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Light Microscopy: Instrumentation and Principles

Author(s): David R. Caprette, PhD

Depth of Focus

Some specimens, such as stained animal or plant tissue, will be obvious to the naked eye and not hard to find at all. Others require some searching, especially if they are very small, sparsely distributed, and/or very light in color. Students often have trouble finding Gram negative bacteria, for example, because they are typically less than a micrometer in diameter and because they stain a very light pink. For hard-to-find objects, it is important to remember that to locate your target, you must bring it into focus, or at least close.

When we look for a particular object, we are looking within a volume of space. The volume of space in which an object will show up depends on the object itself and the choice of objective. Many objects, such as stained bacteria, become invisible when they are far out of focus. Let's define depth of focus as the vertical range over which a very small specimen, such as stained bacteria, remains recognizable.

My microscope has four objectives of 4x, 10x, 40x, and 100x magnification, and oculars that magnify 10x. With the 4x lens in place, the total magnification is 40x and the depth of focus is 160 µm, or 0.16 mm. It is only necessary to position the 4x objective so that the surface of the slide, and thus the specimen, is more or less in focus. The area in view at any one time is that of a circle of 2.5 mm radius. The volume visible with the 4x lens, then, is pi times 2.5 mm squared, times 0.16 mm, or 3 cubic mm. With such a large field of view, specimens should be easy to locate, provided that they have sufficient contrast and that they are recognizable at 40x.

At 100x final magnification, the depth of focus is reduced to 40 µm and the area in view now has radius 0.6 mm. The visible volume of space is now 0.05 cubic mm. At 400x and 1,000x, the depth of focus is 12 and 5 µm respectively, and the areas in view are correspondingly smaller. At 1,000x, you are looking at a volume of space of less than 0.0002 cubic millimeter, which is less than 1 ten thousandth the volume in view at 40x.

Experienced microscopists start at low magnification and work up. Trying to find a tiny object at high magnification without systematically working up from low magnification is not at all unlike looking for a needle in a haystack.