The Principles and Limitations of Scanning and Transmission Electron Microscopes

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The Principles and Limitations of Scanning and Transmission Electron Microscopes

Electron microscopes were first developed due to the limitations of light microscopes (3). The smallest object that can be viewed by any microscope is half the wavelength of light used, and objects smaller than this cannot be seen. This is because the object has to be large enough to interfere with the waves radiation. Light has a wavelength between 400-700nm, so the smallest object that can be viewed using visible light is 200nm(3). By the early 1930’s all the possible scientific progress on understanding the inner parts of cells had been made, and scientists wanted to see more detail. Max Knoll and Ernst Ruska then developed the electron microscopes in 1931 (7).

Electron microscopes use the same principles as light microscopes, but a beam of electrons is used instead of a beam of light. Electron beams have a wavelength of about 0.005nm. This short wavelength means much smaller objects can be seen (3). The resolution of a microscope is its ability to distinguish between two objects that are very close together. Magnification shows the objects as one larger image. The shorter the wavelength, the better the resolution. Therefore the resolution of an electron microscope is better than a light microscope. The magnification is also better. Magnifications of x250 000 can be obtained with an electron microscope compared with x1500 in a light microscope (1).

There are two types of electron microscope- transmission electron microscope (TEM) and scanning electron microscope (SEM). TEMs work on the principle that the beam of electrons is transmitted through a specimen (5). As electrons cannot penetrate very thick materials, the specimens have to be very thin (0.05 to 0.1 um thick). To make the features visible they have to be prepared by (1):

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  1. Fixing the specimen in a solution of fixing chemicals. This hardens the tissue so the structure stays the same (1)
  2. Dehydrating the specimen by passing it through alcohols of gradually increasing concentrations. This prepares the sample for embedding (1).
  3. Embedding the sample in resin and then baking the resin to make it hard. This ensures the cells are not distorted during sectioning (1).
  4. Sectioning. During sectioning, the specimen is cut into very thin slices using an ultramicrotome and glass or diamond knife (1).
  5. Staining the specimen using heavy metal ions, such as lead or osium. These atoms have ...

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