Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Investigation of Protein Structure
Electron Microscopy
Sample Preparation and Staining – Molecular Electron Microscopy

The technique of Three-Dimensional Electron Cell/15.html">Microscopy for "stained" specimens can also be applied to unstained objects to achieve higher resolution. Theoretically, this approach offers the possibility of directly visualizing the atoms that make up a molecule. Attempts to carry out such experiments have been made; however, the results are not yet satisfactory, and the challenges remain formidable. These difficulties are primarily due to the need to protect the specimen from dehydration, which occurs very rapidly under the high-vacuum conditions of the Electron microscope's sample chamber. This problem is solved by embedding the specimen in a thin film of sucrose. Secondly, an "unstained" specimen is vulnerable to direct radiation damage, which can only be avoided by significantly reducing the exposure time of the object to the electron beam. In practice, the specimen can only withstand a very brief pulse, meaning that the resulting image has an extremely low signal-to-noise ratio and poor contrast. Consequently, structural information can only be obtained from highly ordered two-dimensional crystals.

In the case of specimens not stained with heavy metals, the object (protein) consists of light atoms. As a result, its image is formed not through electron absorption, but via electron scattering and phase shift. The latter alters only the phase of the electron wave. Therefore, a single, precisely focused image of "unstained" objects contains no structural information. To obtain such information, one must acquire a focal series of images at varying defocus values (at different depths within the object, roughly speaking). Computer Processing of these micrographs enables the recovery of a phase-contrast image and the reconstruction of the 3D volume Structure.

Crewe microscope. In a conventional electron microscope, the incident electron beam irradiates the entire specimen. There are several types of interaction between the electrons and the sample:

1) no interaction (i.e., passing through interatomic spaces) — the most common;

2) elastic scattering (without energy loss) by the orbital electrons of the sample atoms — also quite frequent;

3) inelastic scattering (with energy loss) by atomic nuclei — less common.

The ratio between these latter Two Types of interaction is characteristic of each element, as every element possesses a specific nuclear target size. Consequently, for heavier elements, the fraction of inelastic scattering increases.

Based on this principle, a novel specialized type of microscope was developed. In this instrument, an electron beam is focused down to an extremely small spot (approximately 5 Å). This probe rapidly scans across the specimen, much like a television raster scan. As the beam moves, the intensity ratio of the latter two electron types is measured at each point using an electron energy spectrometer. The resulting ratio is then converted into a visual image on a TV screen via appropriate electronic circuitry. This represents a significant milestone in electron microscopy, introducing a new analytical dimension by enabling the identification of individual atoms. In certain cases, the ultimate resolution can be pushed down to 2 Å.

Backscattered electron scanning microscope. The Scanning electron microscope is an instrument that has yielded a multitude of stunning micrographs of cell surfaces. Its resolution is limited to 200 Å, and it operates on a fundamentally different principle compared to standard transmission electron microscopes.

Similar to Crewe's microscope, the beam here is focused into a small (100 Å) probe that scans across The surface of a specimen coated with a thick (200 Å) layer of gold or another heavy metal. When the incident beam strikes the metal and penetrates a short distance, the gold begins to emit secondary electrons or backscattered primary electrons. This mechanism makes it possible to generate three-dimensional images of The Cell surfaces under investigation (see Fig. 4.18).

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Fig. 4.18 Scanning electron micrograph of human erythrocytes, obtained by Thomas Hayes