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Recent advances in electron imaging, image interpretation and

applications: environmental scanning electron microscopy
Debbie J. Stokes

Phil. Trans. R. Soc. Lond. A 2003 361, doi: 10.1098/rsta.2003.1279, published 15 December 2003

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10.1098/rsta.2003.1279

Recent advances in electron imaging,

image interpretation and applications:
environmental scanning
electron microscopy
By D e b b i e J. S t o k e s
Polymers & Colloids Group, Department of Physics,
Cavendish Laboratory, University of Cambridge,
Madingley Road, Cambridge CB3 0HE, UK
(djs49@phy.cam.ac.uk)

Published online 3 November 2003

One of the latest developments in electron microscopy is the environmental scanning

electron microscope (ESEM), which enables soft, moist and/or electrically insulating
materials to be viewed without pre-treatment, unlike conventional scanning electron
microscopy, in which specimens must be solid, dry and usually electrically conduc-
tive. Such an advance has significant implications for studies of the ‘native’ surfaces
of specimens including rocks and minerals, polymers, biological tissues and cells, food
and pharmaceutical products, precious artefacts and forensic material, for example.
Previous types of electron microscopes made scientists think carefully about the
physics of electron-beam interactions with specimens and, hence, the interpretation
of images. We now face additional factors influencing the emission and detection
of electron signals, unique to the imaging of specimens in the partial vacuum of
an ESEM. Just as importantly, we must consider the thermodynamic and kinetic
stability of specimens, as appropriate, and explore the possibilities for new applica-
tions, particularly those of a dynamic nature. This paper briefly describes some of
the issues involved and reviews the current state of understanding.
Keywords: environmental scanning electron microscopy; ESEM;
gaseous signal amplification; secondary electron contrast;
dynamic charge-related phenomena; insulators

1. Introduction
An essential component in the pursuit of scientific research is the use and develop-
ment of appropriate experimental techniques and methodologies. A technique that
has become an everyday element in many a researcher’s toolkit is electron microscopy,
playing a significant role in characterizing the structure, properties and behaviour of
a wide range of materials. When the scanning electron microscope (SEM) was intro-
duced in the late 1930s/early 1940s, the transmission electron microscope (TEM)
was already firmly established. Many microscopists in the latter community could

One contribution of 22 to a Triennial Issue ‘Mathematics, physics and engineering’.

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c 2003 The Royal Society
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2772 D. J. Stokes

see little application for an instrument having lower resolution than that already
available. But the SEM’s ability to yield three-dimensional information from the
surfaces of bulk specimens, over a considerable range of length-scales, was quickly
appreciated by many scientists and technologists. Over the intervening years, vari-
ous methods have been devised for the removal or solidification of volatiles and the
coating of electrically insulating specimens. Such measures are necessary in order to
render specimens suitable for the high-vacuum environment of the SEM. However,
frustrated by the restrictions this places on the way certain specimens are treated and
imaged, some scientists began thinking about the possibilities of imaging specimens
in a more ‘natural’ state. As early as 1953, workers were experimenting with differ-
entially pumped, aperture-limited TEMs, or using thin-film windows, with the aim
of creating an ‘environmental chamber’ (Parsons 1975). Research in the 1970s led to
the first SEM capable of maintaining a relatively high pressure, obviating the need
for drying and coating of specimens (Moncrieff et al . 1978, 1979; Danilatos & Robin-
son 1979). The term ‘environmental’ SEM was coined and, by the late 1980s, the
first commercial environmental scanning electron microscopes (ESEMs) were being
produced, opening up a world of possibilities for observing untreated specimens.†
Ironically, it is now the turn of the long-standing SEM community to regard its
new relative with suspicion. Some doubt that specimens can really be imaged with-
out preparation, or that they can be viewed without undue radiation damage, while
others feel that the resolution is inferior to that achievable with conventionally pre-
pared, coated specimens in high vacuum. The extent to which these observations are
justified depends, of course, on the information being sought and on the characteris-
tics of the specimen itself. Hence, this paper reviews the current state of knowledge
with respect to imaging insulators, from solids to liquids, over the variable pressure
range typically used in this type of instrument (0.1–10 torr, or ca. 13–1.3×103 Pa). A
comparatively small but highly international scientific community has been tackling
these issues over the past 10 years or so, with significant progress being made in
the last five years. Issues include specimen stability and control over dynamic pro-
cesses in situ, specimen-dependent secondary electron contrast mechanisms, and the
influence on image interpretation of transient phenomena such as selective electrical
charge build-up, electron–ion interactions and electric fields. Attention focuses on
ways of exploiting the available physics, in order to gain new perspectives about the
materials under investigation.

2. ‘Gaseous’ secondary-electron signal amplification

The main feature distinguishing ESEM from conventional SEM is the presence of a
gas in the specimen chamber, giving a partial vacuum (up to ca. 10 torr) compared
with a high vacuum (ca. 10−6 torr). ‘Gaseous’ secondary-electron detectors have been
specially developed where, typically, positive potentials of just 300–500 V are applied
(in contrast to the 10–12 kV used with a conventional Everhardt–Thornley detector,
which would result in arcing in the presence of gas). Low-energy secondary electrons

† It should be noted that ESEM is a registered trademark of FEI Company. Other manufacturers
(LEO, Hitachi and JEOL) also offer instruments capable of operating over extended pressure ranges.
All of these instruments are generically known as variable-pressure SEMs. The information in this paper
relates specifically to ESEM.

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Environmental scanning electron microscopy 2773

annular detector primary beam

amp
backscattered
electron

E positive ion
V+

secondary
electron

gas molecule
specimen

Figure 1. Schematic of the gaseous amplification process.

(SEs) emitted by the specimen are selectively accelerated in the relatively small elec-
tric field between the specimen and detector. Ionizing collisions with gas molecules
generate additional SEs, causing a ‘cascade’ of electrons, and so the signal is ampli-
fied before reaching the detector. The positive ion by-products resulting from this
cascade process are an essential feature in ESEM: ions drift towards the specimen
surface and thereby help to compensate for negative charge build-up, hence insulating
samples can be imaged without the need for a conductive coating.
Imaging gases may include nitrous oxide, carbon dioxide, helium, argon, nitro-
gen and water vapour, the latter being the most efficient amplifying gas found so far
(Fletcher et al . 1997). For this reason, and because of its useful thermodynamic prop-
erties, water vapour is the most common gas used in ESEM. A simplified schematic
of gaseous amplification in the presence of an on-axis annular electrode detector
is shown in figure 1. The primary electron beam passes through a small aperture
in the detector (final pressure-limiting aperture (PLA), typically 500 µm in diame-
ter), helping to restrict unwanted movement of gas up the column. An alternative
detector geometry is an off-axis planar electrode, useful for amplifying the signal at
low pressures as a consequence of the increased path length of SEs traversing the
specimen–detector gap. In the absence of a PLA, this set-up allows for a larger field
of view, although it does impose an upper pressure limit of ca. 1.1 torr.
The principles underlying the cascade effect are discussed in several papers (see,
for example, Danilatos 1988, 1990; von Engel 1965; Durkin & Shah 1993). More
recently, Thiel et al . (1997) and Fletcher et al . (1997) gave a detailed treatment
of gaseous amplification in ESEM with the emphasis on developing a systematic
approach towards obtaining optimal image intensity and resolution for a given set of
experimental conditions. An important parameter is the distance between the spec-
imen and the detector (of the order of a few millimetres). If the gap is too small,
electrons do not gain sufficient energies to overcome the gas-ionization threshold
(Thiel et al . 1997). If the gap is too large, the electric field becomes rather weak,
and the signal becomes degraded by unwanted scattering events. These extremes are
strongly dependent on the chemistry and partial pressure of the gas being used, as
well as the detector bias. Furthermore, the atomic weight(s) and dielectric proper-

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2774 D. J. Stokes

20
18
16

vapour pressure (torr)

14 100%
12
10 75%
8
50%
6
4
2

0 2 4 6 8 10 12 14 16 18 20
temperature (ºC)
Figure 2. Part of the phase diagram for water, showing data for
100, 75 and 50% relative humidities.

ties of the specimen have a significant part to play. Some of these factors will be
considered in later sections, along with consideration of the influence of the ionized
gas.

3. Criteria for aqueous specimen stability

(a) Thermodynamics
For specimens containing water, consideration must be given to the gaseous and
thermal conditions inside the ESEM chamber. Since the variation in phase behaviour
of water is such an important parameter, it is worth surveying some of the basic
principles involved. These ideas will be extended later, when considering the vapour
pressures of real aqueous specimens.
Water molecules are transported across an air–liquid interface by diffusion and
convection. In a closed system, a thermodynamic equilibrium is set up between a
liquid and its vapour such that the number of molecules escaping across the interface
is matched by those returning: evaporation and condensation occur at equal rates
(see, for example, Tabor (1991) and references therein). Under equilibrium conditions
at a given temperature a specific amount of vapour exists above the liquid, described
as the saturated vapour pressure (SVP). Thermodynamic theory, embodied in the
Clausius–Clapeyron equation, can be used to plot the phase behaviour of a liquid as
a function of temperature. Part of the phase diagram for water, or the SVP curve, is
depicted in figure 2. Equally, such a plot can be produced from tables of experimental
values (Lide 1991). Points that lie on the curve represent thermodynamic equilibria,
where the net liquid–vapour ratio remains constant for a given temperature.
Maintaining an aqueous specimen in thermodynamic equilibrium with its sur-
roundings in the ESEM chamber entails cooling the specimen to a few degrees cel-
sius (using a Peltier-controlled cooling stage) and imaging with an appropriate partial
pressure of water vapour, in accordance with figure 2 (see discussion that follows).
An appropriate purge–flood cycle is employed following pumpdown, to ensure that
air in the chamber is replaced by water vapour (Cameron & Donald 1994).

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Environmental scanning electron microscopy 2775

(b) Kinetics
In ESEM, specimen temperatures and chamber pressures can be controlled inde-
pendently: they can be adjusted in order to attain equilibrium or non-equilibrium
conditions, as required. In a non-equilibrium state the concentration of vapour
molecules above the specimen is either higher or lower than that required for a
stable state, leading to an imbalance in the exchange of molecules between the
liquid and the vapour (condensation or evaporation, respectively). Furthermore, the
phase behaviour of water is a nonlinear function of temperature: by analogy with the
Maxwell distribution of speeds in gases, the probability that an individual molecule
will have a speed much in excess of the average increases with temperature. Hence,
evaporation occurs more readily at higher temperatures. Typically, aqueous speci-
mens tend to be cooled to ca. 2–6 ◦ C in ESEM, where the rate of moisture loss is
quite low (see Cameron & Donald 1994). It is therefore possible to employ pressures
somewhat below the equilibrium vapour pressure given by the SVP curve: speci-
mens can usually withstand slowly dehydrating conditions for a finite period of time
(minutes to hours).

(c) Vapour pressures of aqueous systems

It is tempting, and indeed normal practice, to regard the phase diagram for water
as containing all the thermodynamic information required for stabilizing hydrated
specimens. However, many such specimens consist not of pure water, but of aque-
ous phases containing dissolved solutes. In order to refine our methodologies, we
should therefore consider what constitutes equilibrium conditions for a given speci-
men.
Since the vapour pressure of a solution is proportional to the mole fraction of
solute (Raoult’s law), we note that the vapour pressure of a solution is less than
that of the corresponding pure solvent (Tabor 1991). Because of this, there is a
driving force (osmotic pressure) for solvent to enter a solution, thermodynamically
described by the Van’t Hoff equation, which essentially depends upon the number
of solute molecules contained in the solution. Many solutions have large osmotic
pressures, the magnitudes of which are not always adequately predicted by theory
alone (Weiss 1996). It is assumed that solutions are dilute (solutes occupy negligible
volume) and that their behaviour is ideal (solute molecules do not interact with
each other or with solvent molecules). Real aqueous phases, such as those found in
the interiors of mammalian cells, are neither dilute nor ideal: macromolecules such
as proteins and polysaccharides take up a large volume (ca. 30% of the available
space) and interact strongly with water molecules (protein folding, for example, is
dependent on such interactions) (Ellis 2001). The extent to which these factors lower
the equilibrium vapour pressure of physiological solutions or other aqueous systems
could be significant: some 20–25% lower than for pure water, depending on the
specimen (Stokes et al . 2003b). It is therefore advisable to use vapour pressures below
those corresponding to saturation, since, despite deviating to conditions below 100%
humidity relative to water, the specimen itself is not under dehydrating conditions.
Other workers have made similar empirical observations when studying biological
specimens in ESEM (Tai & Tang 2001), noting that water condenses onto specimens
at humidities of ca. 90%. Additional lowering of the chamber pressure, perhaps by
another 20–25% for a membrane-bound specimen, can be accommodated due to the

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2776 D. J. Stokes

100 µm

Figure 3. ESEM micrograph of a liquid-state vegetable oil-in-water

emulsion, imaged in water vapour at ca. 80% relative humidity.

25 µm

Figure 4. ESEM micrograph of uncoated human bone cells, imaged in water vapour
at ca. 50% relative humidity. (Reproduced with permission from Stokes et al . (2003b).)

kinetic factors previously discussed. Figures 3 and 4 show two examples of the use
of ESEM in this respect.

4. In situ dynamic experiments

Appropriate control over the phase behaviour of the vapour and the specimen is
very useful, in conjunction with specimen cooling, not only for ensuring specimen
stability, but also for enabling dynamic experiments to be carried out in situ. This is
another key aspect of ESEM. Examples of in situ experiments include the swelling
behaviour of textile fibres (Jenkins & Donald 1997), the wetting of polymer substrates
(Stelmashenko et al . 2001), the deformation and failure of biopolymer composites
(Stokes & Donald 2000), gels (Rizzieri et al . 2003) and tissues (Thiel & Donald 1998),
film formation (Keddie et al . 1995) and colloidal crystallization (He & Donald 1996;
Meredith & Donald 1996).
One of the most recent extensions to these methodologies involves experiments at
low temperatures (−150–0 ◦ C, for instance). Conventional high-vacuum cryo-electron
techniques for the study of fast-frozen hydrated specimens are well established and

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Environmental scanning electron microscopy 2777

50 µm

Figure 5. Mixing 0.7 torr N2 vapour with 0.2 torr H2 O vapour and reducing the temperature
to −65 ◦ C results in vapour deposition of ice. The ‘passive’ N2 remains in the gas phase, thus
producing the image.

extremely useful for studying the ‘static’ microstructure of organic materials (Echlin
1992). The development of in situ ‘cryo’ experiments, using ESEM, enables the
observation of dynamic events including structure evolution during freeze-drying,
ice-crystal nucleation, growth and morphology, controlled etching and sublimation,
freeze-thaw or thermal cycling and phase transitions. Applications could include
foods, pharmaceutical products, cosmetics, cement and stone, and could also help
our understanding of biological, environmental and atmospheric systems such as
frost-forming bacteria and the distribution of ice-adsorbed impurities in clouds.
As already discussed, water vapour is commonly associated with ESEM imaging.
However, for very-low-temperature work (less than −80 ◦ C), an alternative imaging
gas is needed with a higher vapour pressure than water, in order to resist precipitation
(e.g. nitrogen or nitrous oxide). Recent work by the present author and co-workers
demonstrates the use of nitrogen to image uncoated, freeze-fractured ice cream at
−95 ◦ C (Stokes et al . 2003a). The very slow rate of sublimation in low vacuum
makes it possible to follow changes over an extended time period (several hours, in
fact), in contrast to the rapid sublimation that would occur in high vacuum at this
temperature.
At temperatures higher than ca. −80 ◦ C, it is possible to begin maintaining very
small partial pressures of water in its vapour phase. This suggests opportunities for
more sophisticated experiments in which two or more gases are present in the cham-
ber, one of which is water vapour. A ‘passive’ gas serves to provide the image, while
water vapour actively participates in the experiment (see figure 5). This means, for
example, that specimens can be stabilized against sublimation at higher tempera-
tures, allowing us to keep frozen specimens intact while getting closer to the temper-
atures at which changes may occur (particularly relevant to systems with sub-zero
glass-transition temperatures). By determining specific humidity and thermal crite-
ria within the chamber, we can also control the freezing/drying of compounds such
as foods and compounds for drug delivery, giving a direct means of observing the
development of characteristic porous microstructures. Similar reasoning applies to in
situ crystallization of solutions, where we can study the onset of ice nucleation and

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2778 D. J. Stokes

1 µm

Figure 6. A physiological apatite layer (essential for bone formation), imaged uncoated
at a magnification of ×20 000. (Specimen courtesy of Jie Huang.)

follow crystal growth and morphology in the presence or absence of growth-inhibiting

additives, or observe tissue damage due to water expansion on freezing.

5. Insulators in general
(a) Dry insulators
The criteria for imaging dry, uncoated insulators are a little different to those for
aqueous systems, since there is no temperature control involved and the imaging
gas does not need to be (although usually is) water vapour. For the present, it will
simply be stated that the basic requirement for observing insulators is that sufficient
gas should be present in the chamber to minimize charging artefacts (it will later
be shown why this is an oversimplification). This is generally termed ‘low-vacuum’
mode, since gas pressures tend to range between 0.1 and 3 torr, rather than up to
the maximum pressure of the instrument.
This mode permits imaging of a broad range of specimens, including polymers,
hard tissues such as wood and bone, rocks, porous materials, minerals and gem-
stones, ceramics and dry foodstuffs. ‘Dry’ can also include specimens in which water
is tightly bound or frozen, or where specimens are protected by a hard or waxy
exterior. An appealing feature of ESEM, and other low-vacuum instruments, is the
high-throughput capability, facilitated by the elimination of preparatory steps. X-ray
microanalysis can be carried out simultaneously, and specimens can be either reused
for further experiments or studied using complementary techniques such as TEM,
atomic force microscopy (AFM) and confocal microscopy.

(b) A question of resolution

It is sometimes said that image resolution in ESEMs does not match that achiev-
able by high-vacuum SEMs, and this is attributed (often erroneously) to excessive
scattering of the primary electron beam on its passage through the gas to the spec-
imen. It is fair to say that there are some issues related to the collection of X-ray
data due to ‘broadening’ of the primary beam (Doehne 1997; Gauvin 1999). How-
ever, from an imaging viewpoint there are some rather more fundamental reasons

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Environmental scanning electron microscopy 2779

for any reduction in resolution, which are, in fact, specimen dependent. Organic
specimens, for example, have low electron-stopping powers, hence primary electron
ranges (i.e. penetration depth and size of interaction volume) within the specimen
are high (Goldstein et al . 1992). Additionally, water or physiological secretions on
the surface may obscure underlying microstructural features. These factors can act to
reduce the apparent ‘crispness’ often associated with dry, coated materials. It should
also be noted that low-vacuum imaging of even moderately soft, uncoated specimens
carries an increased risk of radiation damage, exacerbated at high magnifications
(Farley et al . 1990), particularly in a water-vapour environment (Kitching & Donald
1998; Royall et al . 2001). Hence, some experimentation is usually required to ascer-
tain acceptable beam energies and magnifications for specific specimens. However,
these limitations should not detract from the possibilities for observing untreated
specimens (free from potential preparation artefacts) and for performing in situ
experiments. Moreover, not all uncoated insulators are as sensitive to the factors
mentioned above. Figure 6 is an image obtained in low-vacuum mode, showing phys-
iological apatite (carbonated calcium phosphate) deposited in vitro. The crystals
that can be seen have dimensions of the order of 200 nm × 10 nm, which compares
favourably with the resolution for conventional SEM, yet without the potential for
coating-related artefacts.

6. Image interpretation
(a) Intrinsic SE emission
Variations in signal intensity across a specimen provide image contrast, conveying
information such as composition, surface topography and any crystal orientation.
Compositional information is traditionally associated with the detection of elastically
backscattered electrons, which are highly dependent on the atomic number(s), Z, of
elements in the specimen. Low-energy, surface-sensitive SE signals are more usu-
ally associated with topographic features, particularly in the presence of a coating
(Goldstein et al . 1992). With the ESEM’s ability to image insulating specimens in the
absence of a coating, we have the opportunity to detect intrinsic specimen-dependent
SE signals. SE emission from solid insulators has been extensively discussed in the lit-
erature (see, for example, Reimer & Tollkamp 1980; Burke 1980; Grais & Bastawros
1982; La Verne & Pimblott 1995; Ganachaud & Mokrani 1995; Howie 1995; Scholtz
et al . 1997; Joy & Joy 1998; Cazaux 1999), although much of this work is theoretical
or is based on observations in high vacuum at low beam energies. We must therefore
relate these ideas to the situation in a low-vacuum or ESEM context, especially for
understanding the behaviour of materials having low Z.
SEs are generated by inelastic collisions of primary electrons with atoms within
the sample. The mean depth for emission of SEs (escape depth λ) is equivalent
to their mean free paths in a material. In metals, there is a continuum of states
providing energy-loss mechanisms for all low-energy excited electrons. Hence, metals
exhibit very short escape depths, and λ can be described by relatively simple models.
However, for materials possessing a forbidden energy gap Eg , as exemplified by the
band gap in semiconductors, more sophisticated models are needed. Akkerman et al .
(1996) proposed a model based on the dielectric response function of semiconductors
and insulators, incorporating the energy gap and valence band width. Values of λ
are modelled for excited electrons having energies greater than some threshold for

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2780 D. J. Stokes

electron–hole pair formation (i.e. greater than Eg ). However, this does not account
for excited electrons with energies less than Eg , where diffusion through the material
can occur with minimal energy loss due to the absence of energy-absorbing electronic
states (in fact the figure may be nearer 1.5–2.0 Eg ) (Howie 1995; Bishop 1974). Such
electrons may go on to make a significant contribution to the emitted signal: a cru-
cial factor in determining λ and thereby SE yields for organic materials (noting that
backscatter emission yields are unlikely to be significant for low-Z materials). Fur-
thermore, in order to escape the sample surface, excited electrons must have sufficient
energy to overcome some form of surface potential, which may be approximated by
the electron affinity (Howie 1995) that, for organics and molecular liquids, tends to
be less than 2 eV (Lide 1991).
Optical-energy-loss data (for example, Ashok & Varaprasad 1991) suggest that
Eg has a value of several electronvolts for a substance containing only single (σ)
bonds, whereas the presence of double (π) bonds results in a smaller gap, due to
additional energy-absorbing states arising from delocalized electrons in π orbitals.
Conjugated π bonds lower Eg further still. As a simple rule-of-thumb, therefore, we
can conclude that saturated hydrocarbons will exhibit a more intense SE signal than
unsaturated hydrocarbons or aromatic compounds. Use of these criteria suggests a
means of distinguishing between low-Z materials such as polymers, biological species
and molecular liquids, although the traditional restrictions on direct imaging of soft
matter in electron microscopy mean that very little has been reported about their
intrinsic SE emissive properties. Referring back to the oil-in-water emulsion shown in
figure 3, we have just such an example of a low-Z specimen showing SE contrast due
to heterogeneities in electronic structure. The oil in this case contains π-bonds, and
hence exhibits a less intense SE signal than the aqueous, σ-bonded, phase surrounding
it (Stokes et al . 1998).

(b) Transient phenomena

Electron irradiation of bulk insulators, and associated negative charging, is a well-
known issue in high-vacuum SEM. Accumulated charge is only partly offset by elec-
tron emission, unless a low beam energy is used to give a balance between incoming
electrons and emitted electrons (see, for example, Goldstein et al . 1992; Joy & Joy
1998). Negative potentials within a specimen decelerate (and deflect) the primary
beam, hence reducing its penetration, in addition to accelerating SEs into the vac-
uum. The result is an anomalously high electron emission, which can easily get out
of control, giving distortions and streaking in images. The primary beam may even
be deflected such that it does not impinge on the specimen surface at all.
As previously discussed, positively ionized gas molecules in ESEM help to reduce
or eliminate the effects of excess negative charge, relative to high-vacuum SEM,
although appearances can still be deceptive. ESEM studies of rocks and minerals in
the second half of the 1990s provided hints of phenomena that had not previously
been encountered in electron microscopy (Griffin 1997; Doehne 1998). The highly
complex, interdependent nature of these phenomena has since been studied in detail
by several workers (Toth et al . 2002a–c; Craven et al . 2002; Stokes et al . 2000), and
a very brief description of some of this work follows.
First, consider a pulse of beam current i impinging on a conductor and a perfect
insulator (in high vacuum), where the current pulse represents the dwell time td

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Environmental scanning electron microscopy 2781

poor conductor poor insulator

(a) current pulse (b)
(beam dwell time td)
i i

charge build-up
Q charge decay Q

cumulative SE
δ δ emission δ δ
0 0

(c) (d )

i i

Q Q

δ δ

time
Figure 7. Schematics of charge build-up, decay and SE emission δ for substances with electrical
properties between those of a good conductor and a good insulator, where i is current, Q is
electrical charge and δ is SE yield. The shaded area represents the total SE signal collected
during irradiation of a given region. (a), (b) Short electron-beam dwell times; (c) (d) longer
dwell times.

of the primary electron beam as it scans the specimen. We expect that a good
conductor will immediately dissipate charge and that, conversely, a good insulator
will steadily accumulate charge, up to some limiting value. When the pulse is over
(for example, when the beam moves to another part of the specimen), the time
constant τ for charge decay is very large for a good insulator: the deposited charge
does not dissipate during the scan period and charge accumulates further with each
successive frame. Similar reasoning can be applied to substances with ‘intermediate’
conductivities (poor conductors and poor insulators).
In ESEM, gas pressures and detector biases are user-selected to provide charge
compensation for a given specimen. Residual negative charge is counteracted before
the electron beam returns for the following scan, avoiding charge build-up in the clas-
sical sense. However, we find that the charging and emission behaviours of substances
with specific dielectric properties are sensitive to changes in td and magnification M :
charging occurs during irradiation for selected td and/or M , but dissipates quickly
once the current stops. Figure 7 illustrates the ways in which two substances with
slightly different conductivities might respond to shorter or longer td , and the effect
this might have on their signal intensities. The intrinsic SE emission δ0 is shown at
the start of irradiation. However, the total yield δ (shaded area) will be that arriving
at the detector during td . For short td , δ is not significantly different from δ0 . For
long td , δ for a poor conductor will remain similar to δ0 , but for a poor insulator δ
will appear significantly higher than its corresponding δ0 . This is demonstrated for

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2782 D. J. Stokes

rapid scan (0.5 s per frame) slow scan (30 s per frame)

Figure 8. The effect of varying beam dwell time (frame rate) on SE signal intensity for a het-
erogeneous specimen (oil-in-water emulsion). The contrast between the two phases appears to
invert, with the dispersed oil phase becoming brighter, due to the different dielectric properties
of the phases. (Reproduced with permission from Stokes et al . (2000).)

an actual specimen in figure 8, where the emission of one phase (oil, in this case) is
affected by a change in td , while the emission from the second phase (water) remains
essentially constant, making it seem as though the contrast is ‘inverted’ (Stokes et
al . 2000).
Clearly, the precise electrical or ionic conductivities of phases in a heterogeneous
specimen have a crucial effect on image interpretation, and these ideas generally
apply to both liquids and solids. Far from being a hindrance, dynamic interactions
between the electron beam and the specimen can be a useful additional means of
characterizing specimens, if used appropriately. At the very least, such parameter-
related changes reflect the electrical properties of bulk phases, aiding our interpre-
tation when used in conjunction with the ‘intrinsic’ information described earlier.
Application of a conductive coating would either mask or eliminate such sources of
contrast.
By extension to the ideas outlined above, we should be able to directly visualize
other variations in dielectric properties, caused by localized charge traps, for exam-
ple. Toth and co-workers demonstrated this idea using pre-irradiated GaN (Toth et
al . 2000). This work also showed how subsurface charge could be de-trapped as a
function of increased temperature, becoming re-trapped when the temperature was
lowered, and such experiments could provide valuable insights as to trap depths and
distributions.
Until recently, it was thought that the role of positive ions was simply that of
charge compensation and, although the mechanisms for this had been stirring some
scientific interest, ions had not been paid serious attention. However, workers such
as Toth, Phillips, Thiel, Craven and Baker began to recognize that electric fields set
up below the surface of insulating specimens influence not just the primary beam,
but also the ‘cloud’ of positive ions outside the specimen, and it was acknowledged
that there is a field component due to the ionized gas, modifying the electric field
between the specimen and the detector (Craven et al . 2002; Toth et al . 2002a, b), in
turn influencing gas cascade amplification. Add to this the effect of dynamic surface
potentials on SE energy distributions and, subsequently, on the occurrence and rate
of electron–ion recombination (Toth et al . 2002c), and it can be seen that image

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Environmental scanning electron microscopy 2783

interpretation in low vacuum becomes a tricky business indeed. For a more detailed
explanation of these collective phenomena, the reader is referred to Thiel & Toth
(2003).
The main point is that we have at our disposal a number of unique, potentially use-
ful, sources of contrast. A well-known example where many of these effects come into
play is that of the aluminium hydroxide mineral gibbsite, where crystal growth zones
can be made to appear or ‘disappear’, depending upon specific operating parame-
ters (Baroni et al . 2000; Griffin 2000). A treatment of the physics governing this
phenomenon is discussed in Craven et al . (2002).

7. Summary
Low-vacuum/environmental SEM is a specialized form of electron microscopy, the
physics of which provides much scope for the exploration of parameter space. A wide
range of insulating specimens can be observed, from solid/dry to soft/moist to liquid,
and their properties can be manipulated through in situ dynamic experiments. SE
emission from native surfaces and stable charge-related effects highlight the need for
a systematic new approach to electron imaging in order to harness the information
presented.
In the future, we can expect further development and significant advances in ESEM
technology. One breakthrough would be a means of imaging biological specimens
under conditions closer to those found in their natural states, involving higher tem-
peratures and pressures than currently tenable. Observation of a wide range of living
specimens and physiological processes could then be realized. Meanwhile, the task
at hand is to integrate low-vacuum and ESEM techniques with a range of com-
plementary characterization tools, such as TEM, SEM, AFM, optical and confocal
microscopy and, by means of objective assessment, choose the most appropriate
method(s) for achieving specific results.
The author gratefully acknowledges The Royal Society for provision of a Warren Research
Fund Dorothy Hodgkin Research Fellowship, and useful discussions with many colleagues in
the Polymers & Colloids Group, Cavendish Laboratory, University of Cambridge.

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AUTHOR PROFILE

Debbie J. Stokes
As an undergraduate, Debbie Stokes studied Chemistry and Physics with the Open
University, graduating with a first class honours degree in 1995. She went on to
obtain a PhD in Physics at the University of Cambridge in 1999, and was awarded
a Royal Society Warren Research Fund Dorothy Hodgkin Research Fellowship. Also
in 1999, she was elected a Junior Research Fellow at Newnham College, Cambridge,
becoming a Senior Research Fellow in 2002. Aged 38, she is currently working on
developing ESEM applications at the Cavendish Laboratory, Cambridge, and was
recently co-opted onto the Electron Microscopy Section Committee of the Royal
Microscopical Society. Scientific interests include electron interactions with soft con-
densed matter, such as polymers and biological materials, in a partial vacuum, as
well as the microstructure and dynamic behaviour of these materials. She has two
sons, aged 14 and 16, and her favourite recreation is travel.

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