Three Dimensional Electron Microscopy/Electron microscopes

What is an Electron Microscope?

EM

An electron microscope (EM) is an imaging instrument that uses electrons to see a sample instead of light which is used in the traditional light microscope. In general, an electron microscope works by applying a beam of electrons to a very thinly sliced or diluted sample. The electrons will either bounce off or pass through the sample and an image will be collected depending on the type of microscopy being utilized.

The resolution capability of an electron microscope is much greater than that of a light microscope, generally obtaining a magnification of 100,000X which is 50,000X greater than a traditional light microscope. The resolution difference can be attributed to the energy source of the microscopes as well as the methods of detection by instruments far more sensitive than the human eye. Light microscopes are capable of resolving about 200 nm in size. Electron microscopes in have the ability to resolve approximately 2 Å. There have been reports of electron microscopes reaching a resolution of 0.5 Å.

An Electron Microscope vs. a Traditional Light Microscope

A traditional light microscope and an electron microscope operate on similar principles. Both microscopes contain an energy source, a condenser lens, a specimen holder, an objective lens, and a projector lens. In both cases, energy is directed at a sample and an image is produced. A major differing feature of each microscope is the energy source. In an electron microscope, electrons are emitted from an electron gun, while in the light microscope the energy is generated by a light bulb. Another important difference between the two microscopes is the composition of the lens. The lens of a light microscope are made of glass and are typically spherical. In contrast, an electron microscope’s lenses are electromagnetic and electrostatic. The electromagnetic lens mainly consists of coiled copper wires. In the optical microscope light travels from the source, is split up, and then is refocused to be viewed by the human eye. In an electron microscope the electrons are passed through or bounced off of the sample and are ultimately imaged on film, a fluorescent screen or collected by an electron detector.

A unique feature of the electron microscope is the sample holder and environmental condition of the sample chamber. The sample grid, either suspended in vitreous ice or negatively stained, is placed onto the electron microscope holder. The holder is then inserted into a closed chamber, where it can be rotated at varying degrees to be imaged at specific, user selected, angles. The environment of the microscope chamber is maintained under vacuum to facilitate the directed travel of the electron beam.

Types of Microscopy

There are several types of microscopy where an electron microscope is used. The various electron microscopy techniques are Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Reflection Electron Microscopy (REM), and Scanning Transmition Microscopy (STEM). The most commonly used techniques are SEM and TEM.

Transmission Electron Microscopy works by directing the flow of electrons at a sample. TEM requires a very thin sample specimen that can endure high energy electrons, as the electrons are transmitted through the sample. As the electrons of the beam interact with the sample, an image is formed. In essence, the “shadow” of the image, created in the places were the electrons interacted with the sample, is projected onto the detector.

Bacillus_subtilis imaged using TEM

During SEM, an electron beam scans the surface of the sample and the electrons or X rays that are emitted from the surface are detected. The signals produced are used to derive information about the sample's surface topography. Various types of energy can be used, but the most common is secondary electrons excited by the initial interaction with the electron beam. SEM details the surface of a sample, while TEM details the entire sample.

Pollen imaged by SEM

History

The first model of an electron microscope was built by Ernst Ruska and Max Knoll in 1931. This initial model was only capable of magnification at 400x. Two years later, an electron microscope capable of magnifications greater than the traditional microscope was developed. In 1938 Max Knoll created the first scanning electron microscope which was refined by Manfred Von Ardenne to give better resolution.

Current Applications

The Electron Microscope in currently used in numerous applications such as rapid medical diagnosis, pharmaceutical nano-scale systems and failure mode analysis of components, but is most commonly used in the imaging of molecules. The techniques were most recently used in the imaging of double stranded DNA.