Calibrating an Eyepiece Graticule
An eyepiece graticule is a fixed ruler etched into the eyepiece, so its divisions have no built-in real size — you must calibrate them against a stage micrometer, and you must do it again at every magnification. Use the interactive below to see exactly why the real distance per division changes, then measure a cell for yourself. Written for A-level Biology across every exam board.
See it in action
The circle is the microscope field of view. The top scale is the eyepiece graticule (0–100 eyepiece units, EPU) — it never changes. The bottom scale is a stage micrometer (1 division = 0.1 mm). Change the total magnification and watch the stage micrometer rescale: at each setting, 95 EPU lines up with a different real length, and the dashed line shows that alignment.
Find the actual size
In the exam you read the cell’s width in eyepiece units (EPU), then work out its actual size = EPU × calibration value. Set a cell below, then switch the magnification and watch the EPU and calibration value both change — but the actual size stays the same.
the graticule
(µm per EPU)
(µm) — fixed
Switch between ×40, ×100 and ×400 and watch the equation: the EPU goes up (the cell covers more divisions) while the calibration value goes down (each division is worth less) — they cancel, so the actual size never changes. That is why you must always multiply the EPU reading by the calibration value for the lens you used.
Why does the value change with magnification?
The eyepiece graticule is a fixed ruler etched into the eyepiece. Its 100 divisions never move or change size — so they have no built-in real-world length. What changes is how much of the specimen those divisions are laid across.
When you increase the magnification, the microscope enlarges the specimen’s image. The same small piece of specimen now spreads across more graticule divisions — so each single division covers a smaller real distance. That is exactly what you see in the interactive: the stage micrometer appears to stretch out, and the calibrated value for 1 EPU shrinks.
The relationship is inversely proportional: multiply the total magnification by 10 and the real distance per division falls to one tenth. Compare ×40 → ×400 (a ×10 jump): 26.3 µm becomes 2.63 µm.
| Total magnification | 95 EPU reads | = µm | 1 EPU = µm ÷ 95 |
|---|---|---|---|
| ×40 | 2.5 mm | 2500 | 26.3 µm |
| ×100 | 1.0 mm | 1000 | 10.5 µm |
| ×400 | 0.25 mm | 250 | 2.63 µm |
Total magnification = magnification of the objective lens × eyepiece lens (the eyepiece is usually ×10). So a ×40 objective gives a total magnification of ×400. Always calibrate — and record — at the exact magnification you will use to measure.
How to calibrate — step by step
This is the standard practical technique (OCR Biology B microscopy PAG; specification point 2.1.1: calibration of an eyepiece graticule using a stage micrometer). The same method applies to every exam board.
- Insert the graticule into the eyepiece and place the stage micrometer on the stage. The stage micrometer is a slide engraved with a scale of known real length — commonly 1 division = 0.1 mm = 100 µm.
- Focus and line up the two scales so that the zero of the eyepiece graticule sits exactly over the zero of the stage micrometer.
- Count how many eyepiece divisions line up with a known real length on the stage micrometer. In the interactive above, 95 EPU line up with the stage length shown.
- Calculate the value of one eyepiece division: divide the real length (in µm) by the number of eyepiece divisions it covered.
- Record the magnification. The calibration is only valid at that magnification — swap the objective and you must repeat the whole process.
Worked example (×400). 95 EPU line up with 0.25 mm. Convert: 0.25 mm = 250 µm. Then 250 µm ÷ 95 = 2.63 µm per eyepiece division. A cell that is 8 EPU wide is therefore 8 × 2.63 = 21.1 µm.
Once calibrated, measure any specimen in eyepiece units, then multiply by the calibrated value for that magnification to get the real size in micrometres. To find magnification of a drawing, use: magnification = image size ÷ actual size (keep both in the same units).
Test yourself
Two quick sets. Work each out first, then check.
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