W.J. Przybylowicz

The NAC nuclear microprobe facility

The NAC NMP facility is discussed, showing that it is a well characterised analytical tool, and some novel features are incorporated, such as on-demand beam deflection and a new lid for the sample chamber.

Micro-PIXE in plant sciences: Present status and perspectives

Fundamental processes of plant physiology are affected or regulated by mineral nutrients. Hence understanding the mechanisms of nutrient uptake and their functions in plant metabolism is of fundamental importance in both basic and applied plant studies. The present knowledge of ion uptake mechanisms is based mostly on techniques for bulk analysis, including analysis of small (mg-sized) samples but without spatially resolved results. On the other hand, advanced studies of elemental transport at a cellular level are conducted using techniques with high and very high spatial resolution, but with low sensitivity for elemental analysis. Thus the results obtained are usually restricted to macronutrients or elements present in high quantities. There is a high demand for studies of the functions of trace elements. In addition, it is known that, depending on their concentrations, elements can play different roles in plant life. Studies related to elemental deficiency and toxicity, as well as environmental pollution, require accurate, fully quantitative methods with good spatial resolution. Ideally, these studies should be conducted on organs and tissues as far down as the cellular level. This is where micro-PIXE has been applied until present and can in the near future play a much more important role. Progress is subject to closer collaboration between plant biologists and the PIXE community in terms of addressing problems of specimen preparation, refinement of analytical protocols such as quantitative elemental mapping and the interpretation of results.

Biological applications of the NAC nuclear microprobe

The nuclear microprobe (NMP) is an established analytical instrument for the determination of minor and trace elements. It allows measurements with a spatial resolution of the order of 1 μm and minimum detection limits down to few ppm by weight, with excellent scanning capabilities. The nuclear microprobe of the National Accelerator Centre, South Africa, is being used in a wide number of applications in the biosciences. The complementarity of proton-induced x-ray emission and backscattering spectrometry in a wide range of biological applications is shown. The advantages and restrictions of true elemental imaging are also discussed.

Heavy metal distribution in Suillus luteus mycorrhizas – as revealed by micro-PIXE analysis

Abstract Suillus luteus/Pinus sylvestris mycorrhizas, collected from zinc wastes in Southern Poland, were selected as potential biofilters on the basis of earlier studies carried out with energy dispersive spectrometry (EDS) microanalytical system coupled to scanning electron microscope (SEM) and transmission electron microscope (TEM). Using the National Accelerator Centre (NAC) nuclear microprobe, elemental concentrations in the ectomycorrhiza parts were for the first time estimated quantitatively. Micro-proton-induced X-ray emission (PIXE) true elemental maps from freeze-dried and chemically fixed mycorrhizas revealed strong accumulation of Ca, Fe, Zn and Pb within the fungal mantle and in the rhizomorph. Vascular tissue was enriched with P, S and K, while high concentrations of Si and Cl were present in the endodermis. Cu was the only element showing elevated concentrations in the cortex region. Elemental losses and redistributions were found in mycorrhizas prepared by chemical fixation. Some problems related to elemental imaging are discussed.

Investigation of Ni hyperaccumulation by true elemental imaging

Abstract The newly implemented Dynamic Analysis method for on-line elemental imaging was used to study Ni hyperaccumulation in Senecio coronatus (Thunb.) Harv. Asteraceae, one of only nine Ni hyperaccumulating plants found in the African continent. Elemental maps were obtained from samples with thicknesses varying from 0.4 to 5 mg/cm 2 by assuming cellulose (C 6 H 10 O 5 ) as constant matrix composition for the whole scanned area. The agreement between point analyses and results inferred from maps is good for small thickness variations within scanned regions. Maps of very inhomogeneous samples require a more time-consuming approach of thickness corrections in every pixel.

Quantitative micro-PIXE comparison of elemental distribution in Ni-hyperaccumulating and non-accumulating genotypes of Senecio coronatus

The Ni hyperaccumulator, plant species Senecio coronatus (Thunb.) Harv., Asteraceae is an example of plant adaptation mechanisms to different ecological conditions. This widespread species can inter alia be found on serpentine outcrops and the genotypes growing in serpentine soils show different ways of adaptation. The populations from two distant localities take up and translocate Ni in concentrations which are normally phytotoxic, while plants growing on a different site, in the vicinity of another hyperaccumulating species, absorb amounts which are typical for most of the plants found on serpentine soils. The NAC nuclear microprobe was used to compare the distribution of Ni and other elements in selected organs and cells with simultaneous use of PIXE and proton BackScattering (BS). Quantitative maps of stems showed large differences in concentrations and distributions of major and trace elements. In hyperaccumulating genotypes Ni is present everywhere within stem tissues, but the highest concentrations were found in the epidermis, cortex and phloem. In non-accumulating plants Ni was concentrated in the phloem. In the leaf epidermis Ni was concentrated in the cell walls for both accumulating and non-accumulating plants. These results suggest that biochemical diversity is more than morphological, because investigated genotypes belong to the same taxon.

Botanical applications in nuclear microscopy

Abstract An overview of botanical applications in the field of nuclear microscopy is presented. A wide range of applications has been studied up to now and included an array of very thin to thick samples. The relevant methods of sample preparation which are critical to many botanical applications are discussed. Quantitative mapping techniques allowing simultaneous quantitative studies of major and trace elements offer exciting possibilities in various fields of application, from science to industry and agriculture. In these studies PIXE and RBS were typically used to obtain complementary information. Recent examples are presented.

Elemental distribution in lichens transplanted to polluted forest sites near Kraków (Poland)

This study describes elemental distribution in the epiphytic lichen Hypogymnia physodes (L.) Nyl., with emphasis on heavy metals.Micro-PIXE measurements were performed using the nuclear microprobe at the iThemba LABS, South Africa.Detailed information from different thallus layers was obtained by performing true elemental mapping using the dynamic analysis method, complemented by analyses of selected smaller areas and point analyses.Cl and K concentrations were high near the algal layer whilst S concentrated mostly in the algal and lower cortex layers.The highest concentrations of P were found in the lower cortex.Mn and Zn were mostly concentrated in the algal layer and lower cortex while high concentrations of Fe were noted in the lower cortex.The highest concentrations of Ca and Pb were found in the medullary layer.Mapping of lichen samples soaked in Pb(NO 3)2 solution proved that the highest Pb concentration occurred in the upper and lower cortex. 2002 Published by Elsevier Science B.V.

Heavy metal localisation in mycorrhizas ofEpipactis atrorubens (Hoffm.) Besser (Orchidaceae) from zinc mine tailings

SummaryThe metal distribution within mycorrhizal and nonmycorrhizal roots ofEpipactis atrorubens collected from zinc mine tailings and an area rich in heavy metal ores (both located in southern Poland) was investigated. The tailings, consisting of postflotation material, were characterised by high levels of toxic elements such as Zn, Pb, and Cd, while soil outside the tailings was also strongly enriched in heavy metals. Atomic absorption spectrometry and proton-induced X-ray emission analysis revealed that heavy metals were mostly accumulated within orchid roots. Elemental maps from proton-induced X-ray emission showed that plant root epidermis and fungal coils which had developed within cortical cells of roots collected from the zinc mine tailings were the main places of Zn and Pb accumulation, associated with increased concentrations of Fe, Cd, Ti, Mn, Si, Ca, and S. The mean content of Pb and Zn in the coils was 4 to 5 times higher than in the root epidermis. In mycorrhizal roots from the tailings a statistically significant decrease in Pb and Zn content towards the inside of the root was observed. The mean content of Pb in coils from roots of plants growing outside the tailings was about 1% of the concentration in root coils from the tailings. Coils selected from orchid roots originating from a site outside the tailings contained comparatively high concentrations of Zn, Cd, and Cu, which was probably due to the high content of these elements in the soil. The results presented suggest a biofiltering effect against heavy metals by orchid mycorrhizal fungi.

Nickel biopathways in tropical nickel hyperaccumulating trees from Sabah (Malaysia)

The extraordinary level of accumulation of nickel (Ni) in hyperaccumulator plants is a consequence of specific metal sequestering and transport mechanisms, and knowledge of these processes is critical for advancing an understanding of transition element metabolic regulation in these plants. The Ni biopathways were elucidated in three plant species, Phyllanthus balgooyi, Phyllanthus securinegioides (Phyllanthaceae) and Rinorea bengalensis (Violaceae), that occur in Sabah (Malaysia) on the Island of Borneo. This study showed that Ni is mainly concentrated in the phloem in roots and stems (up to 16.9% Ni in phloem sap in Phyllanthus balgooyi) in all three species. However, the species differ in their leaves – in P. balgooyi the highest Ni concentration is in the phloem, but in P. securinegioides and R. bengalensis in the epidermis and in the spongy mesophyll (R. bengalensis). The chemical speciation of Ni2+ does not substantially differ between the species nor between the plant tissues and transport fluids, and is unambiguously associated with citrate. This study combines ion microbeam (PIXE and RBS) and metabolomics techniques (GC-MS, LC-MS) with synchrotron methods (XAS) to overcome the drawbacks of the individual techniques to quantitatively determine Ni distribution and Ni2+ chemical speciation in hyperaccumulator plants.