Operating Principles and Optics
The main difference between an electron microscope and a light microscope is the use of a beam of electrons as the radiation source. The wavelength of electrons is significantly shorter than that of visible light. This short wavelength easily bypasses minute obstacles, providing the highest possible resolving power.
Instead of glass lenses, electromagnetic coils are used to focus the electron beam. A high vacuum must be maintained inside the microscope column to prevent electrons from losing energy through collisions 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 completely through the specimen. The resulting output is a two-dimensional (flat) image of the internal cell structure.
- Scanning Electron Microscopes (SEM): A thin metal coating is applied to the surface of the specimen. An electron beam scans this surface sequentially, knocking out secondary electrons (beta rays). Detecting these rays yields a three-dimensional (3D) image of the surface topography.
Optical Path of the Transmission Electron Microscope (TEM)
In a TEM, electrons are emitted by the cathode and accelerated via a potential difference toward the anode. After passing through an aperture in the anode, the beam enters a vacuum system of electromagnetic lenses:
- Condenser lens directs and focuses the beam onto the specimen.
- Objective lens gathers the rays that have passed through the tissue, producing the primary magnification.
- Projector lens (acting analogously to an eyepiece) forms the final image.
The image is visualized on a fluorescent screen, which emits light wherever electrons strike it.
Specimen Preparation for TEM
The specimen preparation process has strict requirements. Very small tissue pieces (about 1 mm³) are collected. Fixation is carried out in two stages:
- First with glutaraldehyde (to crosslink and fix proteins).
- Then with osmium tetroxide (to fix membrane phospholipids and impart initial electron density).
After dehydration, the material is embedded not in paraffin, but in epoxy resins. The blocks are polymerized in an incubator.
Sectioning is performed using ultramicrotomes. The resulting sections, 30–50 nm thick, are placed on special metal grids.
Contrasting (Staining)
In electron microscopy, the term "staining" is avoided; instead, the process is called contrasting. Heavy metal salts (tungsten, lead, uranium) are used for this purpose.
Heavy metals deposit on cellular structures (primarily membrane phospholipids) and scatter or absorb electrons. Areas of the specimen where salts accumulate block the electron beam and appear dark on the fluorescent screen.