Alveolar Microdynamics during Tidal Ventilation in Live Animals Imaged by SPring-8 Synchrotron.

It is self-evident that our chests expand and contract during breathing but, surprisingly, exactly how individual alveoli change shape over the respiratory cycle is still a matter of debate. Some argue that all the alveoli expand and contract rhythmically. Others claim that the lung volume change is due to groups of alveoli collapsing and reopening during ventilation. Although this question might seem to be an insignificant detail for healthy individuals, it might be a matter of life and death for patients with compromised lungs. Past analyses were based on static post-mortem preparations primarily due to technological limitations, and therefore, by definition, incapable of providing dynamic information. In contrast, this study provides the first comprehensive dynamic data on how the shape of the alveoli changes, and, further, provides valuable insights into the optimal lung volume for efficient gas exchange. It is concluded that alveolar micro-dynamics is nonlinear; and at medium lung volume, alveoli expand more than the ducts.

Propagation-based phase-contrast imaging method for full-field X-ray microscopy using advanced Kirkpatrick-Baez mirrors.

We demonstrate a propagation-based phase-contrast imaging method for full-field X-ray microscopy based on advanced Kirkpatrick-Baez (AKB) mirrors to achieve high-contrast observations of weak phase objects and correct field curvature aberrations. Through a demonstration performed at SPring-8, the phase contrast of weak phase objects such as polystyrene spheres and chemically fixed cells was successfully observed with high sensitivity (∼0.03 rad). Furthermore, the field of view of the AKB mirrors was expanded to the full area of the obtained images (25 × 30 µm) by correcting the field curvature aberration using reconstructed complex wavefields.

Experimental probing of dynamic self-organized columnar assemblies in colloidal liquid crystals.

Self-organized supramolecular assemblies are widespread in nature and technology in the form of liquid crystals, colloids, and gels. The reversible nature of non-covalent bonding leads to dynamic functions such as stimuli-responsive switching and self-healing, which are unachievable from an isolated molecule. However, multiple intermolecular interactions generate diverse conformational and configurational molecular motions over various time scales in their self-assembled states, and their specific dynamics remains unclear. In the present study, we have experimentally unveiled the static structures and dynamical behaviors in columnar colloidal liquid crystals by a coherent X-ray scattering technique using refined model samples. We have found that controlling the size distribution of the colloidal nanoplates dramatically changed their static and dynamic properties. Furthermore, the resulting dynamical behaviors obtained by X-ray photon correlation spectroscopy have been successfully decomposed into multiple distinct modes, allowing us to explore the dynamical origin in the colloidal liquid-crystalline state. The present approaches using a columnar liquid crystal may contribute to a better understanding of the dynamic nature of molecular assemblies and dense colloidal systems and bring valuable insights into rational design of functional properties of self-assembled materials such as stimuli-responsive liquid crystals, self-healing gels, and colloidal crystals. For these materials, the motion of constituent particles and molecules in the self-assembled state is a key factor for structural formation and dynamically responsive performance.

Deep 3D reconstruction of synchrotron X-ray computed tomography for intact lungs.

Synchrotron X-rays can be used to obtain highly detailed images of parts of the lung. However, micro-motion artifacts induced by such as cardiac motion impede quantitative visualization of the alveoli in the lungs. This paper proposes a method that applies a neural network for synchrotron X-ray Computed Tomography (CT) data to reconstruct the high-quality 3D structure of alveoli in intact mouse lungs at expiration, without needing ground-truth data. Our approach reconstructs the spatial sequence of CT images by using a deep-image prior with interpolated input latent variables, and in this way significantly enhances the images of alveolar structure compared with the prior art. The approach successfully visualizes 3D alveolar units of intact mouse lungs at expiration and enables us to measure the diameter of the alveoli. We believe that our approach helps to accurately visualize other living organs hampered by micro-motion.

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.

The new X-ray/visible microscopy MAXWELL technique for fast three-dimensional nanoimaging with isotropic resolution.

Microscopy by Achromatic X-rays With Emission of Laminar Light (MAXWELL) is a new X-ray/visible technique with attractive characteristics including isotropic resolution in all directions, large-volume imaging and high throughput. An ultrathin, laminar X-ray beam produced by a Wolter type I mirror irradiates the sample stimulating the emission of visible light by scintillating nanoparticles, captured by an optical system. Three-dimensional (3D) images are obtained by scanning the specimen with respect to the laminar beam. We implemented and tested the technique with a high-brightness undulator at SPring-8, demonstrating its validity for a variety of specimens. This work was performed under the Synchrotrons for Neuroscience-an Asia-Pacific Strategic Enterprise (SYNAPSE) collaboration.

Quantitative analysis of the effect of radiation on mitochondria structure using coherent diffraction imaging with a clustering algorithm.

Radiation damage and a low signal-to-noise ratio are the primary factors that limit spatial resolution in coherent diffraction imaging (CDI) of biomaterials using X-ray sources. Introduced here is a clustering algorithm named ConvRe based on deep learning, and it is applied to obtain accurate and consistent image reconstruction from noisy diffraction patterns of weakly scattering biomaterials. To investigate the impact of X-ray radiation on soft biomaterials, CDI experiments were performed on mitochondria from human embryonic kidney cells using synchrotron radiation. Benefiting from the new algorithm, structural changes in the mitochondria induced by X-ray radiation damage were quantitatively characterized and analysed at the nanoscale with different radiation doses. This work also provides a promising approach for improving the imaging quality of biomaterials with XFEL-based plane-wave CDI.

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.

High-resolution fast-tomography brain-imaging beamline at the Taiwan Photon Source.

The new Brain Imaging Beamline (BIB) of the Taiwan Photon Source (TPS) has been commissioned and opened to users. The BIB and in particular its endstation are designed to take advantage of bright unmonochromatized synchrotron X-rays and target fast 3D imaging, ∼1 ms exposure time plus very high ∼0.3 µm spatial resolution. A critical step in achieving the planned performances was the solution to the X-ray induced damaging problems of the detection system. High-energy photons were identified as their principal cause and were solved by combining tailored filters/attenuators and a high-energy cut-off mirror. This enabled the tomography acquisition throughput to reach >1 mm3 min-1, a critical performance for large-animal brain mapping and a vital mission of the beamline.

X-ray adaptive zoom condenser utilizing an intermediate virtual focus.

We propose an extended X-ray adaptive zoom condenser that can form an intermediate virtual focus. The system comprises two deformable mirrors for focusing within a single dimension and can vary its numerical aperture (NA) without changing the positions of the light source, mirrors, or final focus. The desired system NA is achieved simply by controlling the mirror surfaces, which enables conversion between convex and concave forms, by varying the position of the intermediate virtual focus. A feasibility test at SPring-8 under a photon energy of 10 keV demonstrated that the beam size can be varied between 134 and 1010 nm.

Optimal deformation procedure for hybrid adaptive x-ray mirror based on mechanical and piezo-driven bending system.

A hybrid deformable x-ray mirror consisting of a mechanical bender and a bimorph deformable mirror has been developed to realize adaptive optical systems, such as zoom condenser optics, for synchrotron-radiation-based x-ray microscopy. In the developed system, both bending mechanisms comprehensively contribute to the formation of the target mirror shape and can narrow the role of piezoelectric actuators, thereby enabling a more stable operation. In this study, the behavior of the bimorph mirror under the clamped condition was investigated, and the sharing of the deformation amount for each bending mechanism was optimized to minimize the amplitude of the voltage distribution of the bimorph mirror.

X-ray microscope for imaging topological charge and orbital angular momentum distribution formed by chirality.

The distribution of topological charges on X-ray vortices was measured by differential Fourier space filtering microscope, differential radial Hilbert transform microscope. It was experimentally verified for the first time using a Spiral Fresnel zone plate objective lens. This X-ray microscope is highly sensitive to X-ray topological defects, such as edges and vortices, at the exit-face wave field of objects. Its efficient use is also discussed.

Dynamical Heterogeneity near Glass Transition Temperature under Shear Conditions.

We experimentally studied the shear effect on dynamical heterogeneity near glass transition temperature. X-ray photon correlation spectroscopy was utilized to study the dynamics of polyvinyl acetate with tracer particles near its glass transition temperature, to determine the local shear rate from the anisotropic behavior of the time autocorrelation function and to calculate the dynamical heterogeneity using higher-order correlation function. The obtained results show a decrease in the dynamical heterogeneity and faster dynamics with increasing shear rate. This is the first experimental result that proved the predictions of previous molecular dynamics simulations.

Full-field X-ray fluorescence microscope based on total-reflection advanced Kirkpatrick-Baez mirror optics.

A novel full-field X-ray fluorescence microscope based on total-reflection advanced Kirkpatrick-Baez mirror optics was developed. The total-reflection imaging mirror optics arrangement, with four reflections, has the advantage of being able to function both as a powerful low-pass energy filter, completely rejecting incident excitation X-rays, and as an achromatic optical imaging system. Isolated X-ray fluorescence signals can be imaged, avoiding imaging-detector saturation, with low background noise. A prototype fluorescence microscope constructed at SPring-8 demonstrated the capability to simultaneously image elemental distributions using various X-ray fluorescence signals (Ni, Cu, Zn, Ge, and Bi). A half-period spatial resolution of ~0.5-1 µm (1000-500 LP/mm) was achieved, owing to the achromaticity of the imaging mirrors and the photon-counting scheme of the CCD camera used for fluorescence detection.

Development of an X-ray imaging detector to resolve 200 nm line-and-space patterns by using transparent ceramics layers bonded by solid-state diffusion.

A high-resolution lens-coupled X-ray imaging detector equipped with a thin-layer transparent ceramics scintillator has been developed. The scintillator consists of a 5 µm thick Ce-doped Lu3Al5O12 layer (LuAG:Ce) bonded onto the support substrate of the non-doped LuAG ceramics by using a solid-state diffusion technique. Secondary electron microscopy of the bonded interface indicated that the crystal grains were densely packed without any pores in the optical wavelength scale, indicating a quasi-uniform refractive index across the interface. This guarantees high transparency and minimum reflection, which are essential properties for X-ray imaging detectors. The LuAG:Ce scintillator was incorporated into an X-ray imaging detector coupled with an objective lens with a numerical aperture of 0.85 and an optical magnification of 100. The scintillation light was imaged onto a complementary metal-oxide-semiconductor image sensor. The effective pixel size on the scintillator plane was 65 nm. X-ray transmission images of 200 nm line-and-space patterns were successfully resolved. The high spatial resolution was demonstrated by X-ray transmission images of large integrated circuits with the wiring patterns clearly visualized.

Compact reflective imaging optics in hard X-ray region based on concave and convex mirrors.

We demonstrated that the combination of a hyperbolic convex and elliptical concave mirrors works as a compact reflective X-ray imaging system with a short optical focal length and large magnification factor. We performed an experiment to form a one-dimensional demagnified image with a demagnification factor of 321 within an approximately 2-m-long optical setup at an X-ray energy of 10 keV. The results showed that this imaging optics system is capable of providing a resolution of ~40 nm. From wavefront analysis, it was confirmed that the optics possessed a wide field-of-view with a significant reduction of comatic aberration.

Static structure and dynamical behavior of colloidal liquid crystals consisting of hydroxyapatite-based nanorod hybrids.

Biominerals such as bones and teeth have elaborate nanostructures composed of aligned anisotropic hydroxyapatite (HAp) nanocrystals, which results in excellent mechanical properties. Construction of such ordered structures of HAp nanocrystals in synthetic materials is challenging. Recently, we reported that HAp-nanorod-based colloidal liquid crystals could be obtained. In the present study, the static structure and dynamics of liquid-crystalline (LC) colloidal dispersions of HAp nanorods are investigated by using small-angle X-ray scattering (SAXS) and X-ray photon correlation spectroscopy (XPCS). The SAXS results reveal that the interparticle distance decreases with increasing HAp concentration, φHAp, and the decrease of the interparticle distance for the short-axis direction is significantly smaller in the LC phase than the interparticle distance in the isotropic phase. In the dynamical studies of the LC phase using XPCS, we observe the diffusive motion of the HAp colloids, with the diffusion coefficient being dependent on the wave number. The diffusive motion slows down with increasing φHAp. We observe anisotropic dynamics after long-term storage (160 days after sealing), whereas only isotropic dynamics are observed in the initial XPCS measurements after short-term storage (14 days after sealing). Moreover, we have found that the dynamics slows down with increasing storage time.

Diffraction apparatus and procedure in tomography X-ray diffraction imaging for biological cells at cryogenic temperature using synchrotron X-ray radiation.

X-ray diffraction imaging is a technique for visualizing the structure of biological cells. In X-ray diffraction imaging experiments using synchrotron radiation, cryogenic conditions are necessary in order to reduce radiation damage in the biological cells. Frozen-hydrated biological specimens kept at cryogenic temperatures are also free from drying and bubbling, which occurs in wet specimens under vacuum conditions. In a previous study, the diffraction apparatus KOTOBUKI-1 [Nakasako et al. (2013), Rev. Sci. Instrum. 84, 093705] was constructed for X-ray diffraction imaging at cryogenic temperatures by utilizing a cryogenic pot, which is a cooling device developed in low-temperature physics. In this study a new cryogenic pot, suitable for tomography experiments, has been developed. The pot can rotate a biological cell over an angular range of ±170° against the direction of the incident X-ray beam. Herein, the details and the performance of the pot and miscellaneous devices are reported, along with established experimental procedures including specimen preparation. The apparatus has been used in tomography experiments for visualizing the three-dimensional structure of a Cyanidioschyzon merolae cell with an approximate size of 5 µm at a resolution of 136 nm. Based on the experimental results, the necessary improvements for future experiments and the resolution limit achievable under experimental conditions within a maximum tolerable dose are discussed.

Nearly diffraction-limited hard X-ray line focusing with hybrid adaptive X-ray mirror based on mechanical and piezo-driven deformation.

We have developed the new hybrid adaptive X-ray mirror based on mechanical and piezo-driven deformation to realize precise shape controllability on a long-length mirror. The mechanical bender approximately provides the required ellipse, while the piezoelectric actuators attached to the mirror correct very small residual errors to satisfy the diffraction-limited condition. The mechanical bender significantly reduces the role of the piezoelectric actuator, resulting in the suppression of accuracy degradation due to the drift and/or junction effect of the piezoelectric actuators. In addition, line focusing was demonstrated with two different numerical apertures at SPring-8, and the obtained beam sizes were 127 and 253 nm (FWHM), which agree well with the diffraction-limited sizes.