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Measuring and Counting with a Light Microscope

Author(s): David R. Caprette, PhD

Measuring with an Eyepiece Reticule

Once an eyepiece reticule is calibrated, it can be used to measure the length or width of any visible object within the limits of resolution. The reticule is simply superimposed over the object to be measured. The translational stage controls are used to move the specimen itself since the reticule is in a fixed position. To line up the reticule, the eyepiece is rotated in its tube without changing its focus.

In the present example, the investigator wanted to know the diameter of a filament of the alga Spirogyra. First the investigator centered the image using the stage controls. The angle of the filament cannot be changed, however, so it then was necessary to rotate the eyepiece bearing the reticule so that the scale was perpendicular to the filament. The investigator estimated the filament to be 13 divisions wide. With a calibration of 20 micrometers per division, the estimated diameter is 260 µm, or 0.26 mm.

Note that the edges of the filament are indistinct. A different investigator might estimate its diameter as 12, or even 14 divisions. Limits of resolution must be taken into consideration when reporting such measurements. In addition, it can be useful to set criteria for standardizing the measurement. For example, always measuring to the outside edge on both sides of the specimen, or measuring one outside edge and one inside edge.