Microscopy

Unit Conversion
1 m = 100 cm

1 cm = 10 mm

1 mm = 1000 µm

There are two fundamentally different types of microscopes that are being used.

1. Light microscope

2. Electron microscope

Both use a form of radiation to form an image of the specimen.

The light microscope uses light as a form of radiation while the electron microscope uses
electrons.

Magnification

Ratio of an object’s real size to its image size.

How to calculate the magnification?

The diagram given below are plant cells seen under a microscope. The cell P when measured
from the photomicrograph was found to be 60 mm whilst the actual length was 80 µm.

Calculate the magnification of the cell P. Show your working.

1
Calculating magnification from a scale bar

0.6µm

2
Parts of a Compound Microscope

3
How to calibrate an eyepiece graticule?

An eyepiece graticule is a little scale bar that you can place in the eyepiece of your
light microscope. When you look down the microscope, you can see the graticule
as well as the specimen.

The graticule units have to be converted to real units, such as mm or µm. This is
called calibration.

1. Understand the length of one unit shown on the stage micrometer.

4
2. Place a stage micrometer scale (a miniature transparent ruler) on the
microscope stage.

Superimpose the images of the eyepiece graticule and the stage micrometer
scale.

5
How to measure using an eyepiece graticule?

1. Place the eyepiece graticule on the eyepiece of the microscope and observe
the specimen.

2. Measure the diameter of the specimen by counting the graticule units.

6
NB: Every time you switch the objective lens from one to the other, the graticule
needs to be measured.

7
Resolution

Resolution can be defined as the ability to distinguish between two separate

points.

If the two points cannot be resolved, they will be seen as one point. In practice,
resolution is the amount of detail that can be seen – the greater the resolution,
the greater the detail.

Maximum resolution of a light microscope - 200 nm.

This means that if two points or objects are closer together than 200 nm they
cannot be distinguished as separate.

Maximum resolution of an electron microscope - 0.5 nm

8
Electromagnetic Spectrum
Light travels in waves. Wave length of visible light varies from 400 nm to 700 nm.

The whole range of different wavelengths is called the electromagnetic spectrum. Visible light is
only one part of this spectrum.

The longer the wave length is the smaller the frequency.

Note that all waves travel at the same speed.

Objects which are large enough to interfere with the pathway of a wave can only be seen
clearly.

The general rule is that the limit of resolution is about one half the wavelength of the radiation
used to view the specimen. In other words, if an object is any smaller than half the wavelength
of the radiation used to view it, it cannot be seen separately from nearby objects. This means
that the best resolution that can be obtained using a microscope that uses visible light (a light
microscope) is 200 nm, since the shortest wavelength of visible light is 400 nm (violet light).

e.g. Diameter of a mitochondrion is about 1000 nm and the diameter of a ribosome is about 25
nm. As the shortest wave length of visible light is 400 nm, a mitochondrion could be observed
but not ribosomes.

9
*

Q. Make a list of structures in a cell which could be observed using a powerful light
microscope.

If an object is transparent, it will allow light waves to pass through it and therefore will still not
be visible. This is why many biological structures have to be stained before they can be seen.

10
Electron Microscope
An electron microscope uses electrons instead of light.

Why are electrons very suitable forms of radiation for microscopy?

1. Their wavelength is extremely short.

2. They are negatively charged. Therefore they can be focused easily using electromagnets.

Two types of electron microscopes

1. Transmission electron microscope (TEM)

2. Scanning electron microscope (SEM)

11
Light Microscope Electron Microscope

SEM TEM

12