The lamps for both bright-field (tungsten or halogen light) and fluorescent light sources must be correctly centered and focused to achieve optimal imaging. For older microscopes with a reflecting mirror under the substage condenser, the light source is focused directly onto the specimen ("critical illumination"). For the majority of research microscopes, which are designed for Köhler illumination (see Subheading 3.4.2.), the filament image is focused onto the aperture diaphragm of the substage condenser by a collector lens system which produces a homogeneous illuminated field. Apart from a variable control on the voltage passing to the light source, the field of view illuminated by the lamp is regulated by the field diaphragm, which is situated before the substage condenser in the light path (Fig. 2).
For epifluorescence, the image of the filament is focused directly onto the specimen (critical illumination). On some fluorescence microscopes, the images of the filament and a second reflected, virtual image can be viewed and aligned on a focusing window. On others, an image of the lamp can be focused by placing a piece of paper where the slide would be and observing the lamp directly. As with tungsten/halogen light sources, the field of illumination can be regulated via a field diaphragm. Since fluorescence may fade rapidly, it may be desirable to use the field diaphragm to reduce the scatter of light outside the area of interest. Although lamps vary, mercury vapor bulbs, once lit, should burn for at least 30 min and should not be reignited for 30 min after they are switched off. Each time the lamp is switched on and off, approx 2 h are lost from the life-span of the lamp.
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