Electron Microscopy Investigation

Electron Microscopy Investigation

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Electron Microscopy Investigation

The electron microscope was first made when biologists found a problem
with the resolution of a light microscope. Resolution means “the
ability to distinguish between two points”; the problem is that the
maximum resolution of a light microscope is 200nm. So if two points
are closer together than 200nm they are seen as one. One example of
this problem is that under a light microscope the centrioles in an
animal cell appear as one, however when viewed under an electron
microscope we can see that there are two centrioles at right angles to
each other. Magnifying the image or using a higher magnification
cannot overcome this problem, as we increase the magnification we do
not get a better resolution just an enlarged image. In fact if we
increase the magnification beyond a point it only decreases the
resolution.

The light a light microscope uses is between 400nm wavelength to 700nm
wavelength as this is the visible light spectrum. 400nm being the
smallest wavelength of visible light (violet light). Cells like
ribosomes are far to small to interfere with light waves, ribosomes
are about 22nm in diameter. The limit of resolution is half the
wavelength of the radiation used, in this case 400nm (violet light),
to view the specimen with. So anything below 200nm cannot be seen
clearly, if at all. Another flaw of the light microscope that that if
an object is transparent it will allow light to pass through it
therefore cannot be seen using a light microscope. This is why samples
are dyed with chemicals like iodine before they can be viewed.

One solution is to use a radiation with a shorter wavelength than the
smallest visible light. Ultra violet and x-ray microscopes have been
built but faced many problems; one problem is that x-rays are very
hard to focus. A better solution was to use electrons, an electron is
a negatively charged particle witch orbits an atoms nucleus. When a
metal becomes very hot, meaning it has an increase in energy, its
electrons also gain energy some gain so much energy they break free of

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the nucleuses pull and escape their orbits. When free electrons behave
like electromagnetic radiation. A free electron has a very short
wavelength, the more energy an electron has the shorter its wavelength
will be. The reason that electrons are so good for microscopy is
because firstly they have an incredibly small wavelength and secondly
because they have a charge they can be focused easily using
electromagnets, meaning that electromagnets can be used to alter the
path of a beam of electrons.

There are two types of electron microscope, the transmission electron
microscope and the scanning electron microscope.

The transmission electron microscope forms an image by accelerating a
beam of electrons that pass through the specimen. The electrons are
then scattered at different angles depending on the density of the
specimen they encounter. An electron can either be undeflected,
deflected without loosing energy or looses a significant amount of
energy after being deflected. The different scattered angles produce a
contrasting image because all electrons that are scattered more then
0.5 degrees are stopped by a objective aperture lens just below the
specimen itself. The image is projected on a fluorescent screen where
every single feature that is two to three angstroms across can be
seen.

The transmission electron microscope gives an internal representation
of the specimen like the light microscope but dose no show the objects
actual appearance.

So the scanning electron microscope was made, in this electron
microscope a fine beam of electrons, about 200 angstroms across, is
used to scan the specimen. As the electron beam moves across the
specimen some electrons are reflected, also secondary electrons are
emitted form the specimen as they are bombarded by the electron beam.
The image displayed on the cathode comes form these reflected and
secondary electrons.

It is not possible to see an electron beam as its wavelength is well
below the 400nm of visible violet light, so it has to be projected
onto a fluorescent screen. The areas hit by electrons shine brightly
while the areas that weren’t hit do not giving a black an white
picture. To gain a better contrast scientists use stains that contain
heavy metal atoms that stop the electrons path.

Another problem of electron microscopy is that the specimen and the
electrons have to be in a vacuum so we cannot scan living organisms,
the reason the electrons have to be in a vacuum is because if they
collided with air molecules witch makes it impossible to achieve a
clear picture. Also water boils at room temperature in a vacuum so
specimens have to be dehydrated before an electron microscope can be
used on them. This means that material that is going to be scanned has
to be preserved in a life like state.

Overall the electron microscope is very useful because it allows us to
gain a higher resolution and a more accurate view of the structure and
appearance of cells however as the specimen has to be one dehydrated
and two dead before we can scan them using an electron microscope we
cannot use an electron microscope to see thing such as cell division.
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