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.

Eyepiece graticule

The eyepiece graticule is a glass disc fitted into the eyepiece of the microscope. These can be fitted to existing eyepieces or eyepieces can be purchased with graticules already fitted. The disc is marked with a fine scale from 0 to 100. The absolute size of the scale is not important as this is what will be calibrated.

Stage micrometer

The stage micrometer is used to calibrate the eyepiece graticule. A stage micrometer consists of a microscope slide on which is engraved a fine and accurate scale. Because the scale has to be accurately produced to give reference dimensions, stage micrometers are much more expensive than eyepiece graticules.

Method

When carrying out calibration, each objective lens has to be separately calibrated. This will result in separate calibration factors for each objective.

Start with the lowest power objective on the microscope. The scale on the stage micrometer is aligned with the scale of the eyepiece graticule and then a reading is taken from the scales. These readings are then used to calculate the calibration factor for the objective lens in use. 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 = 1mm (this information is 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 measure the diameter of the grain using the eyepiece graticule. As an example, the grain measures 12 divisions on the graticule. As each division corresponds to 2.59 µm, the pollen grain is 12*2.59 = 31 µm in diameter.

 Spreadsheet

Calibration tables for microscope objectives

A lookup table showing the µm corresponding to each reading from the graticule simplifies the process and avoids the need for repeated calculations. Such a lookup table can easily be produced using a spreadsheet. Separate tables are required for each objective. You can download my calibration spreadsheet here.