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Measuring and Counting with a Light Microscope

Author(s): David R. Caprette, PhD

Calibrating an Eyepiece Reticule

An ocular micrometer scale, or reticule, is a scale etched on a glass disk and placed within an eyepiece. The scale is superimposed over any image seen in the microscope, allowing the user to measure any object in the field of view. Such measurement does require the reticule to be calibrated.

To calibrate and/or use an eyepiece reticule, start by focusing the eyepiece itself on the reticule. Looking through that eyepiece only, focus the microscope, then make the adjustment for the other eye as described previously. Then place and center a stage micrometer in the light path. To line up the ocular reticule with the stage micrometer, the eyepiece can be rotated in its tube (without changing focus) and the mechanical stage controls used so that the two images are superimposed. Then measure the distance over which the eyepiece reticule extends and divide by the number of divisions to determine distance per division.

For example, suppose that at 100x an ocular micrometer scale with 50 evenly spaced divisions is superimposed over a portion of stage micrometer that is 1.0 mm (1000 µm) long. In this case, each division of the reticule represents 1000 ÷ 50 = 20 µm per division. The calibration for other magnifications is inversely proportional to objective magnification. For example, if you have 20 µm per division at 100x, you have 5 µm per division at 400x.

(field of view 1) X (magnification 1) = (field of view 2) X (magnification 2)