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Using a Bright Field Light Microscope

Author(s): David R. Caprette, PhD

Mount a Specimen

Prior to mounting a specimen, one should put the lowest available magnification in the light path. An objective of low magnification is shorter than one of high magnification, giving one more room for placing the slide. More importantly, when we look for an object on a slide, we search for it in three dimensions, namely the x-y plane and the vertical dimension (i.e., the focal plane). At low magnification, we see a much greater area of specimen and have a much deeper focal plane than at high magnification.
    
A mechanical stage makes it convenient to search an area systematically for objects of interest and to collect replicate data. Using the translational controls, one can manually “chase” a fairly fast moving living organism around a microscope slide without losing it from view.
    
Whether you have a prepared slide, wet mount, or a smear with no coverslip, it is critical to mount the slide with the specimen toward the objective lens. Usually, that means the specimen will be facing up, although some microscopes (inverted microscopes) have the stage above the objectives.
    
If the slide is upside-down, you may be able to focus at low magnifications without compromising the view. You will not be able to focus at a high magnification, though. High resolution requires that the half angle at which the cone of light enters the objective (alpha in the equation for resolution) be as large as is practical. Proximity to a specimen is necessary to obtain a large enough half angle when the light comes from a very small area. It follows, then, that to obtain the necessary resolution, a high magnification objective lens must be brought very close to the specimen.
    
Coverslips are made of very thin glass or plastic for two reasons. One is to allow an objective to approach within a very short distance of a specimen. The other to prevent the thickness of the glass, which is not optically perfect, from significantly compromising contrast or resolution.