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Using a Bright Field Light Microscope

Author(s): David R. Caprette, PhD

Looking “Through” a Specimen

The thickness of a specimen limits the maximum functional magnification with which we can expect to obtain useful information. Objective lenses, as you may recall, have limited depth of focus, so while one part of a thick specimen is in focus, the other parts that are out of focus make it difficult to see detail. But the structures of some specimens are arranged in such a way that one can focus on one level at a time and obtain much more information than by using low magnification to see everything at once.

For example, a strand of the filamentous alga Spirogyra is, for the most part, in focus at 40 power. At 100 power, some part of the cell, either toward or away from the objective lens, is out of focus at any given distance. At 400 power, so little of a cell is in focus at any given time that one can begin with the specimen below the focal plane and optically section the filament by slowly raising the stage. First, the chloroplasts just beneath the cell wall appear in focus. As the stage is raised, chloroplasts come into and go out of focus in a spiral pattern. A middle view of a cell shows chloroplasts in focus only at the edges, revealing that the chloroplasts of Spirogyra are arranged near the cell walls and are not found in the centers of the cells. Raising the stage further brings the bottommost chloroplasts into focus. Unless you are aware of the direction in which you are moving the stage, you cannot tell the difference between top and bottom.