Localization of chemical elements and isotopes in the leaf of soybean (Glycine max) by secondary ion mass spectrometry microscopy: Critical choice of sample preparation procedure

Secondary ion mass spectrometry (SIMS) is one of the few microscopical methods that potentially can detect and in situ localize the various isotopes of virtually all elements. Recent work with SIMS has demonstrated the possibility of imaging the distribution of various elements in plant cell and tissues. However, in these studies, the elements were incorporated in cell macromolecules or associated with structural polymers, precipitated or immobilized in dry seeds. The localization of mineral ions is of particular significance for the physiology of higher plants owing to their quantitative importance and the impact of their cellular distribution on metabolic regulation. Here we analyse the possibility of mapping different elements (K, Ca, Mg, P, S, N-15 and N-14) present as soluble and/or bound forms in highly vacuolated leaf cells. Cryoprocedures to prepare samples for SIMS detection are described and discussed. The quality of the results is assessed at each step of the sample preparation and analysis. Various methodologies are used, including photonic and electronic microscopies, and the agreement of the observed ion distribution with current knowledge of ion compartmentalization in plant cells. The K/Ca emission ratio is proposed as an index of the degree of preservation of the natural ion distribution to critically evaluate the results and identify where artefacts are likely to occur.