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...
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