The introduction of the electron microscope as a tool for the biologist brought about a complete reappraisal of the micro-anatomy of biological tissues, organisms and cells. In the early days of its application to biological materials, it was the tool of anatomists and histologists, and many previously unimagined structures in cells were revealed. More recent developments in biological specimen preparation have come from biochemists and physicists who have used the electron microscope to examine cells and tissue in many different ways. The fact that electron micrographs appear in most text books and research papers on cell biology and anatomy emphasize the importance of electron microscopy to the biologist faced with the enormous variety of experimental techniques available for the study of cells.
The two most common types of electron microscopes available commercially are the TRANSMISSION ELECTRON MICROSCOPE (TEM) and the SCANNING ELECTRON MICROSCOPE (SEM). In the SEM, the specimen is scanned with a focused beam of electrons which produce "secondary" electrons as the beam hits the specimen. These are detected and converted into an image on a television screen, and a three-dimensional image of the surface of the specimen is produced.
Specimens in the TEM are examined by passing the electron beam through them, revealing more information of the internal structure of specimens.
Although the SEM is used in biology, the TEM is currently the favored tool of the biologist. Here is an attempt at a simple explanation of how the TEM works.
The Transmission Electron Microscope (TEM)
The TEM is an evacuated metal cylinder (the column) about 2 meters high with the source of illumination, a tungsten filament (the cathode), at the top. If the filament is heated and a high voltage (the accelerating voltage) of between 40,000 to 100,000 volts is passed between it and the anode, the filament will emit electrons. These negatively charged electrons are accelerated to an anode (positive charge) placed just below the filament, some of which pass through a tiny hole in the anode, to form an electron beam which passes down the column. The speed at which they are accelerated to the anode depends on the amount of accelerating voltage present.
Electro-magnets, placed at intervals down the column, focus the electrons, mimicking the glass lenses on the light microscope. The double condenser lenses focus the electron beam onto the specimen which is clamped into the removable specimen stage, usually on a specimen grid.
As the electron beam passes through the specimen, some electrons are scattered whilst the remainder are focused by the objective lens either onto a phosphorescent screen or photographic film to form an image. Unfocussed electrons are blocked out by the objective aperture, resulting in an enhancement of the image contrast. The contrast of the image can be increased by reducing the size of this aperture. The remaining lenses on the TEM are the intermediate lens and the projector lens. The intermediate lens is used to control magnification. The projector lens corresponds to the ocular lens of the light microscope and forms a real image on the fluorescent screen at the base of the microscope column.
The human eye can recognize two objects if they are not closer than 0.1 mm at a normal viewing distance of 25 cm. This ability to optically separate two objects is called resolving power. Any finer detail than this can be resolved by the eye only if the object is enlarged. This enlargement can be achieved by the use of optical instruments such as hand lenses, compound light microscopes and electron microscopes.
Resolution in the light microscope
In the light microscope, the quality of the objective lens plays a major role in determining the resolving power of the apparatus. The ability to make fine structural detail distinct is expressed in terms of numerical aperture (NA). The numerical aperture can be expressed as n sine where n is the refractive index for the medium through which the light passes (n air =1.00; n water = 1.33; n oil = 1.4), and is the angle of one half of the angular aperture of the lens. Light microscope objective and condenser lenses are usually designated by this NA value.
The limits of the objective lens are such that cannot be greater than 90 degrees, and the object space, even if filled with oil, can only reach an NA of 1.4. The resolving power of the light microscope is also limited by the wavelength of the light used for illumination.
Changes in resolution with wavelength (light microscope)
Resolution improves with shorter wavelengths of light
It can be seen from the above table that resolving power improves as the wavelength of the illuminating light decreases. To explain this more fully, the resolving power of the optical system can be expressed as
The optimal resolving power for a light microscope is obtained with ultraviolet illumination ( = 365) if a system with the optimal NA is used (1.4).
R = 130.4 nm
In the visible region of the spectrum, blue light has the next shortest wavelength, then green and finally red. If white light is used for illumination then the applicable wavelength is that for green. This is in the middle range of the visible spectrum and the region of highest visible sharpness.
Improvement of resolving power
Due to this limitation of resolving power by light microscopy, other sources of illumination, with shorter wavelengths than visible light, have been investigated. Early experiments using X-rays of extremely short wavelength were not pursued further because of the inability to focus these rays. The first breakthrough in the development of the electron microscope came when Louis de Broglie advanced his theory that the electron had a dual nature, with characteristics of a particle or a wave. The demonstration, in 1923 by Busch, that a beam of electrons could be focused by magnetic or electric fields opened the way for the development of the first electron microscope, in 1932, by Knoll and Ruska. Although the initial development of the electron microscope, in Germany, was followed by technical improvements in America, the first commercially available apparatus was marketed by Seimens.
The first uses of electron microscopes were restricted to material science applications. It wasn't until the late 1950's and early 1960's, when specimen preparative techniques for biological material were developed, that electron microscopes became popular with biologists.
Resolution in the electron microscope
The resolving power of the TEM is approximately three orders of magnitude (10,000) greater than that of the light microscope. The very short wavelength of electrons and modern technology makes it easy to obtain a resolution of 3 nm on a TEM. As electrons are accelerated through a potential difference of V volts, they have a wavelength ( ) equal to
then the resolution is 0.27nm.
Depth of field in the light microscope
In addition to the poor resolving power, the light microscope has another limiting factor which is overcome in the TEM. The depth of field, a term used in photomicroscopy as well as ordinary photography, is the distance from the nearest part of the subject to the farthest part of the subject that is in focus when the picture is being taken. (Do not confuse with "depth of focus," which is the distance that the image can be moved and still be in focus). In photomicroscopy, the higher the magnification and the higher the NA, the shorter the depth of field. As the NA is increased, the aperture of the objective lens widens and the depth of field is reduced, much as occurs when the lens of a normal camera is "opened up" by a photographer. For example, an objective lens with a NA of 0.25 will give a depth of field of only 100 nm.
The obvious answer to the limitation on depth of field is to use only objectives with a low NA. Unfortunately, it is not that simple because the resolution of the lens is dependant upon the NA. This means that the useful magnification obtainable by an objective lens is also dependent upon the NA. If a high magnification is needed this must also be accompanied by an increase in resolution. Although it is possible to enlarge a microscopic image to any size, it is not practicable. As a rule, the useful magnification of any objective lens is approximately 1000 times its NA. Any magnification higher than this will not resolve more detail but will only give "empty magnification." If higher magnification is needed, then an objective with a higher NA must be used, even though this results in a decrease in the depth of field.
Depth of field in the electron microscope
The focal length of the objective lens of an electron microscope operating at 80 kV can be up to 1 mm. In photographic terms, this is the same as a stopped-down lens and thus enhances the depth of field. In fact the depth of field of the electron microscope is several hundred times greater than that of a light microscope equipped with a high NA objective lens. The result of this is that, within limits, it is not critical to have specimens on one plane to remain in focus.
Also increased in the electron microscope is the depth of focus. This means that the position of the image recording apparatus (the photographic film) is not critical and is often placed several centimeters below the viewing screen.
Specimen preparation for TEM
The greatest obstacle to examining biological material with the electron microscope is the unphysiological conditions to which specimens must be exposed.
Since the material must be exposed to a very high vacuum ( to Torr) when being examined, it must be dried at some stage in its preparation. The biological specimen must be stabilized (or fixed) so that its ultrastructure is as close to that in the living material when exposed to the vacuum.
The limited penetrating power of electrons means that the specimens must be very thin or must be sliced into thin sections (50 - 100 nm) to allow electrons to pass through.
Contrast in the TEM depends on the atomic number of the atoms in the specimen; the higher the atomic number, the more electrons are scattered and the greater the contrast. Biological molecules are composed of atoms of very low atomic number (carbon, hydrogen, nitrogen, phosphorus and sulphur). Thin sections of biological material are made visible by selective staining. This is achieved by exposure to salts of heavy metals such as uranium, lead and osmium, which are electron opaque.