Ⅲ-5

Life, Earth andPlanetary SciencesⅢ -5. Life, Earth and Planetary Sciences
BL4U
BL4U

Drug Release and Probing of Thermoresponsive
oresponsive Nanogels in Human Skinby Soft X-Ray Spectromicroscopy
K. Yamamoto1, A. Klossek1, T. Ohigashi2, F. Rancan3, R. Flesch1, M. Giulbudagian1, A. Vogt3,
U. Blume-Peytavi3, P. Schrade4, S. Bachmann4, M. Calderón1, M. Schäfer-Korting5,
N. Kosugi2 and E. Rühl1
1

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

The transport of drugs into human skin facilitated bytriggered drug release from polymeric nanocarriers isreported. Label-free detection by X-ray microscopy isused to probe both, the drug and the drug nanocarriers.
Dexamethasone (DXM) is used as a drug and thethermoresponsive gel was Poly(N- isopropylacrylamide)(nanogel). The stratum corneum (SC) is the top skinlayer. It is the main barrier, which hinders drugs topenetrate into the viable epidermis. We recently
published first results using X-ray microscopy forselectively probing DXM [1, 2] and core-multishell
nanocarriers penetrating human skin [3]. It was shownthat nanocarriers release the drug in the SC without
penetrating into deeper skin layers. This yields higher
local drug concentrations in the viable epidermis thanfor conventional drug formulations [3].
The experiments made use of excised human skin,which was exposed to DXM-loaded nanogels forpenetration times of 100 and 1000 min. The nanogels
were dissolved in water with a concentration of 33.5
mg/mL and had a drug load of 10%. Excised humanskin was tape-stripped 30 times prior to topical drug
application. In total 120 µL of this nanogel suspension
containing 400 µg of DXM was applied per cm² ofskin. Triggered drug release was initiated after 10 min
by exposure of the skin samples to infrared radiationfor 30 s, leading to a contraction of the nanogel anddrug release.
Spectromicroscopy studies were performed at the
BL4U beamline at UVSOR III using a scanning X-ray
microscope. The chemical selectivity for probing either
the thermoresponsive nanogel or DXM in fixed skin
was achieved by exciting the samples selectively at theoxygen K-edge in the pre-edge regime (cf. [1-3]).
Differential absorption maps were derived, similar toour previous work (see Fig. 1).
Figure 1 shows a compilation of the results, where
only the top skin layers are shown (SC and top part ofthe viable epidermis). Fig. 1 (a) - (d) show results for the
nanogel distribution, whereas Fig. 1 (e) - (h) show the
drug distribution. Triggered and non-triggeredconditions result in distinct differences for both, thedrug and the drug nanocarrier distributions. The
nanogel appears to penetrate only efficiently the SC, if
triggered by IR radiation (Fig. 1 (b), (d)), but cannot
reach the viable epidermis. Moreover, significant drug

release and penetration into deeper layers only occurs,
if the release is thermally triggered (Fig. 1 (f), (h)). The
nanogel also provides in the SC access for the drug
into the corneocytes, which has not been observed
before using other vehicles, then DXM remained in thelipid regions between the corneocytes [1-2]. Thisnanogel-induced effect goes along with swelling of thecorneocytes, which has been recently observed byelectron microscopy [4]. Stimulated Raman studies
provide additional evidence that this is related tochanges the organization of proteins and lipids [4].
These results underscore that label-free detection athigh spatial resolution provides novel insights that are
required for the optimization of topical drug delivery
strategies facilitated by polymeric drug nanocarriers.

Ⅲ- 5

Fig. 1. The differential absorption of dexamethasone
loaded nanogels penetrating the top skin layers for
100 and 1000 min, respectively: (a) - (d) selective
probing of the nanogel; (e) - (h) selective probing of
dexamethasone. Triggered ((b), (d), (f), (h)) and nontriggered ((a), (c), (e), (g)) drug release conditions
show different distributions of the selected species.
[1] K. Yamamoto et al., Anal. Chem. 87 (2015) 6173.
[2] K. Yamamoto et al., Eur. J. Pharm. Biopharm., inpress (2017). DOI: 10.1016/j.ejpb.2016.12.005.
[3] K. Yamamoto et al., J. Control. Release 242 (2016)
64.
[4] M. Giulbudagian et al., J. Control. Release 243(2016) 323.

155

Ⅲ -5. Life, Earth and Planetary Sciences
BL1U
BL1U

Optical Activity Emergence in Glycine by Circularly Polarized Light
J. Takahashi1, 2, N. Suzuki1, Y. Kebukawa1, K. Kobayashi1, Y. Izumi3, K. Matsuo3,
M. Fujimoto4 and M. Katoh4
Fucalty of Engineering, Yokohama National University, Yokohama 250-8501, Japan
2
Institute of Laser Engineering, Osaka University, Suita 565-0871, Japan
3
Hiroshima Synchrotron Radiation Center, Higashi-Hiroshima 739-0046, Japan
4
UVSOR Facility, Institute for Molecular Science, Okazaki 444-8585, Japan

1

Ⅲ- 5

The origin of homochirality in terrestrial bioorganic
compounds (L-amino acid and D-sugar dominant) is
one of the most mysterious issues that remainunresolved in the study of the origin of life. Because
bioorganic compounds synthesized in abiotic
circumstances are intrinsically racemic mixtures of
equal amounts of L- and D-bodies, it is hypothesizedthat chiral products originated from asymmetricchemical reactions induced by a “chiral impulse”.
These types of asymmetric reactions could havepossibly been derived from physically asymmetric
excitation sources in space and the chiral products
would have been transported to primitive Earthresulting in terrestrial homochirality (Cosmic Scenario)[1]. In space, asymmetric excitation would be mainly
effectuated by polarized photons of synchrotron
radiation (SR) from kinetic electrons captured bystrong magnetic field around neutron stars or whitedwarfs in supernova explosion area, or by polarized
photons scattered by interstellar dust clouds in starformation areas. Eventually, several terrestrial
observations of polarized photon radiation from space
due to scattering by interstellar dust clouds in starformation regions have been reported [2].
Several experiments have already examinedasymmetric photochemical reactions in simple
biochemical molecules by using circularly polarized
light (CPL). We have reported optical activity
emergence in solid-phase films of racemic mixturesof amino acids by using CPL from UVSOR-II freeelectron laser (FEL) [3].
In this paper, optical activity emergence into theachiral (optically inactive) amino acid film by using
CPL is reported. We formed thin solid films of glycine
on fused quartz substrates from crystal powders ofglycine using a thermal-crucible vacuum evaporator.
Sublimation temperature was 150 ~ 200 °C and pressureof the vacuum chamber was approximately 10-2 Pathroughout the evaporation process. To introduce opticalasymmetry into the racemic film, we irradiate them with
CPL introduced from undulator beam line BL1U of
UVSOR-III (Fig.1). The irradiated CPL wavelength was
215 nm and total irradiation energies were 12 and 20
mW hour.
In order to clarify the optical anisotropy of thefilms, we measured the circular dichroism (CD) andphoto absorption spectra using a commercial CD

spectrometer (JASCO J-720WI). As a result, almost
completely symmetric spectra were observed with
left- and right-handed CPL irradiation. The
dependence on sample rotation angle of the spectra of0- and 90-degree rotation was measured. If linear
dichroism (LD) and/or linear birefringence (LB)components are dominantly effective, the signs ofappearance CD peaks should be inverted. We ensured
that rough spectrum profile and at least signs of two
CD peaks were not changed. Therefore, observed
appearance CD should be derived dominantly fromchiral CD component. We are planning CD spectra
measurement over the shorter wavelength region
using SR-CD beamline of Hiroshima Synchrotron
Radiation Center (HiSOR).

Fig. 1. Photo of the irradiation experiment of circularly
polarized light on BL1U.
[1] Bonner, Origins Life Evol. Biosphere 21 (1991) 59.
[2] T. Fukue et al., Origins Life Evol. Biosphere 40(2010) 335.
[3] J. Takahashi, H. Shinojima, M. Seyama, Y. Ueno,
T. Kaneko, K. Kobayashi, H. Mita, M. Adachi, M.
Hosaka and M. Katoh, Int. J. Mol. Sci. 10 (2009)
3044.

156
Ⅲ-5-2

Ⅲ -5. Life, Earth and Planetary Sciences
BL1U
BL1U

Emergence of Biological Homochirality by Irradiation of Polarized
Quantum Beams
K. Matsuo1, Y. Izumi1, J. Takahashi2, M. Fujimoto3 and M. Katoh3

Hiroshima Synchrotron Radiation Center, Hiroshima University, Higashi-Hiroshima 739-0046, Japan
2
Faculty of Engineering, Yokohama National University, Yokohama 240-8501, Japan
3
UVSOR Facility, Institute for Molecular Science, Okazaki 444-8585, Japan1

Origin of homochirality in terrestrial biomolecules(L-amino acids and D-sugars dominant) remains anunresolved problem. One of the hypotheses for the
emergence of homochirality is Cosmic Scenario, in
which the space materials on the surface of meteorites
are transformed into the complex organic molecules(for example amino acid precursors) by the asymmetric
reactions due to the irradiation of circularly polarized
light (CPL). To validate this scenario, as a pioneer
research, Takahashi et al. investigated the emergenceof optical anisotropy in solid films of racemic aminoacids (L/D = 1) with the left- and right-CPL (LCPL and
RCPL) irradiation of 215 nm, and suggested that the
CPL caused the preferential conformational changes
between the two enantiomers in racemic amino acidfilms [1].
Circular dichroism (CD) spectroscopy can easily
detect the emergence of optical anisotropy due to theasymmetric reaction because it sensitively reflects thesteric structures of chiral molecules such as aminoacids. Further the theoretical calculation of CD
spectrum of L-alanine molecule revealed that thechromophores such as carboxyl and amino groups
had many characteristic electronic transitions (n-π*,
π-π* and n-σ*) below 230 nm [2], suggesting that thephotoreaction might depend on the irradiation energy.
In this study, the thin solid films of racemic alanine(L/D = 1) were irradiated by the two types of CPL
wavelengths (230 and 202 nm) and the emergence ofamino acid homochirality were investigated by the
CD measurements.
The thin solid films on SiO2 substrates were
produced by evaporating the powder of DL-alanine
using the vacuum-evaporation system in Hiroshima
Synchrotron Radiation Center (HiSOR). Sublimation
temperature was about 150 ~ 200 °C and the pressure in
the vacuum chamber during the evaporation wasapproximately 5 x 10-2 Pa. The CD spectra of these thinsolid films were measured from 260 to 170 nm using avacuum-ultraviolet circular dichroism instrument in
HiSOR and confirmed to be mostly zero before theirradiation, showing that the spurious CD due to thecontamination of film surface were negligible. The
LCPL and RCPL irradiation for these films were carriedout in the BL1U of UVSOR at the wavelengths of 230
and 202 nm. After the irradiation, these CD spectra
were measured again, in order to confirm the emergenceof homochirality.
Figure 1 shows the CD spectra of thin films after the

LCPL and RCPL irradiation of two wavelengths. As
shown in Fig. 1, the LCPL and RCPL irradiation
exhibited symmetrically inverted spectra in the both
irradiation wavelengths (230 and 202 nm), indicating
the emergence of homochirality at the both irradiation
wavelengths. Further, the spectral shape observed in
Fig.1a were largely different from those in Fig.1b,
suggesting that the asymmetric reactions of DL-alanine
thin solid film depend on the irradiation wavelength.
To further validate these results, we are planning toirradiate the CPL in the vacuum-ultraviolet region.
Accumulations of these experimental results would
be useful for determining the effective emergence ofhomochirality in solids by the CPL irradiation.

Ⅲ- 5

Fig. 1. CD spectra of DL-alanine thin films after the
LCPL and RCPL irradiation at 230 nm (a) and 202 nm(b).
[1] J. Takahashi, H. Shinojima, M. Seyama, Y. Ueno, T.
Kaneko, K. Kobayashi, H. Mita, M. Adachi, M.
Hosaka and M. Katoh, Int. J. Mol. Sci. 10 (2009) 3044.
[2] T. Fukuyama, K. Matsuo and K. Gekko, J. Phys.
Chem. A 109 (2005) 6928.

157
Ⅲ-5-3

Ⅲ -5. Life, Earth and Planetary Sciences
BL4U
BL4U

STXM Study on the Microbial Alteration ofIron Sulfide Mineral at the Submarine Seafloor
2

Ⅲ- 5

S. Mitsunobu1, 2, 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, Japan
1

Recent studies have revealed that various
microorganisms are alive on the submarine seafloor andcontribute alteration of oceanic crust [1, 2]. It isconsidered that the microorganisms (e.g., bacteria,
archaea) gain their life-energy from the redox reaction
using inorganic active elements such as ferrous iron andsulfur involved in the oceanic crust (basaltic rocks andsulfidic minerals). The microbial activities would
accelerate the alteration of the oceanic crust, which
would influence the carbon dynamics in the earth
significantly. However, little is known on mechanismof the microbial alteration of the minerals, because direct
chemical speciation of both metals and biomolecules atmineral-microbe interface has been crucial due to highspatial resolution in analysis. Moreover, difficulty on thefield access is also an obligate reason that no enoughstudies on that has been done so far.
Here, we performed in situ bio-alteration experimentusing mineral substrates composed of the ocean crust,
fresh sulfidic mineral (pyrite) and basalt rock, atoxidative submarine seafloor in Izu-Bonin region, where
oxidative alteration of the basalt and pyrite naturally
occur. Then, we investigated the mechanisms of thebio-alteration process using scanning transmission X-ray
microscopy (STXM) based C and Fe near edge X-ray
absorption fine structure (NEXAFS) analyses at
UVSOR BL4U.
Before STXM analysis, bacteria attached on the pyrite
were stained with a minimum fluorescent dye andmarked for the STXM analysis. In the analysis, weapplied the STXM analysis to determine in situ carbon(C) and iron (Fe) species at the interface of target
bacteria cell and mineral (pyrite).
STXM-based
imaging and C 1s NEXAFS analysis directly showedthat the attached bacterial cell produce somepolysaccharide-rich extracellular polymeric substances(EPS) at the cell-pyrite interface (data not shown). The
polysaccharide substances generally have a metalchelating effect and enhance the dissolution of mineral
involving the metals [3]. Hence, the bacteria may
produce the polysaccharide-rich EPS on the pyrite
actively to dissolve the pyrite and gain energy
efficiently.
Furthermore, we measured STXM-based Fe imaging
and Fe 4p NEXAFS at the same area. Figure 1 (a)shows the STXM-based merged Fe/C image. The
image shows the several particles yielding Fe withdiameter of 50-100 nm on the cells (orange circles inFig. 1 (a)). The Fe NEXAFS analysis of the particles

indicate that Fe species in these particles were Fe(III)hydroxides such as ferrihydrite and schwertmannite on
the whole (Fig. 1 (b)). Because the Fe species insubstrate (pyrite) is Fe(II), observed Fe(III) resultsfrom oxidation of Fe(II) originally involved in pyrite.
These findings indicate that some of bacteria attachedon the pyrite gain energy by Fe oxidation at thesubmarine seafloor and that have a metabolicmechanism to excrete subsequent insoluble-Fe(III)formed by oxidizing reaction.

Fig. 1. STXM-based C and Fe images (a) and Fe 4p
NEXAFS spectra (b).
[1] T. M. McCollom, Deep-Sea Res. 47 (2000) 85.
[2] K. J. Edwards et al., Geochim. Cosmochim. Acta
67 (2003) 2843.
[3] S. Mitsunobu et al., Microbes Environ. 31 (2016)
63.

158
Ⅲ-5-4

Ⅲ -5. Life, Earth and Planetary Sciences
BL4U
BL4U

NEXAFS Measurements of Biomolecules at the C-K edge for Molecular
Mapping in Biological Specimens by STXM
A. Ito1, T. Ohigashi2, 3, K. Shinohara1, S. Tone4, M. Kado5, Y. Inagaki2 and N. Kosugi2, 3

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
Graduate School of Science & Engineering, Tokyo Denki University, Hatoyama 350-0394, Japan
5
Kansai Photon Science Institute, Nat. Inst. Quantum and Radiological Sci. Technol., Kizugawa 619-0215, Japan
1

2

2.5
67

Absorption

2

12

3

4

8

5

DNA

1.5

Histone1
0.5

Phosphatidylcholine
0

282

284

286

288

290

292

294

Photon energy (eV)Fig. 1. NEXAFS spectra of DNA, histone andphosphatidylcholine at the C-K absorption edge.

1.8

56

1.6

DNA
1

1.4

Absorption

Spectromicroscopy using scanning transmission
X-ray microscope (STXM) has been applied to DNA
and protein distributions in biological specimens suchas chromosome and sperm at the C-K absorption edge
[1, 2]. In our previous studies using STXM at BL4U,
we obtained DNA and histone, a nuclear protein,
distributions in mammalian cells using characteristic
NEXAFS peaks of DNA and histone which can beclearly observed at the N-K absorption edge [3].
Furthermore quantitative mapping of these molecules
was developed [4], but the results showed that thecontribution of biomolecules other than DNA andhistone should be taken into account.
In this study we measured NEXAFS of phospholipids,a major component of cellular membrane, in addition to
nucleic acids and various kinds of proteins at the C-K
absorption edge. Figure 1 shows NEXAFS for typicalbiomolecules of nucleic acid, protein and phospholipid
at the C-K absorption edge.
Ade et al. used the slight shift of a peak as shown by
1 and 2, which was assigned to 1s to π* transition of
C=C bond, to image of DNA and protein inchromosome separately [1]. The significant differencein peaks among nucleic acid, protein and phospholipid
can be observed at the peak 6, 7 and 8 as indicated inFig. 1. These peaks were assigned to 1s to π* transitionof C=O bond [5, 6]. Peak 6 and 8 corresponds to
N-C(=O)-C and N-C(=O)-N, respectively. On the other
hand phospholipid does not have such amide bonds,
but has O-C=O. This bond may result in the uniquepeak 7. The different location of peaks due to C=Obond may be applicable to separate imaging of thesemolecules.
Figure 2 shows the comparison of DNA and RNA
in the NEXAFS at the C-K edge. Peaks 5 and 6, 1s to
π* transition of C=O bond, exhibits a slight difference,suggesting the possibility for imaging nucleic acids
separately.
For the difference among proteins, we measured
spectra of bovine serum albumin (BSA), histone,
cytochrome C and actin. NEXAFS peaks have thesame energies except actin. Interestingly actin has thesignificant peak shift to higher energies. This findingneeds to be confirmed in future experiments.

2

3

4

1.2
1

Ⅲ- 5

RNA

0.8
0.6
0.4
0.2
0

282

284

286

288

290

292

294

Photon energy (eV)Fig. 2. NEXAFS spectra of DNA and RNA at theC-K absorption edge.
[1] H. Ade et al., Science 258 (1992) 972.
[2] X. Zhang et al., J. Struct. Biol. 116 (1996) 335.
[3] A. Ito,T. Ohigashi, K. Shinohara, S. Tone, M. Kado,
Y. Inagaki and N. Kosugi, UVSOR Activity Report 43(2016) 143.
[4] K. Shinohara, T. Ohigashi, S. Tone, M. Kado and A.
Ito, Proc. 13th Intl. Conf. X-Ray Microsc. (XRM2016)(in press).
[5] Kummer et al., J. Phys. Chem. B 114 (2010) 9645.
[6] Stewart-Ornstein et al., J. Phys. Chem. B 111 (2007)
7691.

159
Ⅲ-5-5

Ⅲ -5. Life, Earth and Planetary Sciences
BL4U
BL4U

Chemical Speciation of Single Aerosol Particles: A Reconstruction of
Hygroscopicity for Sulfate Aerosols
1

Ⅲ- 5

Y. Takahashi1, C. Miyamoto1 and K. Sakata2

Department of Earth and Planetary Science, Graduate School of Science, the Univ. of Tokyo, Tokyo 113-0033, Japan2
Department of Earth and Planetary Systems Science, Graduate School of Science, Hiroshima Univ.,
Higashi-Hiroshima 739-8526, Japan

Hygroscopic sulfate aerosol such as ammonium
sulfate (AS: (NH4)2SO4) contributes to indirect
cooling effect by acting as cloud condensation nuclei(CCN) [1]. On the other hand, gypsum (Gyp: CaSO4･
2H2O), non-hygroscopic sulfate, suppresses CCN
activity of AS, which are formed by chemical reactionof calcite (Cal: CaCO3) in mineral dust with H2SO4 in
atmosphere [2]. Thus, non-sea-salt sulfate (nssSO42-)
species is one of the important information for
estimation of CCN activity of sulfate aerosol.
Ice sheet in polar region preserves atmosphericparticles and gases at the time of its deposition [3]. In
the case of Greenlandic ice sheet, they preserve
mineral dust and anthropogenic aerosol fromcontinental region in the Northern hemisphere [3]. In
this study, chemical speciation experiments of trapped
particles in ice sheet were conducted. In addition,
chemical speciation in sea spray aerosol (SSA) withhigh CCN activity was also conducted to distinguishnon-sea-salt sulfate (nssSO42-: AS and Gyp) and SO42from seawater. The aim of this study is to determine
Gyp/AS ratio in trapped particles in ice sheet toestimate CCN activity of nssSO42- aerosols in the past.
Trapped particles in Greenlandic ice sheet wereextracted by sublimation of ice samples in low
temperature room (-20oC), which can extract trapped
particles with minimal chemical alteration by waterfrom melting ice samples [4]. Trapped particles in ice
sheet with ages of 1987, which is a part of ice coredrilled at SE-Dome (67.2°N, 36.4°W, 3170 m above
sea level), were analyzed. SSA sampling wereconducted at the Antarctic Ocean to obtain
non-alteration SSA by anthropogenic gases (R/V
Hakuho-Maru cruise, KH-14-6, GEOTRACES).
Chemical speciation experiments were conducted by
STXM/NEXAFS with image stack [5].
Sulfate and Ca in SSA were enriched in edge positionof aerosol particles (Fig. 1). Carbonate distribution insingle SSA particles, for which we measured carbon
K-edge NEXAFS, were similar between sulfate and Ca(Fig. 1). The spectral analysis showed that Ca species
were aragonite (Arg: CaCO3) and Gyp. Sulfate in SSA
was also present as Na2SO4, which was hygroscopic
sulfate. Considering that Na and Ca concentration in
SSA, Na concentrations is much higher than that of Ca.
Therefore, dominant sulfate species in SSA can be
Na2SO4. On the other hand, center part of SSA wascomposed of NaCl, which result is consistent with aprevious study [5]. Thus, SSA surface and center werecomposed of sulfate with Ca, Na and C, and NaCl,

respectively (Fig. 1).
Sulfate and Ca are distributed heterogeneously intrapped particle in Greenlandic ice sheet (Fig. 2).
Distributions of sulfate and Ca in trapped particles
were different from those in SSA (Figs. 1 and 2).
Therefore, mineral dust and SSA can be distinguishedbased on distributions of sulfate and Ca. Unfortunately,
AS-bearing particles cannot be detected fromGreenlandic ice sheet due to the small number ofmeasured particles. Previous studies reported that AS
particle contains sulfate homogeneously in the single
particle [6]. Thus, SSA, mineral dust, and AS particles
can be distinguished by sulfate distributions in theirsingle particles. Therefore, S speciation for trapped
particles by STXM/NEXAFS and estimation of AS
and Gyp amount of based on optical density (OD) of
SO42- lead to reconstruction of CCN activity of sulfate
aerosol in the past.

Fig. 1. Results of image stack of SSA. (a) OD imageat 295 eV, (b-e) sulfate, Ca, carbonate, and NaCl
distributions, respectively.

Fig. 2. Results of image stack of two trapped
particles in Greenlandic ice sheet: (a) OD image at
168.0 eV, (b) sulfate distribution, (c) OD image at
352.5 eV, and (d) Ca distribution.
[1] C. D. O’Dowd et al., Atmos. Environ. 31 (1997) 73.
[2] Y. Takahashi et al., Environ. Sci. Technol. 43 (17)(2009) 6535.
[3] R. J. Delmas, Rev. Geophys. 30 (1) (1992) 1.
[4] Y. Iizuka et al., J. Glaciology 55 (191) (2009) 552.
[5] R. C. Moffet et al., Anal. Chem. 82 (2010) 7906.
[6] W. Li et al., J. Clean. Prod. 112 (2016) 1330.

160
Ⅲ-5-6

Ⅲ -5. Life, Earth and Planetary Sciences
BL4U
BL4U

Evaluation of Influence on Organic Matters by Dehydrationof Hydrous Asteroids
1

A. Nakato1 and M. Uesugi2

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

Extraterrestrial organics in the carbonaceouschondrites show wide variety and complex structures.
Those characteristics of the structure could be
evidences of the evolution of parent body since thestructure would be modified due to thermal
metamorphism and aqueous alteration [e.g., 1, 2].
Many previous studies have investigated thestructural change of the insoluble organic matters(IOM) extracted from the meteorites, but lacked ‘insitu’ investigation of organics in meteorites. XANESand high resolution STXM installed in UVSOR
BL4U are useful tools for such ‘in-situ’ analysis ofmolecular structure of the organics in bulk rock of
unknown carbonaceous chondrites, and observationof their micro- distribution. We prepared two types
carbonaceous chondrites, one is Murchison thatexperienced aqueous alteration, and the other isAllende that was suffered thermal metamorphism on
the parent body, respectively. Both samples weremade by focused ion beam (FIB), and analyzed by
STXM/XANES. In addition, we examined thatsample contamination during the storage [3], andsample damage due to sample preparing methods [4]suggested by previous studies.
We fibbed 8 samples from the matrix of Murchison
polished thick section. The size is about 15x10 μm
and 100 nm thickness. An Allende fragment wasembedded into sulfur and then sliced about 70 nm
thick each by ultramicrotome. After that, theremaining portion was picked up from the sulfur andembedded in epoxy to be fibbed into 5x5 μm and 100
nm thickness sections. These processes of Allendewere performed for comparison of the sample damage
between the preparing methods. STXM and C, N,
O-NEXAFS was carried out for all samples formed
by FIB and ultramicrotome.
For evaluation of the sample contamination during
the storage, Murchison samples were kept in 4
different conditions for 6-8 months. The storage
atmospheres were pure Nitrogen and approximately
102 Pa vacuumed conditions. The sample holder
made by Si and metal, respectively.
Several characteristic peaks of aromatic carbon at
285 eV, aliphatic carbon at 287.5 eV, carboxyl at
288.5 eV, and carbonate 290.5 eV were detected fromMurchison samples as same as the previous studies of
IOM [e.g., 5]. Most of the Murchison samples show
similar peaks except for those of carbonate. It
suggests that the molecular structure of organics in
Murchison is relatively primitive and homogeneous.
On the other hand, it is well known that Allende IOM

shows unique chemical structure of 1s-σ* exciton at
291.7 eV of graphene structures, that is acharacteristic of thermally metamorphosed meteorites[5]. However, we could not detect the peak in both
fibbed and microtomed samples in this study (Fig. 1).
It may reflect that the graphene can be concentrated
in extracted sample, or apparently masked by featuresof other organic components in in situ observation of
bulk sample. In other words, we found fine–scaled
heterogeneity of organic matter in Allende matrix
using XANES and high resolution STXM, thoughfurther investigation is required. C-XANES of
Murchison and Allende did not show any differencesof the organic molecular structures. Thus, any
influences on the organic matter by thermal
metamorphism were not found in our ‘in-situ’ bulk
analysis and observation so far.
C-XANES spectrum of Murchison samples stored
for 6-8 months do not show any changes irrespectiveof the storage environment. In addition, we can notdetect clear N-XANES transition in all samples, evensome samples were kept in pure N2 atmosphere. We
can conclude from these results that sample storage
does not give any contaminations, under the conditions
tested in our study.

Ⅲ- 5Fig. 1. C-XANES spectra of Allende.
Peaks indicated with letters A-E. A: aromatic carbon,
B: aliphatic carbon, C: carboxyl moiety, D: alcohol or
ether moieties, E: carbonate.
[1] Y. Kebukawa and G. D. Cody, Icarus 248 (2015) 412.
[2] H. Yabuta et al., Meteorit. Planet. Sci. 42 (2007) 37.
[3] M. Uesugi and A. Nakato, UVSOR activity report(2015)[4] N. D. Bassim et al., J. Microsc. 245 (2012) 288.
[5] G. D. Cody et al., Earth Planet. Sci. Lett. 272 (2008)
446.

161
Ⅲ-5-7

Ⅲ -5. Life, Earth and Planetary Sciences
BL4U
BL4U

Carbon Materials and Alteration Products in Martian Meteorites
H. Suga1, N. Sago1, M. Miyahara1, T. Ohigashi2, Y. Inagaki2, A. Yamaguchi3 and E. Ohtani4
Department of Earth and Planetary Systems Science, Graduate School of Science, Hiroshima University,
Higashi-Hiroshima 739-8526, Japan
2
UVSOR Facility, Institute for Molecular Science, Okazaki 444-8585, Japan
3
National Institute of Polar Research, Tokyo 190-8518, Japan
4
Department of Earth Sciences, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan

1

Ⅲ- 5

It is now widely accepted that water existed on theMars. Martian surface materials were launched from theMars by an impact event, and only a few of them weredelivered to the Earth, which are so called Martian
meteorites. It is expected that one group of Martina
meteorite, nakhlite records a water-rock reaction(alteration) occurred on the Mars [e.g., 1]. Iddingsite isone of pervasive alteration textures found in nakhlites. A
small amount of carbon materials is accompanied with
the iddingsite texture [2]. The constituents, chemical
species, and theirs coordination in the iddingsite texturesstrongly depend on pH, temperature, rock/water ratio,
and pressure during the alteration process. The scrutinyof iddingsite in nakhlites will become a clue forelucidating environment on the Mars during a wetperiod. However, the sample volume of nakhliteavailable for a study is limited because nakhlite is very aprecious sample. Iddingsite texture is only about several
10 μm across. Accordingly, we adopted a FIB-assisted
site-specificcarbon/water-free
STXM/NEXAFS
technique [3] to investigate iddingsite texture in naklite.
Yamato 000593 and Nakhlite samples were prepared
for this study. We observed the Yamato 000593 andNakhlite using a FE-SEM. A laser micro-Raman
spectroscopy was employed for phase identification.
Several target portions for a STXM analysis wereexcavated by a FIB technique and became ultra-thinfoils. A STXM analysis was conducted at BL4U.
Finally, the foils were observed with a FE-STEM.
Both Nakhla and Yamato 000593 consist mainly ofaugite and minor olivine [(Mg,Fe)2SiO4]. FE-SEM
observations revealed that iddingsite textures wereobserved in and around the olivine grains. Based on
Raman spectroscopy analysis, the iddingsite texturesin the olivine grains mainly appear to consist of anonstoichiometric distorted olivine-type mineral,laifunite [(Fe2+Fe3+)2(SiO4)2], which is the alteration
product of original olivine. The iddingsite textures
around the olivine grains are complex and varied.
Silica-rich, sulfur-rich, and chlorine-rich veinlets inaddition to laifunite occurred around the olivine
grains. Slices for STXM measurements wereexcavated from these iddingsite textures. Fe- and
O-XANES indicate that most iron in original olivine
is ferrous (Fe2+/Fe3+ = 0.72), whereas most iron inlaifunite is ferric (Fe2+/Fe3+ = 0.52). Fe2+/Fe3+ ratio
decreases discontinuously from olivine to laifunite(Fig. 1). Combined FE-SEM, XANES, and STEM

analysis allowed us to follow the alteration sequence
from olivine to laifunite for the first time. S-XANES
revealed that sulfur in the sulfur-rich veinlet existed
as hexavalent rather than zero-valent by comparing
with reference materials (sulfur, iron sulfate (iii)hydrate, and iron (II) sulfate heptahydrate). Although
Si-XANES was obtained from the silica-rich veinlet,
the spectrum was ambiguous (probably due to strong
adsorption). Cl-XANES could not be obtained fromthe Cl-rich veinlet due to lack of an adequate
reference material. Alternatively, C- and O-XANES
taken showed that the Cl-rich veinlet included bothcarbon and oxygen, and aromatic hydrocarbons(C=C) was detected. Although we have not completed
all measurements, our site-specific STXM/NEXAFS
technique is now uncovering complex and variedalteration processes occurred on the early Mars.

Fig. 1. a) Fe2+/Fe3+ ratio distribution in iddingsite
texture, b) STEM image of a box in a), c) and d) are
the Fe- and O-XANES for olivine (Ol), olivinelaifunite intermediate (Ol-Laith), and laifunite (Laih)domains with standard (STD) materials, respectively.
[1] T. Noguchi et al., J. Geophy. Res. 114 (2009)
E10004.
[2] L. M. White et al., Astrobiology 14 (2014) 170.
[3] H. Suga et al., in prep.

162
Ⅲ-5-8

Ⅲ -5. Life, Earth and Planetary Sciences
BL4U
BL4U

Chemical Evaluations of Sample Preparation Methods for
C-, N-, O-XANES/STXM
Y. Kebukawa1, E. Uchimura 2, T. Ohigashi3, Y. Inagaki3 and K. Kobayashi1

Faculty of Engineering, Yokohama National University, Yokohama 240-8501, Japan
School of Engineering Science, Yokohama National University, Yokohama 240-8501, Japan
3
UVSOR Synchrotron, Institute for Molecular Science, Okazaki 444-8585, Japan1

0.85(A) Murchison

Absorbance

0.8
0.75
0.7

FIB

0.65
0.6

UMT
a

0.55
280
1

b c d ef285
290
Energy [eV]295(B) Humic acid
UMT

0.8

Absorbance

Carbon, nitrogen, oxygen X-ray absorption nearedge structure (XANES) with scanning transmission
X-ray microscope (STXM) became a powerful tool for
extraterrestrial organic matter analyses in this decade.
Some problems may still exist due to small amount oforganic matter that usually exists in extraterrestrial
samples, such as meteorites, and its susceptibility todamage, degradation and contamination. Here wecompared the sample preparation methods for STXM
and evaluate whether correct organic chemistry would
be reflected and reproduced.
We prepared ~100 nm-thick thin sections ofMurchison meteorite and humic acid (IHSS standard
Leonardite humic acid, as an analog of organic matterin meteorites) using a focused ion beam (FIB) and an
ultramicrotome (UMT) equipped with a diamondknife. For UMT, the sample grains were mixed withsulfur powder and heated to melt the sulfur (~150°C
for less than one minute). Solidified sulfur droplet
was attached to an epoxy stub. The ultramicrotomed
sections were transferred to SiO-coated TEM grids
and gently heated until the sulfur sublimed off. C, N,
O-XANES spectra were obtained at BL4U, UVSOR.
Figure 1 shows C-XANES spectra of the Murchison
and humic acid prepared with UMT and FIB.
C-XANES spectra of the Murchison show somedifferences between UMT and FIB. Ketone C=O
appeared only in FIB sample and C-O (alcohol, ether)appeared only in UMT sample. Ketone might beincreased due to FIB beam damage, while C-O in UMT
sample might be a contamination during sample
preparation. Note that the Murchison has someheterogeneity so we examined C-XANES from several
different locations. While humic acid prepared in either
UMT or FIB showed no significant difference in
C-XANES, indicating that slight damage/contamination
affects when one intend to detect small amount oforganic matter embedded in mineral matrix. Figure 2
shows elemental abundances calculated using
computational fit of C-, N-, and O-XANES spectra [1].
The O/C ratio obtained with XANES are underestimatedcompared to elemental composition data for the humic
acid standard provided by International Humic
Substances Society (IHSS) [2]. These results indicatethat XANES data interpretations must be made withcautions.

FIB
0.6
0.4
0.2

a

0
280

b

d

285
290
Energy [eV]295

Fig. 1. C-XANES spectra of (A) Murchison and (B)humic acid. Peak assignments [1]; a: 285.0 eV,aromatic C=C, b: 286.5 eV, ketone C=O, c: 287.5 eV,
aliphatic C-C, d: 288.5 eV, carboxyl O=C-O, e: 289.7
eV, alcohol, ether C-O, f: 290.3 eV, carbonate CO3.
0.6
0.5
0.4
O/C

2

0.3
0.2
UMT
FIB
STXM
IHSS

0.1
0

0

0.01

0.02
0.03
N/C

0.04

0.05

Fig. 2. Atomic N/C versus O/C derived from C-, N-,
and O-XANES of humic acid. An average of 5 UMT
data points and one FIB data point is plotted in a black
circle. IHSS reference data [2] is included in the plot
(red circle).
[1] G. D. Cody et al., Meteorit. Planet. Sci. 43 (2008) 353.
[2] http://www.humicsubstances.org

163
Ⅲ-5-9

Ⅲ- 5

Ⅲ -5. Life, Earth and Planetary Sciences
BL4U
BL4U

Speciation of Carbon in Prokaryotic Organelles of Pseudanabaena foetida(Phormidium tenue)
K. Takemoto1, M. Yoshimura2, Y. Inagaki3 and T. Ohigashi 3

Department of Physics, Kansai Medical University, Hirakata 573-1010, Japan2
SR center, Ritsumeikan University, Kusatsu 525-8577, Japan
3
UVSOR Synchrotron, Institute for Molecular Science, Okazaki 444-8585, Japan1

Ⅲ- 5

Lake Biwa is the most important water reservoir in
Kyoto-Osaka-Kobe region in Japan. In 1969, themusty-odor occurred due to sudden propagation of acertain cyanobacterium in Lake Biwa. Since then, themusty-odor problem in Lake Biwa is reported almost
every year. The matter of the musty-odor was identified
as 2-methylisoborneol (2-MIB) [1]. Although themusty-odor-causing cyanobacterium was reported as
Phormidium tenue (Menegh.) Gomont, in 2016, thecyanobacterium was proposed as a new species
producing 2-MIB: Pseudanabaena foetida Niiyama, Tujiet Ichise sp. nov. [2]. Now, the study how the
microstructure and growth condition are related to the2-MIB productivity has proceeded. In our previous
study based on full field transmission soft X-ray
microscope observation, two interesting intracellular
structure were observed. One is oxygen-enriched
granules and the other is carbon-enriched structures.
Oxygen distribution and K-edge XANES spectra werecollected on the scanning transmission X-ray
microscope (STXM) at UVSOR BL4U [3]. Several
oxygen-enriched granules were confirmed. Though thepre-peak intensity of oxygen-enriched granules was
smaller than that of cytoplasm, the pre-peak energies
were almost similar.
However, the main edge energy of the granule shifted
toward lower than that of cytoplasm. In this study, thecarbon-enriched structures were examined using STXM.
P. foetida sp. collected from Lake Biwa were
subcultured. The culture was maintained on CT
medium (pH 8.0) under a 12 h light/12 h dark cycle at
20 °C. The cultures were illuminated with fluorescentlamps, which provided about 20 μmol photons m-2 s-1of photosynthetically active radiation.
Figures 1 a and b show XM images of air-dried P.
foetida cells. Oxygen was localized around the centerof the cells like a belt (Fig. 1a). Since a blank area
was seen in the belt-like oxygen localizing area, blackarrow, a carbon image was taken at 285 eV (Fig. 1b).
At the oxygen blank area, area 1, a structure withhigh X-ray absorption characteristic was seen. As
shown in Fig. 1c, the carbon XANES spectra weresimilar, but only the spectrum from area 1 showed apeak at 297 eV assigned to potassium, asterisk. It is
well known that in bacteria, sodium, potassium,
calcium, and magnesium have all been shown toeffect cell division [4]. This result suggests that
localization of potassium is implicated as the site ofregulation of cell division of P. foetida.

Fig. 1. STXM images of P. foetida cell taken at 532
eV (a) and 285 eV (b) and carbon XANES spectra (c).
The blue and grey spectra correspond to the area 1
and 2 in (b). The peak around 285 eV, 288.2 eV and
288.5 eV were assigned to C=C, C=N and C=O,
respectively. Scale bar is 1 μm.
[1] M. Yagi et al., Water Sci. Technol. 15 (1983) 311.
[2] Y. Niiyama et al., Fottea 16 (2016) 1.
[3] T. Ohigashi et al., J. Phys. Conf. Ser. 463 (2013)
012006.
[4] L. O. Ingram et al., J. Bacteriol. 125 (1976) 369.

164
Ⅲ-5-10

Ⅲ -5. Life, Earth and Planetary Sciences
BL6B
BL6B

Observation of Infinitesimal Optical Isomers in Homochiral
Aspartic Acid Films by Infrared MicroMicro-Spectroscopic Imaging
2

J. Hibi1, S. Kamei1 and S. Kimura1, 2

Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan
Department of Physics, Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan
1

Proteins, which play an important role in living
bodies, are composed of many kinds of amino acids.
Among amino acids except for glycine, there are two
types of optical isomers, L- and D-types, whose
physical and chemical properties are equal to eachother, but, most of amino acids in living bodies are
L-type. However, in recent years, it has been revealed
that L-amino acids change to D-amino acids in proteinsby aging and the change from L- to D-type(racemization) is considered to be related to aging
diseases such as cataract and Alzheimer's disease. Evenamong amino acids, racemization of aspartic acid
(Asp) is relatively easy to occur in proteins of aged
human lens [1]. In crystallin that is a protein in Humanlens, for example, the amount of D-Asp increases byaging but is infinitesimal in comparison with L-Asp [1].
To detect the D-Asp as well as D-amino acid in L-typeone, special complex techniques are needed, forinstance, a special chromatography with asymmetriccatalysts, are used at present. In addition, the spatial
distribution of D-Asp cannot be detected easily.
To verify whether we can detect infinitesimal D-Asp
in the majority of L-Asp and also the spatial distribution,
we performed an infrared micro-spectroscopic imaging
of aspartic acids thin film.. Thin film samples of L- andD-Asp on potassium bromide (KBr) substrates were
fabricated by a vacuum evaporating method. We madetwo kinds of samples, one is a pure L-Asp film (namely
D:0%) and the other contains D-Asp by 1% (namely
D:1%). The D:1% thin film of was fabricated by the
mixture using a pestle in a mortar beforehand. Infrared
micro-spectroscopic imaging (IR imaging) were
performed by a reflection mode at BL6B using theinfrared microscope (FT/IR 6100 + IRT 7000, JASCO
Co., Ltd.) Wavenumber and spatial resolutions were set
as 2 cm-1 and 100 µm, respectively. The interval of eachstep was set to the same as the spatial resolution. The
wavenumber range was 600-4000 cm-1.
Figure 1a indicates normalized reflectivity spectra of
D:0% and D:1% in the wavenumber range at around
1400 cm-1. The observed peak shifts to the higher
wavenumber side by the mixture of D-type. This result
suggests that we can detect a tiny amount of D-Asp(1%) mixed in the majority of L-Asp by infrared
spectroscopy. To check whether the spatial image of DAsp can be detected, we measured reflectivity imagingof the sample that contains the D:0% and D:1% areas(Fig. 1b) and obtained the peak wavenumbers at eachpoint by the fitting using the Lorentz model. Figure 1cshows the spatial distribution of peak wavenumber.
Comparing with visible image of Fig. 1b, the border of
the areas of D:1% and D:0% can be observed.
Therefore, we can conclude that the presence of thetiny amount of D-Asp can be visualized by the IR
imaging. Moreover, we can perform the IR imaging
with the high spatial resolution and high contrast using
infrared synchrotron radiation (IRSR). Therefore, moredetailed information will be expected by using IRSR.

Ⅲ- 5

Fig. 1. (a) Normalized reflectivity spectra of thin film
samples of pure L-Asp (D:0%) and 1-% D-Asp in
L-Asp (D:1%). (b) Photo of the sample (D:0% andD:1% films on KBr). (c) Spatial distribution of peakwavenumber of the sample. The peak position
corresponds to that of (a).
[1] N. Fujii et al., Biol. Pharm. Bull. 28 (2005) 1585.
[2] S. Kimura and Y. Ikemoto, Hoshako 18 (2005) 5[in Japanese].

165
Ⅲ-5-11UVSOR User 9