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Calibration of the Ocular Micrometer

*Measuring Cells Microscopically*
In order to measure the dimensions of a cell (or other specimen) microscopically, one must first
understand how to properly use and interpret the ocular micrometer. The ocular
micrometer is a measuring device (i.e. ruler) that is positioned inside one of your ocular lenses.
The appearance of the ocular micrometer does not change when objective lenses change. The
entire ocular scale is divided into 100 smaller units that are visible while using the scanning, low-
power, high-power, and oil immersion lenses. Therefore, one must understand the true length
of each ocular unit (in micrometers; µm) when using a particular objective lens. Specimen
measurements will be multiplied by a specific number for each objective lens to adjust for the
magnification. Calibration of ocular scales allows scientists to communicate the true sizes of
specimens with each other regardless of the specific microscope they use.

Laboratory Objectives:
1. Determine the length of one ocular scale unit in micrometers (μm) [also called “microns”]
at the four magnifications on your microscope.

EXERCISE:
Materials:
Compound light microscope fitted with an ocular micrometer scale in one eyepiece
Stage micrometer slide [with a scale of 2 millimeters (mm) or 2000 micrometers (µm)]
Paramecium prepared slide

Procedure:
1. Place the stage micrometer scale slide on the stage; adjust the nosepiece so that the scan
objective lens is in working position. Bring the image of the stage micrometer scale into
sharp focus on scan power. As you look through the microscope, you should also see the
ocular micrometer scale as well as the stage scale. Remember that the ocular scale is in
only one eyepiece. If you cannot see the ocular scale at first, close one eye and look
through each eyepiece with the other eye until you spot the ocular scale. Note: The ocular
scale may be in either the right or the left eyepiece on your microscope. Note: The ocular
scale is broken down into 10 large units, each of which contains 10 smaller units. One
ocular unit is the smallest unit visible on the scale. There are 100 of these in total.

2. Using the mechanical stage controls, line up the left-hand (zero) end of the ocular scale with
the left-hand (zero) end of the 2 mm stage scale.

The overall length of the stage scale is 2 mm, which is equal to ___________ μm.

3. SCANNING LENS: Measure the length of the entire ocular scale (100 ocular scale units) on
scanning power and record your results below.

100 ocular scale units = _____________mm = _____________ µm

1 ocular scale unit = _______________μm on scanning power.

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4. LOW-POWER LENS: Measure the length of the entire ocular scale (100 ocular scale units)
on low-power and record your results below.

100 ocular scale units = _____________mm = _____________μm

1 ocular scale unit = _______________μm on low power.

5. HIGH-POWER LENS: Measure the length of the entire ocular scale (100 ocular scale units)
on high-power and record your results below. (Hint: It may be useful to return to scanning
power and sketch a picture of the stage scale as you may not be able to see unit numbers
on high-power.)

100 ocular scale units = _____________mm = _____________μm

1 ocular scale unit = _______________μm on high power.

6. OIL IMMERSION LENS: Measure the length of the entire ocular scale (100 ocular scale
units) on oil immersion-power and record your results below. **DO NOT USE OIL WITH
THE OIL IMMERSION LENS FOR THIS CALIBRATION**

100 ocular scale units = _____________mm = _____________μm

1 ocular scale unit = _______________μm on oil immersion.

7. Record the values in the table below:

VALUE OF ONE OCULAR

OBJECTIVE LENS TOTAL MAGNIFICATION SCALE UNIT
Scanning ( X) X µm
Low Power (10X) 100X µm
High Power (40X) 400X µm
Oil Immersion (100X) 1000X µm

8. Examine the table above. Note the mathematical relationship between increasing
magnification and the decreasing size of an ocular unit. Could you have completed the table
after determining only one value?

9. Now that you know what the values are for the ocular units on the four different powers of
your microscope, you can use the ocular scale to measure the dimensions of specimens you
examine. Simply line up the image of the specimen with the ocular scale by using the
mechanical stage and/or by rotating the eyepiece that holds the ocular scale. Then count
the number of ocular units along the dimension of the specimen and multiply by the value of
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the ocular unit for that particular power. For example, if an object appears to be 20 ocular
units long when you observe it on high power, and if your calibration showed one ocular unit
to be equal to 2.5 µm on high power, then the object’s length is 20 X 2.5 µm or 50 µm long.
This is the actual size of the specimen (for this example).