Electron Microscopy.

Electron Microscopy.

Electron microscopy is a method of imaging that uses an electron microscope to enlarge small specimens by a greater magnification and resolution than conventional light microscopes. The photographs produced of specimens viewed with an electron microscope are call electronmicrographs. Magnification is the increase in apparent size of the specimen and resolution (also called resolving power) is the ability of the microscope to distinguish and produce separate images of closely placed objects. These two primary properties of electron microscopes make them extremely useful in the analysis and study of specimens.

The obvious difference between electron microscopes and light microscopes is the medium through which each operates. Light microscopes work by using photons to produce an image whereas electron microscopes use electrons to produce an image. The magnifying power of a light microscope is limited by the wavelength of visible light so electrons are used instead because they have a much smaller wavelength so can therefore resolve much smaller structures. The resolving power of a microscope depends on the wavelength of the electromagnetic radiation used. However, the benefits gained by using an electron microscope also bring specific problems that have to be tackled.

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Electron beams cannot pass through glass because electrons are physical matter. Therefore there cannot be any lenses used to focus the electrons like there are with optical microscopes. Instead the microscope uses electromagnets to focus the image onto a fluorescent screen. Another problem is that the electrons are very easily interrupted and scattered by molecules in the air so their path is distorted. To overcome this there has to be a very high vacuum inside the microscope so that there is the absence of any other particles in the path of the electron beams. Consequently electron microscopes can only be used to look at dead cells. If a vacuum is present then life cannot exist within it and so the microscope cannot look at living cells. Also the tissue needs to be preserved using substances that will prevent enzyme action. The tissue also needs to be soaked in alcohol to dehydrate it and make the organelles in cells more visible.

There are two types of electron microscopes, the scanning electron microscope and the transmission electron microscope, both of which have different purposes and require different preparation.

The transmission electron microscope (TEM) allows us to see as separate structures, particles which are as close together as 2 nanometres. TEM can achieve magnifications up to around 1000000x and a resolution of about 2 nanometres. The TEM produces sharp definition at low magnifications but it can be used at high as well as low magnifications if preferred. The electronmicrograph from a TEM is dependent on electrons that do and do not pass through the specimen being sampled for it to be produced. Light areas on an electronmicrograph are produced when electrons have been able to pass through the specimen. However, if any electrons are to pass through the specimen then it would have to be a very thin specimen. The cells being imaged are not thin enough so they have to be cut into very thin sections, about the thickness of the wall of a soap bubble. Very small specimens such as viruses and large molecules are an exception and do not have to be sectioned because they are already thin enough.

There are two methods of cutting the tissue into thin sections depending on the resultant image required. The first is simply cutting the tissue into thin sections using a microtome. First the tissue has to be embedded in a resin which makes the whole specimen become hard. This treatment allows the tissue to be cut by the microtome which is a knife made out of glass. Since TEMs can attain such high resolutions, it is necessary to stain the specimen in order to improve the contrast of the image. After the tissue has been sectioned it is stained using heavy metals.

The second method used to section the tissue is called the freeze facture technique. This process is carried out so that surface detail can be seen by the TEM rather than just a flat plane like the previous method. First the specimen is frozen using liquid nitrogen. It is then placed inside a vacuum chamber and is then split along the line which has the least resistance such as a division of the lipid bilayer. The split surface is then coated with heavy metal ions; a treatment which may introduce artefacts. When a thin section has been created using either method, it is then mounted on copper grids which provide support.

The scanning electron microscope (SEM) creates a magnified image of the surface of a specimen. A SEM can achieve up to around 100000x magnification. It is called a scanning microscope because it scans the surface of the object bit by bit by using beams spanning to and fro across the surface of the complete object. Unlike the TEM, the SEM does not have such a high resolving power, however, there is no need for the specimens to be cut into thin sections prior to scanning and the SEM can also accommodate larger specimens than the TEM. The SEM works on the principle of scanning the electrons that bounce off the surface of the sample as well as any electrons that are emitted too. However, the atoms in organic molecules do not have very high atomic numbers and so do not scatter electrons. To adjust this, the surface of the specimen has to be coated with a thin film of gold. Electron-dense areas on the specimen scatter the electrons from the microscope and produce dark areas on the electronmicrograph.

As you can see the advent of electron microscopy was a great feat. It allows theories based on the molecular scale to be proven and so many more findings and conclusions to be found.