X-RAY SPECTROMETRY X-Ray Spectrom. 2008; 37: 133–136 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/xrs.1035 Localisation of trace metals in metal-accumulating plants using m-PIXE † R. Siegele, 1* A. G. Kachenko, 2 N. P. Bhatia, 1 Y. D. Wang, 3 M. Ionescu, 1 B. Singh, 2 A. J. M. Baker 3 and D. D. Cohen 1 1 Australian Nuclear Science and Technology Organisation (ANSTO), PMB1, Menai, NSW 2234, Australia 2 Faculty of Agriculture, Food and Natural Resources, The University of Sydney, NSW 2006, Australia 3 School of Botany, The University of Melbourne, VIC 3010, Australia Received 29 May 2007; Revised 10 October 2007; Accepted 15 November 2007 Particle induced x-ray emission (PIXE) is a very sensitive technique that can quickly and reliably measure a wide range of elements simultaneously with high sensitivity. Using a focused microbeam, elemental distributions can be mapped with high spatial resolution. We demonstrate high-resolution mapping of metals in plant leaves at 5 μm resolution and its application in detecting sites of metal accumulation in metal-accumulating plant tissues. The importance of biological sample preparation is discussed by direct comparison of freeze-substitution and freeze-drying techniques routinely used in biological sciences. The advantages and limitations of quantitative elemental imaging using these techniques are also discussed. Copyright  2008 John Wiley & Sons, Ltd. INTRODUCTION Particle induced x-ray emission (PIXE) is a powerful analytical technique that is widely used in many research areas. When combined with a microbeam the distribution of elements across a specimen can be mapped, which leads to a wide range of new applications. -PIXE has been extensively used in environmental and biological research to map trace elements in plant and animal tissues. -PIXE has been used to study the localisation of trace metals in metal indicator and hyperaccumulating plant species. A plant is said to be a metal hyperaccumulator, if it concentrates trace metals above a minimum threshold concentration in above-ground tissues. This threshold varies according to the metal involved, for example more than 1000 mg/kg dry weight (DW) for cobalt, copper, lead or nickel or more than 10 000 mg/kg DW for zinc or manganese. 1,2 In contrast, metal uptake in indicator plants to above-ground tissues is relatively unregulated, thus internal concentrations are a passive reflection of external levels. 3 To understand the mechanisms that allow these metal- accumulating plants to tolerate high concentrations of normally toxic metals, the spatial localisation of the metal accumulation has to be known. The objective of this study was to demonstrate the use of -PIXE in mapping the cellular and sub-cellular localisation of trace metals in metal-accumulating plant tissues using freeze-drying and freeze-substitution sample preparation techniques. L Correspondence to: R. Siegele, Australian Nuclear Science and Technology Organisation (ANSTO), PMB1, Menai, NSW 2234, Australia. E-mail: rns@ansto.gov.au † Presented at 11th International Conference on Particle Induced X-ray Emission and its Analytical Applications, PIXE 2007, Puebla, Mexico, 25–29 May 2007. EXPERIMENTAL Plant samples were analysed using the ANSTO High Energy Heavy Ion Microprobe (HIMP). 4 Ion beams with an ME/q 2 of up to 100 can be focused at the HIMP with spot sizes down to 3 μm, providing sufficient current to use it for various ion beam analytical techniques. The samples were analysed using a 3-MeV proton beam with a typical spot size between 3 and 5 μm. At this spot size beam currents between 0.1 and 0.5 nA can be achieved, which is sufficient for PIXE analyses. A high-purity Ge detector was used with a 100 mm 2 active area, located 33 mm from the sample. A 100 μm Mylar foil was used to reduce low energy x-rays and thus pile-up in the -PIXE spectrum. This setup allowed the detection of the accumulated trace metals such as Ni and Cu with high sensitivity. SAMPLE PREPARATION AND RESULTS In the microanalysis of biological tissues, sample preparation is one of the most important steps, which is also the case for -PIXE. Typically, plant samples are dried and thin sectioned for -PIXE analysis. In the case of plant material exposed to metals, it has to be ensured that this process does not result in the redistribution of the metal and that the cell ultrastructure is preserved. On account of the high spatial resolution of -PIXE even small movements have to be avoided. In previous experiments, we have employed a simple technique that involved the hand-sectioning of the samples followed immediately by snap freezing of the sections in liquid nitrogen. The sections were subsequently freeze- dried. 5 However, with this technique the best sections of 50 μm can be achieved. This limits the lateral resolution in -PIXE, because of the possible overlap of the distribution of Copyright  2008 John Wiley & Sons, Ltd.