Development of the multiplex imaging chamber at PAL-XFEL.

Various X-ray techniques are employed to investigate specimens in diverse fields. Generally, scattering and absorption/emission processes occur due to the interaction of X-rays with matter. The output signals from these processes contain structural information and the electronic structure of specimens, respectively. The combination of complementary X-ray techniques improves the understanding of complex systems holistically. In this context, we introduce a multiplex imaging instrument that can collect small-/wide-angle X-ray diffraction and X-ray emission spectra simultaneously to investigate morphological information with nanoscale resolution, crystal arrangement at the atomic scale and the electronic structure of specimens.

Stochastic chromatin packing of 3D mitotic chromosomes revealed by coherent X-rays.

DNA molecules are atomic-scale information storage molecules that promote reliable information transfer via fault-free repetitions of replications and transcriptions. Remarkable accuracy of compacting a few-meters-long DNA into a micrometer-scale object, and the reverse, makes the chromosome one of the most intriguing structures from both physical and biological viewpoints. However, its three-dimensional (3D) structure remains elusive with challenges in observing native structures of specimens at tens-of-nanometers resolution. Here, using cryogenic coherent X-ray diffraction imaging, we succeeded in obtaining nanoscale 3D structures of metaphase chromosomes that exhibited a random distribution of electron density without characteristics of high-order folding structures. Scaling analysis of the chromosomes, compared with a model structure having the same density profile as the experimental results, has discovered the fractal nature of density distributions. Quantitative 3D density maps, corroborated by molecular dynamics simulations, reveal that internal structures of chromosomes conform to diffusion-limited aggregation behavior, which indicates that 3D chromatin packing occurs via stochastic processes.

Ultrafast Energy Transfer Process in Confined Gold Nanospheres Revealed by Femtosecond X-ray Imaging and Diffraction.

Femtosecond laser pulses drive nonequilibrium phase transitions via reaction paths hidden in thermal equilibrium. This stimulates interest to understand photoinduced ultrafast melting processes, which remains incomplete due to challenges in resolving accompanied kinetics at the relevant space-time resolution. Here, by newly establishing a multiplexing femtosecond X-ray probe, we have successfully revealed ultrafast energy transfer processes in confined Au nanospheres. Real-time images of electron density distributions with the corresponding lattice structures elucidate that the energy transfer begins with subpicosecond melting at the specimen boundary earlier than the lattice thermalization, and proceeds by forming voids. Two temperature molecular dynamics simulations uncovered the presence of both heterogeneous melting with the melting front propagation from surface and grain boundaries and homogeneous melting with random melting seeds and nanoscale voids. Supported by experimental and theoretical results, we provide a comprehensive atomic-scale picture that accounts for the ultrafast laser-induced melting and evaporation kinetics.

Three-Dimensional Quantitative Coherent Diffraction Imaging of Staphylococcus aureus Treated with Peptide-Mineralized Au-Cluster Probes.

Characterizing interactions between microbial cells and their specific inhibitory drugs is essential for developing effective drugs and understanding the therapeutic mechanism. Functional metal nanoclusters can be effective inhibitory agents against microorganisms according to various characterization methods, but quantitative three-dimensional (3D) spatial structural analysis of intact cells is lacking. Herein, using coherent X-ray diffraction imaging, we performed in situ 3D visualization of unstained Staphylococcus aureus cells treated with peptide-mineralized Au-cluster probes at a resolution of ∼47 nm. Subsequent 3D mass-density mapping and quantitative structural analyses of S. aureus in different degrees of destruction showed that the bacterial cell wall was damaged and cytoplasmic constituents were released from cells, confirming the significant antibacterial effects of the Au-cluster probe. This study provides a promising nondestructive approach for quantitative imaging and paves the way for further research into microbe-inhibitor drug interactions.

Oxidation-induced three-dimensional morphological changes in Ni nanoparticles observed by coherent X-ray diffraction imaging.

Three-dimensional structures of Ni nanoparticles undergoing significant morphological changes on oxidation were observed non-destructively using coherent X-ray diffraction imaging. The Ni particles were oxidized into Ni1O1 while forming pores of various sizes internally. For each Ni nanoparticle, one large void was identified at a lower corner near the interface with the substrate. The porosity of the internal region of the agglomerated Ni oxide was about 38.4%. Regions of high NiO density were mostly observed at the outer crust of the oxide or at the boundary with the large voids. This research expands our understanding of general catalytic reactions with direct observation of oxidation-induced nanoscale morphological changes.

Direct observation of bond formation in solution with femtosecond X-ray scattering.

The making and breaking of atomic bonds are essential processes in chemical reactions. Although the ultrafast dynamics of bond breaking have been studied intensively using time-resolved techniques, it is very difficult to study the structural dynamics of bond making, mainly because of its bimolecular nature. It is especially difficult to initiate and follow diffusion-limited bond formation in solution with ultrahigh time resolution. Here we use femtosecond time-resolved X-ray solution scattering to visualize the formation of a gold trimer complex, [Au(CN)2(-)]3 in real time without the limitation imposed by slow diffusion. This photoexcited gold trimer, which has weakly bound gold atoms in the ground state, undergoes a sequence of structural changes, and our experiments probe the dynamics of individual reaction steps, including covalent bond formation, the bent-to-linear transition, bond contraction and tetramer formation with a time resolution of ∼500 femtoseconds. We also determined the three-dimensional structures of reaction intermediates with sub-ångström spatial resolution. This work demonstrates that it is possible to track in detail and in real time the structural changes that occur during a chemical reaction in solution using X-ray free-electron lasers and advanced analysis of time-resolved solution scattering data.

Characterizing the intrinsic properties of individual XFEL pulses via single-particle diffraction.

With each single X-ray pulse having its own characteristics, understanding the individual property of each X-ray free-electron laser (XFEL) pulse is essential for its applications in probing and manipulating specimens as well as in diagnosing the lasing performance. Intensive research using XFEL radiation over the last several years has introduced techniques to characterize the femtosecond XFEL pulses, but a simple characterization scheme, while not requiring ad hoc assumptions, to address multiple aspects of XFEL radiation via a single data collection process is scant. Here, it is shown that single-particle diffraction patterns collected using single XFEL pulses can provide information about the incident photon flux and coherence property simultaneously, and the X-ray beam profile is inferred. The proposed scheme is highly adaptable to most experimental configurations, and will become an essential approach to understanding single X-ray pulses.

Comparing the spatial coherence of the natural and focused X-rays from a free electron laser.

The degree of spatial coherence, as basic characteristics of the radiation, becomes an important guide to evaluate the performance of X-rays from newly introduced advanced light sources including the X-ray free electron laser (XFEL). Often the modification of the X-ray wavefronts to fulfill various applications is necessary, but also there is the need to preserve its coherence property. However, experimental investigation directly comparing the coherence property of focused X-ray radiations with the unmodified ones has not been available. We have performed Young's double-slit experiments by recording diffraction patterns both from slit apertures for unfocused XFEL radiation and from pairs of Au nanoparticles for one-micron focused XFEL radiations. The results confirm that the degree of spatial coherence is preserved for well-built K-B focusing mirrors.

Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography.

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme-substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.

Grease matrix as a versatile carrier of proteins for serial crystallography.

Serial femtosecond X-ray crystallography (SFX) has revolutionized atomic-resolution structural investigation by expanding applicability to micrometer-sized protein crystals, even at room temperature, and by enabling dynamics studies. However, reliable crystal-carrying media for SFX are lacking. Here we introduce a grease-matrix carrier for protein microcrystals and obtain the structures of lysozyme, glucose isomerase, thaumatin and fatty acid-binding protein type 3 under ambient conditions at a resolution of or finer than 2 Å.

Three-dimensional reconstruction for coherent diffraction patterns obtained by XFEL.

The three-dimensional (3D) structural analysis of single particles using an X-ray free-electron laser (XFEL) is a new structural biology technique that enables observations of molecules that are difficult to crystallize, such as flexible biomolecular complexes and living tissue in the state close to physiological conditions. In order to restore the 3D structure from the diffraction patterns obtained by the XFEL, computational algorithms are necessary as the orientation of the incident beam with respect to the sample needs to be estimated. A program package for XFEL single-particle analysis based on the Xmipp software package, that is commonly used for image processing in 3D cryo-electron microscopy, has been developed. The reconstruction program has been tested using diffraction patterns of an aerosol nanoparticle obtained by tomographic coherent X-ray diffraction microscopy.

Nanosecond pump-probe device for time-resolved serial femtosecond crystallography developed at SACLA.

X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.

Native sulfur/chlorine SAD phasing for serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Šis successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

Diverse application platform for hard X-ray diffraction in SACLA (DAPHNIS): application to serial protein crystallography using an X-ray free-electron laser.

An experimental system for serial femtosecond crystallography using an X-ray free-electron laser (XFEL) has been developed. It basically consists of a sample chamber, fluid injectors and a two-dimensional detector. The chamber and the injectors are operated under helium atmosphere at 1 atm. The ambient pressure operation facilitates applications to fluid samples. Three kinds of injectors are employed to feed randomly oriented crystals in aqueous solution or highly viscous fluid. Experiments on lysozyme crystals were performed by using the 10 keV XFEL of the SPring-8 Angstrom Compact free-electron LAser (SACLA). The structure of model protein lysozyme from 1 µm crystals at a resolution of 2.4 Šwas obtained.

Analytic 3D imaging of mammalian nucleus at nanoscale using coherent x-rays and optical fluorescence microscopy.

Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens.

Resolution enhancement in coherent x-ray diffraction imaging by overcoming instrumental noise.

We report that reference objects, strong scatterers neighboring weak phase objects, enhance the phase retrieval and spatial resolution in coherent x-ray diffraction imaging (CDI). A CDI experiment with Au nano-particles exhibited that the reference objects amplified the signal-to-noise ratio in the diffraction intensity at large diffraction angles, which significantly enhanced the image resolution. The interference between the diffracted x-ray from reference objects and a specimen also improved the retrieval of the phase of the diffraction signal. The enhancement was applied to image NiO nano-particles and a mitochondrion and confirmed in a simulation with a bacteria phantom. We expect that the proposed method will be of great help in imaging weakly scattering soft matters using coherent x-ray sources including x-ray free electron lasers.

Halloysite nanotubes enhanced polyimide/oxidized-lignin nanofiber separators for long-cycling lithium metal batteries.

The high energy density and robust cycle properties of lithium-ion batteries contribute to their extensive range of applications. Polyolefin separators are often used for the purpose of storing electrolytes, hence ensuring the efficient internal ion transport. Nevertheless, the electrochemical performance of lithium-ion batteries is constrained by its limited interaction with electrolytes and poor capacity for cation transport. This work presents the preparation of a new bio-based nanofiber separator by combining oxidized lignin (OL) and halloysite nanotubes (HNTs) with polyimide (PI) using an electrospinning technique. Analysis was conducted to examine and compare the structure, morphology, thermal characteristics, and EIS of the separator with those of commercially available polypropylene separator (PP). The results indicate that the PI@OL and PI-OL@ 10 % HNTs separators exhibit higher lithium ion transference number and ionic conductivity. Moreover, the use of HNTs successfully impeded the proliferation of lithium dendrites, hence exerting a beneficial impact on both the cycle performance and multiplier performance of the battery. Consequently, after undergoing 300 iterations, the battery capacity of LiFePO4|PI-OL@ 10 % HNTs|Li stays at 92.1 %, surpassing that of PP (86.8 %) and PI@OL (89.6 %). These findings indicate that this new bio-based battery separator (PI-OL@HNTs) has the great potential to serve as a substitute for the commonly used PP separator in lithium metal batteries.

Inverted nucleation for photoinduced nonequilibrium melting.

Ultrafast photoinduced melting provides an essential platform for studying nonequilibrium phase transitions by linking the kinetics of electron dynamics to ionic motions. Knowledge of dynamic balance in their energetics is essential to understanding how the ionic reaction is influenced by femtosecond photoexcited electrons with notable time lag depending on reaction mechanisms. Here, by directly imaging fluctuating density distributions and evaluating the ionic pressure and Gibbs free energy from two-temperature molecular dynamics that verified experimental results, we uncovered that transient ionic pressure, triggered by photoexcited electrons, controls the overall melting kinetics. In particular, ultrafast nonequilibrium melting can be described by the reverse nucleation process with voids as nucleation seeds. The strongly driven solid-to-liquid transition of metallic gold is successfully explained by void nucleation facilitated by photoexcited electron-initiated ionic pressure, establishing a solid knowledge base for understanding ultrafast nonequilibrium kinetics.

Imaging fully hydrated whole cells by coherent x-ray diffraction microscopy.

Nanoscale imaging of biological specimens in their native condition is of long-standing interest, in particular with direct, high resolution views of internal structures of intact specimens, though as yet progress has been limited. Here we introduce wet coherent x-ray diffraction microscopy capable of imaging fully hydrated and unstained biological specimens. Whole cell morphologies and internal structures better than 25 nm can be clearly visualized without contrast degradation.

Three-dimensional coherent x-ray diffraction imaging of molten iron in mantle olivine at nanoscale resolution.

We report quantitative 3D coherent x-ray diffraction imaging of a molten Fe-rich alloy and crystalline olivine sample, synthesized at 6 GPa and 1800 °C, with nanoscale resolution. The 3D mass density map is determined and the 3D distribution of the Fe-rich and Fe-S phases in the olivine-Fe-S sample is observed. Our results indicate that the Fe-rich melt exhibits varied 3D shapes and sizes in the olivine matrix. This work has potential for not only improving our understanding of the complex interactions between Fe-rich core-forming melts and mantle silicate phases but also paves the way for quantitative 3D imaging of materials at nanoscale resolution under extreme pressures and temperatures.