Some Notes for Biological Electron Microscopy
The importance of the electron microscope lies in its ability
to break through the resolution barrier of the light microscope
(c.a.100nm) into the world of biological ultrastructure. The pioneering
work of Robley Williams in 1946 gave the first images of individual
viruses. Since then, the technique has been constantly improved
to the point where resolutions for biological structures in the
2-3nm range are routine. For a small number of specimens, using
a combination of EM and computer image processing (electron crystallography)
it has been possible to reach the level of resolution (0.3nm)
previously possible only by X-ray crystallography and NMR.
The two main types of electron microscopes used in biology are
the scanning electron microscope and the transmission electron
microscope. As the latter is more common, we will go into more
detail here into its use.
The scanning electron microscope (SEM).
In the SEM the specimen is scanned with a focussed electron spot
which produces "secondary" electrons as it hits the specimen surface.
These secondary electrons are detected and converted into a video
image of the surface. The specimen is usually coated with a thin
layer of evaporated metal to prevent electrostatic charging. This
coating and the size of the scanning spot limits the normal resolution
of the SEM to about 5-10nm.
The Transmission Electron Microscope (TEM)
The TEM has much in common with the light microscope in that
images are produced by transmission of the illuminating radiation
through a thin slice of material. This can be an ultra thin section
(less than 0.2mm in thickness), dispersed particles or (in cryo-em)
frozen suspensions. The specimens are normally supported by a
thin carbon film which is analogous to the glass specimen slide
in the light microscope. Contrast is produced either by staining
the specimen with heavy metal salts (e.g. uranyl acetate, lead
citrate), by evaporating a layer of metal on to the surface or
by the use (as in light microscopy) of phase contrast for unstained
specimens.
Resolving Power
The human eye can distinguish two objects if they are not closer
than 0.1 mm at a normal viewing distance of 25 cm. This ability
to separate two objects optically is called resolving power. Any
finer detail than this can be resolved by the eye only if the
object is enlarged. This enlargement can be achieved by the use
of optical instruments such as hand lenses, compound light microscopes
and electron microscopes.
Resolution in the light microscope
The resolving power of the light microscope is mainly limited
by the wavelength of the light (l) used for illumination.
The Abbe "diffraction limit" for image resolution is given by:
d= l/2*N.A.
Where N.A. (numerical aperture) is a measure of the resolving
power of the lens. The best oil immersion lenses have an N.A.
of about 1.4, thus for visible light with a wavelength of 400nm,
the resolution would be about 140nm.
It can be seen that resolving power improves (d decreases) as
the wavelength decreases.
Due to this limitation of resolving power in light microscopy,
other sources of illumination with shorter wavelengths than visible
light, have been investigated. Early experiments using X-rays
of extremely short wavelength were not persued further because
of the inability to focus these rays although there have been
recent attempts to revive X-ray microscopy using synchrotron radiation.
So-called "lensless" imaging systems such as the scanning tunneling
microscope (STM) and the atomic force microscope (AFM) have also
been developed to overcome the limitation of wavelength.
Resolution in the electron microscope
The development of electron imaging became a possibility when
Louis de Broglie proposed in 1924 that just as light has a dual
nature behaving both as particles and waves, there could be a
wave-like quality to the propagation of atomic particles.
l = h/mv where h is Planck's constant, m the mass and
v the velocity
In the case of electrons which are electrically charged and can
be accelerated by an electric field:
l = h/(2meV)-2 where V is the accelerating voltage, e the electron
charge
In addition to this wave-like nature of moving electrons, they
can be easily deflected (focussed) by magnetic or electrostatic
lenses, opening up the possibility of constructing a magnifying,
imaging system with very high resolution - the transmission electron
microscope, first developed by Ernst Ruska in 1935. Using the
formula above we can calculate that for electrons accelerated
by 100 kilovolts, l = 0.0037nm. In practice, the resolution of
the best electron microscopes is limited, mainly by lens defects
(small N.A., spherical aberration etc.), to about 0.2nm.
Construction of the TEM
In its normal form the TEM is like an inverted light microscope.
The illumination source , usually a tungsten filament (cathode)
is at the top of the evacuated column. The filament is heated,
causing it to emit electrons (thermionic emission). A high voltage
(the accelerating voltage) of up to 400,000 volts is passed between
it and the anode, so that the negatively charged electrons are
accelerated towards the anode (positively charged) which is placed
just below the filament. Some electrons pass through a small hole
in the anode, forming an electron beam which passes down the column.
The speed of the electrons and hence their wavelength depends
upon the accelerating voltage.
Electro-magnetic coils, placed at intervals down the column,
act as lenses to focus the electrons. Condenser lenses focus the
electron beam on to the specimen which is mounted on a movable
stage above the objective lens. As the electron beam passes through
the specimen some electrons are scattered whilst the remainder
are focussed first by the objective lens and then by two further
stages of magnification (intermediate lens and projector lens)
to give an image on a fluorescent screen or on photographic film.
Electron lenses (unlike optical lenses) can be varied in focal
length by varying the lens current through the coils which simplifies
focussing (by the objective lens) and zooming of magnification
(by the intermediate and projector lenses).
An aperture is placed below the objective lens to block electrons
scattered to high angles (particularly by the heavy metal atoms
used for staining). The removal of these electrons from the image
produces amplitude contrast. In the case of unstained specimens,
where there is very little amplitude contrast, it is necessary
to use phase contrast, produced by defocussing (under-focussing)
the image.
Specimen preparation for TEM
Most of the improvement in resolution of biological specimens
has resulted from better specimen preparation techniques. There
are four main problems:
a) The need to maintain a high vacuum in the microscope column
The biological specimen must be stabilized in some way so that
its ultrastructure, when exposed to the vacuum, is as close as
possible to that in the wet living material. This can done by
chemical fixation of bulk specimens followed by dehydration and
plastic embedding or by embedding a particulate specimen in a
layer of stain (negative staining). Unstained specimens for electron
crystallography (see below) are embedded in a layer of glucose
or tannic acid which replaces the water. Alternatively, the specimen
is frozen very rapidly to prevent ice-crystal damage and then
transferred, still frozen, at liquid nitrogen or liquid helium
temperature into the EM for observation (cryo-EM).
b) The low intrinsic contrast of biological specimens.
As mentioned above, amplitude contrast depends on the type of
atoms in the specimen; electron scattering increases in proportion
to the atomic number of the scattering atom. Biological specimens
are composed of atoms of low atomic number (carbon, hydrogen,
nitrogen, phosphorus and sulphur) and their contrast can be increased
by staining with heavy metal salts or shadowing with evaporated
heavy metal (e.g. platinum). This can have an added advantage
that the heavy metal is usually less sensitive to irradiation
by electrons (see below). The simplest method is ?negative staining?
in which a sample in suspension is dried down in a layer of uranyl
acetate or sodium phosphotungstate. This limits the resolution
to about 2nm which is, however, sufficient to visualise the subunit
structure of proteins and is useful as a routine technique which
does not require specialised equipment.
c) The specimen must be thin.
The limited penetrating power of electrons means that the specimens
must be very thin or must be sliced into thin sections (50 - 100
nm) with an ultramicrotome to allow electrons to pass through.
d) The damaging effect of electron irradiation of the specimen.
The radiation dose experienced by the specimen during observation
in the TEM can be sufficient to cause covalent bond breakage,
leading to mass loss, shrinkage and other structural changes.
This can be reduced by so-called ?low-dose? techniques where the
minimum electron dose is used to photograph a ?fresh? part of
the specimen - all focussing and other adjustments to the microscope
are made on an adjacent area. Image averaging techniques, either
combining data from many single particles or from unit cells in
a crystal, are also important in order to improve the signal-to-noise
ratio for images taken with very low electron doses. There is
also a reduction in damage (by a factor of 5-10 times) by cooling
the specimen to low temperatures.
3-Dimensional Image reconstruction.
Klug in 1968 showed that because of the high depth of focus of
electron lenses, images are 2-D projections of the whole object
density (or the stain density) on to the image plane. The 3-D
information can be recovered if a number of views of the object
are recorded at different angles of observation. This can be done
using a ?tilt stage? or goniometer, normally up to a maximum tilt
angle of about 60 degrees. For special cases, e.g. helices or
structures of high rotational symmetry it may be possible to use
just a single view or a much smaller number of tilted views to
reconstruct a 3-D model. In all cases, it is necessary to digitise
the micrographs and then use either ?fourier? or tomographic computer
algorithms to calculate the 3-D object density. The fourier methods
(often referred to as ?electron crystallography?, see below) are
analogous to those used in X-ray crystallography with the important
difference that there is no ?phase problem? in electron crystallography
since it is possible to calculate phases directly from the images.
Electron Crystallography
Parsons in 1971 showed that unstained, thin catalase crystals
will give electron diffraction patterns to atomic resolution if
they are maintained in a wet state in an electron microscope equipped
with a hydration chamber. Although this demonstrated that very
high resolution information is present in unstained specimens,
it was not possible to obtain high resolution images by this method.
In 1975 Unwin & Henderson showed that similar results could be
obtained for bacterial rhodopsin (purple membrane) by drying the
specimen in glucose. Electron diffraction gave diffraction intensities
extending to about 0.3nm and the first images gave phase information
extending to about 0.7nm. Combining these data for a series of
images of specimens at different tilt angles they obtained the
first 3-D structure of a membrane protein showing trans-membrane
helices. The method has now been improved to the point where phases
are also measurable to about 0.3nm allowing amino acid side-chains
to be resolved. For this technique, very large single layer crystals
are necessary - it is thus particularly suitable for crystals
of membrane proteins confined within a lipid bilayer. Beam damage
can be reduced by a factor of about 5 times by cooling the specimen
to liquid nitrogen temperature (see below).
Cryo-EM.
Cryo methods were developed partly to reduce the electron beam
damage which decreases at low temperatures and partly to reduce
the damage (drying artefacts) caused by removal of water from
biological specimens prepared for EM. In 1978 Glaeser & Taylor
constructed the first cryo-transfer holder which enabled a frozen
hydrated biological specimen to be observed in the EM. In 1982
Dubochet and co-workers at EMBL improved the technique by introducing
fast freezing in liquid ethane to give vitreous (as opposed to
crystalline) ice films. Cryo-electron microscopy has now become
a routine technique for obtaining well preserved hydrated specimens
with resolutions almost as good as those obtained by electron
crystallography of glucose embedded material. In both techniques,
low dose imaging is necessary to avoid disruption of the specimen
and contrast is enhanced by defocussing the objective lens. Phase
artefacts introduced by defocus - the contrast transfer function
(CTF) - can be corrected during the computer image reconstruction
procedure.
A disadvantage of the electron microscope compared with the light
microscope is that it is not possible to observe dynamic processes.
Rapid freezing and Cryo-EM, however, provide a method for "trapping"
dynamic processes at millisecond time intervals, which can be
useful for many biological processes.
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Literature:
Good introductions to the methodology are provided by the two
multivolume series (which are in the EMBL library)
Hayat, M.A (editor) . Principles and techniques of electron
microscopy:
Glauert, A. Practical methods in electron microscopy
A review on Cryo-EM:
Dubochet et al, 1988: Cryo-electron microscopy of vitrified
specimens. Q Rev Biophys, 21:2, 129-228
A review on 3-D image processing of crystals and electron crystallography:
Amos et al (1982) , Prog. Biophys. Molec. Biol. 39, 183-231