When you look down the microscope with the eyepiece graticule in place, the distance between the divisions does not change. It doesn’t matter which magnification objective lens is used, the eyepiece graticule will always appear the same.

The Stage Micrometer

The stage micrometer is a slide with a very accurate scale engraved into the glass. It is placed on the stage of the microscope. Stage micrometers can come in varying lengths, but most are 2mm long and subdivided into 0.01mm (10µm) divisions.

The stage micrometer used in the example below is a slide with a line 10mm long etched onto it. There are 100 stage micrometer divisions on the 10mm line. So, each stage micrometer division is 0.1mm or 100μm.

When the stage micrometer is viewed down the microscope it will look like the diagram shown in Figure 3 on the lowest power. This photograph was taken at a x40 magnification.

If you increase the magnification, the image looks larger but the distance between the lines remain the same. Figure 4 was taken at a magnification of x100 and Figure 5 is using a x400 magnification (see Page 2). Note that for Figure 5’s micrometer, the distance measured by the lines is 100µm (100µm from the right-hand side of the line to the right-hand side of the next line).

................................Bio Factsheet

Eyepiece graticules and stage micrometers are used to measure the actual sizes of cells and tissues when viewed using a light microscope. It is one of the techniques you are required to use in your practical work and so it can also appear in exam questions.

This Factsheet:

• Describes how to calibrate the eyepiece graticule using a stage micrometer.

• Explains how the eyepiece graticule is used to measure the size of objects when viewed down a light microscope.

• Looks at mistakes candidates make in examination questions on this topic.

The Eyepiece Graticule

The eyepiece graticule is a circular piece of glass or plastic that sits on a ledge in the eyepiece. It has a scale engraved onto it. When you look down the microscope it will look something like the diagram shown in Figure 1.

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© Curriculum Press 2019 Bio Factsheets, 2019/20 Series, Issue 1 of 3, September 2019. ISSN: 1351-5136

Using Eyepiece Graticules and Stage Micrometers

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Figure 1 The scale on an eyepiece graticule

Figure 2 A light microscope with the lenses labelled

Figure 3 The micrometer when viewed down the microscope with the low-power objective lens

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Bio Factsheet 371 - Eyepiece Graticules and Stage Micrometers

Calibration of the Eyepiece Graticule

When calibrating the eyepiece graticule, you must use both the eyepiece graticule and the slide micrometer at the same time.

You can rotate the eyepiece graticule by turning the eyepiece. Move the stage until you superimpose the lines of the graticule upon those of the stage micrometer.

You are looking for where the eyepiece lines and the micrometer lines coincide. In the example in Figure 6, the two arrows show where the eyepiece divisions and the slide divisions coincide.

We know that the micrometer slide distance between those two points is 4 divisions, and so is 400µm.

There are 12 eyepiece units (the small ones) in that distance.

So, 1 eyepiece unit = = 30µm

At the higher magnifications, the slide micrometer lines look thick. Look for where the eyepiece lines and the right-hand side of the slide micrometer lines coincide in order to be accurate.

Using the Calibrated Graticule

We have calculated that 1 eyepiece unit = 30µm.

Now when we look down the microscope using the same magnification we know that each eyepiece unit is equivalent to 30µm.

We can now calculate the actual length of any structure viewed down the same microscope with the same magnification. For example, if we were asked to calculate the distance from X to Y on the image of the leaf cross section shown in Figure 7.

We look at the image and count the number of eyepiece units between the two marks. This one has 13 eyepiece units as shown in Figure 8 (Page 3).

© Curriculum Press 2019 Bio Factsheets, 2019/20 Series, Issue 1 of 3, September 2019. ISSN: 1351-5136

Figure 4 The micrometer when viewed down the microscope with the medium-powered objective lens

Figure 5 The micrometer when viewed down the microscope with the high-powered objective lens

Figure 6 The micrometer when viewed down the microscope with the low power objective lens and the eyepiece graticule in place

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Figure 7 A leaf cross section as viewed down the microscope with the low power objective lens

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Bio Factsheet

If each eyepiece unit represents 30µm, we calculate 13 x 30. The actual length between X and Y is 390µm.

The eyepiece graticule must be calibrated for each microscope and for each magnification.

Questions

1) A student wants to know the thickness of the wall of an arteriole that she has viewed down a microscope with an eyepiece graticule. She decides to use a x400 magnification.

She places a slide micrometer on the stage and this is the image she can see (the smaller scale is the eyepiece graticule):

The stage micrometer the student uses has a 1mm scale divided into 0.1mm divisions.

a) i) How many micrometers (µm) are there in 0.1mm?

ii) Using your answer to a) i) and the photograph above, calculate how many micrometres (µm) each eyepiece unit represents.

371 - Eyepiece Graticules and Stage Micrometers

Acknowledgements: This Biology Factsheet was researched

and written by Cliff Sharp and published in September 2019

by Curriculum Press. Bio Factsheets may be copied free of

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© Curriculum Press 2019 Bio Factsheets, 2019/20 Series, Issue 1 of 3, September 2019. ISSN: 1351-5136

Figure 8 The micrometer as viewed down the microscope with the low power objective lens and eyepiece graticule in place

b) The student removes the slide micrometer and places the slide of the arteriole onto the stage. This is the image she sees:

Using your answer to a) ii. calculate the thickness of the arteriole wall from X to Y in micrometers (µm). Show your working.

To show your working, make it clear what you are doing. This question asks you to use the answer from an earlier part of the question. If you got that part wrong you can still pick up marks using your incorrect answer. It’s called “error carried forward” and it is there to help you gain marks for other parts of the question.

Answers

a) i) There are 1000µm in 1mm and so 0.1mm x 1000 = 100.

There are 100µm in 0.1mm.

ii) 28 eyepiece units = 2 stage micrometer divisions.

28 eyepiece units = 200μm.

1 eyepiece unit = 200/28 = 7.14μm.

b) Thickness of wall = 18 eyepiece units.

18 x 7.14 = 128.52μm.

Exam Hint: Some students will use this figure for the magnification in their calculations. It is not needed. Remember that the distance between the bars on the micrometer slide is always the same regardless of the magnification.

Exam Hint: At the higher magnifications, you are looking for where the eyepiece lines and the right-hand side of the slide micrometer lines coincide.

Comment from Chief Examiner’s Report on This Question: Many students carried out the calculations incorrectly. For example, they didn’t know how many μm there are in 1mm, or they divided 28 by 200 giving the calibration of 1 eyepiece graticule unit as 0.14μm. Even if they calculated this incorrectly, candidates could still gain marks from “error carried forward”.