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The biological research programme of the nuclear microprobe at the National Accelerator Centre, Faure
Nuclear Instruments and Methods in Physics Research B 161±163 (2000) 852±859
www.elsevier.nl/locate/nimb
The biological research programme of the nuclear microprobe at the National Accelerator Centre, Faure V.M. Prozesky a,*, C.A. Pineda b, J. Mesjasz-Przybylowicz a, W.J. Przybylowicz a,1, C.L. Churms a, K.A. Springhorn a, Ph. Moretto c, C. Michelet a,c, U. Chikte d, P. Wenzl e b
a Van de Graa Group, National Accelerator Centre, P.O. Box 72, Faure 7130, South Africa Department of Medicine, Groote Schuur Hospital, Observatory 7925, Cape Town, South Africa c Centre d'Etudes Nucl eaires de Bordeaux Gradignan, France d Department of Community Dentistry, University of Stellenbosch, Tygerberg, South Africa e CAMBIA, Canberra, Australia
Abstract The nuclear microprobe (NMP) unit of the National Accelerator Centre (NAC) has initiated a focused research programme on studies of biological material, ranging from applications in medicine to agriculture and botany. During this period a state-of-the-art cryo-preparation laboratory was also developed. This research programme has resulted in a wide range of projects, and has shown how well suited the NMP is for studies of biological material in general. This paper reports on some of the problems and demands in this ®eld, as well as some of the results obtained using particle induced X-ray spectroscopy (PIXE) and Rutherford backscattering (RBS). True elemental imaging is routinely performed using the dynamic analysis (DA) method, which forms part of the GeoPIXE suite of programmes. A collaborative project, together with the CENBG group of Bordeaux±Gradignan in France, on the development of a facility with the aim of studying eects of single-events of radiation in living cells was recently established and is discussed. Ó 2000 Published by Elsevier Science B.V. All rights reserved. PACS: 07.79.-v; 87.64.F; 82.80.Yc; 87.22.-q Keywords: Scanning probe microscopes; X-ray spectroscopy
1. Introduction
*
Corresponding author. Fax: +27+21-8433543. E-mail address: [email protected] (V.M. Prozesky). 1 On leave from the Faculty of Physics and Nuclear Techniques, The Academy of Mining and Metallurgy, 30-059 Cracow, Poland.
The NMP has become an established tool in a variety of applications. In the last two decades it has been applied extensively in areas such as materials science, geology and minerals, and the biological sciences have found increasing application, see [1] for example. Although the
0168-583X/00/$ - see front matter Ó 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 9 9 ) 0 0 8 9 5 - 2
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NMP, with a spatial resolution of the order of 1 lm, cannot compete with the spatial resolution of the electron microscope, it has better detection limits for trace element analysis and oers the possibility of quantitative mapping. The modern NMP (often called proton microprobe, due to the use of protons in many cases) is a highly computerised instrument, with on-line data-acquisition and analysis. In most cases the sampling stage is movable via a control computer, and the microprobe is ®tted with an optical microscope, with a maximum magni®cation of around 100±250. The full array of ionbeam analytical techniques is also available to the nuclear microscopist, with the added advantage of a small beam-size for lateral spatial resolution, and an imaging capability. The NMP competes with many other techniques available for high-resolution characterisation of a multitude of materials, and therefore it has become essential that it is equipped with the latest technologies to stay competitive. It has also become clear that the NMP has superior performance in a few niche areas in each area of application. For instance, in the geological domain the relative small beam size and the imaging capabilities are probably the only advantage that the NMP has above that of the more commonly used laser ablation ICP-MS [2] technique. It has also become necessary to have at least semi-quantitative on-line imaging available, such as the dynamic analysis (DA) [3] technique, which is a part of the GeoPIXE suite of programmes. The important requirements to draw attention from users in the biological sciences community are its user-friendliness, as well as in the technical backup provided by the institution involved. For this reason there is an actual interest to operate the microprobe over long distances via a computer network or even through the internet [4]. In the application of such an instrument to the biological sciences it is helpful to have complementary skills and techniques available on-site, such as a sample preparation capability and coating of insulating samples to avoid charging during irradiation.
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2. The NAC NMP The NAC NMP is an established facility, which has been described before, see [5±7], and it is not the intention of this paper to focus on the instrument. This paper aims to demonstrate the capabilities of the NMP through the description of some of the wide range of projects in the biological sciences that are pursued at NAC. The NAC NMP is equipped with the Oxford OM150 triplet set of magnetic lenses, and the standard Oxford target chamber has been modi®ed to allow essential features, such as a stepper±motor driven sample stage and a genuine multiple-sample holder. The 6 MV single-ended Van de Graa accelerator routinely delivers proton- and a-particle beams for the microprobe, which is the largest user of beams at the accelerator by far. Beam sizes of around 1±2 lm at 100 pA for analytical purposes, and 100±200 nm for direct counting techniques, such as scanning transmission ion microscopy (STIM), are routinely available. Scanning sizes can vary from a few microns to a maximum of around 2500 lm by beam scanning, and can be extended to some centimetres by combining several scanned areas into one picture through sample movement. On-demand beam de¯ection [6] allows higher beam currents to be used in PIXE experiments, typically currents of up to few hundred picoamperes are used with X-ray count-rates around 2±3 kHz with a system dead-time of a few percent. Naturally, the on-demand beam de¯ection also optimises ecient use of beam in terms of sample preservation, the beam is only on target when needed to provide a signal, not during the processing of signals. List mode data-acquisition is available in the multi-purpose XSYS system [8], and simultaneous use of PIXE, RBS, elastic recoil detection analysis (ERDA) and secondary electron detection is possible. At 0° the Faraday cup can be exchanged with a STIM detector without breaking vacuum, and the STIM detector can be used either for on- or o-axis STIM measurements, valuable for measuring thin biological sample integrity during analytical measurements. The acquisition control software [4] allows for quick modi®cation of input parameters, beam-size
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determination, zooming during imaging, line-scans and multiple point analyses with auto-start and auto-stop, some of the many utility functions. The most important part of analysis of biological samples is the preparation phase [9±11] which should prevent any changes of elemental concentration and distribution. A cryo-preparation unit has been recently developed at NAC which includes a Leica EM CPC Universal cryoworkstation, a Leica EM cryosorption freeze drying system and a cryo-ultramicrotome (Reichert-Jung FC4E). The equipment allows plunge cryo®xation in liquid propane and ethane, metal mirror cryo®xation, freeze-substitution, freeze drying with controlled temperature and time, low temperature resin embedding, UV-polymerisation and cryosectioning. The samples are mounted on formvar or thin mylar ®lms and carbon-coated to prevent charge build-up during analysis. Types of specimens analysed at NAC vary from thin samples (such as single cells grown in culture), targets of intermediate thickness to thick samples. A wide range of research work focused primarily on applications in the ®elds of botany [12±15], medicine [16±18] and zoology [19±21] have recently been undertaken at NAC. For true elemental analysis care is taken to control and monitor beam damage and elemental losses. These experimental variables should guarantee a continuous eort to determine ways to improve the accuracy and characterization of every material analysed as well as its response to ion beam irradiation [22]. A few of the wide-ranging projects being pursued at the NAC NMP are discussed. 3. Optimisation of the NAC NMP for the determination of light elements in bio-medical materials The NMP allows measurements with excellent scanning capabilities, particularly for elements heavier than phosphorus. In bio-medical research there is a need to know the concentration of lighter elements, and therefore a suitable approach to determine Na, Mg, Al and Si by micro-PIXE was investigated. Beam energies of 0.6, 1.0 and 1.5 MeV protons were used with the most suitable energy
found to be 0.6 MeV [22]. Experimental runs were performed on standard materials, as well as unknown biological samples and special attention was given to the inherent electronic noise of the Si(Li) detector which interfered drastically with the detection of the low-energy X-ray signals. Since the most important factor in the optimisation procedure is to maximise the signalto-background ratio of the K X-ray signal for these elements, without detrimentally aecting the detector response, a 25 lm Al absorber was used (for irradiation with 1.5 MeV) to prevent scattered protons from reaching the detector volume. Although the ionisation cross-sections are much higher for irradiation with this energy than is the case with 0.6 MeV protons, the transmission of Xrays is far better for the latter, especially for the lightest elements: Na, Mg, Al and Si. The eective depth of analysis (maximum depth at which X-rays generated by the proton beam are detected) is also closer to the total proton range for 0.6 MeV, compared to 1.5 MeV, which applies well for the analysis of biological materials. This causes the matrix correction factors (MCF) to be relatively constant for most light elements (particularly for Na, Mg, Al and Si [23]), simplifying the analysis. 4. Development of a single ion irradiation facility There is a need to understand and quantify the damage sustained by an individual cell when exposed to a single ionising particle of a few megaelectron-volts. At present, the study of the response of individual cells to very weak irradiation doses can only be performed in three research centres outside South Africa, two of them located in the USA [24,25] and one in the UK [26]. Since 1998, the Van de Graa Group at NAC and the Centre dÕEtudes Nucleaires de Bordeaux Gradignan (CENBG), France, are developing an irradiation facility as part of the international research agreement between CNRS and NAC. First implemented in Bordeaux, the system will be at the later stage available for both laboratories. The initial stage of the project requires the design and testing of the dierent components of the new
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beam line, which must satisfy the following requirements: · to deliver single ions in the mega-electron-volt energy range; · to extract the beam in air through an ultra-thin exit window; · to control the positioning of the ion beam to achieve a spatial resolution of the order of microns. This will allow studies of the biological damage induced by the irradiation of speci®c parts within the cell structure (nucleus, cytoplasm, etc.). The ®rst stage of the experimental work has been devoted to the characterisation of a suitable exit window. A thin foil made of Si3 N4 was selected as a possible candidate and tested at NAC, using the NMP in STIM and RBS modes, with a 2 MeV aparticle beam. The data were stored in event-byevent mode in order to assess the thickness and the homogeneity of the window (see Fig. 1). A fast single-ion detector, based on a thin scintillator ®lm optically coupled with a pair of photomultipliers, has been constructed in Bordeaux and tested for eciency and background noise using the NMP at NAC. Consecutive tests were performed at CENBG, at very low beam ¯uence. Results of these experiments showed that the response signal remained constant throughout the duration of the experiment. Further experiments will involve: (a) the characterisation of other possible candidates for exit windows such as CVD diamond ®lms and thin plastic scintillators; (b) the optimisation of the fast ion detector to improve the eciency for single ion detection. 5. Elemental distribution in young leaves of the Ni hyperaccumulator ± Berkheya coddii Very few plant species growing on soil enriched with heavy metals have the ability to take up exceptionally high amounts of these metals. A plant that contains more than 0.1% of Ni per dry weight of material in any above ground part is de®ned as a Ni hyperaccumulator. Such amounts are highly toxic for normal plants and so far around 300
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Fig. 1. (A) STIM map of a Si3 N4 window: The energy of the a particles transmitted through the window was recorded at each pixel of the map 64 64 pixels on a 180 180 lm2 area. The transmitted energy is directly related to the thickness of the window, which was found to be roughly homogeneous all over the surface. (B) Energy spectra of the incoming 2.0 MeV aparticle beam and of the beam transmitted through the Si3 N4 windows. The thickness of the windows, calculated from the value of the energy loss DE measured on the energy spectra, is around 0.06 lm.
species of Ni hyperaccumulators have been discovered in the world ¯ora [27]. One of ®ve nickel hyperaccumulators from South Africa is Berkheya coddii Roessler (Asteraceae), an endemic herbaceous plant growing on Ni-rich outcrops (Mpumalanga Province, South Africa). The highest enrichment of Ni in the leaves
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Fig. 2. Quantitative elemental maps showing the distribution of Ni, Ca, K and Zn in young leaf cross-sections of Berkheya coddii. Concentrations are in micro-grams per gram (lg=g) dry mass.
is typical for hyperaccumulators and in B. coddii it reaches 3.6% [28] of the dry mass. Dierences of elemental distributions in a young leaf cross-section are clearly visible (see Fig. 2). Ni is present in all tissues, but the highest amount of Ni is found in the outer tissues ± in the epidermis of the leaf midrib (1.25%) and in the leaf tip (2.2%). The distribution pattern shows the storage locations for most of the Ni (in an unknown, probably non-toxic form). The maximum quantity in the epidermis is similar to other Ni hyperaccumulators from South Africa [29±32]. Macro-nutrients are allocated speci®cally to tissues: Ca is concentrated mainly in the midrib cortex whereas K is allocated in the vascular bundles and in the leaf parenchyma. Zn ± an essential micro-nutrient ± is present in the epidermis, vascular bundle and leaf blade. Mn and Fe reach maximum values in the epidermis. The unique response of these plants to elevated soil concentrations of Ni may be practically used in the future as an invaluable tool in the bio-
remediation of metal polluted soils and toxic wastes. Berkheya coddii has already been tested on dierent soil types with very promising results [33,34]. 6. Tolerance of Brachiaria to Al at toxic levels The genus Brachiaria is the source of the most important and widely sown tropical forage grasses. Among the Brachiaria cultivars, B. decumbens Stapf cv. Basilisk is planted on over 30 million ha and is very well adapted to nutrient-poor and Al toxic acid soils of Latin America. However, the mechanisms that contribute to tolerance to Al in this cultivar are not yet de®ned. An understanding of these mechanisms may assist the genetic improvement of other Brachiaria species. Seeds of B. decumbens were germinated on moist paper and seedlings were cultivated for three weeks in a low ionic strength nutrient solution designed to simulate nutrient-de®cient conditions in soil solutions of typical acid soils from the
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Colombian savannas [14]. A growth analysis in the presence and absence of Al indicated that this species is highly Al-tolerant [35]. Root apices (ca. 5 mm) of adventitious roots were cryo-®xed and lyophilized. Since toxic Al3 -ions act primarily on the root apical meristem, micro-analysis (micro-PIXE) was used to study the in¯uence of Al (added to the nutrient solution) on the concentration and distribution of nutrients in root apices. Preliminary results obtained by elemental mapping with 1.5 MeV protons indicated that: 1. Al was preferentially located in the root cap and epidermis; 2. growth in the presence of Al induced an accumulation of P in the meristematic region (0.2±1.2 mm) of the root apex, which was most pronounced in the central vascular tissue; 3. the level of Cl, and to a less extent S, within the meristematic region decreased signi®cantly (see Fig. 3). Recently, the exudation of phosphate by root apices has been suggested to act as an Al exclusion mechanism in wheat [36]. The Al stimulated P accumulation in root apices of B. decumbens could thus re¯ect physiological events leading to exudation of phosphate. Experiments are underway to determine the extent of phosphate exudation as a function of the distance from the root tip.
7. Detection of early caries lesion in human teeth Prevention of demineralization and enhancement of remineralization of caries is a primary goal of preventive dentistry. Current caries detection criteria are unable to measure the disease process as a continuum, which progresses from an increase in the micro-porosity of the subsurface enamel (white spot) to the occurrence of a frank cavitation [37,38]. The criteria are limited in that they detect caries at an advanced stage, they lack the ability to quantify mineral loss or gain and they are unable to observe small changes in mineral loss or gain occurring as a result of demineralization or remineralization. The purpose of this study is to investigate the detection and quanti®cation of mineral loss in
Fig. 3. Quantitative elemental contour maps for (a) Al, (b) P and (c) Cl of a root apex of Bracharia decumbens, obtained with 1.5 MeV protons. Beam current was 100 pA; and the total collected charge was 200 nC. Concentrations are given in lg=g dry mass.
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early arti®cial carious lesions, with particular reference to variability in the spatial elemental distribution and their correlation with the demineralization and remineralization processes, especially for Ca and P. This is achieved by using nuclear microscopy to determine the levels of Ca
and P in human enamel and dentine. Mechanisms (processes) occurring in the surface and subsurface of the enamel are studied as well as areas in and around the interface between dentine and enamel. The eect of ¯uorine protection will also later be tested as well as the eect of demineralization, which is thought to be happening mainly on the subsurface and is represented by voids in the spatial distribution of elements. Elemental maps performed in a test series of experimental samples, determined by DA, showed that some voids are present in the surface and subsurface areas. A number of voids in the distribution of Ca and P were observed on the surface and in the body of the tooth, which appear to be related to the extent of the mineral loss (demineralization). This loss is illustrated in Fig. 4(a) for P, and Fig. 4(b) for Ca, in the subsurface area. The ability of the NMP to detect and possibly quantify mineral loss after exposure to demineralizing solutions suggests that the technique has the potential to detect mineral losses in human teeth at a very early stage and may have the possibility to initiate improved preventive therapies. 8. Discussion The use of the NMP in studies of biological material has proliferated during the last number of years, and is one of the niche areas of application where the techniques provided are often superior than other available techniques. For this reason this ®eld of application for the NMP will grow further in the foreseeable future. Acknowledgements The assistance of the technical sta of the Van de Graa Group at NAC is essential to the research programme, and therefore well appreciated.
Fig. 4. Quantitative elemental maps of dental samples obtained with micro-PIXE; proton energy 0.6 MeV; beam current was 200 pA; total collected charge 800 nC. A number of voids in the distribution of Ca and P appear to be related to the extent of the mineral loss (demineralization). This particular loss is evident for both elements in and around the subsurface area. Concentrations are given in lg=g.
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[21] V.M. Prozesky, E.J. Raubenheimer, W.P. Grotepass, W.F.P. Van Heerden, W.J. Przybylowicz, C.A. Pineda, R. Swart, Nucl. Instr. and Meth. B 104 (1995) 638. [22] W.J. Przybylowicz, C.A. Pineda, A.D. Barnabas, J. Mesjasz-Przybylowicz, Nucl. Instr. and Meth. B 150 (1999) 282. [23] C.A. Pineda, Thick target PIXE analysis, Ph.D. Thesis, University of Cape Town, 1993. [24] L.A. Braby, A.L. Brooks, N.F. Metting, Radiat. Resear. 148 (1997) S108. [25] R.C. Miller, G. Randers-Pehrson, C.R. Geard, E.J. Hall, D.J. Brenner, Proc. Natl. Acad. Sci. USA 96 (1999) 19. [26] M. Folkard, K.M. Prise, B. Vojnovic, A.G. Bowey, C. Pullar, G. Schettino, B.D. Michael, Microdosimetry, in: D T Goodhead, P.O.ÕNeill, H.G. Menzel (Eds.), An Interdisciplinary Approach, Royal Society of Chemistry, Cambridge, UK, 1997, p. 323. [27] R.R. Brooks, B.H. Robinson, in: R.R. Brooks (Ed.), Plants that Hyperaccumulate Heavy Metals, CAB International, 1998. [28] T.R. Anderson, A.W. Howes, K. Slatter, M.F. Dutton, in: Proceedings of the Second International Conference on Serpentine Ecology, Centre ORSTOM de Noumea, 1997, New Caledonia, pp. 111±112. [29] J. Mesjasz-Przybylowicz, K. Balkwill, W.J. Przybylowicz, H.J. Annegarn, Nucl. Instr. and Meth. B 89 (1994) 208. [30] J. Mesjasz-Przybylowicz, K. Balkwill, W.J. Przybylowicz, H.J. Annegarn, D.B.K. Rama, in: L.J.G. van der Maesen, X.M. van der Burgt, J.M. van Medenbach de Roy (Eds.), The Biodiversity of African Plants, Kluwer Academic Publishers, Dordrecht, 1996, p. 331. [31] J. Mesjasz-Przybylowicz, W.J. Przybylowicz, V.M. Prozesky, C.A. Pineda, Proc. Microscopy Society of Southern Africa 26 (1996) 68. [32] J. Mesjasz-Przybylowicz, W.J. Przybylowicz, D.B.K. Rama, C.A. Pineda, Proc. Microscopy Society of Southern Africa 27 (1997) 89. [33] B.H. Robinson, R.R. Brooks, A.W. Howes, J.H. Kirkman, P.E.H. Gregg, J. Geochem. Explor. 60 (1997) 115. [34] R.R. Brooks, B.H. Robinson, in: R.R. Brooks (Ed.), Plants that Hyperaccumulate Heavy Metals, CAB International, 1998, pp. 327±356 (Chapter 15). [35] P. Wenzl, CAMBIA, Canberra (personal communication). [36] D.M. Pellet, D.L. Grunes, V.L. Kochian, Plant Physiol. 112 (1996) 591. [37] F. Manji, O. Fejerskov, N.J. Nagel Herke, V. Baelum, Community Dent. Oral Epidemiol. 19 (1991) 324. [38] A.I. Ismail, Community Dent. Oral Epidemiol. 15 (1997) 13.
www.elsevier.nl/locate/nimb
The biological research programme of the nuclear microprobe at the National Accelerator Centre, Faure V.M. Prozesky a,*, C.A. Pineda b, J. Mesjasz-Przybylowicz a, W.J. Przybylowicz a,1, C.L. Churms a, K.A. Springhorn a, Ph. Moretto c, C. Michelet a,c, U. Chikte d, P. Wenzl e b
a Van de Graa Group, National Accelerator Centre, P.O. Box 72, Faure 7130, South Africa Department of Medicine, Groote Schuur Hospital, Observatory 7925, Cape Town, South Africa c Centre d'Etudes Nucl eaires de Bordeaux Gradignan, France d Department of Community Dentistry, University of Stellenbosch, Tygerberg, South Africa e CAMBIA, Canberra, Australia
Abstract The nuclear microprobe (NMP) unit of the National Accelerator Centre (NAC) has initiated a focused research programme on studies of biological material, ranging from applications in medicine to agriculture and botany. During this period a state-of-the-art cryo-preparation laboratory was also developed. This research programme has resulted in a wide range of projects, and has shown how well suited the NMP is for studies of biological material in general. This paper reports on some of the problems and demands in this ®eld, as well as some of the results obtained using particle induced X-ray spectroscopy (PIXE) and Rutherford backscattering (RBS). True elemental imaging is routinely performed using the dynamic analysis (DA) method, which forms part of the GeoPIXE suite of programmes. A collaborative project, together with the CENBG group of Bordeaux±Gradignan in France, on the development of a facility with the aim of studying eects of single-events of radiation in living cells was recently established and is discussed. Ó 2000 Published by Elsevier Science B.V. All rights reserved. PACS: 07.79.-v; 87.64.F; 82.80.Yc; 87.22.-q Keywords: Scanning probe microscopes; X-ray spectroscopy
1. Introduction
*
Corresponding author. Fax: +27+21-8433543. E-mail address: [email protected] (V.M. Prozesky). 1 On leave from the Faculty of Physics and Nuclear Techniques, The Academy of Mining and Metallurgy, 30-059 Cracow, Poland.
The NMP has become an established tool in a variety of applications. In the last two decades it has been applied extensively in areas such as materials science, geology and minerals, and the biological sciences have found increasing application, see [1] for example. Although the
0168-583X/00/$ - see front matter Ó 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 9 9 ) 0 0 8 9 5 - 2
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NMP, with a spatial resolution of the order of 1 lm, cannot compete with the spatial resolution of the electron microscope, it has better detection limits for trace element analysis and oers the possibility of quantitative mapping. The modern NMP (often called proton microprobe, due to the use of protons in many cases) is a highly computerised instrument, with on-line data-acquisition and analysis. In most cases the sampling stage is movable via a control computer, and the microprobe is ®tted with an optical microscope, with a maximum magni®cation of around 100±250. The full array of ionbeam analytical techniques is also available to the nuclear microscopist, with the added advantage of a small beam-size for lateral spatial resolution, and an imaging capability. The NMP competes with many other techniques available for high-resolution characterisation of a multitude of materials, and therefore it has become essential that it is equipped with the latest technologies to stay competitive. It has also become clear that the NMP has superior performance in a few niche areas in each area of application. For instance, in the geological domain the relative small beam size and the imaging capabilities are probably the only advantage that the NMP has above that of the more commonly used laser ablation ICP-MS [2] technique. It has also become necessary to have at least semi-quantitative on-line imaging available, such as the dynamic analysis (DA) [3] technique, which is a part of the GeoPIXE suite of programmes. The important requirements to draw attention from users in the biological sciences community are its user-friendliness, as well as in the technical backup provided by the institution involved. For this reason there is an actual interest to operate the microprobe over long distances via a computer network or even through the internet [4]. In the application of such an instrument to the biological sciences it is helpful to have complementary skills and techniques available on-site, such as a sample preparation capability and coating of insulating samples to avoid charging during irradiation.
853
2. The NAC NMP The NAC NMP is an established facility, which has been described before, see [5±7], and it is not the intention of this paper to focus on the instrument. This paper aims to demonstrate the capabilities of the NMP through the description of some of the wide range of projects in the biological sciences that are pursued at NAC. The NAC NMP is equipped with the Oxford OM150 triplet set of magnetic lenses, and the standard Oxford target chamber has been modi®ed to allow essential features, such as a stepper±motor driven sample stage and a genuine multiple-sample holder. The 6 MV single-ended Van de Graa accelerator routinely delivers proton- and a-particle beams for the microprobe, which is the largest user of beams at the accelerator by far. Beam sizes of around 1±2 lm at 100 pA for analytical purposes, and 100±200 nm for direct counting techniques, such as scanning transmission ion microscopy (STIM), are routinely available. Scanning sizes can vary from a few microns to a maximum of around 2500 lm by beam scanning, and can be extended to some centimetres by combining several scanned areas into one picture through sample movement. On-demand beam de¯ection [6] allows higher beam currents to be used in PIXE experiments, typically currents of up to few hundred picoamperes are used with X-ray count-rates around 2±3 kHz with a system dead-time of a few percent. Naturally, the on-demand beam de¯ection also optimises ecient use of beam in terms of sample preservation, the beam is only on target when needed to provide a signal, not during the processing of signals. List mode data-acquisition is available in the multi-purpose XSYS system [8], and simultaneous use of PIXE, RBS, elastic recoil detection analysis (ERDA) and secondary electron detection is possible. At 0° the Faraday cup can be exchanged with a STIM detector without breaking vacuum, and the STIM detector can be used either for on- or o-axis STIM measurements, valuable for measuring thin biological sample integrity during analytical measurements. The acquisition control software [4] allows for quick modi®cation of input parameters, beam-size
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determination, zooming during imaging, line-scans and multiple point analyses with auto-start and auto-stop, some of the many utility functions. The most important part of analysis of biological samples is the preparation phase [9±11] which should prevent any changes of elemental concentration and distribution. A cryo-preparation unit has been recently developed at NAC which includes a Leica EM CPC Universal cryoworkstation, a Leica EM cryosorption freeze drying system and a cryo-ultramicrotome (Reichert-Jung FC4E). The equipment allows plunge cryo®xation in liquid propane and ethane, metal mirror cryo®xation, freeze-substitution, freeze drying with controlled temperature and time, low temperature resin embedding, UV-polymerisation and cryosectioning. The samples are mounted on formvar or thin mylar ®lms and carbon-coated to prevent charge build-up during analysis. Types of specimens analysed at NAC vary from thin samples (such as single cells grown in culture), targets of intermediate thickness to thick samples. A wide range of research work focused primarily on applications in the ®elds of botany [12±15], medicine [16±18] and zoology [19±21] have recently been undertaken at NAC. For true elemental analysis care is taken to control and monitor beam damage and elemental losses. These experimental variables should guarantee a continuous eort to determine ways to improve the accuracy and characterization of every material analysed as well as its response to ion beam irradiation [22]. A few of the wide-ranging projects being pursued at the NAC NMP are discussed. 3. Optimisation of the NAC NMP for the determination of light elements in bio-medical materials The NMP allows measurements with excellent scanning capabilities, particularly for elements heavier than phosphorus. In bio-medical research there is a need to know the concentration of lighter elements, and therefore a suitable approach to determine Na, Mg, Al and Si by micro-PIXE was investigated. Beam energies of 0.6, 1.0 and 1.5 MeV protons were used with the most suitable energy
found to be 0.6 MeV [22]. Experimental runs were performed on standard materials, as well as unknown biological samples and special attention was given to the inherent electronic noise of the Si(Li) detector which interfered drastically with the detection of the low-energy X-ray signals. Since the most important factor in the optimisation procedure is to maximise the signalto-background ratio of the K X-ray signal for these elements, without detrimentally aecting the detector response, a 25 lm Al absorber was used (for irradiation with 1.5 MeV) to prevent scattered protons from reaching the detector volume. Although the ionisation cross-sections are much higher for irradiation with this energy than is the case with 0.6 MeV protons, the transmission of Xrays is far better for the latter, especially for the lightest elements: Na, Mg, Al and Si. The eective depth of analysis (maximum depth at which X-rays generated by the proton beam are detected) is also closer to the total proton range for 0.6 MeV, compared to 1.5 MeV, which applies well for the analysis of biological materials. This causes the matrix correction factors (MCF) to be relatively constant for most light elements (particularly for Na, Mg, Al and Si [23]), simplifying the analysis. 4. Development of a single ion irradiation facility There is a need to understand and quantify the damage sustained by an individual cell when exposed to a single ionising particle of a few megaelectron-volts. At present, the study of the response of individual cells to very weak irradiation doses can only be performed in three research centres outside South Africa, two of them located in the USA [24,25] and one in the UK [26]. Since 1998, the Van de Graa Group at NAC and the Centre dÕEtudes Nucleaires de Bordeaux Gradignan (CENBG), France, are developing an irradiation facility as part of the international research agreement between CNRS and NAC. First implemented in Bordeaux, the system will be at the later stage available for both laboratories. The initial stage of the project requires the design and testing of the dierent components of the new
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beam line, which must satisfy the following requirements: · to deliver single ions in the mega-electron-volt energy range; · to extract the beam in air through an ultra-thin exit window; · to control the positioning of the ion beam to achieve a spatial resolution of the order of microns. This will allow studies of the biological damage induced by the irradiation of speci®c parts within the cell structure (nucleus, cytoplasm, etc.). The ®rst stage of the experimental work has been devoted to the characterisation of a suitable exit window. A thin foil made of Si3 N4 was selected as a possible candidate and tested at NAC, using the NMP in STIM and RBS modes, with a 2 MeV aparticle beam. The data were stored in event-byevent mode in order to assess the thickness and the homogeneity of the window (see Fig. 1). A fast single-ion detector, based on a thin scintillator ®lm optically coupled with a pair of photomultipliers, has been constructed in Bordeaux and tested for eciency and background noise using the NMP at NAC. Consecutive tests were performed at CENBG, at very low beam ¯uence. Results of these experiments showed that the response signal remained constant throughout the duration of the experiment. Further experiments will involve: (a) the characterisation of other possible candidates for exit windows such as CVD diamond ®lms and thin plastic scintillators; (b) the optimisation of the fast ion detector to improve the eciency for single ion detection. 5. Elemental distribution in young leaves of the Ni hyperaccumulator ± Berkheya coddii Very few plant species growing on soil enriched with heavy metals have the ability to take up exceptionally high amounts of these metals. A plant that contains more than 0.1% of Ni per dry weight of material in any above ground part is de®ned as a Ni hyperaccumulator. Such amounts are highly toxic for normal plants and so far around 300
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Fig. 1. (A) STIM map of a Si3 N4 window: The energy of the a particles transmitted through the window was recorded at each pixel of the map 64 64 pixels on a 180 180 lm2 area. The transmitted energy is directly related to the thickness of the window, which was found to be roughly homogeneous all over the surface. (B) Energy spectra of the incoming 2.0 MeV aparticle beam and of the beam transmitted through the Si3 N4 windows. The thickness of the windows, calculated from the value of the energy loss DE measured on the energy spectra, is around 0.06 lm.
species of Ni hyperaccumulators have been discovered in the world ¯ora [27]. One of ®ve nickel hyperaccumulators from South Africa is Berkheya coddii Roessler (Asteraceae), an endemic herbaceous plant growing on Ni-rich outcrops (Mpumalanga Province, South Africa). The highest enrichment of Ni in the leaves
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Fig. 2. Quantitative elemental maps showing the distribution of Ni, Ca, K and Zn in young leaf cross-sections of Berkheya coddii. Concentrations are in micro-grams per gram (lg=g) dry mass.
is typical for hyperaccumulators and in B. coddii it reaches 3.6% [28] of the dry mass. Dierences of elemental distributions in a young leaf cross-section are clearly visible (see Fig. 2). Ni is present in all tissues, but the highest amount of Ni is found in the outer tissues ± in the epidermis of the leaf midrib (1.25%) and in the leaf tip (2.2%). The distribution pattern shows the storage locations for most of the Ni (in an unknown, probably non-toxic form). The maximum quantity in the epidermis is similar to other Ni hyperaccumulators from South Africa [29±32]. Macro-nutrients are allocated speci®cally to tissues: Ca is concentrated mainly in the midrib cortex whereas K is allocated in the vascular bundles and in the leaf parenchyma. Zn ± an essential micro-nutrient ± is present in the epidermis, vascular bundle and leaf blade. Mn and Fe reach maximum values in the epidermis. The unique response of these plants to elevated soil concentrations of Ni may be practically used in the future as an invaluable tool in the bio-
remediation of metal polluted soils and toxic wastes. Berkheya coddii has already been tested on dierent soil types with very promising results [33,34]. 6. Tolerance of Brachiaria to Al at toxic levels The genus Brachiaria is the source of the most important and widely sown tropical forage grasses. Among the Brachiaria cultivars, B. decumbens Stapf cv. Basilisk is planted on over 30 million ha and is very well adapted to nutrient-poor and Al toxic acid soils of Latin America. However, the mechanisms that contribute to tolerance to Al in this cultivar are not yet de®ned. An understanding of these mechanisms may assist the genetic improvement of other Brachiaria species. Seeds of B. decumbens were germinated on moist paper and seedlings were cultivated for three weeks in a low ionic strength nutrient solution designed to simulate nutrient-de®cient conditions in soil solutions of typical acid soils from the
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Colombian savannas [14]. A growth analysis in the presence and absence of Al indicated that this species is highly Al-tolerant [35]. Root apices (ca. 5 mm) of adventitious roots were cryo-®xed and lyophilized. Since toxic Al3 -ions act primarily on the root apical meristem, micro-analysis (micro-PIXE) was used to study the in¯uence of Al (added to the nutrient solution) on the concentration and distribution of nutrients in root apices. Preliminary results obtained by elemental mapping with 1.5 MeV protons indicated that: 1. Al was preferentially located in the root cap and epidermis; 2. growth in the presence of Al induced an accumulation of P in the meristematic region (0.2±1.2 mm) of the root apex, which was most pronounced in the central vascular tissue; 3. the level of Cl, and to a less extent S, within the meristematic region decreased signi®cantly (see Fig. 3). Recently, the exudation of phosphate by root apices has been suggested to act as an Al exclusion mechanism in wheat [36]. The Al stimulated P accumulation in root apices of B. decumbens could thus re¯ect physiological events leading to exudation of phosphate. Experiments are underway to determine the extent of phosphate exudation as a function of the distance from the root tip.
7. Detection of early caries lesion in human teeth Prevention of demineralization and enhancement of remineralization of caries is a primary goal of preventive dentistry. Current caries detection criteria are unable to measure the disease process as a continuum, which progresses from an increase in the micro-porosity of the subsurface enamel (white spot) to the occurrence of a frank cavitation [37,38]. The criteria are limited in that they detect caries at an advanced stage, they lack the ability to quantify mineral loss or gain and they are unable to observe small changes in mineral loss or gain occurring as a result of demineralization or remineralization. The purpose of this study is to investigate the detection and quanti®cation of mineral loss in
Fig. 3. Quantitative elemental contour maps for (a) Al, (b) P and (c) Cl of a root apex of Bracharia decumbens, obtained with 1.5 MeV protons. Beam current was 100 pA; and the total collected charge was 200 nC. Concentrations are given in lg=g dry mass.
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early arti®cial carious lesions, with particular reference to variability in the spatial elemental distribution and their correlation with the demineralization and remineralization processes, especially for Ca and P. This is achieved by using nuclear microscopy to determine the levels of Ca
and P in human enamel and dentine. Mechanisms (processes) occurring in the surface and subsurface of the enamel are studied as well as areas in and around the interface between dentine and enamel. The eect of ¯uorine protection will also later be tested as well as the eect of demineralization, which is thought to be happening mainly on the subsurface and is represented by voids in the spatial distribution of elements. Elemental maps performed in a test series of experimental samples, determined by DA, showed that some voids are present in the surface and subsurface areas. A number of voids in the distribution of Ca and P were observed on the surface and in the body of the tooth, which appear to be related to the extent of the mineral loss (demineralization). This loss is illustrated in Fig. 4(a) for P, and Fig. 4(b) for Ca, in the subsurface area. The ability of the NMP to detect and possibly quantify mineral loss after exposure to demineralizing solutions suggests that the technique has the potential to detect mineral losses in human teeth at a very early stage and may have the possibility to initiate improved preventive therapies. 8. Discussion The use of the NMP in studies of biological material has proliferated during the last number of years, and is one of the niche areas of application where the techniques provided are often superior than other available techniques. For this reason this ®eld of application for the NMP will grow further in the foreseeable future. Acknowledgements The assistance of the technical sta of the Van de Graa Group at NAC is essential to the research programme, and therefore well appreciated.
Fig. 4. Quantitative elemental maps of dental samples obtained with micro-PIXE; proton energy 0.6 MeV; beam current was 200 pA; total collected charge 800 nC. A number of voids in the distribution of Ca and P appear to be related to the extent of the mineral loss (demineralization). This particular loss is evident for both elements in and around the subsurface area. Concentrations are given in lg=g.
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