Microscope Workshop - Calibrating an eye piece graticule

Eye piece graticules are useful for measuring the actual size of a
specimen. They are etched onto the eye piece and do not change in
size
Each division on the eye piece graticule will therefore represent different
values depending on the magnification being used.
Each division has to be calculated using a STAGE MICROMETER – this
does change size as it is place on the stage!
This is what a typical stage micrometer looks like

Worked example:

Example 1 – Low power objective used (x4)

The stage micrometer has 100 divisions, each are 0.1mm apart.

Suppose the full length of the eye piece graticule scale (which has 100 divisions) covered 25
divisions of the stage micrometer.

Then the full length of the eye piece scale is therefore equivalent to (25 x 0.1mm) = 2.5mm or 2500
µm long.

The eyepiece graticule has 100 divisions, so each division will measure 25000/100 = 25µm for this
magnification.

Each division of the eyepiece graticule (µm) = Length of stage micrometer (µm)

Number of eye piece divisions

Imagine eyes (eye piece

graticule) looking up at
the stage (micrometer)!
Silly way to remember…
 Using the tutorial on https://www.microscopyu.com/tutorials/reticlecalibration

X and
You Ycan
translation
change the
Switch to specimen sliders allow
colour of theyouscale
to to
once you have position
contrasttheagainst the
calibrated all the background Choose
eyepiece when an a specimen
objective
objective powers viewing theorpower
horizontally to measure
from after
the drop
you
vertically
specimens have calibrated
down
in order menu the
eyepiece.
line it up with the
Change to fine focus stage micrometer
to make the
movements smaller.
Start on coarse. TASK 1
Choose each of the objective magnifications, calculate what each small
division on the eye piece measures at:

Magnification Number of divisions Total length What each eye

of eye piece graticule measured using piece graticule
the stage division represents
micrometer (µm) (µm)
Remember each
division = 0.1mm or
100 µm
X10

X20

X40

X60

X100

TASK 2 Measuring the specimens

Specimen Actual size (µm)

Arrowroot starch
 grain at x20

Human
erythrocyte (RBC)
at x40
Pollen grain
At x60
Zygnema green
algae at x100

TASK 3 Measure the mean diameter for each structure

In order to complete this task, you will need to calibrate each objective lens using a
stage micrometer!

Objective Number of divisions of Length (of EPG) What each eye

eye piece graticule measured using the stage piece graticule
used micrometer (µm) division represents
Remember each division = (µm)
0.1mm or 100 µm
X4 Low

X10 Medium

X40 High

Use the microscopes to view the following slides:

 Insect tracheae (High power)

 Artery (medium power)
 Dicot. Root TS (low power)

Slide Insect tracheae Artery (medium Root TS (low power

(High power x 40) power x 100) x400)

EPG division value

at the objective
magnification (µm)
Number of EPG
divisions

Actual size (µm)