Observation of femtosecond X-ray interactions with matter using an X-ray-X-ray pump-probe scheme.

Resolution in the X-ray structure determination of noncrystalline samples has been limited to several tens of nanometers, because deep X-ray irradiation required for enhanced resolution causes radiation damage to samples. However, theoretical studies predict that the femtosecond (fs) durations of X-ray free-electron laser (XFEL) pulses make it possible to record scattering signals before the initiation of X-ray damage processes; thus, an ultraintense X-ray beam can be used beyond the conventional limit of radiation dose. Here, we verify this scenario by directly observing femtosecond X-ray damage processes in diamond irradiated with extraordinarily intense (∼10(19) W/cm(2)) XFEL pulses. An X-ray pump-probe diffraction scheme was developed in this study; tightly focused double-5-fs XFEL pulses with time separations ranging from sub-fs to 80 fs were used to excite (i.e., pump) the diamond and characterize (i.e., probe) the temporal changes of the crystalline structures through Bragg reflection. It was found that the pump and probe diffraction intensities remain almost constant for shorter time separations of the double pulse, whereas the probe diffraction intensities decreased after 20 fs following pump pulse irradiation due to the X-ray-induced atomic displacement. This result indicates that sub-10-fs XFEL pulses enable conductions of damageless structural determinations and supports the validity of the theoretical predictions of ultraintense X-ray-matter interactions. The X-ray pump-probe scheme demonstrated here would be effective for understanding ultraintense X-ray-matter interactions, which will greatly stimulate advanced XFEL applications, such as atomic structure determination of a single molecule and generation of exotic matters with high energy densities.

X-ray irradiation induces local rearrangement of silica particles in swollen rubber.

X-ray photon correlation spectroscopy (XPCS) of swollen rubber containing spherical silica nanoparticles is reported. It is shown that irradiation by intense X-rays leads to the breakdown of cross-links, thereby inducing the local rearrangement of silica nanoparticles. This rearrangement process depends on the cross-link density and is characterized by a compressed exponential relaxation with aging behaviour, which resembles a common feature of complex fluids observed with XPCS.

Dynamic photoinduced realignment processes in photoresponsive block copolymer films: effects of the chain length and block copolymer architecture.

A series of block copolymers composed of an amorphous poly(butyl methacrylate) (PBMA) block connected with an azobenzene (Az)-containing liquid crystalline (PAz) block were synthesized by changing the chain length and polymer architecture. With these block copolymer films, the dynamic realignment process of microphase separated (MPS) cylinder arrays of PBMA in the PAz matrix induced by irradiation with linearly polarized light was studied by UV-visible absorption spectroscopy, and time-resolved grazing incidence small angle X-ray scattering (GI-SAXS) measurements using a synchrotron beam. Unexpectedly, the change in the chain length hardly affected the realignment rate. In contrast, the architecture of the AB-type diblock or the ABA-type triblock essentially altered the realignment feature. The strongly cooperative motion with an induction period before realignment was characteristic only for the diblock copolymer series, and the LPL-induced alignment change immediately started for triblock copolymers and the PAz homopolymer. Additionally, a marked acceleration in the photoinduced dynamic motions was unveiled in comparison with a thermal randomization process.

Pinhole-type two-dimensional ultra-small-angle X-ray scattering on the micrometer scale.

A pinhole-type two-dimensional ultra-small-angle X-ray scattering set-up at a so-called medium-length beamline at SPring-8 is reported. A long sample-to-detector distance, 160.5 m, can be used at this beamline and a small-angle resolution of 0.25 µm(-1) was thereby achieved at an X-ray energy of 8 keV.

Hydrophobic molecules infiltrating into the poly(ethylene glycol) domain of the core/shell interface of a polymeric micelle: evidence obtained with anomalous small-angle X-ray scattering.

Polymeric micelles have been extensively studied as nanoscale drug carriers. Knowing the inner structure of polymeric micelles that encapsulate hydrophobic drugs is important to design effective carriers. In our study, the hydrophobic compound tetrabromocathecol (TBC) was chosen as a drug-equivalent model molecule. The bromine atoms in TBC act as probes in anomalous small-angle X-ray scattering (ASAXS) allowing for its localization in the polymeric micelles whose shape and size were determined by normal small-angle X-ray scattering (SAXS). Light scattering measurements coupled with field flow fractionation were also carried out to determine the aggregation number of micelles. A core-corona spherical model was used to explain the shape of the micelles, while the distribution of bromine atoms was explained with a hard-sphere model. Interestingly, the radius of the spherical region populated with bromine atoms was larger than the one of the sphere corresponding to the hydrophobic core of the micelle. This result suggests that the TBC molecules infiltrate the PEG hydrophilic domain in the vicinity of the core/shell interface. The results of light scattering and SAXS indicate that the PEG chains at the shell region are densely packed, and thus the PEG domain close to the interface has enough hydrophobicity to tolerate the presence of hydrophobic compounds.

Combined measurement of X-ray photon correlation spectroscopy and diffracted X-ray tracking using pink beam X-rays.

Combined X-ray photon correlation spectroscopy (XPCS) and diffracted X-ray tracking (DXT) measurements of carbon-black nanocrystals embedded in styrene-butadiene rubber were performed. From the intensity fluctuation of speckle patterns in a small-angle scattering region (XPCS), dynamical information relating to the translational motion can be obtained, and the rotational motion is observed through the changes in the positions of DXT diffraction spots. Graphitized carbon-black nanocrystals in unvulcanized styrene-butadiene rubber showed an apparent discrepancy between their translational and rotational motions; this result seems to support a stress-relaxation model for the origin of super-diffusive particle motion that is widely observed in nanocolloidal systems. Combined measurements using these two techniques will give new insights into nanoscopic dynamics, and will be useful as a microrheology technique.

Effect of shot noise on X-ray speckle visibility spectroscopy.

X-ray speckle visibility spectroscopy (XSVS) is a method for studying dynamics in disordered systems. This method was originally developed in the visible-light region, in which an intense laser can be used. When applied in the X-ray region, where the number of photons is much smaller than in the visible-light region, it suffers from photon statistics. In this paper, we quantitatively discuss the effect of photon shot noise on XSVS analyses. The effect is experimentally confirmed using sequential speckle patterns from Brownian polystyrene nanospheres in glycerol.

Indirectly illuminated X-ray area detector for X-ray photon correlation spectroscopy.

An indirectly illuminated X-ray area detector is employed for X-ray photon correlation spectroscopy (XPCS). The detector consists of a phosphor screen, an image intensifier (microchannel plate), a coupling lens and either a CCD or CMOS image sensor. By changing the gain of the image intensifier, both photon-counting and integrating measurements can be performed. Speckle patterns with a high signal-to-noise ratio can be observed in a single shot in the integrating mode, while XPCS measurement can be performed with much fewer photons in the photon-counting mode. By switching the image sensor, various combinations of frame rate, dynamic range and active area can be obtained. By virtue of these characteristics, this detector can be used for XPCS measurements of various types of samples that show slow or fast dynamics, a high or low scattering intensity, and a wide or narrow range of scattering angles.

Changes in structure and geometric properties of human hair by aging.

To clarify hair changes by aging, the effect of age on hair properties was investigated from macro- to microscopic view points. Sensory hair luster tests were performed on 230 Japanese females from 10 to 70 years of age, revealing that hair luster decreases with age. The age dependence of the hair diameter and the ellipticity of the hair cross section could not explain luster reduction by aging. It has been determined that an irregular increase in fiber curvature occurs with age and is a cause of luster reduction with aging. A detailed structural analysis by synchrotron radiation microbeam X-ray diffraction revealed that the inhomogeneity in the lateral distribution of the hair microstructure increased with age and relates to the irregular increase in curvature. Such an increase in curvature is one of the important factors that leads to a poor alignment of hairs and luster reduction, and is related to the appearance of aging hair.

Influence of nucleotide effectors on the kinetics of the quaternary structure transition of allosteric aspartate transcarbamylase.

We report the effects of allosteric effectors, ATP, CTP and UTP on the kinetics of the quaternary structure change of Escherichia coli ATCase during the enzyme reaction with physiological substrates. Time-resolved, small-angle, X-ray scattering of solutions allows direct observation of structural transitions over the entire time-course of the enzyme reaction initiated by fast mixing of the enzyme and substrates. In the absence of effectors, all scattering patterns recorded during the reaction are consistent with a two-state, concerted transition model, involving no detectable intermediate conformation that differs from the less active, unliganded T-state and the more active, substrate-bound R-state. The latter predominates during the steady-state phase of enzyme catalysis, while the initial T-state is recovered after substrate consumption. The concerted character of the structural transition is preserved in the presence of all effectors. CTP slightly shifts the dynamical equilibrium during a shortened steady state toward T while the additional presence of UTP makes the steady state vanishingly short. The return transition to the T conformation is slowed significantly in the presence of inhibitors, the effect being most severe in the presence of UTP. While ATP increases the apparent T to R rate, it also increases the duration of the steady-state phase, an apparently paradoxical observation. This observation can be accounted for by the greater increase in the association rate constant of aspartate, promoted by ATP, while the nucleotide produces a lesser degree of increase in the dissociation rate constant. Under our experimental conditions, using high concentrations of both enzyme and substrate, it appears that this very mechanism of activation turns the activator into an efficient inhibitor. The scattering patterns recorded in the presence of ATP support the view that ATP alters the quaternary structure of the substrate-bound enzyme, an effect reminiscent of the reported modification of PALA-bound R-state by Mg-ATP.

Helical and expanded conformation of equine beta-lactoglobulin in the cold-denatured state.

The thermal unfolding transition of equine beta-lactoglobulin (ELG) was investigated by circular dichroism (CD) over a temperature range of -15 degrees C to 85 degrees C. In the presence of 2 M urea, a cooperative unfolding transition was observed both with increasing and decreasing temperature. The CD spectrum indicated that the heat and cold-denatured states of ELG have substantial secondary structures but lack persistent tertiary packing of the side-chains. In order to clarify the relation between the heat or cold-denatured state and the acid-denatured (A) state characterized previously, we have attempted to observe the temperature dependence of the CD spectrum at pH 1.5. The CD spectrum in the heat-denatured state is similar to that in the A state. The CD spectrum in the A state does not change cooperatively with increasing temperature. These results indicate that the heat-denatured state and the A state are the same structural state. On the other hand, the CD intensity at acid pH cooperatively increased with decreasing temperature. The CD spectrum at low temperature and acid pH is consistent with that in the cold-denatured state. Therefore, the cold-denatured state is distinguished from the heat-denatured state or the A state, and ELG assumes a larger amount of non-native alpha-helices in the cold-denatured state. Small angle X-ray scattering and analytical ultracentrifugation have indicated that ELG assumes an expanded chain-like conformation in the cold-denatured state in contrast to the compact globular conformation in the A state. The relation between the molecular size and the helical content in the partially folded states is discussed.

Volume Phase Transitions of Slide-Ring Gels.

The volume phase transition of slide-ring gels with freely-movable cross-linking junctions was investigated. Ionic chemical gels with fixed cross-linking junctions undergo volume phase transitions when they have higher than the critical degree of ionization. However, the experimentally-observed critical ionization value for slide-ring gels is much higher than theoretical values for chemical gels. This difference indicates that the volume phase transition is significantly suppressed in slide-ring gels. The mesoscale structure at various swollen or shrunken states was also investigated by small angle X-ray scattering. Changes in the scattering patterns with shrinking slide-ring gels suggest microphase separation due to the sliding of cyclic molecules threaded along the axis of the polymer chains, which may suppress the volume phase transition. In addition, slide-ring gels absorbed/desorbed greater than equilibrium volumes in the shrinking/swelling processes and showed slow dynamics; these observations are also related to their sliding properties.

Fast compaction of alpha-lactalbumin during folding studied by stopped-flow X-ray scattering.

To monitor the fast compaction process during protein folding, we have used a stopped-flow small-angle X-ray scattering technique combined with a two-dimensional charge-coupled device-based X-ray detector that makes it possible to improve the signal-to-noise ratio of data dramatically, and measured the kinetic refolding reaction of alpha-lactalbumin. The results clearly show that the radius of gyration and the overall shape of the kinetic folding intermediate of alpha-lactalbumin are the same as those of the molten globule state observed at equilibrium. Thus, the identity between the kinetic folding intermediate and the equilibrium molten globule state is firmly established. The present results also suggest that the folding intermediate is more hydrated than the native state and that the hydrated water molecules are dehydrated when specific side-chain packing is formed during the change from the molten globule to the native state.

Equilibrium and kinetics of the allosteric transition of GroEL studied by solution X-ray scattering and fluorescence spectroscopy.

We have studied the ATP-induced allosteric structural transition of GroEL using small angle X-ray scattering and fluorescence spectroscopy in combination with a stopped-flow technique. With X-ray scattering one can clearly distinguish the three allosteric states of GroEL, and the kinetics of the transition of GroEL induced by 85 microM ATP have been observed directly by stopped-flow X-ray scattering for the first time. The rate constant has been found to be 3-5s(-1) at 5 degrees C, indicating that this process corresponds to the second phase of the ATP-induced kinetics of tryptophan-inserted GroEL measured by stopped-flow fluorescence. Based on the ATP concentration dependence of the fluorescence kinetics, we conclude that the first phase represents bimolecular non-cooperative binding of ATP to GroEL with a bimolecular rate constant of 5.8 x 10(5)M(-1)s(-1) at 25 degrees C. Considering the electrostatic repulsion between negatively charged GroEL (-18 of the net charge per monomer at pH 7.5) and ATP, the rate constant is consistent with a diffusion-controlled bimolecular process. The ATP-induced fluorescence kinetics (the first and second phases) at various ATP concentrations (< 400 microM) occur before ATP hydrolysis by GroEL takes place and are well explained by a kinetic allosteric model, which is a combination of the conventional transition state theory and the Monod-Wyman-Changeux model, and we have successfully evaluated the equilibrium and kinetic parameters of the allosteric transition, including the binding constant of ATP in the transition state of GroEL.

Characterizing transverse coherence of an ultra-intense focused X-ray free-electron laser by an extended Young's experiment.

Characterization of transverse coherence is one of the most critical themes for advanced X-ray sources and their applications in many fields of science. However, for hard X-ray free-electron laser (XFEL) sources there is very little knowledge available on their transverse coherence characteristics, despite their extreme importance. This is because the unique characteristics of the sources, such as the ultra-intense nature of XFEL radiation and the shot-by-shot fluctuations in the intensity distribution, make it difficult to apply conventional techniques. Here, an extended Young's interference experiment using a stream of bimodal gold particles is shown to achieve a direct measurement of the modulus of the complex degree of coherence of XFEL pulses. The use of interference patterns from two differently sized particles enables analysis of the transverse coherence on a single-shot basis without a priori knowledge of the instantaneous intensity ratio at the particles. For a focused X-ray spot as small as 1.8 µm (horizontal) × 1.3 µm (vertical) with an ultrahigh intensity that exceeds 10(18) W cm(-2) from the SPring-8 Ångstrom Compact free-electron LAser (SACLA), the coherence lengths were estimated to be 1.7 ±â€…0.2 µm (horizontal) and 1.3 ±â€…0.1 µm (vertical). The ratios between the coherence lengths and the focused beam sizes are almost the same in the horizontal and vertical directions, indicating that the transverse coherence properties of unfocused XFEL pulses are isotropic. The experiment presented here enables measurements free from radiation damage and will be readily applicable to the analysis of the transverse coherence of ultra-intense nanometre-sized focused XFEL beams.

Denaturation and reassembly of chaperonin GroEL studied by solution X-ray scattering.

We measured the denaturation and reassembly of Escherichia coli chaperonin GroEL using small-angle solution X-ray scattering, which is a powerful technique for studying the overall structure and assembly of a protein in solution. The results of the urea-induced unfolding transition show that GroEL partially dissociates in the presence of more than 2 M urea, cooperatively unfolds at around 3 M urea, and is in a monomeric random coil-like unfolded structure at more than 3.2 M urea. Attempted refolding of the unfolded GroEL monomer by a simple dilution procedure is not successful, leading to formation of aggregates. However, the presence of ammonium sulfate and MgADP allows the fully unfolded GroEL to refold into a structure with the same hydrodynamic dimension, within experimental error, as that of the native GroEL. Moreover, the X-ray scattering profiles of the GroEL thus refolded and the native GroEL are coincident with each other, showing that the refolded GroEL has the same structure and the molecular mass as the native GroEL. These results demonstrate that the fully unfolded GroEL monomer can refold and reassemble into the native tetradecameric structure in the presence of ammonium sulfate and MgADP without ATP hydrolysis and preexisting chaperones. Therefore, GroEL can, in principle, fold and assemble into the native structure according to the intrinsic characteristic of its polypeptide chain, although preexisting GroEL would be important when the GroEL folding takes place under in vivo conditions, in order to avoid misfolding and aggregation.

X-ray four-quadrant diamond phase-retarder system to compensate for off-axis and chromatic aberrations.

An X-ray transmission-type double phase-retarder system that can compensate for off-axis aberration (phase-shift inhomogeneity due to angular divergence of incident X-rays) has been developed and its advantage over a conventional single transmission-type X-ray phase retarder was demonstrated. However, it was noticed that the transmission-type X-ray phase retarder suffers from not only off-axis aberration but also chromatic aberration (phase-shift inhomogeneity due to energy spread of incident X-rays). In this paper, a transmission-type X-ray four-quadrant phase-retarder system is proposed that can compensate for both off-axis and chromatic aberrations. The X-ray four-quadrant phase-retarder system is composed of four transmission-type X-ray phase retarders. The scattering planes of four phase-retarder diamond crystals are set to be inclined by 45 degree, 135 degree (= 45 degree + 90 degree), 225 degree (= 45 degree + 180 degree) and 315 degree (= 45 degree + 270 degree), respectively, with respect to the plane of incident polarization. Under the conditions of 7709 eV photon energy, 1.5 eV energy spread and 45" angular divergence (FWHM) of incident X-rays, the X-ray four-quadrant phase-retarder system created 0.98 degree of vertical-linear polarization from horizontal-linear polarization. This value was favorably compared to 0.89 and 0.96, which were obtained by the single and double phase-retarder systems, respectively. The principle of the X-ray four-quadrant phase-retarder system will be described together with its advantage over the single and double phase-retarder systems.

Cross Nucleation in Polyethylene with Precisely Spaced Ethyl Branches.

In cross nucleation, an early nucleating crystalline polymorph (A) nucleates another crystalline polymorph (B) of higher or lower thermodynamic stability without undergoing a polymorphic transformation. Although this phenomenon was recently observed in the crystallization process of several small molecules, there has been insufficient evidence for cross nucleation in a crystalline polymer. In this paper, we report cross nucleation behavior during an isothermal crystallization of a crystalline polymer with precisely spaced branches. Polyethylene with ethyl branches on every 21st carbon exhibited growth of new spherulites at the growth front of an initially formed spherulite. The radial growth rate of the initially formed spherulite and the newly grown spherulite calculated from polarized optical microscope data were 0.76 µm/min and 1.01 µm/min, respectively. The growth rate of the newly grown spherulite is faster than that of the initially formed spherulite, which meets a required condition for cross nucleation. Scanning microbeam wide-angle X-ray scattering (WAXS) confirmed that the crystalline polymorphs of the two kinds of spherulites are not the same.