The development of the electron microscope
It was the outstanding work of the German Ernst Ruska (1906-1987) in the early 1930’s, which led to the development of the electron microscope. Ruska had shown in his PhD theory the potential for electrons to be used in a microscope. Backed initially by Max Knoll, he put theory into practice and constructed the first electron microscope in 1933. In this type of a microscope, a high vacuum is required in which electrons can be speeded up so that their wavelength is extremely short, which creates an electron beam. Electro-magnetic lenses focus the electron beam on a cell sample and an image is formed on an electron-sensitive photographic plate. In 1986 Ernst Ruska was eventually awarded the Nobel Prize in Physics for his early achievements and fundamental work in electron optics.
By the contact between the electron beam and the cell sample a variety of electrons are produced. Individual scattered electrons that are formed after the electron beam has interacted with the cell sample reveal the internal structures of biological tissues and cells. These are observed in a Transmission Electron Microscope. Secondary and backscattered primary electrons reflected off the surface of cell samples, after an electron beam has scanned such surfaces, reveal the exterior layer of biological tissues and cells. These are observed in a Scanning Electron Microscope.
Electron microscopes can, when pushed to their boundaries, view objects as small as the diameter of an atom. It can magnify up to 1 million times, but electron microscopes have a severe drawback, which is that no living specimen can survive under their high vacuum. This means that they cannot display the movements that occur within a living cell.
A Light Microscope
How the electron microscope has given us a greater understanding of cellular structure
Electron microscopes have proven powerful research tools for investigating the basic structure of matter, specifically in the fields of biology and solid-state science. They have, for example, helped to reveal the surface structures of various materials and confirmed the shapes and behaviours of bacteria as well as animal and human cells and cell components. They are important in research examining the effects of various manipulations or treatments of these various types of subject matter. Scientists and publishers often add colour to the highly detailed electron micrographs to increase interest, to help distinguish portions of the image, and to highlight important areas. Electron microscopes have given scientific and lay media remarkable pictures, such as the “faces” of insects, the shapes of microscopic organisms, and the surface structure of molecules of new, high-tech alloys and other substances. They are also becoming important to regular clinical pathology at medical centres.
A clear explanation of the terms magnification and resolution (with quoted figures)
Magnification:
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The seeming increase in size of an object viewed under the microscope; when noted, this increased size is expressed by a figure preceded by ×, indicating the number of times its diameter is enlarged.2
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Apparent increase in size as under the microscope.3
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The process of making something appear larger, as by use of lenses.3
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The ratio of apparent (image) size to real size.3
Magnification is the number of times larger an image is than the specimen. For example, if a cell is 10 nm in diameter, and a microscope produces an image of it, which is 1 mm (1000Mm) in diameter, then the microscope has magnified the image 100 times:
Magnification = Size Of Image
Size of Specimen
The magnification produced by the light microscope depends on the strengths of the objective lens and the eyepiece lens. If you are using ×40 objective lens and the ×10 eyepiece lens is being magnifies 400 times.
Resolution:
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The optical ability to distinguish detail such as the separation of closely adjacent objects.2
- The perception as separate of two adjacent objects or points. In microscopy, it is the minimal distance at which two adjacent objects can be distinguished as separate. The resolving power of an instrument depends on the wavelength of the radiation used and the numerical aperture of the system; it is expressed in microns distance or lines per millimetre.
Resolution is the degree of detail that can be seen. The limit of resolution of a microscope is the minimum distance by which two points can be separated and still be seen as two separate points and not one fuzzy one. The limit of resolution depends on the wavelength of the light. The resolution limit is about 0.45times the wavelength. Short Wavelengths give the best (smallest) resolution .The shortest wavelength light that we can see is blue light, which has a wavelength of about 450nm. This gives a resolution of about 0.45 ×450nm, which is close to 200 nm.
The difference between transmission and scanning electron microscopes
Commercial TEM’s were developed around 1938 and SEM’s from around 1965.
Transmission Electron Microscope (TEM)
- Beam of electrons is directed through the sample similar to light microscope but with much greater resolution.
- Image produced of the interaction of the electrons with the entire volume of the sample.
- Image can then be digitised by feeding it into a computer, but can also be recorded on film without using a computer.
- All new TEM’s now have built-in computers to digitise images, although film is still being used for the highest of resolution images.
- Resolution is near atomic levels.
- Magnification maximum for standard TEM’s is between 500,000X and 1,000,000X.
- Specimen preparation requires dried specimens, which in the case of biological tissue specimens must be fixed, dehydrated, embedded, and sectioned to be able to preserve and see through them. Thick material samples must be sectioned or thinned. Particles and viruses can be looked at with minor preparation.
Scanning Electron Microscope (SEM)
- Scans a very fine probe of electrons over a specimen surface similar to that used in a TV.
- The SEM collects the electrons emitted from the sample and builds up an image of the surface of the sample.
- This can be digitised with a computer but also can be recorded on film without a computer.
- All new SEM’s now have built-in computers to digitise images, although film is still being used for the highest of resolution images.
- Resolution is near atomic levels.
- Magnification maximum for standard SEM’s is 100,000X.Specimen preparation is simpler in SEM than in TEM, but must be conductive in some manner. In the new SEM’s that can operate at almost atmospheric pressure (minimal vacuum required), wet samples can be viewed with reasonable resolution.
“The two types of electron microscope have some similarities and some differences. They are similar in that they both examine specimens under vacuum; but they differ in how the images are produced. The TEM, as the name implies, actually relies on the transmission of the beam through the specimen: the direct physical interposition of the specimen into the beam is necessary to produce the image. The SEM, on the other hand, can examine specimens much too thick for the beam to pass through, and its image is produced electronically by modulation of the CRT output based on the detector signal. Therefore, no physical connection exists between the object and its image; the column can be in one room and the display CRT in another. So long as the electronics are intact, the image can be produced. Furthermore, contrast and brightness can be adjusted, as on a TV set, with console controls. By actually making images of the CRT screen photographic records are made. Alternatively, the output can be fed to a videotape recorder.” 4
How specimens are prepared for viewing with the light and electron microscope 4
The advantages and disadvantages of the light and electron microscopes
Light Microscopes
Advantages:
1) Easy to use.
2) Inexpensive (relative to electron microscopes).
3) Can look at live samples.
4) Can magnify up to 2000 times.
Disadvantages:
1) Can't magnify more than 2000 times.
Electron Microscopes
Advantages:
1) Can magnify up to 200,000 times.
Disadvantages:
1) Very expensive (100 times more than Light Microscope price).
2) Complicated to use.
3) Can't look at live specimen, as most specimens (biological) must be dehydrated (i.e. dead!).
4) Electron Microscope specimens (biological) are rapidly damaged by the electron beam.
Referencing
1
2 Stedman’s Induction task
3 Dorland’s Illustrated Medical Dictionary
4 Martha G. Shapp, Cathleen FitzGerald, Ben Feder, Dr. Lowell A. Martin. (1996). The New Book of Knowledge (12-M). U.S.A. Grolier Incorporated. 281-286
Bibliography
1.
2. Stedman’s Induction task
3. Dorland’s Illustrated Medical Dictionary
4. Martha G. Shapp, Cathleen FitzGerald, Ben Feder, Dr. Lowell A. Martin. (1996). The New Book of Knowledge (12-M). U.S.A. Grolier Incorporated. 281-286
6. Mary Jones, Geoff Jones. (1997). Advanced Biology. Cambridge. Cambridge University Press
7.
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