Time-resolved Small-angle X-ray Research Articles

Molecular Weight Dependence of Block Copolymer Micelle Fragmentation Kinetics.

The effect of molecular weight (M) on the fragmentation kinetics of micelles formed by 1,2-polybutadiene-block-poly(ethylene oxide) (PB-PEO) copolymers was studied in the ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide. A series of six samples, with total M ranging from 104 to 105 g mol-1 and nearly constant composition (fPEO ≈ 0.4), were examined; all six formed spherical micelles with PEO coronas. Nonequilibrium PB-PEO micelles were prepared by direct dissolution, a process that systematically produces nanoparticles with mean aggregation numbers more than twice the equilibrium values. When subjected to high temperature annealing (170 °C), the average micelle radius was found to decrease substantially, as determined by temperature-jump dynamic light scattering (T-jump DLS) and time-resolved small-angle X-ray scattering (TR-SAXS). The characteristic fragmentation times (τ) were found to increase strongly with increasing degree of polymerization N, as τ ∼ N1.8. This result compares favorably with the prediction of a previously untested model.

Synchrotron Microbeam Diffraction Studies on the Alignment within 3D-Printed Smectic-A Liquid Crystal Elastomer Filaments during Extrusion

3D printing of novel and smart materials has received considerable attention due to its applications within biological and medical fields, mostly as they can be used to print complex architectures and particular designs. However, the internal structure during 3D printing can be problematic to resolve. We present here how time-resolved synchrotron microbeam Small-Angle X-ray Diffraction (μ-SAXD) allows us to elucidate the local orientational structure of a liquid crystal elastomer-based printed scaffold. Most reported 3D-printed liquid crystal elastomers are mainly nematic; here, we present a Smectic-A 3D-printed liquid crystal elastomer that has previously been reported to promote cell proliferation and alignment. The data obtained on the 3D-printed filaments will provide insights into the internal structure of the liquid crystal elastomer for the future fabrication of liquid crystal elastomers as responsive and anisotropic 3D cell scaffolds.

Shear-Modulated Rates of Phase Transitions in Sphere-Forming Diblock Oligomer Lyotropic Liquid Crystals.

Hydration of the amphiphilic diblock oligomer C16H33(CH2CH2O)20OH (C16E20) leads to concentration-dependent formation of micellar body-centered cubic (BCC) and Frank-Kasper A15 lyotropic liquid crystals (LLCs). Quiescent thermal annealing of aqueous LLCs comprising 56-59 wt % C16E20 at 25 °C after quenching from high temperatures established their ability to form short-lived BCC phases, which transform into long-lived, transient Frank-Kasper σ phases en route to equilibrium A15 morphologies on a time scale of months. Here, the frequency and magnitude of applied oscillatory shear show the potential to either dynamically stabilize the metastable BCC phase at low frequencies or increase the rate of formation of the A15 to minutes at high frequencies. Time-resolved synchrotron small-angle X-ray scattering (TR-SAXS) provides in situ characterization of the structures during shear and thermal processing. This work shows that the LLC morphology and order-order phase transformation rates can be controlled by tuning the shear strain amplitude and frequency.

"Host in Host" Supramolecular Core-Shell Type Systems Based on Giant Ring-Shaped Polyoxometalates.

Herein, we show how the chaotropic effect arising from reduced molybdate ions in acidified aqueous solution is able to amplify drastically weak supramolecular interactions. Time-resolved Small Angle X-ray Scattering (SAXS) analysis suggests that molybdenum-blue oligomeric species form huge aggregates in the presence of γ-cyclodextrin (γ-CD) which results in the fast formation of nanoscopic {Mo154 }-based host-guest species, while X-ray diffraction analysis reveals that the ending-point of the scenario results in an unprecedented three-component well-ordered core-shell-like motif. A similar arrangement was found by using preformed hexarhenium chalcogenide-type cluster [Re6 Te8 (CN)6 ]4- as exogenous guest. This seminal work brings better understanding of the self-assembly processes in general and gives new opportunities for practical applications in the design of complex multicomponent materials via the simplicity of the non-covalent chemistry.

Interplays of electron and nuclear motions along CO dissociation trajectory in myoglobin revealed by ultrafast X-rays and quantum dynamics calculations

Ultrafast structural dynamics with different spatial and temporal scales were investigated during photodissociation of carbon monoxide (CO) from iron(II)-heme in bovine myoglobin during the first 3 ps following laser excitation. We used simultaneous X-ray transient absorption (XTA) spectroscopy and X-ray transient solution scattering (XSS) at an X-ray free electron laser source with a time resolution of 80 fs. Kinetic traces at different characteristic X-ray energies were collected to give a global picture of the multistep pathway in the photodissociation of CO from heme. In order to extract the reaction coordinates along different directions of the CO departure, XTA data were collected with parallel and perpendicular relative polarizations of the laser pump and X-ray probe pulse to isolate the contributions of electronic spin state transition, bond breaking, and heme macrocycle nuclear relaxation. The time evolution of the iron K-edge X-ray absorption near edge structure (XANES) features along the two major photochemical reaction coordinates, i.e., the iron(II)-CO bond elongation and the heme macrocycle doming relaxation were modeled by time-dependent density functional theory calculations. Combined results from the experiments and computations reveal insight into interplays between the nuclear and electronic structural dynamics along the CO photodissociation trajectory. Time-resolved small-angle X-ray scattering data during the same process are also simultaneously collected, which show that the local CO dissociation causes a protein quake propagating on different spatial and temporal scales. These studies are important for understanding gas transport and protein deligation processes and shed light on the interplay of active site conformational changes and large-scale protein reorganization.

Restoring size of detonation nanodiamonds from small-angle x-ray scattering of polychromatic synchrotron radiation beam

Over the past two decades, LIH SB RAS and BINP SB RAS have jointly conducted experiments on time-resolved (TR) small-angle x-ray scattering (SAXS) with detonating high explosives. The purpose of these experiments is to restore the dynamics of carbon species condensation to diamond nanoparticles by analyzing series of SAXS patterns behind the detonation front measured in real time with fast detectors. This knowledge is crucial for the development of reliable detonation models. In this paper, we compare SAXS patterns of identical nanodiamond samples measured at the TR-SAXS extreme state of matter end-station (BINP SB RAS) in the static mode under realistic conditions simulating fast real-time measurements with polychromatic SR beam and traditional SAXS BioMUR beamline at the Kurchatov Synchrotron Radiation Source (NRC “Kurchatov Institute”) with monochromatic synchrotron radiation (SR) beam. These experiments confirm that the size of scattering inhomogeneities determined in dynamic experiments with single bunch exposure with polychromatic SR beam is correct.

Kinetic and Equilibrium Measurement of Nucleation of Prion-Like Domain Phase Separation

Compartmentalization by liquid-liquid phase separation (LLPS) has emerged as a unifying theme in biology and as a mechanism for organizing biomolecules in space and time. LLPS can provide a pathway for cells to rapidly respond to changes in their environment. Under biologically relevant conditions, LLPS likely proceeds through nucleation-and-growth and the rate at which phase separated compartments can form is determined by the nucleation barrier. To characterize the nature of the energetic barrier to nucleation, we have combined equilibrium techniques that define the phase boundaries of a prototypical prion-like domain and the size distribution of clusters below the nucleation barrier with rapid mixing time-resolved small-angle x-ray scattering (SAXS).

Time-Resolved Small-Angle X-ray Scattering Studies during Aqueous Emulsion Polymerization.

The persulfate-initiated aqueous emulsion polymerization of 2,2,2-trifluoroethyl methacrylate (TFEMA) is studied by time-resolved small-angle X-ray scattering (SAXS) at 60 °C using a stirrable reaction cell. TFEMA was preferred to styrene because it offers much greater X-ray scattering contrast relative to water, which is essential for sufficient temporal resolution. The evolution in particle size is monitored by both in situ SAXS and ex situ DLS in the absence or presence of an anionic surfactant (sodium dodecyl sulfate, SDS). Post-mortem SAXS studies confirmed the formation of well-defined spherical latexes, with volume-average diameters of 353 ± 9 nm and 68 ± 4 nm being obtained for the surfactant-free and SDS formulations, respectively. 1H NMR spectroscopy studies of the equivalent laboratory-scale formulations indicated TFEMA conversions of 99% within 80 min and 93% within 60 min for the surfactant-free and SDS formulations, respectively. Comparable polymerization kinetics are observed for the in situ SAXS experiments and the laboratory-scale syntheses, with nucleation occurring after approximately 6 min in each case. After nucleation, scattering patterns are fitted using a hard sphere scattering model to determine the evolution in particle growth for both formulations. Moreover, in situ SAXS enables identification of the three main intervals (I, II, and III) that are observed during aqueous emulsion polymerization in the presence of surfactant. These intervals are consistent with those indicated by solution conductivity and optical microscopy studies. Significant differences between the surfactant-free and SDS formulations are observed, providing useful insights into the mechanism of emulsion polymerization.

Time-resolving small angle X-Ray scattering analysis of melt crystallization of mixtures of regular and irregular isotactic polypropylene samples

The melting/crystallization properties of blends obtained by mixing two isotactic polypropylene (iPP) samples synthesized using single-site metallocene catalyst systems and containing a high and low concentration of rr triads as stereo-defects, are studied. The changes occurring at lamellar length scale during a heating/cooling cycle at constant scanning rate are followed in situ by performing time-resolved small angle X-ray scattering (SAXS) measurements. Data analysis demonstrates that the evolution of the SAXS intensity with increase/decrease of the temperature is controlled by the separate melting/crystallization of the two components, the differences in the thermal expansion (contraction) coefficient of the amorphous and crystalline phases and the role of thermal fluctuations in electron density. The two components give rise to different populations of intermixed lamellar stacks in the blends which originate from the good miscibility of the low and high stereoregular samples in the melt.

Assembly of cellulose nanocrystals and clay nanoplatelets studied by time-resolved X-ray scattering.

Time-resolved small-angle X-ray scattering (SAXS) was used to probe the assembly of cellulose nanocrystals (CNC) and montmorillonite (MNT) over a wide concentration range in aqueous levitating droplets. Analysis of the SAXS curves of the one-component and mixed dispersions shows that co-assembly of rod-like CNC and MNT nanoplatelets is dominated by the interactions between the dispersed CNC particles and that MNT promotes gelation and assembly of CNC, which occurred at lower total volume fractions in the CNC:MNT than in the CNC-only dispersions. The CNC dispersions displayed a d ∝ φ-1/2 scaling and a low-q power-law exponent of 2.0-2.2 for volume fractions up to 35%, which indicates that liquid crystal assembly co-exists and competes with gelation.

Effect of Light Intensity on the Aggregation Behavior of Primary Particles during In Situ Photochemical Synthesis of Gold/Polymer Nanocomposites.

Metal/polymer nanocomposites have attracted much attention in recent years due to their exceptional properties and wide range of potential applications. A key challenge to obtain these materials is to stabilize the metal nanoparticles in the matrix, avoiding uncontrolled aggregation processes driven by the high surface free energy of nanosized particles. Here, we investigate the aggregation mechanism of primary particles in gold-epoxy nanocomposites prepared via light-assisted in situ synthesis, under different irradiation conditions. The growth and aggregation of gold nanoparticles were monitored in situ by time-resolved small-angle X-ray scattering experiments, whereas spectroscopic measurements were performed to interpret how matrix polymerization influences the aggregation process. It was found that light intensity has a greater influence on the reduction rate than on the polymerization rate. Under irradiation, gold nanostructures evolve through five time-defined stages: nuclei-mass fractals-surface fractals-spherical nanoparticles-aggregates. If the maximum in the polymerization rate is reached before the aggregation step, individual primary nanoparticles will be preserved in the polymer matrix due to diffusional constraints imposed by the reaction medium. Because the light intensity has a different influence on the reduction rate than on the polymerization rate, this parameter can be used as a versatile tool to avoid aggregation of gold nanoparticles into the polymer matrix.

Diatom-inspired self-assembly for silica thin sheets of perpendicular nanochannels

HypothesisMultistage silicate self-organization into light-weight, high-strength, hierarchically patterned diatom frustules carries hints for innovative silica-based nanomaterials. With sodium silicate in a biomimetic sol-gel system templated by a tri-surfactant system of hexadecyltrimethylammonium bromide, sodium dodecylsulfate, and poly(oxyethylene-b-oxypropylene-b-oxyethylene) (P123), mesoporous silica nanochannel plates with perpendicular channel orientation are synthesized. The formation process, analogous to that of diatom frustules, is postulated to be directed by an oriented self-assembly of the block copolymer micelles shelled with charged catanionic surfactants upon silication. ExperimentsThe postulated formation process for the oriented silica nanochannel plates was investigated using time-resolved small-angle X-ray and neutron scattering (SAXS/SANS) and freeze fracture replication transmission electron microscopy (FFR-TEM). FindingsWith fine-tuned molar ratios of the anionic, cationic, and nonionic surfactants, the catanionic combination and the nonionic copolymer form charged, prolate ternary micelles in aqueous solutions, which further develop into prototype monolayered micellar plates. The prolate shape and maximized surfactant adsorption of the complex micelles, revealed from combined SAXS/SANS analysis, are of critical importance in the subsequent micellar self-assembly upon silicate deposition. Time-resolved SAXS and FFR-TEM indicate that the silicate complex micelles coalesce laterally into the prototype micellar nanoplates, which further fuse with one another into large sheets of monolayered silicate micelles of in-plane lamellar packing. Upon silica polymerization, the in-plane lamellar packing of the micelles further transforms to 2D hexagonal packing of vertically oriented silicate channels. The unveiled structural features and their evolution not only elucidate the previously unresolved self-assembly process of through-thickness silica nanochannels but also open a new line of research mimicking free-standing frustules of diatoms.

Nonsymmetrical Dynamics of the HBV Capsid Assembly and Disassembly Evidenced by Their Transient Species.

As with many protein multimers studied in biophysics, the assembly and disassembly dynamical pathways of hepatitis B virus (HBV) capsid proteins are not symmetrical. Using time-resolved small-angle X-ray scattering and singular value decomposition analysis, we have investigated these processes in vitro by a rapid change of salinity or chaotropicity. Along the assembly pathway, the classical nucleation-growth mechanism is followed by a slow relaxation phase during which capsid-like transient species self-organize in accordance with the theoretical prediction that the capture of the few last subunits is slow. By contrast, the disassembly proceeds through unexpected, fractal-branched clusters of subunits that eventually vanish over a much longer time scale. On the one hand, our findings confirm and extend previous views as to the hysteresis phenomena observed and theorized in capsid formation and dissociation. On the other hand, they uncover specifics that may directly relate to the functions of HBV subunits in the viral cycle.

Spatiotemporal Formation and Growth Kinetics of Polyelectrolyte Complex Micelles with Millisecond Resolution.

We have directly observed the in situ self-assembly kinetics of polyelectrolyte complex (PEC) micelles by synchrotron time-resolved small-angle X-ray scattering, equipped with a stopped-flow device that provides millisecond temporal resolution. A synthesized neutral-charged diblock polycation and homopolyanion that we have previously investigated as a model charge-matched, core-shell micelle system were selected for this work. The initial micellization of the oppositely charged polyelectrolytes was completed within the dead time of mixing of 100 ms, followed by micelle growth and equilibration up to several seconds. By combining the structural evolution of the radius of gyration (Rg) with complementary molecular dynamics simulations, we show how the self-assemblies evolve incrementally in size over time through a two-step kinetic process: first, oppositely charged polyelectrolyte chains pair to form nascent aggregates that immediately assemble into spherical micelles, and second, these PEC micelles grow into larger micellar entities. This work has determined one possible kinetic pathway for the initial formation of PEC micelles, which provides useful physical insights for increasing fundamental understanding self-assembly dynamics, driven by polyelectrolyte complexation that occurs on ultrafast time scales.

Open-Closed Structure of Light-Responsive Protein LOV2 Regulates Its Molecular Interaction with a Binding Partner.

Optogenetic approaches have broad applications, including regulating cell signaling and gene expression. Photoresponsive protein LOV2 and its binding partner ZDK represent an important protein caging/uncaging optogenetic system. Herein, we combine time-resolved small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) to reveal different structural states of LOV2 and the light-controlled mechanism of interaction between LOV2 and ZDK. In response to blue light within a time frame of ca. 70 s, LOV2 has a significantly higher value of radius of gyration Rg (29.6 ± 0.3 vs 26.4 ± 0.4 Å) than its dark state, suggesting unwinding of the C-terminal Jα-helix into an open structure. Atomic force microscopy was used to characterize molecular interactions of LOV2 in open and closed states with ZDK at a single-molecule level. The closed state of LOV2 enables strong binding with ZDK, characterized by a 60-fold lower dissociation rate and a ∼1.5-times higher activation energy barrier than for its open state. In combination, these data support a light-switching mechanism that is modulated by the proximity of multiple binding sites of LOV2 for ZDK.

Two-Stage Assembly of Mesocrystal Fibers with Tunable Diameters in Weak Magnetic Fields.

Controlling the morphology and crystallographic coherence of assemblies of magnetic nanoparticles is a promising route to functional materials. Time-resolved small-angle X-ray scattering (SAXS) was combined with microscopy and scaling analysis to probe and analyze evaporation-induced assembly in levitating drops and thin films of superparamagnetic iron oxide nanocubes in weak magnetic fields. We show that assembly of micrometer-sized mesocrystals with a cubic shape preceded the formation of fibers with a high degree of crystallographic coherence and tunable diameters. The second-stage assembly of aligned cuboidal mesocrystals into fibers was driven by the magnetic field, but the first-stage assembly of the oleate-capped nanocubes was unaffected by weak magnetic fields. The transition from 3D growth of the primary mesocrystals to the second stage 1D assembly of the elongated fibers was related to the size and field dependence of isotropic van der Waals and directional dipolar interactions between the interacting mesocrystals.

Myosin dynamics during relaxation in mouse soleus muscle and modulation by 2'-deoxy-ATP.

Skeletal muscle relaxation has been primarily studied by assessing the kinetics of force decay. Little is known about the resultant dynamics of structural changes in myosin heads during relaxation. The naturally occurring nucleotide 2-deoxy-ATP (dATP) is a myosin activator that enhances cross-bridge binding and kinetics. X-ray diffraction data indicate that with elevated dATP, myosin heads were extended closer to actin in relaxed muscle and myosin heads return to an ordered, resting state after contraction more quickly. Molecular dynamics simulations of post-powerstroke myosin suggest that dATP induces structural changes in myosin heads that increase the surface area of the actin-binding regions promoting myosin interaction with actin, which could explain the observed delays in the onset of relaxation. This study of the dATP-induced changes in myosin may be instructive for determining the structural changes desired for other potential myosin-targeted molecular compounds to treat muscle diseases. Here we used time-resolved small-angle X-ray diffraction coupled with force measurements to study the structural changes in FVB mouse skeletal muscle sarcomeres during relaxation after tetanus contraction. To estimate the rate of myosin deactivation, we followed the rate of the intensity recovery of the first-order myosin layer line (MLL1) and restoration of the resting spacing of the third and sixth order of meridional reflection (SM3 and SM6 ) following tetanic contraction. A transgenic mouse model with elevated skeletal muscle 2-deoxy-ATP (dATP) was used to study how myosin activators may affect soleus muscle relaxation. X-ray diffraction evidence indicates that with elevated dATP, myosin heads were extended closer to actin in resting muscle. Following contraction, there is a slight but significant delay in the decay of force relative to WT muscle while the return of myosin heads to an ordered resting state was initially slower, then became more rapid than in WT muscle. Molecular dynamics simulations of post-powerstroke myosin suggest that dATP induces structural changes in myosin that increase the surface area of the actin-binding regions, promoting myosin interaction with actin. With dATP, myosin heads may remain in an activated state near the thin filaments following relaxation, accounting for the delay in force decay and the initial delay in recovery of resting head configuration, and this could facilitate subsequent contractions.

Growth Kinetics of Core–Shell Au/Ag Nanoparticles

We study by synchrotron-based time-resolved small angle x-ray scattering the growth of silver shells on gold nanorods in the presence of hexadecyltrimethylammonium chloride (CTAC) as a function of ...

Interfacial and Activation Energies of Environmentally Abundant Heterogeneously Nucleated Iron(III) (Hydr)oxide on Quartz.

Poorly crystalline iron(III) (hydr)oxide nanoparticles are ubiquitous in environmental systems and play a crucial role in controlling the fate and transport of contaminants. Yet, the thermodynamic and kinetic parameters, e.g., the effective interfacial (α') and apparent activation (Ea) energies, of iron(III) (hydr)oxide nucleation on earth-abundant mineral surfaces have not been determined, which hinders an accurate prediction of iron(III) (hydr)oxide formation and its interactions with other toxic or reactive ions. Here, for the first time, we report experimentally obtained α' and Ea for iron(III) (hydr)oxide nucleation on quartz mineral surfaces by employing a flow-through, time-resolved grazing incidence small-angle X-ray scattering (GISAXS). GISAXS enabled the in situ detection of iron(III) (hydr)oxide nucleation rates under different supersaturations (σ, achieved by varying pH 3.3-3.6) and temperatures (12-35 °C). By quantitative analyses based on classical nucleation theory, α' was obtained to be 34.6 mJ/m2 and Ea was quantified as 32.8 kJ/mol. The fundamental thermodynamic and kinetic parameters obtained here will advance our fundamental understanding of the surface chemistry and nucleation behavior of iron(III) (hydr)oxides in subsurface and water treatment systems as well as their effects on the fate and transport of pollutants in natural and engineered water systems. The in situ flow-through GISAXS method can also be adapted to quantify thermodynamic and kinetic parameters at interfaces for many important solid-liquid environmental systems.

Assembly of Capsids from Hepatitis B Virus Core Protein Progresses through Highly Populated Intermediates in the Presence and Absence of RNA.

The genetic material of viruses is protected by protein shells that are assembled from a large number of subunits in a process that is efficient and robust. Many of the mechanistic details underpinning efficient assembly of virus capsids are still unknown. The assembly mechanism of hepatitis B capsids has been intensively researched using a truncated core protein lacking the C-terminal domain responsible for binding genomic RNA. To resolve the assembly intermediates of hepatitis B virus (HBV), we studied the formation of nucleocapsids and empty capsids from full-length hepatitis B core proteins, using time-resolved small-angle X-ray scattering. We developed a detailed structural model of the HBV capsid assembly process using a combination of analysis with multivariate curve resolution, structural modeling, and Bayesian ensemble inference. The detailed structural analysis supports an assembly pathway that proceeds through the formation of two highly populated intermediates, a trimer of dimers and a partially closed shell consisting of around 40 dimers. These intermediates are on-path, transient and efficiently convert into fully formed capsids. In the presence of an RNA oligo that binds specifically to the C-terminal domain the assembly proceeds via a similar mechanism to that in the absence of nucleic acids. Comparisons between truncated and full-length HBV capsid proteins reveal that the unstructured C-terminal domain has a significant impact on the assembly process and is required to obtain a more complete mechanistic understanding of HBV capsid formation. These results also illustrate how combining scattering information from different time-points during time-resolved experiments can be utilized to derive a structural model of protein self-assembly pathways.