Department of Biomedical Sciences of Cells and Systems, Groningen, The Netherlands.
Department of Biomedical Sciences of Cells and Systems, Groningen, The Netherlands.
Methods Cell Biol. 2021;162:89-114. doi: 10.1016/bs.mcb.2020.10.023. Epub 2020 Dec 15.
Traditional electron microscopy (EM) can be complemented with analytical EM to increase objective sample information enabling feature identification. Energy dispersive X-ray (EDX) imaging provides semi-quantitative elemental composition of the sample with high spatial resolution (~10nm) in ultrathin sections. However, EDX imaging of biological samples is still challenging as a routine method because many elements are at the detection limit for this technique. Moreover, samples undergo extensive preparation before analysis, which can introduce disruptive X-ray cross-talk or artifacts. EDX data can, for instance, be skewed by (i) osmium interference with endogenous phosphorus, (ii) chlorine present in EPON-embedded tissues, (iii) lead interference with endogenous sulfur, and (iv) potential spectral overlaps with grid material, contrast agents, and the in-microscope sample holder. Here, we highlight how to circumvent these potential pitfalls and outline how we approach sample preparation and analysis for detection of different elements of interest. Utilization of well-considered a priori sample preparation techniques will best ensure conclusive EDX experiments.
传统电子显微镜（EM）可以与分析电子显微镜结合使用，以增加客观样本信息，从而实现特征识别。能量色散 X 射线（EDX）成像可在超薄片中提供具有高空间分辨率（约 10nm）的样品半定量元素组成。然而，由于许多元素处于该技术的检测极限，因此 EDX 成像仍然是生物样品分析的一项挑战。此外，在分析之前，样品需要经过广泛的准备，这可能会引入破坏性的 X 射线串扰或伪影。例如，EDX 数据可能会受到以下因素的影响：（i）锇对内源性磷的干扰，（ii）EPON 包埋组织中的氯，（iii）铅对内源性硫的干扰，以及（iv）与网格材料、对比剂和显微镜内样品支架的潜在光谱重叠。在这里，我们将重点介绍如何规避这些潜在的陷阱，并概述我们如何进行样品准备和分析以检测不同感兴趣的元素。利用经过深思熟虑的先验样品制备技术将最好地确保 EDX 实验的结论性。
