ABSTRACT
Many important specimens, most particularly living unstained biological cells, are almost completely transparent. A typical preparation for study will consist of small membrane-bounded compartments containing aqueous fluid (the organelles), enclosed within a larger compartment of aqueous fluid of slightly different composition (the cell itself), the whole mounted either in fresh water, seawater, or an appropriate medium capable of supporting the life of cultured cells. The differences in light absorption between the various components of this preparation are negligible, and they will thus be quite invisible to the eye in bright-field transmitted-light microscopy. It is common practice in these circumstances to close down the illuminating aperture and defocus a little, as discussed in Section 3.1; there is a resulting increase in contrast (which can be considered to be a form of phase contrast) but the resolving power of the system will be seriously reduced. Although such specimens appear invisible in normal bright-field microscopy, they have in fact caused some small changes in the phase relationships between the rays of light as they emerge from the specimen, and they are thus frequently known as ‘phase specimens’. The amount of this phase change, to which our eyes are not sensitive, is dependent upon the product of the refractive index of the object and its thickness. Only if such phase changes can be converted into amplitude changes will our eyes be able to appreciate the morphology of the object and contrast be generated.
