Measuring Objects using a light microscope To measure

Measuring Objects using a light microscope • To measure objects with a light microscope we use an eyepiece graticule. The eyepiece graticule is a glass disc that is placed into the eyepiece • The eyepiece graticule has a scale on it which is divided into 100 sub-divisions – the divisions are often called ‘eyepiece units’. Field of view Eyepiece graticule scale 100 sub-divisions. An eyepiece graticule has to be calibrated so we know what each unit represents as the units on this eyepiece depend on the magnification used.

Calibrating an eyepiece graticule with a stage micrometer • To calibrate an eyepiece graticule we use a stage micrometer. This is essentially a microscope slide with a scale etched into the central circular region – rather like a tiny ruler. It has known measurements. • The stage micrometer is 1 mm long and has 100 sub-divisions. So each sub-division = 1/100 = 0. 01 mm. This is often marked on the micrometer. or 0. 01 x 1000 = 10µm. These sub-divisions are often called ‘stage units’. Stage micrometer – the scale is often labelled on the slide. 1 mm long 100 divisions 0. 01 mm (10µm) each divsion Outer circular region. Inner circular region has etched scale Scale 1 mm long with 100 sub-divisions. 0. 1 mm

• The micrometer is lined up with the eye-piece graticule – e. g. at 100 x magnification as shown below…. Field of Vision x 100 Eyepiece graticle 100 ‘eyepiece units’ eyepiece graticule stage micrometer Stage Micrometer 1 mm long with 100 ‘stage units’ smallest division on stage micrometer is 0. 01 mm = 10µm

Stage micrometer 67 - 42 = 25 ‘stage units’ x 100 We know each ‘stage unit’ = 10µm eyepiece graticule So in total we have 25 x 10µm = 250µm = 100 ‘eyepiece’ units stage micrometer So each eyepiece unit = 250/100 = 2. 5µm The micrometer can now be removed as the eyepiece graticule is now calibrated.

Now measure specimen using calibrated eyepiece graticule x 100 Specimen = Paramecium Extends from 8 -85 units 85 -8 = 77 eyepiece units. Each unit = 2. 5µm So specimen is: 77 x 2. 5 = 192. 5µm