Review
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Applications of ESEM on Materials Science: Recent
Updates and a Look Forward
Zhouyang Zhang, Yangbo Zhou, Xinli Zhu, Linfeng Fei,* Haitao Huang,* and Yu Wang
The microscopic understanding of materials relies on the development of
microscopy. Historically, the technological evolution from optical micro-
scopes to electron microscopes has directly stimulated many discoveries
of new physical and chemical fundamentals as well as advanced materials.
Scanning electron microscopy (SEM) is, undoubtedly, the most widely used
tool for microscopic characterization in modern materials science. Especially,
the incorporation of a gas pressure around the sample area of conventional
SEM, i.e., the environmental scanning electron microscope (ESEM), has
opened new possibilities in observing samples in their natural states, leading
to unprecedented chances for studying the details of biological, organic,
and hydrated samples. What is more, in recent years, in situ ESEM studies
concerning materials change and chemical reactions have played a more
important role in the field of materials science. Herein, the recent applica-
tions of ESEM in materials science are comprehensively reviewed, in terms of
common static observations for various materials and in situ investigations
of dynamic processes in controlled experimental environments. Moreover,
the problems concerning state-of-the-art ESEM experiments are discussed,
as well as possible solutions. Looking forward, the rapid developments on
instrumentation and fundamental science of ESEM can bring a clear vision of
materials in the near future.
1. Introduction
About 300 years ago, when optical microscope was successfully
invented by Leeuwenhoek for studying the microstructures
of organisms, it is the first time that human was empowered
with the capability to explore unprecedented details of this
Z. Zhang, Prof. Y. Zhou, Prof. L. Fei, Prof. Y. Wang
School of Materials Science and Engineering
Nanchang University
Nanchang, Jiangxi 330031, China
E-mail: flf@ncu.edu.cn
Dr. X. Zhu
Thermal Fisher Scientific
Building 8, 399 Shengxia Road, Zhangjiang Hi-Tech Park
Shanghai 201210, China
Prof. L. Fei, Prof. H. Huang
Department of Applied Physics
The Hong Kong Polytechnic University
Hung Hom, Kowloon, Hong Kong, China
E-mail: aphhuang@polyu.edu.hk
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smtd.201900588.
DOI: 10.1002/smtd.201900588
Small Methods 2020, 4, 1900588 1900588  (1 of 21)
world, which is also an essential step for
understanding the development of new
materials and science. However, due
to the limit of light wavelength, optical
microscope, with the best resolution at
≈200 nm, is evidently not enough for
materials science. Therefore, a new micro-
scope technology with a shorter-wave-
length light source for better resolution is
needed. In 1931, German physicists Ruska
and Knoll fabricated the first transmis-
sion electron microscope (TEM). Although
with a magnification of only 16, it is still
an epoch-making invention, for electrons
was first employed as the light source for
a microscope.[1] Since then, great efforts
have been made to improve the spatial
resolution of electron microscope (EM),
as well as to develop new applications for
such instrumentation. For example, based
on the EM technique, scanning electron
microscope (SEM) was designed later to
obtain microscopic topological informa-
tion of bulk samples. In 1942, Zworykin
constructed the first SEM with a resolu-
tion of ≈50 nm.[1,2] In the design of an
SEM, the requirement of high vacuum for
observation, typically around 10−3 to 10−5  Pa, is needed to focus
the electron beam and minimize the electron scattering through
the column. As a result, an issue was raised in the application
of SEM, i.e., the sample needs to be “pre-treated” to enhance
its surface conductivity to avoid surface charging during the
SEM observation. To explore the “real” surface of a sample in
SEM and for experimental convenience, since 1953, a number
of hardware configurations, including differential pumping
system, aperture-limited system, and thin-film windows, were
proposed to achieve an “environmental chamber” in EM.[2–4] It
was until the late 1980s that the first commercial environmental
SEM (ESEM) was demonstrated by Electroscan Corporation for
observations of wet or insulated samples in their native states
without any surface treatments, whose nominal specimen
chamber pressure could go up to 25 Torr (3300 Pa).[1,5] By far,
the most advanced ESEM can reach a secondary electron image
resolution of ≈1.3 nm at an accelerating voltage of 30 kV, and
the maximum chamber pressure can be up to 4000 Pa.
The ESEM has been defined as an SEM capable of main-
taining a minimum water vapor pressure of at least 609 Pa
in its specimen chamber.[6] Nowadays, depending on the
different vacuum levels in the chamber area, there are usu-
ally three working modes for ESEMs: high vacuum (HiVac)
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mode, low vacuum (LoVac) mode, and the ESEM mode. For
the HiVac mode, the typical chamber pressure is within the Zhouyang Zhang is currently
range of 10−2 to 10−4  Pa, and the operating conditions of an a Ph.D. candidate in the
ESEM in this mode are very much similar to that of a con- School of Materials Science
ventional SEM (CSEM). On the other hand, pressure ranges and Engineering, Nanchang
in LoVac and ESEM modes are 10–130 and 10–4000 Pa, University, under the supervi-
respectively, in the sample area (either the water vapor from a sion of Prof. Linfeng Fei.
built-in water reservoir, up to 2600 Pa, or a gas from an aux- Her current research mainly
iliary gas inlet can be used as the imaging gas for different focuses on in situ obser-
experiments), while the column part is still working at a high vations of dynamic phys-
vacuum of less than 10−2 Pa to generate and focus the electron icochemical processes by
beam. In principle, the LoVac mode is designed for observing electron microscope.
naked (noncoated) and nonconductive specimens, whereas
the ESEM mode is more appropriate for observing natural
samples (contain volatile components). In line with a CSEM,
ESEM has the identical advantages of good field depth, long Linfeng Fei received his B.S.
focal length, and simple preparation procedures for bulk sam- degree from University of
ples. Furthermore, the unique advantage of ESEM is the exist- Science and Technology of
ence of a gas environment in the chamber, so that samples of China, followed by a Ph.D.
any states (wet or dry, conductive or insulated) can be directly degree from The Hong
tested without surface pre-treatment. This “gas environment” Kong Polytechnic University.
usually refers to water vapor for ESEM observation and, there- Afterward, he worked as a
fore, hydrated or “wet” samples can be maintained in their postdoctoral fellow in The
native state. In this perspective, the development of ESEM Hong Kong Polytechnic
has greatly expanded the applications of SEM in materials University and has recently
research.[7,8] moved to Nanchang
Therefore, it is not surprising that hundreds of papers University. His research is
using ESEM as their major research tool are published each focused on in situ observa-
year, which cover almost every corner of modern materials tions of dynamic processes and advanced characteriza-
science (see Figure  1). However, we recently noted that, the tions at the atomic-scale for emerging materials using
latest holistic review of ESEM and its applications was pub- electron microscopy.
lished over 16 years ago.[7] More importantly, as the research
focus has changed a lot during this period, a great amount of
seminal papers have been published with the help of ESEM. Haitao Huang is a professor
For example, Kiselev et al. reported the direct observations of in the Department of Applied
deposition growth of aligned ice crystals on feldspar;[9] the in Physics, The Hong Kong
situ observations performed by Huang et al. demonstrated the Polytechnic University. His
formation of nanowire arrays proceeds via a selective vapori- current research interests
zation process of the bulk crystals;[10] and Toth et al. achieved include electrochemical energy
fabrication and slimming of nanostructure by beam-induced storage and conversion, and
deposition and etching in ESEM.[11] Actually, the total number ferroelectric materials for
of papers is over 3000 in the last 16 years. These significant energy applications. He is
achievements and contributions have inspired us to present currently the board committee
a comprehensive summary of ESEM applications on mate- member of the International
rials science, focusing mainly on the breakthroughs in recent Academy of Electrochemical
years. Energy Science (IAOEES).
Our review will be organized as follows. First, we will
briefly introduce the operation principle and structural con-
figuration of ESEM, together with some functional acces-
sories. Second, we will emphasize the recent developments
in application scenarios of ESEM. These applications will be water, wetting, water transportation, and so on), 2) observa-
categorized into common static observations (ex situ) and in tions on material changes with the participations of water (del-
situ dynamic observations. For static observations, we will iquescence, metallic corrosion, fabrication of nanostructure,
focus on biological hydrated samples (e.g., organisms, cells, hydration process of cements, and so on), and 3) in situ obser-
bacteria, proteins, etc.) and insulated samples (e.g., fossils, vations on other dynamic processes in ESEM (reactions in bat-
cements, skeletons, polymers, etc.). The in situ observations, teries, growth of 2D materials and nanomaterials, crystalliza-
which is the most important application of ESEM nowadays, tion of oxides at high temperature, and so on). Finally, we will
is classified into three parts: 1) observations on phase changes try to give our outlook on both technical perspectives of ESEM
of water (morphological changes during phase transition of and their possible applications in future materials science.

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Figure 1. a) Number of publications identified using the topic word
“ESEM,” showing a marked increase after 2006 and a steady tendency
over the past decade. b) The distribution of publications across different
research areas. The data are acquired from the Web of Science Core Col-
lection (accessed in June 2019).
2. Operational Principle
Generally, there are three main components to ensure the
normal operation of ESEMs, which are the electron source,
lens and scanning system, and detection unit. Table  1 shows
the functions for each component. Meanwhile, to obtain the
“environmental chamber” with the existence of gas, pressure
limiting apertures (PLAs) are used to form a pressure gra-
dient along with the electron beam motion from electron gun
Table 1.  Main components of ESEM.
Component
Electron source The electron beam is emitted within a small spatial volume at a small angular spread and selectable energy.
Lens and scanning system The electron beam enters the lens system consisting of several electromagnetic lenses and exits to hit the specimen surface. The scan-
ning system generated signal, fed to the deflection systems, moves the beam in a raster pattern over the specimen area.
Detection unit Electrons striking the specimen react with its surface, producing three basic types of signal: backscatter electrons, secondary electrons,
and X-rays. The detection system picks up these signals, converts them into an amplified electrical signal which is sent to the control PC
and displayed on the monitor.
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to specimen chamber. As shown in Figure  2a, the final pres-
sure limiting aperture PLA1 separates the specimen chamber
from the rough vacuum region, which is further separated by
aperture PLA2 from the column part where the electron beam
moves in a high vacuum with a high energy. The maximum
pressure allowed in the specimen chamber at LoVac or ESEM
mode is thus influenced by the design of the PLAs.[12] The
gas flows freely along sideways between these two apertures.
The most commonly used gas in ESEM experiments is water
and, of course, other gases (nitrogen, carbon dioxide, nitrogen
dioxide, helium, argon, CxHy, and so on) can also be used as
the imaging gas. Compared with environmental TEM (ETEM)
which provides observations at a higher resolution in gaseous
atmosphere (up to 2000 Pa for N2), ESEM still holds its advan-
tages including an easy sample preparation procedure, a good
depth of field, and a higher permitted gas pressure (which is
extremely important for biological samples). It is worthy of
noticing that the gas not only can offer a gaseous environ-
ment for stabilization of the sample but also play a significant
role in signal amplification for imaging. Considering that the
widely used Everhardt–Thornley detector in CSEM would lead
to arcing in the presence of gas, a special gaseous secondary-
electron detector (GSED) was developed for usage in ESEM.
As shown in Figure 2b, a positive potential of 300–500 V is
applied on the GSED during imaging, and an electric field was
established between specimen and detector to attract those sec-
ondary electrons (SEs) emitted from the specimen after inter-
acted with the incident electron beam. Electron collisions with
gas molecules occur during the acceleration of SEs, causing the
partial ionization of gas molecules and generating additional
electrons (so-called “cascade” of electrons). In this way, signals
obey the cascade amplification process before reaching the
detector.[13] Furthermore, the positive ions resulting from these
cascades can move around the sample surface to compensate
for negative charge build-up of the insulated sample so it does
not need to carry out any conductive treatments on samples in
the ESEM test. Besides, ESEM also supports the backscattered
electron imaging mode beyond the secondary electron imaging
mode.[5]
As indicated above, the insulated samples without surface
preparations can be observed in ESEM under a notable gas
pressure to maintain their native state including the hydrated
state, in contrast to CSEM. Additionally, the specimen chamber
has enough space to guarantee the operation of a range of
functional accessories, as shown in Table  2 (the relevant data
are provided by the Thermal Fisher Scientific). Besides, in spe-
cific experiments, one or more accessories can be used inde-
pendently or simultaneously in ESEM. In fact, the specimen
Function
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Figure 2.  Schematic illustrations of ESEM. a) The optical layout of the ESEM column. b) The cascade amplification process during the imaging of
hydrated/insulated samples.

chamber has become a micro-processing laboratory which
can satisfy various testing requirements by supplying specific
gas environment and combining with functional accessories.
Hence, it is now available for scientists to accomplish a series
of in situ observations on dynamic processes with rational
experimental designs, by controlling specific gas, variable tem-
peratures, variable pressures, variable humidity, and so on.
3. Recent Applications
3.1. Common Static Observations
As mentioned above, there are generally three working modes
in ESEMs (HiVac, LoVac, and ESEM modes). When samples
are to be observed under ESEMs at HiVac mode, they must
be vacuum-friendly (no volatile components) and electrically
conductive (especially the surfaces). Samples as biological
materials, food, cells, etc., which contain water and oils that
can volatilize at a certain vacuum degree and may break the
vacuum as well as contaminate the electron source, are not
allowed to be observed unless they have gone through a series
of pre-treatments (chemical fixing, dehydration, freeze-drying,
and so on). Additionally, in order to enhance the surface con-
ductivity, less conductive or insulated samples must be treated
via sophisticated procedures (to be coated with a thin layer of
conductive metallic material such as gold, platinum, palla-
dium, and chromium, etc.). The subsequent observation can
be very similar to that in CSEM. However, the aforementioned
“treatment” may usually interfere with scientific judgments on

Table 2.  Common functional accessories for ESEM.

Accessory Function Applications Examples

EDS Analyze elemental composition for microstructures Qualitative/quantitative distribution of elements across [14–18]
isolated/“wet” surfaces for archaeology/geology/material
science
EBSD Characterize the crystallography Obtain quantitative crystallographic (grain size, domains [19–22]
orientation, etc.) and microstructural information
STEM Extract information on internal structure of samples by trans- Observation on nanoscale objects in complex environment [1,23–25]
mission imaging (liquid cells, etc.)
Gas supply system Provide low pressure gases in sample chamber (usually water Achieve specific experimental environment for [26–29]
vapor, also including H2, O2, CO2, N2, NH3, and so on) observations
Cooling stage Control the sample temperature around the ambient (usually Accurately cool samples for observing hydrated samples, [30–33]
from −25 to +55 °C) and performing in situ experiments at low temperature
Heating stage Control the sample temperature up to > 1000 °C In situ heating observations (metallic corrosion, ceramic [34–36]
crystallization, etc.)
Mechanical test system Apply static/dynamic compress/tensile stresses on samples Achieve in situ observations on samples during mechan- [37–40]
ical process
Homemade accessories Satisfy specific requirements for individual experiment Nanorobotic system, etc. [41,42]

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Figure 3.  ESEM images of biological materials under water vapor. a) The surface of a raw eggshell is composed of beads and pits on the intact cuticular
colloid layer. The inset is the CSEM image in which the cuticular colloid layer cannot be found. Reproduced with permission.[48] Copyright 2011, Springer
Nature. b) The structures of spider silk. 1,2) Structures of the dry spider silk. 1) Low-magnification image of periodic puffs and joints surrounding two
main-axis fibers. 2) Magnified image of puff composed of countless nanofibers. 3) Periodic spindle-knots structure linking with slender joints of spider
silk after wet-rebuilding. 4) Magnified image of random nanofibers on the spindle-knot. 5) Magnified image of aligned nanofibers on the joint region.
Reproduced with permission.[50] Copyright 2010, Springer Nature. c) A cell captured on the hierarchical nanowire structures with both horizontal and
vertical nanowire branches of ITO nanowire array. The structures make the substrate topographically match better with cell filopodia and provides
more binding sites for cell capture. Reproduced with permission.[56] Copyright 2016, American Chemical Society. d) ESEM cross-sectional image of
pork biceps femoris muscle of raw meat. Reproduced with permission.[71] Copyright 2012, Institute of Food Technologists.

surface features because the damage or cover of structural and
chemical details may be likely generated on the native sample
surface during manual handling. Moreover, longtime prepara-
tion from sampling to fixation, dehydration, drying, and metal
coating can raise the risk of introducing various undesirable
artifacts. In addition, samples through coating treatments could
induce the loss of valuable compositional contrast and give only
a morphologic contrast. Thanks to the development of ESEM,
samples with no prior preparation could be directly imaged and
analyzed in its environmental chamber with the existence of
a certain atmosphere.[43] ESEM can be used to image samples
(damp, filthy, or greasy), and contaminations (from the sample)
do not damage the instrument and do not reduce the quality
of imaging. Insulators can also be well observed without any
coating layer. In this review, except for those observations in
HiVac,[44–47] we focus on observations of samples in the gaseous
modes (LoVac or ESEM). Note that in most ESEM experiments,
water vapor is used as the background gas.
Up to now, there is no doubt that biological samples account
for a large proportion in the application of ESEM. Due to the
intrinsic advantage of ESEM, direct characterization of bio-
logical samples which usually contain something volatile (like
water), including organisms, microorganisms, and biomole­
cules, is allowed. The comparative study of ESEM and CSEM
on biological samples suggested that, ESEM observations
can provide new information and data for understanding the
morphology and structure. For example, as shown in Figure 3a,
Zhou et al. used ESEM to observe the colloid membrane of raw
eggshell and found that there are some roughness of beads and
pits on the amorphous colloid layer, which cannot be observed
under CSEM because the layer was destroyed during sam-
pling. Compared to CSEM image, the ESEM results, appar-
ently, give more details of the raw eggshell.[48] Of course, other
natural organism samples, from either plants or animals, are
also very common in ESEM study, such as spider silks,[49,50]
leaves,[51] insects/mites,[52] etc. Zheng et al. investigated the
relationship between the microstructures of spider silk and
its directional water-collecting ability from humid air. The
structures of dry and wetted silk were observed by ESEM at
an accelerating voltage of ≈4 kV. The structures of dry spider

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silk (Figure 3b-1,2) were composed of two main-axis fibers
covered by joints and periodic puffs of countless highly hydro-
philic nanofibers. The spider silk showed a directional water-
collecting ability when it was placed in humid environment.
The nanofibers could generate a unique fiber structure after
wetting, and the process was called structural wet-rebuilding.
Under condensed water droplets, the puffs were made to
shrink into periodic spindle-knots and joints (Figure 3b-3).
The periodic spindle-knots were made of random nanofibrils
and separated by joints which were made of aligned nanofibers
(Figure 3b-4,5). These ESEM results clarified the mechanism of
directional water collection behavior of spider silk which relied
on the changes of its microstructures.[50] Similarly, in order to
analyze the morphology of lotus leaf surface under different
humid conditions, Zheng et al. used ESEM to carry out the
observation under both low and high relative humidity (RH).
The observation under low RH of ≈70% showed that the leaf
surface is composed of numbers of papillae which were cov-
ered with countless nanohairs. The observation under high RH
(≈100%) revealed that some thin layers of liquid are on the tops
of papillae and accompanied by liquid immerging in the inter-
spaces among nanohairs, which indicated that wettability gra-
dient is formed along the exterior surface of papilla and the top
might be a more wettable region.[51]
Besides, for investigation of microorganisms and biomacro-
molecules, such as cells,[53–59] bacteria,[60–64] and proteins,[65–67]
it is more appropriate to image these samples with an ESEM
than a CSEM. In order to improve the imaging quality, these
samples sometimes are treated through a simple processing
before observation, e.g., fixing and drying, which is still sim-
pler than the preparation for CSEM. Zhang et al. discussed
the effect of hierarchical structures of indium tin oxide (ITO)
nanowire arrays on their cell-capturing ability using ESEM.
To observe morphologies of cells captured on anti-EpCAM-
coated different substrates, the cells were fixed with glutar-
aldehyde (2.5% in phosphate-buffered saline) for more than
4 h at room temperature after the cell-capture experiments. The
substrates were immersed in a series of alcohol aqueous solu-
tion with different volume fractions (0, 30%, 50%, 75%, 85%,
95%, and 100%) in sequence before drying in carbon dioxide
under supercritical condition. Based on the ESEM observa-
tions at low vacuum (93 Pa), they found that these arrays with
both horizontal and vertical nanowire branches, which could be
regarded as a new 3D fractal nanobiointerface, could enhance
the efficient capture of cancer cells, as shown in Figure 3c.[56]
Figure 4.  ESEM images of material systems with nonconductive surfaces in water vapor. a) The morphology of pH-sensitive water-soluble imprinted
hydrogels under pH = 2.0. Reproduced with permission.[77] Copyright 2010, Wiley–VCH. b) Microstriations indicative of cutting with a stone tool
on fossil rib bone. The direction of the rib head is indicated by the black arrows. Reproduced with permission.[81] Copyright 2010, Springer Nature.
c) Microstructures of virgin bitumen. Reproduced with permission.[100] Copyright 2017, Taylor & Francis.
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Also, Pollitt et al. observed the structure of the Staphylococcus
aureus comets in ESEM considering that ESEM could enable
the observation of the comets structure to perform at a high
magnification and without disrupting the comet structure. So
that the ESEM data could be a convictive information to com-
plete their work with the light microscope.[61]
For food systems, such as potato,[68] chocolate,[69] apple,[70]
and pork,[71] ESEM could also facilitate the microstructural
analysis, so as to contribute to the development of food science.
These samples have complex surface topologies as well as water
inside, and ESEM observation is a convenient way to discover
their native structures. As shown in Figure 3d, this microscopic
technique has been successfully employed to characterize the
microstructures of pork muscle. In order to investigate the effect
of high pressure and salt on pork meat quality, Duranton et al.
imaged the pork under at a low temperature (3.4–3.9 °C), with
84% to 86% RH and a pressure of 650 to 680 Pa in the ESEM
chamber to prevent dehydration of the samples. The observa-
tions show that the negative effects of texture and water holding
capacity on pressure-treated/cooked pork were caused by the
general shrinkage of the structure, and the addition of 1.5% salt
could counteract these negative effects by inducing the break-
down of structure.[71] The research shows that the integrity of the
muscle after treatment can be evaluated by ESEM which repre-
sents a new development in the field of meat quality assessment.
Furthermore, for deformable or “soft” particles, while the
samples must endure tedious preparation of drying, fixing, and
coating to maintain their “original” morphologies in CSEM obser-
vations, it is very suitable for them to be observed under ESEM
for imaging their “real” morphologies. Polymer particles, as the
classic representative of these soft particles, can usually shrink
or swell in response to environmental conditions such as tem-
perature, pH, salt concentration, light, solvent composition, etc.
Many studies related to polymers have been done with the help
of ESEM.[72–78] Figure 4a shows the morphology of pH-sensitive
water-soluble imprinted hydrogels under pH = 2.0, the hydrogel
is in a largely swollen state under acidic conditions as compared
with its relatively compact state. The particles containing water
were directly mounted without drying on metal stubs and meas-
ured under a low vacuum (≈10-3 atm.) and relatively low tem-
perature (near 4 °C in ESEM to obtain this snapshot.[77]
Additionally, in archaeological research, high-resolu-
tion images on morphologies of precious fossils should be
preferentially captured by ESEM, so as to prevent the native
surface from damage by either the conductive coating or the
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high vacuum environment in CSEM. Moreover, in order to
obtain the elemental distribution on the natural (“real”) sur-
faces which have not been destroyed or covered by sample
preparation, energy dispersive X-ray spectrometer (EDS) can
be also integrated in ESEM for those electrically insulated
samples. However, the EDS analysis must be performed at the
lowest possible chamber pressure, considering the formation of
the “skirt electrons” caused by the interaction between the elec-
tron beam and the chamber gas.[4] In this case, the information
of shape/arrangement of microstructures and element distribu-
tions on fossils can be extracted by ESEM so that to provide
paleogeographic environmental data of stratigraphic formation
for determining geological age and stratigraphic correlation.
Many studies about fossils based on ESEM have hence made
significant contributions to cultural heritage conservation.[79–83]
For example, McPherron et al. observed the fossilized bones
under ESEM and found unambiguous stone-tool cut marks for
flesh removal and percussion marks for marrow access. This
discovery of bones was the oldest direct evidence of stone tool
manufacture. Figure 4b shows microstriations indicative of
cutting with a stone tool on the fossil surface in ESEM under
water vapor.[81] Also, Rybczynski et al. performed the mineral
analysis on the unprepared fossil camel bone using an ESEM
equipped with an energy dispersive spectrometer. The results
provide geochemical details of preservation by the analysis of
surface morphology and chemistry.[83] Similarly, ESEM provides
a nondestructive examination of surface morphology observa-
tion and compositional contrast on various other samples, such
as rocks and minerals,[84–87] skeletons,[88–90] teeth,[91–94] etc.
One more thing, for building materials (such as cements
and bitumen), it is difficult to handle the bulk samples as they
are always insulated and may contain volatile contaminants.
As mentioned above, in ESEM, the SEs collide with the gas
to produce a large number of positive ions and electrons, and
the positive ions move on the sample surface and interact rap-
idly with the accumulated electrons. Thus, the phenomena
of charge and discharge can be eliminated in ESEM and
provide a chance for a direct observation on these materials
(cements[95–97] and bitumen[98–100]). Ye et al. characterized the
morphological changes of the pore structures during cement
hydration process which was comparable with the stimulation
results. They concluded that both ESEM technique and ste-
reology theory in the study of cement-based materials on the
relationship between the microstructure and their macroscopic
properties can contribute to the understanding and designing
cement materials with improved performance.[95] Also,
Mikhailenko et al. studied the effects of oxidation, blending,
and freezing/heating on bitumen fibril structure. The obser-
vation was carried out at accelerating voltage of 20 kV and a
chamber pressure of 0.8 mbar. Figure 4c shows the micro-
structure of virgin bitumen, where the fibril diameter was
relatively large at around 15–20 µm, and the structure itself
was relatively loose and sparse. Then they compared the rela-
tive density, organization, and fibril size of this sample with
other structures after treatments and the results showed the
potential use of ESEM for the observation of bitumen micro-
structure.[100] These ESEM studies advance the understanding
of the building materials and contribute to the development in
practical applications.
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3.2. In Situ Observations
In recent years, in situ analysis has become an important
research direction in the field of materials science, i.e., to observe
and record dynamic physicochemical processes of materials in
real-time, so as to reflect more “process” information rather than
traditional static observations either from “start point” or “end
point.” The aim of in situ analysis is to clarify the mechanisms
of interactions between materials and the environment that have
not been clearly understood in the past. The in situ monitoring
of dynamic processes, including in situ spectrum and in situ
imaging, has gradually replaced the post-mortem analysis or
static observation and been applied more and more widely.[101–103]
Of course, in situ studies in materials research based on ESEM
were also remarkably expanded in the past few years.
The main purpose of sample preparation in CSEM imaging
is to fix the sample to ensure that it does not change during
the vacuum process. In this regard, however, one of the con-
sequences of those preparations is that it is almost impossible
to perform any in situ observations under CSEM. Thanks to
the development of ESEM, the directly exposed sample sur-
face (without coating) makes it possible for various potential
applications of in situ observations on the interaction between
the sample and various environments, especially in case of the
availability of those functional accessories (see Table 2). Actu-
ally, the chamber of ESEM armed with multifarious functional
accessories can now be looked as an independent micro-
laboratory where the samples can be processed in precisely
controllable experimental conditions (including temperature,
gas environment, humidity, etc.) throughout the observations.
Consequently, in situ observations based on dedicated ESEMs
have played an important role in revealing the relevant truths
during dynamic physical–chemical processes in materials sci-
ence, such as material growth, chemical reaction, phase trans-
formation, dissolution, crystallization, adsorption, humidifica-
tion, dehydration, corrosion, etching, etc.
Considering that the most significant part of ESEM is the
chamber with a controllable gas environment, and the most
common gas being used in ESEM experiment is water vapor,
the following discussions on in situ ESEM observations will be
categorized into three sections, roughly according to whether
or how the water interacts with the samples during the whole
dynamic process.
3.2.1. Phase Transitions of Water
Water is considered as a good imaging gas in the ESEM with a
built-in water reservoir. Meanwhile, it is also the most common
and important substance in nature, which participates in every
aspect of people’s daily life. Moreover, because of its unique
thermal properties, a large number of ESEM studies focused on
the phase changes of water. Water normally has three phases of
solid, liquid, and gas, and phase transitions could occur under
certain conditions (temperature, pressure, etc.). Therefore,
dynamic processes related to the physical phase changes of
water under various circumstances can be directly visualized
under ESEM to provide detailed evidence to the mechanism of
these phase transitions.
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Apparently, the configuration of ESEM makes it feasible to
design in situ experiments of phase transitions of water, in
case of the rational controlling over the chamber temperature
and pressure. For example, the icing process of water and the
melting process of ice, which can provide the fundamentals
for phase transitions of water, are frequently observed and
examined by in situ ESEM experiments in terms of structural
and morphological changes.[9,104–106] Kiselev et al. investigated
the nucleation and growth of aligned ice crystals from water
vapor on feldspar (an atmospherically important component of
mineral dust) under ESEM to clarify the dominate reason in
regulating the ice-nucleation properties on specific substrates.
The observation was conducted in an ESEM with a double-stage
Peltier element, with its low temperature limit down to 213 K.
The results gave a direct experimental evidence to indicate that
the active sites for ice-nucleation on the surface of feldspar
are the surface patches with (100) crystallographic orientation.
These patches occurred exclusively at surface steps, cracks, and
cavities, which are thought to be responsible for the prefer-
ential nucleation of ice. As shown in Figure  5, the ice crystal

Figure 5.  Dynamic ESEM observation of gas–solid phase transition of water at 251 K. a–e) An ice crystal nucleated on the patch with (100) orienta-
tion of feldspar. The patch was thought as an ice-nucleating active site which was on the inner wall of a cavity in the (010) face of the specimen. The
relative orientation of crystal planes is shown in yellow, with (010) facing up. f) Schematic drawing of ice crystals nucleating from their (0110) planes
on the steps with (100) orientation [red filled surface and the hidden face of the step on the (010) face of feldspar; the relative position of ice crystals
to each other and with respect to crystal faces of feldspar specimen corresponds to the case shown in (e)]. Reproduced with permission.[9] Copyright
2017, American Association for the Advancement of Science (AAAS).

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nucleated with its primary prism plane (1010) on the (100)

crystallographic plane of feldspar, and the ice always seemed
to grow at defect sites (as ice-nucleation active sites) where
(100) patches may be exposed.[9] This research shows that the
strict experimental conditions of water vapor pressure and tem-
perature could be readily controlled in ESEM to provide direct
and visual investigations on the reaction process, particularly
in those studies related to phase transitions of water on the
fundamental issues of atmospheric and environmental sci-
ence. Recently, Zhang et al. also recorded the process of direct
ice formation from a supersaturated water vapor environment
under ESEM.[106] They revealed a step-by-step growth pathway
of hexagonal ice crystals (with the steps coming from screw dis-
locations and initial steps), and demonstrated the quantitative
relationship between the edge-length (of hexagonal ice crystals)
and the growth time. On the other hand, dynamic observations
on ice melting process can also be performed with the help of
ESEM. Yang et al. obtained the first series of in situ dynamic
micrographs during evaporation of frost flowers with the exist-
ence of NaCl in both temporal and spatial dimensions. The
chamber temperature ranged from −5 to −18 °C and the pres-
sure was controlled above 500 Pa. Here, the major heat source
for ice melting was the energy from the scanned electrons.
Their results well explained several previous observations: the
frost flowers were not a direct source of sea-salt aerosols and
the saline ice crystals under evaporation could accelerate the
heterogenous reactions of bromine liberation.[104] With the help
of ESEM, these fundamental studies exhibit potential signifi-
cance in a variety of application scenarios, including the prepa-
ration of advanced materials (e.g., anti-icing materials[107]), the
solutions for practical problems (e.g., elimination of freezing-
induced disasters[108]), and a thorough understanding of atmos-
pheric science.[109]
On the other side, the gas–liquid phase transition of water,
namely, condensation and evaporation, can also be realized
and observed in situ in ESEM. Interestingly, this transition
was always characterized to reveal the properties of other mate-
rials. For instance, wettability, which is a common liquid–solid
interaction, is related to microstructural features of materials
surfaces. Therefore, many work based on ESEM have demo­
nstrated the relationships between surface microstructures
and wettability by observing the evolution of water droplets
on the material surface during condensation, aiming to obtain
a comprehensive evaluation of surface wetting of the mate-
rial rather than traditional contact angle measurement.[110–116]
For example, Cheng et al. reported an in situ observation of
water condensation and evaporation on lotus leaf surfaces to
reveal the evolution of contact angle and droplet size during
the process.[112] Moreover, such in situ observations can show
a further real-time kinetic information of the wetting pro- Figure 6.  In situ observation of gas–liquid phase transition of water at
a water vapor pressure P  = 1200 ± 12 Pa and substrate temperature
cesses. Miljkovic et al. investigated in situ water condensation
TS  = 9 ± 1.5 °C. a–h) Time lapse images of both partially wetted (PW)
on superhydrophobic nanostructured surfaces using ESEM. and suspended (S) droplets during water condensation which show the
Droplet growth on the surfaces was characterized at a water difference in growth behaviors including droplet morphology and growth
vapor pressure P  = 1200 ± 12 Pa and substrate temperature rate. Droplets A, B, C, and D are in the PW state, whereas E and F are in
TS  = 9 ± 1.5 °C with backscattered detection mode. As shown the S state. The scale bar in (h) also applies to (a–g). Reproduced with
in Figure 6, they observed the changes of morphologies of dif- permission.[114] Copyright 2012, American Chemical Society.
ferent condensed droplets, including suspended droplets on top
of the nanostructures and partially wetted droplets on the base than that of suspended ones. Based on the ESEM observations,
of the nanostructures. Consequently, they found initial droplet the growth dynamics of droplets were discussed and a droplet
growth rates of partially wetted droplets were six times larger growth model was developed to explain the experimental

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Figure 7.  ESEM observation on nucleation of dew on a moss awn surface at 4 °C. Time and pressure are increased from (a) to (f). a–c) The pressure in
chamber increases from 706.6 to 879.9 Pa and the water vapor pressure of water is 813.5 Pa at this temperature. The nanogrooves (purple arrowheads)
are initially filled by nucleated water. d–f) The vapor pressure is slowly increased, and the developing thin water film covers the nanogrooves (purple
arrowheads) and fills the microgrooves (yellow arrowheads) of the awn. The scale bar in (a) also applies to (b–f). Reproduced with permission.[118]
Copyright 2016, Springer Nature.

observations.[114] Recently, Zhang et al. demonstrated in situ
ESEM observation, as a straightforward pathway, to under-
stand the wetting behavior on adaxial and abaxial surfaces of a
bamboo leaf. They microscopically and quantitatively identified
the surface-structure-dependent water droplet growth dynamics
of the bio-related materials and provided the evidence that the
ESEM observation is a convenient and efficient tool for quanti-
tative and qualitative study of this phase transition.[117]
It must be mentioned that, specific surfaces/structures/
materials, which are designed for collecting water from air,
could be easily characterized on in situ ESEM observations. For
example, plants grown in desert usually have unique leaf struc-
tures, which are adapted to collecting water by promoting water
droplet nucleation from humid air. Pan et al. investigated the
leaves of desert mosses, which are featured with ample awns
and can collect water rapidly in humid environment (such as
dew, fog, mist, and rain). Nucleation of dew on the surface of
a moss awn at 4 °C was observed by ESEM, while the pres-
sure in ESEM chamber was controlled to simulate the different
levels of RH. Figure  7 shows their observation of nucleation
process with respect to the increasing vapor pressure and time
in ESEM. First, water molecules were condensed on the nano-
grooves within a microgroove or a shallow cavity on the leaf sur-
face (Figure 7c). After a period, more water was appeared in the
nanogrooves and eventually filled the microgrooves (Figure 7d–
f). Moreover, based on further ESEM observations, they also
revealed the mechanisms of water collection from limited water
sources and transportation on the mosses, and presented the
physics-based evidence of how bryophytes adapt to scarce water
environments by efficiently using their small structures.[118]
Besides, morphological or structural changes of materials
caused by gas–liquid transition of water can also be character-
ized using in situ ESEM observations. The volume of hygro-
scopic materials, especially cellular materials, usually swell
after absorbing water. The structural changes of these samples
against the changes of humidity or processes of water conden-
sation/evaporation can, of course, be studied by in situ obser-
vations in the ESEM chamber.[119–124] Ha et al. investigated the

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Figure 8.  ESEM observations of morphological changes on the mate-
rials during the water condensation. a) Merging of micropores due
to hygroscopic expansion of the cellulose sheet. The pores start to
grow when the RH exceeds 90%, and they coalesce with their neigh-
bors. The scale bar in (1) also applies to (2) and (3). Reproduced with
permission.[123] Copyright 2018, AAAS. b) A film at different humidity
levels. 1) The initial film thickness of 2.9 µm at 0% and 10% RH and
2) the film thickness of 18.6 µm at 100% RH. The scale bar in (1)
also applies to (2). Reproduced with permission.[120] Copyright 2007,
Springer Nature.
evolution of microscale wall pores in hygroscopically responsive
multiscale porous materials during the wetting process by in
situ observation under ESEM. As shown in Figure 8a, when the
RH was increased by suppling water vapor to the chamber at
2 °C, the sponge underwent drastic shape changed by absorbing
water molecules as the environmental humidity reached approx-
imately 90%. They found that the deformation underwent two
steps, which were the growth of pore size and the coalescence
of the expanded pores. On the contrary, the deformation of
sponge was insignificant when RH was below 90%, indicating
that water molecules hardly infiltrated into the sponge until the
vapor pressure reaches a critical value.[123] Similarly, Kang et al.
directly imaged the swelling of a film by ESEM to study the
effect of humidity on swelling behavior. Figure 8b shows such
a swelling of the film. The thickness of film increased from an
initial value of 2.9 to 18.6 µm as the RH increased form 0%
to 100%.[120] Hiranuma et al. also determined the hygroscopic
properties of agricultural aerosols in ESEM, which was used to
acquire not only a qualitative description of the shape and mor-
phology but also the detailed images of samples as a function
of RH. They found that most of the agricultural particles did
not take up significant amounts of water when exposed up to
96% RH.[121] These observations demonstrated that ESEM is a
simple yet powerful tool to characterize the material response to
water or other liquids. It has a wide significance in the study of
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Figure 9.  ESEM observation of micro-transportation behavior based on
the gas–liquid phase transition of water. a) Condensation of water at
the closed end of the tube with two bubbles trapped inside the liquid.
b–f) Dynamic behavior of a water plug close to the open end of the tube.
The meniscus shape changes when, at a constant stage temperature, the
vapor pressure of water in the chamber is changed: b) 5.5 Torr, c) 5.8 Torr,
d) 6.0 Torr, e) 5.8 Torr, and f) 5.7 Torr, where the meniscus returns to
the shape seen in (b). The shapes of the meniscus are asymmetrical,
especially the complex shape of the meniscus on the right side in
(b) and (f). Reproduced with permission.[125] Copyright 2004, American
Chemical Society.
water/humidity-sensitive materials and will make further con-
tributions to biological and medical fields.
In addition, water micro-transportation, which is very impor-
tant in the development of nanofluidic devices, can also be
well defined using in situ observations on gas–liquid transi-
tion. Rossi et al. investigated the condensation, evaporation,
and transportation process of water inside carbon nanotubes
(CNTs) by in situ fluidic experiment in ESEM. They observed
complex meniscus shapes and slow liquid dynamics caused by
water confinement and strong interaction with the hydrophilic
tube walls. Due to the heating effect of electron beam on the
liquid inclusions, complex interfaces form between the liquid
and the gas inside the nanotube, which move under the action
of bubbles formed in the bulk liquid (Figure  9a). Figure 9b–f
shows the dynamic behavior in meniscus (shape and size) of
water plug in response to varied pressures at a constant tem-
perature of 4 °C. The contact angle (formed between water and
the CNT) was approximately 5°–20° (Figure 9b). The shape of
menisci within the CNT behaved asymmetric on both sides,
especially for the complex shape of the meniscus on the right
side in Figure 9b,f (which was caused by different vapor pres-
sures on different sides of water plug, resulting from the closed
right end of the tube and presence of liquid at the tube tip in
contrast with the open left end of the tube (Figure 9a)).[125] This
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work demonstrates that ESEM has a potential application for in

situ fluidic experiments at the nanoscale, by using the ability
to see through the nanotube walls, and to study the liquid flow,
condensation, and evaporation inside these nanotubes.

3.2.2. Material Changes with the Participations of Water

As reviewed above, water itself is so important in materials sci-

ence and engineering that its three-phase transitions have been
frequently highlighted in literatures. However, in many circum-
stances water can also influence, or even participate in physico-
chemical processes of other materials, causing notable changes
in morphology, structure, or property of these materials. Plenty
of examples can be found across the fields of biology, materials
science, and chemical industry. Therefore, by adopting similar
experimental protocols to those in the above part, people can
also design and conduct in situ ESEM experiments concerning
dynamic processes of advanced materials (even micro- or nano-
materials), especially in the cases that water can exist in the
environment (act as both imaging gas and reactant in these
processes). Actually, a large number of achievements have been
done using ESEM as a tool to observe relevant processes of
materials changes with the participation of water, and we will
summarize them in this section.
First of all, many chemical reactions induced by water
molecules, and the corresponding changes in microstructures
of materials have been investigated by in situ ESEM, which
mainly include metal corrosion,[126–129] hydration process of
building materials (cement),[130,131] and decomposition/deli-
quescence/degradation of materials.[132–136] For example, Chen
et al. performed in situ ESEM observation aiming to charac-
terize the process of hydrogen generation around micro-drop-
lets on as-cast AZ91 magnesium alloy in an atmosphere of
water vapor.[128] Jonsson et al. investigated the oxidation of iron
Figure 10.  Applications of ESEM in imaging material chemical changes
under three O2/H2O mixtures (water vapor, wet air, and dry air)
with the participations of water. a) Plan-view images of iron surface upon
at 500 °C using ESEM in secondary electron mode to under- exposing to water vapor. The experiment was carried out at 500 °C in low
stand the effect of water on the oxidation. Figure 10a shows the vacuum level (2.5 Torr). (1) and (2) show the surface morphology after
development of surface morphology of iron in water vapor after 17 and 24 min exposure, respectively. The black dotted line represents the
17 and 24 min. It can be seen that both thin and thick oxide boundary between the two regions with different thickness. Reproduced
regions are created. The thin oxide region has much smoother with permission.[126] Copyright 2009, Elsevier. b) In situ monitoring of
the hydration process of K-PS geopolymer cement with ESEM, (1) and
surface with less faceted oxide grains (Figure 10a-1), and more
(2) show microstructures after 30 min and 1.5 h of hydration, respectively.
faceted oxide grains can be seen after longer time exposure Reproduced with permission.[130] Copyright 2004, Elsevier. c) Dissolution
(Figure 10a-2). In combination with further in situ observa- and melting of the chrysotile fibers in the presence of low melting glass
tions under dry and wet air, different oxide growth modes were with water vapor as the chamber gas (2.5–3.6 Torr), recorded at a tem-
observed under different water contents.[126] In addition, Rossil perature of 1) 155 °C, 2) 630 °C, 3) 691 °C, and 4) 750 °C. Reproduced
et al. monitored the initial stages of the corrosion process (pro- with permission.[133] Copyright 2008, Elsevier.
moted by sodium chloride solution) occurred on zinc and mag-
nesium surfaces using ESEM. The water vapor pressure was paste from 10 min to 4 h after mixing have been recorded during
changed in ESEM chamber (and consequently, the RH was also the experiment. Figure 10b shows the detailed information in
changed), so as to obtain direct information of corrosion pro- this process. In the hydration, sponge-like amorphous gels were
cesses. By further conduction of ESEM experiments under the gradually generated and filled the voids among the metakaolin
humidostatic chamber, the authors demonstrated that in situ particles, and hence the microstructure of K-PS geopolymer
ESEM technology could indeed simulate the corrosion behav- cement paste became more and more compacted.[130] Simi-
iors (as that occurs in traditional accelerated tests).[129] larly, Zhang et al. observed the paste microstructure at different
Sun et al. carried out another in situ study on the hydration hydration periods in ESEM to explain the modification mecha-
process of K-PS geopolymer cement under 4.2 Torr, 80% RH nism of epoxy resin on cement-based materials.[131]
environment in ESEM with an accelerating voltage of 20 kV. The The hydration capabilities of the ESEM can also contribute
morphological changes of the fixed location in K-PS geopolymer to the study of the mechanism of discoloring interactions

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between paint pigments. White et al. investigated the interac-
tion (between cadmium yellow and malachite pigments) in
aqueous media and the in situ imaging showed the growth of
the discoloration along the interface.[132] What is more, in situ
ESEM analysis has also been a powerful tool in studying the
dehydroxylation of chrysotile present in cement-asbestos and
the subsequent recrystallization into nonhazardous minerals.
Gualtieri et al. studied the effect of gas type (He and water
vapor) on the kinetics of thermal transformation of chrysotile
fibers using an ESEM equipped with a 1500 °C hot stage. The
experiments were carried out at 20 kV acceleration voltage and
a working distance of about 20 mm. The comparative obser-
vations under different atmospheres were performed in He
pressure (1.9−3.5 Torr) at 25–1150 °C and water vapor pres-
sure (2.5−3.4 Torr) at 25–1300 °C, respectively. The secondary
electron images provided a direct evidence of the negative effect
of water vapor on the dehydroxylation of chrysotile. Moreover,
the decomposition of cement-asbestos with the addition of a
low melting glass was further investigated by in situ observa-
tion at relatively low temperature (600−750  °C). As shown in
Figure 10c, the images demonstrated the use of a low melting
glass may greatly decrease the melting temperature of chryso-
tile as a result of rapid decomposition inside the glassy phase
induced by unstable contact of the asbestos fibers with B- and
Si-rich forming glass.[133]
It should be noted that, the incident electron beam and water
gas in the ESEM chamber have chances to promote interesting
changes of samples, especially during the investigations on
sensitive nanomaterials and 2D materials. Toth et al. realized
the fabrication of functional nanostructures and slimming pro-
cess by electron beam-induced deposition (EBID) and etching
(EBIE) in ESEM, respectively. By overcoming the charging and
delocalized deposition issues in CSEM (or focused ion beam,
etc.), high-resolution nanostructures were produced. First, car-
bonaceous structures deposited on bulk SiO2 was constructed
by EBID using a mixture of styrene and N2. Then, slimming
of the EBID structures can be done by EBIE using H2O pre-
cursor. During EBIE process, the working pressure (P), acceler-
ating voltage (V0), current (I0), and dwell time (tD) were in the
range of 60–81 Pa, 10–30 kV, 77–446 pA, and 3–6 µm pixel−1,
respectively. As shown in Figure  11, there are two types of
etching methods regarding the different EBID structures. For
the EBID nanowires, Figure 11a shows the slimming process
of them where the decrease in nanowire width is largely driven
by secondary electrons emitted from the wire sidewalls during
imaging. For EBID film, Figure 11b shows an alternative slim-
ming process. The structures can be controlled by pre-etching
pits into deposits by growth of these pits in a H2O environ-
ment due to EBIE driven by electrons emitted from the pit
sidewalls.[11] The process of sequential EBID–EBIE nanofabri-
cation methodology based on the ESEM provides an inspiring
insight of decreasing the minimum dimensions of structures
or removal of thin films produced unintentionally during EBID.
Additionally, it is also very practical to study the degradation
behavior of 2D materials in ESEM (using simulated environ-
ment), as these materials are usually sensitive to the environ-
ment (water, temperature, oxygen, etc.). Walia et al. used ESEM
to monitor the degradation rate of few-layered black phos-
phorous (BP) in real time while they were exposed to a small
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Figure 11.  Applications of ESEM in slimming process on the nanostruc­
tures by using H2O precursor under the electron beam irradiation.
a) Carbonaceous nanowires fabricated by EBID on a bulk SiO2 substrate.
1,2) Two frames of a H2O etch process used to slim the wire in (1) (depo-
sition, t  = 60 s, I0  = 400 pA; etch, P  = 81 Pa, V0  = 15 kV, I0  = 77 pA,
tD = 6 µm pixel−1). 3–5) Similar nanowires slimmed to produce diameters
in the range of 13–17 nm. b) A carbonaceous film and a nanowire fabricated
by EBID on a SiO2 substrate. 1–3) Pits pre-etched into the carbonaceous
film using 1,2) a scanned and 3) a stationary electron beam. Reproduced
with permission.[11] Copyright 2007, American Chemical Society.
amount (≈8 Pa) of H2O vapor. Two types of samples (pristine
BP and ionic liquid-treated BP flakes including [BMIM][PF6]-
treated BP and [BMIM][BF4]-treated BP flakes) on the SiO2/Si
substrate were imaged using an electron beam of 20 kV and
1.35 nA, with a scan rate of 3.36 ms per line and 1452 lines
per frame. The background mechanism of this process is, obvi-
ously, a typical chemical etching of BP flakes, resulting from
the highly reactive radicals which were generated by elec-
tron irradiation under the imaging conditions (these radicals
can certainly give rise to oxidation in the vicinity of the elec-
tron beam). As shown in Figure  12, the radicals gave rise to
the rapid degradation of pristine BP within 15 min, while the
[BMIM][PF6]-treated BP and [BMIM][BF4]-treated BP flakes
can withstand up to 120 and 180 min, respectively. The images
indicated that the ionic liquid (IL)-treated samples are much
more robust and hence can withstand more aggressive elec-
tron exposures. This in situ ESEM study demonstrates that the
reactive oxygen species play a dominant role in BP degradation,
and ionic liquid treatment can effectively improve the stability
and prevent the photooxidative degradation of BP flakes under
ambient conditions.[137]
3.2.3. Other Dynamic Processes
Except for the above in situ observations concerning the changes
of water itself or the interactions between water and samples,
there are still many other dynamic processes that have been per-
formed and analyzed by in situ ESEM technique. As discussed
above, ESEM can be equipped with a wide range of accesso-
ries, such as cooling, heating, stretching, gas supplying as well
as homemade systems. Two or more accessories may work
together for complex experiments. Thus, the functions of ESEM
can be further extended, which is extremely suitable for in situ
experiments. Therefore, controllable environmental parameters,
including different gases, pressure, temperature, and humidity
can be readily tuned to match other in situ experimental con-
ditions (such as force or electrical field, etc.), enabling direct
visual analysis on very complex dynamic processes, which is an
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Figure 12.  In situ ESEM study on ambient degradation of few-layered BP at a low water vapor under electron beam irradiation. Sequential images of
BP flakes when they are exposed to a small amount (≈8 Pa) of H2O vapor: a) Pristine BP, b) [BMIM][PF6]-treated BP, and c) [BMIM][BF4]-treated BP
flakes. The observation was made in ESEM with a 20 kV, 1.35 nA electron beam. Reproduced with permission.[137] Copyright 2017, Wiley–VCH.

undisputed advantage for ESEM (rather than CSEM et al.). In
recent years, more and more in situ ESEM observations have
been highlighted across the literatures of materials science,
including physical changes and chemical reactions, as well as the
interactions between materials and environment. Some repre-
sentative examples will be sequentially reviewed in this section.
First, for chemical reactions or dynamic processes which are
sensitive to ambient air (or require strict control over the envi-
ronment), it is extremely difficult to perform comprehensive
studies concerning the segmented mechanisms during reac-
tion/process using post-mortem characterizations. Instead, in
situ ESEM observations have an ability to follow the growth,
morphological change, evolution, and disappearance of mate-
rials during reactions/processes. A typical example of these
processes is the cell reaction.[138,139] In an attempt to elucidate
the detailed dynamic process of electrochemical reaction in the
all-solid-state Li-O2 battery, which was regarded as the potential
candidate to meet the ever-increasing demand for high-energy
storage in electric vehicles, Zheng et al. realized the direct visu-
alization of discharge and charge processes of a microscale all-
solid-state lithium-oxygen battery with an ESEM equipped with
a nanoprobe system. The battery was composed of Li metal
(anode), a native oxide layer Li2O (solid state electrolyte), and
super aligned CNTs (SACNTs, cathode). They first observed the
discharge process of a Li-O2 battery under a constant potential
of −3 V (on SACNT vs Li) with an O2 partial pressure of 200 Pa
and imaged the growth and morphological changes of dis-
charge product (Li2O2). Moreover, an electrochemical cycle was
conducted to follow the formation and decomposition of Li2O2.
As shown in Figure 13, a Li2O2 particle was formed at the CNT-
solid-state electrolyte-oxygen three-phase interface (TPI) and
grew up to 1.5 µm after 3000 s during the discharge process
(Figure 13a). Then the decomposition of Li2O2 particle initiated
from its surface at the charge cycle with a constant potential of
8 V (on SACNT vs Li) (Figure 13b), because the electronic and
ionic conductivities of surface were much higher than those in
the bulk and the oxygen release caused by Li2O2 decomposi-
tion was also much easier on the surface. The results showed
detailed morphological evolution of Li2O2 products during the
charge/discharge process of the all-solid-state Li-O2 battery,
which shed light on the understanding of battery reactions at
the microscale. This experimental design may find its applica-
tion in revealing similar processes.[138]
Second, in recent years, there are also increasing interest
in the fabrication of low-dimensional materials using high-
resolution ESEM, considering that low-dimensional materials
have received extensive attention in many fields due to their
unique structural and electronic properties, such as graphene
flakes,[28,29] nanowires,[10,26,140] etc. For example, aiming at the
exploration of interlayer coupling and the illustration of stacking
sequence of few-layer graphene (FLG), Wang et al. took in situ
imaging on isothermal growth and etching experiments of FLG
under ESEM. After isothermal chemical vapor deposition (CVD)
using C2H4 performed in ESEM chamber, graphene layers were
grown on polycrystalline Pt foils. Then, in situ monitoring of
etching on FLG at 900 °C in an atmosphere of 25 Pa hydrogen
gas was performed by characterizing the shape and size evo-
lution of graphene layers. As shown in Figure  14, shrinking

Small Methods 2020, 4, 1900588 1900588  (14 of 21) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
Figure 13.  In situ ESEM observation toward the formation and decom-
position of Li2O2 in an all-solid-state Li-O2 battery during an electro-
chemical cycle. a) During discharging, the spherical particle grows up at
a CNT-solid-state electrolyte-oxygen TPI which is indicated by the yellow
arrows. b) The decomposition of the same particle initiates locally at the
surface and proceeds into the bulk upon charging process. Red arrows
indicate the position where the particle decomposes. Reproduced with
permission.[138] Copyright 2014, American Chemical Society.
behavior took place simultaneously at the periphery of each indi-
vidual layer during hydrogen etching, and diffusion of etching
products from graphene edges in each layer to the surrounding
atmosphere was not hindered by the other layers. This behavior
indicated that new layers grow on the topmost layer, and the
isothermal CVD growth of FLG on Pt substrates followed the
wedding cake type stacking. Moreover, the dynamic data of time-
depended perimeters of each individual layer were given by the
in situ observation, which indicated that the interlayer coupling
strength between the second and third layers was significantly
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Figure 14. In situ observation on shape evolution of graphene layers
during H2 etching at 900 °C in an atmosphere of 25 Pa hydrogen.
a–d) Time-lapse image series show the etching of single-layer graphene
(SLG) and the topmost layers in bi-layer graphene (BLG) and tri-layer gra-
phene (TLG). The green arrows indicate the up-step and down-step direc-
tions of Pt terraces. The scale bar in (a) also applies to (b–d). Reproduced
with permission.[28] Copyright 2016, Springer Nature.
weaker than the coupling strength between first layer and the
Pt substrate, according to the comparison of etching rates. This
in situ ESEM study gave a promising insight to guide the con-
trolled production of FLG with defined number of layers.[28]
Similarly, Huang et al. performed an in situ heating experiment
in ESEM equipped with a home-made laser heating stage and a
gas supply unit to directly monitor the growth dynamics of CdS
nanowire arrays. Under 700 °C, using a flow of H2/Ar (5%:95%)
gas mixture at a chamber pressure of 60 Pa, at an acceleration
voltage of 12.5 kV, they captured the secondary electron images
of the formation of nanowire arrays during in situ observa-
tion. The results revealed the morphological evolution of the
CdS from the particle shape to the nanowire array architecture.
The in situ observation clearly demonstrated that the nanowire
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com www.small-methods.com

Figure 15.  Reaction between Na3RuO4 and SBN glass occurred during in
situ heat treatment in ESEM. The reaction started at 520 °C and a) was
recorded at T = 400 °C (no reaction). The arrow in (b) shows the interme-
diate phase formed at 660 °C. Incorporation of the Na3RuO4 crystals in
the SBN melt at c) 733 °C and d) 746 °C. Reproduced with permission.[144]
Copyright 2012, American Chemical Society.

arrays formed through a selective vaporization process of the

bulk crystals, which validated the formation mechanism of the
nanowire arrays. [10]
Third, for crystallization/sintering processes occurred
at either high or low temperature (relative to room temperature,
≈25 °C), the corresponding morphological evolution and growth
mechanism can, of course, be monitored under ESEM by using
different temperature-controlling stages. For example, the evo-
lution of morphology and size distribution of reaction products
during crystallization or sintering of glass/ceramic materials can
be imaged by ESEM at high temperature.[141–144] Boucetta et al.
observed the changes in morphology and composition of the
ruthenium compounds formed during an in situ heat treatment
in ESEM operated at 30 kV. Figure  15 shows a direct incorpo-
ration of ruthenates in a glass frit. There was no obvious reac-
tion until the temperature reached 520 °C (Figure 15a) while
an intermediate phase began to form at the Na3RuO4−SBN
(SiO2−B2O3−Na2O) glass interface induced by the initial reaction
between Na3RuO4 crystals and the glass at 520 °C (Figure 15b).
With increasing temperature, the reaction became faster and
local melting of SBN glass frit took place at temperatures higher
than 670 °C (Figure 15c). Finally, at about 750 °C, the Na3RuO4
crystals clearly started to dissolve in the melt (Figure 15d).[144]
On the other hand, local crystallizations at relatively low
temperature have also attracted considerable attentions. For
example, an interesting phenomenon on large single crystals
of calcite with smooth convoluted fenestrated morphologies
was shown in the skeletons of adult echinoderms. In order to
explain this phenomenon, Politi et al. focused on a sample sea
urchin (one of the echinoderms) which is able to regenerate
broken spines by transforming a thin covering layer of amor-
phous calcium carbonate (ACC) into crystalline calcite as time
Figure 16. ESEM observation toward crystallization over a period of
≈15 min for regenerated fresh microspines with an ACC sheath. The
spine was observed under a humid atmosphere (pH2O = 6.6 Torr, 5 °C),
and the electron beam (10 kV) provides the energy required to induce
crystallization. a) Microspine at the beginning of the observation. b–h)
Pictures taken in succession every 2–5 min at the same magnifica-
tion. Between (b) and (c) and between (g) and (h), the viewing frame
was moved along the axis of the microspine to compare the condi-
tion of a nonirradiated region of the microspine relative to the irradi-
ated region. Lines in (c) and (h) show the boundaries of the previous
frames. Arrows in (d) show the growing crystal faces. The scale bar in
(a) also applies to (b–h). Reproduced with permission.[145] Copyright
2004, AAAS.
goes on. The crystallization of the amorphous sheath and the
tip of microspines into calcite was directly captured by in situ
observation with an ESEM in a wet atmosphere (water vapor
pressure of 6.6 Torr) at 5 °C. In this experiment, the electron
beam provided the energy required to initiate crystallization. As
shown in Figure  16, at initial stage, the tip of the microspine
underwent a reduction in volume (Figure 16a; compared with
Figure 16b,c). Then, a single crystal gradually formed as thin
plates with well-defined edges (Figure 16d,e). Later, the crystal

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Figure 17.  In situ tensile fracture process of an as-deposited LDMD Ti-6Al-4 V specimen at 650 °C for the displacements of: a) 900 µm, b) 1150 µm,
and c) 1204 µm for fracture. The loading direction was horizontal. Reproduced with permission.[148] Copyright 2019, Elsevier.
grew at the expense of amorphous material that was consumed
in the electron-irradiated region (Figure 16f,g), whereas the
nonirradiated region remained unchanged (Figure 16c,h). The
results showed that the sea urchin spine regenerated through
the initial deposition of ACC and indicated that single crystals
with complex morphology could be produced by the deposition
of transient amorphous phases which may promote the devel-
opment of sophisticated materials.[145]
Finally, specific accessories, such as various mechanical test
systems, can be equipped on the ESEM to achieve the obser-
vations of deformation behavior of samples under the action
of elongation or compression. For example, Bos and Donald
investigated the compressive behavior of elementary flax
fibers under a modified loop test with ESEM. They showed
that the flax fibers presented very different deformation
behaviors due to the difference in chemical composition and
morphology, and the lateral strength of the fibers was lower
than its tensile strength,[146] Similarly, Wang et al. studied the
effect of microstructure and corrosion on the fracture behavior
of Sn-3.0Ag-0.5Cu solder alloys using a three-point bending
test in ESEM.[38] Besides, Koester et al. performed an in situ
measurement in ESEM to identify the mechanism of propa-
gation of physiologically pertinent short (< 600 µm) cracks in
both the transverse and longitudinal orientations in cortical
bone. The in situ test of samples soaked in Hanks’ balanced
salt solution was carried out at 25 °C using a Gatan Microtest
2 kN three-point bending stage. The instrument was operating
in backscattering mode at 15 kV and a pressure of 35 Pa to
monitor the crack path. The results revealed the crack initia-
tion and growth behavior and the development of the extrinsic
toughening mechanisms in cortical bone.[147] Additionally, to
reveal the deformation mechanisms of laser direct melting
deposited (LDMD) Ti-6Al-4 V alloys (which are mainly used
in aerospace fields), Wang et al. used an in situ tensile test
based on ESEM to identify the initiation of cracks and pro­
pagation mode of slip bands. They compared the microstruc-
tural evolution during the fracture process at 23 and 650 °C.
Figure  17 shows the in situ macroscopic deformation at
650  °C. When the displacement was increased from 900 µm
(Figure 17a) to 1150 µm (Figure 17b), the specimen showed
uniform deformation and smooth necking; while, as the elon-
gation attained 1204 µm (Figure 17c), the sample fractured.
The results showed that the grain structure was greatly elon-
gated with good plastic flow behavior over long stretching
distance, and the elongation of the alloy was higher at 650 °C
than at 23 °C.[148] Moreover, such in situ mechanical experi-
ments can be combined with the characterization of crystal-
lographic orientation information with electron backscatter
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diffraction (EBSD) in ESEM. For instance, Reichmann et al.
performed in situ compression test on ferroelectric BaTiO3
material, and EBSD measurements were performed before
and after the experiments to determine the misorientation
between two neighboring domains.[20] These mechanical tests,
apparently, can be further extended by combining other envi-
ronmental controlling systems in the chamber of ESEM and
therefore, extracting more fascinating mechanical properties
for metal/ceramic/glass/organic materials as well as emerging
nano and 2D materials.
4. Summary and Prospect
As we reviewed above, in situ/ex situ ESEM studies on mate-
rials and physicochemical processes of materials have indeed
made significant contributions to the understanding of funda-
mental science and mechanisms by providing direct, visual,
and dynamic evidences. Yet, we have to point out that there
are still several urgent challenges awaiting to be resolved in the
development of the microscope/microscopy, including achieve-
ment of a better resolution, minimizing beam-induced damage
during observation, and more hardware designs on in situ
experiments for widening the applications of ESEM, etc.
First, ESEM is regarded as one of the best solutions for
imaging insulated and hydrated samples, even under different
harsh conditions (including water and oil). It is quite feasible
to obtain true and noninvasive images concerning the micro-
morphological, structural, and compositional characteristics
of materials in their native states, because ESEM has a spe-
cial capability to allow gases to be existed in the chamber, and
opens up a new field of application in observations of noncon-
ductive samples without complex preparation that were previ-
ously impossible in CSEM. However, the chamber gas is also
a double-edged sword in ESEM in consideration of its negative
effects as a result of compensating the charges induced by the
environmental gas. The most common problem is that, the
high ambient pressure in the chamber intensifies the scattering
of electrons by gas, which then increases the astigmatism of
the image and reduces the contrast and signal-to-noise ratio.
For in situ observations, the presence of gas in the chamber
can severely degrade the image resolution and contrast, and it
is a challenge to perform a high-resolution and high-sensitivity
imaging during dynamic interactions at high-pressure condi-
tion. The resolution of CSEM can reach sub-nanometer scale
in the latest models, while the resolution of ESEM remains at
1–3 nm by far. Considering this factor, in practical scenes for
ESEM, the pressure is usually kept below 1000 Pa to obtain a
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
good spatial resolution and image contrast.[7] In this regard,
significant improvement in resolution under environmental
gas pressure is urgently needed in the future development
of ESEM. Fortunately, pioneer contributions have been done
by using state-of-the-art software technologies. For example,
Zhang et al. presented an ESEM technique for enhanced reso-
lution using phase reconstruction to improve the image quality
with the operation pressure ranging from 1000 to 2500 Pa in
dynamic processes during the water droplet condensation. This
work suggested a practical method to achieve high-resolution
observation at high pressure without changes on the hardware,
and is expected to expand the application of ESEM in static and
dynamic processes.[149] However, we are also looking forward
to modifications on current ESEM hardware that can push the
resolution limit further.
Besides, during the imaging on biological samples or ultra-
thin nanostructures in ESEM, undesirable morphological
changes have been frequently reported which are resulted
from the interactions between the electron beam and sam-
ples.[150–152] Moreover, the exposure of specimen under elec-
tron beam sometimes causes irreversible damages, which is a
serious problem for in situ analysis. To minimize such risk of
induced sample damage by electron beam, we should pay spe-
cial attention to the choice of observation parameters of ESEM,
such as reducing the acceleration voltage and the spot size. In
view of this situation, we also suggest that a trail experiment
regarding the electron beam damage on these samples should
be performed to evaluate the impact before the observation.
For example, two samples can be put in the same environ-
ment, with one of them irradiated by the electron beam while
the other is not. This comparative experiments of two samples
can be conducted to analyze the morphological changes within
a specified time limit to help us know the degree of damage
and give us the hint on how to avoid it. Furthermore, quantita-
tive evaluation scheme of beam-induced damage on interested
samples is essentially needed for comprehensive ESEM study,
either static or dynamic ones.[153]
Last but not least, compared with the technological advance-
ments arisen from other types of microscopes, the hardware/
software improvements in ESEM in the last one or two decades
seem to be unsatisfactory, at least from scientists’ view (e.g., for
TEMs, there have been rapid developments on achieving better
images in the past 20 years, including spherical and temporal
resolutions, as well as new types of sample holders for control-
ling of the sample’s micro-environment[154,155]). We anticipated
that these relevant technical advancements on similar electron-
optic systems can inspire more contributions on ESEM, so that
ESEM can finally evolve into a “micro-environmental lab” for
materials characterization. In this perspective, we are glad to
find that initial attempts have been made. For example, one
long-lasting problem in ESEM is that the electron beam cur-
rent loss inside and above the pressure-limiting aperture (PLA,
which separates the high vacuum and low vacuum regions in
ESEM, see Figure 2a), which is caused by the electron beam
collides with gas molecule before entering the chamber, signifi-
cantly reduces the image quality during observation. Danilatos
et al. compared the electron beam loss above the PLA on three
different ESEMs (each with a different PLA geometry) and the
results showed that an optimized PLA design can be useful to
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decrease the electron beam losses and hence improve the image
quality.[156] On the other hand, more functional accessories could
also be developed to solve complex physical/chemical processes
which is useful in modern materials science (e.g., the electric/
magnetic field assisted sintering process[157]) In addition, a wise
combination of three working modes (HiVac, LoVac, and ESEM)
may create extra possibilities in realizing novel experimental
techniques through rational design. Wan et al. have adopted
an innovative method to directly observe the effect of hydrogen
(H) on fatigue crack growth behavior of pre-cracked single-edge
notched tensile (SENT) specimen.[158] They performed in situ
H-plasma charging on the SENT in the low vacuum environ-
ment in ESEM, and then imaged it in HiVac mode when the
H-plasma was switched off (the normal GSED cannot work in
H-plasma). So, the “in situ imaging” here was actually “in situ
in position” plus “ex situ in environment.” Moreover, new hard-
ware and software technologies, such as MEMS (micro-electro-
mechanical system) and AI (artificial intelligence), are also
very promising to be incorporated into next-generation ESEM
machines to play their roles in materials science.
In summary, ESEM observations have offered significant
opportunities in materials characterization over the past few
years. The including of various gases in the sample chamber
has not only allowed the direct observations on wet and insu-
lated samples without extensive preparation procedures, but
also provided unprecedented chances in investigating complex
physicochemical processes under controlled environments
(temperature, pressure, humidity, etc.). Assisted by modern
ESEM, scientists and engineers have achieved a wide range
of successes in either static or dynamic characterizations of
almost all sorts of materials. In the era of nanotechnology, we
believe that ESEM will play an even greater role in materials
science in the near future with rational experimental designs
and hardware/software evolutions.
Acknowledgements
The authors acknowledge the financial support from the National
Science Foundation of China (Project no.: 51972159) and Jiangxi’s
Natural Science Foundation Key Projects (Project nos.: 20192ACB21018
and 20171ACB20006). Y.Z. acknowledges the funding support from the
National Science Foundation of China (Project no.: 11864022). This work
was also supported by the Research Grants Council of the Hong Kong
Special Administrative Region, China (Project no.: PolyU152140/19E)
and the Hong Kong Polytechnic University (1-YW2W).
Conflict of Interest
The authors declare no conflict of interest.
Keywords
ESEM, in situ observations, materials science, static observations
Received: August 24, 2019
Revised: November 3, 2019
Published online: December 4, 2019
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
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