Ⅲ-5

Life, Earth andPlanetary SciencesⅢ -5. Life, Earth and Planetary Sciences

BL2A

Mo LIII-Edge XANES Study of Active Mo Species on H-GaAlMFI Catalysts
for Methane Dehydroaromatization
1

H. Aritani1, T. Sugawara1, N. Naijo1, S. Mogi1, Y. Takayama1 and A. Nakahira2

Department of Life Science & Green Chemistry, Saitama Institute of Technology, Fukaya 369-0293 Japan
2
Graduate School of Engineering, Osaka Prefecture University, Sakai 599-8531 Japan

MoO3-modified H-MFI (Mo/H-MFI) is a typicalcatalyst for methane dehydroaromatization, which isan important reaction for direct GTL (Gas to Liquid)
processes.
Manyworkers
revealed
that
MoO3-modified H-MFI zeolite (SiO2/Al2O3=28-40)
shows catalytically high activity for MTB (Methane
to Benzene) reaction, which is so-called methane
dehydroaromatization. The unique activity is basedon strong acidity and sieving effects onto MFI zeolite
support. During the reaction, MoOx species must be
reduced and carbonized to form carbide-like species(MoCxOy). But catalytic deactivation is brought aboutat the same tome by carbon contamination on MFI.
Because the deactivation strongly depend on thestrong acidity on H-MFI, an improvement of surface
property onto H-MFI is called for. In our recent study,
Ga-containing H-MFI (GaAl-MFI) supports havebeen synthesized hydrothermally,[1] and Momodified GaAl-MFI catalysts have been applied to
employ the methane dehydroaromatization. By
partially substitution of Ga ions onto H-MFI
framework (Ga/Al=50-100), the strong acidity isslightly suppressed. The effect of Ga ion onto H-MFI,
highly active and durable catalysts can be expected.
In the present report, Mo LIII-edge XANES study isapplied to characterize the Ga incorporation or
surface modification onto Mo/H-MFI to evaluate theactive Mo species after the MTB reaction.
Catalysts were prepared by impregnation of
H-GaAlMFI support with MoO2(acac)2!CHCl3
solution, and followed by drying overnight andcalcination at 773 K for 3 h. The amount of
MoO3-loading is 5.0 wt% in this study. H-GaAlMFI
supports were synthesized hydrothermally at 413 K
for a week, and followed by ion-exchanging with
NH4Cl and calcination at 873 K. The catalytic activityof methane dehydroaromatization was evaluated bymeans of fixed bed flow reaction, as described in a
separate paper [2]. The Ga-incorporated catalyst
samples are denoted as Mo/GaMFIn, in which
n=Si/Al2 atomic ratios. The Ga-modified ones (inextraframework) are denorted as Mo/Ga/MFIn. Mo
LIII-edge XANES spectra were measured in BL2A of
UVSOR-IMS in a total-electron yield mode using
InSb double-crystal monochromator. Photon energy
was calibrated by using Mo metal-foil at Mo LIII-edge,
and normalized XANES spectra and their second
derivatives are presented. REX-2000 (Rigaku)
software was used by normalization of each XANES
spectrum.
Figure 1 shows the Ga-incorporated or modified
Mo/MFI28 (upper). By Ga modification, edge energy

155

of XANES becomes lower, suggesting the promotionof Mo reduction to form Mo2C species. In case of
Mo/MFI56 catalysts (Fig. 1, lower), similar feature of
the results can be shown. But Mo/GaMFI in
Mo/Ga=50-100 catalysts only enhance the catalytic
MTB activity by Ga incorpotration. It is likely themodified Mo species are only present on MFI
extraframework, and thus, Ga incorporation brings
about the both inhibition of acidity and reduction of
Mo species. Ga modification gives similar effect only
for Mo species on MFI, but the acid site is poisoned.
Thus the catalytic activity becomes lower.

Ⅲ- 5Fig. 1. Mo LIII-edge XANES of Ga- incorporated or
modified Mo/MFI28 (upper) and Mo/MFI56 (lower)
catalysts after MTB reaction at 1023 K.
[1] K. Nagashima, S. Nakamura, K. Okada, A.
Nakahira and H. Aritani, Bull. Chem. Soc. Jpn., 82(2009) 1203.
[2] H. Aritani, H. Shibasaki, H. Orihara and A.
Nakahira, J. Environm. Sci. 21 (2009) 736.

Ⅲ -5. Life, Earth and Planetary Sciences

BL4U

Uptake of Dexamethasone into Human Skin
Investigated by Soft X-Ray Spectromicroscopy
R. Flesch1, T. Ohigashi2, S. Küchler3, K. Yamamoto1, S. Ahlberg4, F. Rancan4, A. Vogt4,
U. Blume-Peytavi4, P. Schrade5, S. Bachmann5, M. Schäfer-Korting3, N. Kosugi2 and E. Rühl1
1

Ⅲ- 5

Physical Chemistry, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany
2
UVSOR Facility, Institute for Molecular Science, Okazaki 444-8585, Japan
3
Institut für Pharmazie, Freie Universität Berlin, 14195 Berlin, Germany
4
Charité Universitätsmedizin, 10117 Berlin, Germany
5
Abteilung für Elektronenmikroskopie at CVK, 13353 Berlin, Germany

The uptake of drugs, such as dexamethasone,
topically applied onto human skin is investigatedby soft X-ray spectromicroscopy. Dexamethasone
is a widely used for the treatment of inflammatory
skin diseases such as atopic dermatitis. It is aimed
to study the depth profile of dexamethasone, sothat specific information on the uptake process isderived. Dexamethasone was dissolved in ethanol
and this 0.5% solution was applied onto the skinsample for 4 h. Subsequently, the sample was fixedand sliced into 300 nm thick sections.
The experiments were performed at the BL4U
beamline at UVSOR III using a scanning X-ray
microscope (STXM) [1]. Chemical selectivity is
obtained from excitation at the O 1s-edge (525-560
eV). Figure 1 shows a comparison of the O
1s-absoption of fixed human skin and dexamethasone.
Both spectra are similar in shape, showing an intense
O 1s! !* resonance dominating the pre-edge regime.
This resonance occurs at slightly lower energy indexamethasone (E=530.5 eV) than in skin (E=532.2
eV), providing chemical selectivity for probing the
drug uptake into skin.
Figure 2 shows a comparison of a skin sample
exposed to dexamethasone probed by optical
microscopy and soft X-ray microscopy. Figure 2(a)clearly shows the layered structure of the stratum
corneum, the outermost skin layer, probed by optical
microscopy. It is followed by the viable epidermis
and the dermis. Figure 2(b) shows for the same
section of the skin sample the spatial distribution ofabsorption, which is obtained from a difference imagein X-ray absorption measured at 528 eV (pre-edge
regime) and on the O 1s!!*-transition (530.5 eV) of
dexamethasone (cf. Fig. 1) providing chemical
selectivity. The spatially resolved results indicate thathighest absorption contrast is found in the stratum
corneum, as indicated by red color. In contrast, lower
concentration is observed in the viable epidermis andno change in absorption contrast occurs in the dermis.
It is also evident that the cells nuclei in the viableepidermis (circular structures in Fig. 2(a)) do notshow any evidence for drug uptake.

156

Fig. 1. O 1s excitation of fixed human skin (black
curve) and dexamethasone (red curve).

Fig. 2. (a) optical microscopy image of human skin;(b) spatial distribution of dexamethasone in the same
skin section, as shown in (a). See text for further
details.
[1] T. Ohigashi, H. Arai, T. Araki, N. Kondo, E.
Shigemasa, A. Ito, N. Kosugi and M. Katoh, J. Phys.
Conf. Ser. 463 (2013) 012006.

Ⅲ -5. Life, Earth and Planetary Sciences

BL4U

Nanoscale Analysis of Microbe-Mineral Interface in Bioleaching Process
by STXM Technique
1

2

S. Mitsunobu1, S. Ming1, H. Makita2 and T. Ohigashi3

Institute for Environmental Sciences, University of Shizuoka, Shizuoka 422-8526, Japan
Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka 237-0061, Japan
3
UVSOR Facility, Institute for Molecular Science, Okazaki 444-8585, Japan

Some kind of microbe can dissolve and leach “solidmineral” to obtain life energy from the solid phase.
This biotic leaching rate of minerals is much fastcompared with its abiotic leaching rate [1].
Accordingly, the bacterial mineral leaching is
recently used as a save-cost and save-energy
technique for extracting metals (eg., Cu, Ni, Zn) from
sulfide ores such as pyrite [2]. Previous papers imply
that the microbes would produce some powerfulextracellular substances to promote dissolution of theminerals effectively. On the other hand, the leachingof sulfide minerals leads to the formation of acidmine drainage. It is also important subject tounderstand the bioleaching process from a viewpointof environmental sciences. However, little is known
about the interfacial process at mineral-bacteriainterface leading to the degradation of metal sulfides,
because the interface is microscopic region and it isdifficult to analyze it directly.
Here, we have tried to analyze the extracellular
substances in bacteria-mineral interface in pyrite(FeS2) bioleaching with a high spatial resolution byscanning transmission X-ray microscopy (STXM)
installed at UVSOR BL4U, where is the first STXM
beamline in Japanese synchrotron facilities. In thisstudy, powdered natural pyrite (Navajún mine, Spain)
and Acidithiobacillus ferrooxidans (JCM 7812 andATCC 23270) were used for the sulfide mineral andleaching bacteria, respectively. The acidophilic,
iron(II)-oxidizing bacteria A. ferrooxidans is one of
the most important mesophiles for the extraction of
metals from sulfidic ores by the bioleaching [1-2].
Samples for the STXM analysis were collected after
30 days incubation. The sample was dropped on
Si3N4 membrane and air-dried at RT.
Figure 1 shows STXM images of A. ferrooxidans
cells accreted on the pyrite particle for oxygen (O)and nitrogen (N). Characterization of extracellularmatter in the bacteria-mineral interface was
performed by near edge X-ray absorption finestructure (NEXAFS) at the O K-edge (Fig. 2).
Features around the absorption edge and post-edge oflipid, DNA, sugar, and protein clearly varied. Spectralfeatures of A. ferrooxidans cell are mostly similar to
those of alginate and albumin representative for sugar
and protein, respectively. Thus, O NEXAFS results
indicate that A. ferrooxidans forms the extracellular
substances on the pyrite surface containing the sugar
such as polysaccharide addition to the protein, which
was also supported by N NEXAFS and lectin-staining

results(data
notshown).
The
biogenic
polysaccharides often form a strong complex with Fe
ion under wide pH region [3-4]. Thus, our findings
imply that A. ferrooxidans could produce thepolysaccharides to accelerate the dissolution of pyrite
and/or mediate contact between the cell and pyrite
surface.

Normalized absorbanceFig. 1. STXM-derived elemental maps for oxygen(550 eV) and nitrogen (410 eV) of cells andsurrounding pyrite particles.

Lipid
DNA
Alginate
ProteinSpot 1 in cell

Spot 2 in cell

530 535 540 545 550
Energy (eV)Fig. 2. STXM-based oxygen 1s NEXAFS spectra ofstandards and samples. Standards contain bovine
serum
albumin (protein), alginate (acidic
polysaccharide),
E.
coli
DNA
and
1,2-dipalmitoyl-sn-glycero-3-phospho ethanolamine(lipid).
[1] H. Tributsch, Hydrometallurgy 59 (2001) 177.
[2] W. Sand et al., Appl. Environ. Microbiol. 58(1992) 85.
[3] C. Chan et al., Geochim. Cosmochim. Acta 73(2009) 3807.
[4] S. Mitsunobu et al., Environ. Sci. Technol. 46(2012) 3304.

157

Ⅲ- 5

Ⅲ -5. Life, Earth and Planetary Sciences

BL4U

Observation of DNA and Protein Distributions in Mammalian Cell Nuclei
Using STXM
T. Ohigashi1,2, A. Ito1,3, K. Shinohara3,4, S. Tone5, M. Kado4, Y. Inagaki1, Y. F. Wang1
and N. Kosugi1,2
1

2

Ⅲ- 5

UVSOR Facility, Institute for Molecular Science, Okazaki 444-8585, JapanThe Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8585, Japan
3
Tokai University, Hiratsuka 259-1292, Japan
4
Japan Atomic Energy Agency, Kizugawa 619-0215, Japan
5
Kawasaki Medical School, Kurashiki 701-0192, Japan

To observe the structure of biological samples, anelectron microscope and a fluorescence microscopeare extensively used. However, the former requires
specimen in vacuum and the latter has relatively
lower spatial resolution. Moreover, the electron
microscope usually requires several preparation
processes for the samples, such as fixing, slicing andstaining. On the other hand, a soft X-ray microscopy
is applicable to relatively thick specimen even underhydrated condition at high resolution, and is expected
to be complementary to these two types of
microscopes. A scanning transmission X-ray
microscope (STXM) must be a powerful tool for thispurpose [1]. The STXM has high spatial resolution,high transmittance and lower radiation damage thanthe electron beam. Especially, chemical analysis
combined with near edge X-ray absorption finestructure (NEXAFS) enables us to obtain
2-dimensional chemical information of the sample [2].
In this study, nuclei of cultured human cells wereobserved with the STXM installed on UVSOR BL4U
to image the distributions of DNA and protein
separately.
NEXAFS spectra of the DNA and histone, anuclear protein, were measured by the STXM as
reference data. Their suspensions were dropped onto
100 nm-thick silicon nitride membranes and weredried in the air. Their NEXAFS spectra aroundnitrogen 1s are shown in Fig. 1. In these spectra, aremarkable feature to discriminate the DNA from theprotein is seen on a peak at 400.8 eV as nitrogen
1s→π* resonance arising from C=N double bonds inthe DNA.
Human A549 cells derived from lung cancer were
cultured directly on the silicon nitride membrane,
fixed with glutaraldehyde, and dried in the air. The
sample was placed in the STXM chamber, which wasthen evacuated and was filled by helium to 30 mbar.
The 51 X-ray transmission images (an energy stack)were acquired with changing the X-ray energies from
399 to 404 eV. The dwell time and the scanning pitchof the specimen were 5 ms and 0.2 µm step,
respectively. The reference spectra of the DNA andthe histone in Fig. 1 were fitted to the energy stack byusing aXis2000 software [3] and their distributions
are shown in Fig. 2. Figures 2 (a) and (b) show
distributions of the DNA and the histone (protein),158

respectively. Figure 2 (c) shows the distribution of
constant profile with no spectral feature, suggestingthat in the nucleolus molecules other than the DNA
and/or the histone (protein) are densely accumulated.
The results show that the DNA was distributed overthe nucleus, while the histone was poorly distributedin the nucleolus. Considering that RNA is rich in the
nucleolus, the RNA may be present with less proteinin the nucleolus.
DNA

HistoneFig. 1. X-ray absorption spectra of the DNA and thehistone (protein) around nitrogen 1s resonance.
(a)

(c)

(b)

Nucleus
NucleoliFig. 2. Distributions of the DNA (a), the histone(protein) (b) and constant (c) in the cell. Bright color
shows high density. Scale bars are 2 µm.
[1] J. Kirz et al., Nucl. Instr. and Meth. B 87 (1994)
92-27.
[2] T. Ohigashi et al., J. Phys. Conf. Ser. 463 (2013)
012006.
[3] http://unicorn.mcmaster.ca/aXis2000.html

Ⅲ -5. Life, Earth and Planetary Sciences

BL4U

Nitrogen- and Oxygen-XANES Spectral Analysis of Mineral Olivine andOrganic Material from Meteorite: Preliminary Measurements for
Implementation Planning of a Synchrotron Cosmochemistry Research Baseat UVSOR BL4U
H. Yabuta

Department of Earth and Space Science, Osaka University, Osaka 560-0043, JapanThe PI has eight years of experiences of a scanningtransmission x-ray microscope (STXM) operations
and analyses in cosmo- and geochemistry [e.g., 1-3].
Making great use of her experiences, she is planningto construct a synchrotron cosmochemistry researchbase at BL4U, UVSOR, under cooperation with abeamline manager. Because of the first use of the
STXM (Bruker) built at BL4U, UVSOR, this year,
the initial purpose of this study was to acquire theoperation of the STXM and to confirm thereproducibility of the STXM measurements oforganic materials from meteorites. However,
unfortunately, it was impossible to measure Carbon
K-edge XANES spectra, due to a bad condition of the
STXM stage motions and contamination of the mirror.
This report summarizes the preliminary result of
Nitrogen and Oxygen K-edge XANES.
As samples, mineral olivine (magnesium ironsilicate) and acid-insoluble organic solids (IOM)
isolated from carbonaceous meteorite Asuka 881458
were used. Thin sections (100 nm thickness) of theindividual samples were prepared by a focused ion
beam (FIB) - scanning electron microscope (SEM).
FIB sections of olivine Nos. 1 and 2 were prepared
before and after that of IOM was prepared,
respectively. Nitrogen- and Oxygen-X-ray absorption
near edge structure (XANES) spectra of the FIB
sections were acquired using a scanning transmission
x-ray microscope (STXM) at the BL 4U, UVSOR.
Nitrogen- and Oxygen-XANES spectra of olivine(Nos. 1 and 2) and IOM are shown in Fig. 1.
O-XANES spectrum of meteorite Asuka (A) 881458
IOM exhibits a peak of organic carbonyl group(C=O) at 532 eV. This peak was not identified fromthe O-XANES spectra of olivine samples 1 and 2,
demonstrating that the previous sample did notcontaminate the latter sample during the FIB
procedures. Likewise, N-XANES spectrum of
meteorite Asuka (A) 881458 IOM exhibits absorption
peaks of organic imine (C=N), nitrile (CN), andamide (NHx(C=O)C) groups at 399, 400, 402 eV,
respectively, while those of olivine samples 1 and 2
do not.

159

Ⅲ- 5

Fig. 1. N- and O-XANES spectra of mineral olivine
samples Nos. 1 (black) and 2 (blue) andacid-insoluble organic solids isolated fromcarbonaceous meteorite Asuka (A) 881458 (red).
[1] S. A. Sandford et al., Science 314 (2006)
1720-1724.
[2] G. D. Cody et al., Meteoritics & Planetary
Science 43 (2008) 353 – 365.
[3] H. Yabuta et al., The 44th Lunar and Planetary
Science Conference. Abstract (2013) #2335.

Ⅲ -5. Life, Earth and Planetary Sciences

BL4U

Organelle-Spectra of a Leydig Cell in Cultural Fluid
T. Ejima1, R. Hirose1, M. Ishino2, M. Kado2, M. Aoyama3, K. Yasuda3 and T. Tamotsu3
1

IMRAM, Tohoku University, Sendai 980-8577, Japan
Japan Atomic Energy Agency, Kizugawa 619-0215, Japan
3
Dept. of Biological Sciences, Fac. of Science, Nara Women’s University, Nara 630-8263, Japan2

Ⅲ- 5

To investigate suitable wavelength for observing
organelles in a bio-cell with fluid in Water-window
wavelength region, contrast of each organelle in a
bio-cell to fluid or the other organelles will be
determined by absorption spectra of each component.
For this purpose, soft X-ray absorption images ofLeydig cells in cultural fluid were observed
controlling the thickness of the fluid. Spectra of bothorganelles and fluid will be obtained from the images
that irradiate wavelength was changed.
At first, Leydig cells of mouse testes cultured andfixed on a SiN membrane were preserved in thecultural fluid during the X-ray observation. Thicknessof the cultural fluid was controlled by a SU8 film
deposited around the membrane. The membrane withthe bio-cells was placed in a holder and the holder
was set to the stage under He atmosphere to avoid
leak of the fluid during the observation. The
observation was made at 2.93.1nm (395430eV)
wavelength region under the conditions that the step
width was 300nm, exposure time, 5 msec/pixel, andwavelength resolution !/"!, 5000.
Organelle structures in a cell and spectra
corresponding to the structures were obtained
applying PCA and Cluster analyses to the observation
results [1]. Figure 1 (a) shows an eigen-image thatshows maximum value of the contribution ratio
obtained from the PCA analysis. The eigen-image
represents that several large organelles exist in thestar-shaped bio-cell and many dot-structures arearound the large organelles.
According to Cluster analysis, these organelles are
categorized by the degree of similarity as the map
represented in Fig. 1 (b). In the map, blue area
represents the culture fluid and the other areas, a
bio-cell and organelles in the cell.
Figure 1 (c) shows that the absorption spectrum
that corresponds to the yellow area of Fig. 1(b). In the
spectrum, there is a sharp peak at 400eV and a broad
peak with a shoulder structure at 405 eV. In addition,
small structures exist at 398 eV, 402 eV, and 404 eV.
The sharp peak at 400 eV and the broad one at 405
eV should be originated from the #* and $*structures, respectively, of C-N bonds in theorganelles [2]. Small and shoulder structures will besatellite structures of C-N bonds and the details of theorigin need further investigation.
Small organelles such as dot structures in Fig. 1(a)are recognized in the eigen-images, but the spectra of
the small orgranelles are not clearly separated fromthe other organelles. The experimental conditions andimage-analysis techniques will be improved.
This work was supported by JSPS KAKENHI
Grant Numbers 23241038 and by Nanotechnology
Platform Program (S-13-MS-1002) of MEXT, Japan.

(b)
(a)
(c)

Fig. 1. (a) Optical density image of a Leydig cell. (b)Clusters obtained from cluster analysis on the basis of
the results of PCA analysis. (c) Optical density
spectrum of the yellow area represented in (b) (inset).
[1] M. Lerotic, C. Jacobsen, J. B and Gillow, A. J.
Francis, S. Wirick, S. Vogt and J. Maser, J. Elec.
Spectro. Rel. Phenom. 144-147 (2005) 1137-1143.
[2] Y. Zubavichus, A. Shaporenko, V. Korolkov, M.
Grunze and M. Zharnikov, J. Phys. Chem. B 112(2008) 13711-13716.

160