Walther P, Müller M
Laboratories for Electron Microscopy I, Swiss Federal Institute of Technology, Zurich, Switzerland.
Scanning. 1997 Aug;19(5):343-8. doi: 10.1002/sca.4950190501.
Imaging of fast-frozen samples is the most direct approach for electron microscopy of organic material. It prevents chemical fixation and drying artifacts. Frozen samples can be replicated and imaged in the transmission electron microscope (TEM), or they can be directly visualized in the cryo-scanning electron microscope (cryo-SEM). Double-layer coating combines these two techniques and many of their advantages. With this method, the frozen bulk sample is coated similar to the TEM-replica technique with, for example, a shadow of platinum (at an angle of 45 degrees) and an additional layer of carbon. Then, the sample is cryo-transferred to an SEM equipped with a cold stage and imaged with the material-dependent backscattered electron signal that shows the platinum distribution. With this method, charging artifacts and the effects of beam damage are significantly reduced. Although currently the resolution of the replica technique cannot be surpassed, the method greatly facilitates the processing of brittle, rapidly frozen samples because no replica cleaning is necessary. This makes the method especially suitable for high-pressure frozen samples.
快速冷冻样品的成像技术是对有机材料进行电子显微镜观察的最直接方法。它可以避免化学固定和干燥过程中产生的假象。冷冻样品可以在透射电子显微镜（TEM）中进行复制和成像，或者可以在低温扫描电子显微镜（cryo-SEM）中直接观察。双层镀膜结合了这两种技术及其许多优点。使用这种方法，冷冻的块状样品的镀膜方式类似于TEM复制技术，例如，先镀一层铂（以45度角）形成阴影，再镀一层碳。然后，将样品低温转移到配备有冷台的扫描电子显微镜中，利用与材料相关的背散射电子信号成像，该信号显示铂的分布情况。通过这种方法，充电假象和束流损伤的影响显著降低。虽然目前复制技术的分辨率无法被超越，但该方法极大地简化了脆性快速冷冻样品的处理过程，因为无需进行复制清洗。这使得该方法特别适用于高压冷冻样品。
