Download Electron Microscope

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Sizes
10 0
10 -3
10 -6
10 -9
10 -10
10 -12
1 metre
1 millimetre
1 micron
1 nanometre
1 angstrom
1 picometre
(m)
(mm)
(µm)
(nm)
(Å)
(p)
Electron Microscope
(a)
High voltage (e.g. 50,000 volts) is passed through a “tungsten filament” at the top
of the column. This makes the filament emit a stream or beam of electrons.
(b)
Electrons are focussed by electromagnets (rather than glass lenses – as in the
optical microscope)
(c)
The image produced by the electron microscope cannot be detected by the naked
eye – instead, the electron beam is directed onto a fluorescent screen or
photographic film. The black and white image, which is produced, is called an:
“electron micrograph”.
(d)
The inside of the electron microscope is under a high vacuum. This minimises the
electrons scattering due to collisions with air particles and the subsequent
heating, which would occur.
There are two types of Electron Microscope:
1.
Transmission electron microscope
-
a beam of electrons is passed through thin sections (10 – 100 nm)
-
where electrons are absorbed by the material, they do not reach the screen
the image formed is DARK – these areas are called “electron dense areas”
where electrons pass through, the specimen, the screen appears BRIGHT –
-
these are called “electron transparent areas”
the image formed is flat and therefore gives no idea of the natural contours of
the specimen.
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2.
Scanning electron microscope
Biology AS Level
-
a fine beam of electrons is passed “to and fro” across the specimen. This
specimen does not have to be thin, as the electrons do not have to pass through
it.
-
some electrons bounce off the specimen and are scattered
others are absorbed by the specimen
the electrons, which are scattered are amplified and transmitted onto a screen
the result is that the image shows “holes and depressions” as DARK areas
-
the “ridges and extensions” are shown as bright areas
this gives a 3D image
the resolution for the scanning electron microscope is poorer than for the
transmission electron microscope (5-20nm)
Transmission Electron Microscope
Scanning Electron Microscope
flat
3D
× 500,000
× 10,000 - × 100,000
(i.e. higher magnification)
(i.e. lower magnification)
no depth
greater depth
Electrons
pass through / absorbed
scattered / absorbed
Resolving power
0.5 nm
(i.e. greater resolving power)
5 – 20 nm
(i.e. lower resolving power)
Image
Magnification
Preparation of material for Electron Microscope
Fixation
-
chemical is added to stop biochemical reactions
-
to make sections sensitive to staining agents
-
section without fixing causes the structures to oxidise in air
-
thus the structure is affected by fixation
-
fixation also acts as a preservative. e.g.:
Osmium tetroxide (for animal cells)
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KMnO4 (for plant cells)
Embedding
-
sections must be very thin
-
they are sliced with an ultramicrotone (20 – 100 nm)
-
to do this, the specimen is embedded in Araldite or Epoxyresin to make it
rigid: (i.e. too flimsy or fragile to be sectioned on its own)
-
may need to make replica / cast to fill the crevices before sectioning
Sectioning
-
using glass blade / diamond
ultramicrotone
Dehydration
-
soak section in acetone / organic solvent
-
to remove water slowly
-
water evaporates on contact with electrons and would destroy the vacuum and
the focussed electron beam.
Staining
-
stains which scatter electrons must be used rather than those which reflect
different wavelengths of light. e.g.:
uranium
tungsten
lead
-
used as salts (i.e. uranyl acetate, sodium phosphotungstate)
Mounting
-
specimens are mounted on a copper grid
-
rather than a glass slide – which would absorb the electrons
Artefact
-
treatments which specimens undergo are very harsh
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this, therefore, may change the cell – and produce “artefacts” (structures
which are not part of the original cell, but which appear as a result of the
treatment
-
it is necessary to make many different sections, from different angles, using
different stains to ensure that the structure appears regularly – and is not an
artefact.
Cell membranes
-
very delicate
freeze with liquid nitrogen
cut with a glass blade
knife goes along the path of least resistance (i.e. the membrane)
Advantages of an electron microscope
(a)
Magnifies up to × 500,000
(b)
High resolution (0.5 nm / 0.005µ) (low wavelength)
Disadvantages of an electron microscope
(a)
expensive to purchase and operate:
i. up to £1 million to buy 1990
ii. uses lots of electricity
(b)
large – and needs a special room
(c)
affected by magnetic fields
(d)
preparation of material takes a long time – requires expertise and complex
equipment
(e)
preparation of material causes artefacts (most of the time) (i.e. distortions)
(f)
living material cannot be observed
(g)
images are black and white