cambriedge
the shorter the wavelength (rather like squashing a spring!). now look at figure 1.10, which shows a mitochondrion, some very small cell organelles called ribosomes and light of 400nm wavelength, the shortest visible wavelength. The mitochondrion is large enough to interfere with the light waves. However, the ribosomes are far too small to have any effect on the light waves. The general rule is that the limit of resolution is about one half the wavelength of the radiation used to view the specimen. In other words, if an object is any smaller than half the wavelength of the radiation used to view it, it cannot be seen separately from nearby objects. This means that the best resolution that can be obtained using a microscope that uses visible light (a light microscope) is 200 nm, since the shortest wavelength of visible light is 400 nm ( violet light ). In practice, this corresponds to a maximum useful magnification of about 1500 times. Ribosomes are approximately 22 nm in diameter and can therefore never be seen using light.
If an object is transparent it will allow light waves to pass through it and therefore will still not be visible. This is why many biological structures have to be stained before they can be seen.
The electron microscope
Biologists, faced with the problem that they would never see anything smaller than 200 nm using a light microscope, realized that the only solution would be to use radiation of a shorter wavelength than light. Both ultraviolet and x-ray microscopes have been built, the latter with little success partly because of the difficulty of focusing x-rays. A much better solution is to use electrons. Electrons are negatively charged particles which orbit the nucleus of an atom. When a metal becomes very hot, some of its electrons gain so much energy that they escape from their orbits, like a rocket escaping from earth’s gravity. Free electrons behave like electromagnetic radiation. They have a very short wavelength: the greater the energy, the shorter the wavelength. Electrons are a very suitable form of radiation for microscopy for two major reasons. Firstly, their wavelength is extremely short (at least as short as that of X-rays); secondly, because they are negatively charged, they can be focused easily using electromagnets (the magnet can be made to alter the path of beam, the equivalent of a glass lens bending light).
Electron Microscopes were developed during the 1930s and 1940s but it was not until after the second world war that techniques improved enough to allow cells to be studied with the electron microscope.
Transmission and scanning electron microscopes
Two types of electron microscope are now common use. The transmission electron microscope was the type originally developed. Here the beam of electrons is passed through the specimen before being viewed. Only those electrons that are transmitted (pass through the specimen) are seen. This allows us to see thin sections of specimens, and thus to see inside cells. In the scanning electron microscope, on the other hand, the electron beam is used to scan the surfaces of structures, and only the reflected beam is observed. An example of a scanning electron micrograph is shown in figure 1.11. the advantage of this microscope is that surface structures can be seen. Also, great depth of field is obtained so that much of the specimen is in focus at the same time. Such a picture would be impossible to obtain with a light microscope, even using the same magnification and resolution, because you would have to keep focusing up and down with the objective lens to see different parts of the specimen. The disadvantage of the scanning electron microscope is that it cannot achieve the same resolution as a transmission electron microscope.
Viewing specimens with the electron microscope
It is not possible to see an electron beam, so to make the image visible the electron beam has to be projected onto a fluorescent screen. The areas hit by electrons shine brightly. Giving overall a ‘black and white’ picture. The stains used to improve the contrast of biological specimens for electron microscopy contain heavy metal atoms which stop the passage of electrons. The resulting picture is therefore similar in principle to an X-ray photograph, with the more dense parts of the specimen appearing blacker. ‘false-colour ’ images are created by processing the standard black and white image using computer.
(reliable source)
the shorter the wavelength (rather like squashing a spring!). now look at figure 1.10, which shows a mitochondrion, some very small cell organelles called ribosomes and light of 400nm wavelength, the shortest visible wavelength. The mitochondrion is large enough to interfere with the light waves. However, the ribosomes are far too small to have any effect on the light waves. The general rule is that the limit of resolution is about one half the wavelength of the radiation used to view the specimen. In other words, if an object is any smaller than half the wavelength of the radiation used to view it, it cannot be seen separately from nearby objects. This means that the best resolution that can be obtained using a microscope that uses visible light (a light microscope) is 200 nm, since the shortest wavelength of visible light is 400 nm ( violet light ). In practice, this corresponds to a maximum useful magnification of about 1500 times. Ribosomes are approximately 22 nm in diameter and can therefore never be seen using light.
If an object is transparent it will allow light waves to pass through it and therefore will still not be visible. This is why many biological structures have to be stained before they can be seen.
The electron microscope
Biologists, faced with the problem that they would never see anything smaller than 200 nm using a light microscope, realized that the only solution would be to use radiation of a shorter wavelength than light. Both ultraviolet and x-ray microscopes have been built, the latter with little success partly because of the difficulty of focusing x-rays. A much better solution is to use electrons. Electrons are negatively charged particles which orbit the nucleus of an atom. When a metal becomes very hot, some of its electrons gain so much energy that they escape from their orbits, like a rocket escaping from earth’s gravity. Free electrons behave like electromagnetic radiation. They have a very short wavelength: the greater the energy, the shorter the wavelength. Electrons are a very suitable form of radiation for microscopy for two major reasons. Firstly, their wavelength is extremely short (at least as short as that of X-rays); secondly, because they are negatively charged, they can be focused easily using electromagnets (the magnet can be made to alter the path of beam, the equivalent of a glass lens bending light).
Electron Microscopes were developed during the 1930s and 1940s but it was not until after the second world war that techniques improved enough to allow cells to be studied with the electron microscope.
Transmission and scanning electron microscopes
Two types of electron microscope are now common use. The transmission electron microscope was the type originally developed. Here the beam of electrons is passed through the specimen before being viewed. Only those electrons that are transmitted (pass through the specimen) are seen. This allows us to see thin sections of specimens, and thus to see inside cells. In the scanning electron microscope, on the other hand, the electron beam is used to scan the surfaces of structures, and only the reflected beam is observed. An example of a scanning electron micrograph is shown in figure 1.11. the advantage of this microscope is that surface structures can be seen. Also, great depth of field is obtained so that much of the specimen is in focus at the same time. Such a picture would be impossible to obtain with a light microscope, even using the same magnification and resolution, because you would have to keep focusing up and down with the objective lens to see different parts of the specimen. The disadvantage of the scanning electron microscope is that it cannot achieve the same resolution as a transmission electron microscope.
Viewing specimens with the electron microscope
It is not possible to see an electron beam, so to make the image visible the electron beam has to be projected onto a fluorescent screen. The areas hit by electrons shine brightly. Giving overall a ‘black and white’ picture. The stains used to improve the contrast of biological specimens for electron microscopy contain heavy metal atoms which stop the passage of electrons. The resulting picture is therefore similar in principle to an X-ray photograph, with the more dense parts of the specimen appearing blacker. ‘false-colour ’ images are created by processing the standard black and white image using computer.
(reliable source)
