. Here, the synthesis and characterization of two unichiral BC-LCs and one racemic mixture with tris-biphenyl-diester cores featuring chiral (R,R) and (S,S) or racemic 2-octyloxy aliphatic side chains are presented. In comparison to the achiral compound with linear side chains forming an intralayer modulated HNF phase (HNFmod ), synchrotron small angle X-ray diffraction indicates that the unichiral derivatives form a dual modulated HNF phase with intra- as well as interlayer modulations (HNFmod2 ) suggesting a columnar local structure of the nanofilaments. Transmission electron microscopy and circular dichroism spectropolarimetry confirm that the unichiral materials exclusively form homochiral HNFs with a twist sense-matching secondary twist. A contact preparation provides the first example of two identical chiral liquid crystal phases only differing in their handedness that do not mix and form an achiral liquid crystal phase with an entirely different structure in the contact zone.

Force generation by skeletal muscle is controlled by mechanosensing in myosin filaments.

Contraction of both skeletal muscle and the heart is thought to be controlled by a calcium-dependent structural change in the actin-containing thin filaments, which permits the binding of myosin motors from the neighbouring thick filaments to drive filament sliding. Here we show by synchrotron small-angle X-ray diffraction of frog (Rana temporaria) single skeletal muscle cells that, although the well-known thin-filament mechanism is sufficient for regulation of muscle shortening against low load, force generation against high load requires a second permissive step linked to a change in the structure of the thick filament. The resting (switched 'OFF') structure of the thick filament is characterized by helical tracks of myosin motors on the filament surface and a short backbone periodicity. This OFF structure is almost completely preserved during low-load shortening, which is driven by a small fraction of constitutively active (switched 'ON') myosin motors outside thick-filament control. At higher load, these motors generate sufficient thick-filament stress to trigger the transition to its long-periodicity ON structure, unlocking the major population of motors required for high-load contraction. This concept of the thick filament as a regulatory mechanosensor provides a novel explanation for the dynamic and energetic properties of skeletal muscle. A similar mechanism probably operates in the heart.

Thermomyces lanuginosus lipase-catalyzed hydrolysis of the lipid cubic liquid crystalline nanoparticles

In this study well-ordered glycerol monooleate (GMO)-based cubic liquid crystalline nanoparticles (LCNPs) have been used as substrates for Thermomyces lanuginosus lipase in order to establish the relation between the catalytic activity, measured by pH-stat titration, and the change in morphology and nanostructure determined by cryogenic transmission electron microscopy and synchrotron small angle X-ray diffraction. The initial lipase catalyzed LCNP hydrolysis rate is approximately 25% higher for large 350nm nanoparticles compared to the small 190nm particles, which is attributed to the increased number of structural defects on the particle surface. At pH 8.0 and 8.4 bicontinuous Im3m cubic LCNPs transform into “sponge”-like assemblies and disordered multilamellar onion-like structures upon exposure to lipase. At pH 6.5 and 7.5 lipolysis induced phase transitions of the inner core of the particles, following the sequence Im3m cubic → reversed hexagonal → reversed micellar Fd3m cubic → reversed micelles. These transitions to the liquid crystalline phases with higher negative curvature of the lipid/water interface were found to trigger protonation of the oleic acid produced during lipase catalyzed reaction. The increase curvature of the reversed discrete micellar cubic phase was suggested to cause an increase in the oleic acid pKa to a larger value observed by pH-stat titration.

Preparation and characterization of quercetin-loaded lipid liquid crystalline systems

The aim of the present study was to investigate mixtures of soy phosphatidylcholine (SPC) and glycerol dioleate (GDO) as encapsulation matrices for antioxidant quercetin. The effects of quercetin loading into non-aqueous formulations, non-lamellar liquid crystalline phases and their colloidal dispersions were studied by using synchrotron small angle X-ray diffraction, dynamic light scattering, cryogenic electron microscopy and high performance liquid chromatography. Quercetin incorporation is discussed in the context of lipid aggregation behavior, self-assembled nanostructure and chemical stability. The obtained results show that SPC/GDO-based formulations can incorporate relatively high amounts of quercetin and serve as liquid crystalline delivery vehicles in the form of bulk phases or colloidal dispersions.

Intrafibrillar plasticity through mineral/collagen sliding is the dominant mechanism for the extreme toughness of antler bone

The inelastic deformability of the mineralised matrix in bones is critical to their high toughness, but the nanoscale mechanisms are incompletely understood. Antler is a tough bone type, with a nanostructure composed of mineralised collagen fibrils ∼100nm diameter. We track the fibrillar deformation of antler tissue during cyclic loading using in situ synchrotron small-angle X-ray diffraction (SAXD), finding that residual strain remains in the fibrils after the load was removed. During repeated unloading/reloading cycles, the fibril strain shows minimal hysteresis when plotted as a function of tissue strain, indicating that permanent plastic strain accumulates inside the fibril. We model the tensile response of the mineralised collagen fibril by a two – level staggered model – including both elastic – and inelastic regimes – with debonding between mineral and collagen within fibrils triggering macroscopic inelasticity. In the model, the subsequent frictional sliding at intrafibrillar mineral/collagen interfaces accounts for subsequent inelastic deformation of the tissue in tension. The model is compared to experimental measurements of fibrillar and mineral platelet strain during tensile deformation, measured by in situ synchrotron SAXD and wide-angle X-ray diffraction (WAXD) respectively, as well as macroscopic tissue stress and strain. By fitting the model predictions to experimentally observed parameters like the yield point, elastic modulus and post-yield slope, extremely good agreement is found between the model and experimental data at both the macro- and at the nanoscale. Our results provide strong evidence that intrafibrillar sliding between mineral and collagen leads to permanent plastic strain at both the fibril and the tissue level, and that the energy thus dissipated is a significant factor behind the high toughness of antler bone.

Electrochemical dissolution of chalcopyrite: Detection of bornite by synchrotron small angle X-ray diffraction and its correlation with the hindered dissolution process

The formation of bornite (Cu 5FeS 4), an iron-deficient sulfide, and its correlation with the slow oxidation rate of chalcopyrite (CuFeS 2) in acidic media under atmospheric conditions is demonstrated. Chalcopyrite electrodes oxidized in 0.1 mol/L H 2SO 4 solutions at room temperature (25 ± 1 °C) were analyzed by micro Raman spectroscopy and synchrotron small angle X-ray diffraction (S-SAXRD), techniques indicated for thin films analysis. Anodic polarization curves of chalcopyrite electrodes showed two well-defined behaviors: quasi-potential-independent regime and potential-dependent regime. When the critical potential (Ec) is attained, which ranged from 0.75 to 0.90 V vs. Standard Hydrogen Electrode (SHE), the mineral oxidation rate becomes strongly dependent on potential. Potentiostatic current-time profiles at 0.60 and 0.70 V vs. SHE indicated a current decay, which suggests the formation of a progressively thickening protective layer. The profiles at 0.80 V vs. SHE showed a similar current decay, but also an active oxidation process for some samples. After chronoamperometry at 0.70 and 0.80 V vs. SHE, respectively, for 6 and 2 h, analyses by using S-SAXRD revealed peaks of bornite on oxidized chalcopyrite electrodes. Elemental sulfur (S 8) was also detected by S-SAXRD at 0.80 and 1.00 V vs. SHE, respectively, for 2 and 0.5 h. An unidentified metal-deficient phase and covellite (CuS) were detected on chalcopyrite by micro Raman spectroscopy after chronoamperometry at 0.60 and 0.80 V vs. SHE, respectively. The formation or absence of these product phases under a constant applied potential correlated well with a hindered dissolution or active oxidation processes. The results of this work support the hypothesis that the formation of intermediate iron-deficient sulfides contributes to the slow oxidation rate of chalcopyrite under atmospheric conditions.

Inhomogeneous fibril stretching in antler starts after macroscopic yielding: Indication for a nanoscale toughening mechanism

Antler is a unique mineralized tissue, with extraordinary toughness as well as an ability to annually regenerate itself in its entirety. The high fracture resistance enables it to fulfill its biological function as a weapon and defensive guard during combats between deer stags in the rutting season. However, very little is quantitatively understood about the structural origin of the antler's high toughness. We used a unique combination of time-resolved synchrotron small angle X-ray diffraction together with tensile testing of antler cortical tissue under physiologically wet conditions. We measured the deformation at the nanoscale from changes in the meridional diffraction pattern during macroscopic stretch-to-failure tests. Our results show that on average fibrils are strained only half as much as the whole tissue and the fibril strain increases linearly with tissue strain, both during elastic and inelastic deformation. Most remarkably, following macroscopic yielding we observe a straining of some fibrils equal to the macroscopic tissue strain while others are hardly stretched at all, indicating an inhomogeneous fibrillar strain pattern at the nanoscale. This behavior is unlike what occurs in plexiform bovine bone and may explain the extreme toughness of antler compared to normal bone.