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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 rather than light to scatter off
material, magnifying at levels up to 500,000 times.
History
The first electron microscope was built in 1931 by Ernst Ruska and Max Knoll. It was greatly developed through the
1950s and has allowed great advances in the natural sciences. The advantage of an electron beam is that it has a much smaller wavelength (see wave-particle duality),
which allows a higher resolution - the measure of how close together two things
can be before they are seen as one. Light microscopes allow a resolution of
about 0.2 micrometres, whereas electron microscopes can have resolutions as low
as 0.1 nanometers.
Process
High voltage electron beams from a cathode are focused by magnetic lenses on to the specimen. They are then
magnified by a series of magnetic lenses until they hit photographic plate or light sensitive sensors - which transfer the image
to a computer screen. The image produced is called an electron micrograph (EM).
Types
The Transmission electron
microscope (TEM) produces images by detecting electrons that are transmitted through the sample, while 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.
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.
Treatment
Samples viewed under an electron microscope have to be treated in many ways:
- Cryofixation - is freezing a specimen so rapidly, to liquid nitrogen temperatures, that the water forms vitreous
(non-crystalline) ice. This preserves the specimen in a snapshot of it's solution state. This technique produces the best
specimen preservation, but isn't applicable to all specimens.
- Fixation - is preserving the sample to make it more realistic. Glutaraldehyde - for hardening - and osmic acid - which
stains lipids black - are used.
- Dehydration - is the removing of water to be replaced with an embedding
medium such as ethanol or propanone.
- Embedding - supports the tissue for sectioning in a resin such as araldite.
- Sectioning - produces thin slices for mounting. These can be cut on an ultramicrotome with a diamond knife to produce very thin slices.
- Staining - uses metals such as lead and uranium to reflect electrons to give contrast between different structures.
- 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.
Disadvantages
The samples have to be viewed in vacuums, as air would scatter the electrons. This
means that no living material can be studied.
The samples have to be prepared in many ways to give proper detail, which may result in artifacts - objects purely the
result of treatment, and this gives the problem of distinguishing artifacts from material, particularly in biological samples.
There have been a few scientists, such as Dr Harold Hillman, who believe that such artefacts are responsible for all the
structures observed in biological samples by electron microscopy, rendering the techniques useless for these materials.
Mainstream scientists maintain that the results from various preparation techniques have been compared, and as there is no reason
that they should all produce similar artefacts, 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 techique.
Electron microscopes are also very expensive to buy and maintain.
Wikipedia articles containing electron microscope images:
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