Electron Microscope: Principle, Types, Applications

Electron microscope as the name suggests is a type of microscope that uses electrons instead of visible light to illuminate the object. Electromagnets function as lenses in the electron microscope, and the whole system operates in a vacuum.

Since electrons have a very short wavelength, the resolving power of electron microscopes is very high and produces a high-resolution image on a fluorescent screen, like a television screen.

Electron microscopes are used for detailed investigation of the ultrastructure of a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals.

German physicist Ernst Ruska invented electron microscope in 1931.

Components of an Electron Microscope

Like any ordinary microscope, the electron microscope also uses a light source, a combination of lenses to produce a magnified image, however, this vary slightly as compared to ordinary light microscope.

1. Source of light: The source of light is replaced by a beam of very fast-moving electrons. These electron beams are obtained when the tungsten filament in an electron microscope, is heated by applying a high voltage current, which is used as a light beam.
2. Electromagnetic fields (instead of lenses): The lenses that make the specimen seem bigger are replaced by a series of coil-shaped electromagnets through which the electron beam travels also known as electromagnetic lenses. There are 3 sets of electromagnetic lenses in an electron microscope as compared to two in light microscopes. In addition to the condenser and objective lenses, a third projector lens is also present. The magnetic lens of an electron microscope can have different powers (focal lengths and magnification) depending on the amount of current flowing through the electrical coils. In an ordinary microscope, the glass lenses bend (or refract) the light beams passing through them to produce magnification. In an electron microscope, the coils bend the electron beams the same way.
3. Image viewing and recording system: The magnified image of the specimen is formed as a photograph (called an electron micrograph) or as an image on a TV screen.

In electron microscope, the entire illuminating imaging system is usually referred to as the microscope column and is constructed upside down as compared to a light microscope.

Types of Electron Microscope

There are two types of electron microscopes with different operating styles: the transmission electron microscope (TEM) and the scanning electron microscope (SEM).

Transmission Electron Microscope (TEM)

As the name suggests, this type uses transmitted electrons as light sources. It provides a detailed view of the internal structure of the prepared test samples. Tissues must be cut in thin sections for viewing under TEM. Its magnifying power is very high as compared to SEM and shows a 2-dimensional image. It is the most commonly used type of electron microscope.

Scanning Electron Microscope (SEM)

It uses scattered electrons as the light source. SEM scans the surface of the objects and provides three-dimensional views of the surface structures. In SEM sample is prepared by coating it with a thin layer of metal such as gold or palladium. It has low magnifying power compared to TEM.

The major differences between scanning electron microscope (SEM) and transmission electron microscope (TEM) are available here.

For Transmission Electron Microscope

Specimen Preparation

The specimen suitable for electron microscopes should be very thin (20-100 nm thickness) so the bacterial cells and any other biopsy materials should be slice into thin layers. The general flow of sample preparation is as follows:

Unlike visible light, electron beams can not penetrate even a single cell.

• Fixation: Cells are fixed by using glutaraldehyde or osmium tetroxide for stabilization
• Dehydration: Specimen is then dehydrated with organic solvents (e.g. acetone or ethanol)
• Embedding: Specimen is embedded in plastic polymer and then, is hardened to form a solid block.
• Slicing: Specimen is cut into thin slices by an ultramicrotome knife. Slices thus prepared are then mounted on a metal slide.

Principle

The electronic beam is obtained from a heated tungsten filament which generates electrons. A large voltage is applied between the cathode (the tungsten filament) and the anode, which excites the electrons and it travels with high velocity towards the condenser lens. The condenser lenses focus the electronic beams through the specimen and electrons are scattered depending upon the thickness or refractive index of different parts of the specimen.

The denser regions in the specimen scatter more electrons and therefore appear darker in the image since fewer electrons strike that area of the screen. In contrast, transparent regions are brighter. The electron beam coming out of the specimen passes to the objective lens, which has high power and forms the intermediate magnified image. The projector throws its image onto a fluorescent screen which may be substituted by a photographic plate to make a permanent record.

Advantages of Electron Microscope

1. Magnification and higher resolution –Electron microscopes provide an image resolution in the range of up to 0.2 nm. An electron microscope can achieve magnification in excess of 100,000x compared with 1000X magnification with light microscopy.
2. High-quality images – Electron microscopes produce highly detailed images of structures which are of high quality, revealing complex and delicate structures.

Limitations of Electron Microscope

Electron microscope is a major capital investment and is not needed for the laboratory diagnosis of most infectious diseases. The major limitations of this microscope are as follows:

1. It is expensive, requires a large space in the laboratory, and is highly sensitive equipment.
2. Require trained manpower for proper sample preparation to avoid artifacts and proper handling of the microscope.
3. Electron microscopes operate in a vacuum; therefore, live specimens cannot be observed.
4. Since the penetration power of the electron beam is very low, the object should be ultra-thin. For this, the specimen is dried and cut into ultra-thin sections before observation (for TEM). This could lead to distortions in the structures.
5. Magnetic fields and vibrations caused by other lab equipment may interfere with their operation.
6. Only black and white images can be produced by an electron microscope.

Applications of Electron Microscope

Electron microscopes are powerful research tools. We are able to discover many new morphological features of bacteria, bacterial components, fungi, viruses, and parasites using electron microscopes.

Apart from Microbiology, electron microscopy have a diverse range of applications in many different fields such as technology, industry, biomedical science, and chemistry.

• In pathology, it is used to examine microscopic features of different diseases including tumors.
• In chemistry, it is used for quality control and assurance, analysis of atomic structures, and drug development.
• In the technology industry, it is used for high-resolution 2D and 3D imaging, semiconductor inspection, computer chip manufacture.
• In forensics SEM is widely used for gunshot residue analysis, firearm identification (bullet markings comparison), investigation of gemstones and jewelry, counterfeit banknotes, examinations of non-conducting materials, etc.
• In geology, SEM is a routine technology employed in the study of rocks and minerals.

References and further readings

1. Tille, P. (2017). Bailey & Scott’s Diagnostic Microbiology (14 edition). Mosby.
2. Madigan Michael T, Bender, Kelly S, Buckley, Daniel H, Sattley, W. Matthew, & Stahl, David A. (2018). Brock Biology of Microorganisms (15th Edition). Pearson.