FLUORESCENCE MICROSCOPY
Fluorescence is the luminescence of a substance excited by radiation. Luminescence can be subdivided into phosphorescence, which is characterized by long lived emission, and fluorescence, in which the emission stops when the excitation stops. The wavelength of the emitted fluorescence light is longer than that of the exciting radiation. In other words, a radiation of relatively high energy falls on a substance. The substance absorbs and/or converts (into heat, for example) a certain, small part of the energy. Most of the energy that is not absorbed by the substance is emitted again. Compared with the exciting radiation, the fluorescence radiation has lost energy, and its wavelength will be longer than that of the exciting radiation. Consequently, a fluorescing substance can be excited by near-UV invisible radiation, and its fluorescent components (fluorophores) are seen in the visible range. In a fluorescence microscope, the specimen is illuminated with light of a short wavelength, for example, ultraviolet or blue. Part of this light is absorbed by the specimen and re-emitted as fluorescence. To enable the comparatively weak fluorescence to be seen, despite the strong illumination, the light used for excitation is filtered out by a secondary (barrier) filter placed between the specimen and the eye. This filter, in principle, should be fully opaque at the wavelength used for excitation, and fully transparent at longer wavelengths so as to transmit the fluorescence. The fluorescent object is therefore seen as a bright image against a dark background. It follows that a fluorescence microscope differs from a microscope used for conventional light microscopy mainly in that it has a special light source and a pair of complementary filters. The lamp should be a powerful light source, rich in short wavelengths. A primary or excitation filter is placed somewhere between the lamp and the specimen. The filter, in combination with the lamp, should provide light over a comparatively narrow band of wavelengths corresponding to the absorption maximum of the fluorescent substance. The secondary, barrier, or suppression filter prevents the excitation light from reaching the observer’s eye and is placed anywhere between the specimen and the eye. A fluorescence microscope and filter sets are shown in Figure 4.14.
FIGURE 4.14 A fluorescence microscope uses various filters to exclude and excite specific wavelengths of light to induce fluorescence. Most microscope companies now package filters in sets, or cubes, to make choosing combinations easier.
