Your beginning microscope may be equipped with a scale, called a reticule, that is built into one eyepiece. The reticule is usually used in measuring any planar dimension in a beginning microscope field since the ocular can be turned in any direction and the object of interest can be repositioned with the stage manipulators. The magnification level of a microscope is measured theoretically by multiplying the magnification of the ocular lens by the magnification of the objective lens. In actual practice, each beginning microscope used and each lens on the same beginning microscope will differ a little from the stated magnification level. When using a reticule for the first time, it is necessary to calibrate the scale by focusing on a second micrometer scale (a stage micrometer) placed directly on the stage.Calibrate the beginning microscope first, before accurate measurements can be made, using both an eyepiece or ocular micrometer and a stage micrometer. The size will not change when the objectives are changed since the ocular micrometer is inside the ocular lens. For that reason, each objective lens must be calibrated separately.

The calibration mark of a beginning microscope’s stage micrometer has a known interval, usually 0.01 mm. Focus on the stage micrometer with the lowest objective in place. The ocular micrometer is rotated until it is exactly overlaid on the stage micrometer with the forward edge of both scales being exactly even. Afterwards, look for another point where the two scales again overlap, as far from the beginning as possible. The spaces on each scale from the start to the second point of overlap are then counted. Finally, divide the number of ocular units into the stage units or mm to find out the number of mm in each ocular unit. The measurements of microscopic structures are usually given in micrometers or microns (µm). The mm figure can be multiplies by 1000, since there are 1000 µm in each mm, to reveal the number of µm in each ocular unit.

Using a stage micrometer, properly calibrate your reticle by aligning the zero line or beginning of the stage micrometer with the zero line or beginning of the reticle. Carefully scan over until the lines line up again. Use a simple ratio to determine the value that each line that represents your reticle. For example, you have 30 divisions of the reticle (eyepiece micrometer) which equals to 200 micrometers. So what does one division on the reticle equal? Put simply, 30 is to 200 as one is to X. There will be two fractions, 30 over 200 equals 1 over X. By cross multiplication, you get 30X=200um. Solve for X by dividing both sides by 30 and X equals 6.7 um. As you can see, they line up again at 60 but alignment is off by one at 90. If we use 90 and 61 (610um) we get 6.8 um. The wider the interval is, the more accurate your results should be.

It must be remembered that this distance between reticle lines is only good for that particular beginning microscope objective lens. It may not emerge as a nice round number. You must recalibrate your beginning microscope objective lens when you switch to a different objective.