Wall-free droplet microfluidics for probing biological processes by high-brilliance X-ray scattering techniques.

Here we review probing biological processes initiated by the deposition of droplets on surfaces by micro- and nanobeam X-ray scattering techniques using synchrotron radiation and X-ray free-electron laser sources. We review probing droplet evaporation on superhydrophobic surfaces and reactions with substrates, basics of droplets deposition and flow simulations, droplet deposition techniques and practical experience at a synchrotron beamline. Selected applications with biological relevance will be reviewed and perspectives for the latest generation of high-brilliance X-ray sources discussed.

Microstructural metrology of tobacco mosaic virus nanorods during radial compression and heating.

We determined stress-induced deformations and the thermal stability of nanorod-shaped tobacco mosaic virus (TMV) capsids in coffee-ring structures by X-ray nanodiffraction. The hexagonal capsids lattice transforms under compression in the outer boundary zone of the coffee-ring into a tetragonal lattice. The helical pitch of the nanorods increases by about 2.5% across the outer boundary zone while the lateral distance between nanorods decreases continuously across the whole coffee-ring structure by about 2% due to compressive forces. The diffraction patterns show a mixture of helical scattering and Bragg peaks attributed to a lattice of nanorods interlocked by their helical grooves. Thermo-nanodiffraction reveals water loss up to about 100 °C resulting in a reduction of the helical pitch by about 6% with respect to its maximum value and a reduction of the nanorods separation by about 0.5 nm. Up to about 200 °C the pitch is increasing again by about 2%. Secondary crystallization in the bulk reaches a maximum at 150-160 °C. At higher temperatures the crystallinity is continuously decreasing up to about 220 °C. Above about 200 °C and depending on the heating history, the nanorods start disintegrating into small, randomly oriented aggregates.

Persistence and variation in microstructural design during the evolution of spider silk.

The extraordinary mechanical performance of spider dragline silk is explained by its highly ordered microstructure and results from the sequences of its constituent proteins. This optimized microstructural organization simultaneously achieves high tensile strength and strain at breaking by taking advantage of weak molecular interactions. However, elucidating how the original design evolved over the 400 million year history of spider silk, and identifying the basic relationships between microstructural details and performance have proven difficult tasks. Here we show that the analysis of maximum supercontracted single spider silk fibers using X ray diffraction shows a complex picture of silk evolution where some key microstructural features are conserved phylogenetically while others show substantial variation even among closely related species. This new understanding helps elucidate which microstructural features need to be copied in order to produce the next generation of biomimetic silk fibers.

Identification and dynamics of polyglycine II nanocrystals in Argiope trifasciata flagelliform silk.

Spider silks combine a significant number of desirable characteristics in one material, including large tensile strength and strain at breaking, biocompatibility, and the possibility of tailoring their properties. Major ampullate gland silk (MAS) is the most studied silk and their properties are explained by a double lattice of hydrogen bonds and elastomeric protein chains linked to polyalanine ß-nanocrystals. However, many basic details regarding the relationship between composition, microstructure and properties in silks are still lacking. Here we show that this relationship can be traced in flagelliform silk (Flag) spun by Argiope trifasciata spiders after identifying a phase consisting of polyglycine II nanocrystals. The presence of this phase is consistent with the dominant presence of the -GGX- and -GPG- motifs in its sequence. In contrast to the passive role assigned to polyalanine nanocrystals in MAS, polyglycine II nanocrystals can undergo growing/collapse processes that contribute to increase toughness and justify the ability of Flag to supercontract.

Minor ampullate silks from Nephila and Argiope spiders: tensile properties and microstructural characterization.

The mechanical behavior and microstructure of minor ampullate gland silk (miS) of two orb-web spinning species, Argiope trifasciata and Nephila inaurata, were extensively characterized, enabling detailed comparison with other silks. The similarities and differences exhibited by miS when compared with the intensively studied major ampullate gland silk (MAS) and silkworm (Bombyx mori) silk offer a genuine opportunity for testing some of the hypotheses proposed to correlate microstructure and tensile properties in silk. In this work, we show that miSs of different species show similar properties, even when fibers spun by spiders that diverged over 100 million years are compared. The tensile properties of miS are comparable to those of MAS when tested in air, significantly in terms of work to fracture, but differ considerably when tested in water. In particular, miS does not show a supercontraction effect and an associated ground state. In this regard, the behavior of miS in water is similar to that of B. mori silk, and it is shown that the initial elastic modulus of both fibers can be explained using a common model. Intriguingly, the microstructural parameters measured in miS are comparable to those of MAS and considerably different from those found in B. mori. This fact suggests that some critical microstructural information is still missing in our description of silks, and our results suggest that the hydrophilicity of the lateral groups or the large scale organization of the sequences might be routes worth exploring.

Structure and flow of droplets on solid surfaces.

The structure and flow of droplets on solid surfaces is investigated with imaging and scattering techniques and compared to simulations. To access nanostructures at the liquid-solid interface advanced scattering techniques such as grazing incidence small-angle x-ray scattering (GISAXS) with micro- and nanometer-sized beams, GISAXS and in situ imaging ellipsometry and GISAXS tomography are used. Using gold nanoparticle suspensions, structures observed in the wetting area due to deposition are probed in situ during the drying of the droplets. After drying, nanostructures in the wetting area and inside the dried droplets are monitored. In addition to drying, a macroscopic movement of droplets is caused by body forces acting on an inclined substrate. The complexity of the solid surfaces is increased from simple silicon substrates to binary polymer brushes, which undergo a switching due to the liquid in the droplet. Nanostructures introduced in the polymer brush due to the movement of droplets are observed.

Raster microdiffraction with synchrotron radiation of hydrated biopolymers with nanometre step-resolution: case study of starch granules.

X-ray radiation damage propagation is explored for hydrated starch granules in order to reduce the step resolution in raster-microdiffraction experiments to the nanometre range. Radiation damage was induced by synchrotron radiation microbeams of 5, 1 and 0.3 µm size with ∼0.1 nm wavelength in B-type potato, Canna edulis and Phajus grandifolius starch granules. A total loss of crystallinity of granules immersed in water was found at a dose of ∼1.3 photons nm(-3). The temperature dependence of radiation damage suggests that primary radiation damage prevails up to about 120 K while secondary radiation damage becomes effective at higher temperatures. Primary radiation damage remains confined to the beam track at 100 K. Propagation of radiation damage beyond the beam track at room temperature is assumed to be due to reactive species generated principally by water radiolysis induced by photoelectrons. By careful dose selection during data collection, raster scans with 500 nm step-resolution could be performed for granules immersed in water.

Colloidal silver nanoparticle gradient layer prepared by drying between two walls of different wettability.

A one-dimensional silver (Ag) nanoparticle gradient layer is prepared from an aqueous colloidal solution upon a polystyrene (PS) coated silicon (Si) substrate. For preparation two walls of different wettability are used. The 40 nm PS-layer exhibits a locally constant film thickness due to the strong roughness correlation with the underlying Si-substrate and is less wettable as compared to the glass plate placed above. The Ag nanoparticles have a triangular prism-like shape. The structural characterization of the obtained complex gradient formed by drying is performed with microbeam grazing incidence small-angle x-ray scattering based on compound refractive lenses. Due to the adsorption from aqueous solution in the selective geometry a double gradient type structure defined by two areas with characteristic lateral lengths and a cross-over regime between both is observed.

Synchrotron radiation microdiffraction of ballistic molten wax microdrops.

Using stroboscopic techniques, diffraction patterns of ballistic paraffin wax microdrops have been observed. The microdrops, generated by a high-temperature ink-jet system, travel through the 1 mum synchrotron radiation beam with a speed of about 1.4 m/s. Diffraction patterns were recorded in flight by a charge couple device with a microchannel plate image intensifier stage, which was activated with the microdrop generation frequency of 1000 Hz during 2 mus. The data show liquid microdrops with a constant temperature up to 8 mm from the ink-jet system capillary exit. The general technique could be adapted for studying fast structural processes, such as protein conformational changes in aqueous microdrops.

Coherent x-ray diffraction imaging with nanofocused illumination.

Coherent x-ray diffraction imaging is an x-ray microscopy technique with the potential of reaching spatial resolutions well beyond the diffraction limits of x-ray microscopes based on optics. However, the available coherent dose at modern x-ray sources is limited, setting practical bounds on the spatial resolution of the technique. By focusing the available coherent flux onto the sample, the spatial resolution can be improved for radiation-hard specimens. A small gold particle (size <100 nm) was illuminated with a hard x-ray nanobeam (E=15.25 keV, beam dimensions approximately 100 x 100 nm2) and is reconstructed from its coherent diffraction pattern. A resolution of about 5 nm is achieved in 600 s exposure time.

Thermal behavior of Bombyx mori silk: evolution of crystalline parameters, molecular structure, and mechanical properties.

The thermal behavior up to degradation of Bombyx mori silk has been studied by scanning synchrotron radiation microdiffraction, gel electrophoresis, and mechanical testing. The diffraction patterns from single baves can be separated into scattering from anisotropic crystalline beta-sheet domains and random short-range order. In contrast to dragline silk, scattering from oriented, short-range-order fibroin is not observed. The sheath of sericin proteins can be selectively probed by a microbeam and shows also principally random short-range-order domains with a small crystalline beta-sheet fraction. Microdiffraction experiments on single baves from 100 to 573 K show an increase in lattice expansion along the [010] chain-stacking direction above 200-250 K, which could be due to an increase in side-chain mobility. Degradation of the crystalline fraction commences at approximately 500 K, and the fibers have become amorphous at about 570 K with an onset of carbonization. Gel electrophoresis shows that the degradation of FibH molecules starts already at about 350 K, while FibL molecules start degrading at about 400 K. The mechanical properties of single baves such as strain-to-failure and tensile strength also start degrading at about 400 K, while the initial modulus increases up to about 475 K. It is proposed that this is due to the development of cross-linking in the short-range-order chain fraction.

High-pressure potato starch granule gelatinization: synchrotron radiation micro-SAXS/WAXS using a diamond anvil cell.

Potato starch granules have been examined by synchrotron radiation small- and wide-angle scattering in a diamond anvil cell (DAC) up to 750 MPa. Use of a 1 microm synchrotron radiation beam allowed the mapping of individual granules at several pressure levels. The data collected at 183 MPa show an increase in the a axis and lamellar period from the edge to the center of the granule, probably due to a gradient in water content of the crystalline and amorphous lamellae. The average granules radius increases up to the onset of gelatinization at about 500 MPa, but the a axis and the lamellar periodicity remain constant or even show a decrease, suggesting an initial hydration of amorphous growth rings. The onset of gelatinization is accompanied by (i) an increase in the average a axis and lamellar periodicity, (ii) the appearance of an equatorial SAXS streak, and (iii) additional short-range order peaks.

Na-cellulose formation in a single cotton fiber studied by synchrotron radiation microdiffraction.

A cotton fiber was kept under slight tension and exposed locally to a stream of aqueous 1 N NaOH microdrops of 50 microm diameter. The resulting "macrodrop" of about 300 microm size was at the origin of the formation of Na-cellulose I domains extending about 550 microm from the center of the macrodrop along the fiber. The phase transformation zone between cellulose I and Na-cellulose I was mapped by scanning synchrotron radiation microdiffraction using a 300 nm x 300 nm beam. A stitching technique was used to limit radiation damage. Subsequent exposure of the NaOH containing macrodrop to a stream of H2O or HCl microdrops converted part of the Na-cellulose I back into cellulose I.

Characterizing the decay of ancient Chinese silk fabrics by microbeam synchrotron radiation diffraction.

Scanning synchrotron radiation microdiffraction with an approximately 1 x 1 microm(2) beam has been used as a novel method for characterizing the decay of several T'ang dynasty (618-907 AD) silk fabrics. The crystalline fraction could be visualized based on beta-sheet 210 reflection intensities, extracted by recursive peak fits from several thousand diffraction patterns recorded during mesh scans. The azimuthal width of the 210 reflection, which is related to the orientation distribution of the crystalline domains within nanofibrils and the macroscopic orientation of the fibers traversed by the beam, was found to be sensitive to the overall state of decay of the fabric. The fine structure of the histogram of azimuthal width was related to the fiber hierarchical microstructure and the fabric morphology. SAXS/WAXS analysis supports the assumption of an initial loss of the random chain network with decay. At a subsequent state of aging, decay proceeds into the nanofibrils and the silk fibers break up into even smaller fractions.

Direct observation of nanocrystallite buckling in carbon fibers under bending load.

Single carbon fibers are deformed in bending by forming loops with varying radius. Position-resolved x-ray diffraction patterns from the bent fibers are collected from the tension to the compression region with a synchrotron radiation nanobeam of 100 nm size from a waveguide structure. A strain redistribution with a shift of the neutral axis is observed. A significant increase of the misorientation of the graphene sheets in the compression region shows that intense buckling of the nanosized carbon crystallites is the physical origin of different tensile and compressive properties.

Structural processes during starch granule hydration by synchrotron radiation microdiffraction.

Starch granule hydration has been examined on the level of a single potato starch granule by static and dynamic synchrotron radiation (SR) microdiffraction techniques. A cryofrozen, hydrated granule was mapped through a 5 microm SR-beam in order to investigate its internal organization. The edge of the granule showed fiber texture scattering due to radially oriented amylopectin helices. The variation of fiber texture across the granule center supports the model of concentric shells. The crystalline phase appears, however, to increase strongly toward the granule center due to a random amylopectin fraction, which could be related to crystallization of short-range ordered amylopectin during hydration. During gelatinization, the shell structure breaks down and remaining fiber-textured amylopectin domains belong probably to the swollen starch granule envelope. Hydration of a granule was initiated by a microdrop generator and followed in situ by SR-microdiffraction. A fast hydration process with a half time of about 7 s seems to reflect the porous nature of starch granules. The size of the hydrated domains suggests that this process is limited to the level of amylopectin side chain clusters. Longer hydration times are assumed to involve remaining short-range ordered amylopectin and results in larger domains.

Recent synchrotron radiation microdiffraction experiments on polymer and biopolymer fibers.

The status of synchrotron radiation (SR) microdiffraction techniques developed at the ID13 beamline of the European Synchrotron Radiation Facility (ESRF) is reviewed for polymer and biopolymer fiber applications. Beam sizes in the micrometer-range have been used to study the local structure of whole fibers such as viscose-rayon or poly(p-phenylene terephthalamide). The possibilities for in situ studies during stretching, extrusion, or indentation will be discussed.

Two-dimensional x-ray waveguides and point sources.

We show that resonant coupling of synchrotron beams into suitable nanostructures can be used for the generation of coherent x-ray point sources. A two-dimensionally confining x-ray waveguide structure has been fabricated by e-beam lithography. By shining a parallel undulator beam onto the structure, a discrete set of resonant modes can be excited in the dielectric cavity, depending on the two orthogonal coupling angles between the beam and the waveguide interfaces. The resonant excitation of the modes is evidenced from the characteristic set of coupling angles as well as the observed far-field pattern. The x-ray nanostructure may be used as coherent x-ray point sources with a beam cross section in the nanometer range.

Spider silk fibre extrusion: combined wide- and small-angle X-ray microdiffraction experiments.

The major and minor ampullate silks from live Nephila senegalensis (Tetragnathidae) and the major ampullate silk from Euprostenops spp. (Pisauridae) spiders were investigated in situ by X-ray diffraction during forced silking. Wide- (WAXS) and small-angle (SAXS) scattering patterns were obtained at the same time. WAXS data show that the thread at the exit of the spigots already contains beta-sheet poly(alanine) crystallites. SAXS data suggest the presence of microfibrils with an axial repeating period of approximately 8 nm for both Nephila and Euprostenops. Minor ampullate (MI) Nephila silk, however, does not show this axial repeat which is probably due to a higher amount of crystal forming poly(alanine). A microfibrillar morphology, connected by a network of random polymer chains, can explain the presence of highly oriented crystallites, an oriented halo and a diffuse background in the WAXS patterns. At high reeling speeds, bound water is co-extruded with the fibre. It can be squeezed out of the fibre by friction at a needle. Under natural conditions it is the spider's tarsal claws which might serve to squeeze out the water to improve the mechanical properties of the thread during dragline production.

Microstructural homogeneity of support silk spun by Eriophora fuliginea (C.L. Koch) determined by scanning X-ray microdiffraction.

Scanning X-ray microdiffraction (SXD) permits the 'imaging' in-situ of crystalline phases, crystallinity and texture in whole biopolymer samples on the micrometre scale. SXD complements transmission electron microscopy (TEM) techniques, which reach sub-nanometre lateral resolution but require thin sections and a vacuum environment. This is demonstrated using a support thread from a web spun by the orb-weaving spider Eriophora fuliginea (C.L. Koch). Scanning electron microscopy (SEM) shows a central thread composed of two fibres to which thinner fibres are loosely attached. SXD of a piece of support thread approximately 60 microns long shows in addition the presence of nanometre-sized crystallites with the beta-poly(L-alanine) structure in all fibres. The crystallinity of the thin fibres appears to be higher than that of the central thread, which probably reflects a higher polyalanine content of the fibroins. The molecular axis of the polymer chains in the central thread is orientated parallel to the macroscopic fibre axis, but in the thin fibres the molecular axis is tilted by about 71 degrees to the macroscopic fibre axis. A helical model is tentatively proposed to describe this morphology. The central thread has a homogeneous distribution of crystallinity along the macroscopic fibre axis.