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Microscopy Practical

Cambridge Biology AL Microscopy Practical

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Mangetsu Hozuki
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33 views4 pages

Microscopy Practical

Cambridge Biology AL Microscopy Practical

Uploaded by

Mangetsu Hozuki
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Microscopy: Callibrating a Microscope

The purpose of a microscope is to magnify small objects. In everyday speech, the word magnification means
how much bigger an image is than the actual object. It’s the same in microscopy, except that you can put
numbers on it (it is quantifiable).

𝐢𝐦𝐚𝐠𝐞 𝐬𝐢𝐳𝐞
magnification =
𝐨𝐛𝐣𝐞𝐜𝐭 𝐬𝐢𝐳𝐞

The object on the slide is first magnified by the objective lens. If all you had was an objective lens, then the
image would be four times, or ten times or forty times the object size, depending on which lens you use. The
objective lens also determines the resolution of the image. The higher power the objective, the greater the
resolution. This means that you can distinguish two points that are close together, so a higher power objective
allows you to see more detail.

Resolution is defined as the smallest distance between two points that can be separately distinguished.

Resolution for:
 Light microscope = ~ 200 nm
 Electron microscope = ~ 0.5 nm

Eyepiece lenses generally have x10 or x15 etched on the side so you can see the power of your eyepiece. The
image from the objective lens is magnified by the eyepiece, so the total magnification of the image you see is
the product of the two separate magnifications. The eyepiece magnification does not affect the resolution of the
image. It just makes it bigger.

Calculating the magnification of image size with a x40 objective lens and x10 eyepiece lens:

magnification = objective x eyepiece

= 40 X 10

= 400

1
Calibrating a Microscope
Calibrating a microscope lets you measure the actual size of structures on the slide.

You need an eyepiece graticule, which is the ruler or scale inside the eyepiece that you can see. It looks like
this:

It looks the same through every objective because it is in the eyepiece. So with objectives of different
magnifications, the divisions on the graticule represent different lengths. The higher the power of the objective,
the smaller length each division of the graticule represents. So calibrating has to be done for each objective
lens.

Notice that the eyepiece graticule has no units written on the scale itself. This is because the units, or length,
between each division depends on the power of the objective being used.

Each division is called an eyepiece unit (epu).

2
To know what length each division represents, you need a stage micrometer. This is a microscope slide on
which the object is a line 1 mm long, divided into 100 divisions.

Let’s call each division on the stage


micrometer a stage micrometer unit
(smu).

1 smu = 1 mm ÷ 100 = 0.01 mm

From the beginning to the end of this scale,


the total length is 1 mm (this is written on the
slide itself, or on the scale).

The entire scale is divided into 100 divisions.

Therefore, the length of one division (1 smu)


is 1 mm ÷ 100 = 0.01 mm

3
In the next step, line up the zero of the eyepiece graticule with the zero of the stage micrometer, making sure
that the scales are absolutely parallel. Look along the scales and see where they coincide again.

In this example, you can see that on a x40 objective, 20 stage micrometer divisions may line up exactly with 80
eyepiece divisions:
0 and 8 of the eyepiece graticule (80 epu)
lines up perfectly with 0 and 20 of the stage
micrometer scale (20 smu)

Eyepiece graticule

Stage micrometer

Therefore, at a x40 objective lens:

80 epu = 20 smu

1 epu = 20/80 smu = 0.25 smu

But we know that 1 smu = 0.01 mm, so:

1 epu = 0.25 x 0.01 mm

= 0.0025 mm

= 2.5 μm

You can work out similar calibrations for all the objective lenses that you use.

You have to do it all over again for every microscope that you use, too. Therefore it is generally good practice
to use the same microscope each time, to avoid having to calibrate a different one each time you use one.

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