Ⅲ-5

Life, Earth andPlanetary Sciences

961BL4U 光延聖.docx

Ⅲ -5. Life, Earth and Planetary Sciences

BL4U

A Key Mechanism of Bacterial Pyrite Leaching
Identified by Direct STXM Analysis at Cell-Pyrite Interface
S. Mitsunobu1, 2, M. Zhu2, Y. Ohashi2 and T. Ohigashi3

Department of Agriculture, Ehime University, Matsuyama 790-8566, Japan
Institute for Environmental Sciences, University of Shizuoka, Shizuoka 422-8526, Japan
3
UVSOR Facility, Institute for Molecular Science, Okazaki 444-8585, Japan1

2

Microbial bioleaching of metal sulfides has beenused as a low-cost engineering process for extracting
metals from sulfidic ores due to its fast dissolution rate[1]. The microbial bioleaching of metal sulfide also
contributes to formation of environmentally
detrimental acid mine drainage (AMD), whose acidic
nature and heavy-metal constituents cause serious
contamination of soil and groundwater in the world.
Thus, a better understanding of the mechanisms is of
crucial importance for improvement of both industrial
bioleaching and AMD formation.
There have been considerable efforts to identify
mechanisms of bioleaching.
However, little is
known on the microbial bioleaching mechanism,
because direct chemical speciation of metals andbiomolecules at mineral-microbe interface has beendifficult due to high spatial resolution in analysis.
Here, we investigated the mechanisms of thebioleaching process in bacterial pyrite leaching byleaching bacteria (Acidithiobacillus ferrooxidans) by
scanning transmission X-ray microscopy (STXM)
based C and Fe near edge X-ray absorption finestructure (NEXAFS) analyses at UVSOR BL4U.
Carbon NEXAFS analysis directly showed that
attached A. ferrooxidans produces polysaccharideabundant extracellular polymeric substances (EPS) at
the cell-pyrite interface (data not shown, [2-3]).
Figure 1 shows the STXM-based merged Fe/C image
and Fe 4p NEXAFS of bacteria cells attached to pyrite
particles in 2 weeks incubation. The image in Fig. 1ashows that Fe was localized around the surface of thebacteria cells.
Considering that C NEXAFS
demonstrated the appearance of a polysaccharide-rich
EPS layer at the cell-pyrite interface, this suggests that
Fe had accumulated in this polysaccharide layer. In
the Fe NEXAFS spectra, both the spectra of whole celland cell-pyrite interface (interface 1 and 2 in Fig. 1b)
consist of Fe(II) and Fe(III) peaks. Thus, the Fe
species in both cell and cell-pyrite interface were
Fe(II) addition to Fe(III) [3].
In previous study, researchers have implied apossible role for the Fe(III) in EPS in whole pyrite
bioleaching process, an oxidizing agent for sulfides [1].
That is the Fe(III) in EPS would abiotically oxidize
sulfides in pyrite according to following equations.
FeS2 + 6Fe3+ + 3H2O → S2O32- +7Fe2+ + 6H+
S2O32- + 8Fe3+ + 5H2O → 2SO42- +8Fe2+ +10H+
However, this interfacial process remained speculative
because no Fe(II) in the EPS subsequently formed by141

this oxidation process had been observed yet.
Previous studies determined the Fe species in EPS byindirect methods [1]. The Fe(II) detected by direct
STXM based NEXAFS analyses in this study is a first
direct evidence supporting the oxidative attack by the
Fe(III) in EPS. These findings could be important
information for understanding key mechanism of bothbacterial mineral leaching and AMD formation.

Ⅲ- 5

Fig. 1. STXM-based C and Fe images (a) and Fe 4p
NEXAFS spectra (b).
[1] W. Sand and T. Gherke, Res. Microbiol. 157 (2006)
49.
[2] S. Mitsunobu et al., Chem. Lett. 44 (2015) 91.
[3] S. Mitsunobu et al., Microbes Environ. (in press).

2003-2019.docx
Ⅲ -5. Life, 965BL4U
Earth and上椙真之Planetary
Sciences

BL4U

Application of XANES to the Analysis of Carbonaceous Materials in
Hayabusa-Returned Samples for Determination of Their Origin
1

Ⅲ- 5

M. Uesugi1 , A. Nakato1 , T. Ohigashi2 and Y. Inagaki2

Institute of Space and Astronautical Science, Japan aerospace exploration agency, Sagamihara, 252-5210, Japan
2
UVSOR Synchrotron, Institute for Molecular Science, Okazaki, 444-8585, Japan

Carbonaceous particles, those have been found in
the sample catcher of Hayabusa spacecraft together
with silicate particles, were still under theinvestigations, including precise determination of
their origin. Results of isotopic analysis of H, C and
N of the carbonaceous materials by NanoSIMS did
not show any signatures of their extraterrestrial origin,e.g. isotopic anomalies against the terrestrial
composition [1]. Some of the results of analyses by
Transmission Electron Microscopy (TEM, [2]) and
X-ray absorption near edge structure (XANES, [3])indicated relation to the terrestrial material. Thus,
those particles might indicate the contamination ofterrestrial material into the sample catcher before,
during and/or after the operation of Hayabusa
spacecraft.
In previous study, a particle from the witness plate
exposed to the clean room of Hayabusa2 spacecraft
construction, where Hayabusa1 spacecraft wasconstructed, was investigated by XANES/STXM
installed in ALS/LBNL, Berkeley. The shape of
XANES spectrum of the particle (green, WP1 in Fig.
2) closely relates to that of a Hayabusa-returned
sample, RA-QD02-0180-03 (blue line in Fig. 2), andindicate the relation of them.
In this work, we investigated a particle newly
picked up from the same witness plate using
XANES/STXM installed in UVSOR, in order toconfirm the result of previous study.
Figure 2 shows C-XANES spectra of the newparticle (red, WP2) and Hayabusa-returned samples(blue, RA-QD02-0180-03). The shape of the spectra
was similar, but position of the peaks of WP2 weredifferent from others.
N-XANES spectrum of the WP2 was also largely
different from other two samples. Though particles
were picked up from the same witness plate andchemical composition of them was similar, thosedifference of XANES spectrum indicate the different
origin of WP1 and WP2. The fact indicate several
paths of the contamination to the sample catcher werepossible, and should be considered for the precise
determination of the origin of the contaminant.
In future work, we will apply the XANES/STXM
analysis to further possible contaminants such as
particles of biological materials.

142

Fig. 1. STXM image of the ultrathin section of WP2
in Fig. 2. The particle was pressed on a gold plate andcut by Focused ion beam to extract ultrathin sections.

Fig. 2. C-XANES spectra of the particles from thewitness plate of clean room of the Hayabusa2
spacecraft construction (green and red), andHayabusa-returned sample (blue). Peak positions of
WP2 were different from those other two samples.
[1] M. Ito et al., Earth planet. Space 66 (2014) 91.
[2] M. Uesugi et al., Earth planet. Space 66 (2014)102.
[3] H. Yabuta et al., Earth planet. Space 66 (2014)156.

971BL4U 伊藤敦.docx

Ⅲ -5. Life, Earth and Planetary Sciences

BL4U

Distribution of DNA and Protein in Mammalian Cell Nuclei Calculated
Using NEXAFS Profiles at the N-K and O-K Absorption Edges
A. Ito1, T. Ohigashi2, 3, K. Shinohara1, S. Tone4, M. Kado5, Y. Inagaki2 and N. Kosugi2
1
School of Engineering, Tokai University, Hiratsuka 259-1292, JapanUVSOR Synchrotron, Institute for Molecular Science, Okazaki 444-8585, Japan
3
The Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8585, Japan
4
Department of Biochemistry, Kawasaki Medical School, Kurashiki 701-0192, Japan
5
Nat. Inst. Quantum and Radiological Sci. Technol., Kizugawa 619-0215, Japan
2

Spectromicroscopy using scanning transmission Xray microscope (STXM) has been applied to DNA andprotein distributions in biological specimens such as
chromosome and sperm. Our previous study on the
DNA mapping in mammalian cell nuclei was
successfully carried out for cultured A549 cells withthe aid of different NEXAFS (Near Edge X-ray
Absorption Fine Structure) profiles between DNA andprotein at the N-K absorption edge as shown in the left
panel of Fig. 1 [1]. The results gave rather unexpected,
different distributions of DNA and histone in nucleoli.
In this study, to confirm these results we extended theanalysis to the O-K edge, and also to obtain
quantitatively consistent results at different absorption
edges, we developed the correction of reference
NEXAFS spectra used for the “stack fit” method to
obtain molecular distribution.
Human HeLa cell was used because of its large
nuclear area. Figure 2 shows transmission and opticaldensity (OD) images of a whole cell taken at 400 eV.
Four large nucleoli in the nucleus were identified, andfor the enlarged area DNA and histone distributions
were calculated using aXis2000 software to fit the
specimen spectra to the reference spectra. In the centerof nucleoli histone content was rather poor comparedwith DNA, which was consistent with our previous
report [1].
For the extension of the analysis to the O-K edge,
reference spectra at the O-K edge (Fig. 1) should becorrelated to those at the N-K edge, since the fitting
results do not necessarily assure the same quantitative
distribution due to the independent measurements ateach absorption edge. As a trial of the correlation, first
we calculated mass thickness, which is defined as a
product of the density and thickness of specimen, from
OD by considering mass absorption coefficients at 395
eV. Then the OD of reference spectra at the O-K edge
was corrected using this mass thickness at 525 eV.
Based on these corrected spectra, DNA and histone
distributions were obtained as shown in the lower
panels of Fig. 3, indicating similar distributions tothose at the N-K edge. Table 1 shows OD range of
DNA and histone at the N-K and O-K edges. OD
differences and the ratio of DNA and histone were also
listed. Similar ratios between the N-K and O-K edges
were obtained, although the absolute OD values were
not totally the same.

N-K edge

O-K edge

Fig. 1. NEXAFS spectra of DNA and histone at theN-K and O-K edge.
Transmission image

OD image

Fig. 2. Transmission and OD images of human
HeLa cell at 400 eV. White circle in OD image isenlarged in Fig. 3. Scale bar corresponds to 5
microns.
DNA

HistoneN-K edge

O-K edge
Fig. 3. DNA and histone distributions of nucleus of
HeLa cell obtained at the N-K and O-K edges. OD
range bar is shown in the right. Scale barcorresponds to 2 microns.
Table 1. Comparison of DNA and histone contents
between the N-K and O-K edges.
DNA(N-edge)
Histone(N-edge)
DNA(O-edge)
Histone(O-edge)

OD range-0.2561.80
0.4431.26-0.06943.44-0.004131.48

Difference DNA/Histone
2.06
2.52
0.817
3.51
2.36
1.48

[1] T. Ohigashi et al., AIP Conference Proceedings
1696 (2016) 020027.

143

Ⅲ- 5

Ⅲ -5. Life, 981BL4U
Earth and高橋嘉夫.docx
Planetary Sciences

BL4U

Speciation of Sulfur and Calcium in Aerosols by STXM
C. Miyamoto1, H. Suga2, T. Ohigash3, Y. Inagaki3 and Y. Takahashi1

2

Ⅲ- 5

1
Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan
3
UVSOR Synchrotron, Institute for Molecular Science, Okazaki 444-8585, Japan

Aerosols, defined as small particles or droplets inthe atmosphere, have important effects on theenvironment. Sulfate is the main compound in aerosols(in particular anthropogenic ones) and is emitted
primarily by human activities. As suggested by theIntergovernmental Panel on Climate Change, sulfate
aerosols can cool the earth in two ways: directly byreflecting sunlight, and indirectly by acting as cloud
condensation nuclei. The latter cooling role is madepossible by the hygroscopic nature of some sulfates. In
the atmosphere, sulfate occurs in several forms
including ammonium salts (NH4HSO4, (NH4)2SO4),
calcium salt (gypsum; CaSO4·2H2O), and hydrated
species (Takahashi et al., 2006). Among these species,
ammonium salts are hygroscopic, while calcium salts
are not. Thus, sulfate species should be well identified
to precisely evaluate the global cooling effect ofsulfate aerosols. Thus, this study conducted speciationof sulfur in aerosols by Scanning Transmission X-ray
Microscopy installed at BL4U, UVSOR.
In this study, distributions of sulfur and calcium
species within aerosol particles were examined foraerosols collected in Higashi-Hiroshima in May, 2014,
which can be a basis for understanding chemical
processes to form sulfate aerosols. First, distribution of
calcium in a particle with a few μm diameter was
examined. Although calcium is distributed in thewhole particle, calcium oxalate or calcium sulfate wasmainly found at the rim of the particle based on the
calcium LIII- and LII- edge XANES (Figs. 1 and 2). It
is known that concentrations of sulfate and oxalate
correlate each other [1]. The present results suggested
that both sulfuric acid and oxalic acid react withcalcium particle, particularly calcite, in the atmosphere.
Distribution of sulfur was also examined by thesulfur LIII edge absorption. It was found that calcium
sulfate wad distributed within the rim where calcium
oxalate and/or calcium sulfate were found by calcium
L-edge absorption (Fig. 1).
All these results support that secondary calcium
species produced by the reactions of calcium carbonate
with sulfuric or oxalic acids were formed in theatmosphere. In particular, formation of calcium oxalate
at the rim of the particle is found for the first time by
any microscopic analyses.
Sulfur speciation was also conducted for theparticles collected as a fine particle ranging from 0.69
to 1.3 μm. As a result, sulfur species was almost
exclusively ammonium salt within the particles basedon the XANES analysis, showing that sulfate aerosols
smaller than 1.3 μm is not the products of reactions

144

with minerals, but sulfate aerosol possibly produced indroplet mode.

Fig. 1. STXM images of total calcium, Ca-oxalate
and/or sulfate, and total sulfate by absorption at Ca andS L-edges.

Fig. 2. Calcium LⅢ and LⅡ edges XANES for Caoxalate, Ca-sulfate, and Ca in various spots in Fig. l (b).
[1] T. Furukawa and Y. Takahashi, Atmos. Chem. Phys.
11 (2011) 4289.

985BL4U 藪田ひかる 2008-2022.docx

Ⅲ -5. Life, Earth and Planetary Sciences

BL4U

Chemical Evolution of Meteoritic Organics during Impact Metamorphism
H. Yabuta1, T. Atou2, T. Ohigashi3, 4, Y. Inagaki3, P. Beck5, L. Bonal5 and E. Quirico5

1
Department of Earth and Space Science, Osaka University, Osaka 560-0043, Japan
Laboratory for Materials and Structures, Institute of Innovative Research, Tokyo Institute of Technology, Tokyo
226-8503, Japan
3
UVSOR Facility, Institute for Molecular Science, Okazaki 444-8585, Japan
4
School of Physical Sciences, The Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8585,
Japan
5
Institut de Planétologie et d’Astrophysique de Grenoble (IPAG), UJF-Grenoble 1 / CNRS-INSU, UMR 5274,
Grenoble, F-38041, France

2

The primitive solar system materials such as
meteorites, comets, and these-derived cosmic dusts
were formed 4.5 billion years ago by accretion of gas
and dusts in interstellar cloud. The interstellar dusts areabout ten micron-sized solid particles consisting ofsilicate mineral core, refractory organics, andamorphous ice mantle. Therefore organic molecules inspace can be described as one of the major building
blocks of our Solar System and Life. To date,molecular and isotopic compositions of organics fromthe meteorites ranging various groups and petrologic
types have been frequently studied in relation to thechemical history in the early Solar System [1, 2].
Shock metamorphism is one of the important
processes in the early Solar System, but its relationshipwith chemical evolution of meteoritic organic
compounds has been less frequently studied thanaqueous alteration and long-term heating due toradiogenic elements on a meteorite parent body.
Yabuta et al. (2010) [3] has revealed that molecularand isotopic characteristics of organic macromolecularsolids in the shocked meteorites are distinct from thosein the long-term heated meteorites. In this study, forunderstanding chemical behaviors of organic matter inmeteorites, molecular analyses of organic matter theexperimentally shocked meteorite were conducted.
As a sample, Murchison meteorite powder was usedin this study. The shock experiments of a meteorite
powder pellet (~180 mg) in a SUS collecting vessel
were conducted at 5 and 10 GPa by a single-stage
powder gas gun and at 20 and 40 GPa by a doublestage light gas gun at Tokyo Institute of Technology(Fig. 1). After the experiment, the meteorite sample
was recovered and treated with CsF/HF-dioxane [4] topurify the insoluble organic macromolecular solid(IOM). The insoluble organic solids from theexperimentally shocked meteorites were analyzed by ascanning x-ray transmission microscope (STXM) - xray absorption near edge structure (XANES) at
beamline 4U, UVSOR.
Carbon-XANES spectrum of organics theexperimentally shocked Murchison IOM at 5 GPa(blue spectrum in Fig. 1) showed a peak of aromatic
carbon (285 eV) and a peak of carboxyl carbon (288eV) with a shoulder of aliphatic carbon (297.6 eV),which was very similar to non-shocked Murchison
IOM. The C-XANES spectra of IOM at 10 and 20 GPa

were also similar to that at 5 GPa. On the other hand,in the C-XANES spectrum of organics at 40 GPa, apeak intensity of carboxyl carbon largely decreased
and that of aromatic carbon was slightly developed.
This result indicates that modification of IOM byshock heating occurs at 40 GPa and is comparable totheir Raman spectra [5].

a)b)Fig. 1.(a) Double-stage light gas
gun, (b) Single-stage powder gas
gun at Tokyo Inst. Technology.
Credit: T. Atou.

Ⅲ- 5

Fig. 2. C-XANES spectra of organic macromolecularsolids from the experimentally shocked meteorites.
Blue: 5 GPa, Green: 40 GPa.
[1] C. M. O’D. Alexander et al., Geochim. Cosmochim.
Acta 71 (2007) 4380.
[2] G. D. Cody et al., Earth. Planet. Sci. Lett. 272(2008) 446.
[3] H. Yabuta et al., Meteor. Planet. Sci. 45 (2010)
1446.
[4] G. D. Cody and C. M. O’D. Alexander, Geochim.
Cosmochim. Acta 69 (2005) 1085.
[5] P. Beck et al., Meteor. Planet. Sci. 45 (2012) 1446.

145

Ⅲ -5. Life, 988BL4U_Ruhl_2011-2027_V.docx
Earth and Planetary Sciences

BL4U

Probing Tacrolimus in Human Skin by Soft X-Ray Spectromicroscopy
K. Yamamoto1, A. Klossek1, T. Ohigashi2, F. Rancan3, R. Flesch1, A. Vogt3,
U. Blume-Peytavi3, P. Schrade4, S. Bachmann4, R. Schulz5, R. R. Netz5, S. Hedtrich6,
M. Schäfer-Korting6, N. Kosugi2 and E. Rühl1

1

Ⅲ- 5

Institute for Chemistry and Biochemistry, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany
2
UVSOR Synchrotron, Institute for Molecular Science, Okazaki 444-8585, Japan
3
Charité Universitätsmedizin, 10117 Berlin, Germany
4
Abteilung für Elektronenmikroskopie at CVK, 13353 Berlin, Germany
5
Fachbereich Physik, Freie Universität Berlin, 14195 Berlin, Germany
6
Institut für Pharmazie, Freie Universität Berlin, 14195 Berlin, Germany

Lipophilic drugs, such as dexamethasone andtacrolimus, are commonly used to treat by topicalapplication inflammatory skin diseases. We haveinvestigatedrecentlythepenetrationofdexamethasone (C22H29FO5), a drug that penetrates
readily into human skin, as probed by X-ray
microscopy [1]. Adequate penetration is due to its
moderate lipophilicity and the relatively low molecularweight of 392 Da, which is well below the 500 Da limit[2]. This limit is known to be of relevance for
distinguishing low molecular weight drugs that easily
penetrate into skin from heavier ones, such as
tacrolimus (C44H69NO12, M=804 Da), requiring
advanced formulations for sufficient penetration [3].
We have investigated by label-free X-ray
spectromicroscopy tacrolimus in the O 1s-regime. The
experiments were performed at the BL4U beamline at
UVSOR-III using a scanning transmission X-ray
microscope (STXM). Intact skin samples from healthy
donors as well as tape-stripped skin for thinning thestratum corneum (SC), the top skin penetration barrier,were investigated. For intact skin the drug wasdissolved in aqueous ethanol (50%) with aconcentration of 10 mg/mL, where 40 μL were applied
per cm2. Tacrolimus was applied to tape-stripped skinby using Protopic ointment 0.1% (Astellas Pharma,
Munich, Germany), 30 µg/cm2. Penetration times
ranged between 10 and 1000 min. Chemical selectivity
for probing the drug in fixed human skin slices isgained by excitation of the O 1s→π*-transition at 531.3
eV, which is suitable to suppress the backgroundabsorption of fixed human skin (see Fig. 1). This
indicates in addition to recent work [1], that X-ray
microscopy is a general approach to probe drugs in
fixed human skin slices.
The results indicate that in normal skin tacrolimus
can only penetrate into the stratum corneum. The drug
was not detected in deeper skin layers, such as in theviable epidermis and dermis. However, if the skin ismechanically removed by repeated tape-stripping (50times) prior to drug exposure, then penetration oftacrolimus is observed, as indicated in Fig. 2. Clearly,
the drug is detected in the viable epidermis. It is
anticipated that this finding will also apply to drug
penetration in barrier deficient inflamed skin, as
known for the improvement of atopic dermatitis

following the use of tacroliumus ointment [3]. The
shape of local tacrolimus distribution is similar toearlier work on dexamethasone [1]. This implies thatthe transport of both drugs is similar, independent of
the molecular weight, once the stratum corneum ispassed. We also observe that the drug concentration
drops to the detection limit at the basal membrane,
implying that in the dermis different transport
properties occur. Thus, the basal membrane may form
another barrier for transdermal drug penetration, as
suggested by present X-ray spectromicroscopy studies.

Fig. 1. O 1s-excitation regime of tacrolimus incomparison with fixed human skin. The resonancenear 531 eV (O 1s→π*-transition) is used for selective
probing of the drug.

Fig. 2. Penetration of tacrolimus into human skinafter tape-stripping. Two different penetration times
were studied (10 min: red and 1000 min: blue).
[1] K. Yamamoto et al., Anal. Chem. 61 (2015) 6173.
[2] J.D. Bos et al., Exp. Dermatol. 9 (2000) 165.
[3] M. Lapteva et al., Mol. Pharm. 11 (2014) 2989.

146

991BL4U 中藤亜衣子.docx

Ⅲ -5. Life, Earth and Planetary Sciences

BL4U

Evaluation of FIB Sample Contamination Depends on the Storage
Environment Based on XANES Analysis and Application to Organics in
Carbonaceous Chondrites
A. Nakato1and M. Uesugi2

Division of Earth and Planetary Sciences, Kyoto University, Kyoto 606-8502, Japan,
2
JASRI/SPring-8, Sayo-gun 679-5198, Japan

1

XANES and high resolution STXM installed in
UVSOR BL4U are useful tools for the characterizationof molecular structure of the unknown carbonaceous
material, and understanding of their micro-distribution.
We expected to apply these analyses to the hydrous
carbonaceous chondrites, since the extraterrestrialorganics have some variety and complex structures
those could be evidences of the evolution of parent
body of the chondrites. In this aspect, samples
preparation must avoid the change of their original
microstructure.
It is well known that electron beam of the TEM
observation could modify the C-XANES spectrum [1].
In contrast, we reported the sample damage induced by
C-XANES in the report of the previous study [2]. TEM
observations just before and after XANES analysis
showed that NaCl and KCl inclusions in the sample
were damaged heavily by the XANES analysis. Before
further precise description of the carbonaceous
materials in carbonaceous chondrite, damage during
XANES analyses and contamination during the sample
storage must be evaluated. In this study, we evaluatedthe contamination during the sample storage bychanging the storage environment and the period by
XANES/STXM.
We fibbed 8 samples from the matrix of Murchison,
one of the hydrous carbonaceous chondrites. The size
is about 15x10 um and 100 nm thickness. The samples
from #1 to #4 were kept in 4 different conditions for 1
week, and the remaining samples from #5 to #8 were
kept for 2 months until the XANES/STXM analysis.
#1-4 samples were analyzed again after storage for 3
months. The storage atmospheres were pure Nitrogenand approximately 102 Pa vacuumed conditions, andthe sample holder made by Si and metal, respectively(Table. 1). C, N-XANES analyses were performed for#1-4 samples after 1 week storage. N, O-XANES
spectrum were obtained for #5-8 samples after 2
months storage and for #1-4 samples after 3 months
storage.
C-XANES spectrum of #1-4 samples stored for 1
week do not show any changes irrespective of thestorage environment. In addition, we can not detect
clear N-XANES transition in all samples, even somesamples (#1-2, #5-6) were kept in pure N2 atmosphere(Fig. 2). These results suggest that sample storage does
not give any contaminations, under the conditions
tested in this study.
In future work, we plan to perform C-XANES

147

analysis for these samples to confirm the molecular
structures of the carbonaceous materials to beunchanged by a few months storage. Besides, we will
apply the XANES/STXM analysis to various hydrous
carbonaceous chondrites.
#

1
2
3
4
5
6
7
8

Table 1. Samples and storage conditions.
Atmosphere Holder Storage
periodandanalyzed spectrum
12
3
week months months
Si
C, N
O, N
N2
Metal
C, N
O, N
Si
C, N
O, N
Vacuum
Metal
C, N
LOST
Si
O, N
N2
Metal
O, N
Si
O, N
Vacuum
Metal
O, N

Ⅲ- 5

Fig. 1. N-XNAES spectrum of samples #1-8.
[1] H. Yabuta et al., Earth planet. Space 66 (2014) 156.
[2] M. Uesugi and A. Nakato, UVSOR activity report
42 (2015) 148.

Ⅲ -5. Life, 1001BL6B
Earth and 木村真一.docx
Planetary Sciences

BL6B

Visualization of Spatial Distribution of the Optical Isomers of Amino Acids
by Infrared Micro-Spectroscopic Imaging
S. Kamei1, J. Hibi2, H. Yokoyama 2, Y. Ohtsubo 1,2, H. Watanabe 1,2 and S. Kimura1,2
1

Ⅲ- 5

Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan
2
Graduate School of Science, Osaka University, Osaka 565-0043, Japan

Protein, which is one of important components ofhuman bodies, consists of essential amino acids. Although amino acids have optical isomers of L- and Dtypes except for glycine, living bodies mainly consistof L-amino acids. However, in recent years, it has been
revealed that the ratio of D-amino acids increases inliving body by racemization reaction with aging. It isconsidered that an increase of D-amino acids causes
the change of the higher-order structure of protein,which is the origin of aging sicknesses such as cataract
and Alzheimer's disease [1]. For early prevention of
those aging diseases, a simple detection method of Damino acids is required.
Since physical and chemical properties of D-aminoacids are the same as those of L-types, special techniques to separate these isomers, for instance a special
chromatography with asymmetric catalysts, are used atpresent.
In the case of the mixing of L- and D-amino acidmolecules, so-called racemates, however, the crystal
structure is different from those of homochiral L- andD-molecules. This implies that some infrared (IR) absorption peaks owing to molecular vibrations of racemates are different from those corresponding peaks ofthe homochiral molecules [2]. Therefore, the IR absorption peak as well as the micro-spectroscopic imaging is a good probe to detect the area and density ofracemates, that is of D-amino acids, in the backgroundof L-amino acid molecules.
In this paper, we developed the methodology to detect D-amino acid molecules in L-molecules by using
different IR absorption peaks of racemates from thoseof homochiral molecules as a probe.
Thin film samples of L- and D-alanine, which is oneof essential amino acids, on potassium bromide (KBr)
substrates were fabricated by a vacuum evaporating
method, where L- and D-alanine powders were evaporated separately. A part of evaporation area of thesematerials was overlapped as shown in Fig. 1(c). In theoverlapped area, namely L+D area, the interfacial
layer between L- and D-alanine becomes racemates.
The IR micro-spectroscopic imaging measurement
was performed at the infrared beamline 6B. The imaging measurement was carried out at about 55 mm2
area with 100-μm step (about 3,000 points in total) on
the sample.
Figure 1(a) shows an IR absorption peak of L- andD-alanine and L+D overlapping areas. Although theabsorption peak shape of pure L and D areas are similar to each other, but the peak of L+D area is different,
that looks to have two components, one is the same aspure areas and the other is an extra peak. To clarify the
speculation, the peak of the L+D area is well fitted by
two Lorentz components as shown in Fig. 1(b). The
extra peak originate from the racemate because thesame peak has been observed in racemic alanine of apellet yet. Figure 1(d) shows the spatial distribution of
racemic ratio (R) evaluated from the ratio of the absorption peak area of the racemic alanine by the fitting
at each point performed as the same as that in Fig. 1(b).
The image suggests that the R values in the L+D area
are much different from other homochiral area. This
result pronounces that it is possible to visualize thespatial distribution and evaluation of the ratio of D-alanine in L-alanine by the IR imaging. In addition, we
could evaluated higher spatial resolution up to 10 μm
by using the high-brilliant IR synchrotron radiation(IR-SR). Therefore, the micro-spectroscopic imaging
using IR-SR is a suitable tool to detect optical isomers
in homochiral amino acids.

Fig. 1. (a) Infrared absorption spectra of homochiral
L-, D-alanine and these overlapping area (L+D). (b)Fitting of the L+D absorption by two Lorentz functions, one is the homochiral spectrum and the other racemic one. (c) Visible image of L-alanine (horizontal
line part) and D-alanine (vertical line part) film fabricated on a KBr substrate. (d) Spatial distribution of racemic ratio (R) evaluated by the fitting as shown in(b).
[1] Y. Kaji et al., Invest. Ophthalmol. Vis. Sci. 48(2007) 3923.
[2] V. S. Minkov et al., J. Struct. Chem. 51 (2010)
1052.

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