- Fixing the specimen in a solution of fixing chemicals. This hardens the tissue so the structure stays the same (1)
- Dehydrating the specimen by passing it through alcohols of gradually increasing concentrations. This prepares the sample for embedding (1).
- Embedding the sample in resin and then baking the resin to make it hard. This ensures the cells are not distorted during sectioning (1).
- Sectioning. During sectioning, the specimen is cut into very thin slices using an ultramicrotome and glass or diamond knife (1).
- Staining the specimen using heavy metal ions, such as lead or osium. These atoms have large, positively charged nuclei which do not allow electrons to pass through. This means the electrons do not arrive on the screen, so there is a dark area on the image. Therefore, the structures which absorb these heavy metal stains appear dark
The specimen is finally mounted on a copper grid, ready to be viewed (1).
A beam of electrons is produced using an electron gun. This consists of a tungsten filament, a cathode, and an anode with a hole in it. The tungsten filament is heated to about 3000oC, and this emits electrons (1). These electrons are thrown out of their orbits using high voltages between 50kv and 100kv (3), and as they pass through the negative cathode, the density of electrons is increased. The anode helps to accelerate the electrons down the microscope because the electrons are attracted to the positive anode but pass through the hole in it rather than sticking to the plate. The higher the voltage used, the shorter the wavelength of the electron beam, so the better the resolution of the microscope (1).
The electrons are focused on to the specimen by a condenser. In electron microscopes this is an electromagnet which straightens and intensifies the beam of electrons (5). Electromagnets are used to focus the beam because they can deflect the negatively charged electrons (2). After passing through the specimen, the radiation is focused by an objective lens (5). The human eye cannot see electrons, so the projector lens focuses the final image on to a fluorescent screen which emits visible light where the electrons hit (4). This gives a black and white picture. It is possible to obtain a photograph of the final image by allowing the electrons to pass on to a photographic film. This produces an electron micrograph (3).
The main advantage of a TEM is the high resolution and magnification that can be obtained. This allows very detailed images of the cell structure to be seen. However one major disadvantage of TEMs is that electrons are easily scattered or absorbed by air molecules. Therefore, the specimens must be viewed in a vacuum. This, and the fact that the specimens must be cut very thinly means there are limitations on what can be viewed using an electron microscope. Usually only dead material can be viewed (2). The preparation of the specimen is time consuming, and it is difficult to ensure that the specimen resembles a living cell because it might be affected during preparation. Once the specimen is being viewed, it gradually deteriorates in the electron beam. This means photographs have to be taken have to be taken if the specimen needs to be studied further. As well as these disadvantages, the microscope is expensive to buy and run, and training is needed to be able to prepare the specimens and use the microscope effectively (3).
Scanning electron microscopes were invented after TEMs. Here, electrons do not pass
through the specimen (2). Electrons are emitted by an electron gun, and a beam is formed by the first condenser lens. The second condenser lens focuses the beam further to from a thin, tight beam. This beam passes through a set of coils which scan the beam to and fro over the specimen. The final lens, the objective, focuses the beam on to the desired part of the specimen (7). When the electron beam hits the specimen, it causes atoms at its surface to emit secondary electrons (2). These electrons are collected and counted by an electron counter at the other side if the microscope. This counter is positively charged to attract the electrons (1). Each scanned point on the specimen appears as a pixel, or spot, on the monitor of a cathode ray tube. The spot is brighter with the more electrons the counting deice detects. As the electron beam scans over the sample, the spot scans over the television screen to create a complete three-dimensional image of the specimen on the monitor of the cathode ray tube (6).
The resolving power of a SEM is approximately 5-20nm, less than a TEM, because the
electron waves do not have such a short wavelength. However, the SEM has other
advantages (2). The specimen needs less preparation than in a TEM. Organisms with hard
outer coats do not need any preparation, and freeze etching can be used on more delicate
materials. In freezing etching, the specimen is frozen in liquid nitrogen and then fractured
along a line of weakness using a razor. This line of weakness is usually at the cell
membranes. The specimen is kept in a vacuum where the ice sublimes away to leave an
etched surface. A layer of carbon is then deposited on the surface to form a replica, and this
is coated in a layer of heavy metals. The heavy metals are used because they are good
emitters of secondary electrons. The specimen can then be destroyed and the replica
viewed (1).
SEMs show the surface of the structure and have great depth of field which enables them to
give a very detailed three dimensional image(3). They can be used to study relatively large
objects. The images are likely to look more lifelike because the replica is formed before any
chemical damage is done to the specimen (5). It is possible to view some organisms alive-
some insects can withstand the vacuum.(3). However, SEMs do not have the resolving
power of TEMs (5). External disturbances such as a stray magnetic field and mechanical
vibrations can cause image distortion, jagged edge lines and other phenomena (7).
References
1. Adds J. (1992) Tools, techniques and Assessment in Biology Nelson
Larkcom, E.
Miller, R.
Sutton, R.
2. Arms, K. (1988) Biology, a journey through life Saunders
Camp, P.S.
3. Green, N. P. O (1997) Biological Science 1 (Third Edition) Cambridge
Soper, R.
Stout, G. W.
Taylore, D.J.
4. Jones, G. (1997) Advanced biology Cambridge
Jones, M.
5.Marcus Barbor (2000) Biology (2000 Edition) Collins
Mike Boyle Advanced Science
Mike Cassidy
Kathryn Senior