Takao Kuwada

Advanced applications of PXR at LEBRA, Nihon University

The monochromatic X-ray source based on parametric X-ray radiation (PXR) was developed by using Si(111) perfect crystals and the electron beam from the 125-MeV linac at Nihon University. Since the X-ray beam from the PXR system has a large exposure area with uniform flux density, the PXR-based source is suited for X-ray radiography. In addition to ordinary radiography, X-ray absorption spectroscopy and phase-contrast imaging have been developed as advanced applications of PXR. The absorption spectra of several samples were obtained using the energy dispersion of PXR, and the X-ray absorption fine structures (XAFS) were actually found in the spectra. With respect to phase-contrast imaging, refraction-contrast images have been obtained by using the X-ray diffraction in the (+, −, +) arrangement of perfect crystals. The high-contrast and the phase-reversal of the images taken in the experiment suggest that LEBRA-PXR has a high spatial coherence sufficient for phase-contrast imaging.

X-ray imaging using a tunable coherent X-ray source based on parametric X-ray radiation

A novel X-ray source based on parametric X-ray radiation (PXR) has been employed for X-ray imaging at the Laboratory for Electron Beam Research and Application (LEBRA), Nihon University. Notable features of PXR are tunable energy, monochromaticity with spatial chirp, narrow local bandwidth and spatial coherence. Since the X-ray beam from the PXR system has a large irradiation area with uniform flux density, the PXR-based source is suited for X-ray imaging, especially for application to phase-contrast imaging. Despite the cone-like X-ray beam, diffraction-enhanced imaging (DEI) can be employed as a phase contrast imaging technique. DEI experiments were performed using 14- to 34-keV X-rays and the phase-gradient images were obtained. The results demonstrated the capability of PXR as an X-ray source for phase-contrast imaging with a large irradiation field attributed to the cone-beam effect. Given the significant properties of the LEBRA-PXR source, the result suggests the possible construction of a compact linac-driven PXR-Imaging instrument and its application to medical diagnoses.

Crystallization And Preliminary X-Ray Diffraction Study Of Hemoglobin D From The Aldabra Giant Tortoise, Geochelone Gigantea

Hemoglobin D (Hb D) from the Aldabra giant tortoise, Geochelone gigantea, was crystallized by the hanging drop vapor diffusion technique with a precipitant solution containing 10% polyethylene glycol 3350 and 50 mM HEPES-Na, pH 7.5. The Hb D crystals of G. gigantea, which diffract to at least a 2.0 A resolution, belong to the monoclinic space group C2 with unit cell dimensions of a = 112.1 A, b = 62.4 A, c = 54.0 A, and beta = 110.3 degrees. One alphabeta dimer molecule of Hb D existed in an asymmetric unit, with a calculated value of Vm of 2.77 A(3)Da(-1).

Phase Contrast Imaging of Biological Materials using LEBRA‐PXR

Phase contrast x‐ray imaging is an important technique for investigation of materials consisted of light atoms, such as soft biological tissues. The tunable monochromatic x‐ray source based on Parametric X‐ray Radiation (PXR), which was developed at Laboratory for Electron Beam Research and Application (LEBRA) in Nihon University, provides x‐rays with a high spatial coherence which is an essential property required for phase contrast imaging. In preliminary experiment, refraction contrast images for leaf tissues of a tree and animal specimen have been obtained successfully with the LEBRA‐PXR x‐rays. In the imaging system, the x‐ray that passed through the sample once reflects off the silicon perfect‐crystal x‐ray analyzer at the Bragg angle, and then enters the imaging plate. The bright‐field and the dark‐field phase contrast images have been obtained by infinitesimal rotations of the analyzer, showing the evidence of contrast reversal. Although the conventional radiograph by absorption contrast was also taken with the LEBRA‐PXR, significant differences are found between the radiograph and the phase contrast images.Phase contrast x‐ray imaging is an important technique for investigation of materials consisted of light atoms, such as soft biological tissues. The tunable monochromatic x‐ray source based on Parametric X‐ray Radiation (PXR), which was developed at Laboratory for Electron Beam Research and Application (LEBRA) in Nihon University, provides x‐rays with a high spatial coherence which is an essential property required for phase contrast imaging. In preliminary experiment, refraction contrast images for leaf tissues of a tree and animal specimen have been obtained successfully with the LEBRA‐PXR x‐rays. In the imaging system, the x‐ray that passed through the sample once reflects off the silicon perfect‐crystal x‐ray analyzer at the Bragg angle, and then enters the imaging plate. The bright‐field and the dark‐field phase contrast images have been obtained by infinitesimal rotations of the analyzer, showing the evidence of contrast reversal. Although the conventional radiograph by absorption contrast was also ...

Tunable Monochromatic X‐ray Source Based on Parametric X‐ray Radiation at LEBRA, Nihon University

The monochromatic X‐ray source based on parametric X‐ray radiation (PXR) was developed by using the electron beam from the 125‐MeV linac at Nihon University. The X‐ray generating system consists of two silicon perfect‐crystal plates to offer a wide tunability. The system has actually been providing the energy dispersive monochromatic X‐ray beam in the region of 6 to 20 keV, using Si(111)‐plane for the target and the second crystals. Since the X‐ray beam from the PXR generator has rather high energy resolution and coherency, X‐ray absorption fine structure (XAFS) measurement and phase‐contrast imaging are possible applications of PXR. Actually, preliminary experiments on energy dispersive XAFS measurement and refraction‐contrast imaging have been successfully carried out using the PXR beam.

Side-necked turtle (Pleurodira, Chelonia, reptilia) hemoglobin: cDNA-derived primary structures and X-ray crystal structures of Hb A

Red blood cells of yellow‐spotted river turtles (Podocnemis unifilis, Pleurodira, Chelonia, REPTILIA) have two hemoglobin (Hb) components, Hb A and Hb D. We purified the hemoglobin component homologous to amniote (reptiles, birds, and mammals) adult Hb A which comprises two identical αA‐globin polypeptides and two identical β‐globin polypeptides. To establish the crystal structure of Podocnemis Hb A, we first determined the globin primary structures using cDNA nucleotide sequencing with the assistance of protein sequencing. The purified Podocnemis Hb A produced a different form of crystal for each of the two different buffer systems used: form A, tetragonal crystals (space group, P41212), produced under neutral pH (pH 7–8) conditions; and form B, hexagonal crystals (space group, P6122), produced under high alkaline pH (pH 11–13) conditions. Single crystals of the two forms were examined by Raman microscopy with an excitation of 532 nm, indicating their structural differences. The crystal structures of the two forms were constructed by X‐ray crystallographic diffraction at a resolution of 2.20 Å for form A and 2.35 Å for form B. The differences of the tertiary and quaternary structures of the two forms were marginal; however, one clear difference was found in helix structure. When comparing Podocnemis Hb A with Hb A from specimens in other taxa, such as Anser indicus (birds) and Homo sapiens (mammals) by SHELXPRO, the root mean square deviation (RMSD) between the corresponding Cα atoms of the two globins does not exceed 2.0 Å. These low values indicate the crystal structures resemble each other. Our data on X‐ray crystal structures and Raman spectra not only reveal the first findings on the two crystal forms of Podocnemis unifilis Hb A but also provide the first refined models for reptilian adult Hb A.

Involvement of the distal Arg residue in Cl⁻ binding of midge larval haemoglobin.

Monomeric haemoglobin component V (Hb V) from the larva of the midge Propsilocerus akamusi shows high Cl⁻ affinity under high salt concentrations at acidic pH. In order to understand the structural changes that depend on Cl⁻ binding, crystal structures of Hb V were determined under acidic high-salt conditions and the structural changes arising from different haem-bound ligands were simulated. Crystal structures of Hb V under acidic high-salt conditions indicated that the side chain of ArgE10 on the distal face of the haem contributes to stabilizing haem-bound Cl⁻. The conformation of the Arg side chain in the Cl⁻-bound form was almost identical to that in ligated Hb V at neutral pH but not to that in met Hb V under acidic salt-free conditions. Furthermore, preliminary molecular-dynamics simulations also indicated that the swinging of the Arg side chain into the haem pocket depends on Cl⁻ ligation. This result suggests that, like pH change, Cl⁻ binding affects the location of the distal Arg residue. Owing to the increased positive electrostatic potential observed in the haem pocket at acidic pH, it was concluded that electrostatic changes caused by pH change and anionic ligand binding may affect the behaviour of the polar Arg residue.

Phase‐contrast imaging with a novel X‐ray source

A novel X‐ray source based on Parametric X‐ray radiation (PXR) has been employed for phase‐contrast imaging at the Laboratory for Electron Beam Research and Application (LEBRA), Nihon University, Japan. The PXR X‐rays were generated by the 100 MeV electron beam passing through a Si single crystal. The X‐rays in the 16∼34 keV range were chosen for imaging of biological samples. The quasi‐monochromatic, tunable, and coherent X‐ray source is appropriate for this application. In addition, the large X‐ray beam irradiation field of approximately 100 mm in diameter, which was achieved without special optics, suggests that the PXR is applicable to imaging for medical diagnostics.

GEOMETRICAL EFFECT OF TARGET CRYSTAL ON PXR GENERATION AS A COHERENT X-RAY SOURCE

The experiments of the PXR performance carried out for the target crystals with different cutting planes have shown significant difference in the PXR property, which suggests another way to increase the PXR intensity than by increasing the electron beam current. In order to investigate the effect of the geometrical condition of the crystal surface on the PXR property, the experiments have been carried out for the target crystal with a knife-edge-shaped cut surface. For the case with symmetric Bragg geometry on the front surface and asymmetric condition on the rear surface, the rather low intensity X-ray beam has shown considerably good spatial coherence. The X-ray beam with narrow line width has made it possible to obtain X-ray absorption spectra with a high resolution. In contrast, relatively high intensity, which enabled taking an absorption image with the exposure for several tens of seconds, has been obtained for the geometry with asymmetric front surface and symmetric rear surface. This configuration, however, has raised a problem of degradation in the spatial coherence of the X-rays due to the superposition of two different X-ray beams.

pH-dependent structural changes in haemoglobin component V from the midge larva Propsilocerus akamusi (Orthocladiinae, Diptera)

Haemoglobin component V (Hb V) from the midge larva Propsilocerus akamusi exhibits oxygen affinity despite the replacement of HisE7 and a pH-dependence of its functional properties. In order to understand the contribution of the distal residue to the ligand-binding properties and the pH-dependent structural changes in this insect Hb, the crystal structure of Hb V was determined under five different pH conditions. Structural comparisons of these Hb structures indicated that at neutral pH ArgE10 contributes to the stabilization of the haem-bound ligand molecule as a functional substitute for the nonpolar E7 residue. However, ArgE10 does not contribute to stabilization at acidic and alkaline pH because of the swinging movement of the Arg side chain under these conditions. This pH-dependent behaviour of Arg results in significant differences in the hydrogen-bond network on the distal side of the haem in the Hb V structures at different pH values. Furthermore, the change in pH results in a partial movement of the F helix, considering that coupled movements of ArgE10 and the F helix determine the haem location at each pH. These results suggested that Hb V retains its functional properties by adapting to the structural changes caused by amino-acid replacements.