The History of EM
By the middle of the 19th century, microscopists had accepted
that it was simply not possible to resolve structures of
less than half a micrometre with a light microscope because
of the Abbe’s formula, but the development of the cathode
ray tube was literally about to change the way they looked
at things; by using electrons instead of light! Hertz
(1857-94) suggested that cathode rays were a form of wave
motion and Weichert, in 1899, found that these rays could
be concentrated into a small spot by the use of an axial
magnetic field produced by a long solenoid. But
it was not until 1926 that Busch showed theoretically that
a short solenoid converges a beam of electrons in the same
way that glass can converge the light of the sun, that a
direct comparison was made between light and electron beams.
Busch should probably therefore be known as the father of
electron optics.
In 1931 the German engineers Ernst Ruska and Maximillion
Knoll succeeded in magnifying and electron image. This
was, in retrospect, the moment of the invention of the electron
microscope but the first prototype was actually built by
Ruska in 1933 and was capable of resolving to 50 nm. Although
it was primitive and not really fit for practical use, Ruska
was recognised some 50 years later by the award of a Nobel
Prize. The first commercially available electron microscope
was built in England by Metropolitan Vickers for Imperial
College, London, and was called the EM1, though it never
surpassed the resolution of a good optical microscope. The
early electron microscopes did not excite the optical microscopists
because the electron beam, which had a very high current
density, was concentrated into a very small area and was
very hot and therefore charred any non-metallic specimens
that were examined. When it was found that you could
successfully examine biological specimens in the electron
microscope after treating them with osmium and cutting very
thin slices of the sample, the electron microscope began
to appear as a viable proposition. At the University
of Toronto, in 1938, Eli Franklin Burton and students Cecil
Hall, James Hillier and Albert Prebus constructed the first
electron microscope in the New World. This was an effective,
high-resolution instrument, the design of which eventually
led to what was to become known as the RCA (Radio Corporation
of America) range of very successful microscopes.
Unfortunately, the outbreak of the Second World War in
1939 held back their further development somewhat, but within
20 years of the end of the war routine commercial electron
microscopes were capable of 1 nm resolution.
Types of Electron Microscopes
All electron microscopes use electromagnetic and/or electrostatic
lenses to control the path of electrons. Glass lenses,
used in light microscopes, have no effect on the electron
beam. The basic design of an electromagnetic lens is
a solenoid (a coil of wire around the outside of a tube)
through which one can pass a current, thereby inducing an
electromagnetic field. The electron beam passes through the
centre of such solenoids on its way down the column of the
electron microscope towards the sample. Electrons are very
sensitive to magnetic fields and can therefore be controlled
by changing the current through the lenses.
The faster the electrons travel, the shorter their wavelength. The
resolving power of a microscope is directly related to the
wavelength of the irradiation used to form an image. Reducing
wavelength increases resolution. Therefore, the resolution
of the microscope is increased if the accelerating voltage
of the electron beam is increased. The accelerating voltage
of the beam is quoted in kilovolts (kV). It is now possible
to purchase a 1,000kV electron microscope, though this is
not commonly found.
Although modern electron microscopes can magnify objects
up to about two million times, they are still based upon
Ruska's prototype and the correlation between wavelength
and resolution. The electron microscope is an integral part
of many laboratories such as The John Innes Centre. Researchers
can use it to examine biological materials (such as microorganisms
and cells), a variety of large molecules, medical biopsy
samples, metals and crystalline structures, and the characteristics
of various surfaces. Nowadays, electron microscopes
have many other uses outside research. They can be
used as part of a production line, such as in the fabrication
of silicon chips, or within forensics laboratories for looking
at samples such as gunshot residues. In the arena of
fault diagnosis and quality control, they can be used to
look for stress lines in engine parts or simply to check
the ratio of air to solids in ice cream!
Transmission Electron Microscope (TEM)
The original form of electron microscopy, Transmission electron
microscopy (TEM) involves a high voltage electron beam emitted
by a cathode and formed by magnetic lenses. The electron
beam that has been partially transmitted through the very
thin (and so semitransparent for electrons) specimen carries
information about the structure of the specimen. The spatial
variation in this information (the "image") is
then magnified by a series of magnetic lenses until it is
recorded by hitting a fluorescent screen, photographic plate,
or light sensitive sensor such as a CCD (charge-coupled device)
camera. The image detected by the CCD may be displayed in
real time on a monitor or computer.
Transmission electron microscopes produce two-dimensional,
black and white images.
Resolution of the TEM is also limited by spherical and
chromatic aberration, but a new generation of aberration
correctors has been able to overcome or limit these aberrations.
Software correction of spherical aberration has allowed the
production of images with sufficient resolution to show carbon
atoms in diamond separated by only 0.089 nm and atoms in
silicon at 0.078 nm at magnifications of 50 million times.
The ability to determine the positions of atoms within materials
has made the TEM an indispensable tool for nano-technologies
research and development in many fields, including heterogeneous
catalysis and the development of semiconductor devices for
electronics and photonics. In the life sciences, it
is still mainly the specimen preparation which limits the
resolution of what we can see in the electron microscope,
rather than the microscope itself.
At JIC we have a high voltage (200kV) TEM, which was installed in 2008. We have two digital cameras on it, one is higher resolution than the other, so that the need for developing and printing film has been negated. Our TEM is designed for use with biological samples and is capable of resolving to better than 1nm. It is also capable of 3-D tomography which involves taking a succession of images whilst tilting the specimens through increasing angles, which can then be combined to form a three-dimensional image of the specimen.
Scanning Electron Microscope (SEM)
Unlike the TEM, where the electrons in the primary beam
are transmitted through the sample, the Scanning Electron
Microscope (SEM) produces images by detecting secondary electrons
which are emitted from the surface due to excitation by the
primary electron beam. In the SEM, the electron beam is scanned
across the surface of the sample in a raster pattern, with
detectors building up an image by mapping the detected signals
with beam position.
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| SEM image of a fly's foot taken at JIC in 2006 |
From "Micrographia", by Robert Hooke, 1665:
plate showing the drawing of a fly's foot |
TEM resolution is about an order of magnitude better than the SEM resolution. Our TEM can easily resolve details of 0.2nm. Our two SEMs at JIC are both relatively recent acquisitions and are high-resolution instruments capable of about 2 nm resolution on biological samples. Because
the SEM image relies on electron interactions at the surface
rather than transmission it is able to image bulk samples
and has a much greater depth of view, and so can produce
images that are a good representation of the 3D structure
of the sample. SEM images are therefore considered
to provide us with 3D, topographical information about the
sample surface but will still always be only in black and
white.
In the SEM, we use much lower accelerating voltages to
prevent beam penetration into the sample since what we require
is generation of the secondary electrons from the true surface
structure of a sample. Therefore, it is common to use
low KV, in the range 1-5kV for biological samples, even though
our SEMs are capable of up to 30 kV.
At JIC we currently have two SEMs, both with high-resolution
capabilities, digital imaging facilities and cryo-systems
which enable them to be used for looking at frozen-hydrated
specimens.
Sample Preparation
Materials to be viewed in an electron microscope generally
require processing to produce a suitable sample. This is
mainly because the whole of the inside of an electron microscope
is under high vacuum in order to enable the electron beam
to travel in straight lines. The technique required
varies depending on the specimen, the analysis required and
the type of microscope:
Cryofixation - freezing a specimen rapidly, typically
to liquid nitrogen temperatures or below, that the water
forms ice. This preserves the specimen in a snapshot of its
solution state with the minimal of artefacts. An entire field
called cryo-electron microscopy has branched from this technique.
With the development of cryo-electron microscopy, it is now
possible to observe virtually any biological specimen close
to its native state.
Fixation - a general term used to describe the process
of preserving a sample at a moment in time and to prevent
further deterioration so that it appears as close as possible
to what it would be like in the living state, although it
is now dead. In chemical fixation for electron microscopy,
glutaraldehyde is often used to crosslink protein molecules
and osmium tetroxide to preserve lipids.
Dehydration - removing water from the samples. The
water is generally replaced with organic solvents such as
ethanol or acetone as a stepping stone towards total drying
for SEM specimens or infiltration with resin and subsequent
embedding for TEM specimens.
Embedding - infiltration of the tissue with wax
(for light microscopy) or a resin (for electron microscopy)
such as araldite or LR White, which can then be polymerised
into a hardened block for subsequent sectioning.
Sectioning - the production of thin slices of the
specimen. For light microscopy, the sections can
be a few micrometres thick but for electron microscopy they
must be very thin so that they are semitransparent to electrons,
typically around 90nm. These ultra-thin sections for electron
microscopy are cut on an ultramicrotome with a glass or diamond
knife. Glass knives can easily be made in the laboratory
and are much cheaper than diamond, but they blunt very quickly
and therefore need replacing frequently.
Staining - uses heavy metals such as lead and uranium
to scatter imaging electrons and thus give contrast between
different structures, since many (especially biological)
materials are nearly "transparent" to the electron
beam. By staining the samples with heavy metals, we add electron
density to it which results in there being more interactions
between the electrons in the primary beam and those of the
sample, which in turn provides us with contrast in the resultant
image. In biology, specimens can be stained "en
bloc" before embedding and/or later, directly after
sectioning, by brief exposure of the sections to solutions
of the heavy metal stains.
Freeze-fracture and freeze-etch - a preparation
method particularly useful for examining lipid membranes
and their incorporated proteins in "face on" view.
The fresh tissue or cell suspension is frozen rapidly (cryofixed),
then fractured by simply breaking or by using a microtome
while maintained at liquid nitrogen temperature. The cold,
fractured surface is generally "etched" by increasing
the temperature to about -95°C for a few minutes to let
some surface ice sublime to reveal microscopic details. For
the SEM, the sample is now ready for imaging. For the
TEM, it can then be rotary-shadowed with evaporated platinum
at low angle (typically about 6°) in a high vacuum evaporator.
A second coat of carbon, evaporated perpendicular to the
average surface plane is generally performed to improve stability
of the replica coating. The specimen is returned to room
temperature and pressure, and then the extremely fragile "shadowed" metal
replica of the fracture surface is released from the underlying
biological material by careful chemical digestion with acids,
hypochlorite solution or SDS detergent. The floating replica
is thoroughly washed from residual chemicals, carefully picked
up on an EM grid, dried then viewed in the TEM.
Sputter Coating - an ultra-thin coating of electrically-conducting
material, deposited by low vacuum coating of the sample.
This is done to prevent charging of the specimen which would
occur because of the accumulation of static electric fields
due to the electron irradiation required during imaging.
It also increases the amount of secondary electrons that
can be detected from the surface of the sample in the SEM
and therefore increases the signal to noise ratio. Such coatings
include gold, gold/palladium, platinum, chromium etc.
Disadvantages of Electron Microscopy
Electron microscopes are very expensive to buy and maintain.
They are dynamic rather than static in their operation: requiring
extremely stable high voltage supplies, extremely stable
currents to each electromagnetic coil/lens, continuously-pumped
high/ultra-high vacuum systems and a cooling water supply
circulation through the lenses and pumps. As they are very
sensitive to vibration and external magnetic fields, microscopes
aimed at achieving high resolutions must be housed in buildings
with special services.
A significant amount of training is required in order to
operate an electron microscope successfully and electron
microscopy is considered a specialised skill.
The samples have to be viewed in a vacuum, as the molecules
that make up air would scatter the electrons. This means
that the samples need to be specially prepared by sometimes
lengthy and difficult techniques to withstand the environment
inside an electron microscope. Recent advances have allowed
some hydrated samples to be imaged using an environmental
scanning electron microscope, but the applications for this
type of imaging are still limited.
Artefacts
It must be emphasised from the outset that every electron
micrograph is, in a sense, an artefact. Changes in
the ultra-structure are inevitable during all the steps of
processing that samples must undergo: material is extracted,
dimensions are changed and molecular rearrangement occurs. The
best thing we can do is to keep these changes to a minimum
by understanding the processes involved so that we make informed
choices of the best preparative procedures to use for each
sample. Artefacts present themselves in
many ways: there could be loss of continuity in the membranes,
distortion or disorganisation of organelles, empty spaces
in the cytoplasm of cells or sharp bends or curves in filamentous
structures that are usually straight, such as microtubules
and so on. With experience, microscopists learn to recognise
the difference between an artefact of preparation and true
structure, mainly by looking at the same or similar specimens
prepared in the same or a different way.
Scanning electron microscopes usually image conductive
or semi-conductive materials best. Non-conductive materials
can be imaged, either by an environmental scanning electron
microscope or more usually by coating the sample with a conductive
layer of metal. A common preparation technique is to coat
the sample with a layer of conductive material, a few nanometers
thick, such as 10nm of gold, from a sputtering machine. This
process does, however, have the potential to disturb delicate
samples and cover some detail. When using chemical
fixation and dehydration as part of the sample preparation,
there is often much shrinkage and collapse of delicate structures
and so, especially for our interests at JIC in botanical
specimens which are highly hydrated, we tend to use the cryo-fixation
technique which is far less prone to artefacts.
For the TEM, samples are generally prepared by exposure
to many nasty chemicals, in order to give good ultra-structural
detail which may result in artefacts purely as a result of
preparation. This gives the problem of distinguishing artefacts
from genuine structures within the specimen, particularly
in biological samples. Scientists maintain that the results
from various preparation techniques have been compared, and
as there is no reason that they should all produce similar
artefacts, it is therefore reasonable to believe that electron
microscopy features correlate with living cells. In addition,
higher resolution work has been directly compared to results
from X-ray crystallography, providing independent confirmation
of the validity of this technique. Recent work performed
on unfixed, vitrified (rapidly frozen, without the use of
any chemicals, to form ice without any crystallisation) specimens
has also been performed, further confirming the validity
of this technique. However, even cryo-fixation techniques
are not without their own artefacts of preparation and ice
crystal damage, due to the fact that as water freezes it
expands, is a common problem when trying to image a large
specimen (greater than 200 µm) which cannot be
frozen rapidly enough to vitrify the water
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