| Contents for this page | Related topics |  |
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General principles of electron microscopy The scanning electron microscope (SEM) The transmission electron microscope (TEM) Differences between TEM and SEM |
The wave nature of matter |
 Data Glossary |
| Learning Outcomes | ||
| After studying this section, you will understand the principles underlying the electron microscope. | ||
There is a limit to the resolution that one can obtain in microscopes that operate with visible light. The resolution increases with increasing frequency of the light beam, and electron microscopes use a beam of electrons to examine specimens on a much smaller scale than a light or optical microscope. The interaction of the electron beam with the specimen gives information concerning its morphology, topography, crystallographic arrangement and elemental composition.
Electron microscopes are very costly instruments (usually costing upwards of a couple of million rands), and require extensive facilities and highly trained and skilled staff in order to operate them. Their major applications are in the fields of health sciences, molecular and cellular biology, biotechnology, structural biology and engineering.
The electron microscope consists of an electron source, an anode, magnetic lenses, apertures, specimen stage and image recording system all of which operate in a high vacuum.
The electron source can be made of various types of materials. The most common is the tungsten filament which, when heated, produces electrons which are attracted by the anode and are accelerated down the column and interact with the specimen. The electrons are focused using magnetic lenses in the column and the apertures filter out scattered electrons so the resulting beam is monochromatic. This monochromatic beam is focused and interacts with the specimen in different ways, depending on the type of electron microscope. These interactions are detected and converted into an image by the image recording system. This system converts the radiation into a permanent image either onto photographic film, or into a digital image.
Two types of electron microscopes are normally available, the SCANNING ELECTRON MICROSCOPE, (SEM), and the TRANSMISSION ELECTRON MICROSCOPE, (TEM). Both these types are discussed in some detail below.
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In the SEM, a set of scan coils moves the electron beam across the specimen in a 2 dimensional grid fashion. When the electron beam scans across the specimens, different interactions take place. These interactions are decoded with various detectors situated in the chamber above the specimen. Some electrons from the surface material are knocked out of their orbitals by the electron beam, and are called SECONDARY ELECTRONS. These electrons are detected by the secondary electron detector. Different interactions give images based on topography, elemental composition or density of the sample. A SEM can magnify up to about 100,000x.
 Budding yeast cell, original magnification 32 000X |
 E. coli bacteria, original magnification 30 000X |
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In the TEM, the focused, monochromatic electron beam interacts with and is transmitted through the sample, focused into an image and projected onto a phosphor coated screen which emits visible light. The brighter areas of the image represent areas where more electrons have passed through the sample. The darker areas represent areas where fewer electrons have passed through as a result of higher specimen density. A TEM can magnify up to about 500,000x
 Thin section of budding yeast cell |
 Thin section of E. coli bacteria |
| TEM | SEM |
|---|---|
| Electron beam passes through thin sample. | Electron beam scans over surface of sample. |
| Specially prepared thin samples or particulate material are supported on TEM grids. | Sample can be any thickness and is mounted on an aluminum stub. |
| Specimen stage halfway down column. | Specimen stage in the chamber at the bottom of the column. |
| Image shown on fluorescent screen. | Image shown on TV monitor. |
| Image is a two dimensional projection of the sample. | Image is of the surface of the sample. |
| Microscope | Resolution | Magnification |
|---|---|---|
| Optical | ± 200 nm | ± 1000X |
| TEM | ± 0.2 nm | ± 500 000X |
| SEM | ± 2 nm | ± 200 000X |
The RESOLUTION or RESOLVING POWER of a microscope is the instrument's ability to separate two objects that are close together. The minimum separation, d, that can be resolved by a microscope is given by the formula shown below:
where λ is the wavelength of the light used, n the refractive index of the medium separating the specimen and the objective lens, and θ the angle subtended by the objective lens. |
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The resolution therefore is inversely proportional to the wavelength (that is, directly proportional to the frequency) - for a small d (a desirable outcome), λ should be correspondingly small. With visible light, this imposes a limit to the resolution that can be obtained. A beam of electrons can have energies that equate to wavelengths down to 5x10-3 nm, or about 100 000 times less than visible light, reducing d to values around 0.2 nm.
