Transmission Electron

Microscopy: Preparation
of Specimens
Bettina Bottcher, University of Edinburgh, Edinburgh, UK
Sample preparation of biological specimens for electron
microscopy aims at bringing the sample into a suitable
size (5500nmthick, accommodatedona 3mmdiameter,
round sample carrier) and to strengthen these samples
against the adverse conditions inthe electron microscope
(beam damage and vacuum). There are two types of
samples, one are large cellular structures, which require
further sectioning and the other are individual assem-
blies, which are small enough to be imaged in suspension.
Any preparation method of these types of samples has to
fulfil four basic requirements: (1) to avoid collapse of the
structures in the vacuum of the electron microscope; (2)
to provide a sample sufficiently thin to avoid multiple
scattering of electrons; (3) to minimise structural alter-
ations as a consequence of the damage by the electron
beam; (4) to maximise the contrast inthe resolutionband
of interest. The relative importance of each constraint
varies with the type of specimen and with the level of
resolution sought.
Introduction
Electron microscopy targets dierent types of biological
material. One class of objects (here referred to as class I
objects) are protein assemblies, which exist in multiple
identical copies. This group includes large (4200 kDa)
biological complexes, such as ribosomes or proteasomes,
helical assemblies like microtubules or actin laments,
regular viruses like Parvoviruses and 2D-crystals of mem-
brane proteins or soluble proteins bound to a matrix such
as a lipid monolayer. Typically, the objects in this class are
thin enough (5100 nm) to be imaged directly without
further sectioning. In most cases micrographs of these
objects are further analysed by image processing, which
uses averaging to improve the signal-to-noise ratio and to
restore the 3Dimage information. Image processing is very
powerful andcandeliver image informationclose toatomic
resolution. To realise the potential of the method, sample
preparation needs to preserve the molecular structure as
precisely as possible without distortions or alterations of
the objects.
The second class of objects (here referred to as class II
objects) are unique, large sub-cellular, cellular and supra-
cellular structures, which are too thick to be imaged as a
whole. These objects require sectioning for trimming the
objects to an appropriate thickness (50500 nm), which
minimises multiple scattering of electrons during imaging.
Further analysis of micrographs of these objects aims at
identifying their overall morphology and the distribution
and properties of class I objects in their native environ-
ment. Thus sample preparation needs to preserve the large-
scale morphology of the class II object without disturbing
the spatial relationship between the class I objects within.
In general, sample preparation of all objects aims at
preserving the structures against the adverse conditions
inside the electron microscope. The main challenges for the
samples are (1) the damage by the bombardment with
electrons and (2) the vacuuminside the electron microscope.
(1) The dose, which is experienced by the sample during a
3 s observation at high magnication, is in the order of
4000 Mrad, which is similar to the dose in the centre of an
atomic bomb explosion. The deposition of energy during
bombardment leads to bond breakage, which is considered
as a primary event of beamdamage. The resulting fragments
are often no longer stable in the places where they are gen-
erated and start moving around. In addition the fragments
contain highly reactive radicals, which react easily with
reaction partners. The movement of fragments and further
chemical reactions are considered as the secondary events of
beamdamage. Althoughthe primaryevents of beamdamage
are largely independent of the temperature, the secondary
events are temperature dependant and thus can be reduced
by cooling the sample. Therefore, at lower temperatures
high-resolution information is preserved at a higher electron
dose. As a consequence many strategies for sample prepar-
ation stabilise the sample at cryogenic temperatures.
Another possibility to harden samples against beam
damage is to preserve the structure in a state, which only
responds very little to the dose challenge. One option is to
use imprints in inorganic salts or metal coatings, which
change only slowly in response to the dose even at room
temperature. This strategy is used in negative staining and
in metal shadowing.
Introductory article
Article Contents
. Introduction
. Sample Carriers
. Preparation of Class I Objects
. Preparation of Class II Objects
Online posting date: 15
th
June 2012
eLS subject area: Structural Biology
How to cite:
Bottcher, Bettina (June 2012) Transmission Electron Microscopy:
Preparation of Specimens. In: eLS. John Wiley & Sons, Ltd: Chichester.
DOI: 10.1002/9780470015902.a0002998.pub2
eLS & 2012, John Wiley & Sons, Ltd. www.els.net 1
(2) The other adverse eect in the electron microscope is
the vacuum. At room temperature the vacuum leads to
immediate evaporation of all structural water of the sam-
ple. Without water the sample collapses and attens.
There are various ways to reduce attening eects. In
principle the water can be replaced by a substance, which
satises the hydrogen bonds of the protein similarly well as
water but which has a lower vapour pressure and thus, does
not evaporate. This property is satised by sugars such as
glucose (Unwin and Henderson, 1975) or trehalose (Hirai
et al., 1999), which have been successfully used for pre-
paring class I objects in 2D-protein crystals. However, the
density of the sugar matches approximately the density of
the protein. Therefore, there is almost no contrast fromthe
envelope of the particles and contrast arises mainly by
internal density dierences inside the protein (only visible
at high resolution).
For class II objects embedding in sugar is no longer
sucient for the eective replacement of the large volumes
of structural water. Instead, resins are used for replacing
the water. This preserves the large scale morphology to a
certain degree but also introduces various types of artefacts
such as aggregation, swelling of cells and organelles or
disturbance of the lipidic structures. The class I objects in
these resin substituted class II samples are only poorly
preserved and have often changed their relative positions.
Thus this preparation method is not suitable to address
questions which require the identication of class I objects
at their native surroundings.
Insteadof replacing the water, the water canbe stabilised
inside the microscope. This is achieved by lowering the
vapour pressure of the water belowthe ambient pressure of
the vacuum. In practice this is achieved by cooling the
sample below 21008C were the water does not evaporate.
Inorder toavoidthe formationof ice crystals, whichdistort
the ultra structure of the biological objects, samples
are vitried by rapid freezing or by high-pressure freezing.
See also: Single Particle EM
Sample Carriers
For electron microscopy, all samples have to be brought
into a format that ts into the holders of commercial
electron microscopes. The commonly used electron
microscopes accept thin circular carriers with a diameter
of 3.05 mm.Typical carriers are metal grids (e.g. copper,
molybdenum, gold, nickel, Figure 1), which have dierent
sizes of holes that are specied by the number of holes per
inch. For example the commonly used 400 mesh grid has
400 holes per inch giving a repeating distance of 63 mm
between the holes. Depending on the purpose of the grids
the mesh varies between 50 and 2000. The holes come in
dierent shapes such as round, hexagonal, squared or slit
shaped and can have numbers or letters on themin order to
nd a specic position. For most applications squared
holes are used. The girds can be obtained in dierent
materials. Copper is most commonly used because it is
most cost eective. For applications, which require long
incubation times on corrosive buers gold grids are used.
Molybdenum is sometimes used if grids are imaged at low
temperatures, because it has a lower expansion coecient
than the other materials and thus leads to atter objects
and less strain in the sample.
In some cases the samples are prepared as thin lms (e.g.
sections). These can be directly applied to the bare grids.
For most other preparation methods, the holes of the grids
have to be covered with a support lm, which is translucent
for electrons. Typical support lms are carbon lms, or
plastic lms (e.g. formvar) which are optionally coated
with an additional carbon lm. The carbon lms are gen-
erated by evaporation either directly onto a plastic support
lm or onto mica. Freshly cleaved mica has a clean,
atomically at surface and therefore is anideal recipient for
the generation of very smooth, at lms. For further pro-
cessing the carbon lm is oated o the mica on a clean
water surface.
Whereas freshly prepared carbon lms are hydrophilic,
they become hydrophobic over time. This interferes with
sample absorption and wetting properties. Therefore,
carbon support lms are treated by glow discharge (high
voltage discharge in a low vacuum), which generates rad-
icals that react with the carbon surface and restore the
hydrophilicity.
Nowadays other materials such as graphene oxide
(Wilson et al., 2009) or silicone nitride, which also have
good properties in terms of conductivity and translucence
for electrons are also occasionally used.
Support lms are either continuous or containholes. The
latter is important for lowcontrast objects such as vitried,
unstained samples, which are imaged over the holes in
suspensiontoreduce the noise introducedby the additional
support lm and to avoid distortion of the objects by
absorption to the support lm.
Preparation of Class I Objects
Staining
The most frequently used method for sample preparation
of class I objects is negative staining, which was introduced
in the late 1950s (Brenner and Horne, 1959). Here the
sample is dried and the biological object is surrounded by a
Tweezers Grid
3

m
m
Rim
Figure 1 400 mesh copper grid held by a pair of tweezers. The grid has a
diameter of 3mm.The grid is somewhat translucent due to the holes and it
has a wider rim (arrow) for handling.
Transmission Electron Microscopy: Preparation of Specimens
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cast of electron dense material. Typical stains contain high
atomic number elements, which have a larger cross section
for electrons than the low atomic number elements in the
biological samples. Stains are typically heavy metal salts
such as uranyl acetate, uranyl formiate, phosphotungstic
acid and silicotungstic acid or ammonium molybdate.
Stains can either interact directly with the biological
material or are just excluded fromareas where the sample is
located. The former event gives rise to positive staining
whereas the latter is used in negative staining. In positive
staining direct interaction of the stain with the object leads
to an enrichment of the stain at the sample. Owing to the
accumulation of the stain, the sample appears dark. Posi-
tive staining is dicult to control and very variable. Thus it
is not used for structure determination on purpose but is an
occasional artefact. Positive staining often occurs in highly
charged areas such as at the RNA in ribosomes and other
ribo-protein complexes.
In negative staining there is no direct interaction of the
sample with the stain. So the stain forms a more or less
amorphous layer and is excluded from areas where the
protein is located (Figure 2). Thus the sample leaves an
imprint in the stain and depletes stain from areas, which it
occupies. Because the heavy metal ions have high atomic
numbers, they scatter electrons much stronger than the low
atomic number biological material. Thus contrast arises
mainly from the pattern in the stain and to a much lesser
extent fromthe sample itself. The areas, whichare occupied
by the sample, appear bright (less scattering at the bio-
logical material) andthe backgrounddark(more scattering
at the electron dense stain, Figure 2).
For staining, an aqueous solution of the protein is
incubated on a carbon lm (30 s2 min). During incu-
bation some of the proteins adsorbs to the carbon lm. The
buer is then exchanged by washing the surface of the
carbon lm with a few droplets of aqueous staining solu-
tion (12%stain) and incubating with the last drop of stain
(for 30 s5 min; staining for longer gives better staining
and less positive staining). Then, most of the solution is
removed by absorbing it to a lter paper. Only a thin layer
of the staining solution remains on the surface and dries by
slow evaporation of the water. After complete drying, the
stainforms a crust at the stainaccessible areas leaving a cast
of the protein. This cast is quite robust, keeps for months
and can be imaged in the electron microscope at room
temperature.
The advantages of staining are the high contrast and the
resistance to beam damage. Therefore, this method is well
suited for looking at the sample and making assessments
of the sample quality. Furthermore, the superior contrast
of the stain make small proteins and small complexes
(5300 kDa) accessible to electron microscopy, which do
not generate sucient contrast for image processing if they
are unstained.
The disadvantages of staining are: (1) it is an indirect
method, where the protein is not observed directly but only
its cast in the stain. This ultimately limits the resolution
(1.52 nm). (2) In most cases the protein is exposed to high
salt concentrations during staining and often also to low
pH (e.g. uranyl acetate), which can lead to structural
damage or alterations of the structure. (3) At room tem-
perature, further structural water is lost when exposed to
the vacuum inside the electron microscope. As a con-
sequence the sample attens. (4) If the sample is not com-
pletely embedded in the stain, the resulting images
represent only parts of the structure. Flattening and partial
embedding are especially cumbersome if images are used
for 3D image reconstruction (dierently oriented particles
are dierently distorted and thus are no longer compatible
Biological
object
Negatively stained sample
Electron microscope
Projection of density
Carbon support film
Electron dense
stain
Figure 2 Schematic drawing of a negatively stained sample
(perpendicular to the direction of imaging) and its projection image
(shown in the direction of imaging) generated by the electron microscope.
Top, the biological object (white circle) is supported by a carbon support
film (brown line) and surrounded by the electron dense stain (black). The
stain accumulates at the edges of the object and is less thick further away
from the object. The electron microscope generates a projection of the
object. Stain is excluded by the object. Thus these areas appear bright.
Stain accumulates close to the object (giving a dark rim) and is less thick
further away from the object. This gives rise to a density gradient around
the object.
Transmission Electron Microscopy: Preparation of Specimens
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with a unique 3D shape). See also: EM Analysis of Protein
Structure
Sugar embedding
Well ordered 2D-crystals have the potential to deliver
structures closetoatomic resolution. Inorder topreserve the
structures well, these crystals are typically embedded in
glucose or trehalose. The procedure is very similar to
staining. Acarbonlm(ca. 4 4 mm) is oatedona suitable
buer solution, picked up with a grid (e.g. molybdenum),
transferred to a drop of buer containing 7% trehalose.
Then excess liquid is removed and a drop of sample is
incubated for 3060 s. Afterwards the liquid is removed and
the sample is plungedintoliquidnitrogen. Inavariant of this
procedure, the sample is sandwiched between two layers of
carbon. Therefore, a second piece of carbon is oated on a
buer, picked up with a loop and transferred onto the
sample after the last blotting step. Then again, excess liquid
is removed and the sample is frozen in liquid nitrogen
(Gyobu et al., 2004). The latter procedure keeps the 2D-
crystals very at, which is required for obtaining high-
resolution information from tilted specimens.
The advantage of the method is that the sugar provides a
relatively stable embedding medium, which stabilises the
protein well at cryogenic temperatures. The contrast arises
mainly from the protein. Thus it is a direct method which
delivers high resolution information. The disadvantage of
the method is that the sugar approximately matches the
density of the protein. Thus the envelopes of the individual
proteins (low resolution information) become almost
invisible. Therefore, sugar embedding is not used for
individual particles and only for extended objects such
as 2D-crystals. See also: Crystallization of Proteins: Two-
dimensional; Two-dimensional Electron Crystallography
Vitrification
To retain the water in the vacuum and thus to avoid
structural collapse, the vapour pressure of the water can be
loweredbelowthe pressure inside the columnby decreasing
the temperature below approximately 21008C. At this
temperature, the most stable modication of water is
crystalline, hexagonal ice. Hexagonal ice requires more
space than liquid water. This is why the ice oats on the
lakes in winter. While ice crystals grow, they expand and
put tremendous pressure onto the sample. This damages
larger structures (e.g. lamentous structures become bent).
In addition, crystalline ice in certain orientations reects
the electron beam giving rise to Bragg-reections. These
Bragg-reections give an uneven background with dark
and white areas that distract from the features in the
sample.
To avoid Bragg-reections and to reduce damage to the
sample by growing ice crystals, it is desirable to maintain
the liquid properties of the water (amorphous solid or
glass-like). This can be achieved by rapid cooling, which
prevents the water molecules to rearrange into a crystal
before they become rigid in a glass-like state (vitrication,
Dubochet, 1981; Dubochet et al., 1988). The required
cooling rates for vitrication are in the order of 10
4
k/s.
Although the density of vitried water is lower than of uid
water (0.93 g/cm
3
compared to 1 g/cm
3
), the water does not
form ice crystals and probably ows around the biological
sample while it expands. Thus, vitrication does not cause
any obvious damage to the structures of biological
specimens.
The vitried state is only stable at temperatures below
21508Cand starts to convert slowly into a cubic crystalline
form at temperatures above (no vitried state above
approximately 21308C). This means that after vitrication
the sample needs to be handled, stored and imaged below
21508C by cooling it with liquid nitrogen.
Rapid cooling rates below the vitrication temperature
are the key for obtaining vitrication. To achieve these
cooling rates, cryogens need to have a relatively high
boiling point to prevent lm boiling, when the hot sample
enters the cryogen. Film boiling generates an insulating
gas-layer, which slows down cooling despite the low tem-
perature of the cryogen. This eect is called Leidenfrost
eect. Therefore, for vitrication liquid ethane (T
b
=
2898C) or less commonly also liquid propane (T
b
=
2428C) are used as cryogens instead of liquid nitrogen
(T
b
=21968C), which is used for cooling the cryogens. In
addition vitrication only works for thin lms (512 mm
thick), because for thicker lms the thermal conductivity of
the sample is not sucient to remove the heat from the
sample quickly enough to obtain vitrication.
The most commonly used vitrication devices are guil-
lotine-like machines (Figure 3), where a grid is mounted in a
pair of tweezers, which is hold by the guillotine. Approxi-
mately 25 ml of sample is applied to the grid. Then, most
of the sample is removed by blotting with lter paper
(115 s). Afterwards, the grid is plunged into liquid ethane.
For more reproducible results, variations of this setup
(Figure 3b) use a computer to control blotting times, blot-
ting forces, humidity and temperature. In addition many
set-ups employ environmental chambers (Bellare et al.,
1988; Figure 3a), which keep the humidity before freezing at
100%. This prevents evaporation of the sample, which
would lead to uncontrolled, slow cooling and would
increase the concentrations of the solutes including the
biological objects.
As an alternative to blotting, the sample is sprayed in a
thin layer onto the grid, while the grid is plunged into the
cryogen. This setup is less commonly used, because it
requires more sample and the thickness of the sample is
dicult to control. Here, the thickness of the sample
depends onthe size of the droplet andthe surface properties
of the solvent that control how a droplet spreads. Despite
these experimental diculties, spraying is a very useful
method for time-resolved experiments, where reaction
events 1 s are trapped (Berriman and Unwin, 1994;
White et al., 1998).
The advantage of vitrication is that it is the gentlest
method for sample preparation and gives the least
Transmission Electron Microscopy: Preparation of Specimens
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artefacts. The unstained sample is directly imaged. There-
fore, there is no limitation in the achievable resolution and
close to atomic resolution structures have been obtained
(e.g. Zhang et al., 2008). The disadvantages of the method
are that the contrast is low (due to the low amplitude
contrast and the similar scattering cross-section of vitried
water and biological material), that the sample must be
kept at lowtemperatures (to avoid devitrication) and that
the sample is more beam sensitive (due to the higher cross-
section of low atomic number material (water and protein)
for inelastic scattering of electrons than for elastic scat-
tering). A comparison of a vitried and a negatively
stained viral capsid is shown in Figure 4. See also: Electron
Cryomicroscopy; Electron Cryomicroscopy and Three-
dimensional Computer Reconstruction of Biological
Molecules
Cryo negative staining
Whereas negative staining provides superior contrast, vit-
rication gives excellent specimen preservation. The latter
is due to keeping the structural water in the sample. To take
advantage of both merits, a combination of the two
methods has been developed (Adrian et al., 1998; De Carlo
et al., 2002), which is called cryo negative staining. In the
original set-up the sample was stained with 16% ammo-
nium molybdate, mounted in a guillotine-like apparatus,
then blotted to form a thin lm, partly dried for 23 s and
nally plunged into liquid ethane. The drying step is
necessary to obtain the typical contrast reversal which is
required for negative staining. However, the sample retains
enough water that its structure is fully supported and does
not collapse. Similarly to the unstained vitried samples,
the frozen samples have to be kept and imaged below the
devitrication temperature.
Alternate methods for cryo negative staining (De Carlo
and Stark, 2010) have in common that the stained grids
are frozen before the transfer into the vacuum of the
microscope and are kept below the devitrication tem-
perature during imaging. However, the amount of struc-
tural water, which is kept with the sample, varies. An
example for cryo negative staining, which retains much
Insulating box
Stand
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S
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Nebulizer
(a) (b)
Filter paper
for blotting
Mount for
tweezers
Computer
for control
Mount for
tweezers
Pot for
ethane
H
u
m
i
d
i
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i
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Figure 3 Apparatus for vitrification. (a) Manual freezing apparatus with an environmental chamber. The humidification of the chamber is achieved with
water soaked sponges. If hot water is used for soaking, the air inside the chamber is saturated with water. The grid is held by tweezers, which are mounted to
a movable rod, which is accelerated by a spring (not visible) during plunging. The formation of a thin sample suspension is achieved by blotting with filter
paper, which is mounted inside a chamber. A rod connects the filter paper to the outside and allows the user to handle it, without disturbing the humidity
inside the chamber. The cryogen is placed in an insulating box outside the chamber. (b) Shows a fully computer controlled freezing apparatus. Blotting is
done from both sides of the grid. The humidity inside the chamber is measured and adjusted with a nebuliser that generates small droplets of water with
ultrasound.
Transmission Electron Microscopy: Preparation of Specimens
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less water is the sandwich cryo negative staining (Golas
et al., 2003). Here a small piece of carbon is oated from
mica onto a droplet of sample picked up again and
oated on a staining solution (typically 2% uranyl for-
miate). A second small piece of carbon is oated on a
second drop of staining solution. The rst carbon lm
with the stained sample is picked up with a bare grid. The
grid is then turned around and the second carbon lm is
picked up in a way that the sample is sandwiched between
the two lms. Excess liquid is removed and the sand-
wiched sample is dried for 12 min before freezing it in
liquid nitrogen. The little remaining water that is retained
by the sample does not form crystalline ice despite the
slow freezing in nitrogen.
The advantages of cryo negative staining are that it
preserves the sample better than room temperature nega-
tive staining; it gives good contrast and is less beam sensi-
tive than vitrication alone. The disadvantages are that it is
an indirect method, thus the resolution is limited; details
are only visible in stain accessible areas and the stain can
interfere with the integrity of the sample.
Metal shadowing
An alternative approach to contrasting of particulate spe-
cimens is metal decoration (shadow casting). In this
approach, a very thin, discontinuous coating of heavy
metal atoms (usually platinum or tungsten) is deposited
under high vacuum and at an angle to the support lm
(Figure 5). Metal atoms accumulate on surface features of
the particles, producing a high level of topographic con-
trast. The termshadowcasting refers tometal depositedat
a xed angle, producing a metal-free region that resembles
an optical shadow. This shadowcan be used to estimate the
height of the object (Figure 5). In a variant of this procedure
the specimen is rotated during the metal deposition (rotary
shadowing).
Metal coating provides a high-contrast image in which
particles are at least partially protectedfrombeamdamage.
The obvious diculty is that images of particles are dis-
torted by an accumulation of metal. Molecular masses,
asymmetries and shapes of macromolecules estimated
from measurements of coated particles agree well with
values obtained in solution (e.g. by ultracentrifugation or
light scattering). The metal grain(crystallite) size is reduced
in proportion to the amount of metal applied and/or the
substrate temperature. Estimated coating mass thicknesses
are usually of the order of 10
26
g cm
22
, but are useful
down to 10
27
g cm
22
on the side of a specimen particle or
macromolecule. The latter value corresponds to an average
thickness approaching monoatomic dimensions. Because
of its inherently high contrast, metal coating can visualise
small molecules and domains more reliably than negative
staining. Shadowing gives anaccurate representationof the
surface of the particles, which canbe useful for determining
the absolute hand of an object. Best resolutions are
achieved if the coating is directly coupled to the specimen
transfer into the microscope. In this case the metal surface
is not exposed to the oxygen in the air and corrosion of the
metal is omitted as well as repeated rehydration and
dehydration. In such a dedicated setup resolutions of 10 A

in the plane and 5 A

vertically can be achieved (Walz et al.,

1996). However, because shadowing only represents
the surface of an object it is unsuitable for 3D image
Vitrified, unstained viral capsid Negatively stained viral capsid
Figure 4 Viral capsids negatively stained (left) and vitrified (right). The negatively stained capsids appear bright against a darker background, because the
stain in the background is more electron dense than the capsids. The contrast is high and the capsids can be easily recognised. The spikes of the capsid
appear to have collapsed on the body of the capsids. In the vitrified sample (right) capsids are dark against a brighter background, because the protein is
more electron dense than the vitrified buffer. The contrast is low, which makes it more difficult to recognise the particles. The spikes are better preserved
giving the particles a more spherical appearance.
Transmission Electron Microscopy: Preparation of Specimens
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reconstructions of the whole object. See also: EM Analysis
of Protein Structure
Preparation of Class II Objects
Cells and tissues are very large and fragile structures. Their
morphology changes withchanges in the water content, the
state of the water and the concentration of solutes in the
water. Thus these structures have to be stabilised against
changes in the water content before the objects can be
sectioned to a suitable thickness (50500 nm) for electron
microscopy.
Fixation and freezing
The rst step in sample preparation of cells and tissues is
the xation of the sample. This can be done either chem-
ically by cross-linking structures or by rapid freezing,
which leads to the vitrication of the sample. The xation
process must preserve morphological details at the level of
resolution sought. Ideally the xation process is rapid and
does not allow any restructuring after it is initiated. For
chemical xation, aldehydes are used as primary xatives.
They cross link amines of proteins. Glutaraldehyde gives
the most rapid cross-linking, whereas formaldehyde is
somewhat slower. Often mixtures of formaldehyde and
glutaraldehyde are used (total concentration of aldehydes
27%). The aldehydes are mixed in a 1:1 ratio with the
cellular material. During xation by cross-linking protons
are released. Therefore, buering in order to avoid a drop
in pH is essential.
Another xative, which is mainly used as secondary
xative is Osmiumtetroxide. Osmiumtetroxide reacts with
unsaturated acyl chains in lipid membranes. While it sta-
bilises the retention of lipids in the membranes during
embedding, it can be destructive to the proteins. Osmium
tetroxide is also added to increase the contrast of
membranes, which is enhanced by the incorporation of the
heavy osmium atoms into the lipid.
Fixation by cross-linking has the problemthat molecules
are interconnected, which do not necessarily interact. Thus
cross-linking can lead to none native aggregation of
structures and segregation of structures.
Alternatively to chemical xation cryo xation is used.
Freezing lowers the viscosity of the water and xes the
relative positions of the morphological units. To avoid
damage to the delicate structures, it is important that
freezing generates vitried water rather than crystalline
water. For small cells andorganelles this canbe achievedby
plunge freezing and jet freezing as described for the class I
objects and by slam freezing. For larger cells and tissues,
the cooling rates are not sucient to achieve vitrication of
the whole object. Here, high pressure freezing is used. In
high pressure freezing the sample is rapidly cooled down
and at the same time exposed to a pressure of approxi-
mately 2000 Bar. The high pressure lowers the melting
temperature to 2208C and the nucleation temperature at
which pure water nucleates to form ice crystals to
approximately 2908C. Thus ice formation at high pressure
is slowenough that no nucleation occurs during cool-down
and proper vitrication is achieved. High pressure freezing
allows sample with a thickness of 200600 mm to be vitri-
ed. The vitried, cryo-xed material can be either sec-
tioned directly followed by imaging in the vitried state or
it can be further processed by freeze substitution, embed-
ding and staining before imaging at room temperature.
Embedding in resin
In samples, which are not imaged in the vitried state the
water is replaced by a medium that is rigid and nonvolatile
in the vacuum of the microscope. An ideal material is
plastic such as epoxy resins or acrylics which are inltrated
in the monomeric form and then polymerised. For chem-
ically xed cells, this is done at room temperature, whereas
for cryo-xed material a freeze substitution process is
Height of
object: d
D
ir
e
c
t
io
n

o
f

s
h
a
d
o
w
in
g
Metal coat
Metal cost
Length of shadow
l=d/tan

Figure 5 Schematic drawing of unidirectional metal shadowing. The object is shown as a grey circle, the carbon support film as a brown line and the metal
coat as a red line. The height of the object d can be calculated from the length of the shadow l and the shadowing angle a.
Transmission Electron Microscopy: Preparation of Specimens
eLS & 2012, John Wiley & Sons, Ltd. www.els.net 7
initiated. In freeze-substitution, the water is replaced by
methanol or acetone at low temperatures over several
hours to days. Then the freeze-dried tissue is inltrated
with embedding medium hopefully avoiding collapse of
structures. In freeze-drying the sublimation of the water
happens at lowtemperature, fromthe frozen state (starting
2908C slowly increasing to room temperature). This pro-
vides a less traumatic route for dehydrationthanair drying.
Generation of thin sections
Plastic embedded samples can be sectioned with com-
mercially available ultramicrotomes that use a diamond
knife as the cutting edge (fractured glass edges may also be
used). Depending on the purpose, sections are generated
that are between 50 nm and 300 nm thick. The very fragile
sections are then mounted on grids on a thin support lm
(typically carbon or carbon-coated plastic).
Vitried samples can also be sectioned with a diamond
knife at temperatures belowthe devitrication temperature.
These sections are oated on liquid nitrogen and then they
are directly mounted to a grid and imaged below the devit-
rication temperature. The method is referred to as
CEMOVIS(Al-Amoudi et al., 2004; McDowall et al., 1983).
Vitried sections are at least disturbed by the preparation
process, because the water is fully retained and unspecic
aggregation due to drying, embedding and staining is
avoided. Thus the interior of the cells appear much more
homogeneous and the membranes more smooth than in
samples prepared by conventional methods which use
plastic embedding. However, the vitried sections usually
have a signicant compression in the direction of sectioning
(up to approximately 50%) and show knife marks and cre-
vasses at the surface (Al-Amoudi et al., 2005).
These limitations of sectioning vitried material can be
overcome with a dierent sectioning approach, which
makes use of a focused ion beam (FIB). In FIB the bom-
bardment of the specimen with ions (typically Gallium)
ablates the surface of the frozen material and leaves thin
slices at dened locations. Because ion milling is slow, only
small areas can be thinned in a reasonable time. In order to
target the FIB more precisely it can be combined with
uorescent microscopy (Rigort et al., 2010) that identies
areas of interest via the presence of uorescent markers.
See also: Electron Tomography
Contrasting and immune labelling of
sections
The contrast of plastic embedded sections is further
enhanced by staining procedures, whereas vitried sections
are imaged unstained. Contrasting of tissue sections begins
often with osmium xation. Application of any compound
containing heavy atoms tothe cut sections has the potential
for improving contrast. Useful materials include 1% ura-
nyl acetate and alkaline lead citrate. When this is combined
withosmiumtetroxide xations, ultra structural features of
cells are revealed. Alarge variety of staining methods are to
be found in the literature.
Immune labelling (immunocytochemistry) extends the
positive-staining approach, and has as its goal the location
of specic antigenic sites (epitopes) or other biochemically
distinct features. Osmium xation, which destroys epi-
topes, must be avoided. Typically, frozen sections are
thawed, and subjected to the immunocytochemical pro-
cedure in the unfrozen, wet state. Sites on the specimen are
thus coupled with specic antibodies, the latter being ren-
dered visible by attachment of heavy atoms, usually in the
form of colloidal gold particles of identiable dimensions.
See also: Immuno-electron Microscopy
Freeze fracture
Freeze-fracture techniques visualise internal fracture
planes within cells. Here, frozen whole cells are cleaved
inside a vacuum chamber and freeze-etched by limited
sublimation of ice from the newly exposed surface. The
surface is then replicated by coating with a platinumcar-
bon mixture similar as described for the shadowing of class
I objects. After digestion of remaining biological materials,
the replica is oated onto a grid and observed. Details of
the cells interior appear at high contrast. Freeze fracture
often occurs within membranes and leaves transmembrane
protein complexes, giving interesting insights into the
organisation of the membrane systems.
References
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Al-Amoudi A, Studer D and Dubochet J (2005) Cutting artefacts
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Further Reading
Glauert AM and Lewis PR (1998). Biological Specimen Prepar-
ation for Transmission Electron Microscopy. In: Glauert AM
(ed.) Practical Methods in Electron Microscopy, vol. 17.
Princeton: Princeton University Press.
Griths G (1993) Fine Structure Immunocytochemistry. Berlin,
Heidelberg and New York: Springer-Verlag.
Jensen G (ed.) (2010) Methods in Enzymology Cryo-EM, Part A
Sample Preparation and Data Collection, vol. 481. Amsterdam
and New York: Elsevier Science Publishing Co Inc.
Lewis PRand Knight DP (1977). Staining Methods for Sectioned
Material. In: Glauert AM (ed.) Practical Methods in Electron
Microscopy, vol. 5. Amsterdam, NY: Elsevier Science Pub-
lishing Co Inc.
Reid N and Beesley JE (1991). Sectioning and Cryosectioning for
Electron Microscopy. In: Glauert AM (ed.) Practical Methods
in Electron Microscopy, vol. 13. Amsterdam, NY: Elsevier
Science Publishing Co Inc.
Transmission Electron Microscopy: Preparation of Specimens
eLS & 2012, John Wiley & Sons, Ltd. www.els.net 9