Comparison Between Light and Electron Microscope

The introduction of the microscope as a tool for the biologist brought about a complete reappraisal of the micro- composition of biological tissues, organisms and cells. In the infancy of its application to organic materials, it was the implement of anatomists and histologists in particular, where previously unimagined structures in cells were revealed. More recent developments in biological specimen preparation have come from biochemists and physicists who have used the microscope to examine cells and tissue, utilizing a diverse range of techniques available. The fact that electron micrographs appear in most text books and research papers on cell structure and constituents, emphasize the importance of microscopy to the biologist faced with the enormous variety of experimental practices existing today for the analysis of cells.

The microscope itself is a device used to produce a magnified image of an object or specimen. Ever since Anton Von Leeuwenhoek's (1632-1723) invention of a device powerful enough to explore the world of microbes, an explosion of interest has been enthused in the scientific possibilities of microscopes. The fascination of the microscopic world that opened up in biology inspired rapid progress both in microscope design and, equally more importantly, in preparing material for examination - both of which played a vital role in shaping the microscopes of today.

There are two fundamentally different microscopes now in use today: the light microscope and the electron microscope- both of which utilize different forms of radiation in order to create an image of the specimen being examined.

The light microscope, so called because it employs visible light to detect small objects, is probably the most well-known and most commonly used research tool in the laboratory. Here, Specimens are illuminated with light which then passes through two sets of lenses known as the objective lens and the ocular (or eyepiece) lens. The lenses present, help to refract the light to give a magnified image of the specimen to be viewed through the eyepiece directly into the viewer's eyes, or projected onto photographic film (light micrograph). However, before the specimen can be observed, focusing the image is a necessity. This is simply the verifying of the position of the objective lens in relation to the distance from the specimen. In addition, the focus is related to focal length and can be controlled with the focus knobs that work in conjunction with one another. The focus knob brings the object into the focal plane of the objective lens whereas the fine-focus is used to make minor adjustments to the resulting image.

Specimens that be viewed with such types of microscopes include both living and dead cells, requiring staining with a coloured dye to make them visible. Many different stains are available that blemish specific parts of the cell such as DNA, lipids and cytoskeleton.

All light microscopes today are compound microscopes, where essentially several lenses are employed to obtain high magnification. Light microscopy has a resolution of about 200 nm, which is sufficient enough to appreciate cells, but not the features of cell organelles.

By 1900 almost all the simple cell structures had been discovered. There followed a time of frustration for microscopists because they realized that no matter how much the design of light microscopes improved, there was a limit to how much could ever be seen using light. In order to understand the problem, it is necessary to understand the nature of light itself and the difference between magnification and resolution.

The magnification of an instrument is the increase in the apparent size of the object. The total magnification of a light microscope is worked out by multiplying the magnification of the objective lens by that of the ocular lens.

There is virtually no limit to the magnification produced by a light microscope (typically 1500x magnification); it depends on the power of the lenses used. However, above a certain magnification the image becomes blurred and it is impossible to distinguish structures lying close together - this limit of effective magnification is called the resolving power or resolution of the microscope. It is defined as the ability of a microscope to show two different objects as separate or simply the ability to distinguish between two points on an image.

The resolution of an image is limited by the wavelength of radiation used to view the sample. Ultimately, the resolving power of the light microscope is limited by the wavelength of light (400-600nm for visible light), and as such cannot resolve detail finer than 0.2 micro-metres. In order to improve the resolving power, a shorter wavelength of light is needed, and sometimes microscopes have blue filters for this purpose (because blue has the shortest wavelength of visible light). By using a microscope with a more powerful magnification, it will have no bearing on the resolution. Magnification will increase the size of the image, but objects closer than 200nm will still only be seen as one point.

Biological specimens may be examined in a living or preserved form. In the latter case material can be sectioned for closer examination and treated with a wide variety of stains to reveal and identify different structures.

Fixation: This is the preservation of material in a life-like condition. Tissues must be killed rapidly and this is best achieved with small pieces of living material.

Dehydration: The removal of water using ethanol is done to prepare the material for filtration with an embedding material, or mounting material with what water will not mix. Also bacterial decay would eventually occur if water were present. This is particularly important in electron microscopy because water molecules deflect the electron beam which blurs the image.

Embedding: Very thin sections can be cut if the material is embedded in a supporting medium. For Light microscopy, embedding involves impregnating the material with molten wax which is then allowed to set. A harder material (plastic or resin) must be used for electron microscopy because thinner sections are required, and so more rigid support is needed during cutting.

Sectioning: Most pieces of material are too thick to allow sufficient light to pass through for microscopic investigation. It is usually necessary to cut very thin slices of the materials (sections) and this may be done with a microtome or an ultramicrotome to make them either a few micrometres (light microscopy) or nanometre (electron microscopy) thick.

Staining: Most biological material is transparent, so that some means of obtaining contrast between