Tuesday, October 13, 2009

The Dark-Field Microscope:-


Living, unstained cells and organisms can be observed by simply changing the way in which they are illuminated. A hollow cone of light is focused on the specimen in such a way that unreflected and unrefracted rays do not enter the objective. Only light that has been reflected or refracted by the specimen forms an image. The field surrounding a specimen appears black, while the object itself is brightly illuminated; because the background is dark, this type of microscopy is called dark-field microscopy. Considerable internal structure is often visible in larger eucaryotic microorganisms. The dark-field microscope is used to identify bacteria like the thin and distinctively shaped Treponema pallidum, the causative agent of syphilis.

The simplest way to convert a microscope to dark-field microscopy is to place 
(a) a dark-field stop underneath 
(b) the condenser lens system. 
The condenser then produces a hollow cone of light so that the only light entering the objective comes from the specimen.

Microscope Resolution:-
The most important part of the microscope is the objective, which must produce a clear image, not just a magnified one. Thus resolution is extremely important. Resolution is the ability of a lens to separate or distinguish between small objects that are close together. Much of the optical theory underlying microscope design was developed by the German physicist Ernst Abbé in the 1870s. The minimum distance between two objects that reveals them as separate entities is given by the Abbé equation, in which lambda  is the wavelength of light used to illuminate the specimen and n sin theta is the numerical aperture (NA).


As d becomes smaller, the resolution increases, and finer detail can be discerned in a specimen. The preceding equation indicates that a major factor in resolution is the wavelength of light used. The wavelength must be shorter than the distance between two objects or they will not be seen clearly. Thus the greatest resolution is obtained with light of the shortest wavelength, light at the blue end of the visible spectrum.

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