Practical Protein Chemistry - A. Darbre 1989

X-ray Crystallography and Electron Microscopy
Electron Microscopy
Technical Challenges

Since molecular images are formed through their interaction with electrons, it is expected that the presence of any foreign atoms will lead to a reduction in image quality. Therefore, a high vacuum is maintained inside the Microscope Column. Under these conditions, the sample placed in the microscope inevitably undergoes dehydration. The Methods allowing to prevent structural Changes in the studied samples during dehydration are discussed below.

Another undesirable effect arises from the interaction of electrons with the sample itself. It is caused not only by the destructive action of high-energy electrons, but also by the generation of highly reactive chemical radicals, leading to sample contamination during Cell/15.html">Microscopy. The impact of chemical radicals can be minimized by placing a copper aperture cooled to liquid nitrogen Temperature near the sample, which sorbs contaminating particles onto its surface. The destructive effect of the electrons themselves can only be reduced by lowering the radiation dose. This is discussed in more detail in the section dedicated to Molecular Microscopy.

The impact of dehydration, contamination, and radiation damage effects can be mitigated by 'staining' the samples with heavy metal salts. Such staining is performed prior to sample drying, and the bound 'stain' is significantly less sensitive to dehydration and electron bombardment than the light atoms forming the object. In addition, heavy metal salts possess a substantially higher electron density, which diminishes METABOLISM/18.html">The Influence of contamination on image quality. However, even in this case, it is necessary to achieve maximum cleanliness of the internal Structure/83.html">Structural elements of the microscope and the copper grids supporting the sample, as well as to employ various technical means to reduce the radiation dose delivered to the sample.