Lesson 5 of 5 / Cells and microscopy
Calibrate a microscope measurement
What does one eyepiece division represent at this objective?
In this lesson: Use a stage micrometer, graticule, scale bars and cautious image interpretation.
About 7 min
The key ideaCalibrate the eyepiece scale at each objective before converting a specimen measurement to a real length.
Explore the idea
Calibrate first, then measure
Align the zero marks. Read a second pair of coincident marks to connect eyepiece divisions to a known length.
The stage micrometer here has 10-micrometre divisions. The eyepiece scale has arbitrary units. The orange guides mark the two positions used for calibration.
The drawn cell is illustrative. Calculate with the calibrated scales, never the number of pixels or centimetres on your screen. Actual slide mounting, focusing and calibration require laboratory practice.
Explanation
An eyepiece graticule has arbitrary divisions. A stage micrometer has a known physical scale. Superimpose the scales at the chosen objective, align one pair of marks, and find another matching pair. Divide the known micrometer distance by the corresponding eyepiece divisions.
If 40 eyepiece divisions span 100 micrometres, one division represents 2.5 micrometres. A structure spanning 12 divisions is then 30 micrometres long. Changing objective changes the relationship; recalibrate instead of reusing the old conversion.
Begin focusing at low power, centre the specimen, then increase magnification and use fine focus. A thin mount and carefully lowered coverslip reduce overlap and air bubbles. Select appropriate staining, record observations accurately and distinguish what is visible from what a textbook says should be present.
Magnification equals image size divided by actual size, in the same units. A scale bar remains useful when an image is resized because the specimen and bar scale together. Greater magnification without greater resolution merely enlarges an unresolved image; it does not reveal a ribosome.
Step by step
- 1
Align the scales
Use matching marks sufficiently far apart to reduce proportional reading error.
- 2
Calculate one division
Keep the physical unit throughout the conversion.
- 3
Measure the specimen
Multiply its eyepiece divisions by the calibrated length per division.
Worked example
Work through the evidence
25 eyepiece divisions align with 0.10 mm. A cell spans 18 eyepiece divisions. Find its length.
One way to explain it
0.10 mm = 100 micrometres. One division = 100/25 = 4 micrometres. The cell length = 18 x 4 = 72 micrometres.
Why this answer works
- Convert mm to micrometres before dividing.
- Use the objective at which the calibration was made.
Is this true? "Every eyepiece division always equals one micrometre."
Eyepiece divisions have no fixed specimen length until calibrated at the selected objective.