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).

中文翻译：

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迈向单细胞离子组学：一种用于纳克级生物样品多元素分析的新型微尺度方法。

背景为了解在单细胞水平上调节营养稳态的过程，需要新的方法来对生物样品进行多元素分析，最终只能作为分离的组织或细胞获得，通常是纳克大小的样品。除了组织分离外，此类分析的主要挑战是获得每个样品的完全和均匀的消化，将样品稀释度保持在最低限度，并产生准确和可重复的结果。特别是当样本量减少时，确定小样本的重量变得越来越具有挑战性。结果 我们开发了一种用于纳克级植物组织的采样、消化和多元素分析的新方法，以及量化太小而无法称重的样品中元素浓度的策略。该方法基于通过激光捕获显微切割 (LCM) 进行组织分离，然后进行加压微消化和 ICP-MS 分析，后者使用稳定的 µL min-1 样品抽吸系统。该方法允许从大麦根中分离、消化和分析微解剖组织，估计样品重量仅为 ~ 400 ng。在收集和分析步骤中，确定了许多污染源。在消除这些来源后，可以量化包括镁 (Mg)、磷 (P)、钾 (K) 和锰 (Mn) 在内的几种元素。通过测量每个显微解剖组织的确切面积和厚度，计算它们的体积。结合估计的样本密度，随后可以计算样品重量，从而可以避免这些样品太小而无法称重的事实。通过分析拟南芥种子 (~ 20 µg) 以及此类种子的组织部分 (~ 10 µg) 进一步记录了该方法。结论 所提出的方法能够收集和多元素分析小尺寸生物样品，范围低至纳克级。因此，该方法为单细胞和组织特异性定量离子组学铺平了道路，允许未来的转录、蛋白质组学和代谢组学数据与离子组学特征相关联。这样的分析将加深我们对植物元素组成如何被调节的理解，例如通过转运蛋白和物理屏障（即根内皮层中的里海带和木栓质薄片）。