Becker R P, Sogard M
Scan Electron Microsc. 1979(2):835-70.
A fraction of the beam electrons which interact with a specimen scatter back. The number of backscattered electrons (BE's) increases with the atomic number of the elements encountered. Cell and tissue structures lacking a heavy metal content yield few BE's compared to structures affixed with heavy metals, either vitally or by means of staining methods applied after fixation. The BE imaging mode of a scanning electron microscope (SEM) provides an intensity map of the BE yield from the specimen. BE imaging of selectively stained structures in cells and tissues renders these structures visible in contrast to the unstained surround. Since BE's can emerge from a significant depth within the material, BE imaging can be used to view such heavy metal stained structures beneath intact cell surfaces. The microcontours of the overlying surface can be viewed concurrently by using the surface scanning (i.e., the secondary electron imaging; SEI) mode of the microscope. Methods for selectively contrasting subsurface structures can be adapted from existing light microscope (LM) and transmission electron microscope (TEM) methods. Staining methods have been devised for subsurface viewing of cell organelles, including nuclei, mitochondria, peroxisomes, lysosomes, and phagosomes. A physical model is presented which describes these observations and suggests future possible trends in this subject. Specifically the image contrast and resolution are described in terms of the physical properties of the stain and specimen and of the SEM operating conditions of energy and current. Finally a summary of instrumentation considerations describes present and potential BE detectors, their ancillary electronics, and image processing.
与样本相互作用的一部分电子束会反向散射。背散射电子（BE）的数量会随着所遇到元素的原子序数增加而增多。与通过活体方式或固定后染色方法附着重金属的结构相比，缺乏重金属含量的细胞和组织结构产生的背散射电子较少。扫描电子显微镜（SEM）的背散射电子成像模式可提供样本背散射电子产额的强度图。对细胞和组织中选择性染色结构进行背散射电子成像，能使这些结构在未染色的背景衬托下清晰可见。由于背散射电子可从材料内部较深的深度射出，因此背散射电子成像可用于观察完整细胞表面下方的此类重金属染色结构。通过使用显微镜的表面扫描（即二次电子成像；SEI）模式，可同时观察覆盖表面的微观轮廓。用于选择性突出亚表面结构差异的方法可借鉴现有的光学显微镜（LM）和透射电子显微镜（TEM）方法。已设计出用于亚表面观察细胞器（包括细胞核、线粒体、过氧化物酶体、溶酶体和吞噬体）的染色方法。本文提出了一个物理模型，该模型描述了这些观察结果，并指出了该领域未来可能的发展趋势。具体而言，图像对比度和分辨率是根据染色剂和样本的物理特性以及扫描电子显微镜的能量和电流操作条件来描述的。最后，对仪器方面的考虑进行了总结，介绍了当前和潜在的背散射电子探测器、它们的辅助电子设备以及图像处理。
