Instrumentation and Examples

The required instrumentation for micro-PIXE includes an ion accelerator, ion lenses, a scanning system, detectors and acquisition software. The method is becoming readily available to external users in several laboratories as a standard technique for quantitative element mapping in biological tissue.

Although micro-PIXE is a multielement technique, where all of the elements are detected simultaneously, the duration of any single measurement depends strongly on the targeted trace element. When the interest is primarily in the distribution of light or mid-Z elements, the time required for a single measurement ranges from 10 min to several hours. In extreme cases, such as with cadmium distribution in plant tissue (Vogel-Mikus et al. 2008b), a single measurement may take up to 2 days (Fig. 6.6). Figure 6.7 illustrates an example of the use of micro-PIXE for element localization in plant tissues. This includes element maps of a leaf cross-section from the Cd/Zn hyperaccumulator Thlaspi praecox (Wulf.) growing on soil highly polluted with Pb, Cd and Zn. The Zn map illustrates the epidermal cells that are rich in Zn, while the K map shows the regions of the vascular bundles with their abundances of K. Finally, the Cd and Pb maps demonstrate the tolerance to the uptake of both Cd and Pb of the palisade and spongy mesophyll tissue. The black-and-white maps show the selected positions of the xylem, phloem and collenchyma regions of the vascular tissues, where as the spectral data extracted provide the quantified concentrations given in Table 6.1.

Fig. 6.6 PIXE spectrum measured in a section of a leaf of Thlaspi praecox (Wulf.) grown on highly polluted soil. For Cd detection, the Cd Ka line at 23 keV is used for evaluation, as the Cd L lines overlap with the strong K Ka line. This drastically increases the required detection time for Cd quantification (Vogel-Mikus et al. 2008b)

Fig. 6.6 PIXE spectrum measured in a section of a leaf of Thlaspi praecox (Wulf.) grown on highly polluted soil. For Cd detection, the Cd Ka line at 23 keV is used for evaluation, as the Cd L lines overlap with the strong K Ka line. This drastically increases the required detection time for Cd quantification (Vogel-Mikus et al. 2008b)

The typical running costs of the micro-PIXE method range from ~200€ per hour to ~1,500-4,000€ per day. At several micro-PIXE facilities, access is granted after evaluating and selecting scientific proposals.

6.4 Element Complexation Analyses 6.4.1 X-Ray Absorption Spectroscopy

High-resolution X-ray absorption spectroscopy (XAS) became available with the development of synchrotron radiation sources. The advent of this form of spectroscopy has introduced powerful experimental methods for the investigation of atomic and molecular structures of materials that have enabled the identification of local structures around atoms of a selected type in the sample. In X-ray absorption near-edge structure (XANES), the valence state of the selected type of atom in the sample and the local symmetry of its unoccupied orbitals can be deduced from the information hidden in the shape and energy shift of the X-ray absorption edge. In extended X-ray absorption fine structure (EXAFS), the number and species of neighboring atoms, their distances from the selected atom, and the thermal or structural disorder in their positions can be determined from the oscillatory part of

ijflpw A* X -

STIM

K

Ca

If If

0 '

Zn

Cd

Pb

Fig. 6.7 Elemental distributions in a section of a leaf of Thlaspipraecox (Wulf.) grown on highly polluted soil. C, Collenchyma; Xy, xylem; Ph, phloem; STIM, scanning ion transmission microscopy image

Fig. 6.7 Elemental distributions in a section of a leaf of Thlaspipraecox (Wulf.) grown on highly polluted soil. C, Collenchyma; Xy, xylem; Ph, phloem; STIM, scanning ion transmission microscopy image the absorption coefficient above the K or L absorption edges (Koningsberger and Prins 1988; Rehr and Albers 2000; Wong et al. 1984). This analysis can be applied to crystalline, nanostructural or amorphous materials, as well as to biological samples, liquids and molecular gases. EXAFS is often the only practical way to study the arrangements of atoms in materials without long-range order, where traditional diffraction techniques cannot be used. In the case of biological samples, the method can provide specific structural information at the atomic level, especially about metal contaminants in ecosystems. The information on the valence states and the local structures around metal cations that are bound in different plant tissues or in the soil, or are dissolved in water, can provide direct evidence of metal complexation with organic molecules. This can help to resolve important questions about the bioavailability of toxic metals in the soil, and about soil-plant interactions and metal accumulation. The information obtained using XANES and EXAFS can explain complexation mechanisms in different plant tissues at a biochemical level, and can provide insights into tolerance mechanisms in metal (hyper)accumulating species.

A basic XAS experiment is shown in Fig. 6.8. A thin homogeneous sample of the material to be investigated is prepared with an optimal absorption thickness

Table 6.1 Elemental concentrations obtained by analyzing spectroscopic data from the xylem, collenchyma and phloem regions of the vascular tissues

Table 6.1 Elemental concentrations obtained by analyzing spectroscopic data from the xylem, collenchyma and phloem regions of the vascular tissues

Xylem Collenchyma Phloem

Conc.

Stat.

LOD

Conc.

Stat.

LOD

Conc.

Stat.

LOD

El.

(^g g-1)

err. (%)

(^g g-1)

(^g g-1)

err. (%)

(^g g-1)

(^g g-1)

err. (%)

(^g g-1)

Cl

2,890

6.33

407

10,174

1.78

381

4,122

5.96

541

K

26,477

0.34

70.5

43,286

0.15

60.7

33,573

0.35

89.7

Ca

5,189

1.27

116

10,177

0.71

129

5,421

1.53

150

Mn

66.5

9.25

9.6

104

3.69

6.1

70

11

12.3

Fe

66.1

9.35

9.2

25.8

15.3

7.5

41.1

18.9

13.5

Ni

11.6

33.2

4.3

14

17.7

4.3

£lod

/

7.5

Cu

20.5

23.5

5.9

£lod

/

3.5

9.4

46.3

7.0

Zn

872

2.28

9.7

1,742

0.78

4.6

870

2.79

14.2

Cd

832

33.5

417

2,440

7.52

169

1,438

27.3

703

Pb

261

12.7

19.3

1,466

2.08

14.1

411

11.5

53.3

El., element; conc., concentration; LOD, limit of detection; stat. err., statistical error. Phosphorus and sulfur were below the limit of detection

El., element; conc., concentration; LOD, limit of detection; stat. err., statistical error. Phosphorus and sulfur were below the limit of detection

Flúores, detector 1

Fig. 6.8 Schematic representation of an X-ray absorption spectroscopy beamline

(pd) of about 2, and the intensities of the incident and the transmitted X-ray beams are recorded with a stepwise progression in the incident photon energy. In a typical synchrotron radiation experimental setup performed in transmission detection mode, ionization cells monitor the intensities of the incident (I0) and transmitted (Ij) monochromatic photon beams through the sample. Since the exponential attenuation of X-rays in a homogeneous medium is given by the well-known relation I=/exp(-md), where d is the sample thickness, the absorption coefficient m(E) can be obtained at a given photon energy E. The energy dependence of the absorption coefficient is obtained by a stepwise scan of the photon energy in the monochromatic beam that is provided by a double-crystal Bragg monochromator. The exact energy calibration of the monochromator is established with simultaneous absorption measurements on a reference metal foil placed between the second and third ionization chambers.

With diluted samples that contain small amounts of the element of interest, which is often the case in biological samples, and for thin films that are deposited on thick substrates, the standard transmission detection mode cannot be used. Instead, we can exploit the fluorescence detection mode, where fluorescence photons from the sample are monitored instead of the transmitted beam. The intensities of the detected fluorescence signals are linearly proportional to the absorption coefficient of the element under investigation. The investigated thickness of the sample is proportional to the penetration depth of the X-ray beam.

The dominant process in X-ray absorption at photon energies below 100 keV is the photoelectric effect, whereby the photon is completely absorbed, transferring its energy to the ejected photoelectron. The photoelectric cross-section, and hence the absorption coefficient, decreases monotonically with increasing photon energy. When the photon energy reaches one of the deep inner-shell ionization energies of the atom, there is a sharp jump (absorption edge) that marks the opening of an additional photoabsorption channel. For practical purposes, in structural analysis, the K and L absorption edges are most important. An example of an absorption spectrum for Cr metal in the energy region of the Cr K edge is shown in Fig. 6.9.

XANES

Cr metal

EXaps

6600

5800

6000

6600

6800

5980 6000 6020 6040 6060 E (eV)

Fig. 6.9 X-ray absorption spectrum of Cr metal in the energy range of the Cr K edge (5.989 keV), showing rich EXAFS structure. The K-edge energy region, magnified in the right graph, reveals details of the Cr K edge XANES structure

The analysis mainly focuses on two energy regions that are usually treated separately: the energy region of the absorption edge and pre-edge structures within about 30 eV of the threshold, denoted XANES, and the energy region in a range from about 30 eV up to about 1,000 eV above the threshold, which shows oscillatory variations of the X-ray absorption coefficient, and is known as EXAFS.

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