Electrons are suitable for use in microscopy for two main reasons, their wavelength is equally as short as that of X rays and they can be easily focused through the use of electromagnets. The electromagnets work in the same way as lenses for light, by altering the path of the beam. Electron microscopes were first being used in the 1930’s and 1940’s, but it wasn’t until after WW2 that they were sophisticated enough for viewing cells.
There are two different types of electron microscope, the transmission electron microscope (TEM) and the scanning electron microscope (SEM). The transmission electron microscope was the first to be invented, and works by passing a beam of electrons through the specimen. Only electrons that pass through or are transmitted by the specimen can be seen. This allows us to view thin sections of specimens and view inside the cells.
The scanning electron microscope uses and interprets a reflected beam from the specimen. This microscope provides advantages in terms of being able to view surface structures and provides a large depth of field so large areas of the specimen can be viewed at once. However the SEM does not have the same resolving power as the TEM. The SEM is a lot simpler to use than the TEM as the preparation is a lot easier. Specimens usually do not need to be sectioned. However most specimens do need to be coated in gold to prevent them becoming negatively charged and this can affect the accuracy of the result.
Electron beams do not stimulate the human eye, and so to view the image created by the electron microscope the beam must be projected onto fluorescent screens. This produces a “black and white” image. Stains are often used to improve the contrast of biological specimens. However these stains must be caused by very heavy metals such as lead because they are the only substances dense enough to stop the electron beam. This results in an X ray type image, where denser parts of the specimen appear darker. “False colour” images can now be created by computer.
Whilst the electron microscope was an immense step forward in microscopy it too has it limitations. Electron beams can only be focussed accurately in vacuums. This is because in air they would interact with all the other atoms and molecules present in the atmosphere, making it impossible to focus them. This means that the entire process of using the electron microscope must take place within a vacuum. This presents problems when preparing specimens as:
- All specimens have to be dead as nothing can survive in a vacuum.
- All specimens must be fully dehydrated as water boils at room temperatures in a vacuum.
- All specimens must be encased in resin-type substances, as there is no pressure in a vacuum and this would cause most fragile specimens to explode, the resin is an attempt to prevent this.
Obviously these requirements bring about their own problems and add limitations to the electron microscope. For instance it is not possible to measure the growth rate of a live specimen in an electron microscope. It is also virtually impossible to view specimens as they were in life, specifically because they must be dehydrated. There are also other difficulties with the electron microscope mainly relating to the preparation of specimens and the presence of artefacts. Specimens have to endure a very rigorous preparation. There are six stages of preparation.
- Fixing. Specimens are first fixed in very strong chemicals such as osmic acid to prevent any post mortem denature of the specimens. These chemicals bring about large chemical changes and it is not clear how that affects the specimens.
- Dehydration. The specimen is dehydrated slowly by using increasing concentrations of ethanol or propanone (alcohol has a dehydrating effect as it absorbs water)
- Clearing. The specimen is immersed in xylol as this makes the specimen transparent.
- Embedding. The specimen is embedded in a substance such as araldite. This is for two reasons, it helps to protect the specimen during sectioning, and it also helps to provide strength to the specimen in the vacuum.
- Sectioning. In order for electrons to be able to pass through the specimen the specimen needs to be approximately 20-100nm thick. Sectioning is done using a machine called an ultra microtome; this moves the embedded sample forward in 20nm blocks. A diamond or glass knife is then used to cut sections. These sections are collected in water, to prevent damage.
- Staining. Electrons are not distorted by biological samples. Therefore it is necessary to stain the samples to differentiate between different structures. However electrons can only be prevented from passing through substances with very high densities, so very heavy metals are used.
It is impossible to say with precise accuracy how this process affects the specimen. However it is possible to conclude that the end result will not be exactly the same as the initial specimen. There are often artefacts in the specimens; frequently these are from the heavy metal staining, but they may be caused by a number of things, from chemical changes caused by the fixing chemicals to structures physically altering in the dehydrating and embedding phases. This has made it difficult to understand whether or not the conclusions found from electron micrographs are real, or if they are interpretations of artefacts.
Another limitation of the electron microscope is its cost and size. They are prohibitively expensive and as such are only usually found in universities and industrial laboratories. This obviously limits the amount of people who have access to them.
The advantages of electron microscopy are clear, in that it allows a far more detailed knowledge of the microscopic world than has ever been possible before. However it does bring many difficulties, and whilst ways have been found to overcome these the result is far from perfect.
Here's what a star student thought of this essay
Quality of writing
The spelling and grammar are sound, with a few places of questionable punctuation problems. Namely, I question the lack of the use of a comma after the word however (I was always taught this was imperative). The technical terms used in this piece of work is that which is expected of students at A level, however I would suggest putting in a glossary which would help further demonstrate the understanding of the topic and therefore, the question set. The student follows the general expectations of an A level student. To make this piece of work 'stand out from the crowd', extra explanation would be needed to demonstrate further understanding of the microscopes. Whilst such explanations are not required to obtain high marks, it will impress the examiner and arguably, will make them more likely to award the candidate a higher mark.
Level of analysis
The analysis in this piece of work would be the knowledge of understanding how the microscopes work. The level of understanding is very high in this piece of work and the student clearly understands the topic. Yet, I feel that the wording of the explanations are tending towards a 'textbook' style. Whilst it is hard to avoid this assertion (which occurs a lot in textbooks), it is best to avoid copying the style of writing of a textbook as this means that the work is more likely to become just a paraphrased essay of the textbook. This said, I do not feel that this piece of work is plagarised at all. The appropriate conclusion was reached but it should be clearer such that the examiner can easily identify the advantages and disadvantages to award the marks.
Response to question
The introduction was spot on and introduced the student's work well. It addressed the question and guides the reader well into the essay. The response is clearly set out and is set out logically. However, in assessing the advantages and disadvantages, I would say that more explicit points need to be made. Whilst most of these advantages and disadvantages are implied whilst the student explains the science behind the technology, I feel that more explicit reference to the question is needed. This reduces all doubts that the examiner should have about whether the student has explicitly answered the question. Nevertheless, the understanding of the topic is clearly shown in this piece of work.