To find the length of one eyepiece unit under low power, a 10 mm stage micrometer is used. This is a scale 10 mm long, divided up into tenths of one millimetre. When viewed under a microscope using the eyepiece graticule, the zeros of both scales are lined up. When a mark on both the micrometer scale and the eyepiece graticule scale lined up, a measurement in millimetres is obtained for a certain number of eyepiece units. The stage micrometer value is then divided by the number of eyepiece units to give the length of one eyepiece unit in millimetres. The same is then done under medium and high power, using a 1 mm stage micrometer divided into hundredths of a millimetre, so that a value for the length of one eyepiece unit is obtained for each magnification enabling dimensions of minute objects to be measured.
There are two types of preparation for viewing freshly killed biological material under a compound microscope - permanent preparation and temporary preparation. Temporary preparation is quicker and so more suitable for a preliminary investigation, while permanent preparation results in a slide that can be used several times and over a long period of time.
The following are the stages involved in the preparation of a permanent slide:
1. Fixation:
Obviously, it is necessary that the freshly killed biological material be preserved in a life-like condition. This is done using a fixative, a chemical, such as formalin, which hardens the tissue so that the structure is retained when sections are cut.
2. Dehydration:
The removal of water is necessary because firstly, if it were present bacterial decay would occur, and secondly, the embedding medium, see embedding, would not mix if any water were present. Dehydration is usually accomplished by gradually increasing the concentration of added alcohol, usually ethanol, until ‘absolute alcohol’ is reached – nearly pure alcohol.
3. Clearing:
In most cases alcohol is immiscible with the embedding, see next stage, or mounting media, so it is replaced by a clearing agent, such as xylol or xylene, which does mix. The agent also makes the material transparent, which is necessary, as light rays must travel through the specimen into the eye.
4. Embedding:
Before the material can be cut into thin sections to allow light through, it must be embedded using a supporting medium so that the slices do not collapse. In optical microscopy, the material is impregnated with molten wax, which consequently sets.
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5. Sectioning:
Once the wax has settled, the material can be cut up into thin sections. This is either done using a razor or a microtome. The latter being a machine that cuts extremely thin slices, usually between 3 and 20 micrometres. A freezing microtome may be used which avoids the need of embedding as the specimen is frozen and therefore firm. A razor can also be used in sectioning although this method is more difficult as it relies on manual dexterity – sections are cut in swift, horizontal actions, drawing the razor to the body.
6. Staining:
In order to distinguish between transparent structures under a light microscope, it is necessary to artificially colour them, so that sufficient contrast is achieved. This is done using stains or dyes, such as methyl blue. Counterstaining, or double staining, generates even better contrast, as different stains colour different structures in tissues. Note that when wax-embedded sections are stained, the wax is firstly dissolved away and the specimen is partially rehydrated. Living material is stained using vital stains as these are non-toxic and so do not kill the specimen.
7. Mounting:
Finally, the thin sections are mounted on a microscope slide in a mounting medium, such as Canada balsam or euparol, which excludes air so that no air bubbles are present. The mounted specimen is then covered with a glass cover slip, and is ready to be used.
As mentioned before the preparation of a temporary slide is quicker as only fixation, sectioning, staining and mounting are involved. Sectioning is done using a razor, and only temporary stains can be added to the specimen. As before, the specimen is mounted on a microscope slide, to which some drops of alcohol may be added, and a cover slip is then placed over the specimen in order to exclude air.
Electron Microscopy:
As mentioned before in the introduction, the electron microscope (EM) has greater magnification and resolution than the compound microscope. The resolution, or resolving power, of a microscope is dependant upon the wavelength of illumination being used. As a beam of electrons has a wavelength of approximately 0.005 nm, shorter than that of light, which is approximately 500 nm, the resolution of an electron microscope is far greater. At best a compound microscope can distinguish two points which are 200 nm apart while an electron microscope can resolve points 1 nm apart.
There are two types of electron microscopy, transmission electron microscopy and scanning electron microscopy.
Transmission Electron Microscopy: (TEM)
In a transmission electron microscope, a high voltage is applied to a heated tungsten filament, which as a result generates electrons. To accelerate the electrons, a high negative voltage is applied to a cathode assembly, under which an anode plate, positive charge, is positioned. The electric field generated accelerates the electrons, which then pass through a slit in the anode plate forming a beam. This collection of apparatus is known as an electron gun.
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The electron beam is focussed onto the specimen by magnetic coils, which act the condenser lenses, see figure 2. The specimen is positioned between the condenser lens and the next magnetic coil, the objective lens, which magnifies the image and resolves structure. Varying the current passing through this magnetic coil focuses the specimen. The final magnetic coils, also known as the projector lenses, enlarge the image, projecting it onto a fluorescent screen so that the human eye, which is insensitive to electrons, can see the image, as the fluorescent screen is sensitive to electrons. The final image will show different tones of black and white depending on the densities of the various parts of the specimen. In those areas of the specimen that are dense, more electrons are absorbed resulting in a darker tone on the image, and those areas which are less dense show up on the image as lighter tones as more electrons pass through. Note that the electron beam can also be focussed onto a photographic plate for permanent recording, resulting in a photograph known as an electronmicrograph.
As electrons travelling through the microscope column have low energy, a vacuum is necessary to prevent any scattering and deflecting of electrons caused by collisions with the nuclei of atoms composing air, which would distort the final image.
Figure 2, a transmission electron microscope:
Preparation of biological material for TEM:
Firstly, the biological material is killed and chemically ‘fixed’ in a life-like condition using glutaraldehyde, which strengthens the subcellular structure. During fixation, one must be careful not to cause any artefacts, objects which appear in the image but aren’t present in the specimen. Plasmolysis, rupture of membranes, coagulation of proteins all cause artefacts. The material is then treated with osmium tetroxide, another fixative.
As a vacuum is required in the electron microscope, the material is dehydrated using acetone.
Next, the specimen is embedded in acrylic resin to provide support when it is cut into very thin sections. For electron microscopy slices must be less than 2 μm thick. If the sections were too thick, electrons would not pass through and there would be no contrast in the image. The
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slices are cut using an ultramicrotome, a cutting machine similar to a microtome used for sectioning in optical microscopy.
The fragile sections are then placed on a supporting copper grid and ‘stained’. In electron microscopy electron-dense substances, such as lead and uranyl salts, are used to ‘stain’ sections, as these absorb a lot of electrons thus improving the contrast on the final image.
Another method of preparation of biological material for electron microscopy is freeze-etching. This procedure avoids the need to fix, section and stain before EM examination, and also allows us to check for any artefacts by comparing the transmission electronmicrograph with the electronmicrograph of the specimen prepared using freeze-etching. To prepare a specimen using freeze-etching, it is firstly fast-frozen using liquid nitrogen. The specimen is then fractured into sections with a knife along lines of weakness. Because solid ice evaporates as water vapour from the surface of each section, structures within the cell are exposed. A copy of the surface is then made by depositing a layer of carbon onto it. The copy is then shadowed by condensing heavy metal atoms onto the carbon layer. The specimen is thawed and the carbon layer replica is removed and examined in TEM. As heavy atoms are opaque to electrons, a shadow effect is produced, allowing detail to be seen in the image.
Scanning Electron Microscopy: (SEM)
In SEM the whole specimen is exposed to a beam of electrons. An image is then created from the electrons reflected from the surface of the object. Although the resolution isn’t as good as with TEM, scanning electronmicrographs show depth of focus and the object is three-dimensional. Another advantage is that SEM allows larger specimens to be examined.
When comparing optical microscopy and electron microscopy, it is clear that each has its own advantages and disadvantages over the other. Obviously, an electron microscope produces an image of greater magnification and resolution than optical microscopes due to the very nature of light, but electron microscopy doesn’t produce coloured images. EM is also very expensive and preparation takes a lot of time. Another disadvantage in EM is that the specimen has to be nonliving, which can be a cause for irritation as studying living material under high magnification and resolution would help us to learn more about cell structure and function, for example.
To conclude, the choice of one type of microscopy over the other is dependant on the requirements of the given study. For example, if one wanted to observe the structure of mitochondria one would choose EM over optical microscopy because it has greater magnification and resolving powers.