The optical(light) microscope
A basic optical microscope is shown in Fig 1.
A basic optical microscope employs a mirror and condenser lens to illuminate a specimen by projecting a beam of light onto it. (In the above
picture the condensor lens cannot be seen since it is hidden under the stage).
The image is focussed and magnified by objective and eyepiece lenses.
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The total magnification is calculated by multiplying together the number on the eyepiece (e.g. x10) and the number on the objective lens (e.g.
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x40). In this case the image would have been magnified × 400.
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Magnification is the number of times the image of an object is increased
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The maximum magnification available with optical microscopes is about × 1500.
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However, creating an enlarged image does not necessarily provide the observer with more information. To achieve that, the image not only
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has to be enlarged but must also show detail.
How successfully this is achieved depends on the resolving power (resolution) of the microscope.
The resolving power of a microscope is its ability to distinguish between two
points that are close together. It is a measure of the clarity of the image.
Resolving power of a microscope depends upon the wavelength of the
illumination source (radiation) i.e. light or electrons
Resolution of a microscope is inversely proportional to the wavelength of the
illumination source i.e. the shorter the wavelength, the greater the resolving
power.
Light has a longer wavelength than electrons, that is why electron microscopes
have a greater resolving power.
The electron microscope
The specimen is illuminated in an electron microscope by an electron beam.
Electrons are very small, negatively charged particles that show wave-like
properties.
The electron beam is focused using electromagnets arranged around the path of
the electron beam. Electrons produce an image when focused onto a fluorescent
screen.
Modern electron microscopes can produce images with a magnification of up to
×500 000.
Table 1. Compares the basic components, advantages and disadvantages of
optical and electron microscopes.
There are two types of electron microscope, transmission electron microscopes (TEMs) and scanning electron microscopes (SEMs).
The electrons pass through or past a thin section of the specimen in a TEM on their way to the fluorescent screen or photographic film.
In SEMs the electrons are reflected off the prepared surface of the specimen. SEMs are very useful for detailed study of surfaces.
The SEMs resolution is about 5-20nm
The TEMs resolution is about 0.5nm
Units of measurement. The standard SI units of measurement used in microscopy are:
Size and Magnification
Photomicrographs, electron-micrographs and biological drawings usually have either a statement of magnification (e.g. x100) or a linear
scale of some description (a bar of defined length e.g. 50 nm).
Actual size can be determined using the following equation:
Magnification can be determined using the same relationship, rearranged as follows:
Estimation of size
It is possible to estimate the size of a structure seen with a microscope by comparing the image with a known linear scale.
Two pieces of apparatus are commonly used:
• a graticule (eyepiece micrometer); and
• a stage micrometer.
A stage micrometer is a slide with a fine scale of known dimension etched onto it. It
can also be a transparent ruler
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An graticule is a fine scale that fits inside an eyepiece lens. The absolute size of the
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scale is unknown hence is the one that is calibrated
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This is shown in Fig 3.
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The graticule can be calibrated using a stage micrometer. This is shown in Fig 4.
Preparation of material for viewing under a microscope
1. Fixation
Organism/tissues are killed rapidly to preserve them in a life-like condition. For use under light microscope, it is killed using 70% ethanol
and for viewing under electron microscope, glutaraldehyde is used
2. Dehydration
Is the removal of water to prepare material for infiltration with an embedding medium or mounting medium which will not mix with water.
For light microscope, ethanol or propanone series are used and for electron microscope ethanol or propanone series are also used
3. Clearing
Is used where alcohol does not mix with some common embedding and mounting media. Xylol is used and it also makes the material
transparent
4. Embedding
Enables very thin sections to be cut if embedded in a supporting medium. For light microscopy, molten wax is used. Plastic or resin is used
for electron microscopy
5. Sectioning
The material is then cut very thinly to allow electrons and light to pass through it. For light microscopy, a microtome or metal knife are used.
For electron microscopy, a diamond cutter or an ultra-microtome are used
6. Staining
This provides contrast between different structures. For light microscopy, coloured dyes are used because they reflect light and for
electron microscopy heavy metals e.g. lead is used because they reflect electrons.
7. Mounting
A chemical is used to fix the material on the slide which will exclude air and preserve the material. For light microscopy, Canadian Balsam or
euparol is used. No mounting is required for electron microscopy.
What is the estimate diameter of the cell in micrometers?
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