Light microscopes have many important uses, especially for medical professionals and researchers who use them to help make a diagnosis and make new discoveries.
However light microscopes have limited resolving power which is the ability to distinguish small or closely adjacent images. The reason for this is due to the nature of light itself.
Therefore the obvious solution was to use a beam of electrons instead of light, giving rise to electron microscopy, an extremely powerful microscope that has revolutionized science.
The electron microscope can achieve a resolution of about 2 nanometers (incredibly small) and a mind-blowing magnification of x 1,000,000. There are two distinct types of electron microscopy, called Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM).
History of the Electron Microscope (EM)
1924, Louis De Broglie introduced the theory of electron waves, the foundation of electron microscopy.
In 1931, German engineers Ernst Ruska and Max Knoll developed the transmission electron microscope, capable of four-hundred-power magnification.
Ruska realized that electron wavelengths are far shorter than light wavelengths and understood that, if he was able to discover a technique to apply this knowledge, he could develop a far more superior microscope.
Both Knoll’s and Ruska worked together to develop the first-ever electromagnetic lens, which worked by focusing a beam of electrons instead of a traditional illuminator to generate a magnified image.
In 1939, the first EM was produced commercially by Siemens under the guidance of Ruska who worked as an electrical engineer and in 1986 Ruska was awarded the Nobel Prize for Physics.
How does the Electron Microscope work?
An Electron Microscope works by generating an electron beam which enables the user to examine objects at the nano-scale. They work on the same principle as a light microscope except instead of light or photons they utilize electrons.
The basic operation of an electron microscope involves an electron gun producing a beam of electrons from an electric current, the electrons are accelerated using an anode plate to focus on the object.
All EM’s are composed of electromagnetic and/or electrostatic lenses, which are made up of a solenoid, which is a coil of wire wrapped around the exterior of a tube. The specimen is then placed into a vacuum chamber as electrons do not travel well in the air.
In a light microscope, the glass lenses refract the light passing through them causing magnification, and in an electron microscope the coils bend the electron beams in a similar way.
An image is then produced, called an electron micrograph. This is usually seen on a computer screen as electron microscopes typically come accompanied with software for image analysis and observation.
Specimens used in an Electron Microscope need specialized preparation before being placed in the air free chamber. The exact method depends on the kind of specimen and analysis, which include:
- Cryofixation
- Fixation
- Dehydration
- Embedding
- Sectioning
- Staining
The use of an electron microscope and the preparation of specimens requires specialist training and knowledge as inexperience can lead to contamination with other artefacts. Specimens are typically embedded in plastic epoxy resin and sectioned using glass knives on an ultramicrotome.
Transmission Electron Microscope (TEM)
The Transmission Electron Microscope (TEM), was the very first type of Electron Microscope developed and is the primary EM used in diagnosis. It is an incredibly powerful microscope that works on the same principle as the light microscope, except photons are replaced by an electron beam. It can produce images at the nanoscale.
The electron beam is passed through the specimen at high speeds (transmission) which produces a high-resolution, black and white 2D image of the specimen surface (topography). The image can be viewed via a fluorescent screen.
Scanning Electron Microscope (SEM)
The scanning electron microscope (SEM) has no relation to light microscopy and has a lower resolution than the TEM. The main advantage of the SEM is that it can produce 3D images of samples.
The SEM microscope utilizes multiple solenoids which scan the surface of the specimen and the specimen reflects electrons back when irradiated with the electron beam.
The electrons are detected by converting energy into light energy which is measured by a photomultiplier. The magnification can be increased by increasing the strength of the electron beam. The backscattered electrons give information of the subsurface and an image is produced.
Specimen processing for the SEM is highly specialized. Firstly the specimen is dried with alcohol, then soaked with liquid CO2, and the CO2 is converted into gas and slowly released. It is then sputter coated with a film of metal causing the specimen to become electrically conductive.