Evaluation of specimen preparation techniques for micro-PIXE localisation of elements in hyperaccumulating plants

Hybanthus floribundus subsp. floribundus, a rare Australian Ni-hyperaccumulating shrub and Pityrogramma calomelanos var. austro-americana, an Australian naturalized As-hyperaccumulating fern are promising species for use in phytoremediation of contaminated sites. Micro-proton-induced X-ray emission (mu-PIXE) spectroscopy was used to map the elemental distribution of the accumulated metal(loid)s, Ca and K in leaf or pinnule tissues of the two plant species. Samples were prepared by two contrasting specimen preparation techniques: freeze-substitution in tetrahydrofuran (THF) and freeze-drying. The specimens were analysed to compare the suitability of each technique in preserving (i) the spatial elemental distribution and (ii) the tissue structure of the specimens. Further, the mu-PIXE results were compared with concentration of elements in the bulk tissue obtained by ICP-AES analysis. In H. floribundus subsp. floribundus, mu-PIXE analysis revealed Ni, Ca and K concentrations in freeze-dried leaf tissues were at par with bulk tissue concentrations. Elemental distribution maps illustrated that Ni was preferentially localised in the adaxial epidermal tissues (1% DW) and least concentration was found in spongy mesophyll tissues (0.53% DW). Conversely, elemental distribution maps of THF freeze-substituted tissues indicated significantly lower Ni, Ca and K concentrations than freeze-dried specimens and bulk tissue concentrations. Moreover, Ni concentrations were uniform across the whole specimen and no localisation was observed. In P. calomelanos var. austroamericana freeze-dried pinnule tissues, mu-PIXE revealed statistically similar As, Ca and K concentrations as compared to bulk tissue concentrations. Elemental distribution maps showed that As localisation was relatively uniform across the whole specimen. Once again, THF freeze-substituted tissues revealed a significant loss of As compared to freeze-dried specimens and the concentrations obtained by bulk tissue analysis. The results demonstrate that freeze-drying is a suitable sample preparation technique to study elemental distribution of ions in H. floribundus and P. calomelanos plant tissues using mu-PIXE spectroscopy. Furthermore, cellular structure was preserved in samples prepared using this technique. (C) 2007 Elsevier B.V. All rights reserved.