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Measuring and Counting with a Light Microscope

Author(s): David R. Caprette, PhD

Using the Fine Focus to Measure Depth

To measure a vertical dimension using the fine focus, one must be able to focus separately on the top, and on the bottom of a specimen. For example, to measure the depth of liquid under a cover slip, it is necessary to measure the distance from the top of the slide to the bottom of the cover slip. To do so while lowering the focus requires one to focus on the bottom of the cover slip (or on a small object attached to it). While keeping track of the distance traveled, one then focuses on the top of the slide or on a small object on top of the slide.

It is easiest to measure the depth of an object that provides distinctly different cross sectional views from top to bottom. For example, the amoeboid protists known as Difflugia develop a shell, called a test, that encloses the cell. The test is usually textured and curved, and it is semi-transparent. When the organism is active, pseudopodia protrude from an aperture in the base of the test.

Depending on the type of microscope you are using, you will either raise the stage or lower the nosepiece until the object comes into view. When the central part of the test comes into focus, you are looking at the very top. When the aperture appears in focus, you are viewing the bottom of the test. The tips of the pseudopodia are attached to the surface of the slide. When you measure the distance traveled in the vertical direction from the top of the test to the tip of the pseudopodia, you have measured the height of the organism.

I would not rely too heavily on the precision of measurements using a fine focus control. For one thing, depth of focus limits the accuracy with which one can estimate a position. There also is the matter of hysteresis. We have hysteresis when the direction of change has a significant effect on a result. If I focus precisely on a specimen, then de-focus by moving the stage down a precise number of turns, the specimen is not in focus when I move the stage back up the exact number of turns. No machinery is perfect. Hysteresis results from slippage, for example, as a knob is turned. To minimize the effect, one should approach calibrated positions by moving a control in the same direction each time.