ABSTRACT: Background:To understand processes regulating nutrient homeostasis at the single-cell level there is a need for new methods that allow multi-element profiling of biological samples ultimately only available as isolated tissues or cells, typically in nanogram-sized samples. Apart from tissue isolation, the main challenges for such analyses are to obtain a complete and homogeneous digestion of each sample, to keep sample dilution at a minimum and to produce accurate and reproducible results. In particular, determining the weight of small samples becomes increasingly challenging when the sample amount decreases. Results:We developed a novel method for sampling, digestion and multi-element analysis of nanogram-sized plant tissue, along with strategies to quantify element concentrations in samples too small to be weighed. The method is based on tissue isolation by laser capture microdissection (LCM), followed by pressurized micro-digestion and ICP-MS analysis, the latter utilizing a stable µL min-1 sample aspiration system. The method allowed for isolation, digestion and analysis of micro-dissected tissues from barley roots with an estimated sample weight of only?~?400 ng. In the collection and analysis steps, a number of contamination sources were identified. Following elimination of these sources, several elements, including magnesium (Mg), phosphorus (P), potassium (K) and manganese (Mn), could be quantified. By measuring the exact area and thickness of each of the micro-dissected tissues, their volume was calculated. Combined with an estimated sample density, the sample weights could subsequently be calculated and the fact that these samples were too small to be weighed could thereby be circumvented. The method was further documented by analysis of Arabidopsis seeds (~?20 µg) as well as tissue fractions of such seeds (~?10 µg). Conclusions:The presented method enables collection and multi-element analysis of small-sized biological samples, ranging down to the nanogram level. As such, the method paves the road for single cell and tissue-specific quantitative ionomics, which allow for future transcriptional, proteomic and metabolomic data to be correlated with ionomic profiles. Such analyses will deepen our understanding of how the elemental composition of plants is regulated, e.g. by transporter proteins and physical barriers (i.e. the Casparian strip and suberin lamellae in the root endodermis).
