Operation principles and optics
The main difference between an electron microscope and a light microscope is the use of an electron beam as the radiation source. The wavelength of electrons is significantly shorter than the wavelength of visible light. A short wavelength easily bypasses the smallest obstacles, providing the highest resolving power of the instrument.
Instead of glass lenses, electromagnetic coils are used to focus the electron beam. A high vacuum must be maintained inside the instrument column—this is necessary so that electrons do not lose energy when colliding with gas molecules.
Classification of electron microscopes
Depending on how the electron beam interacts with the specimen, there are two main types of instruments:
- Transmission electron microscopes (TEM): The electron beam passes straight through the object. The output is a two-dimensional (flat) image of the internal structure of the cell.
- Scanning electron microscopes (SEM): A thin metal coating is applied to the surface of the object being studied. The electron beam successively scans this surface, knocking secondary electrons (beta rays) out of the metal. Registration of these rays yields a volumetric (three-dimensional) image of the relief.
Optical scheme of a transmission electron microscope (TEM)
In a TEM, electrons are emitted by the cathode and accelerated due to the potential difference with the anode. After passing through the aperture in the anode, the flux enters the vacuum system of electromagnetic lenses:
- The condenser directs and focuses the beam on the sample.
- The objective lens collects the rays that have passed through the tissue, providing the initial magnification.
- The projection lens (acting as the eyepiece) forms the final image.
The image is visualized on a luminescent screen, which begins to glow where the electrons strike.
Sample preparation for TEM
The sample preparation process has a number of strict requirements. Very small pieces of tissue are taken (about 1 mm³). Fixation takes place in two stages:
- First, with glutaraldehyde (to fix proteins).
- Then with osmium tetroxide (to fix membrane phospholipids and give them initial electron density).
After dehydration, the material is embedded not in paraffin, but in epoxy resins. The blocks are polymerized in a thermostat.
Sectioning is performed on ultramicrotomes. The resulting sections, 30–50 nm thick, are placed on special metal grids.
Contrasting (staining)
In electron microscopy, the term "staining" is not used—instead, they speak of contrasting. Heavy metal salts (tungsten, lead, uranium) are used for this purpose.
Heavy metals deposit on cellular structures (mainly on membrane phospholipids) and absorb electrons. Those areas of the specimen where many salts have accumulated block the electron beam and appear dark on the luminescent screen.
Briefly: answers to topical questions
What reagents are used for tissue dehydration prior to embedding in epoxy resins?
Alcohols of increasing concentration are used to dehydrate tissue prior to embedding.
- Alcohol is used for the gradual dehydration of the material in the form of solutions with a concentration ranging from 70° to 100°.
The process consists in the sequential processing of the sample through a "battery" of alcohols. After complete removal of water from the tissue, the next stage begins—infiltration and embedding of the material in embedding media, which are synthetic resins.
Class and exam questions
Why is a vacuum necessary in an electron microscope?
A high vacuum is necessary so that the electron beam does not scatter and lose energy when colliding with air molecules inside the column.
What is the difference between a transmission microscope and a scanning microscope?
A transmission electron microscope (TEM) passes electrons through a thin section, yielding a flat image of the cell interior. A scanning microscope "feels" the surface of the object, coated with metal, creating a three-dimensional picture of the relief.
What is used instead of microscope slides in electron microscopy?
Instead of glass microscope slides, special metal grids are used to hold the ultrathin sections.
Why is osmium needed during sample preparation?
Osmium tetroxide is used for double fixation: it stabilizes membrane phospholipids and simultaneously contrasts structures, imparting electron density to them.
What else to study on this topic
- Mechanisms of image formation in a scanning electron microscope
- Comparison of tissue processing steps for light and electron microscopy
- Principle of operation of the ultramicrotome
- Features of the application of glutaraldehyde and epoxy resins
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