THE LIGHT MICROSCOPE
CALIBRATION OF A MICROSCOPE
AS Bio
CALIBRATION OF A LIGHT MICROSCOPE
• Light microscopes are used to increase the magnification
and resolution of an image.
• They must be calibrated to enable the accurate measurement
of the size of a specimen.
• An eyepiece graticule and stage micrometer are used to do
this.
CALIBRATION OF MICROSCOPE
• An eyepiece graticule and stage micrometer are used to measure the
size of the object when viewed under a microscope
• Each microscope can vary slightly so needs to be calibrated when
used
• The calibration is done with a stage micrometer, this is a slide with a
very accurate scale in micrometres (µm)
• The eyepiece graticule is a disc placed in the eyepiece with 100
divisions, this has no scale (arbitrary scale)
• To know what the divisions equal at each magnification the eyepiece
graticule is calibrated to the stage micrometer at each magnification
Apparatus required
● Light microscope
● Eyepiece graticule (square grid fitted into eyepiece, size of
graduations calibrated)
● Stage micrometer (slide with divided scale, used to calibrate eyepiece
graticule)
● Microscope slide
Calibration of a microscope
• Choose the magnification (high/low) by rotating the nosepiece and
selecting the magnification lens on the objective lens
• Place the stage micrometer on the stage of the microscope
• Align the eyepiece graticule (arbitrary scale ) with the stage
micrometer (known scale) by rotating the eyepiece lens
( make sure there are two coincidence points in both scales)
• Count how many eyepiece graticule micrometer divisions corresponds
to a set number of stage micrometer divisions
• The size of one eyepiece graticule division can be calculated
EXAMPLE
•In the diagram, the stage micrometer has three lines each 10 µm apart
•Each 10 µm division has 40 eyepiece graticule divisions
40 graticule divisions = 10 µm
1 graticule division = number of micrometres ÷ number of graticule division
•1 graticule division = 10 ÷ 40 = 0.25 µm this is the magnification factor
•The specimen slide would be used to replace the stage micrometer and the eyepiece graticule at
the same magnification would be used to measure the length of the object
•The number of graticule divisions can then be multiplied by the magnification factor:
graticule divisions x magnification factor = measurement (µm
CALIBRATION OF THE EYEPIECE
• Stage micrometer is 1 cm long and divided into 100 divisions
• ∴ each division: 1 ÷ 100 = 0.01 cm = 100 µm 5.
• 20 eyepiece graticule divisions = 9 stage micrometer divisions
• ∴ 20 eyepiece graticule divisions= 9 × 100
= 900 µm
• ∴ 1 eyepiece graticule division= 900 ÷ 20
= 18 µm