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

Methods for experimental study of protein structure
Electron microscopy

Currently, There are two primary Methods for directly studying The Structure of Proteins in the solid state: Electron Cell/15.html">Microscopy and X-Ray Diffraction Analysis. Let us examine them briefly.

Under normal conditions, the unaided human eye can distinguish two separate points if the distance between them is about 0.1 mm (100 µm). At smaller distances, spatially separated objects merge into a single continuous image to the human eye.

Optical microscopes have a theoretical resolution limit of about 2000 Å. This limit arises because a microscope of any design (ideally, with perfect lenses) can resolve two points only if the distance between them is at least 1/2 the wavelength of the light used to illuminate the object. Such are the laws of optics. If, for instance, we use the visible region of the optical spectrum (= 5000Å, 0.5µm), the theoretical limit of resolving power for an optical microscope cannot be less than 2500Å (0.25µm). The Use of UV light and specialized quartz lenses can improve the resolution down to 0.17µm. While optical microscopes allow us to observe intact Cells 1-20 µm in diameter, they are clearly unsuitable for visualizing individual protein molecules. Significantly higher resolving powers are achieved using electron microscopes.

In terms of its operating principle, an Electron microscope is no different from an optical one. The only distinction is that it uses a high-energy electron beam as the radiation source, replacing physical (Glass) lenses with electromagnetic focusing coils (electromagnetic lenses).

Let us estimate the theoretical resolution limit of an electron microscope. According to de Broglie, the energy of a moving material particle and its wavelength are related by the equation:

Class="center">λ = h/m_eV

where m_e and V are the mass and velocity of the electron, respectively. If an electron is accelerated in an electric field with a potential difference U, its kinetic energy is given by:

m_eV²/2 = eU

where e is the elementary charge of the electron.

Substituting the fundamental constants with their numerical values, we obtain the following simple relationship for the wavelength of an accelerated electron:

λ = 12.3/U^1/2(ß)

At U = 50000V (50kV), λ = 0.05Å, meaning the resolving power would be about 0.025Å (0.0000025µm). This is an exceptionally high resolution, sufficient to visualize individual hydrogen atoms. In practice, however, this cannot be achieved due to inhomogeneities in the focusing fields and several other factors. The best modern electron microscopes provide a resolution of about 2Å (0.0002µm), making it possible not only to study cell ultrastructure but also to observe individual large macromolecules. The Operating Principle and general appearance of a modern electron microscope are illustrated in Figure 4.13.

Fig. 4.13 General view and schematic diagram illustrating the operating principle of an electron microscope

Electrons passing through the specimen form its image, which is recorded on a special photographic film to produce an electron micrograph of the sample (object). In electron microscopy, image contrast is determined by the mass-density distribution within the specimen. Because protein molecules lack sufficient inherent contrast, samples are "stained" by Treatment with electron-dense substances—salts of tungsten, osmium, or platinum.

The sample under investigation in an electron microscope is placed on a thin carbon film mounted on a circular, fine-mesh copper grid approximately 3 mm in diameter (see the inset in Fig. 4.14).

Fig. 4.14 Electron microscope sample holder (grid): fragment, top view (A) and side view (B).

1 - carbon support film; 2 - sample; 3 - metal grid