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Auditory ossicles in the middle ear and bony labyrinth of the inner ear are highly mineralized in adult mammals. Cellular mechanisms underlying formation of dense bone during development are unknown. Here, we found that osteoblast‐like cells synthesizing highly mineralized hearing‐related bones produce both type I and type II collagens as the bone matrix, while conventional osteoblasts and chondrocytes primarily produce type I and type II collagens, respectively. Furthermore, these osteoblast‐like cells were not labeled in a “conventional osteoblast”‐specific green fluorescent protein (GFP) mouse line. Type II collagen‐producing osteoblast‐like cells were not chondrocytes as they express osteocalcin, localize along alizarin‐labeled osteoid, and form osteocyte lacunae and canaliculi, as do conventional osteoblasts. Auditory ossicles and the bony labyrinth exhibit not only higher bone matrix mineralization but also a higher degree of apatite orientation than do long bones. Therefore, we conclude that these type II collagen‐producing hypermineralizing osteoblasts (termed here auditory osteoblasts) represent a new osteoblast subtype. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)., Type II collagen‐producing auditory osteoblasts highly mineralize hearing related bones.

Gadolinium absorption gratings are critically important devices for grating-based neutron dark-field and phase-contrast imaging. The evaporation process is an excellent candidate for preparing Gd gratings large enough for imaging applications. However, there is some concern about the lower average density of obliquely evaporated Gd, which sometimes grows with many voids that are caused by shadowing effects. We have fabricated Gd gratings with 9-µm and 4-µm pitches as part of our design and measured the density distributions of the resulting Gd deposits by three-dimensional X-ray microtomography. The obtained tomographic image data is helpful not only for fabrication process design but also estimating device performance without performing neutron irradiation experiments.

Owing to the development of X‐ray focusing optics during the past decades, synchrotron‐based X‐ray microscopy techniques allow the study of specimens with unprecedented spatial resolution, down to 10 nm, using soft and medium X‐ray photon energies, though at the expense of the field of view (FOV). One of the approaches to increase the FOV to square millimetres is raster‐scanning of the specimen using a single nanoprobe; however, this results in a long data acquisition time. This work employs an array of inclined biconcave parabolic refractive multi‐lenses (RMLs), fabricated by deep X‐ray lithography and electroplating to generate a large number of long X‐ray foci. Since the FOV is limited by the pattern height if a single RML is used by impinging X‐rays parallel to the substrate, many RMLs at regular intervals in the orthogonal direction were fabricated by tilted exposure. By inclining the substrate correspondingly to the tilted exposure, 378000 X‐ray line foci were generated with a length in the centimetre range and constant intervals in the sub‐micrometre range. The capability of this new X‐ray focusing device was first confirmed using ray‐tracing simulations and then using synchrotron radiation at BL20B2 of SPring‐8, Japan. Taking account of the fact that the refractive lens is effective for focusing high‐energy X‐rays, the experiment was performed with 35 keV X‐rays. Next, by scanning a specimen through the line foci, this device was used to perform large FOV pixel super‐resolution scanning transmission hard X‐ray microscopy (PSR‐STHXM) with a 780 ± 40 nm spatial resolution within an FOV of 1.64 cm × 1.64 cm (limited by the detector area) and a total scanning time of 4 min. Biomedical implant abutments fabricated via selective laser melting using Ti–6Al–4V medical alloy were measured by PSR‐STHXM, suggesting its unique potential for studying extended and thick specimens. Although the super‐resolution function was realized in one dimension in this study, it can be expanded to two dimensions by aligning a pair of presented devices orthogonally., A new X‐ray focusing device generates hundreds of thousands of line foci, periodically spaced in the sub‐micrometre range, with centimetre length. It enables to achieve large FOV pixel super‐resolution scanning transmission hard X‐ray microscopy. image

Four-dimensional phase computed tomography (4D phase CT) by an X-ray Talbot interferometer (XTI) with white synchrotron radiation has ever been demonstrated at a temporal resolution of about 1 s for soft-matter samples. However, the radiation damage to samples caused by white synchrotron radiation occasionally hampers our understanding of the sample dynamical properties. Based on the fact that XTI functions with X-rays of a bandwidth up to ca. 10% with performance comparable to that by monochromatic X-rays, filtering white synchrotron radiation to generate a ‘pink-beam’ of a 10% bandwidth is effective to reduce radiation damage without degrading the image quality and temporal resolution. We have therefore developed pink-beam 4D phase CT at SPring-8, Japan by installing a multilayer mirror with a 10% bandwidth and a 25 keV central photon energy. XTI optimal at this photon energy was built downstream, and a CMOS-based X-ray detector was used to achieve fast image acquisitions with an exposure time of 1 ms (or 0.5 ms) per moiré image. The resultant temporal resolution of pink-beam 4D phase CT was 2 s (1 s). We applied the pink-beam 4D phase CT to in-situ observation of polypropylene, poly(methyl methacrylate), and polycarbonate under infrared laser irradiation (1064 nm). The dynamics of melting, bubbling, and ashing were successfully visualized in 3D movies without problematic radiation damage by synchrotron radiation.

An x-ray phase tomographic microscope has been developed by optimizing the optical system for 9-keV synchrotron x rays at BL37XU, SPring-8, Japan. The system consists of a full-field x-ray imaging microscope and a Talbot interferometer. In contrast with our previous system, a Fresnel zone plate for the microscope objective and transmission gratings of the interferometer was designed to improve the refraction sensitivity, with a larger field of view and a higher spatial resolution, utilizing a 28-m space available at BL37XU. An x-ray phase tomographic microscope with a field of view of 375 µm and a spatial resolution of 560 nm was thus constructed, even with a considerably low x-ray dosage. By applying this tomographic microscope to auditory bone specimens of young mice, it was shown that soft tissues in addition to bone tissue could be observed simultaneously without serious radiation damage.

In conventional grating-based X-ray interferometry, it is necessary to repeatedly translate one of the gratings with high accuracy in regular submicron steps and to ensure a constant dose delivery during each step. However, stepping errors and dose fluctuations inevitably occur due to mechanical inaccuracies and/or thermal drift of the interferometer during the stepping process. As a result of these stepping errors and dose fluctuations, the standard reconstruction procedure without considering them causes artifacts in the images as stripes of specific frequencies. In this report, we propose an improved reconstruction method to process phase stepping data with stepping errors and dose fluctuations. The approach can be used to estimate the stepping errors and dose fluctuations, and reconstruct virtually artifact-free images. Based on numerical simulations and experimental data including stepping errors and dose fluctuations, we demonstrate that the proposed method is more effective to other previously reported approaches.

X-ray phase contrast imaging offers a complementary modality next to conventional absorption. To reveal internal structures of soft materials in high resolution, an X-ray phase imaging microscope has been developed by installing a Lau interferometer into a commercially available ZEISS Xradia 800 Ultra-nano computed tomography X-ray microscope. On this system, a twin phase image is once generated through measurements of interference fringes. In order to produce a quantitative X-ray phase map, the twin phase image should be treated by deconvolution. However, conventional deconvolution results showed a reduction in image quality due to noise and artifacts. Therefore, we propose and evaluate the performance of an iterative deconvolution method. The results show that the proposed deconvolution method does not affect spatial resolution, increases the signal to noise ratio compared to that of the twin phase image, and is capable of imaging complex structures.

A grating interferometer (GI) system has been installed in an X-ray microscope equipped with a Zernike phase contrast (ZPC) system and a Cu rotating anode X-ray source. The GI and ZPC systems are switchable, and their performances of phase information extraction have been compared. The GI system is based on a Lau interferometer consisting of an absorption grating and a π/2 phase grating, which extracts a magnified phase shift map of a sample via a phase-stepping measurement. The ZPC system generates a phase contrast image by using a phase plate and a corresponding condenser device. The ZPC system and the GI system are compared in terms of detectability of phase objects. By the Fourier analysis of images of a logarithmic ruler pattern, the spatial resolution was found to be identical between the two systems. Although the sensitivity depends on the sample size, the signal-to-noise ratio of polystyrene spheres with a few microns in diameter was used for sensitivity comparison, showing the superior sensitivity of the GI system to that of the ZPC system. The quantitativeness of the GI system with the phase-stepping measurement was also demonstrated over the ZPC system, which generates halo and shade-off artifacts. The GI system exhibits twin image artifacts that need to be resolved for practical applications of the technique.

Multilayer zone plate (MZP) technology for hard X-ray focusing was upgraded and its focusing performance was evaluated using 20-keV X-rays at the synchrotron beamline (BL24XU) of SPring-8. The MZP consists of MoSi2 and Si layers alternately deposited on a glass fiber by magnetron sputtering so that all zone boundaries satisfy the Fresnel zone configuration. The focused beam was evaluated using knife-edge scanning in which the measured intensity distribution is identical to the line spread function (LSF) in the focal plane. The focused beamsize of about 30 nm was estimated by oscillation peaks observed in the measured LSF according to Rayleigh’s criterion.

A total-reflection zone plate (TRZP), which is a reflective grating that generates a line focus of hard X-rays, was developed. Newly designed TRZPs, introducing a laminar grating concept, were fabricated with various zone parameters. The focusing performances with regard to the beam size and the diffraction efficiency were evaluated using synchrotron radiation X-rays of 10 keV energy. Although the beam sizes measured are insufficient in comparison with the ideal value, the maximum diffraction efficiency, measured at 20%, exceeds the limitations of conventional TRZPs based on a binary grating.

The experimental procedure for obtaining the point spread function (PSF) of a focusing beam generated using an X-ray multilayer zone plate (MZP) with a narrow annular aperture has been developed. It was possible to reconstruct the PSF by applying the tomographic process to the measured dataset consisting of line spread functions (LSFs) in every radial direction on the focal plane. The LSFs were measured by a knife-edge scanning method of detecting scattered intensity. In the experimental work, quasi-monochromatic undulator radiation with a first harmonic energy of 20 keV was directly focused without a monochromator by the MZP, and the PSF was measured using this procedure. As a result, a near diffraction-limited focused beam size of 46 nm full width at half-maximum was obtained.

Concentric multilayers of alternate high-Z material and low-Z one, deposited on the wire substrate, can be applied to various optical elements for synchrotron radiation (SR) high-brilliance X-ray beamlines. We present new application of the concentric multilayer to optical elements for the SR high-brilliance X-ray beamlines at SPring-8. Three types of Cu/ Al concentric multilayer samples have been fabricated and applied to (1) a spatial resolution test pattern for X-ray computed tomography (CT), (2) a spatial resolution test pattern for the X-ray equipment and (3) a condenser device in the X-ray imaging microscopy. These characteristics have been tested. For the (1), alternate opaque and transparent concentric ring pattern with 2 micron film thickness has been clearly resolved. For the (2), the test pattern can work well in the high energy X-ray region. For the (3), absorption contrast image of a test pattern without modulation by the coherence of the source has been obtained by using the condenser device.

The performance of a Fresnel zone plate has been tested by observing the focusing property of higher-order diffraction. The Fresnel zone plate was fabricated by the electron-beam lithography technique. The zone material was made from 1 µm-thick tantalum and the outermost zone width was 0.25 µm. The third-order focused spot size measured by the knife-edge scan method was 0.1 µm full width at half-maximum at an X-ray energy of 8 keV, which is exactly equal to one-third of the first-order focal spot size.

We have improved the performance of a previously reported multilayer zone plate by reducing its outermost zone width, using the same multilayer materials (MoSi2 and Si) and fabrication technique. The focusing performance was evaluated at the BL24XU of SPring-8 using 20-keV X-rays. The line spread function (LSF) in the focal plane was measured using a dark-field knife-edge scan method, and the point spread function was obtained from the LSF through a tomographic reconstruction principle. The spatial resolution was estimated to be 30 nm, which is in relatively good agreement with the calculated diffraction-limited value of 25 nm, while the measured diffraction efficiency of the +1st order was 24%.

X線集光素子の1つである多層膜フレネルゾーンプレート (FZP) の作製方法, 開発したFZPを利用した集光実験の成果および関連分野の世界の研究動向を紹介した.大型放射光施設SPring-8における集光実験ではトップレベルのデータが得られ, 多層膜FZPは特に高エネルギー領域 (>25keV) の集光にも有効であることがわかった.FZPは連続100時間以上の使用の間, 放射線損傷は観察されなかった.このことから, 高輝度放射光を利用した硬X線顕微鏡の実用化に大きく前進したと言える.測定された性能値を理論値に限りなく近づけるためには, 成膜工程を中心に一層の工夫が必要である.今後, ゾーン (多層膜界面) の平滑性を改善するために成膜条件の検討, 成膜装置の改良を引き続き行うと共に, 多層膜FZPの結像特性の理論的考察も行いたい.また, マイクロビームを利用して微小領域の元素マッピング, 化学状態のイメージングなどを行う予定である.本報告で紹介した以外にも注目すべき新しい硬X線領域用の集光素子が最近相次いで発表され, 今後の進展が期待される.集光テストを行ったSPring-8での利用研究課題の課題番号は, 1999A0091-NM-np, 1999B0080-CM-np, 1999B0103-NM-np, 2000AO160-NM-npである.

An X-ray fluorescence imaging microscope with a Wolter-type objective mirror (magnification: 13) has been constructed at beamline 39XU of SPring-8. Monochromatic X-rays (DeltaE/E approximately 10(-4)) in the energy range 6-10 keV were used for X-ray fluorescence excitation of the specimens. Using two monochromatic X-rays above and below the absorption edge of a particular element, a two-dimensional image of the element could be obtained. As a result, two-dimensional element mapping of the test specimens (Cu, Co, Ni, Fe and Ti wires) and constituent minerals (Fe, Mn and Ti) of a rock specimen (a piemontite-quartz schist) became possible.

We constructed a soft X-ray imaging microscope on a laboratory scale by using a laser produced plasma source. Two different soft X-rays (wavelength: 3.2 nm and 5.2 nm) were obtained by using a Nd-YAG laser with a power of 1.2 J and a pulse width of 8 ns. Wolter mirrors were used as a condenser and an objective. A back-illuminated CCD camera was introduced as an X-ray imaging detector. Spatial resolution was estimated to be about 60 nm with a transmission grating object. An X-ray micrograph of a stoma at epidermis of vincia faba L. was obtained. A human red blood cell in a solution was also observed and it showed that the soft X-ray microscope is available for living specimens in a wet state.

A Zernike-type phase-contrast X-ray microscope with a zone plate and a phase plate was constructed at the Photon Factory BL3C2. Parallel monochromatic X-rays of 8.97 keV were incident on a specimen and a direct beam transmitted through the specimen was focused on the back focal plane of the zone plate, where an aluminium phase plate was placed. Tantalum line patterns as fine as 0.3 µm could be imaged. Phase-contrast images of polypropylene wires and polystyrene latex beads were obtained, which showed better contrast than that of their bright field images.

A high spatial resolution X-ray microscope was constructed using an X-ray refractive lens as an objective. The spatial resolution was tested using 18 keV X-ray. A 0.4 mm line and 0.4 mm space tantalum test pattern was successfully resolved. Using the similar setup with the addition of a phase plate, a Zernike type phase-contrast microscopy experiment was carried out for the phase retrieval of the samples. Two-dimensional phase-contrast images were successfully taken for the first time in the hard X-ray region. Images of a gold mesh sample were analyzed and the validity of this method was indicated. An improvement of the lens, however, is required for the precise phase retrieval of the samples. # 2001 Elsevier Science B.V. All rights reserved.

A Zernike-type imaging microscope using a sputtered-sliced Fresnel zone plate (SS-FZP) has been developed and tested at an X-ray energy of 25 keV. The SS-FZP was used as an objective. A copper (Cu) phase plate was placed at the back focal plane of the SS-FZP in order to produce phase contrast. The performance of the Zernike-type imaging microscope was tested with a gold (Au) mesh and a resolution test pattern at undulator beamline 47 of SPring-8. The Au mesh and the resolution test pattern could be imaged in transmission with a magnification of ×10.2. Owing to the Cu phase plate, different image contrast was observed compared with the bright-field image contrast. Tantalum microstructures down to 0.5 µm line-and-space have been observed on spatial resolution test patterns.

The first result is presented of an X-ray fluorescence microscope with a Wolter mirror in combination with a CCD camera used as an energy-resolved two-dimensional detector in photon-counting mode. Two-dimensional elemental maps of metallic wires, such as Fe, Co, Ni and Cu, and inclusions of a synthesized diamond could be obtained with an energy resolution of 350 eV.

Hard X-ray microbeam experiments with sputtered-sliced Fresnel zone plates have been performed. Zone plates with an outermost zone width of 0.25 µm (#FZP1) and 0.1 µm (#FZP2) were fabricated and evaluated. In a scanning X-ray microscopy experiment, a line-and-space pattern with structure as fine as 0.1 µm was resolved using #FZP2 at an X-ray wavelength of 1 Å. As an application of the microbeam technique, a two-dimensional distribution of constituent elements in forensic samples has been obtained (e.g. section view of human and elephant hairs) using fluorescent scanning microscopy.

Hard X-ray microscopy with high spatial resolution (≤0.1 µm) using a high-energy and high-brilliance X-ray microprobe is expected to be a promising technology for various types of analysis, imaging etc. in materials science, biology and medicine. A multilayer Fresnel zone plate (FZP) could be a promising approach to focusing optics in the high-energy X-ray region (≥20 keV) because a large thickness (aspect ratio) can be available. Various types of multilayer FZPs have been fabricated by DC sputtering deposition. Their focusing characteristics have been evaluated at the high-brilliance undulator beamline BL47XU of SPring-8. An optical system using a Cu/Al multilayer FZP (with an outermost zone width of 0.25 µm) as the focusing optics fabricated by the optimum deposition condition with precise film (zone) thickness control has attained an almost diffraction-limited microbeam of 0.3–0.35 µm at 8.9 keV. A line-and-space resolution test pattern has been observed: fine structures up to 0.2 µm were clearly observed in the measured image. This FZP has been working since 1995, keeping good focusing characteristics. It can be said from these results that a spatial resolution better than 0.1 µm in the high-energy X-ray region is in prospect by the development of a multilayer FZP with a narrower outermost zone width in the near future.

We have developed a four-dimensional (4D) x-ray microcomputed tomography (CT) system that can obtain time-lapse CT volumes in real time. The system consists of a high-speed sample rotation system and a high-frame-rate x-ray imager, which are installed at a synchrotron radiation x-ray beamline. As a result of system optimization and introduction of a "zoom resolution" procedure, a real-time 4D CT movie with a frame rate of 30 was obtained with a voxel size of 2.5 μm using 10 keV x-rays.

We designed point focusing total-reflection zone plates (TRZPs): a double linear type TRZP in Kirkpatrick-Baez arrangement, a conical TRZP, and an open conical TRZP. In this paper, we show detail characteristics of the open conical TRZP, which is the most promising candidate for practical use, and evaluate the effect of fabrication error and misalignment on focused beam profiles by calculation.

To develop a multilayer Laue lens (MLL), we fabricated depth-graded Mo/Si and MoSi2/Si multilayers with each boundary according to the Fresnel zone configuration. The multilayers were deposited by magnetron sputtering. From the result of SEM image analysis of the multilayer cross sections, MoSi2/Si multilayer had smaller layer-thickness errors than Mo/Si multilayer. In addition, from the result of the focusing test by using 20-keV X-rays, the measured beam size of MoSi2/Si MLL had a small blurring from the diffraction limited beam size. These results suggest that MoSi2/Si multilayer is better suited than Mo/Si multilayer for use as an MLL in hard x-ray nanofocusing.

A new total reflection double-slit for hard X-ray region was developed. It consists of Au narrow stripes deposited on a SiO2 substrate and can function equivalently as a conventional Young's interferometer by operating with a small grazing incident angle. The Young's interferometer using the total reflection double-slit was constructed at Hyogo-ID BL of SPring-8, and the spatial coherence at 10 keV X-rays was evaluated by analyzing the interference fringes.

Fast X-ray computed tomography (CT) system with sub-second order measurement for single CT acquisition has been developed. The system, consisting of a high-speed sample rotation stage and a high-speed X-ray camera, is constructed at synchrotron radiation beamline in order to utilize fully intense X-rays. A time-resolving CT movie (i.e. 4D CT) can be available by operating the fast CT system continuously. Real-time observation of water absorbing process of super-absorbent polymer (SAP) has been successfully performed with the 4D CT operation.

α-Methylstyrene(α-MeSt) and 2, 3-dimethylbutadiene (DMB) were copolymerized in THF and in hydrocarbon solvents with n-BuLi. The copolymer produced at 60°C in THF was composed of almost equimolar amounts of α-MeSt and DMB, if α-MeSt was charged in excess. In the copolymer α-MeSt and DMB were considered to be arranged alternately, and DMB was incorporated in the form of 1, 4-structure. In the copolymerization below 60°C the copolymer composition was affected by the ratio of initial monomer concentrations. The content of α-MeSt in the copolymer increased with decreasing polymerization temperature and reached 91% at −78°C. The copolymerization at 30°C gave the copolymer composed of (2:1) α-MeSt and DMB under the existence of large excess of α-MeSt. It was presumed that the major part of the copolymer was composed of repeated sequences of [α-MeSt·α·MeSt·DMB]. In benzene a copolymer rich in DMB (71%) was obtained at 60°C. The addition of THF to the polymerization system in benzene decreased the content of DMB.

2,3-Dimethylbutadiene was polymerized in tetrahydrofuran with n-BuLi. The content of the 1,2-unit in the polymer varied with the polymerization temperature as follows: 86% at −78°C, 69% at −45°C, 54% at −30°C, and 48% at 0°C. The relationship between log (k1,2/k1,4) and 1/T showed a break at −30°C, where k1,2 and k1,4 are the propagation rate constants leading to the denoted structures, respectively, and T is the polymerization temperature (°K). It was postulated that the continuous arrangement of 1,2-units, which is preferential at low temperature, becomes impossible at higher temperatures due to steric hindrance, and the insertion of the 1,4-unit in the sequence of 1,2-units takes place more frequently, resulting in alternating arrangements of the 1,2- and 1,4-unit above −30°C. The NMR spectra of the polymers showed that the chemical shifts of each monomeric unit varied with the difference of the neighboring monomeric units in both sides. The spectrum of the polymer having a 1,2- and 1,4-unit in 1:1 suggested the 1,2-unit being held between the 1,4-units, i.e., both the units being alternate. The polymerization of 2,3-dimethylbutadiene in hydrocarbon solvent gave a polymer rich in the 1,4-unit with n-BuLi at 30°C, but formed a similar polymer with K-dispersion as that obtained with n-BuLi in tetrahydrofuran at 30°C.

o-Methoxy-α-methylstyrene (o-MeO-α-MeSt) was polymerized under various conditions using BF3OEt2, TiCl4, SnCl2, EtAlCl2, n-butyllithium (n-BuLi), or Na-naphthalene as catalysts, and methylene chloride, n-heptane, toluene, or tetrahydrofuran (THF) as solvents. Generally poly(o-MeO-α-MeSt) was formed below −30°C. The cationic polymerizations proceeded more rapidly than the anionic ones, which gave polymer only in a polar solvent such as THF. α-Methylstyrene (α-MeSt) was also polymerized for comparison with the tacticity of poly(o-MeO-α-MeSt). The stereoregularity of the polymer was investigated by NMR spectroscopy. Assignments of the α-methyl resonances of the polymers were made on the basis of the methylene resonances. In the NMR spectrum of poly(o-MeO-α-MeSt), the methyl groups showed three peaks at 4.45, 4.80, and 5.35 ppm on the high-field side from methylene chloride used as an internal standard; the peaks were assigned to isotactic (I), heterotactic (H), and syndiotactic (S) triad sequences with increasing magnetic field. The polymer obtained at −78°C from the anionic or cationic catalyst showed an S content higher than 74%, and the content decreased with an increasing polymerization temperature. The tacticities of the polymers formed in the cationic polymerizations depended only slightly on the catalyst and the solvent. The stereospecificity in the cationic polymerization was very similar to that of α-methylstyrene (α-MeSt) reported by Ohsumi et al.,1 whereas considerably different results were obtained in the anionic polymerization.