Pivovarova Natalia B, Andrews S Brian
Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health.
J Vis Exp. 2013 Nov 20(81):e50807. doi: 10.3791/50807.
In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires "cryofixation" of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 °C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at ≤-160 °C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 °C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their "characteristic" peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.
本文介绍了适用于使用电子探针微分析（EPMA）技术对细胞内元素含量进行定量测量的工具、技术和仪器。线粒体钙是一个特别关注的焦点，因为线粒体钙超载在神经退行性疾病中起着关键作用。该方法基于对电子显微镜（EM）中电子束与样品相互作用产生的X射线的分析。为了在电子显微镜标本中保持可扩散元素的天然分布，EPMA需要对组织进行“冷冻固定”，然后制备超薄冷冻切片。培养细胞或器官型切片培养物的快速冷冻分别通过在液态乙烷中骤冷或靠在冷金属块上猛击冷冻来进行。用金刚石刀在约-160°C下干式切割名义厚度为80nm的冷冻切片，安装在碳/聚甲醛涂层的铜网上，并使用专门的冷冻样品架将其冷冻转移到冷冻电子显微镜中。在≤-160°C和低电子剂量下进行目视检查和定位映射后，冷冻水合冷冻切片在-100°C下冷冻干燥约30分钟。通过慢扫描电荷耦合器件（CCD）相机记录干燥冷冻切片的细胞器水平图像，同样在低剂量下，并选择感兴趣的亚细胞区域进行分析。由固定、聚焦、高强度电子探针从感兴趣区域发射的X射线由能量色散X射线（EDX）光谱仪收集，由相关电子设备处理，并呈现为X射线光谱，即X射线强度与能量的关系图。额外的软件有助于：1）通过其“特征”峰值能量和指纹识别元素成分；2）通过提取峰面积/背景进行定量分析。本文最后给出了两个例子，说明了EPMA的典型应用，一个例子中线粒体钙分析为兴奋性毒性损伤机制提供了关键见解，另一个例子揭示了缺血耐受性的基础。
