Practical Protein Chemistry - A. Darbre 1989

X-ray Crystallography and Electron Microscopy
Electron Microscopy
Molecular Microscopy

The hardware used in Three-Dimensional Electron Cell/15.html">Microscopy of stained specimens can also be applied to unstained objects for high-resolution studies. Theoretically, this implies the possibility of directly visualizing the atoms that make up a molecule. Such investigations of unstained specimens have indeed been carried out [7, 15]; however, they required The Development of specialized Microscopy Techniques and significantly more complex computer-based Image Processing Methods. This is due to several reasons. First, the specimen must be protected from dehydration in the Microscope vacuum by embedding it in a thin sucrose film. Second, an unstained specimen lacks the protection usually provided by a heavy-metal contrasting agent; consequently, its exposure to electron irradiation must be kept extremely short to minimize radiation damage. In practice, this exposure time is so brief that the resulting images exhibit exceptionally low contrast. Therefore, structural information can only be extracted from ordered two-dimensional crystals.

The Need for specialized image processing techniques stems from the fact that the object consists of light atoms that have not been stained with heavy-metal salts. In this case, image formation is driven primarily not by electron absorption from the specimen atoms, but by electron scattering that alters the electron phases. As a result, a precisely focused image of an unstained specimen yields no structural information. To visualize light atoms, it is necessary to capture a series of micrographs at different focal settings so that computer analysis can be used to select phase-contrast images depicting the relative arrangement of the light atoms.

Reconstructing a three-dimensional Structure requires combining information from numerous micrographs. It is essential that the signal-to-noise ratio in each micrograph be sufficiently high, which is only achievable with highly ordered Periodic structures. This is precisely why high-resolution investigations can only be performed on such objects.