Micron 38 (2007) 390–401
www.elsevier.com/locate/micron
A history of scanning electron microscopy developments: Towards
‘‘wet-STEM’’ imaging
A. Bogner a,b,*, P.-H. Jouneau a,c, G. Thollet a, D. Basset b, C. Gauthier a
a
Groupe d’Etudes de Métallurgie Physique et de Physique des Matériaux, UMR CNRS 5510, INSA de Lyon, Bâtiment B. Pascal,
7 Avenue Jean Capelle, 69621 Villeurbanne Cedex, France
b
Total France, Centre de Recherche de Solaize, BP 22, 69360 Solaize Cedex, France
c
CEA Grenoble, DRFMC/SP2M, Laboratoire d’Etudes des Matériaux par Microscopie Avancée, 15 Rue des Martyrs, 38054 Grenoble, France
Abstract
A recently developed imaging mode called ‘‘wet-STEM’’ and new developments in environmental scanning electron microscopy (ESEM)
allows the observation of nano-objects suspended in a liquid phase, with a few manometers resolution and a good signal to noise ratio. The idea
behind this technique is simply to perform STEM-in-SEM, that is SEM in transmission mode, in an environmental SEM.
The purpose of the present contribution is to highlight the main advances that contributed to development of the wet-STEM technique. Although
simple in principle, the wet-STEM imaging mode would have been limited before high brightness electron sources became available, and needed
some progresses and improvements in ESEM. This new technique extends the scope of SEM as a high-resolution microscope, relatively cheap and
widely available imaging tool, for a wider variety of samples.
# 2006 Elsevier Ltd. All rights reserved.
Keywords: Electron microscopy; STEM-in-SEM; Transmission mode; Scattered electrons; Environmental scanning electron microscopy; ESEM
1. First steps in scanning electron microscopy
In scanning electron microscopy (SEM), a fine probe of
electrons with energies typically up to 40 keV is focused on a
specimen, and scanned along a pattern of parallel lines. Various
signals are generated as a result of the impact of the incident
electrons, which are collected to form an image or to analyse
the sample surface. These are mainly secondary electrons, with
energies of a few tens of eV, high-energy electrons backscattered from the primary beam and characteristic X-rays. This
section reviews the most important steps that have allowed
using such rich physical interaction in a practical tool, making
the SEM the powerful instrument it is today in materials and
life-science.
The history of electron microscopy began with the
development of electron optics. In 1926, Busch studied the
trajectories of charged particles in axially symmetric electric
and magnetic fields, and showed that such fields could act as
particle lenses, laying the foundations of geometrical electron
optics (Oatley, 1982 and references therein). Nearly at the same
* Corresponding author. Tel.: +33 4 72 43 61 30; fax: +33 4 72 43 85 28.
E-mail address: Agnes.Bogner@insa-lyon.fr (A. Bogner).
0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micron.2006.06.008
time, the French physicist de Broglie introduced the concept of
corpuscule waves. A frequency and hence a wavelength was
associated with charged particles: wave electron optics began
(Hawkes, 2004 and references therein). Following these two
discoveries in electron optics, the idea of an electron
microscope began to take shape.
In 1931, independently of the ‘‘material wave’’ hypothesis
put forward by de Broglie several years earlier (1925), Ruska
and his research group in Berlin, were working on electron
microscopy. They were disappointed learning that even with
electrons a wavelength would limit the resolution. But they
found using de Broglie equation that electron wavelengths were
almost five orders of magnitude smaller than the wavelength of
light used in optical microscopy. It was thus considered that
electron microscopes still could prove a better resolution than
light instruments, and no reason existed to abandon this aim. In
1932, Knoll and Ruska tried to estimate the resolution limit of
the electron microscope. Assuming the resolution limit formula
of the light microscope was still valid for material waves, they
replaced the light wavelength by the electrons wavelength at an
accelerating voltage of 75 kV. A theoretical limit of 0.22 nm
resulted, a value experimentally reached only 40 years later.
Although these calculations proved it was possible to reach a
better-than-light-microscope resolution when working at high
A. Bogner et al. / Micron 38 (2007) 390–401
magnifications (Ruska, 1986), many technical limits had to be
to overcome. Ruska and Knoll tried to implement Busch’s lens
formula experimentally. Their work resulted in the construction
of the first transmission electron microscope (TEM) in 1931,
with a magnification of 16 (Haguenau et al., 2003).
Knoll built a first ‘‘scanning microscope’’ in 1935. However,
as he was not using demagnifying lenses to produce a fine
probe, the resolution limit was around 100 mm because of the
diameter of the focused beam on the specimen. In 1938, von
Ardenne clearly expressed the theoretical principles underlying
the scanning microscope, as we know it today. Because it was
difficult to compete with TEM in resolution achieved for thin
specimens, the scanning microscopy development was oriented
more toward observing the surface of samples. The first true
SEM was described and developed in 1942 by Zworykin, who
showed that secondary electrons provided topographic contrast
by biasing the collector positively relative to the specimen. One
of his main improvements was using an electron multiplier tube
as a preamplifier of the secondary electrons emission current.
He reached a resolution of 50 nm, which was still considered
low in comparison with the performance obtainable in TEM
(Goldstein et al., 2003).
Indeed, many microscopists in the TEM community were
seeing only limited applications for an instrument having a
lower resolution than a TEM. Fortunately many scientists and
technologists quickly recognised the SEM ability to yield threedimensional information from the surfaces of bulk specimens
over a large range of length-scales.
In 1948, Oatley began to build an SEM based on Zworykin’s
microscope. Following this development, Smith (1956) shown
that signal processing could be used to improve micrographs.
He introduced nonlinear signal amplification, and improved the
scanning system. Besides, he was also the first to insert a
stigmator in the SEM to correct lens cylindrical imperfections.
In 1960, Everhart and Thornley greatly improved the
secondary electron detection. A new detector was created with
a positively biased grid to collect electrons, a scintillator to
convert them to light, and a light-pipe to transfer the light
directly to a photomultiplier tube (Goldstein et al., 2003).
In 1963, Pease and Nixon combined all of these improvements in one instrument: the SEM V with three magnetic lenses
and an Everhart–Thornley detector (ETD). This was the
prototype for the first commercial SEM, developed in 1965—
the Cambridge Scientific Instruments Mark I ‘‘Stereoscan’’
(Breton, 1999). The SEM we are using today are not very
different from this first instrument.
2. What makes the SEM a high-resolution technique?
2.1. Lens aberrations and source brightness limit the
resolution in SEM
In the SEM, electron optics is used to demagnify the size of
the electron source, usually a small tungsten tip, to form the
smallest possible probe. The demagnification is achieved using
a series of ‘‘condenser lenses’’, and a final ‘‘objective lens’’
also known as the ‘‘probe-forming’’ lens. This last lens
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provides the final demagnification and focuses the electron
beam on the surface. At high magnification, the image
resolution is roughly equal to the width of this probe. It is
limited by a few key parameters: aberrations of the lenses,
especially the objective lens because it works at large
convergence angles, the brightness of the electron source
(Hawkes, 2004), and the interaction volume, especially when
the samples are not very thin.
Aberrations, that are lens imperfections, limit the ability of
focusing the beam, and therefore blur the image. The difficulty
arises from two main aberrations. Due to spherical aberration,
rays travelling far from the optical axis are focused more
strongly than those close to the axis. To reduce this effect, an
objective lens aperture is used to limit the angle of the outer rays
through the lens. Chromatic aberration also blurs the image
since electrons with slightly different wavelengths are focused
more or less strongly. Electron beams with narrow energy
distribution are used to limit this effect. Today, the energy
spread of the beam in a microscope is typically less than 2 eV
for a thermal source and less than 1 eV for field-emission
sources.
The second key parameter limiting the resolution is the
conservation of brightness throughout the microscope column.
It means it is impossible to decrease the size of the electron
probe (by additional optical demagnification) without decreasing the current at the same time. When the electron flux
becomes to low in the probe, the poor signal to noise ratio limits
the resolution.
Two main approaches exist to improve the SEM resolution:
decrease the lens aberrations, or increase the source brightness. Optical aberrations may be reduced by improving lens
design, and by developing electro-optical correcting devices.
Much work is currently being carried out on this subject. But
in recent years, most of the progresses in SEM resolution have
resulted from the development of high brightness sources. The
early thermionic sources, still in use in low cost instruments,
are now superseded by field emission and Schottky electron
guns, which exhibits a higher brightness and a better
monochromaticity, as shown in Table 1. Their use leads to
the emergence of the ‘‘high-resolution SEMs’’ in the 1980s
(Joy, 1991).
2.2. Different technologies of electron sources in the
electron microscope
The first and most widespread electron source in electron
microscopy was the thermionic gun. It consists of a tungsten
filament bent into a V-shaped hairpin, with a tip radius of about
100 mm. This tip spontaneously emits thermionic electrons
when electrically heated to a temperature around 2700 K. The
thermionic electron source presents the advantages to be
relatively inexpensive, and to require a relatively low vacuum.
However, the lifetime of W-cathodes is limited to about 100 h
by evaporation of the cathode material, which result in a failure
when part of the wire becomes too thin. In thermionic LaB6
electron guns developed later, the emitting material has a lower
work function, meaning the same amount of electrons can be
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Table 1
Comparison of different electron sources at 20 kV (Goldstein et al., 2003)
Source
Brightness
(A/cm2 sr)
Lifetime
(h)
Virtual source
size
Energy spread
DE (eV)
Beam current
stability (%/h)
Tungsten hairpin
LaB6
Cold field emission
Thermal field emission
Schottky field emission
105
106
109
108
108
40–100
200–1000
>1000
>1000
>1000
30–100 mm
5–50 mm
<5 nm
<5 nm
15–30 nm
1–3
1–2
0.3
1
0.3–1.0
1
1
2
2
1
emitted at a lower heating temperature. The emitter is a tiny
block of LaB6 single-crystal, about 100 mm in diameter and
about 0.5 mm long, polished to present a 1 mm tip radius.
Depending on the sharpness of the tip, this electron gun exhibits
5–10 times higher brightness and a 10 times longer lifetime
than a tungsten filament.
The second main technology of electron sources, originally
developed in the 1930s, is based on field emission. A fieldemission gun is a wire with a sharp point, supported by a
hairpin. The tip emits electrons by quantum mechanical
tunnelling process when it is brought into close proximity with
a positively biased extraction electrode. Two types of fieldemission sources exist: cold field emission (CFE), and thermal
field emission (TFE), including Schottky field emission (SFE).
In CFE, the high electric field causes electrons to tunnel
through the potential barrier and leave the cathode wire at room
temperature. Electrons are emitted from a virtual source with
an area of only a few nanometers in diameter. TFE are similar
in principle but operate at a higher temperature. This helps to
keep a clean tip and to reduce noise and instability. Finally, in
SFE, a thin ZrO2 layer is deposited on the flattened tip from a
small dispenser to further reduce the work function. SFE is in
fact a thermionic source but with a brightness comparable to
CFE (cf. Table 1). The main benefit of these field-emission
sources is a higher brightness, of the order of 109 A/cm2 sr at
20 keV. As previously said, brightness is a fundamental limit
for the resolution in the SEM, as it determines the current
available at a given probe size, and sets the recording time per
pixel of the image. Despite this obvious advantage, the
development of field-emission sources took time. Many
technical barriers had to be overcome increasing the
instrument cost. An additional delay may have also been
caused by the great success of the early thermionic SEM
instruments (Pawley, 1997). Besides the high brightness, fieldemission guns show another important advantage: the virtual
source is small, usually in the 5–25 nm range. The optics
required to demagnify this probe for high-resolution observations is therefore simplified in comparison with the case of
thermionic emitters, where the effective source size is of the
order of 5 mm. Lower demagnification contribute to reduce
lens aberrations. Moreover, the FEG has a small energy spread,
as low as 0.35 eV for a thermal or Schottky FEG, to compared
with 1.5 eV or more for a thermionic emitter. While chromatic
aberration does not really limit the resolution at high energies
(>10 keV), it is a major factor of improvement for low-energy
imaging (Joy, 1991).
Today, thanks to these new field-emission sources, resolutions on the order of 1–2 nm are routinely achieved for
commercial instruments operating between 1 and 30 keV.
3. The use of STEM mode in SEM
3.1. STEM mode
The STEM concept was described by von Ardenne in the
late 1930s: he was the first to perform a STEM mode
experiment in 1938, by adding scan coils to a transmission
electron microscope (Goldstein et al., 2003). However, the
STEM did not develop at that time due to a lack of electronics
and adequate electron sources. In 1960s, interest in STEM was
revived by Crewe and coworkers with the development of the
cold field-emission electron source and the optimization of
electron-optical components, culminating in the first visualization of single heavy atoms in the electron microscope in 1971.
In a dedicated STEM, the electron optics are designed to
produce an atomic-size beam of electrons that illuminates a
small area on the surface of the specimen. Images are formed by
rastering the subnanometer probe over the surface and
collecting electrons that were transmitted through the sample
(unlike in SEM where secondary electrons are used). A STEM
image may be considered as a collection of individual
scattering experiments. The STEM can be considered a low
dose technique, in comparison with fixed-beam TEM. Various
types of signals discriminated in scattering angle and/or energy
loss yield different structural and chemical information, and
may be captured simultaneously. Quantitative analysis may be
performed, with no limitation of the solid angle and the energy
loss interval over which scattered electrons may be collected. In
fact, nearly all electrons coming through the specimen can be
collected by at least one of the detectors. Biological specimens
are difficult to see in the transmission electron microscope
because of their low contrast. A solution is to increase contrast
by staining, i.e. heavy atoms addition, however a question arises
whether the observed image features are actually artifacts.
Dark-field STEM imaging is a direct approach that has been
developed to circumvent the need for staining. It is for this
reason, and for low dose techniques requirements, that STEM
was deliberately aimed at biological applications (Colliex and
Mory, 1994).
Although STEM can be performed in a dedicated instrument
specifically designed for the technique, it is more often
developed as a hybrid technique performed on modified SEMs
A. Bogner et al. / Micron 38 (2007) 390–401
or TEMs, microscopes more user friendly and widely available
(Tracy and Alberi, 2004).
A STEM system added to a standard SEM is often designated
as ‘‘low-voltage STEM’’, term referring to the 20–30 kV regime,
i.e. low relative to typical TEM operating energies. It is near the
lower limit of energies that will provide sufficient transmission
through the sample. As in a SEM, the beam focuses on a small
spot that scans over the sample. The image is formed by mapping
some signal intensity synchronously with the scan. As in a TEM,
image information is extracted from electrons that have passed
through a thin sample.
Using the transmission mode in a scanning electron
microscope, both contrast and resolution are improved due
to the lower accelerating voltages, which increase the crosssections and reduce the interaction volume of the incident
electron beam (Tracy and Alberi, 2004; Golla-Schindler,
2004). Indeed in transmission electron microscopy, high
electron energies result in a scattering contrast produced by the
narrow cone of transmitted electrons scattered through small
angles and limited by the objective aperture. For contrast
enhancement, the electron energy must be reduced and/or in
the case of low atomic number elements the sections stained
with heavy metal compounds. However, low-voltage fixedbeam TEM is limited by the strong decrease in transmission
with decreasing electron energy and by chromatic aberrations
from more frequent energy losses. Low-voltage scanning
transmission electron microscopy can be an interesting
alternative. The transmission mode in SEM has an advantage
of avoiding chromatic aberration. As there is no projection
lens, no image deterioration occurs due to chromatic
aberrations, even in the case of inelastic interactions at low
voltages. As already suggested by von Ardenne, the potential
advantage of transmission-SEM indeed lays in the fact that
electrons are not required to pass through a lens after crossing
the specimen. The spread of velocities caused by absorption in
393
the specimen therefore do not give rise to chromatic aberration
induced loss of resolution, as it does in a conventional
transmission microscope: it is thus possible to examine a
thicker specimen (Oatley, 1982). It is an argument also cited by
Merli et al. (2004): due to the absence of image-forming lenses,
the energy loss and the large scattering angles do not affect the
resolution as in transmission electron microscopy.
In low-energy STEM, the specimen response will be
proportional to the average scattering power of atoms which
will give rise to mass–thickness contrast, or absorption contrast.
For a good Z-contrast, it is necessary to use large scattering
angles in transmission. In the transmission mode of SEM, the
aperture can be increased to obtain a high transmission and
signal-to-noise ratio.
SEM remains one of the most flexible tools for highresolution imaging (resolution down to 1 nm), however 1 nm
resolution is sometimes not achieved when characterizing
ordinary samples, due to limitations arising from the nature of
beam–sample interactions when secondary electrons signal is
used. STEM-in-SEM is one method that can overcome these
limitations and enable imaging with high resolution (Vanderlinde, 2005). As the samples investigated are thin in comparison
with SEM, the interaction volume is rather small. For this
reason, in STEM mode, the resolution is only limited by the
spatial broadening of the electron probe at the exit surface of the
sample, whereas structures at the entrance surface are resolved
with a resolution of approximately same as the diameter of the
electron probe. This beam broadening phenomenon is called
the ‘‘top–bottom effect’’ (Golla-Schindler, 2004). For illustration, Fig. 1 shows the investigation of small indium cluster on a
formvar film with a polystyrene sphere placed on the indium
layer. This test structure can be investigated with the indium
cluster above the polystyrene sphere (top object) or below
(bottom object) in STEM-in-SEM, mode called ‘‘transmissionSEM’’ by the authors (Fig. 1a and b) or in TEM (Fig. 1c and d).
Fig. 1. Illustration of the top–bottom effect according to Golla-Schindler et al. (Golla-Schindler, 2004).
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A. Bogner et al. / Micron 38 (2007) 390–401
Improved sharpness of the indium cluster can be obtained by
using the top object and the transmission-SEM mode (Fig. 1a).
Due to the parallel illumination in TEM the best object is the
bottom object, but the resolution of the indium cluster is limited
by the chromatic aberration of the post specimen objective lens
(Fig. 1c). Therefore, for TEM investigations thinner sections
and increased accelerating voltages are necessary to improve
the attainable resolution (Fig. 1d).
Today, every microscope manufacturer offers a STEM
detector for SEM. In addition with the success related to SEM
ease of use and availability when compared to TEM, it is indeed
recognised that STEM-in-SEM extends the usefulness of the
SEM (Woolf et al., 1972). On one hand, thicker samples can be
observed thanks to large collection angles, and high contrast is
results from the use of low voltages. On the other hand, high
resolution is available thanks to limited interaction volume and
chromatic aberration.
3.2. STEM-in-SEM detection methods
In the present article, we are interested in the transmission
mode performed in SEM. Several detection strategies for
transmitted 10–30 keV electrons exist, using several types of
STEM detectors.
3.2.1. Transmitted electrons/secondary electrons converter
and Everhart–Thornley detector (Golla et al., 1994; GollaSchindler, 2004; Vanderlinde, 2005)
Various devices have been developed using the following
idea, that does not require the development of a new detector:
transmitted electrons (TE) are converted to secondary electrons
(SE) using a plate below the sample: the converter. SEs can then
be detected by using a conventional Everhart–Thornley
detector (ETD). This is one of the configurations exhibiting
the advantage of TV-rate image.
This principle is applied in the configuration used by Golla
et al. presented in Fig. 2. In their device, a cylindrical shield
stops the ‘‘reflected’’ secondary electrons, so that only
Fig. 2. Scheme of a STEM-in-SEM system: TE/SE converter and ETD configuration (Golla-Schindler, 2004).
transmitted or scattered electrons can be collected by the
ETD. An electron trap also prevents large angle scattered
electrons from reaching the ETD. An adjustable aperture
determines the collection angle for the bright-field mode or the
angle segment for the dark-field mode.
3.2.2. Direct collection of transmitted electrons by a solidstate detector
Transmitted electrons can also be directly collected by a
semiconductor detector. In this case, electrons reaching the
detector are physically detected as they create electron–hole
pairs in the silicon diode (2700 pairs by an 10 keV electron
incident on the detector), resulting electrical charge is collected
from the biased p–n junction of the detector.
The configuration described in Fig. 3 has been used by Merli
et al. Transmitted electrons are collected using the detector
normally used for backscattered electrons (BSE) but placed just
below the sample (Merli and Morandi, 2005). There are several
positions for the detector: (1) by placing the center of the
detector on the optical axis and varying the specimen-detector
distance, it is possible to select the angular distribution for darkfield imaging (Fig. 3a); (2) by removing the center of the
annular detector from the optical axis and defining with an
aperture a portion of the detector itself, it is possible to define
the angular range of transmitted electrons producing the brightfield image (Fig. 3b).
It should be noted that in the case of a dipolar detector, two
diodes are placed on the optical axis. In this way, bright- or darkfield images can be acquired through the collection of transmitted
Fig. 3. Merli et al. STEM-in-SEM detection conditions: (a) BSE annular
detector centered on the optical axis for dark-field imaging; (b) off-axis annular
detector with an aperture on the cover to define the collection angle a for brightfield imaging. Scheme courtesy of Merli et al. (Merli and Morandi, 2005) and
Microscopy and Microanalysis from Cambridge University Press.
A. Bogner et al. / Micron 38 (2007) 390–401
Fig. 4. Scheme of Woolf et al. transmission stage for the SEM, including a
scintillator. Scheme courtesy of David Joy (Woolf et al., 1972) and IOP
Publishing.
or scattered electrons, respectively. This setup is implemented for
example in the FEI high vacuum STEM holder.
3.2.3. Transmitted electrons/photons conversion via a
scintillator
STEM-in-SEM has become an established technique because
it extends the capabilities of the SEM and offers considerable
advantages in signal processing and analysis when compared to
the TEM (Woolf et al., 1972). The detection strategy of Woolf
et al. in their ‘‘transmission stage for the scanning electron
microscope’’ is presented in Fig. 4. It used a scintillator/light-pipe
combination. After their path through the sample, electrons are
converted to photons by a scintillator and pass through a lightguide. The signal is amplified afterwards by a photomultiplier.
This is a relatively expensive standard detector system, but it
allows TV-rate imaging, and the selection of diffracted beams.
Each of the three precedent detection strategies presents
advantages, but practically a type of detector is often adopted
because of financial or availability reasons.
4. Environmental SEM development
The origin of environmental SEM is directly linked to the
high vacuum needed in electron microscopes, that introduce
restrictions on the way that certain specimens are prepared and
imaged. Very early in the history of electron microscopy,
studies were related to the possibilities of imaging specimens
in a more ‘‘natural’’ state. In the 1950s, experiments concerned
differentially pumped, aperture-limited TEMs, or creation of
‘‘environmental chambers’’. The separation of high vacuum
electron gun chamber from a gaseous specimen chamber via
open diaphragms (PLA for pressure limiting apertures) was
first used in several high-voltage TEM. Research by Danilatos
and Robinson in the 1970s led to the first SEM capable of
maintaining a relatively high pressure, removing the need to
dry and coat the specimens (Danilatos, 1991). The term
‘‘environmental’’ SEM was introduced in 1980. As mentioned
in Section 1, during the first development steps of SEM, the
established TEM community was very suspicious. In the same
way, the SEM community was not convinced when the first
environmental SEMs appeared (Stokes, 2003). The introduction of gas in an electron microscope is completely opposed to
395
the generally established ideas of a clean high vacuum. It was
generally perceived that a gaseous environment would
deteriorate the resolution by an assumed broadening of the
electron beam and exclude the SE detection mode of imaging
because highly positive bias of the ETD induces arcing in the
presence of gas. To convince the microscopy community, these
two main problems – i.e. reduced resolution, and exclusion of
SE detection – had to be resolved with the: (1) determination
that the electron probe diameter remained small at elevated
pressure; (2) invention of the gaseous detection device (GDD),
where the principle of gaseous ionization produced by the
signal-gas interaction is used for imaging; this GDD was called
GSED (for gaseous secondary electron detector) to insist on
the fact that it is possible to detect secondary electrons in
ESEM (Danilatos, 1991). Over more than two decades,
Danilatos showed tenacity and developed the ESEM instrument to a point where the rest of the scientific community and
manufacturing world could further benefit. ESEM became a
stable instrument. By the late 1980s, the first commercial
environmental scanning electron microscopes (ESEMs by
Electroscan, a company created for the purpose of gaseous
microscope manufacturing) were being produced, opening up
a world of possibilities for observing untreated specimens. A
few years later in 1996, a traditional electron microscope
manufacturer Philips FEI took over the operation to further
commercialize the instrument. Other manufacturers have in
the meantime followed with low-vacuum systems reaching
100 Pa, the level of pre-ESEM technology, probably due to
patent restrictions (ESEM Research Laboratory, Australia).
The ESEM has rapidly gained acceptance by the scientific,
technical and industrial community as shown by the large
number of publications arising from its use.
To summarize, ESEM differs from conventional SEM
mainly by the presence of a gas in the specimen chamber.
Samples are thus not viewed under high vacuum but under a
deteriorated or ‘‘low’’ vacuum. This is possible thanks to a
special design of electron optics column that allows
differential pumping: the column is divided into different
pressure zones separated by pressure limiting apertures
(Danilatos, 1993). The presence of gas, or environment around
the sample that inspired the term ‘‘environmental’’ SEM, can
play two main roles. The first, common for environmental as
well as low-vacuum SEMs,1 is electronic. The gas acts as an
electrical charge conductor avoiding sample charging and
facilitates signal detection. This first role is described in Fig. 5:
collisions between electrons and gas molecules create positive
ions that can balance the accumulation of negative charges on
the surface of specimens, and ‘‘cascade’’ electrons that help to
amplify the signal collected by the gaseous secondar electrons
detector (GSED). The second role, more specific to environmental SEMs, is thermodynamic, i.e. the gas is a conditioning
medium, preventing evaporation of liquids from a sample
(Thiel and Toth, 2005).
1
ESEM is a trademark from FEI Company, but other constructors have
developed their low-vacuum SEM too.
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A. Bogner et al. / Micron 38 (2007) 390–401
Fig. 5. A schematic of the amplification process due to collisions between the
secondary electrons and gaseous molecules. Image courtesy of D.J. Stokes
(Stokes, 2003) and the Royal Society of U.K.
With this specialized form of electron microscopy a wide
range of insulating materials, oils, liquids, etc., can be observed
at equilibrium or during in situ evolutions. The potential
applications are described in numerous recently published
articles, in particular by Donald (2003).
In this paper, we discuss liquid specimens, most of the time
aqueous. Water vapour is the most common gas used in ESEM,
due to its efficiency in signal amplification, and its useful
thermodynamic properties leading to good quality images and
specimen stability.
Fig. 6 is an example concerning the amplification signal of
water, compared with four other gases: carbone dioxide, nitrous
oxide, nitrogen, helium (Fletcher et al., 1997). The authors
conclude that water vapour gives quantitatively the largest
maximum amplification of all the cases considered in their
study (variable accelerating voltage and partial pressure). It
should also be noted that the maximum of these amplification
curves is around 4–5 Torr for water.
Concerning aqueous specimen stability, it is important to
consider the thermodynamic saturated vapour pressure (SVP)
curve for water as reproduced in Fig. 7 (Stokes, 2003). It can
be seen that the pressure range up to around 10 Torr meets the
SVP when the specimen temperature is lowered to a few
degrees Celsius from ambient. Typically, a water-cooled
Peltier stage is employed to keep the specimen at around
3 8C, within a chamber environment of water vapour at a
pressure in the region of 4.5 Torr, leading to improved
Fig. 6. Dependences of the amplification on the pressure for various primary-beam energies and for five gases: (a) water vapour, (b) carbon dioxide, (c) nitrous oxide,
(d) nitrogen, and (e) helium. Figures courtesy of Bradley Thiel (Fletcher et al., 1997) and IOP Publishing.
A. Bogner et al. / Micron 38 (2007) 390–401
397
Fig. 7. Saturated vapour pressure curve for water. Image courtesy of D.J. Stokes
(Stokes, 2001) and Wiley.
specimen stability and good quality images. It is also essential
to ensure that the specimens’ natural moisture is preserved
during the initial pumpdown of the chamber. It is therefore
usual to perform a sequential pumpdown such that the air in
the chamber is successively replaced with water vapour, and
that evaporation from and condensation onto the sample are
minimized (Cameron and Donald, 1994).
ESEM also extends the possibilities of SEM in term of the
wide variety of samples and of states that can be characterized.
However, in the case of liquids, classical wet mode in ESEM
only allows the observation of the surface, which is often very
smooth so that very little information about the sample can be
collected. For this purpose, the wet-STEM imaging mode
described hereafter may be suitable for aqueous samples, or
other liquids.
5. Wet-STEM imaging: observing a thin liquid film in
transmission mode
It actually refers to the STEM-in-SEM applied to environmental SEM, benefiting from the improved FE SEM
performance in STEM mode and low-vacuum techniques.
5.1. History
Bultreys and Thollet were the pioneers of the ‘‘wetSTEM’’ imaging mode a few years ago. During one of their
experimental collaborations in the GEMPPM laboratory in
Lyon, they placed a TEM grid on a Peltier stage of an FEI XL
30 FEG ESEM, and relocated the dipolar detector usually
used for backscattered electrons detection below the grid: the
same configuration used in the high vacuum STEM detector,
already commercially available in FEI XL series SEMs, but
adapted for the use on a cooling stage.
A series of investigations were conducted by Bogner et al.,
examining the experimental procedure and signal detection
conditions as well as the influence of gas purge, type of grids,
sample-detector distance, annular dark-field detection (Bogner
et al., 2005).
The wet-STEM instrument in our laboratory is described as
follows.
Fig. 8. The wet-STEM device at the GEMPPM laboratory: (a) Peltier stage; (i)
incident convergent electron beam; (b) SEM mount maintaining the TEM grid;
(c) solid-state annular detector.
5.2. Experimental setup of wet-STEM
As the presented imaging mode allows the observation of
wet samples in transmission mode, the term ‘‘wet-STEM’’ is
self-explanatory. The device, described in Fig. 8, is placed in an
FEI XL 30 FEG ESEM. A copper grid is placed on a TEM
sample holder, and positioned on a Peltier cooling stage. A
small amount of liquid containing particles or floating objects
(organic, inorganic, liquid or solid) is dropped on the grid with a
micropipette.
As in the conventional wet mode of ESEM and in relation
with the work described in Cameron and Donald (1994), we use
an optimized pump down sequence in order to prevent
evaporation from, and condensation onto the sample droplet.
Classical ESEM detectors are also available enabling to
control of the sample surface in SE mode and in BSE mode,
using the gaseous SE detector (GSED), and the gaseous
backscattered electron detector (GAD), respectively. This is
very helpful for example to control the presence of liquid until
the thickness is adequate for transmission imaging to perform
transmission observations and to detect whether objects are
submerged in water.
When the required partial pressure of water is reached,
pressure and temperature can be adjusted to evaporate a small
amount of water – if the considered sample is aqueous – from
the droplet. It allows obtaining a water layer thin enough such
that the incident electrons can pass through it, and can be
collected to form a STEM image. Films of wet samples are
thinned in situ in the ESEM chamber, their thickness depends
on the quantity of water evaporated from the initial droplet. As
evaporation is an endothermic reaction, it is then possible to
follow it by checking the difference between the setting
temperature and the measured one. Then, the thickness of the
film is kept constant thanks to an equilibrium water pressure
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A. Bogner et al. / Micron 38 (2007) 390–401
using the (P, T) water diagram (presented in the precedent
section). For instance, a water pressure of 5.3 Torr is required at
a sample temperature of 2 8C, so that objects remain in a water
layer with constant thickness. By controlling the sample
temperature through the Peltier stage, and using water vapour
as the imaging gas at a controlled pressure, samples can be kept
above their saturated vapour pressure during all the experiment.
When the required thickness is obtained, the incident
electron beam is focused on the droplet, and passed through the
liquid layer and floating nano-objects. The signal is then
collected by a detector, usually used for the collection of
backscattered electrons, but in the present configuration located
below the sample. Holey carbon coated TEM copper grids
placed with the carbon layer faced pointing down enable copper
squares to play the role of retention basins. In the carbon layer,
holes of typical diameter ranging from less than 1 to 20 mm
allow maintaining overhanging liquid films on very small areas.
It is important to note that adequate initial droplet parameters –
i.e. volume and solid content – enable to control the amount of
nano-objects on the grid. It is possible, for instance in the case
of a latex emulsion sample, to choose to image only a
monolayer of particles if required. Due to the initial
concentration of the suspension, when the droplet of smallest
volume that can be dropped contains more objects than for a
monolayer on the grid, a dilution step with distilled water can
be performed before placing the liquid on the grid.
As discussed above, several detection strategies can be
implemented for ‘‘transmitted’’ electrons detection in an SEM.
The present device is based on the direct collection of electrons
passed through the sample by a solid-state detector (two semiannular detectors A and B). For the setup usually used in STEM
detectors for FEI SEMs, the incident electron beam arrives on
the border of diodes A and B, and bright- or dark-field images
can be produced with the collection of transmitted or scattered
electrons, respectively. In the latter case, only a small area of
the sample is above the border of diodes A and B; so that we do
not have the choice of the imaging mode (bright- or dark-field)
for the other areas. In the present study, we choose another
possibility: annular dark-field imaging conditions can be
obtained if the dipolar detector is placed on the optical axis so
that the direct transmitted electron beam is not collected and
only scattered beams are detected on a ring constituted by both
of the diodes A and B. Using this method, a more important part
of the scattered electrons available is used to form an image and
higher-contrast images can be obtained. Consequently, all the
ESEM micrographs presented in the following section have
been obtained using the annular dark-field configuration. The
second advantage of this configuration, using the summed
signal, is that imaging conditions are not linked to the area of
the sample imaged. Moreover, in our experimental setup, the
distance between the sample and the detector has been
experimentally investigated in order to optimize the contrast.
Best results have been obtained with a distance of about 7 mm,
corresponding to collection angles between 349 and 820 mrad
for the dark-field mode. Nevertheless, the optimal distance
between the grid and the detector depends on the sample
composition and thickness, and should be a variable parameter.
5.3. Benefits of STEM-in-SEM mode
By using low voltages, which lead to improved contrasts,
and thanks to the absence of a projection lens when compared to
TEM, thicker samples can be imaged using the present STEM
mode. For example, an estimation of the thickness of water that
can be passed through with an incident electron voltage of
30 kV is presented in Fig. 9. It has been estimated using an
evoluted Monte Carlo Model applied by SAMx1 in their
Hurricane software. This powerful tool has been specially
adapted so that it is possible to consider transmitted electrons
and to store them as a function of their energy and their
scattering angle. Here, transmission is defined as the ratio
between the number of electrons scattered with angles from 349
to 820 mrad and the number of the incident electrons. It can be
shown that transmission occurs in as much as several microns
of water. Although it is not within the scope of the present
article, it should be pointed out that this special extension of
Hurricane is also very helpful to progress in wet-STEM images
contrast interpretation.
Theoretically, the resolution of wet-STEM mode should be
better than seen in ESEM wet mode, due to the properties of the
thin sample (STEM-in-SEM a high-resolution mode in SEM
because there is no interaction volume, only top–bottom effect:
see Section 3), and that contrary to TEM, electrons do not have
to pass through a lens after their path through the sample (see
Section 3 also). Unfortunately in both cases (wet mode in
classical reflection ESEM, and wet-STEM), we have observed
that the resolution is limited by the mechanical vibrations
partially due to the cooling flow of the Peltier stage and to the
vacuum system.
We are limited to relatively high beam energies (25–30 kV)
when compared to the range typically used in SEM (a few eV
to 30 kV). Indeed, in ‘‘environmental’’ or ‘‘low-vacuum’’
SEMs, the electrons leaving the specimen are significantly
retarded by their collisions with gas molecules reducing their
energies. The use of a TE/photons conversion via a scintillator,
as proposed by KE company in the Centaurus detector, could
be used to explore other imaging conditions in wet-STEM,
Fig. 9. Evaluation of the scattering transmission through a water layer at 30 kV
with Hurricane (SAMx1) Monte Carlo simulation: scattered electrons are
collected from 349 to 820 mrad.
A. Bogner et al. / Micron 38 (2007) 390–401
Fig. 10. Au particles suspended in water, and imaged in the wet-STEM annular
dark-field mode.
such as using lower. This detector is very sensitive at low
energies (0.5 keV) and offers the advantage of TV-rate
imaging.
As a feature of STEM-in-SEM imaging mode, wet-STEM
mode improves contrast and resolution. This technique also
improves volume information in comparison with classical wet
mode in ESEM, where only the surface of liquids can be
observed.
6. Applications of wet-STEM imaging
Using the wet-STEM imaging mode described previously, a
wide variety of nm- to mm-scale objects suspended in a liquid
layer (not only water) can be investigated (Bogner et al., 2005).
The present imaging conditions correspond to annular darkfield mode, using very large collection angles. An acceleration
voltage of 30 kV has been chosen to optimize resolution and
contrast.
An image of aqueous suspension of gold nanoparticles is
presented in Fig. 10. This image highlights the high resolution
of wet-STEM imaging: particles with diameter of 20 nm are
399
well resolved; the resolution is estimated as low as 5 nm. Au
particles exhibit a very high bright contrast, as expected when
considering their high atomic number.
Even objects constituted of lighter elements induce an
observable contrast in wet-STEM using annular dark-field
detection. Fig. 11 presents images of colloidal clay called
‘‘chocolate’’ referring to the colour of the aqueous solution.
Clay platelets typical sizes are 0.1 mm 0.5 mm 2 mm.
These objects can be observed using a cryo-TEM, but exhibit
little contrast when they are disposed as to be imaged through
their thinner thickness. Wet-STEM imaging mode corresponds to a scattering contrast: whether the flattest face of a
platelet is parallel or perpendicular to the grid, the scattering of
electrons will occur in a different local thickness. In wetSTEM, perpendicular platelets show brighter contrast than
others because of the important local thickness acting for the
scattering of electrons, and some lamellar structures are
observed; even ‘‘parallel’’ platelets exhibit a good contrast.
Contrast interpretation is not always trivial, as suspensions
often contain several additives, that may modify the scattering
of electrons, or the sample sensitivity to the electron beam.
Fig. 12 presents carbon nanotubes in two different solutions: (1)
water with two different concentrations of sodiumdodecylsulfate (SDS) surfactant; (2) pure ethanol. Micrometric holes
present in the carbon layer of the grid are observed as dark
regions. In Fig. 12a, carbon nanotubes are well distinguished,
and some brighter contrasts correspond to superpositions, or
heavy atomic number particles resulting from the carbon
nanotubes synthesis. In Fig. 12b, carbon nanotubes are also well
distinguished but the presence of water introduces a fuzziness,
and some bright nm-scale features are present. The latter
objects are understood as surfactant clusters, and are found to
be larger and more numerous in a solution containing more
surfactant: Fig. 12c.
Biological specimens, typically low in atomic number and
sensitive to beam damage and dehydration, have also been
successfully observed in wet-STEM. Fig. 13 presents an image of
bacteria of the species Pseudomonas syringae, acquired at a
magnification of 50 000. The contrast of the bacteria is very
Fig. 11. ‘‘Chocolate’’ colloidal clay in water imaged in wet-STEM annular dark-field imaging mode.
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A. Bogner et al. / Micron 38 (2007) 390–401
Fig. 12. Carbon nanotubes: (a) dispersed in ethanol without surfactant; (b) in water with surfactant at the concentration C1; (c) in water with surfactant at the
concentration C2 > C1.
good without preliminary staining. Moreover, volumic information is obtained as double membrane structure is distinguished.
Others types of samples have been imaged in wet-STEM
(Bogner et al., 2005) such as mini-emulsions, latices,
bacteriophages, particles in oil, etc. In fact, the term ‘‘wet’’
is restrictive in comparison with the effective imaging
possibilities of the wet-STEM imaging mode: provided that
the liquid is compatible with the microscope, a thin layer of
nonaqueous liquids can also be studied, its stability only
depending on its saturated vapour pressure.
7. Summary and outlook
Fig. 13. Bacteria Pseudomonas syringae, imaged in wet-STEM annular darkfield imaging mode.
The history of electron microscopy presented in this article
highlights the extent of SEM applications. SEM is not in
competition with TEM as it allows different imaging modes.
Wet-STEM, i.e. STEM-in-SEM performed in environmental
SEM, has been presented as an powerful imaging technique
developed thanks to general progress in electron microscopy. It
allows straightforward transmission observations of wet
samples constituted of nano-scale objects in a liquid layer.
With the benefits of field emission and STEM mode applied in
SEM, an excellent resolution can be achieved. For example
5 nm was achieved on a resolution test sample of gold
nanoparticles in colloidal suspensions. Thanks to the low
operating voltage of an SEM and large scattering angle
collection, the contrast is enhanced, this result is especially
A. Bogner et al. / Micron 38 (2007) 390–401
interesting for low atomic number materials. Low-vacuum
technology allows imaging samples in their native state. The
scanning mode allows to image samples at a ‘‘low dose’’, that is
important when examining polymers and biological samples.
Finally, the transmission mode gives access to volume
information since that we do not image only the surface of
the liquid drop. Particularly adapted for suspension-type
samples, and others delicate objects with nanometric features,
wet-STEM allows characterizations in materials science as well
as life-science at the nano-scale.
Acknowledgments
We are very grateful to D. Bultreys from FEI Company
(Brussels) for shared experimental sessions and discussions.
We also would like to acknowledge authors for figures reprint
permissions.
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