Kuniko Takemoto

Scanning X-ray Microscopy of Living and Freeze-Dried Blood Cells in Two Vanadium-Rich Ascidian Species, Phallusia mammillata and Ascidia sydneiensis samea

Abstract Some ascidians (sea squirts) accumulate the transitional metal vanadium in their blood cells at concentrations of up to 350 mM, about 107 times its concentration found in seawater. There are approximately 10 different types of blood cell in ascidians. The identity of the true vanadium-containing blood cell (vanadocyte) is controversial and little is known about the subcellular distribution of vanadium. A scanning x-ray microscope installed at the ID21 beamline of the European Synchrotron Radiation Facility to visualize vanadium in ascidian blood cells. Without fixation, freezing or staining realized the visualization of vanadium localized in living signet ring cells and vacuolated amoebocytes of two vanadium-rich ascidian species, Phallusia mammillata and Ascidia sydneiensis samea. A combination of transmission and fluorescence images of signet ring cells suggested that in both species the vacuoles contain vanadium.

Microstructure and Crystallinity of N-Type Porous Silicon

Microstructure and crystallinity of a porous silicon (PS) layer formed on a highly doped n-type silicon substrate were examined in detail, and the luminescent nature of the PS layer was also studied qualitatively. A layered pore structure developed after about 3000 s of anodization, and pore morphology became simpler with increasing thickness of the PS layer. All luminescent PS layers were found to show spongelike structure near the surface. From the peak width of X-ray rocking curves, the crystallinity of n-type PS was found to be inferior to that of p-type PS. No systemic tendency of the lattice expansion was seen under various forming current densities. Luminescent PS layers showed slightly broader background intensity than nonluminescent ones. It was also shown that the anodization of the PS layer took place through two anodization processes: primary and secondary anodizations; secondary anodization was effective in forming luminescent parts having spongelike structure.

Transmission X-Ray Microscopy with 50 nm Resolution Installed at Ritsumeikan Synchrotron Radiation Center

A transmission X-ray microscope having two zone plates has been developed at BL-12, Ritsumeikan Synchrotron Radiation Center (Rits SR Center). By incorporating a new type of condenser zone plate (diameter 9 mm, zone number 41,890, outermost zone width 53.7 nm) in the microscope, 50 nm resolution has been obtained from both knife edge test and star test pattern at a wavelength of 3.2 nm, and for diatoms, the 0.1 µm inner structures have been clearly resolved.

Transmission X-ray microscopy with 50 nm resolution in Ritsumeikan synchrotron radiation center

Transmission X-ray microscope at Rits SR Center at Ritsumeikan University has been upgraded to get a higher performance. Its optical configuration allows a continuous wavelength change throughout water window region, 2.2–4.3 nm. The present paper describes results observed with the high-performance X-ray microscope, and compared the images taken at two different wavelengths, 2.4 nm and 3.2 nm. Its achieved resolution with each wavelength was below 50 nm. Several specimens such as latex spheres of 0.23 μm diameter, diatoms of 0.1 μm inner structures could be clearly resolved.

Imaging soft X-ray microscope at Rits Synchrotron Radiation Center

An imaging soft X-ray microscope with zone plates has been installed at Rits SR Center (Ritsumeikan University, Kusatsu, Japan). With this microscope, specimens were set in air, which made it possible to investigate the specimens without breaking the vacuum of the microscope. The specimens can be prefocused with an optical microscope. Dry and wet biospecimens in air were observed. A new optical system was designed to improve the resolution.

Estimation of Organic Carbon Content of the Cyanobacterium Synechococcus sp. by Soft X-ray Microscopy

A method for the accurate evaluation of the organic carbon (OC) content of phytoplankton by soft X-ray microscopy (XM) was developed and applied to the picophytoplankton Synechococcus sp., whose cells are covered by extracellular polysaccharides (EPS). Based on the X-ray absorption coefficients and gray levels of the XM images, the OC content of EPS-covered cells of Synechococcus sp. (0.39–0.47 pg/cell) was found to be 2.0–2.4 times larger than that of EPS-removed cells (0.20 pg/cell). These findings suggest that soft XM could be a useful tool for evaluating the OC content of picophytoplankton and EPS without pretreatment steps.

Micro XAFS Study on Vanadium in Ascidians Alive Blood Cells Investigated by Fluorescence Scanning X‐Ray Microscopy at ID21 at ESRF

X‐ray microscopy microscope installed at the beam line ID21 at European Synchrotron Radiation Facility (ESRF) was used for the elucidation of micro‐distribution of vanadium in ascidians. In order to examine chemical states of vanadium in the alive blood cell, a high pressure cryo‐fixation technique was applied to fix its chemical states of vanadium. We have succeeded in observing uniform distribution of vanadium in the vacuole, in identifying the valency of vanadium in the cell, and more importantly in finding granules rich in vanadium of the 4th valency.

Application of soft X-ray microscopy to environmental microbiology of hydrosphere

Microstructures of unprocessed filamentous cyanobacterium, Pseudanabaena foetida sp., producing a musty smell were observed using soft X-ray microscopy. Carbon-enriched structures and granules as well as oxygen-enriched granules which have been already reported were observed. Except for early log growth phase, the oxygen-enriched granules were observed. However, the carbon-enriched structures were observed throughout log growth phase. The result suggests there is a relationship between the oxygen-enriched granules and 2-methylisoborneol (2-MIB) productivity, since the 2-MIB productivity of each cell is increased depending on the culture period in log growth phase.

Imaging of musty-odor producing filamentous cyanobacteria by various microscopies

A method for the determination of filamentous cyanobacteria producing musty-odor substances quickly and accurately by soft X-ray microscopy (XM) was developed and applied to Phormidum tenue sp. (Pseudanabaena sp.) green strain (PTG). To improve the identification accuracy of PTG, PTG was observed by soft XM using two wavelengths above and below the K-edge of oxygen. Two types of granules were confirmed. Since a granule of which oxygen is a major constituent element is identified as polyphosphate granule, polyphosphate granule and carboxysome are identified, respectively.

Improvement of cryogenic 3-dimensional observation system of soft x-ray microscope at the SR center of Ritsumeikan University

The improvements of a soft x-ray microscope beamline (BL-12) at the SR center of Ritsumeikan University are reported. A wedge-shaped slit and Si plane mirror were newly introduced. The better energy resolution was expected and the +2nd order diffraction from the CZP (1.2 nm at 2.4 nm observation) was suppressed. A new sample holding fixture allows the sample to be replaced quickly and accurately. A new sample cooling system allowed a stable cryogenic x-ray imaging.