3D Binary Mesocrystals from Anisotropic Nanoparticles.

Binary mesocrystals offer the combination of nanocrystal properties in an ordered superstructure. Here, we demonstrate the simultaneous self-assembly of platinum and iron oxide nanocubes into micrometer-sized 3D mesocrystals using the gas-phase diffusion technique. By the addition of minor amounts of a secondary particle type tailored to nearly identical size, shape and surface chemistry, we were able to promote a random incorporation of foreign particles into a self-assembling host lattice. The random distribution of the binary particle types on the surface and within its bulk has been visualized using advanced transmission and scanning electron microscopy techniques. The 20-40 µm sized binary mesocrystals have been further characterized through wide and small angle scattering techniques to reveal a long-range ordering on the atomic scale throughout the crystal while showing clear evidence that the material consists of individual building blocks. Through careful adjustments of the crystallization parameters, we could further obtain a reverse superstructure, where incorporated particles and host lattice switch roles.

In Situ Visualization of the Structural Evolution and Alignment of Lyotropic Liquid Crystals in Confined Flow.

Self-assembled materials such as lyotropic liquid crystals offer a wide variety of structures and applications by tuning the composition. Understanding materials behavior under flow and the induced alignment is wanted in order to tailor structure related properties. A method to visualize the structure and anisotropy of ordered systems in situ under dynamic conditions is presented where flow-induced nanostructural alignment in microfluidic channels is observed by scanning small angle X-ray scattering in hexagonal and lamellar self-assembled phases. In the hexagonal phase, the material in regions with high extensional flow exhibits orientation perpendicular to the flow and is oriented in the flow direction only in regions with a high enough shear rate. For the lamellar phase, a flow-induced morphological transition occurs from aligned lamellae toward multilamellar vesicles. However, the vesicles do not withstand the mechanical forces and break in extended lamellae in regions with high shear rates. This evolution of nanostructure with different shear rates can be correlated with a shear thinning viscosity curve with different slopes. The results demonstrate new fundamental knowledge about the structuring of liquid crystals under flow. The methodology widens the quantitative investigation of complex structures and identifies important mechanisms of reorientation and structural changes.

NanoMAX: the hard X-ray nanoprobe beamline at the MAX IV Laboratory.

NanoMAX is the first hard X-ray nanoprobe beamline at the MAX IV laboratory. It utilizes the unique properties of the world's first operational multi-bend achromat storage ring to provide an intense and coherent focused beam for experiments with several methods. In this paper we present the beamline optics design in detail, show the performance figures, and give an overview of the surrounding infrastructure and the operational diffraction endstation.

Nanostructure surveys of macroscopic specimens by small-angle scattering tensor tomography.

The mechanical properties of many materials are based on the macroscopic arrangement and orientation of their nanostructure. This nanostructure can be ordered over a range of length scales. In biology, the principle of hierarchical ordering is often used to maximize functionality, such as strength and robustness of the material, while minimizing weight and energy cost. Methods for nanoscale imaging provide direct visual access to the ultrastructure (nanoscale structure that is too small to be imaged using light microscopy), but the field of view is limited and does not easily allow a full correlative study of changes in the ultrastructure over a macroscopic sample. Other methods of probing ultrastructure ordering, such as small-angle scattering of X-rays or neutrons, can be applied to macroscopic samples; however, these scattering methods remain constrained to two-dimensional specimens or to isotropically oriented ultrastructures. These constraints limit the use of these methods for studying nanostructures with more complex orientation patterns, which are abundant in nature and materials science. Here, we introduce an imaging method that combines small-angle scattering with tensor tomography to probe nanoscale structures in three-dimensional macroscopic samples in a non-destructive way. We demonstrate the method by measuring the main orientation and the degree of orientation of nanoscale mineralized collagen fibrils in a human trabecula bone sample with a spatial resolution of 25 micrometres. Symmetries within the sample, such as the cylindrical symmetry commonly observed for mineralized collagen fibrils in bone, allow for tractable sampling requirements and numerical efficiency. Small-angle scattering tensor tomography is applicable to both biological and materials science specimens, and may be useful for understanding and characterizing smart or bio-inspired materials. Moreover, because the method is non-destructive, it is appropriate for in situ measurements and allows, for example, the role of ultrastructure in the mechanical response of a biological tissue or manufactured material to be studied.

Six-dimensional real and reciprocal space small-angle X-ray scattering tomography.

When used in combination with raster scanning, small-angle X-ray scattering (SAXS) has proven to be a valuable imaging technique of the nanoscale, for example of bone, teeth and brain matter. Although two-dimensional projection imaging has been used to characterize various materials successfully, its three-dimensional extension, SAXS computed tomography, poses substantial challenges, which have yet to be overcome. Previous work using SAXS computed tomography was unable to preserve oriented SAXS signals during reconstruction. Here we present a solution to this problem and obtain a complete SAXS computed tomography, which preserves oriented scattering information. By introducing virtual tomography axes, we take advantage of the two-dimensional SAXS information recorded on an area detector and use it to reconstruct the full three-dimensional scattering distribution in reciprocal space for each voxel of the three-dimensional object in real space. The presented method could be of interest for a combined six-dimensional real and reciprocal space characterization of mesoscopic materials with hierarchically structured features with length scales ranging from a few nanometres to a few millimetres--for example, biomaterials such as bone or teeth, or functional materials such as fuel-cell or battery components.

Validation study of small-angle X-ray scattering tensor tomography.

Small-angle scattering tensor tomography (SASTT) is a recently developed technique able to tomographically reconstruct the 3D reciprocal space from voxels within a bulk volume. SASTT extends the concept of X-ray computed tomography, which typically reconstructs scalar values, by reconstructing a tensor per voxel, which represents the local nanostructure 3D organization. In this study, the nanostructure orientation in a human trabecular-bone sample obtained by SASTT was validated by sectioning the sample and using 3D scanning small-angle X-ray scattering (3D sSAXS) to measure and analyze the orientation from single voxels within each thin section. Besides the presence of cutting artefacts from the slicing process, the nanostructure orientations obtained with the two independent methods were in good agreement, as quantified with the absolute value of the dot product calculated between the nanostructure main orientations obtained in each voxel. The average dot product per voxel over the full sample containing over 10 000 voxels was 0.84, and in six slices, in which fewer cutting artefacts were observed, the dot product increased to 0.91. In addition, SAXS tensor tomography not only yields orientation information but can also reconstruct the full 3D reciprocal-space map. It is shown that the measured anisotropic scattering for individual voxels was reproduced from the SASTT reconstruction in each voxel of the 3D sample. The scattering curves along different 3D directions are validated with data from single voxels, demonstrating SASTT's potential for a separate analysis of nanostructure orientation and structural information from the angle-dependent intensity distribution.

Controlling water evaporation through self-assembly.

Water evaporation concerns all land-living organisms, as ambient air is dryer than their corresponding equilibrium humidity. Contrarily to plants, mammals are covered with a skin that not only hinders evaporation but also maintains its rate at a nearly constant value, independently of air humidity. Here, we show that simple amphiphiles/water systems reproduce this behavior, which suggests a common underlying mechanism originating from responding self-assembly structures. The composition and structure gradients arising from the evaporation process were characterized using optical microscopy, infrared microscopy, and small-angle X-ray scattering. We observed a thin and dry outer phase that responds to changes in air humidity by increasing its thickness as the air becomes dryer, which decreases its permeability to water, thus counterbalancing the increase in the evaporation driving force. This thin and dry outer phase therefore shields the systems from humidity variations. Such a feedback loop achieves a homeostatic regulation of water evaporation.

High-acceptance versatile microfocus module based on elliptical Fresnel zone plates for small-angle X-ray scattering.

High-efficiency microfocusing of multi-keV X-rays at synchrotron sources is highly profitable for spatially resolved structural analysis of many kinds. Because radiation from synchrotron sources is typically elongated along the horizontal dimension, generating a microbeam that is isotropic in size requires a carefully designed optics system. Here we report on using a combination of a horizontally tunable slit downstream of the undulator source with elliptical diffractive Fresnel zone plates. We demonstrate the arrangement in context of small-angle X-ray scattering experiments, obtaining a microbeam of 2.2 µm × 1.8 µm (X × Y) with a flux of 1.2 × 1010 photons/s at an energy of 11.2 keV at the sample position.

Rapid Acquisition of X-Ray Scattering Data from Droplet-Encapsulated Protein Systems.

Encapsulating reacting biological or chemical samples in microfluidic droplets has the great advantage over single-phase flows of providing separate reaction compartments. These compartments can be filled in a combinatoric way and prevent the sample from adsorbing to the channel walls. In recent years, small-angle X-ray scattering (SAXS) in combination with microfluidics has evolved as a nanoscale method of such systems. Here, we approach two major challenges associated with combining droplet microfluidics and SAXS. First, we present a simple, versatile, and reliable device, which is both suitable for stable droplet formation and compatible with in situ X-ray measurements. Second, we solve the problem of "diluting" the sample signal by the signal from the oil separating the emulsion droplets by multiple fast acquisitions per droplet and data thresholding. We show that using our method, even the weakly scattering protein vimentin provides high signal-to-noise ratio data.

Intermicellar Interactions and the Viscoelasticity of Surfactant Solutions: Complementary Use of SANS and SAXS.

In ionic surfactant micelles, basic interactions among distinct parts of surfactant monomers, their counterion, and additives are fundamental to tuning molecular self-assembly and enhancing viscoelasticity. Here, we investigate the addition of sodium salicylate (NaSal) to hexadecyltrimethylammonium chloride and bromide (CTAC and CTAB) and 1-hexadecylpyridinium chloride and bromide (CPyCl and CPyBr), which have distinct counterions and headgroup structures but the same hydrophobic tail. Different contrasts are obtained from small-angle neutron scattering (SANS), which probes differences between the nucleus of atoms, and X-rays SAXS, which probes differences in electron density. If combined, this contrast allows us to define specific intramicellar length scales and intermicellar interactions. SANS signals are sensitive to the contrast between the solvent (D2O) and the hydrocarbonic tails in the micellar core (hydrogen), and SAXS can access the inner structure of the polar shell because the headgroups, counterions, and penetrated salt have higher electron densities compared to the solvent and to the micellar core. The number density, intermicellar distances, aggregation number, and inter/intramicellar repulsions are discussed on the basis of the dependence of the structure factor and form factor on the micellar aggregate morphology. Therefore, we confirm that micellar growth can be tuned by variations in the flexibility and size of the the headgroup as well as the ionic dissociation rate of its counterion. Additionally, we show that the counterion binding is even more significant to the development of viscoelasticity than the headgroup structure of a surfactant molecule. This is a surprising finding, showing the importance of electrostatic charges in the self-assembly process of ionic surfactant molecules.

Ionic micelles and aromatic additives: a closer look at the molecular packing parameter.

Wormlike micellar aggregates formed from the mixture of ionic surfactants with aromatic additives result in solutions with impressive viscoelastic properties. These properties are of high interest for numerous industrial applications and are often used as model systems for soft matter physics. However, robust and simple models for tailoring the viscoelastic response of the solution based on the molecular structure of the employed additive are required to fully exploit the potential of these systems. We address this shortcoming with a modified packing parameter based model, considering the additive-surfactant pair. The role of charge neutralization on anisotropic micellar growth was investigated with derivatives of sodium salicylate. The impact of the additives on the morphology of the micellar aggregates is explained from the molecular level to the macroscopic viscoelasticity. Changes in the micelle's volume, headgroup area and additive structure are explored to redefine the packing parameter. Uncharged additives penetrated deeper into the hydrophobic region of the micelle, whilst charged additives remained trapped in the polar region, as revealed by a combination of 1H-NMR, SAXS and rheological measurements. A deeper penetration of the additives densified the hydrophobic core of the micelle and induced anisotropic growth by increasing the effective volume of the additive-surfactant pair. This phenomenon largely influenced the viscosity of the solutions. Partially penetrating additives reduced the electrostatic repulsions between surfactant headgroups and neighboring micelles. The resulting increased network density governed the elasticity of the solutions. Considering a packing parameter composed of the additive-surfactant pair proved to be a facile means of engineering the viscoelastic response of surfactant solutions. The self-assembly of the wormlike micellar aggregates could be tailored to desired morphologies resulting in a specific and predictable rheological response.

Mastering the magnetic susceptibility of magnetically responsive bicelles with 3ß-amino-5-cholestene and complexed lanthanide ions.

The magnetic susceptibility of lanthanide-chelating bicelles was selectively enhanced by introducing 3ß-amino-5-cholestene (aminocholesterol, Chol-NH2) in the bilayer. Unprecedented magnetic alignment of the bicelles was achieved without altering their size. An aminocholesterol conjugate (Chol-C2OC2-NH2), in combination with different lanthanide ions, offers the possibility of fine-tuning the bicelle's magnetic susceptibility.

Viscoelasticity Enhancement of Surfactant Solutions Depends on Molecular Conformation: Influence of Surfactant Headgroup Structure and Its Counterion.

During the anisotropic growth from globular to wormlike micelles, the basic interactions among distinct parts of the surfactant monomer, its counterion, and additives are fundamental to tune molecular self-assembly. We investigate the addition of sodium salicylate (NaSal) to hexadecyltrimethylammonium chloride and bromide (CTAC and CTAB), 1-hexadecylpyridinium chloride and bromide (CPyCl and CPyBr), and benzyldimethylhexadecylammonium chloride (BDMC), which have the same hydrophobic tail. Their potential to enhance viscoelasticity by anisotropic micellar growth upon salt addition was compared in terms of (i) the influence of the headgroup structure, and (ii) the influence of surfactant counterion type. Employing proton nuclear magnetic resonance ((1)H NMR), we focused on the molecular conformation of surfactant monomers in the core and polar shell regions of the micelles and their interactions with increasing concentration of NaSal. The viscoelastic response was investigated by rotational and oscillatory rheology. We show that micellar growth rates can be tuned by varying the flexibility and size of the surfactant headgroup as well as the dissociation degree of the surfactant counterion, which directly influences the strength of headgroup-counterion pairing. As a consequence, the morphological transitions depend directly on charge neutralization by electrostatic screening. For example, the amount of salt necessary to start the rodlike-to-wormlike micelle growth depends directly on the number of dissociated counterions in the polar shell.

Tailoring Bicelle Morphology and Thermal Stability with Lanthanide-Chelating Cholesterol Conjugates.

Bicelles composed of DMPC and phospholipids capable of chelating lanthanide ions, such as 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA), are highly tunable magnetically responsive soft materials. Further doping of these systems with cholesterol-DTPA conjugates complexed to a lanthanide ion considerably enhances the bicelle's size and magnetic alignability. The high value of these cholesterol conjugates for bicelle design remains largely unexplored. Herein, we examine how molecular structural alterations within the cholesterol-DTPA conjugates lead to contrasting self-assembled polymolecular aggregate structures when incorporated into DMPC/DMPE-DTPA/Tm(3+) bilayers. The nature of the linker connecting the DTPA-chelating moiety to the sterol backbone is examined by synthesizing conjugates of various linker lengths and polarities. The incorporation of these compounds within the bilayer results in polymolecular aggregate geometries of higher curvature. The increasing degrees of freedom for conformational changes conveyed to the chelator headgroup with increasing linker atomic length reduce the cholesterol-DTPA conjugate's critical packing parameter. Consequently, an inverse correlation between the number of carbon atoms in the linker and the bicelle radius is established. The introduction of polarity into the carbon chain of the linker did not cause major changes in the polymolecular aggregate architecture. Under specific conditions, the additives permit the formation of remarkably temperature-resistant bicelles. The versatility of design offered by these amphiphiles gives rise to new and viable tools for the growing field of magnetically responsive soft materials.

Controlling Orientational and Translational Order of Iron Oxide Nanocubes by Assembly in Nanofluidic Containers.

We demonstrate that spatial confinement can be used to control the orientational and translational order of cubic nanoparticles. For this purpose we have combined X-ray scattering and scanning electron microscopy to study the ordering of iron oxide nanocubes that have self-assembled from toluene-based dispersions in nanofluidic channels. An analysis of scattering vector components with directions parallel and perpendicular to the slit walls shows that the confining walls induce a preferential parallel alignment of the nanocube (100) faces. Moreover, slit wall separations that are commensurate with an integer multiple of the edge length of the oleic acid-capped nanocubes result in a more pronounced translational order of the self-assembled arrays compared to incommensurate confinement. These results show that the confined assembly of anisotropic nanocrystals is a promising route to nanoscale devices with tunable anisotropic properties.

Unveiling breast cancer metastasis through an advanced X-ray imaging approach.

Breast cancer is a significant global health burden, causing a substantial number of deaths. Systemic metastatic tumour cell dissemination is a major cause of poor outcomes. Understanding the mechanisms underlying metastasis is crucial for effective interventions. Changes in the extracellular matrix play a pivotal role in breast cancer metastasis. In this work, we present an advanced multimodal X-ray computed tomography, by combining Small-angle X-ray Scattering Tensor Tomography (SAXS-TT) and X-ray Fluorescence Computed Tomography (XRF-CT). This approach likely brings out valuable information about the breast cancer metastasis cascade. Initial results from its application on a breast cancer specimen reveal the collective influence of key molecules in the metastatic mechanism, identifying a strong correlation between zinc accumulation (associated with matrix metalloproteinases MMPs) and highly oriented collagen. MMPs trigger collagen alignment, facilitating breast cancer cell intravasation, while iron accumulation, linked to angiogenesis and vascular endothelial growth factor VEGF, supports cell proliferation and metastasis. Therefore, these findings highlight the potential of the advanced multimodal X-ray computed tomography approach and pave the way for in-depth investigation of breast cancer metastasis, which may guide the development of novel therapeutic approaches and enable personalised treatment strategies, ultimately improving patient outcomes in breast cancer management.

Micro- and nanostructure specific X-ray tomography reveals less matrix formation and altered collagen organization following reduced loading during Achilles tendon healing.

Recovery of the collagen structure following Achilles tendon rupture is poor, resulting in a high risk for re-ruptures. The loading environment during healing affects the mechanical properties of the tendon, but the relation between loading regime and healing outcome remains unclear. This is partially due to our limited understanding regarding the effects of loading on the micro- and nanostructure of the healing tissue. We addressed this through a combination of synchrotron phase-contrast X-ray microtomography and small-angle X-ray scattering tensor tomography (SASTT) to visualize the 3D organization of microscale fibers and nanoscale fibrils, respectively. The effect of in vivo loading on these structures was characterized in early healing of rat Achilles tendons by comparing full activity with immobilization. Unloading resulted in structural changes that can explain the reported impaired mechanical performance. In particular, unloading led to slower tissue regeneration and maturation, with less and more disorganized collagen, as well as an increased presence of adipose tissue. This study provides the first application of SASTT on soft musculoskeletal tissues and clearly demonstrates its potential to investigate a variety of other collagenous tissues. STATEMENT OF SIGNIFICANCE: Currently our understanding of the mechanobiological effects on the recovery of the structural hierarchical organization of injured Achilles tendons is limited. We provide insight into how loading affects the healing process by using a cutting-edge approach to for the first time characterize the 3D micro- and nanostructure of the regenerating collagen. We uncovered that, during early healing, unloading results in a delayed and more disorganized regeneration of both fibers (microscale) and fibrils (nanoscale), as well as increased presence of adipose tissue. The results set the ground for the development of further specialized protocols for tendon recovery.

Iron-carbohydrate complexes treating iron anaemia: Understanding the nano-structure and interactions with proteins through orthogonal characterisation.

Intravenous (IV) iron-carbohydrate complexes are widely used nanoparticles (NPs) to treat iron deficiency anaemia, often associated with medical conditions such as chronic kidney disease, heart failure and various inflammatory conditions. Even though a plethora of physicochemical characterisation data and clinical studies are available for these products, evidence-based correlation between physicochemical properties of iron-carbohydrate complexes and clinical outcome has not fully been elucidated yet. Studies on other metal oxide NPs suggest that early interactions between NPs and blood upon IV injection are key to understanding how differences in physicochemical characteristics of iron-carbohydrate complexes cause variance in clinical outcomes. We therefore investigated the core-ligand structure of two clinically relevant iron-carbohydrate complexes, iron sucrose (IS) and ferric carboxymaltose (FCM), and their interactions with two structurally different human plasma proteins, human serum albumin (HSA) and fibrinogen, using a combination of cryo-scanning transmission electron microscopy (cryo-STEM), x-ray diffraction (XRD), small-angle x-ray scattering (SAXS) and small-angle neutron scattering (SANS). Using this orthogonal approach, we defined the nano-structure, individual building blocks and surface morphology for IS and FCM. Importantly, we revealed significant differences in the surface morphology of the iron-carbohydrate complexes. FCM shows a localised carbohydrate shell around its core, in contrast to IS, which is characterised by a diffuse and dynamic layer of carbohydrate ligand surrounding its core. We hypothesised that such differences in carbohydrate morphology determine the interaction between iron-carbohydrate complexes and proteins and therefore investigated the NPs in the presence of HSA and fibrinogen. Intriguingly, IS showed significant interaction with HSA and fibrinogen, forming NP-protein clusters, while FCM only showed significant interaction with fibrinogen. We postulate that these differences could influence bio-response of the two formulations and their clinical outcome. In conclusion, our study provides orthogonal characterisation of two clinically relevant iron-carbohydrate complexes and first hints at their interaction behaviour with proteins in the human bloodstream, setting a prerequisite towards complete understanding of the correlation between physicochemical properties and clinical outcome.

Alignment of bicelles studied with high-field magnetic birefringence and small-angle neutron scattering measurements.

Birefringence measurements at high magnetic field strength of up to 33 T were used to detect magnetically induced alignment of bicelles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-diethylenetriaminepentaacetate (DMPE-DTPA) with complexed lanthanide ions. These birefringence measurements together with a small-angle neutron scattering (SANS) analysis in a magnetic field showed parallel alignment of the bicelles if the lanthanide was thulium (Tm(3+)), and perpendicular alignment with dysprosium (Dy(3+)). With the birefringence measurements, the order parameter S can be determined as a function of the magnetic field strength, if the magnetic alignment reaches saturation. Additional structural information can be obtained if the maximum induced birefringence is considered. The degree of alignment of the studied bicelles increased with decreasing temperature from 40 to 5 °C and showed a new bicellar structure comprising a transient hole formation at intermediate temperatures (20 °C) during heating from 5 to 40 °C.

Small-angle scattering tensor tomography algorithm for robust reconstruction of complex textures.

The development of small-angle scattering tensor tomography has enabled the study of anisotropic nanostructures in a volume-resolved manner. It is of great value to have reconstruction methods that can handle many different nanostructural symmetries. For such a method to be employed by researchers from a wide range of backgrounds, it is crucial that its reliance on prior knowledge about the system is minimized, and that it is robust under various conditions. Here, a method is presented that employs band-limited spherical functions to enable the reconstruction of reciprocal-space maps of a wide variety of nanostructures. This method has been thoroughly tested and compared with existing methods in its ability to retrieve known reciprocal-space maps, as well as its robustness to changes in initial conditions, using both simulations and experimental data. It has also been evaluated for its computational performance. The anchoring of this method in a framework of integral geometry and linear algebra highlights its possibilities and limitations.