Electron microscope
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The electron microscope is a microscope that can magnify very small details with high resolving power due to the use of electrons as the source of illumination, magnifying at levels up to 500,000 times.
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History
The first electron microscope was built by the German physicist Ernst Ruska, who was awarded the Nobel Prize in 1986 for its invention. He knew that electrons possess a wave aspect, so he believed he could treat them in a fashion similar to light waves. Ruska was also aware that magnetic fields could manipulate electrons, possibly focusing them as optical lenses do to light. After confirming these principles through research, he set out to design an electron microscope. Ruska had deduced that an electron microscope would be much more powerful than an ordinary optical microscope, because he knew that magnification increased with shorter wavelengths. Since electron waves were shorter than ordinary light waves, it followed that they would allow for greater magnification. In 1932 Ruska and a collaborator, German physicist Max Knoll, under whom he obtained his doctorate, built the first crude electron microscope. Despite the fact that it was primitive and not fit for practical use, the instrument was still capable of magnifying objects 400 times.
The first practical electron microscope was built by Eli Franklin Burton and students, Cecil Hall, James Hillier and Albert Prebus, at the University of Toronto, Canada in 1938.
Although modern electron microscopes can magnify an object 2 million times, they are still based upon Ruska's prototype and his correlation between wavelength and magnification. The electron microscope is an integral part of many laboratories. Researchers use it to examine biological materials (such as microorganisms and cells), a variety of large molecules, medical biopsy samples, metals and crystalline structures, and the characteristics of various surfaces.
Types
Electron beam microscopes
Transmission Electron Microscope (TEM)
The Transmission Electron Microscope (TEM) involves a high voltage electron beam emitted by a cathode and formed by magnetic lenses. The electron beam that has been partially transmitted through the very thin (and so semitransparent for electrons) specimen carries information about the inner structure of the specimen. The spatial variation in this information (the "image") is then magnified by a series of magnetic lenses until it is recorded by hitting a fluorescent screen, photographic plate, or light sensitive sensor such as a CCD (charge-coupled device) camera. The image detected by the CCD may be displayed in real time on a monitor or computer.
Resolution of the high-resolution TEM (HRTEM) is limited by spherical and chromatic aberration, but a new generation of aberration correctors has been able to overcome spherical aberration. Software correction of spherical aberration has allowed the production of images with sufficient resolution to show carbon atoms in diamond separated by only 0.89 ångström (89 picometers) and atoms in silicon at 0.78 ångström (78 picometers) at magnifications of 50 million times. The ability to determine the positions of atoms within materials has made the HRTEM an indispensable tool for nano-technologies research and development in many fields, including heterogeneous catalysis and the development of semiconductor devices for electronics and photonics.
Scanning Electron Microscope (SEM)
Unlike the TEM, where electrons are detected by beam transmission, the Scanning Electron Microscope (SEM) produces images by detecting secondary electrons which are emitted from the surface due to excitation by the primary electron beam. In the SEM, the electron beam is rastered across the sample, with detectors building up an image by mapping the detected signals with beam position.
Generally, the TEM resolution is about an order of magnitude better than the SEM resolution, however, because the SEM image relies on surface processes rather than transmission it is able to image bulk samples and has a much greater depth of view, and so can produce images that are a good representation of the 3D structure of the sample.
Scanning Transmission Electron Microscope (STEM)
A Scanning Transmission Electron Microscope (STEM) is a specific sort of TEM, where the electrons still pass through the specimen, but, as in SEM, the sample is scanned in a raster fashion.
Reflection Electron Microscope (REM)
In addition there is a Reflection Electron Microscope (REM). Like TEM, this technique involves electron beams incident on a surface, but instead of using the transmission (TEM) or secondary electrons (SEM), the reflected beam is detected. This technique is typcially coupled with Reflection High Energy Electron Diffraction and Reflection high-energy loss spectrum (REELS). Another variation is Spin-Polarized Low-Energy Electron Microscopy (SPLEEM), which is used for looking at the microstructure of magnetic domains [1].
Scanning Tunneling Microscope (STM)
A Scanning Tunneling Microscope (STM) can be considered a type of electron microscope, but it is a type of Scanning probe microscopy and it is non-optical. The STM employs principles of quantum mechanics to determine the height of a surface. An atomically sharp probe (the tip) is moved over the surface of the material under study, and a voltage is applied between probe and the surface. Depending on the voltage electrons will tunnel or jump from the tip to the surface (or vice-versa depending on the polarity), resulting in a weak electric current. The size of this current is exponentially dependent on the distance between probe and the surface.
Sample Preparation
Samples viewed under an electron microscope may be treated in many ways:
- Cryofixation - freezing a specimen so rapidly, to liquid nitrogen or even liquid helium temperatures, that the water forms vitreous (non-crystalline) ice. This preserves the specimen in a snapshot of its solution state. This technique produces the best specimen preservation, but isn't applicable to all specimens. An entire field called cryo-electron microscopy has branched from this technique.
- Fixation - preserving the sample to make it more realistic. Glutaraldehyde - for hardening - and osmium tetroxide - which stains lipids black - are used.
- Dehydration - replacing water with organic solvents such as ethanol or acetone.
- Embedding - infiltration of the tissue with a resin such as araldite or epoxy for sectioning.
- Sectioning - produces thin slices of specimen, semitransparent to electrons. These can be cut on an ultramicrotome with a diamond knife to produce very thin slices. Glass knives are also used because they can be made in the lab and are much cheaper.
- Staining - uses heavy metals such as lead, uranium or tungsten to block electrons to give contrast between different structures, since many (especially biological) materials are nearly "transparent" to electrons (weak phase objects).
- Freeze-fracture or freeze-etch - a preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face on" view. The fresh tissue or cell suspension is frozen rapidly (cryofixed), then fractured by simply breaking or by using a microtome while maintained at liquid nitrogen temperature. The cold fractured surface (sometimes "etched" by increasing the temperature to about -100°C for several minutes to let some ice sublime) is then shadowed with platinum or gold at an average angle of 45° in a high vacuum evaporator. A second coat of carbon, evaporated normal to the average surface plane is often performed to improve stability of the replica coating. The specimen is returned to room temperature and pressure, then the extremely fragile "pre-shadowed" metal replica of the fracture surface is released from the underlying biological material by careful chemical digestion with acids, hypochlorite solution or SDS detergent. The still-floating replica is thoroughly washed from residual chemicals, carefully fished up on EM grids, dried then viewed in the TEM.
- Ion Beam Milling - thins samples until they are transparent to electrons by firing ions (typically argon) at the surface from an angle and sputtering material from the surface. A subclass of this is Focused ion beam milling, where gallium ions are used to produce an electron transparent membrane in a specific region of the sample, for example through a device within a microprocessor.
- Conductive Coating - Evaporation, Thin-film deposition, or sputtering of carbon, gold, gold/palladium, platinum or other conductive material to avoid charging of non conductive specimens in a scanning electron microscope.
Disadvantages
Electron microscopes are expensive to buy and maintain. As they are sensitive to vibration and external magnetic fields, suitable facilities are required to house microscopes aimed at achieving high resolutions.
The samples have to be viewed in vacuum, as the molecules that make up air would scatter the electrons. Recent advances have allowed hydrated samples to be imaged using environmental scanning electron microscope.
Scanning electron microscopes usually image conductive or semi-conductive materials best. Non-conductive materials can be imaged by an environmental scanning electron microscope. A common preparation technique is to coat the sample with a several-nanometer layer of conductive material, such as gold, from a sputtering machine; however this process has the potential to disturb delicate samples.
The samples have to be prepared in many ways to give proper detail, which may result in artifacts—objects that are purely the result of treatment. This gives the problem of distinguishing artifacts from material, particularly in biological samples. Scientists maintain that the results from various preparation techniques have been compared, and as there is no reason that they should all produce similar artifacts, it is therefore reasonable to believe that electron microscopy features correlate with living cells. In addition, higher resolution work has been directly compared to results from X-ray crystallography, providing independent confirmation of the validity of this technique. Recent work performed on unfixated, vitrified specimens has also been performed, further confirming the validity of this technique.
See also
External links
Archives
- Rubin Borasky Electron Microscopy Collection, 1930-1988 Archives Center, National Museum of American History, Smithsonian Institution.cs:Elektronový mikroskop
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