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While silk fibers produced by silkworms and spiders are frequently described as a network of amorphous protein chains reinforced by crystalline β-sheet nanodomains, the importance of higher-order, self-assembled structures has been recognized for advanced modeling of mechanical properties. General acceptance of hierarchical structural models is, however, currently limited by lack of experimental results. Indeed, X-ray scattering studies of spider’s dragline-type fibers have been particularly limited by low crystallinities. Here we are reporting on probing the local structure of exceptionally crystalline bagworm silk fibers by X-ray nanobeam scattering. Probing the comparable thickness of cross sections with an X-ray nanobeam allows removing the strong scattering background from the outer sericin layer and reveals a hidden structural organization due to a radial gradient in diameters of mesoscale nanofibrillar bundles in the fibroin phase. Our results provide direct support for lateral interactions between nanofibrils.

Phycocyanin molecules, which are part of light-harvesting complexes in cyanobacteria, can be assembled into mesoscale multilayer nanofilms by the Langmuir–Blodgett technique. Results obtained by quartz crystal microbalance and atomic force microscopy confirm the homogeneity and reproducibility of phycocyanin Langmuir–Blodgett multilayer deposition. We show by cryo-electron microdiffraction that amorphous phycocyanin Langmuir–Blodgett multilayers form, after annealing at 150 °C and cooling to room temperature, a layered nanofibrillar lattice with rotational disorder. Scanning X-ray nanodiffraction suggests that structural transformation is not homogeneous through the film but limited to patches of up to about 10 μm diameter.

Amyloid β (Aβ) peptides are the main constituents of Alzheimer’s amyloid plaques in the brain. Here we report how the unique microfluidic flows exerted by droplets sitting on superhydrophobic surfaces can influence the aggregation mechanisms of several Aβ fragments by boosting their fibril self-assembly. Aβ(25–35), Aβ(1–40), and Aβ(12–28) were dried both on flat hydrophilic surfaces (contact angle (CA) = 37.3°) and on nanostructured superhydrophobic ones (CA = 175.8°). By embedding nanoroughened surfaces on top of highly X-ray transparent Si3N4membranes, it was possible to probe the solid residues by raster-scan synchrotron radiation X-ray microdiffraction (μXRD). As compared to residues obtained on flat Si3N4membranes, a general enhancement of fibrillar material was detected for all Aβ fragments dried on superhydrophobic surfaces, with a particular emphasis on the shorter ones. Indeed, both Aβ(25–35) and Aβ(12–28) showed a marked crystalline cross-β phase with varying fiber textures. The homogeneous evaporation rate provided by these nanostructured supports, and the possibility to use transparent membranes, can open a wide range of in situX-ray and spectroscopic characterizations of amyloidal peptides involved in neurodegenerative diseases and for the fabrication of amyloid-based nanodevices.

The evaporation of single droplets of colloidal tobacco mosaic virus (TMV) nanoparticles on a superhydrophobic surface with a hexagonal pillar-pattern results in the formation of coffee-ring type residues. We imaged surface features by optical, scanning electron, and atomic force microscopies. Bulk features were probed by raster-scan X-ray nanodiffraction. At ∼100 pg/μL nanoparticle concentration, the rim of the residue connects to neighboring pillars via fibrous extensions containing flow-aligned crystalline domains. At ∼1 pg/μL nanoparticle concentration, nanofilaments of ≥80 nm diameter and ∼20 μm length are formed, extending normal to the residue-rim across a range of pillars. X-ray scattering is dominated by the nanofilament form-factor but some evidence for crystallinity has been obtained. The observation of sheets composed of stacks of self-assembled nanoparticles deposited on pillars suggests that the nanofilaments are drawn from a structured droplet interface.

The formation of thaumatin crystals by Langmuir-Blodgett (LB) film nanotemplates was studied by the hanging-drop technique in a flow-through cell by synchrotron radiation micrograzing-incidence small-angle x-ray scattering. The kinetics of crystallization was measured directly on the interface of the LB film crystallization nanotemplate. The evolution of the micrograzing-incidence small-angle x-ray scattering patterns suggests that the increase in intensity in the Yoneda region is due to protein incorporation into the LB film. The intensity variation suggests several steps, which were modeled by system dynamics based on first-order differential equations. The kinetic data can be described by two processes that take place on the LB film, a first, fast, process, attributed to the crystal growth and its detachment from the LB film, and a second, slower process, attributed to an unordered association and conversion of protein on the LB film.

In this study, we used microbeam grazing-incidence small-angle x-ray scattering (μGISAXS) to investigate in situ protein nucleation and crystal growth assisted by a protein nanotemplate, and introduced certain innovations to improve the method. Our aim was to understand the protein nanotemplate method in detail, as this method has been shown to be capable of accelerating and increasing crystal size and quality, as well as inducing crystallization of proteins that are not crystallizable by classical methods. The nanotemplate experimental setup was used for drops containing growing protein crystals at different stages of nucleation and growth. Two model proteins, lysozyme and thaumatin, were used under unique flow conditions to differentially probe protein crystal nucleation and growth.

This study reports on the investigation of kink bands within a single high performance fiber. Using microfocus XRD in a scanning acquisition mode, kink bands can be visualized along the length of a fiber in a manner similar to other microscopy techniques. Unlike other techniques, however, µXRD also provides access to internal structural information. For example, from the diffraction data it is possible to reconstruct the complete microgeometry of single kink bands based upon experimental results. This capability opens the door to a range of future studies. This could include tracking the formation and evolution of kink bands in situ, or studying kink bands within fibers embedded within a composite material.

A simple analytical approach to phase space analysis of the performance of x-ray optical setups (beamlines) combining several elements in position-angle-wavelength space is presented. The mathematical description of a large class of optical elements commonly used on synchrotron beamlines has been reviewed and extended with respect to the existing literature and is reported in a revised form. Novel features are introduced, in particular, the possibility to account for imperfections on mirror surfaces and to incorporate nanofocusing devices like refractive lenses in advanced beamline setups using the same analytical framework. Phase space analysis results of the simulation of an undulator beamline with focusing optics at the European Synchrotron Radiation Facility compare favorably with results obtained by geometric ray-tracing methods and, more importantly, with experimental measurements. This approach has been implemented into a simple and easy-to-use program toolkit for optical calculations based on the Mathematica software package.

Summary:The phenomenon of crazing in polymers has received considerable attention in the past as it is thought to play a pivotal role in determining the performance of polymers under load. One aspect of particular interest has been the interconnection between molecular structure, craze characteristics, and macromechanical properties. In the present study, three different grades of polystyrene (PS) with different molecular weights have been systematically investigated in situ with synchrotron radiation microfocus small‐angle X‐ray scattering (µSAXS). The results suggest that there are different mechanisms operating in PS samples with low and very high molecular weights, compared to those of medium‐to‐high molecular weight. Previously it was thought that, above the critical molecular weight of entanglement, the effect of molecular weight on PS's mechanical behaviour at room temperature was negligible.

Amyloid fibrils are found in association with at least two dozen fatal diseases. The tendency of numerous proteins to convert into amyloid-like fibrils poses fundamental questions for structural biology and for protein science in general. Among these are the following:  What is the structure of the cross- spine, common to amyloid-like fibrils? Is there a sequence signature for proteins that form amyloid-like fibrils? What is the nature of the structural conversion from native to amyloid states, and do fibril-forming proteins have two distinct stable states, the native state and the amyloid state? What is the basis of protein complementarity, in which a protein chain can bind to itself? We offer tentative answers here, based on our own recent structural studies.

Combined microfocus Raman and microfocus XRD is employed to probe the in situ deformation of a single poly(p-phenylene terephthalamide), PPTA, or Kevlar fiber. The experimental results are obtained from the same point on the sample simultaneously, using coaxial microfocus X-ray and laser beams. The 1610 cm-1Raman band shifts linearly to a lower wavenumber with macroscopic stress, while the crystal lattice along the fiber axis direction extends linearly. The resulting Raman band shift rate and crystal modulus are −3.3 cm-1/GPa and 214 GPa, respectively. Raman band broadening with increasing stress suggests an increasing stress heterogeneity between the crystalline and amorphous fractions. Meanwhile, meridional layer line narrowing is interpreted as a deformation-induced homogenization of the PPTA fiber's skin-core structure. These results demonstrate the benefits of combining experimental techniques in situ which offer complementary phase and volume selectivity. In this case, macroscopic fiber deformation can be qualitatively explained using low- and high-stress uniform-strain mechanical models. At low stresses, linear Raman band broadening with stress can be attributed to a phase-related difference in mechanical properties. At high stresses, an increasing degree of crystalline domain orientation causes heterogeneous stiffening within the PPTA fiber structure. Consequently, the amorphous fraction remains “understressed” during subsequent deformation.

Summary:The fracture properties of polymers are one of the key parameters that define their service life and limit their applications. One of the most interesting and important questions is how the molecular architecture and the structure of polymers at nanolength scales influence their fracture properties. X‐ray scattering is a powerful means of probing bulk structures at the nanometre scale. It can therefore provide a wealth of information relating to such structure‐property relationships. In the present study, synchrotron radiation microfocus small‐angle X‐ray scattering is used to investigate the damage area ahead and around the crack tip in polyamide 6 (PA6). The results reveal that the damage area propagates far beyond the visible crack, and inside the damaged zone platelet‐shaped cracks/voids are formed.

Abstract: Fibrous proteins found in natural materials such as silk fibroins, spider silks, and viral spikes increasingly serve as a source of inspiration for the design of novel, artificial fibrous materials. The fiber protein from the adenovirus has previously served as a model for the design of artificial, self-assembling fibers. The fibrous shaft of this protein consists of 15-amino-acid sequence repeats that fold into a triple β-spiral motif in their native context. Recombinant proteins based on multimers of simplified consensus shaft repeats were previously reported to form self-assembling fibrils from which filaments could be spun. Here, we describe the structural characterization of these fibrils; X-ray fiber diffraction, Raman spectroscopy, and Congo Red binding strongly suggest an amyloid-type structure for these fibrils, with β-strands arranged perpendicular to the fibril axis. This amyloid structure is distinct from the native β-spiral fold, and similar to amyloid structures formed by short, synthetic peptides corresponding to shaft sequences. We discuss implications for the rational design of novel fibrous materials, based on crystal structure information and knowledge of folding and assembly pathways of natural fibrous proteins.

The mechanical properties of spider dragline fibres are altered by CO 2 exposure under anaesthetizing conditions during the spinning process. In order to relate these macroscopic changes to a microscopic model, the extrusion of dragline silk was studied by synchrotron radiation microdiffraction. A brief exposure of a female Nephila senigalensis spider to CO 2results after an incubation time of less than 7 min in the extrusion of a thread (two fibres) swollen with water. The data are interpreted for a model of crystalline ß-sheet domains containing nanofibrils, which reinforce a network of protein chains. The protein network absorbs water, leaving the nanofibrils unaffected. A continuous flow of CO 2 results in a co-extrusion of a dragline thread and an isotropic silk fraction, which probably has a glycine-rich sequence. Long CO 2 exposure reduces the axial alignment of nanofibrils, presumably due to a partial destruction of the amorphous network.

Intermediate filaments (IFs) impart mechanical integrity to cells, yet IF mechanics are poorly understood. It is assumed that IFs in cells are as stiff as hard α-keratin, F-actin, and microtubules, but the high bending flexibility of IFs and the low stiffness of soft α-keratins suggest that hydrated IFs may be quite soft. To test this hypothesis, we measured the tensile mechanics of the keratin-like threads from hagfish slime, which are an ideal model for exploring the mechanics of IF bundles and IFs because they consist of tightly packed and aligned IFs. Tensile tests suggest that hydrated IF bundles possess low initial stiffness (Ei=6.4MPa) and remarkable elasticity (up to strains of 0.34), which we attribute to soft elastomeric IF protein terminal domains in series with stiffer coiled coils. The high tensile strength (180MPa) and toughness (130 MJ/m3) of IF bundles support the notion that IFs lend mechanical integrity to cells. Their long-range elasticity suggests that IFs may also allow cells to recover from large deformations. X-ray diffraction and congo-red staining indicate that post-yield deformation leads to an irreversible α→βconformational transition in IFs, which leads to plastic deformation, and may be used by cells as a mechanosensory cue.

The distribution and orientation of collagen fibrils, and apatite crystals, in the scales of a bony fish (Leuciscus cephalus) were investigated by X-ray diffraction. The small-angle diffraction patterns obtained with a microfocus scanning setup from most of the examined areas exhibit a distribution of intensity of the collagen reflections according to five preferential orientations, at 36° from one another. It is suggested that the peculiar small-angle X-ray diffraction pattern is due to a plywood arrangement of collagen fibrils in successive layers parallel to the surface of the scale. The fibrils are strictly aligned in each layer and the alignment rotates by 36° in successive layers, according to a discontinuous twist that generates a symmetric plywood pattern. The large spread of the wide-angle reflections does not allow one to distinguish the five directions of orientation in the intensity distribution of the 002 reflection of apatite. However, the patterns recorded from the less ordered regions of the scales display two different orientations of the 002 reflection and allow one to infer a preferential distribution of the apatite crystals with their c-axes parallel to the collagen fibrils. Although much electron microscopic evidence of plywood arrangements in calcified, as well as uncalcified, tissues has been reported, these are the very first diffraction data which unambiguously confirm the presence of these peculiar structures and suggest that this kind of investigation represents a powerful tool with which to study plywood arrangements in biological tissues.

A banded spherulite of bacterial poly(3-hydroxybutyrate) (band spacing 120 μm) was linearly scanned along the radial direction in 3 μm steps by means of microfocus X-ray diffraction using a synchrotron source with a beam diameter of 3 μm. A large number of X-ray patterns was collected inside each band. Those taken at the center of the bands represented the two limiting diffraction patterns from which the exact orientation of the unit cell was inferred. The intensity of the reflections (020) and (002), taken as indicators of parallelism to the X-ray beam of the unit cell's c- or b-axis, respectively, changed periodically along the spherulite radius, alternating maxima with zero-intensity zones. When the reflection (020) showed a maximum, the reflection (002) was practically nil and vice versa. This behavior clearly shows that the unit cell's mean position smoothly rotates around the a-axis with increasing distance from the spherulite center. The identity of the cell rotation period with the band spacing derived from optical microscopy observations provides structural evidence that the extinction bands originate from a regular twisting of the lamellar crystals during their growth.

A chiral side-chain liquid-crystalline polymeric model for the structure and physical properties of starch was reviewed and extended [1—6]. The lamellae in starch are considered in terms of three components: backbone, side-chains and double helices. It is the degree of mobility of these three components, coupled with the helix-coil transition, which gives starch its distinctive properties. The model is used to help explain phenomena involved during hydration, gelatinisation, plasticisation, freezing, and morphogenesis. The new technique of scanning microfocus X-ray diffraction is used to study double helix orientation and the tilt of the lamellae. Using integration of the double helix orientation along closed contours in single potato granules it is shown that they conain a “+1” hedgehog disclination. The effect of the chirality of the double helices on the phase behaviour is highlighted.

There is a growing demand for the examination of protein microcrystals at third‐generation synchrotron sources. After successful pilot experiments at EMBL/ESRF, which proved that protein microcrystals are often suitable for data collection, operation of the microfocus beamline ID13 was made more user‐friendly and suitable for macromolecular crystallography experiments. Given the excellent quality of the beamline microfocusing optics, the key element for successful experiments becomes the handling and visualization of microcrystals. To address this, a microdiffractometer has been designed to allow maximum precision combined with ease of usage and is currently under construction.

With the current interest in the use of transition metal-exchanged phyllosilicates as catalysts for novel organic syntheses, investigations into the factors which affect the movement of reactant, product, and solvent molecules into and out of their interlamellar region are of considerable importance. Mixed organic-water intercalates of a Wyoming montmorillonite, exemplified by the Na-montmorillonitepyridine-water system which can form four different intercalates exhibiting basal spacing of 29.3, 23.3, 19.4, and 14.8 Å depending on the pyridine: water ratio, have been used as a model system. X-ray and neutron diffraction and quasielastic neutron scattering data relating to the interconversion of interlayer species indicate that access to and exit from the interlayer space is hindered at high partial pressures of water by a water-film diffusion barrier in the interparticulate voids which exist between the aggregated silicate layers. At lower water vapor pressures the rate-limiting step for interconversion from one intercalate to another is the rate of transport of reagents and products to and from the clay particles. Under conditions where these rates are fixed, the rate-limiting step is the rate of diffusion of the pyridine molecule in the lower-spacing intercalate. Processes which involve a change in basal spacing do not necessarily proceed via a single discrete step, but are also affected by the amount of water made available to the system. In organic reactions catalyzed in the interlamellar space of various cation-exchanged montmorillonites (e.g., the conversion of alk-1-enes to di-2,2’-alkyl ethers and the reaction of alcohols to form ethers), rate-determining steps similar to those found above are likely to be operative. In particular, for reactions carried out in the liquid phase, where mass transport is facile and where phase-transfer problems are avoided, such reactions are likely to be diffusion controlled.

Small angle x-ray microfocus scattering patterns were obtained from 4 μm regions of hydrated potato starch granules. The 9 nm lamellar reflection was found to be modulated along two flat streaked layer lines symmetrically related, and arranged in a weak four point pattern. This provides evidence for two populations of tilted lamellae in the granule present in equal quantities, but opposite angles of tilt. The paucity of reflections in the scattering patterns implies the existence of correlated disorder between the lamellae, and is a product of either their liquid crystallinity or a well-developed defect structure between the constituent amylopectin helices. The angular position of the four point modulation (17.25°) on the scattering patterns is compared with the values predicted from a superhelical lamellar model containing cumulative disorder along its axis, and good agreement is found with the data. The physical basis for the occurrence of these helical lamellae is examined on the basis of the model of amylopectin as a chiral side chain liquid- crystalline polymer (T. A. Waigh et al., Macromolecules, 1997, Vol. 30, pp. 3813–3820; Faraday Discussions, 1996, Vol. 103, pp. 325–337). © 1999 John Wiley & Sons, Inc. Biopoly 49: 91–105, 1999

Focused X-ray beams from third generation synchrotron sources allow the possibility of data collection from previously unusable protein microcrystals of only a few microns in size.

Micro-beam X-ray diffraction has been used to investigate the texture of banded spherulites of melt-crystallized poly(trimethylene terephthalate), PTT, formed in films of approximately 30 to 50 mm thickness. The WAXS micro-diffraction patterns show that at the local scale, the PTT texture is close to that of a single crystal. In agreement with previous studies using selected-area electron diffraction, it is shown that the crystal growth direction is parallel to the a-axis of the unit cell. When plotted as a function of the distance to the spherulite center, the intensity of different diffraction peaks reveals the same periodicity. This means that the lamellar twist is strictly uniform. The latter observation is more compatible with the model explaining the twist as a result of unbalanced surface stresses than that of isochiral giant screw dislocations. The main features of the experimental diffractograms can be understood using the numerical approach, which is developed in the approximation of a purely geometric broadening of X-ray reflections. In particular, the simulation can predict the sequence of appearance of different diffraction peaks and their shape on the 2D micro-diffraction patterns.

Microbeams are now used routinely for X-ray diffraction experiments and a range of complex sample environments have been developed. Nanobeams, meanwhile, are only just starting to promote new experimental possibilities. This article discusses instrumentation and sample environments as well as selected applications of micro- and nano-diffraction at the ID13 beamline of the ESRF, with particular emphasis on polymer science.

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