PROGRESS ARTICLE

The use of environmental scanning

electron microscopy for imaging wet and
insulating materials
The environmental scanning electron microscope (ESEM) is a direct descendant of the conventional SEM,
but also permits wet and insulating samples to be imaged without prior specimen preparation. A low pressure
(up to around 10 torr) of a gas can be accommodated around the sample. When this gas is water, hydrated
samples can be maintained in their native state. Whether the gas is water or some other gas, ions formed on
collisions between electrons emitted from the sample and the gaseous molecules drift back towards the
sample surface helping to reduce charge build up. This eliminates the need for insulators to be subjected to a
conductive surface coating. These two key advantages of ESEM open up a wide range of materials to the
power of scanning electron microscopy.

ATHENE M. DONALD air scattering of either the incident beam or the

Cavendish Laboratory, University of Cambridge, Madingley Road,
Cambridge CB3 OHE, UK.
e-mail: amd3@phy.cam.ac.uk
INTRODUCTION
What makes environmental scanning electron
microscopy (ESEM) different from conventional
scanning electron microscopy (CSEM), a technique
widely used in materials research? Crucially, as will be
shown below, the answer is that it can deal with difficult
samples — including both wet and insulating ones — in
a straightforward way. It does this by enabling a gas,
frequently water vapour, to be present in the sample
chamber whilst retaining the rather high resolution of
the standard SEM. Despite these obvious advantages,
although ESEM has been commercially available for
over a decade, it is only recently that it has begun to
make significant impact across the diverse field of
materials. Conventional SEM has been around much
longer and is well established, but ESEM turns out to be
much more than just a ‘leaky’CSEM, and much
fundamental work needs to be done before its full
potential can be realized.
CSEM has a long and distinguished record in the
field of materials. It works by scanning an electron beam
across the sample in the manner of a TV raster, and
collecting the electrons emitted from the sample.
Typically, these emitted electrons fall into two
categories: low-energy secondary electrons (≤ 50 eV
usually being taken as the arbitrary cut-off in energy),
produced by inelastic collisions of the incident primary
beam electrons with the sample, and backscattered
electrons (with energies greater than 50 eV) that arise
from elastic collisions in the sample leading to scattering
through angles approaching 180°. The whole column of
the microscope operates under high vacuum to prevent
subsequently produced electrons.
ESEM differs from CSEM in two crucial aspects.
Firstly, instead of the sample being held under a high
vacuum, a gaseous environment is maintained around
the sample whilst imaging is carried out, although the
electron gun itself is kept at the standard pressures of
around 10–6 to 10–7 torr. This is achieved by the use of
differential pumping down the column, so that there is a
series of different pressure zones, with increasing
pressures as the sample chamber is reached (see Fig. 1a).
Around the sample, pressures of up to ~10 torr can be
tolerated, which means that if water vapour is the gas in
the sample chamber, hydrated samples (including those
of biological origin) can be imaged in their native state
without significant specimen preparation. The second
major difference between ESEM and CSEM is that
insulators do not need to be coated with a metallic layer
before imaging in ESEM. Because of the gas in the
chamber, a mechanism exists to help dissipate the build-
up of charge, injected by the incident electron beam.
This will be discussed in more detail below.
As indicated above, a high vacuum is usually
required to stop the electrons being scattered leading to
degradation of image quality. So why should ESEM be
different? The answer is that the total distance through
which electrons travel through a significant presence of
gas is kept as short as possible, so that most incident
electrons do not undergo any large-angle collisions.
The effect of this on the probe shape is that a sharp
central probe is maintained, superimposed on quite a
broad ‘skirt’, which essentially leads to a background d.c.
signal. There is some slight degradation of image quality
due to a reduction in the signal:noise ratio, and in
practice, current resolutions of ~5 nm are about as good
as one can get. But for many classes of samples, this
disadvantage is more than outweighed by the ability to
do completely new experiments.

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PROGRESS ARTICLE
a b
Specimen stage
Pressure Pressure
range zone
10–7 torr Gun chamber
10–6 torr Upper column
10–4 torr EC2
10–1 torr EC1
10 torr Specimen
Figure 1 Schematic of ESEM.
a,The different pressure zones
in the ESEM column.The gun
can be maintained at high
vacuum, while a system of
differential pumping- and
pressure-limiting apertures
allows the chamber to be
maintained at a pressure of a
few torr (1 torr = 133 Pa).
b,The cascade amplification
process.This process occurs
within the chamber, where the
gas molecules are ionized by
the electrons emitted from the
sample. Each ionizing collision
gives rise to a daughter
electron, which, like the original
electron, is accelerated towards
the positively charged detector,
and further collisions can occur
en route to the detector.
The positive ions drift back
towards the sample surface.
512
Positively charged detector
Gun chamber
Anode aperture
Column lining
Projection apertures
Column isolation valve
Upper pressure
limiting aperture
Vacuum manifold
Lower pressure
limiting aperture
and integrated
detector
The gas is not simply a passive participant in the
imaging process, there simply to open up the range of
samples studied, it plays a key role in signal detection1,2.
At its simplest,it can be seen that as electrons (secondary
or backscattered) are emitted, they also have to travel
through the gas. The secondary electrons in particular,
because of their low energies, have a high collision cross-
section with the gas molecules, and ionization of the
molecules has a significant probability of occurring.
Each such ionizing collision leads to the generation of
an additional daughter electron, so that a cascade
amplification occurs, shown schematically in Fig. 1b.
All the electrons produced are drawn towards the
positively biased detector: this is not the standard
Everhardt–Thornley detector used in CSEM, which
could not work under these pressures, but a specially
designed detector appropriate for these conditions.
The original ESEM (which is a trademark of FEI/Philips)
detects charge flow in the gas, but as other
manufacturers enter the market (selling so-called
variable pressure instruments, which typically are not
yet able to operate at such high pressures for technical
reasons, limiting their ability to maintain hydrated
samples in their fully hydrated state), a variety of
different signals are being used for detection. This article
will, for simplicity, concentrate simply on the electronic
detection in the ESEM.
As well as the daughter electrons produced in the
ionizing collisions, positive ions are also produced.
These drift down towards the sample, and hence serve
to compensate charge build-up at the surface of
insulators. It is for this reason that insulators do not
need to be coated to prevent charging artefacts and
consequent loss of image quality. However, it should be
noted that the precise role of ions in affecting the whole
imaging process is still incompletely understood and is
an active field of study; this complexity is beyond the
©2003 Nature Publishing Group
Electron beam
V0
A
Cascade electrons
Positive ion
+
Gaseous atom
V
Earthed sample
scope of this article, and will only be touched upon
briefly in what follows.
Recent research in the ESEM field is of two types.
There is an increasing range of applications in the field
of materials that use the full power of the instrument —
these include a significant amount of work on ceramics
such as cement, which use the ESEM’s ability to image
uncoated insulators. There are also interesting
developments in the study of natural fibres, which are
not only insulators but also may be partially hydrated.
Finally there are (fewer) examples of using the
instrument to look at aqueous dispersions, or actual
water droplets, as well as biological samples maintained
in their native state. This aspect of biological studies is
the least well developed because the number of workers
in biological departments who are entering the field is
still small. For any of these applications there is the
possibility of carrying out dynamic, in situ experiments,
in which the state of the sample is changed whilst
continuing to image3,4. Such changes could be in
temperature5, state of hydration6, or even by carrying
out a chemical reaction7,8.
The second developmental area is in what might be
termed ESEM physics. Few groups are doing this kind of
work, but it is vital to the successful interpretation of
images. Experiments in this area deal with more
quantitative analysis of what happens in the gas cascade,
exploring the effect of charge deposition on electron
yield in different types of experiments, and
consideration of contrast and detection mechanisms.
WET SAMPLE IMAGING
As indicated above, the ESEM can operate with a
significant gas pressure around the sample.In principlethis
means that hydrated samples can be kept fully hydrated,
but in practice achieving this takes some thought.
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 PROGRESS ARTICLE
During the instrument pumpdown it is important to 25
make sure that dehydration does not occur before the
start of imaging. This was not initially recognized,
leading to frustration at the apparent inability to keep 20
samples wet. However, with due attention to the Liquid
pumpdown procedure9, and with the possible
inclusion of a drop of water on the sample near the 15

Pressue (torr)
area to be imaged10, the state of hydration can be
maintained. It is also important during the whole
Condense Evaporate
imaging process to be aware of the state of the sample 10
relative to the chamber pressure. Figure 2 shows the
form of the saturated vapour pressure curve for water
as a function of temperature. If one tries to work at 5 Gas
room temperature, it can be seen that the water vapour
pressure required to maintain a saturated vapour in
the chamber is impossibly high. Imaging under these 0
conditions is currently not practical. To achieve 0 5 10 15 20 25
saturation, it is desirable to drop the sample
Temperature (°C)
temperature to just above freezing, and then only a
modest gas pressure is needed to stabilize water.
Usually this is done by the use of a Peltier-chip-
controlled cooling stage, making it possible to image are still obvious — there is no need for the standard Figure 2 Saturated vapour
samples containing water. type of sample preparation including drying, possibly pressure of water as a function
Figure 3a shows a colloidal dispersion of a fixing, and coating required for CSEM. This removes of temperature.The useful range
poly(methyl methacrylate) latex, containing spherical many of the potential pitfalls of artefacts being of working conditions for the
particles ~250 nm across. In Fig. 3a, the individual introduced during preparation. And such work is ESEM are shown; it can be seen
particles near the sample surface can clearly be seen as beginning to throw up examples of where tried and that the state of the sample can
dark against the light background of the continuous tested routes of sample preparation are now being be changed by small alterations
water phase.Watching such a sample shows the particles shown to have introduced artefacts28,31–33. in temperature.
undergoing constant Brownian motion. During this Will ESEM become the microscopy of choice for
process, collisions between particles can occur and, biologists? At this time the answer to this is far from
depending on the interparticle potential, sticking may clear. The ability to image cells and other biological
occur. This process can be studied in more detail by assemblies including whole organisms without
deliberately setting instrument conditions to favour dehydration or coating — let alone staining or fixing —
aggregation: as Fig. 2 shows, it is a simple matter to cross is clearly very attractive. But as yet it is not known
the saturated vapour pressure curve in either direction whether living cells can be viewed but not killed by the
by slightly increasing or decreasing the temperature. electron beam. Clearly, the lower the magnification used
To study aggregation, dehydrating conditions can be set the lower the electron dose and therefore the greater the
up to pull surface water off the sample. Figure 3b shows
a sample of the same latex but now dispersed in a salt-
containing solution after a short period of dehydration:
in this case a fractal aggregate has started to form. a b
Armed with this knowledge of how to control the
thermodynamic state of the sample, a whole range of 5 µm
new, dynamic experiments become possible (other
kinds of dynamic experiments will be discussed later).
In essence, the sample chamber becomes a
microprocessing unit. Significant work has been done
in this area using ESEM on cement11–14 and related
inorganic materials7,15, as well as colloidal aggregation
leading to film formation, in which the packing of
particles can be interrogated at intermediate steps as the
film dries (Fig. 4)16–18.Another type of experiment has
involved the growth of Langmuir films at an air–water
interface19. In this case, the structure of the films grown 2 µm
under conditions of different concentrations can be
examined to see how the packing alters.As a further
example, Fig. 5 shows how contact angles can be
determined from condensation of water onto a Figure 3 ESEM images of colloidal dispersions under different conditions. a,A colloidal dispersion of
cellulose fibre20. 250 nm poly(methyl methacrylate) latex particles dispersed in water.The particles undergo Brownian
Not all samples of course involve free water; many motion and may collide with each other. b,A dispersion of the same particles now dispersed in a 0.078 M
are simply ‘damp’. Such types of samples include magnesium sulphate solution. In this solution, the interparticle potential is altered such that any collisions
natural biopolymers (as fibres21–24 or gels25, lead to a reasonably high sticking probability, and a fractal structure develops as aggregation proceeds.
biofilms26,27 and many truly biological samples28–30). The exact nature of the aggregate is strongly dependent on the salt concentration; in the absence of any
Nevertheless, the advantages of working in an ESEM salt the sticking probability is low, and colloidal crystals rather than fractals form.

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PROGRESS ARTICLE
Figure 4 ESEM image of a because water will evaporate rapidly, for many
partially dehydrated latex. insulators the ability to follow high-temperature phase
This latex consists of film- transformations has been seen as a key advantage of the
forming particles consisting technique. For ceramics, work has been carried out
of a copolymer of methyl examining phase changes occurring at high
methacrylate and 2-ethyl hexyl temperatures34–36, and such experiments frequently
methacrylate.At this stage of make use of the ability of the chamber to act as a
dehydration there is still a thin reaction vessel. This applies equally well (and has
layer of water surrounding each actually been used more widely) for metals, for which it
particle, and the particles have is the ability to introduce a reactive atmosphere into the
not begun to coalesce. chamber that is so fruitful. In other words,
transformations can occur under oxidative5,8 or
hydrating conditions37, or even with traces of other
gases present to initiate a reaction38,39. In principle, work
could also be carried out at low temperatures, but a
5 µm
search of the literature shows that cryo-ESEM has not
yet been applied in a significant way40,41; this field can be
expected to expand as standard cryo-transfer devices are
interfaced with the ESEM column. Mechanical testing
in the ESEM is a valuable tool because the removal of
chance of survival, but then the advantage over light the need for a coating means that charging does not start
microscopy is less obvious. However, what is clear is that up as soon as fresh, uncoated surfaces of insulators are
when a biologist/biomedical researcher wants to use an opened up during the test42–46.Again the literature is not
SEM they should seek out the nearest ESEM to avoid all yet particularly substantial, and can be expected to grow.
possibilities of artefacts being introduced at any stage So far, no attention here has been paid to image
during preparation.What they see may well not be interpretation. It has been presumed that interpretation
identical to what they are used to seeing28,33 but this does is identical to that for CSEM, and for the sort of work
not mean the ESEM is at fault! described above this is in general true: both standard
topographical information obtained from the
INSULATORS AND THE CHALLENGE OF IMAGE secondary electron signal, and compositional
INTERPRETATION information from the backscattered signal can be seen
(with the additional complication that backscattered
For insulators — dry ceramics, polymers and electrons can give rise to daughter electrons during
semiconductors — the obvious key advantage of the collisions with the gas, although the cross-section for
ESEM is the removal of the need to apply a conductive this process means that the numbers of these are fairly
coating. This means not only that fine detail can be low). The effect of changing accelerating voltage is
observed, but also that dynamic processes can be similar to CSEM, with higher voltages giving greater
followed as the sample is changed — either by penetration into the sample, and less surface detail.
mechanical testing or by chemical or physical However if Fig. 4 is examined in more detail, it is seen
transformations.Whereas for wet samples, high- that as the water is lost from the film, the edges of the
temperature work is unlikely to be particularly useful latex particles display a uniform bright ring. This would
not be expected on the basis of topography alone.
Something else is contributing47.As insulators have
been studied in more detail, it has become apparent that
Figure 5 ESEM image of a understanding the contrast observed in the image is a
water droplet condensed onto a far from trivial matter.
cellulosic fibre of cylindrical A range of studies has indicated that there are
cross-section.The bar sources of contrast in the ESEM not in general seen in
represents 50 µm. Such images the CSEM48–52, at least in part because the presence of
show much clearer definition at the standard metallic coating obscures intrinsic
the edge of the drop (which is contrast.A unifying interpretation is beginning to
unduloidal in shape — that is, emerge, but is not yet complete. Clearly the contrast is
the surface has a constant arising from variations in the electron yield, and it has
non-zero mean curvature), been argued that this is caused by variations in the
permitting an easier energy-loss processes as generated electrons travel to the
determination of the contact sample surface due to local electronic structure
angle than is typically provided differences49. Such variations mean that dopant
by optical microscopy. concentrations can be imaged in semiconductors53,
ferroelectric domains seen in insulators54 as well as
differences between chemically similar polymers55.
An example of the striking contrast that can be seen in
polymers is shown in Fig. 6 for a lamellar phase of a
diblock copolymer. Observation of contrast in minerals
such as gibbsite has been attributed51,56,57 to ‘charge
contrast imaging (CCI)’, implying that charge

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deposition is affecting the secondary yield, and the
extent of this process is attributed to local impurities in
the mineral associated with its deposition and growth.
This statement about secondary-electron yield is of
course true, but contrast can be observed much more
generally and with far more parameters playing a part in
the variation in yield than it implies. The effective
secondary-electron yield is not a uniquely defined
quantity, but depends both on charge movements
within the sample, and also on the ions in the chamber:
it transpires that the ions are not passive in this process
at all, but can actively ‘scavenge’the emitted electrons by
recombination, and hence reduce the effective number
of electrons that enter the cascade amplification
process58. The story of contrast interpretation in the
ESEM is still far from complete.
OUTLOOK
So what of the future? The technique is beginning to
make its mark across the field of materials, with the
examples above demonstrating its applicability to a
wide range of different types of samples and problems.
With modern instruments being capable of rapidly
switching between high vacuum and environmental
mode, every researcher in a department can in principle
have the type of environment needed for their research.
However, not unnaturally the technique has not yet had
a chance to mature and the underlying theory is far
from completely understood.
One clear limitation at the present time lies in the
detectors. Sticking with the true ESEM, which detects
the electron signal, none of the existing detectors are
really capable of providing a pure secondary-electron
detector, and backscattered detectors are even worse.
For other kinds of variable-pressure SEM, the
information about the detectors is even more limited.
Clearly this is an area where one can expect significant
progress from the manufacturers in detectors designed
to improve the situation. The field is yet young.
A second issue is the problem with making the
instrument fully quantitative. In common with CSEM,
the ESEM can be used for chemical analysis, but the
challenges to make the technique quantitative are
severe. Energy-dispersive X-ray spectroscopy can be
straightforwardly performed59–61, as in a CSEM, but
interpretation of the spectra is not straightforward62.
Secondly, for all the reasons given above, the contrast in
the electron image provides clues about local
heterogeneities, but as yet cannot be used in any
quantitative sense to identify phase composition.
Future work will be required to establish exactly how
secondary-electron yield depends on both local sample
properties and instrument operating parameters and,
because current detectors do not detect a pure,
amplified secondary-electron signal, how the yield
translates into detected signal. Nevertheless, the
potential in this area is huge, because the fact that no
surface coating is present to mask the true emission
signal from the sample opens up the field of imaging
heterogeneous insulators (the ESEM in this context can
be seen to perform here similarly to a field-emission gun
SEM, for which coating of insulators is also often
unnecessary, but without the constraint of imaging
under high vacuum). It can also be anticipated that
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PROGRESS ARTICLE
Figure 6 ESEM image of a
block copolymer of
polystyrene–polyisoprene, with
a volume fraction of ~50%
polystyrene.The polystyrene
forms the darker lamellae in this
case.The advantage of this
approach over the conventional
one of staining one of the
phases (for example, with
osmium tetroxide), is that no
sample preparation is required,
and artefacts such as swelling
1 µm due to the staining are avoided.
Micrograph courtesy of A. J. Ryan and
C. Salou, University of Sheffield, UK.
other types of signal may be usefully detected to
complement the electron images and the X-ray maps.
An obvious candidate would be cathodoluminescence,
and work is just beginning to develop such
measurements within the ESEM63.
Finally, general application of the technique to an
increasing number of materials problems can be
expected. Researchers who merely want to extend the
conditions under which simple insulators can be
imaged, have already appreciated the power of the
technique, even if the nuances of contrast have been
largely overlooked. However, the experimental
protocols needed to work with fully wet samples are
perhaps perceived as sufficiently complex and
challenging that the community working on these
materials is still rather small. It is to be hoped that more
people will recognize the power of the technique, and
extend further the range of samples actively studied
through this exciting technology. In summary, this
technique, young by comparison with CSEM and
therefore still with its theoretical challenges, looks set to
make significant inroads in materials research across
many subdisciplines.
doi:10.1038/nmat898
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Acknowledgements
My thanks are due to all of my group who over the years have put so much work
into developing ESEM, and stimulated frequent lively debate.
Competing financial interests
The author declares competing financial interests: details accompany the paper on
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