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Fig. 2

Arsenic hyperaccumulator (Pteris vittata L.) (left) and cad­
mium hyperaccumulator (Arabidopsis halleri ssp. gemmifer­a) (right).

70  úËõ March 2010 Vol.23 No.2Å

é±ÆªzéBµ©µC»ÀÉÍC÷u·ÈÇÌ»wIÈðµÈ¢Å X ü}CNr[i1 mmjöxÌúÝÌA¨ØÐðì¬·é±ÆÍCñíÉïµ¢BÜ½A¨×EÍðà ¡ úËõ X ü}CNr[Éæédà®´~ÏA¨Ì~ÏJjYÌ¤

Table 1Analytical techniques for determining elemental distribution and chemical form in plant
Spatialresolution

Sensitivity

Chemicalspeciation

Place ofexperiment

synchrotron radiation
micro­XRF (SR­m­XRF
imaging, m­XAFS)38)

ca.1 mm

specialized
facility

Elemental distribution and information regarding
chemical form are obtained from one sample at a
spatial resolution of mm.

X­ray uorescencemicroscope (XRF)

10 mm

~

~

laboratory

White X­rays from the X­ray tube give high levels of
background for a wide energy range. This causes a
low S/N ratio for analysis.

Scanning electron
microscopy­energy
dispersive spectrometry(SEM­EDS)

severaldozen nma)~~b)

laboratory

Excitation eciency for the K­line of heavy elements
is generally low. Analysis is carried out in a vacuum.

micro­particle induced
X­ray emission analysis(m­PIXE analysis)39)

ca.1 mm~

specialized
facility

Measurements in the air deteriorate in spatial
resolution due to diusion of the particle beam (k10 mm). Sample damage is sometimes serious.
Chemical state analysis is not possible.

positron emitting tracer
imaging system (PETIS)40)

ca.1 mm¢~

specialized
facility

Element dynamics are obtained for the living
sample. Radioactive isotope with appropriate life­
time is necessary.

Chemiluminescence
spectrometry41)

0.1 mmc)¢d)~laboratory

HPLC/ICP­MS

\e)





laboratory

Nuclear magnetic resonancespectroscopy (NMR)

\e)

~



laboratory

Element dynamics are obtained in a living state.
Chemical treatment is necessary with an appropriate
reagent to detect a target element.
Liquid samples are generally necessary. Extractionof target material should be required.
Any sample condition is analyzable. Target elements
are limited because optimum nuclei are essential.

Analytical techniqueRemarksa) The resolution for elemental analysis is much larger than that for imaging due to penetration of the incident electron beam andcharacteristic x­rays.
b) Transmission Electron Microscopy (TEM) together with Electron Energy­Loss spectrometry (EELS)can provide chemical state
infromation. Some studies of Ca, O, Si, etc. have been reported.42)c) With use of a laser microscope.
d) Easily aected by coexisting material.
e) This technique has no spatial resolution. A target sample can be separated individually before the analysis.

Fig. 3ÜA system of micro X­ray uorescence analysis with high­energy synchrotron radiation (SPring­8 BL37XU). K­Bmirror: fused quartz coated with platinum, fabricated by a bent­polishing method at Canon Co. Ltd. Length ofthe mirror: 100 mm, focal length: 250 mm (1st mirror) and 100 mm (2nd mirror)Caverage glancing angle: 0.8
mrad.

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Fig. 4

Fig. 5

A photo of in vivo XRF analysis of A. halleri (SPring­8
BL37XU).

72  úËõ March 2010 Vol.23 No.2

SEM image for a root section of P. vittata prepared byfreeze­drying (a) and its enlarged view (b).

ðà ¡ úËõ X ü}CNr[Éæédà®´~ÏA¨Ì~ÏJjYÌ¤

Fig. 7

Digital microscope image for the root section of A. halleri
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öxÉ·é±ÆÉæèCæèðxÌ¢³fÌªz}ªFig. 6

Preparation procedure for plant section in a frozen state.
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Elemental distribution in leaves of arsenic accumulator, P. vittata, obtained by micro­XRF imaging. (a) mature
leaf, (b) sterile leaf. Energy of incident X­ray: 16.5 keV, beam size: 200~200 mm2, scan step: 200 mm, measure­ment time: 3 s/point, measurement point: 115(H)~36(V) (a), 125(H)~45(V) (b). Scale bar: 5 mm.

74  úËõ March 2010 Vol.23 No.2

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Fig. 9Results of micro­XRF imaging for marginal area of a leaf for P. vittata. (a) diagram for leaves of the fern, (b)(f) elemen­
SEM image of spore assemblage, (c) optical microscope image of sample and measurement area, (d)_tal map of As, K, and Ca. Energy of incident X­ray: 14.2 keV, beam size: 5.5(H)~3.5(V) mm2, scan step: 5.5 mm(H), 3.5 mm(V), measurement time: 1 s/point, measurement point: 180(H)~125(V). Scale bar: 100 mm.

Fig. 11

Fig. 10

Change in arsenic and potassium distributions for spore as­semblage of P. vittata with the passage of time after arsenic
addition. (a) 1 h, (b) 12 h. Energy of incident X­ray: 12.8
keV, beam size: 3~3 mm2, scan step: 3 mm, measurement
time: 0.3 s/point, measurement point: 201(H)~201(V)(a), 208(H)~151(V) (b). Scale bar: 150 mm.

A photo of in vivo XAFS measurement of P. vittata (PF,BL­12C).

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æèñ¾âJh~Eðç ppm Æ¢¤ZxÅ~ÏµÄ¢é±Æª©o³êÄ¢é28,29)BêûCClÍJ
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Fig. 12 Arsenic K­XANES spectra for P. vittata and referencematerials. (a) As2O3, (b) H3AsO4, (c) arsenic contaminat­ed soil, (d) petiole, (e) middle midrib, (f) upper midrib,(g) base of pinna, (h) apex of pinna, (i) edge of pinna with
spore, (j) old pinna

Fig. 13ÅéBäXÍæO¢ãÌúËõ{Ý SPring­8 Ìõ¹ðp·é±ÆÅCPxÈGlM[ X üÉæè Cd Ì
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Miro­XRF imaging of a leaf of A. halleri. (a) Photograph of the leaf showing the measured imaging area, scalebar: 5 mm. The two­dimensional distributions of (b) Cd, (c) Zn, (d) Ca, and (e) Mn. Imaging area: 6.5 mm(H)~3 mm(V), X­ray energy: 37 keV; beam size: 50 mm~50 mm, scan step: 50 mm~50 mm; measurement
point: 131(H)~61(V); measurement time: 0.5 s/point.

76  úËõ March 2010 Vol.23 No.2

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égCR[É¨¢Ä~Ï³ê¬µ35)CCd Ì Ka üðoµÄ´xÈC[WOð±ÌgCR[ðõw°÷¾ºÅt©çØèæèC}sÁ½BÜ½}CNr[ªÍÉæ§¿C4 ÛÀXbCNr[ÉæévªÉµ½Æ±ëC»ÌtªÌßÉgÅì¬µ½50 mm pÌr[ð¿ÉÆËµÄCtSÌ¨¢ÄJh~E­xª­¢±Æª¦³ê½iFig. 14jB

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Fig. 14Micro­XRF imaging of trichome. (a) photograph of themeasured samples showing the imaging area, (b) Cd, (c)Zn, (d) Ca, and (e) Mn. Imaging area: 204 mm(H)~81mm(V), beam size: 3.8 mm(H)~1.3 mm(V), step size: 3 mm
(H)~1 mm(V), measurement time: 0.3 s/point. (i)_(iii)
points measured by m­XANES of Cd. The scale bar is 50
mm.

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Zn, and (d) Cd vs. Mn in the trichome. The XRF data shown in Figs. 13 and 14 were used in the plots.

úËõ March 2010 Vol.23 No.2  77

ViÌ±ÇtÅÍ Cd Í O © N ÆµCªÅÍ S ÆµÄ¢éÆñ³êÄ¨è36) C±Ìæ¤ÈA¨íÉæé Cd Ì»w`ÔÌá¢ÍC~Ï@\Ì·ð½fµÄ¢é
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Cadmium K­edge m­XANES spectra for the trichome of A.
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Fig. 14(b). (d) PC_
Cd, (e) MT_Cd, (f) CdS, (g) CdO,(h) Cd(NO3)2 aq, (i) AcCd, (j) (Im)6Cd(NO3)2. Beamsize: 3.8 mm(H)~1.3 mm (V), energy step: 1 eV, and meas­
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Study on accumulation mechanism for heavy metal
in hyper­accumulating plants by synchrotron
radiation X­ray analysis
Akiko HOKURA
Nobuyuki KITAJIMA
Yasuko TERADA
Izumi NAKAI

Tokyo DENKI University, School of Engineering, Department of Green
Sustainable Chemistry, 2_
2 Kanda­Nishikicho, Chiyoda­ku,
Tokyo 101_8457, Japan
Fujita Corporation, Technology Development Division,
2025_1 Ono, Atsugi, Kanagawa 243_0125, Japan
Japan Synchrotron Radiation Research Institute (JASRI),
1_1_1, Kouto, Sayo­cho, Sayo­gun, Hyogo 679_5198, JapanTokyo University of Science, Faculty of Science, Department of Applied
Chemistry, 1_3 Kagurazaka, Shinjuku­ku, Tokyo 162_
8601, Japan

Abstract Some plants accumulate heavy metal elements such as As, Cd, and Pb, etc., and these plants
have been focused on from the perspective of their application to phytoremediation. In order to understand theaccumulation mechanism, the distribution and the chemical form of heavy metal should be revealed at cellularlevel. Here, we have introduced the recent works regarding arsenic hyperaccumulating fern (Pteris vittata L.)
and cadmium hyperaccumulating plant (Arabidopsis halleri ssp. gemmifera ). A combination of m­XRF and m­
XANES techniques excited by high­energy X­ray microbeam with 1 mm resolution has proved to be an in­dispensable tool for the study of Cd accumulation in biological samples on a cellular scale. The sample­prepa­
ration techniques were also summarized.

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