Microscope Calibration Guide

How to use Ocular & Stage Micrometer for Calibration

Use an ocular micrometer to measure the specimen image, and use a stage micrometer as the reference scale for calibration. This page explains the basic workflow, calculation method and installation points for practical microscope measurement.

Ocular Micrometer vs. Stage Micrometer — Which to Use?

I want to measure the specimen size under the microscope.
Should I use an Ocular Micrometer ?
or a Stage Micrometer ?

Stage micrometer scale - 1 mm rule with 0.01 mm pitch

Step 3 calibration procedure illustration

Simply put...

●	To measure specimen size under a microscope, use an ocular micrometer.
●	A stage micrometer does not measure specimens directly, but is used to check objective lens magnification errors, ensuring more accurate ocular micrometer measurements.

How to Use an Ocular Micrometer

The magnified specimen image is projected onto the ocular micrometer, allowing simultaneous viewing of both the micrometer scale and the sample through the eyepiece. The specimen size is measured by determining the single pitch width on the micrometer scale, which varies depending on the objective lens magnification.

Single pitch width of an ocular micrometer under the microscope =
[Actual width of a single pitch] ÷ [Objective lens magnification]

Example Measurement

The figure below demonstrates measuring the size of a kidney glomerulus.
1. The ocular micrometer : XY11 (10 mm / 100 divisions / 1 pitch = 100 μm),
2. The objective lens : 20x magnification.

The size represented by one pitch on the specimen is 5 μm (= 100 μm ÷ 20).
Since the glomerulus measures 23 pitches horizontally and 22 pitches vertically,
the specimen size can be calculated as follows:
Horizontal: 23 × 5 μm = 115 μm
Vertical: 22 × 5 μm = 110 μm

Microscope field of view at 20x magnification showing stage micrometer for XY size measurement

100 μm (1 pitch) ÷ 20 (objective) = 5 μm
X : 40 to 63 : 23 pitch x 5 μm = 115 μm
Y : 40 to 62 : 22 pitch x 5 μm = 110 μm

Objective Lens Magnification	Magnified Pitch Width 10mm /100 div / pitch 0.1mm
5 x	20μm (= 100μm ÷ 5)
10 x	10μm (= 100μm ÷ 10)
20 x	5μm (= 100μm ÷20)
40 x	2.5μm (= 100μm ÷ 40)
50 x	2μm (= 100μm ÷ 50)
100 x	1μm (= 100μm ÷ 100)

Ocular Micrometer

How to Use an Ocular & Stage Micrometer for Calibration

Objective lenses may have slight magnification errors. A stage micrometer is used to check these errors in advance, so that microscope measurements can be corrected more precisely.

Example of Magnification Error Measurement for Objective Lenses

Position the micrometers so that the ocular and stage micrometer scales appear parallel when viewed through the eyepiece. This allows for precise magnification error calculation.
The figure below shows an example using a
stage micrometer NOB1 (1 mm / 100 div / pitch = 10 μm) and an
ocular micrometer S11 (10 mm / 100 div / pitch = 100 μm) under a
20x objective lens.

Calibration example showing ocular and stage micrometer alignment under 20x objective

[Expected Measurement]
If the magnification is accurate, 10 pitches on the stage micrometer
should match 20 pitches on the ocular micrometer:
2000 μm = 10 μm (1 pitch of stage micrometer) × 10 pitches × 20x objective

[Observed Measurement]
However, as seen in this example, if the ocular micrometer reads 21 pitches instead of 20, the actual magnification is slightly higher than 20x, indicating a magnification error.

[Correcting Measurement Values]
To obtain more accurate measurements, multiply the ocular micrometer reading by the error correction factor: 0.95 (= 20 ÷ 21).
Thus, the actual measurement value = ocular micrometer reading × 0.95,
ensuring more precise results.

Stage Micrometer Ocular Micrometer

How to Install an Ocular Micrometer

An ocular micrometer is printed in chrome on a glass surface. The printed side is considered the front. Insert it into the eyepiece with the printed side facing the microscope, as shown in the image below.

Each eyepiece model has a different fitting diameter, and compatible micrometer sizes vary depending on the eyepiece manufacturer and model number.
Nikon and Olympus Eyepiece & Ocular Micrometer Compatibility List

Diagram for installing an ocular micrometer into the eyepiece with correct printed side orientation

How to Identify the Front and Back of an Ocular Micrometer

The scale of the ocular micrometer is printed in chrome on the glass. Once the printed side is identified, place it on the mounting bracket with the printed side facing the objective lens (downward). Refer to the installation instructions above for details. This section explains how to distinguish the front (printed side) and back side of the micrometer.

1. Shine a light on the micrometer to reflect the numbers on the scale, making them glow silver. Without light reflection, the front and back cannot be distinguished.	* A light source with a large surface area is recommended for better reflection visibility.

2. If there is no shadow, the reverse side (non- printed side) is facing you.

3. If there is a shadow, the printed side is facing you.

Frequently Asked Questions

Q How do I calibrate a microscope using an ocular micrometer and stage micrometer? ▼

A Place the stage micrometer on the microscope stage and focus on its scale at the magnification you wish to calibrate. Align the ocular micrometer scale with the stage micrometer divisions and count how many ocular divisions correspond to a known stage micrometer distance. Divide the known distance by the number of ocular divisions to determine the value per ocular division at that magnification.

Q What is an ocular micrometer and how is it installed? ▼

A An ocular micrometer is a glass disc with an engraved scale that is placed inside the eyepiece of a microscope. Unscrew the eye lens from the eyepiece, place the micrometer disc on the field-stop diaphragm inside the eyepiece (scale facing down), then re-screw the eye lens. The scale appears superimposed on the sample image.

Q How often should microscope calibration be performed? ▼

A Calibration should be performed whenever the objective or eyepiece is changed, when the tube length is adjusted, and at regular intervals (typically annually) for quality assurance. If measurement results seem inconsistent, recalibration is recommended.

Q Do I need a different calibration for each objective magnification? ▼

A Yes. The value of each ocular micrometer division changes with objective magnification. Calibration must be performed separately for each objective (e.g., 4×, 10×, 40×, 100×) and the results recorded. When switching objectives, use the calibration value corresponding to that magnification.