Graticule Calibration

Eyepiece graticules allow the size of a specimen to be measured. This is valuable when viewing pollen grains for example as size is one of the factors which will help with identification. Before using  a graticule it needs calibration for the objective lenses it will be used with.

Eyepiece graticuleGraticule for measuring object size

The eyepiece graticule is a glass disc in the microscope eyepiece with a printed scale from 0 to 100. The absolute size of the scale is not important as graticule calibration will account for this. It is possible to fit a graticule disc to an existing eyepiece. Alternatively you can purchase an eyepiece with a graticule already in place.

Stage micrometer

To calibrate the eyepiece graticule we use a stage micrometer. A stage micrometer consists of a microscope slide which has a fine and accurate scale engraved on it.  Stage micrometers are much more expensive than eyepiece graticules. This is because the scale on the stage micrometer is manufactured to accurate reference dimensions.

Calibration method

When carrying out graticule calibration, each objective lens has to be separately calibrated. Therefore, this will result in separate calibration factors for each objective.
Start with the lowest power objective on the microscope. Align the scale on the stage micrometer with the scale of the eyepiece graticule and then take a reading from the two scales. Using these readings we can calculate the calibration factor. The following example shows how to calibrate the graticule for the x40 objective lens:

  • Reading from the two scales we find 100 divisions on the eyepiece graticule equals 25.9 divisions on the stage micrometer.
  • For the particular stage micrometer we are using 100 divisions equates to  1mm (you can find this information marked on the stage micrometer).
    • Each division is 1/100 mm = 10µm
    • 100 eyepiece div = 25.9*10 µm
    • 1 eyepiece div = 259/100 µm
    • =2.59 µm

Repeat the calibration for the remaining objectives.

Using the calibrated graticule

Looking at a pollen grain using the x40 objective lens we can measure the diameter of the grain using the eyepiece graticule. For instance, if the pollen grain measures 12 divisions on the graticule the pollen grain is 12*2.59 = 31 µm in diameter. In this example we have used the calibration factor of 2.59 µm/division as calculated above.

Spreadsheet

A lookup table showing the µm corresponding to each reading from the graticule simplifies the measurement process and avoids the need for repeated calculations. Using a spreadsheet is an easy way to produce such lookup tables. Separate tables are necessary for each objective. You can find our calibration spreadsheet here.