The Use and Operation of the Light and Electron Microscope

Kim Sills 12F Biology

Light microscopes were first used in the seventeenth century, famously by the scientist Robert Hooke; the man that first named ‘cells’. It was not until the nineteenth century however that microscopy became popular. Before this time, the lenses had not been of high enough quality to view images clearly, in the early nineteenth century improvements were made and the identification of cell structures began.

Problems with resolution then became apparent; a wave length shorter than light needed to be used in order to improve the clarity of image produced; Electron microscopes were developed as a solution; both Ultraviolet light and X-rays have shorter wave lengths but it was found that these were extremely difficult to focus. Electrons, however, are negatively charged particles; this gives the advantage of being able to focus them easily, by use of electromagnets. These electromagnets act in exactly the same way as a lens would on a Light Microscope.

How light microscopes work

In a compound light microscope; a light source is located underneath the stage. The light goes through a condenser lens and through the specimen, the resulting light is then passed through two more lenses, both used to magnify the image and focus towards the ocular. The lenses used indicate the resulting magnification of the specimen. Lenses are used as these refract the light, and although the image is inversed, the image is much larger, as is illustrated below.

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Electron microscopes

There are two main types of Electron Microscope, the first is a transmission electron microscope (TEM) this was the first developed electron microscope and is used to see inside of cells. Only those electrons which pass through the specimen may be seen, which gives the impression of black/white and shades of grey on the finished image.

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Electron microscopes

The Electron source is a pin-shaped cathode, heated to a degree that causes a ray of electrons to be produced. The vacuum that exists inside the microscope then causes the electron ray to enter through the condenser. The condenser is an electromagnet that focuses the electron rays onto the specimen. Electrons are then deflected when the hit areas of the specimen; this is due to the specimen being stained with heavy metals, the heavy metal will be absorbed by specific parts of the cell that will then deflect the electrons differently and allow contrasts to be seen within the cell.

The Projective (A series of lens-systems) then focuses the resulting electron ray onto a fluorescent screen; where a black and white image is formed.

The other main type of electron microscope is a Scanning electron microscope (SEM) this is used to see only the surface of the specimen, as the image is produced from the reflected electrons.

The specimen is first coated with a heavy metal, usually gold.

The surface of the specimen is scanned point by point by the primary electron ray, this then causes secondary electrons to be set free, their intensity of radiation is dependent upon the angle of inclination upon the object’s surface.

This secondary wave of electrons is then detected by the Collector, the signal is enhanced using computers and an image is produced at the chosen magnification.

Differences in resolution and magnification

Magnification is the number of times larger an image is in comparison to the actual size of the object, whereas resolution is defined as the ability to distinguish between two separate points.

A Light Microscope is not limited by magnification however it is severely limited by the resolution of the image it produces. A light microscope has a maximum resolution of 200nm, due to the smallest wave length of light being 400nm, if two points are closer together than half this distance they cannot be seen on a light microscope because they do not disturb the light waves.

The electron microscope was designed to overcome this problem, as they have a very short wave length which can be altered by the acceleration voltage used within the microscope, at the anode. Electron microscopes can therefore see objects smaller than 200nm and produce images of a much higher resolution. In reality, an electron microscope can be used to view objects that are a minimum of 0.5nm in size.

Electron microscopy must take place within a vacuum, due to the interference that air would provide. In a vacuum, however, water boils at room temperature and living organisms cannot survive. Electron microscopy is therefore limited to non-living organisms.

Slide preparation

Fixation must take place for electron microscopes and depends upon the specimen for light microscopes, the chemical OsO4 is used. The specimen is, next, dehydrated. Then the specimen is embedded using resin or plastic and a cross-section of this is then cut using an Ultramicrotome, no more than 100nm thick for a TEM.

The Electron microscope uses heavy metals as stains, such as lead and uranium, in order to show contrasts between the different structures within the cell.

Light microscopy samples must be stained, in order for transparent objects to be seen and to make structures easier to see, there are many different stains that are used which are specific to different organelles e.g. eosin shows cytoplasm as pink whilst haematoxylin is used to stain nuclei.

Special techniques include Freeze-Fracture Analysis;

The samples are frozen, quickly, using liquid nitrogen, transferring the water to a glass-like state. This is then fractured using a glass knife, the preparation splinters with the break running along the membrane of the cell or between two half membranes.

Contributions to Biology

The light microscope was responsible for allowing cells to be viewed for the first time, not only did it allow cells to be named as ‘cells’, but allowed, later, for structures within the cell to be observed and named. Light microscopy was mainly important in the nineteenth century, until they realised that there was a huge limit to how much could be seen using light; due to limitations of resolution.

Electron microscopy has allowed many more structures within the cell to be observed, with ribosomes and DNA molecules being recognised for the first time with the use of higher resolution.

With the use of radioactive amino acids it has also been possible to identify how cells produce proteins, with the functions of organelles involved being recognised. The same technique; of inserting radioactive substances which are required by the cell, have also allowed all other organelle functions to be discovered.

Microscopy is responsible for all of our current understanding of biological cells, whether directly or indirectly and microscopes are constantly being developed to produce higher resolution images to expand our knowledge of cells.

Bibliography

Biological Science (2nd Edition)

Green, Stout and Taylor

Peter v. Sengbusch

Video

Open University

Peer Reviews

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7th of Aug 2012

Quality of writing

This piece of work is well written and the layout works well, the information is clear and the essay is easy to follow as the candidate first discusses one type of microscope, then the other and then compares the two rather than jumping from one to the other and back again. In addition to this, the candidate has used appropriate terminology and there are no grammatical or spelling mistakes.

Level of analysis

It is evident that the candidate has taken the time to research this topic and they have used the information they have found to make their essay more interesting to read, this is great as it keeps the attention of the reader and ensures that they read all of your essay rather then simply scanning through it. It is important to research a topic for a science based essay as this ensure that you get your facts straight but it can also show enthusiasm and dedication towards your subject which is always a good thing. Furthermore, the candidate has stated the resources they have used in a bibliography, this is essential, it shows the steps that you have taken to prepare for your work and that your work is not plagiarised. On another positive note, the candidate has used appropriate images throughout their work to help aid their discussion, this can be useful when you are talking about concepts that are difficult to visualise, as long as you make sure that the images are relevant and that they don’t detract from what your writing. Finally, the candidate has unfortunately not written a conclusion, this is essential and should link together with your introduction which will help draw your essay to an appropriate close. A good introduction summarises key points from your essay with reference to why these points are important as this helps to tie together any loose ends. It is also important to write a personal response in your conclusion, for this type of essay you could discuss what you have learnt in the process of researching and writing about your topic.

Response to question

Overall this is a good piece of work, the candidate demonstrates a good subject knowledge, the information is clear and the candidate has provided a suitable amount of detail. However, they have not written an introduction, this is a mistake. It is important to write an introduction so that the purpose of your essay is clear to the reader. Your introduction should state what you will cover in your essay. It is sometimes easier to write an introduction when you’ve finished your essay as you will know exactly what you have discussed. Your introduction should also include a few sentences in which you introduce your topic, in this case you could state what a microscope is and what it is used for.

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