Lechene C
Fed Proc. 1980 Sep;39(11):2871-80.
Electron probe microanalysis is a method based upon X-ray spectrometry used for analyzing the chemical element content of very small amounts of material. The concentration of electrolytes in the microenvironment of cells, in cells, and in intracellular organelles can be measured. The main difficulties in using this method in biological soft tissue lie in sample preparation and in proper interpretation of the data. Best tissue preparation seems to be to quench the sample and to analyze it either freeze dried in thin or ultrathin sections, or frozen hydrated in thin sections or bulk samples. In all cases analysis should be performed using a cold stage and an ultra clean vacuum in order to minimize mass loss due to beam damage and mass gain due to contamination trapping. Interpretation of the data relies upon the knowledge of both the localization of the volume excited by the electron beam and the origin of the continuum and characteristic X-ray signals received by the X-ray spectrometer. This knowledge can be complicated by two facts: 1) when the electron beam is used in an analytical mode, viewing of the analyzed microarea can be lost, and 2) the X-ray signals received by the spectrometer can originate not only from the volume directly excited by the electron beam but from areas that can be far apart, excited by the electron beam tail, scattered electrons or secondary fluorescence, particularly when using energy dispersive spectrometers. Theoretical quantitation of the results is well developed. Practical quantitation could be complicated by the possibility of mass gain, mass loss, standard inhomogeneity, non-uniformity of sample thickness, possibility of shrinkage during freeze drying and, when using energy dispersive spectrometry, by the low signal over background for low atomic number elements (Na), the possibility of overlap of characteristic X-ray lines, and the use of complex and empirical methods for background stripping and peak deconvolution. All these difficulties can be overcome, making electron probe microanalysis one of the most powerful tools available to the biologist.
电子探针显微分析是一种基于X射线光谱法的方法，用于分析极少量材料的化学元素含量。可以测量细胞微环境、细胞内以及细胞内细胞器中的电解质浓度。在生物软组织中使用该方法的主要困难在于样品制备和数据的正确解读。最佳的组织制备方法似乎是淬灭样品，然后对其进行分析，分析时可采用冻干的薄切片或超薄切片，或者冷冻水合的薄切片或块状样品。在所有情况下，分析都应使用冷台和超净真空进行，以尽量减少由于电子束损伤导致的质量损失以及由于污染物捕获导致的质量增加。数据解读依赖于对电子束激发体积的定位以及X射线光谱仪接收到的连续谱和特征X射线信号来源的了解。有两个事实会使这种了解变得复杂：1）当电子束用于分析模式时，可能会看不到被分析的微区；2）光谱仪接收到的X射线信号不仅可能来自电子束直接激发的体积，还可能来自相距较远的区域，这些区域是由电子束尾部、散射电子或二次荧光激发的，特别是在使用能量色散光谱仪时。结果的理论定量方法已经很成熟。实际定量可能会因质量增加、质量损失、标准不均匀性、样品厚度不均匀、冻干过程中收缩的可能性而变得复杂，并且在使用能量色散光谱法时，还会因低原子序数元素（钠）的信号背景比低、特征X射线线重叠的可能性以及用于背景扣除和峰去卷积的复杂和经验方法而变得复杂。所有这些困难都可以克服，这使得电子探针显微分析成为生物学家可用的最强大工具之一。
