Volume X-Ray Micro-Computed Tomography Analysis of the Early Cephalized Central Nervous System in a Marine Flatworm, Stylochoplana pusilla.

Platyhelminthes are a phylum of simple bilaterian invertebrates with prototypic body systems. Compared with non-bilaterians such as cnidarians, the bilaterians are likely to exhibit integrated free-moving behaviors, which require a concentrated nervous system "brain" rather than the distributed nervous system of radiatans. Marine flatworms have an early cephalized 'central' nervous system compared not only with non-bilaterians but also with parasitic flatworms or freshwater planarians. In this study, we used the marine flatworm Stylochoplana pusilla as an excellent model organism in Platyhelminthes because of the early cephalized central nervous system. Here, we investigated the three-dimensional structures of the flatworm central nervous system by the use of X-ray micro-computed tomography (micro-CT) in a synchrotron radiation facility. We found that the obtained tomographic images were sufficient to discriminate some characteristic structures of the nervous system, including nerve cords around the cephalic ganglion, mushroom body-like structures, and putative optic nerves forming an optic commissure-like structure. Through the micro-CT imaging, we could obtain undistorted serial section images, permitting us to visualize precise spatial relationships of neuronal subpopulations and nerve tracts. 3-D micro-CT is very effective in the volume analysis of the nervous system at the cellular level; the methodology is straightforward and could be applied to many other non-model organisms.

Extremely Long Chains of Magnetic Particles via Large Plastic Beads Observed in Bimodal Magnetic Elastomers.

The relationship between the magnetorheology of bimodal magnetic elastomers with high concentrations (60 vol %) of plastic beads with diameters of 8 or 200 µm and the meso-structure of the particles was investigated. Dynamic viscoelasticity measurements revealed that the change in storage modulus of the bimodal elastomer with 200 µm beads was 2.8 × 105 Pa at a magnetic field of 370 mT. The change in the storage modulus for monomodal elastomer without beads was 4.9 × 104 Pa. The bimodal elastomer with 8 µm beads hardly responded to the magnetic field. In-situ observation for the particle morphology was performed using synchrotron X-ray CT. For the bimodal elastomer with 200 µm beads, a highly aligned structure of magnetic particles was observed in the gaps between the beads when the magnetic field was applied. On the other hand, for the bimodal elastomer with 8 µm beads, no chain structure of magnetic particles was observed. The orientation angle between the long axis of the aggregation of magnetic particles and the magnetic field direction was determined by an image analysis in three dimensions. The orientation angle varied from 56° to 11° for the bimodal elastomer with 200 µm beads and from 64° to 49° for that with 8 µm beads by applying the magnetic field. The orientation angle of the monomodal elastomer without beads changed from 63° to 21°. It was found that the addition of beads with a diameter of 200 µm linked the chains of magnetic particles, while beads with a diameter of 8 µm prevented the chain formation of the magnetic particles.

Observation of water droplets in microporous layers for polymer electrolyte fuel cells by X-ray computed nano-tomography.

An X-ray computed nano-tomography (nano-CT) system has been established at the BL33XU beamline of SPring-8. The optical system consists of pseudo-Köhler illumination with a sector condenser zone plate, an apodization Fresnel zone plate as the objective lens, and a Zernike phase plate. The imaging detector is a fiber-coupling type X-ray camera. The performance of the X-ray nano-CT system was confirmed by imaging an X-ray test chart. The system was subsequently applied to the observation of a microporous layer for polymer electrolyte fuel cells and a simulated microporous layer including liquid water. The nano-CT system, which can perform a computed tomography measurement in less than 4 min, allowed visualization of a spherical water droplet produced in the microporous layer. In the present study, the shape of water droplets in a nanoscale porous structure is investigated.

In Situ Observation of the Movement of Magnetic Particles in Polyurethane Elastomer Densely Packed Magnetic Particles Using Synchrotron Radiation X-ray Computed Tomography.

In situ observation of the migration and structure formation of magnetic particles in polyurethane elastomers was carried out by X-ray computed tomography using synchrotron radiation. The mean diameter of the magnetic particles was 7.0 µm, and the volume fraction was ϕ= 0.24 at its maximum. The exposure time was 100 ms/frame, and the pixel size was 0.458 µm/pixel. The orientation angle and the volume fraction of the maximum aggregate were analyzed using commercial software for image analysis. The orientation angle for magnetic elastomers with ϕ = 0.24 was approximately 55° at 0 mT and decreased remarkably with the magnetic field. At magnetic fields above 150 mT, the orientation angle gradually decreased with the field and showed a constant value of 38° at 300 mT, suggesting that magnetic particles move and form a chain-like structure although the chains do not align perfectly in the direction of the magnetic field. On the other hand, the volume fraction of the maximum aggregate was constant at magnetic fields below 100 mT, and it significantly increased with the field, indicating that magnetic particles were connected to each other and developed into a macroscopic structure with anisotropy. Dynamic viscoelastic measurements revealed that the storage modulus of the magnetic elastomers cannot be simply scaled by the orientation angle. It was also found that the volume fraction of the maximum aggregate is a good parameter for explaining the huge increase in the storage modulus. The dynamic movement of magnetic particles when a magnetic field of 300 mT was switched on and off was also successfully observed. When the field was switched on, magnetic particles connected instantly and their aggregates were rapidly elongated in the direction of the magnetic field. When the field was switched off, some of the connections between aggregates were broken; however, most of the aggregates did not return to the original position even 5 min after being switched off.

Methods for dynamic synchrotron X-ray respiratory imaging in live animals.

Small-animal physiology studies are typically complicated, but the level of complexity is greatly increased when performing live-animal X-ray imaging studies at synchrotron and compact light sources. This group has extensive experience in these types of studies at the SPring-8 and Australian synchrotrons, as well as the Munich Compact Light Source. These experimental settings produce unique challenges. Experiments are always performed in an isolated radiation enclosure not specifically designed for live-animal imaging. This requires equipment adapted to physiological monitoring and test-substance delivery, as well as shuttering to reduce the radiation dose. Experiment designs must also take into account the fixed location, size and orientation of the X-ray beam. This article describes the techniques developed to overcome the challenges involved in respiratory X-ray imaging of live animals at synchrotrons, now enabling increasingly sophisticated imaging protocols.

Hard x-ray phase-contrast microscopy using a Gabor hologram without a zero-order term.

In order to achieve a nanometer-scale resolution in an x-ray microscopy system, a Gabor-type hologram was produced by eliminating the zero-order term of the object diffraction pattern. In this system, a Fresnel zone plate was used for strong illumination of an object, and the zero-order diffraction was physically eliminated by a center stop. An accurate phase plate of π/2 in the Zernike method was numerically created, and the phase-contrast image was realized. The theoretical resolution was 19.8 nm. We proved that a gold nanocube with a size of 50 nm can be reconstructed with the predicted resolution.

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.

Wavelet processing and digital interferometric contrast to improve reconstructions from X-ray Gabor holograms.

In this work, the application of an undecimated wavelet transformation together with digital interferometric contrast to improve the resulting reconstructions in a digital hard X-ray Gabor holographic microscope is shown. Specifically, the starlet transform is used together with digital Zernike contrast. With this contrast, the results show that only a small set of scales from the hologram are, in effect, useful, and it is possible to enhance the details of the reconstruction.

Fresnel zone plate with apodized aperture for hard X-ray Gaussian beam optics.

Fresnel zone plates with apodized apertures [apodization FZPs (A-FZPs)] have been developed to realise Gaussian beam optics in the hard X-ray region. The designed zone depth of A-FZPs gradually decreases from the center to peripheral regions. Such a zone structure forms a Gaussian-like smooth-shouldered aperture function which optically behaves as an apodization filter and produces a Gaussian-like focusing spot profile. Optical properties of two types of A-FZP, i.e. a circular type and a one-dimensional type, have been evaluated by using a microbeam knife-edge scan test, and have been carefully compared with those of normal FZP optics. Advantages of using A-FZPs are introduced.

Starlet transform applied to digital Gabor holographic microscopy.

In this paper, we show how the starlet transform can be used to process holograms from a digital Gabor holographic microscope. The starlet transform is an undecimated wavelet transform with the property that when performing reconstruction, we only need to add all scales without the use of a synthesis filter bank. When the starlet transform is applied to a hologram, we divide the hologram into a certain number of scales, process them separately, and propagate each one using a numerical diffraction method. After diffraction propagation, we perform processing on complex amplitudes that correspond to individual scales. With the aforementioned procedure, it is possible to reduce the background and effects of parasitic fringes caused by high coherence of a laser, enhance the contrast, and reduce the effects of the twin image. Experimental results confirming the method are presented.

Ideal osmotic spaces for chlorobionts or cyanobionts are differentially realized by lichenized fungi.

Lichens result from symbioses between a fungus and either a green alga or a cyanobacterium. They are known to exhibit extreme desiccation tolerance. We investigated the mechanism that makes photobionts biologically active under severe desiccation using green algal lichens (chlorolichens), cyanobacterial lichens (cyanolichens), a cephalodia-possessing lichen composed of green algal and cyanobacterial parts within the same thallus, a green algal photobiont, an aerial green alga, and a terrestrial cyanobacterium. The photosynthetic response to dehydration by the cyanolichen was almost the same as that of the terrestrial cyanobacterium but was more sensitive than that of the chlorolichen or the chlorobiont. Different responses to dehydration were closely related to cellular osmolarity; osmolarity was comparable between the cyanolichen and a cyanobacterium as well as between a chlorolichen and a green alga. In the cephalodium-possessing lichen, osmolarity and the effect of dehydration on cephalodia were similar to those exhibited by cyanolichens. The green algal part response was similar to those exhibited by chlorolichens. Through the analysis of cellular osmolarity, it was clearly shown that photobionts retain their original properties as free-living organisms even after lichenization.

A method for estimating spatial resolution of real image in the Fourier domain.

Spatial resolution is a fundamental parameter in structural sciences. In crystallography, the resolution is determined from the detection limit of high-angle diffraction in reciprocal space. In electron microscopy, correlation in the Fourier domain is used for estimating the resolution. In this paper, we report a method for estimating the spatial resolution of real images from a logarithmic intensity plot in the Fourier domain. The logarithmic intensity plots of test images indicated that the full width at half maximum of a Gaussian point spread function can be estimated from the images. The spatial resolution of imaging X-ray microtomography using Fresnel zone-plate optics was also estimated with this method. A cross section of a test object visualized with the imaging microtomography indicated that square-wave patterns up to 120-nm pitch were resolved. The logarithmic intensity plot was calculated from a tomographic cross section of brain tissue. The full width at half maximum of the point spread function estimated from the plot coincided with the resolution determined from the test object. These results indicated that the logarithmic intensity plot in the Fourier domain provides an alternative measure of the spatial resolution without explicitly defining a noise criterion.

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.

Internal structure changes of eyelash induced by eye makeup.

To investigate how eye makeup affects eyelash structure, internal structure of eyelashes were observed with a scanning X-ray microscopic tomography system using synchrotron radiation light source. Eyelash samples were obtained from 36 Japanese women aged 20-70 years and whose use of eye makeup differed. Reconstructed cross-sectional images showed that the structure of the eyelash closely resembled that of scalp hair. The eyelash structure is changed by use of eye makeup. There was a positive correlation between the frequency of mascara use and the degree of cracking in cuticle. The positive correlation was also found between the frequency of mascara use and the porosity of the cortex. By contrast, the use of eyelash curler did not affect the eyelash structure with statistical significance.

In situ 3D crystallographic characterization of deformation-induced martensitic transformation in a metastable Fe-Cr-Ni austenitic alloy by X-ray microtomography.

Excellent strength-ductility balance in metastable Fe-Cr-Ni austenitic alloys stems from phase transformation from austenite (fcc structure) to α' martensite (bcc structure) during deformation, namely deformation-induced α' martensitic transformation (DIMT). Here, DIMT in a metastable Fe-17Cr-7Ni austenitic alloy was detected in situ and characterized in three dimensions (3D) by employing synchrotron radiation X-ray microtomography. This technique utilizes refraction contrast, which is attributable to the presence of phase boundaries between the parent austenite and the newly formed α' martensite phase. By combining microtomography and position-sensitive X-ray diffraction, we succeeded in crystallographically identifying multiple α' martensite phases continuously transformed in four groups from a single parent austenitic phase.

Microstructural and chemical features of impact melts on Ryugu particle surfaces: Records of interplanetary dust hit on asteroid Ryugu.

The Hayabusa2 spacecraft delivered samples of the carbonaceous asteroid Ryugu to Earth. Some of the sample particles show evidence of micrometeoroid impacts, which occurred on the asteroid surface. Among those, particles A0067 and A0094 have flat surfaces on which a large number of microcraters and impact melt splashes are observed. Two impact melt splashes and one microcrater were analyzed to unveil the nature of the objects that impacted the asteroid surface. The melt splashes consist mainly of Mg-Fe-rich glassy silicates and Fe-Ni sulfides. The microcrater trapped an impact melt consisting mainly of Mg-Fe-rich glassy silicate, Fe-Ni sulfides, and minor silica-rich glass. These impact melts show a single compositional trend indicating mixing of Ryugu surface materials and impactors having chondritic chemical compositions. The relict impactor in one of the melt splashes shows mineralogical similarity with anhydrous chondritic interplanetary dust particles having a probable cometary origin. The chondritic micrometeoroids probably impacted the Ryugu surface during its residence in a near-Earth orbit.

Three-dimensional network of Drosophila brain hemisphere.

The first step to understanding brain function is to determine the brain's network structure. We report a three-dimensional analysis of the brain network of the fruit fly Drosophila melanogaster by synchrotron-radiation tomographic microscopy. A skeletonized wire model of the left half of the brain network was built by tracing the three-dimensional distribution of X-ray absorption coefficients. The obtained models of neuronal processes were classified into groups on the basis of their three-dimensional structures. These classified groups correspond to neuronal tracts that send long-range projections or repeated structures of the optic lobe. The skeletonized model is also composed of neuronal processes that could not be classified into the groups. The distribution of these unclassified structures correlates with the distribution of contacts between neuronal processes. This suggests that neurons that cannot be classified into typical structures should play important roles in brain functions. The quantitative description of the brain network provides a basis for structural and statistical analyses of the Drosophila brain. The challenge is to establish a methodology for reconstructing the brain network in a higher-resolution image, leading to a comprehensive understanding of the brain structure.

Three-dimensional phase-contrast X-ray microtomography with scanning-imaging X-ray microscope optics.

A three-dimensional (3D) X-ray tomographic micro-imaging system has been developed. The optical system is based on a scanning-imaging X-ray microscope (SIXM) optics, which is a hybrid system consisting of a scanning microscope optics with a one-dimensional (1D) focusing (line-focusing) device and an imaging microscope optics with a 1D objective. In the SIXM system, each 1D dataset of a two-dimensional (2D) image is recorded independently. An object is illuminated with a line-focused beam. Positional information of the region illuminated by the line-focused beam is recorded with the 1D imaging microscope optics as line-profile data. By scanning the object with the line focus, 2D image data are obtained. In the same manner as for a scanning microscope optics with a multi-pixel detector, imaging modes such as phase contrast and absorption contrast can be arbitrarily configured after the image data acquisition. By combining a tomographic scan method and the SIXM system, quantitative 3D imaging is performed. Results of a feasibility study of the SIXM for 3D imaging are shown.

X-ray microtomographic visualization of Escherichia coli by metalloprotein overexpression.

This paper reports X-ray microtomographic visualization of the microorganism Escherichia coli overexpressing a metalloprotein ferritin. The three-dimensional distribution of linear absorption coefficients determined using a synchrotron radiation microtomograph with a simple projection geometry revealed that the X-ray absorption was homogeneously distributed, suggesting that every E. coli cell was labeled with the ferritin. The ferritin-expressing E. coli exhibited linear absorption coefficients comparable with those of phosphotungstic acid stained cells. The submicrometer structure of the ferritin-expressing E. coli cells was visualized by Zernike phase contrast using an imaging microtomograph equipped with a Fresnel zone plate. The obtained images revealed curved columnar or bunching oval structures corresponding to the E. coli cells. These results indicate that the metalloprotein overexpression facilitates X-ray visualization of three-dimensional cellular structures of biological objects.

Robust liquid marbles stabilized with surface-modified halloysite nanotubes.

We have demonstrated the fabrication of fluorine-free liquid marbles from halloysite nanotube. Halloysite is a naturally occurring inorganic nanotube that has a high aspect ratio, and the surface was modified with octadecyltrimethoxysilane. The surface-modified halloysite formed pincushion agglomerates on the surface of the liquid droplets, which create superhydrophobic surface similar to that of the plant gall surface prepared by aphids. As a result, the liquid marbles showed high mechanical strength upon impact without the use of low surface energy fluoroalkyl or fluorine-modified materials. Our results suggest a new strategy for designing novel materials for liquid marbles inspired by nature.