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In use for almost nine decades, electron microscope technology continues to evolve and those using it need to be aware of both the time-honored industry standards and the latest news.
In June, a team of American and Swedish researchers announced the development of an electron microscope capable of detecting magnetism at the atomic level. Meanwhile, Cornell University researchers also announced in June that they had developed new kind of detector that was capable of recording an image in 1/1000 of a second and from 1 to 1,000,000 electrons per pixel.
These are just a couple of the technologies expected to make it into the electron microscopy industry in the coming decades, but there are a lot of tools that should be being utilized currently.
Energy Dispersive X-ray Spectroscopy (SEM-EDX)
Used in chemical analysis, energy dispersive X-ray spectroscopy (SEM-EDX) involves the use of X-rays or a high-energy particle beam to excite atoms in a sample. Every element has a distinct atomic makeup, which allows the SEM-EDX to identify elements based on their distinct set of peaks on an X-ray emission spectrum chart.
While resting an atom inside the sample has unexcited electrons with known, specific energy amounts, or electron shells attached to the nucleus. With SEM-EDX, a high-energy beam excites an electron in an inner shell, popping it from the shell while generating a "hole" where the electron had been. An electron from an outer shell then plugs the hole and the difference in energy between the outer and the inner energy shell may then be released in the form of X-ray emissions. The number and energy of the X-rays released from a sample can be assessed by an energy-dispersive spectrometer, and EDS allows the elemental makeup of the sample to be assessed.
Before a specimen can be analyzed by transmission-electron-microscope (TEM), technicians typically use a refining process called electropolishing.
The technique involves polishing the specimen with electrochemical action, often through the use of twin jet electropolisher. The equipment has a sample holder and sample disc situated between two nozzles.
The process begins by immersing the sample disc and the nozzles in an electrolyte. Streams of electrolyte are then pumped through these nozzles. A current causes the surface of the disc to be oxidized, with the thinning rate in the center of the disc being greater than at the edges. Consequently, a perforation occurs near the central area with a ridge at the edge of the disc. The perimeters close to the perforation have a wedge-shape with those near the perforation getting the desired thickness for electron transparency.
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Cryogenic Sample Preparation
Cryogenic electronmicroscopy (Cryo-EM) is a rapidly growing field that has recently seen massive strides forward in producing higher resolution thanks to better imaging technology, image processing software, and sample preparation techniques.
Sample preparation involves flash-freezing a thin layer of the sample on a small platform. This can be accomplished with a minimal amount of sample, in any buffer and without using any stain, which is very helpful to ascertain the structure and function of macromolecules. When coupled with single-particle image processing, the method has seen extensive usefulness for 3D structural resolution of purified macromolecules.
Electron Backscatter Diffraction (EBSD) Analysis
Materials typically used in engineering, like steel and aluminum, are aggregates of single crystal grains, which means they only have a uniform structure over short distances. The makeup of these crystal structures has implications for the larger material’s strength and other properties.
With electron backscatter diffraction (EBSD) a beam of electrons is fired onto a sample, and the resulting interactions cause a pattern of electrons to emerged from the sample, this pattern can be seen with the help of fluorescent phosphor screen. The pattern can be used to determine crystal orientations and identify materials.
Electron crystallography is often used when protein crystals are too small for X-ray crystallography. This is because radiation damage is the limiting factor in information collection, and electrons typically place less energy in the crystal than X-rays. As a consequence, resolving the framework of two-dimensional crystals just one unit cell thick is achievable with electron diffraction, but not with X-ray diffraction.
However, in the case of three-dimensional protein crystals thicker than one unit cell, electrons scattering in the sample may prevent significant structure resolution.
References and Further Reading
Imaging Techniques and Scanning Electron Microscopy as Tools for Characterizing a Si-Based Material Used in Air Monitoring Applications
TEM Specimen Preparation Techniques
An Introduction to Sample Preparation and Imaging by Cryo-Electron Microscopy for Structural Biology
The Diffraction Pattern
Electron Crystallography - An Introduction