Rsos.191204 LCTEM

Liquid cell transmission
royalsocietypublishing.org/journal/rsos
electron microscopy and
its applications
Shengda Pu†, Chen Gong† and Alex W. Robertson
Review
Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH, UK
Cite this article: Pu S, Gong C, Robertson AW.
Downloaded from https://royalsocietypublishing.org/ on 08 September 2023
AWR, 0000-0002-9521-6482
2020 Liquid cell transmission electron microscopy
and its applications. R. Soc. open sci. 7: 191204. Transmission electron microscopy (TEM) has long been an
http://dx.doi.org/10.1098/rsos.191204 essential tool for understanding the structure of materials. Over
the past couple of decades, this venerable technique has
Received: 10 July 2019 undergone a number of revolutions, such as the development
Accepted: 19 November 2019 of aberration correction for atomic level imaging, the realization
of cryogenic TEM for imaging biological specimens, and new
Subject Category: instrumentation permitting the observation of dynamic systems
Chemistry in situ. Research in the latter has rapidly accelerated in recent
years, based on a silicon-chip architecture that permits a
Subject Areas: versatile array of experiments to be performed under the high
materials science/electron microscopy/ vacuum of the TEM. Of particular interest is using these silicon
electron microscopy chips to enclose fluids safely inside the TEM, allowing us to
observe liquid dynamics at the nanoscale. In situ imaging of
Keywords: liquid phase reactions under TEM can greatly enhance our
understanding of fundamental processes in fields from
transmission electron microscopy,
electrochemistry to cell biology. Here, we review how in situ
in situ transmission electron microscopy,
TEM experiments of liquids can be performed, with a
liquid cell transmission electron microscopy particular focus on microchip-encapsulated liquid cell TEM.
We will cover the basics of the technique, and its strengths and
Author for correspondence: weaknesses with respect to related in situ TEM methods for
Alex W. Robertson characterizing liquid systems. We will show how this technique
e-mail: [email protected] has provided unique insights into nanomaterial synthesis and
manipulation, battery science and biological cells. A discussion
on the main challenges of the technique, and potential means
to mitigate and overcome them, will also be presented.
This article has been edited by the Royal Society 1. Introduction

of Chemistry, including the commissioning, peer
Visualizing liquid phase processes at the nanoscale can yield
review process and editorial aspects up to the essential information for understanding fundamental processes in
point of acceptance. physics, chemistry and biology. Thanks to the development of
liquid cell transmission electron microscopy (TEM) within the last
† 30 years, phenomena such as nanomaterial synthesis [1–6], live
These authors contributed equally to this
work. biological cells [7–12], battery solid–electrolyte interface (SEI)
formation [13–15] and localized corrosion [16,17] have been
visualized with unprecedented spatial and temporal resolution.
© 2020 The Authors. Published by the Royal Society under the terms of the Creative
Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits
unrestricted use, provided the original author and source are credited.
Liquid cell TEM allows the observation of processes that cannot be imaged with conventional TEM or 2
other techniques. For example, biological specimens are destroyed within seconds in conventional TEM due
to the high vacuum. And while cryo-electron microscopy can be used to safely image and determine their
structures [18–20], the specimens are no longer in their native state, with the potential for specimen damage
and artefacts being introduced during sample freezing. Liquid cell TEM not only preserves the liquid state
of the specimens inside the TEM vacuum, but also allows the in situ observation of biological process [9].
In situ TEM has become a popular technique for observing reactions at the nanoscale [21]. Compared
with other in situ techniques like atomic force microscopy (AFM), scanning tunnelling microscopy (STM)
and various X-ray methods, TEM not only has the advantage of having both high temporal and spatial
resolution, it also provides direct visualization of any changes in structural, morphological [22] or
elemental distribution [23,24] at the nanoscale. The development of enclosed liquid cells has allowed
the in situ TEM imaging of liquid phase reactions, opening up fields from electrochemistry to cell
biology for study. This paper reviews in situ TEM experiments of liquid samples, with a focus on
microchip encapsulated liquid cell TEM. We will cover the basics of the technique, its strengths and
R. Soc. open sci. 7: 191204

weaknesses with respect to related in situ TEM methods for characterizing liquid systems, and its
application in biological, chemical and materials science fields.
2. Different TEM configurations for imaging liquid samples

In TEM, a high-energy electron beam is transmitted through a thin specimen, with the potential for elastic
and inelastic scattering of some of the beam. The resultant beam can be collected and analysed in
different ways, such as with high angle annular dark-field detectors (HAADF) to yield atomic number
sensitive images, or the energy loss of inelastically scattered electrons can be measured to yield a
characteristic spectrum (electron energy loss spectroscopy, EELS). There are two crucial requirements
for this technique [25]. First, the TEM column needs to be maintained at a high vacuum, at least 10−5 Pa,
to minimize undesired electron beam scattering and sample contamination. Secondly, specimens need
to be thin enough for electrons to penetrate through, usually of the order of hundreds of nanometres
for nanoscale imaging. These requirements are problematic for imaging liquid phase specimens; most
liquids will simply vaporize under the high vacuum, and manipulating a liquid to remain sufficiently
thin can be challenging. There are various approaches to tackling these issues, with the main methods
for TEM imaging of liquid samples shown and compared in figure 1.
The most straightforward approach is to use low-vapour-pressure liquids where possible, since these
liquids can withstand the high vacuum of the TEM [2,31–34]. These liquids can be imaged in the same
way as solid specimens within TEM, with the ionic liquid simply applied to a standard specimen grid as
shown in figure 1(i). Using micrometre-sized holes for wetting ensures that the ionic liquid droplets are
sufficiently thin to allow for imaging. More complex methods have also been designed to perform in situ
biasing experiments on ionic liquid specimens. For example, as shown in figure 1(ii), a miniature battery
can be built and cycled in situ under TEM using an ionic liquid electrolyte, which provides the ionic
conduction between the two electrodes. Upon applying electric potential, the charging/discharging
processes can be performed as in a real battery, and the corresponding changes at the electrodes can
be imaged using in situ TEM. The electrode material is in the form of nanowires to allow good image
quality under TEM. This technique requires a special sample holder with electrical feedthroughs, so
that a bias may be applied from outside the TEM. However, a major limitation is that this technique
can only be used with low-vapour-pressure liquids. Most liquids cannot survive the high vacuum of
the TEM, and they often cannot be replaced with a low-vapour-pressure alternative without
fundamentally changing the nature of the reaction one plans to observe.
To address this, other approaches have been developed that can image liquids regardless of their
vapour pressure. The ‘open environmental cell’, or environmental TEM (ETEM), maintains the specimen
chamber at a relatively high pressure compared with the rest of the microscope by differential pumping.
This approach has been widely adopted in SEM [35,36] to image liquid specimens. An ETEM is a
special type of TEM that employs a series of pumps and apertures to gradually change the pressure
along the column, as shown in figure 1(iii). Pressure as high as 2000 Pa can be achieved within its
specimen chamber [28]. However, due to the limits of differential pumping, atmospheric pressures
cannot be achieved using such a set-up, which means most liquids with high vapour pressure will still
evaporate. This mean that common solvents like isopropanol, dimethyl carbonate, dichloromethane or
even water cannot be easily used within such system. Any salt dissolved in them will precipitate out
during experiment due to the rapid evaporation of the solvent. Although some liquid phase
imaging liquid phase with TEM 3
open cell closed cell
low-vapour- TEM with differential microchip graphene liquid

pressure liquid pumping liquid cell cell
(iv)
(i) (iii)
(viii)

(v)
(ii)
(vii)
(vi)
advantages: advantages: advantages: advantages:

æ easy sample preparation æ easy sample preparation æ fairly easy preparation æ image with normal TEM
æ image with standard TEM æ can use a standard TEM æ image with standard TEM æ wide liquid choice
æ high resolution sample holder æ wide liquid choice æ fair resolution
æ can use a standard TEM æ electrical biasing with æ controlled liquid flow æ standard TEM holder
sample holder (i) special TEM holder æ electrical biasing
æ or electrical biasing with æ controlled liquid flow æ commercially available
special TEM holder (ii) with special TEM holder
æ high resolution
disadvantages: disadvantages: disadvantages: disadvantages:

æ limited liquid choice æ special TEM required æ compromised resolution æ tricky sample
æ limited liquid choice æ special TEM holder preparation
æ no liquid flow
æ no electrical biasing
æ not commercialized
Figure 1. Comparison of different techniques for liquid phase TEM imaging. (i) Ionic liquid films maintained inside micrometre-sized
holes from surface tension, suitable for TEM imaging [26]. (ii) Design schematic of a miniature battery that may be imaged inside TEM,
using an ionic-liquid-based electrolyte [27]. (iii) Schematic of differentially pumped TEM column with three different pumping stages,
indicated by different coloured pipes, permitting a high pressure sample chamber [28]. (iv) Schematic of an in situ liquid TEM stage on
the tip of a TEM holder [29]. (v) Schematic of an assembled liquid cell from two silicon/silicon nitride chips [23]. (vi) Silicon chip with
microfabricated electrodes [14]. (vii) Magnified SEM image of the red box region in (v), showing electrodes partially over the SiNx viewing
window [14]. (viii) Schematic of a graphene liquid cell for TEM. The specimen is encapsulated between two graphene sheets [30].
experiments were successfully conducted within such systems [31,37], it is mainly used for gas phase
experiments [38–42] and biological specimens [7,43], which do not require very high pressure to maintain.
Another approach can be defined as the ‘closed environmental cell’. Unlike the ‘open environmental cell’
approach with differential pumps, the pressure of the closed cell is maintained by encapsulating the sample
volume in screening membranes. Such a specimen can be maintained at a much higher pressure. The
electron beam penetrates and images the specimen through robust and electron-transparent windows on the
top and bottom of the encapsulated specimen cell. The cell can be in-built within the TEM specimen holder
and is therefore compatible with conventional TEMs. A closed environmental cell can be used for both gas
phase [44] and liquid phase imaging [1,3]. When used for liquid phase, it is usually called a ‘liquid cell’.
A liquid cell is typically only hundreds of nanometres thick, confined by two thin but robust electron-
transparent membranes. The commercially available and by-far most widely adopted liquid cell system is
(i) (ii) 4
Si substrate
deposit SiN layer assemble with top chip
deposit electrodes
flow liquid
deposit spacer

imaging reaction
back-etching
Figure 2. (i) Simplified schematics of the production process of a liquid cell microchip via photolithography. (ii) Schematics of the
assembly of the microchips for liquid cell TEM imaging.
based on silicon microchips, as shown in figure 1(iv–vii). A thin layer of liquid phase specimen of
between tens of nanometres to a micrometre can be confined between two microchips using O-rings
and spacer materials. A thin layer of amorphous silicon nitride (SiN) is most commonly used as the
membrane window material, through which the electron beam can penetrate and be used to image.
(Detailed fabrication and application processes of this system is shown in the next section.) Such
liquid cells can safely contain almost all types of liquid, independent of vapour pressure. In addition,
microfabrication allows additional capabilities to be added, including allowing liquid flow [45],
temperature control (heating and cooling [46]) and applying electrical bias using patterned electrodes.
Avariant of the liquid cell is the ‘graphene liquid cell’, which has been used since 2012. Microencapsulation
using graphene [10,47], or 3 nm thick amorphous carbon films [5], have been shown to well preserve volatile
specimens like biological cells and liquid crystals within the ultra-high vacuum in TEM. This technique can also
be used to image liquid specimens [48–50]. Compared with the microchip cell, the graphene cell can normally
achieve better resolution due to less scattering from the window material and smaller liquid thicknesses. In
addition, it does not require an expensive dedicated sample holder [51–55]. However, this technique is
currently limited in several ways. First, encapsulation of a liquid sample with graphene requires very careful
cell assembly due to the difficulties in handling graphene. Second, only a very small amount of liquid can
be encapsulated and imaged, less than 0.01 picolitre, compared with the nanolitre capacity of a microchip
liquid cell. Third, unlike with microchips, microfabrication cannot be used in this cell, which means that it is
not compatible with functions like liquid circulation or applying electrical biasing. The lack of liquid flow,
combined with the low volume of liquid between the graphene membranes, means studying dynamic
processes is hindered by the limited availability of reactants. This review focuses on the microchip-based
liquid cell, with a detailed discussion of the graphene liquid cell beyond the scope of this review.
3. Liquid cell design and application

Figure 2 illustrates the production and use of a typical liquid cell with electrodes. The liquid cell is usually
fabricated by standard photolithography patterning of silicon microchips. A thin layer of SiN, typically
tens of nanometres thick, is first deposited on top of the Si substrate. Amorphous SiN is the most widely used 5
electron-transparent membrane material in liquid cell TEM. Other membrane material alternatives include
silicon oxide [56] and amorphous carbon [9]. Electrodes and spacers may then be deposited. The spacer is
used to provide enough space between the two chips to ensure enough liquid remains in the cell during
imaging and to allow for liquid flow. Electrodes can be employed to apply an electric potential to
manipulate particle movement within the liquid or to induce electrochemical reactions during imaging.
Finally, the Si is etched away from the back, leaving a small window of only the SiN membrane. The
window is usually in a rectangular shape, as shown in figure 1 (vi,vii), with widths of tens of micrometres
and lengths of over a hundred micrometres. While this limits the viewing area, wider windows are more
susceptible to breaking and bowing, common issues associated with the liquid cell, which will be
discussed in detail later. The electron beam is able to transmit through the windows to allow imaging.
A comprehensive description of the production process of a liquid cell was given by Grogan & Bau [57].
The assembly of a liquid cell is shown in figure 2(ii). A second, flipped, chip is aligned on top of the
bottom chip, normally fixed in place by clamping tight with O-rings and metal frames on the holder tip

(figure 1(iv)), although wafer-bonding and epoxy have also been used by some researchers. The
separation between the windows determines the thickness of the liquid phase specimen being imaged.
Such separation is controlled by the height of the spacer being used. A thicker layer usually means
better liquid flow and allows imaging of relatively large-sized specimens like biological cells [8,56].
However, a thick liquid layer leads to compromised resolution due to increased beam scattering [58].
The optimum resolution of the TEM image after scattering through a liquid layer is estimated to be [59]
AL a C c T
dTEM ¼ , ð3:1Þ
E2
where dTEM is the resolution, which is directly proportional to the objective semi-angle, α, and to the
liquid thickness T, and inversely proportional to the electron beam energy E. (A is a constant that
depends on the liquid and Cc is the chromatic aberration coefficient.) For scanning-mode TEM (STEM)
[58,59] the optimum resolution is also dependent on the liquid thickness:
dSTEM / T 1=2 : ð3:2Þ
As we can see from both equations (3.1) and (3.2), thicker liquid means worse resolution. Another
critical factor limiting the resolution is the dose rate. For both TEM and STEM, the resolution is
dependent on the dose rate as follows [60]:
dTEM,STEM / D1=4 : ð3:3Þ
Dose rate should be carefully monitored and controlled during imaging to avoid beam damage, as
will be discussed in detail later. A comprehensive study of all factors affecting liquid cell resolution in
TEM and STEM was done by de Jonge et al. [60].
4. Liquid research with conventional TEM and open environmental TEM

This review is primarily focused on the TEM study of liquid samples that are encapsulated inside
microchip liquid cells. However, for comparison we will briefly discuss some in situ TEM studies of
open-cell liquid systems. Due to the chamber pressure limit of the conventional TEM and open ETEM,
specimens need to withstand high vacuum during experiments if they are not encapsulated inside a
cell. This limits the options of the liquid being imaged. Most works performed in an open system
employ either a liquid phase alloy or an ionic liquid, both of which are particularly stable under high
vacuum. Many of the pioneer works were performed on alloys heated close to their melting point
[31–33]. These works closely examined the liquid–solid interface during melting and solidification,
imaging processes like phase segregation, crystallization etc.
An interesting application of molten alloys is to serve as the catalyst during nanomaterial growth.
With TEM, in situ growth of these materials can be captured as shown in figure 3a. In 2005, Ross et al.
[2] showed the in situ growth of a Si nanowire in a disilane environment catalysed by a Au-Si alloy
droplet at the wire tip at elevated temperature (500–650°C), which showed a faceting sawtooth
structure. Both the width of the nanowire and the faceting period are highly dependent on the size of
the Au-Si droplet. Based on these observations, it was later found that the Si nanowire growth can be
precisely controlled via manipulating droplet geometry by applying an electric field [62]. This
(a) 6
growth direction (111)
(iv)
11.2°
viewing
direction
–
(110)
p
h
– –
(121) 23.3°
(v)
(i) (ii) (iii) –
(211)
––
(112)
(b)
(ii) (iv)
(i)

460
410°C
Au-Si
420
temperature (°C) 370°C 410°C
380
370°C 270°C
360°C
Au-Si 340
(iii)
250°C
300
Au
260 liquid surface
Au-Si solid surface
250°C solid bulk
220
time
single crystal SnO2 dislocation cloud amorphous
(c)
Li+
conducting epoxy SnO2 nanowires
LiCoO2
200 nm
(ii)
Au rod IL Au rod
(206)
(201)
–– (110)
(110)
(006)
– +
(i) potentiostat (101)
(011) (000)
(200)
(211)
5 nm
(iii) (11–1) (iv) (v) (vi) (301)
(vii)
Figure 3. (a) Growth of saw-tooth faceting Si nanowires using liquid Au-Si droplet as the catalyst [2]. (i–iii) Three Si wires of different
width grown from Au-Si droplets of three different sizes. Scale bar is 100 nm. (iv) Schematic of the three-dimensional structure of the
nanowire. (v) The surface structure with facet angles indicated. p and h denote sawtooth period and amplitude. Scale bar is 50 nm.
(b) Crystalline ordering at the surface of Au-Si liquid [61]. (i) A crystalline bilayer visualized on a 50 nm diameter droplet at 350°C.
(ii) Surface ordering on a 12 nm diameter droplet at 350°C. (iii) A solid Au particle heated on Si at 360°C where the eutectic liquid
starts to cover its surface. Scale bars are 2 nm for (i–iii). (iv) The surface ordering evolution at different temperature. (c) Lithiation of a
single SnO2 nanowire anode at −3.5 V against a LiCoO2 cathode [34]. (i) Schematic of the experimental set-up. (ii) The reacted
(‘amorphous’) and non-reacted (‘single-crystal SnO2’) wire separated by the lithiation interface ‘dislocation cloud’. (iii–vi) Diffraction
patterns taken from different sections of the nanowire in (ii) showing the amorphization upon lithiation. (vii) Sn nanoparticles
dispersed in an amorphous matrix from the lithiated part of the wire.
illustrates how in situ TEM is able to provide unique insights into fundamental materials processes,
which in this case was the relationship between the surface energy and morphology of the
nanomaterial, and how to manipulate the growth of nanomaterials to achieve accurate and direct
nanowire growth in devices [63]. Since then, this system was widely adopted to study in situ the
three-phase growth (solid formed from gas, catalysed by a liquid alloy) of different nanowires [64–66].
A significant advantage of the open-cell system is the high resolution achievable due to the high 7
vacuum and no interceding encapsulating layers. A good example of this is shown in figure 3b, which
shows a stable atomically thin crystalline phase formed from liquid Au-Si and its changes with respect
to different temperatures [61]. The imaged solid–liquid interface atomic structure is of interest to trace
the individual atoms during solidification/melting to help better understand and model these processes.
Ionic liquids are liquid salts consisting of ions and ion pairs [67], which can dissolve many kinds of
substances. They can withstand high vacuum and electron beam irradiation [68,69]. They are also good
ionic conductors, which makes them perfect for use as alternative electrolytes for battery and other
energy-related in situ TEM research. In 2010, the first experimental design for performing biasing
under high vacuum with an ionic liquid in a TEM was published [27]. Within the same year, with
this design, Huang et al. [34] studied the in situ lithiation of SnO2 nanowires within TEM, as shown in
figure 3c. (Lithiation/delithiation are the incorporation/removal process of lithium from an electrode
in a lithium ion battery.) Upon lithiation, the crystalline SnO2 nanowire was converted to amorphous
Li2O with nanocrystalline Sn and LixSn dispersed within it (as shown in figure 3c(vi)). The interface

between lithiated and unlithiated wire contained a ‘dislocation cloud’, which significantly expanded
and distorted the wire as it moved forward. This expansion and distortion was not reversible upon
delithiation. This observation directly showed the degradation process of the electrode material and
helps explain the loss in battery capacity due to cycling.
The same set-up can be used to test the charging/discharging effects within different battery systems
[70] on a wide range of electrode materials [71–75] and to study the effect of coating [76,77] and doping
[78] on their degradation processes. For example, Liu et al. [72] found that graphene nanoribbons showed
greater flexibility and were highly resistant to fracture upon lithiation, unlike multi-wall carbon
nanotubes [71] which always become brittle and prone to fracture after lithiation. The difference was
ascribed to the unconfined stacking of graphene layers, which prevents the interlayer stress build-up
and makes it potentially a highly durable electrode material. Visualization by TEM not only helps us
to understand the degradation mechanisms within different battery systems, it can also help test
different potential electrode materials, which can be beneficial in battery research to improve stability
and capacity. However, using an ionic liquid as the test battery electrolyte means its
representativeness toward real batteries is questionable, as they are not typically employed in
batteries. A common battery electrolyte consists of a lithium salt, for example LiPF6 or LiClO4,
dissolved in organic solvents, for example, dimethyl carbonate (DMC) or diethyl carbonate (DEC).
These solvents have high vapour pressure [79] (DMC: 5300 Pa at 20°C; DEC 1400 Pa at 25°C), which
cannot be maintained in an open-cell configuration. A closed cell is required to image battery systems
with these more relevant electrolytes.
5. Research with liquid cell

5.1. Nanomaterials research
5.1.1. In situ growth of single phase nanoparticles
The microchip-based closed liquid cell has a wide range of applications, due to the ability to pattern
electrodes and liquid flow spacers on them, and their robustness towards a wide variety of liquids. So
far, the most common application is to study the synthesis and manipulation of nanomaterials.
Table 1 shows a selected list of liquid cell experiments on nanomaterials with significant importance.
The first in situ liquid cell TEM nanomaterial synthesis was performed by Williamson et al. in 2003 [1].
This work visualized the in situ electrochemical nucleation and growth of Cu clusters. The same
experiment was later repeated in 2006 [97–99] with better resolution, which allowed its growth
mechanism to be modelled and quantified. This is shown in figure 4a; a negative potential was
applied from +0.3 V to −0.27 V with respect to a Cu reference electrode. Then a positive potential was
applied back to +0.3 V. The deposition was a two-stage process. The first stage started immediately
below 0 V potential. During this stage, the growth was reaction limited (lowering potential gives
higher deposition rate due to higher driving force). Towards −0.1 V, the second stage starts, where the
deposition became mainly kinetically limited (deposition rate starts to decrease upon further lowering
potential). Upon applying positive potential, all the deposited Cu was stripped away. By measuring
the size change of the Cu particle with TEM, such two-stage deposition was for the first time
visualized and well correlated with the electrochemical I–V curve.
Table 1. Selected list of liquid cell experiments on nanomaterials performed since 2003.
materials observation author methods importance
Cu cluster nucleation, growth Williamson et al. 2003 [1] biasing via electrodes first liquid cell experiment
Pt nanoparticle nucleation, growth Zheng et al. 2009 [4] E beam illumination sub-nm resolution allows clear visualization of growth mechanism
PbS nanoparticle nucleation, growth Evans et al. 2011 [6] E beam illumination different morphology from different solution; first atomic resolution
in liquid cell
Ag nanoparticle nucleation, growth Woehl et al. 2012 [80] E beam illumination different morphology formed from different beam current.
iron oxyhydroxide oriented attachment Li et al. 2012 [81] premade in situ crystal movement and attachment with atomic resolution
nanoparticles
Pt3Fe nanorod nucleation, growth and Liao et al. 2012 [82] E beam illumination first in situ nanorod formation
assembly
Au-Pd core–shell growth of the shell onto Jungjohann et al. 2013 E beam illumination first in situ core–shell synthesis by coating the core with shell
nanoparticle the core [83] material
Pt nanoparticle nucleation, growth Liao et al. 2014 [84] E beam illumination first in situ crystal growth with atomic resolution
Pd nanoparticle dissolution Jiang et al. 2014 [85] oxidative etching by E beam first in situ oxidative etching of nanoparticle
Ag-Pd nanocage formation Sutter et al. 2014 [86] galvanic replacement of Ag using a Pd first in situ formation of nanocage
solution
Au nanoparticle dissolve, merge Hermannsdörfer et al. premade particles change pH, NaCl in situ change of particle with respect to environment
2015 [87] concentration, beam dose
Au nanoparticle nucleation, growth Park et al. 2015 [88] E beam illumination quantify the relationship between beam dose with growth rate
Au nanoparticle nucleation, growth Alloyeau et al. 2015 [89] E beam illumination
Fe3Pt–Fe2O3 core–shell nucleation and growth Liang et al. 2015 [90] E beam illumination first in situ growth of the complete core–shell structure
nanoparticle
Pt-Au core–shell growth of the shell onto Wu et al. 2015 [91] E beam illumination quantify the growth kinetics of core–shell structure, showing the
nanoparticle the core three different stages during growth
(Continued.)
8
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Table 1. (Continued.)
materials observation author methods importance
UiO-66(Zr) & ZIF-8 MOF nucleation, growth and Patterson et al. 2015 [92] mixing solutions and heating first liquid cell in situ experiment on MOF
assembly
Pd nanoparticle nucleation, growth, size Abellan et al. 2016 [93] E beam illumination precise size control growth to sub-3 nm using capping agent
control
Ag nanoparticle nucleation, growth Ge et al. 2017 [94] E beam illumination catalytic growth due to the presence of Pt
Ag particle capped with oriented attachment Zhu et al. 2018 [95] premade the role of capping ligands on the oriented attachment process
ligands
Pd nanoparticle nucleation, growth Yang et al. 2019 [96] electrochemical biasing significant effect of HCl on the growth mechanism and resultant
morphology
9
R. Soc. open sci. 7: 191204 royalsocietypublishing.org/journal/rsos

(a) 10
h i a b e
g
5 j
I (10–7 A)
f
0
a f h j
cb
d
e
–5
–0.2 –0.1 0 0.1 0.2 0.3 200 nm
U (V)
(b) 4.0
(i) t (ii)
3.5
D (nm)
3.0
2.5 a: simple growth (iii)
2.0 b: with coalescence
0 20 40 60 80 100 120
t (s)
(c)
(i) (ii) (iii) (iv)

0s 52.75 s 82.25 s 132.25 s
(v) (vi) (vii) (viii)
2 nm
139.5 s 145.5 s 797.5 s 797.5 s
(d) 001 view along [011]

–
2D projection
22 s 55 s 70 s 85 s 107 s 143 s
{111} {111}
100
d100
2 nm
d111
d011
010 (i) (iii)
(ii) 4.0 {111}
{011}
3.5 {100}
D (nm)
3.0
2.5
2.0
1.0
(iv)
0 20 40 60 80 100 120 140
time (s)
(e) (i) 0:00 0:39 1:40 2:34 3:46 9:32
10 nm
(ii) 0:00 4:44 6:13 9:39 12:06 16:06
10 nm
Figure 4. (a) Images recorded during the electrochemical deposition and stripping process of copper; a, b, e were recorded during
deposition; f, h, j were recorded during stripping [97]. The cyclic voltammogram on the left-hand side shows the point in the cycle
at which each image was recorded. (b) Platinum nanocrystal growth trajectories. (i) Particle size versus growth time for these
particles with two different types of growth trajectories. (ii) Coalescence growth. (iii) Monomer addition growth (simple
growth) [3]. (c) Direction-specific oriented attachment of iron oxyhydroxide nanoparticles. (i–vii) Sequential dynamics of the
attachment process of two particles ( particle I and II), the surfaces of particles I and II made transient contact at many points
and orientations (1–1, 1–2, 2–3 and 3–4) before final attachment and growing together (point 3–5), scale bar 5 nm.
(viii) Attachment interface from (vii), showing an inclined twin plane [81]. (d ) In situ facet development of a Pt nanoparticle.
(i) The atomic model of a Pt nanoparticle and its projection along the [011] zone axis. (ii) Average growth profile along three
different directions (100), (011) and (111). (iii) Sequential growth of the Pt nanoparticle. (iv) Simulated TEM images of the Pt
nanoparticle in (iii). (e) [82]: Real-time imaging of Pt3Fe nanorod formation. (i) Nucleation and growth of Pt3Fe particles
followed by their assembly into a nanorod. Particles contributed to the nanorod are highlighted in green. (ii) The growth of a
long Pt3Fe nanorod from the assembly of several short nanorods [84].
By improving the liquid cell design and using intensive electron beam radiation instead as the means to
instigate nucleation and growth of nanoparticles, Zheng et al. [3] managed to improve the resolution of the
liquid cell from 5 nm to sub-nanometre. A high electron beam dose can lead to radiolysis of the liquid,
yielding radiolysis species like hydrated electrons, eh [100], which act as a strong reducing agent and
thus cause the reduction of the solution and the formation of the nanoparticles. Using this technique,
the nucleation and growth of nanosized Pt particles was clearly visualized. With this high-resolution
technique, Zheng et al. revealed two different growth mechanisms during the early stage of nanocrystal 11
growth (coalescence versus monomer attachment), as shown in figure 4b. In addition, by tracing the
growth rate of individual particles, it was found that a monodisperse size distribution could always be
reached regardless of the initial growth path, as shown in figure 4b(i). Zheng et al.’s method of
instigating nanomaterials growth by beam illumination has been widely adopted to study nanomaterials
synthesis with liquid cell TEM. For example, using such method, Evans et al. [6] grew PbS nanoparticles
of different morphologies with precursor solutions of different chemical compositions. In the same
paper, Evans et al., for the first time, showed that lattice resolution could be achieved within liquid cell TEM.
Apart from nanoparticle nucleation and growth, many other processes can also be visualized in situ
with atomic resolution. For example, in 2012, the oriented attachment process of iron oxyhydroxide
nanoparticles was visualized by Li et al. [81], as shown in figure 4c. The nanocrystal kept moving and
rotating around another bigger particle until a point where lattice matching with the adjacent
nanocrystal was achieved. Then they merged into a single crystal. Such oriented attachment process
was later also visualized in Au nanoparticles capped with organic ligands by Zhu et al. [95], who

pointed out the importance of ligands in this case. Ligands guide the rotation of the particles to share
a common {111} orientation. {111} has the lower ligand binding energy and thus it is the preferential
orientation for attachment.
In 2014, the first in situ nanocrystal growth with atomic resolution was performed by Liao et al.
[84], as shown in figure 4d. With such high resolution, the growth rate along different
crystallographic directions of a Pt nanocube could be individually tracked. Such visualization
greatly enhanced the understanding of crystal growth with respect to surface energy of each facet,
which is the key to the manipulation of nanocrystal growth. And apart from synthesis, the
dissolution process of different nanoparticles has also been studied using liquid cell TEM
[85,101,102]. For example, Wu et al. [102] visualized the in situ dissolution of platinum
nanoparticles, from which a quantitative kinetic model was developed, taking into account different
crystal structures and the atoms’ location within them.
5.1.2. The growth of more complex structures

Apart from single-phase nanoparticles, liquid cell TEM has also been used to image the growth of more
complex systems. For example, much research has been done on core–shell structured nanoparticle
synthesis. The first liquid cell experiment on a core–shell structure was performed in 2013 by
Jungjohann et al. [83], who synthesized the Au-Pd core–shell structure by coating Au nanoparticles with
Pd. It was shown that a different core morphology could lead to different shell growth. In 2015, Liang
et al. [90] demonstrated the in situ growth of Fe3Pt-Fe2O3 core–shell structure, during which both the
core and the shell were formed by the action of the electron beam, as described in the previous section.
The depletion of Pt precursor in the solution terminated the core growth and further electron beam
illumination led to the epitaxial growth of the shell. With the same technique, Zheng et al. [103]
demonstrated the in situ growth of the PtNi-Ni core–shell structure, showing the feasibility of applying
a metallic shell as a potential strategy to protect novel catalyst materials during reactions. The growth
kinetics of core–shell structures was studied and quantified by Wu et al. [91]. Using the Pt-Au as the
model system, three different growth stages of the Au shell were revealed; firstly, Au deposited on the
corner sites of Pt icosahedral nanoparticles, then Au diffused from corners to terraces and edges, and
finally Au grew on Au surfaces layer-by-layer to form the final Pt-Au core–shell structure.
The formation of nanorods from the assembly of nanoparticles was demonstrated by Liao et al. [82].
As shown in figure 4e. Pt3Fe nanoparticles were nucleated and grown with the electron beam. A uniform
size distribution was reached and stabilized by the surfactant. A self-assembly process of the particles
(attachment, reorientation and size adjustment) then led to the formation of a single crystal nanorod.
The effect of surfactant concentration [104] on the morphology and stability of the formed Pt3Fe
nanocrystals and nanorods were also studied.
Other in situ liquid cell TEM works on more complex systems include nanocage formation using galvanic
replacement [86], metal–organic framework’s (MOF) nucleation and growth rate with respect to different
metal-to-ligand ratio [92], iron Keggin ion’s building/conversion process to magnetite and ferrihydrite
[105], the growth of various metal–Fe–oxide nanoparticles using various combinations of different
precursor metal solutions [106] etc. These studies demonstrate the power of liquid cell TEM to image a
wide range of nanomaterial structures, which can help us understand their growth mechanisms, and thus
allow us to make informed choices in our synthesis strategies for nanomaterial preparation. With such
knowledge, better control of the growth and tailoring of the morphology can be achieved.
5.1.3. The effects of the growth environment 12
As shown before, applying bias and electron beam irradiation are the most common ways to grow
nanostructures in liquid cell TEM, although temperature control can also be used to induce growth
and dissolution [107]. Regardless of the growth method, two key factors always dominate the growth
kinetics and the resultant material: precursor solution composition and electron beam radiation. Many
works have been performed to assess their effects.
Different precursor solutions can lead to different growth mechanisms. For example, Yang et al. [96]
showed that by adding HCl into the precursor solution, the Pd particles switched from three-
dimensional island growth to aggregative growth. Such visualization helped explain the change in the
resultant morphology (from smooth to porous and open morphology). Abellan et al. [93] visualized
how, by adding a capping agent tri-n-octylphosphine (TOP) to the solution, precise size control of the
grown Pd nanoparticle to sub-3 nm can be achieved. Ge et al. [94] showed anomalously fast growth rates
of Ag nanoparticles when the growth took place adjacent to Pt, providing experimental proof of the

catalytic effect of Pt on the growth of nanomaterials. A future avenue for such in situ TEM experiments
could be to use the liquid flow lines into the liquid cell to inject different reactants at sequential stages,
allowing the controlled growth of more complex nanostructures. Apart from precursor solutions, using
different electron beam currents can also lead to different growth mechanisms, and thus resultant
morphologies, during nanoparticle growth in a liquid cell, as shown by Woehl et al. [80]. Systematic
studies to quantify the beam current effect were later performed by Park et al. [88] and Alloyeau et al.
[89] using Au nanoparticles as the model system. Park et al. showed that a minimum dose rate is
required to nucleate the Au nanomaterials due to the competition between the reduction and oxidation
processes in the solution. Above such limit, particles’ growth rate follows a power law with dose rate.
However, such power law relation does not apply to very high electron beam dose, as shown by
Alloyeau et al. [89]. Alloyeau et al. showed that, at relatively lower electron dose, the growth is
thermodynamically driven, i.e. a higher growth rate under a higher electron beam dose. However, an
upper limit exists, from which the growth becomes dominated by kinetics, i.e. diffusion, and higher dose
cannot further increase growth rate. The morphology of the grown particle is also shown to be different
for thermodynamically dominated and kinetically dominated growth.
These in situ experiments allow us to better understand and model growth mechanisms within
different environments, which is useful to grow, stabilize and manipulate different nanostructures. For
example, Zečević et al. [108] showed that under STEM, the narrowly focused electron beam probe can
elongate silica nanoparticles. Tian et al. [109] showed that the electron beam can split AgCl
nanocrystals, which reassemble when illumination terminates. As we will discuss later, often the
electron beam effect is something that the experimentalist wants to avoid, with beam-induced damage
changing the behaviour of the sample in adverse ways. However, these above experiments show that
the beam can be valuable as the trigger to instigate reactions inside the TEM.
In situ imaging can also be used to test materials for different applications. For example,
Hermannsdörfer et al. [87] performed a comprehensive study of the environment’s effect (pH, NaCl,
beam dose) on Au nanoparticles’ behaviour (dissolution or merging), showing the change in particle
size and morphology in response to the change in environment. Au nanoparticles can be potentially
used for medicine, like imaging and protein targeting drug delivery. Such in situ work is useful since
it is important to know how Au particles behave in different parts of the body.
5.1.4. Nanoparticle movement and interactions within the liquid cell

Liquid cell TEM can be used to image the movement and interactions of nanoparticles directly at nanoscale
resolutions, rather than relying on the indirect measurements of spectroscopy techniques. Zheng et al. [4]
showed the in situ observation of Au nanoparticle and nanorod motion in a liquid thin film driven by
solvent evaporation. Three distinct modes of motion of nanorods were observed: when the particles are
relatively far from the fluid front, its motion is a combination of the slow displacement mode (Brownian
motion) and the occasional jump mode (nanorod rolling). The fastest motion mode took place when the
nanorods were close to the drying patch where they were dragged by the fast-moving fluid front.
However, solvent evaporation is hard to control within a liquid cell. Thanks to the development of
liquid cell with controlled liquid flow [45], nanoparticle movement with respect to liquid flow can be
systematically studied with respect to the flow rate. For example, Verch et al. [110] demonstrated that
the Au nanoparticles’ motion close to the membrane is three orders of magnitude slower than expected
given the flow rate. By quantitatively comparing the nanoparticle motion with respect to electron dose,
particle coating, membrane surface charge, viscosity and liquid thickness, it was found that such slowing is 13
due to the formation of an ordered layer with viscosity five orders of magnitude larger than bulk liquid due
to the surface charge of the silicon nitride windows.
Particle motions due to various interactions can also be studied using liquid cell, including
nanoparticle–nanoparticle interactions [111–113], particle–beam interactions [114], particle–bubble
interaction [115] etc. For example, Park et al. [113] showed how Au nanoparticles may self-assemble
into a superlattice. By tracking the movement of individual nanoparticles with respect to time, he was
able to quantify the driving force for the process [116]: strong long-range anisotropic forces first drive
the formation of chains from particles, then these chains fold to form the closed-pack superlattice due
to close-range van der Waals forces. Using the same technique, processes like nanomaterial growth by
particle attachment [81,117] can also be tracked and better understood.
5.2. Battery research


5.2.1. Dendrite formation
Metallic dendrite formation within a battery can lead to short-circuiting and even explosion. Upon
cycling, Lithium metal can deposit and form dendrites on the anode. These dendrites can pierce
through the battery separator and establish an electronic connection between the two electrodes. Such
short-circuiting can lead to excessive localized heating which can lead to explosion. Further, the
irregular deposition of dendritic lithium can lead to the build-up of ‘dead lithium’ that is not
removed. This reduces the efficiency and capacity of the battery. Understanding this dendrite
formation mechanism is of paramount importance to allow the application of more efficient and safer
batteries with lower capacity loss upon cycling.
Since Li is a very light and reactive element, its in situ dendrite formation experiment was first difficult to
conduct under TEM. Instead, most early in situ TEM dendrite growth experiments with liquid cells were
performed with other metals, including Au, Pd [118], Cu [119] and Pb [120,121]. These experiments
improved the understanding of metallic dendrite growth in general, based on which the in situ study of
the Li system was later realized. The work on Pb dendrite by White et al. [120] is particularly interesting,
as shown in figure 5a. Not only were the plating and stripping process of Pb dendrites clearly revealed,
the change in Pb2+ ionic concentration was also visualized by tracing the change in image contrast after
subtracting two consecutive frames from the in situ movie. Such visualization is useful since it can trace
the ion diffusion process, which is critical to the understanding of the growth kinetics of the dendrite.
With better spatial and temporal resolution, the growth kinetics can then be quantified.
The visualization of Li dendrite formation was realized in 2014, during which a few researchers, Zeng
et al. [15], Mehdi et al. [29] and Sacci et al. [14], managed to capture the Li dendrite formation and
dissolution with liquid cells using common commercialized Li battery electrolytes like LiPF6/EC/DEC
and LiPF6/PC. All three works reported the formation of a solid–electrolyte interface (SEI), which is a
critical factor in battery research, dominating the plating and stripping processes during battery cycles.
Yet the observed SEI by the three groups of researchers are very different in each case: Zeng et al. showed
a uniform layered SEI formed on the Au electrode first, and then Li dendrite deposited on top of the SEI
layer. Sacci et al. showed that the SEI, rather than a uniform layer, formed in a dendritic morphology, after
which Li deposited in between these SEI dendrites. Mehdi et al.’s results can be seen in figure 5b,c.
Figure 5c shows a uniform layered SEI formed on top of the deposited Li. However, such SEI only formed
on Li dendrites that were still in good electric contact with Pt electrode. ‘Dead’ Li no longer in contract
with the Pt did not form such SEI, and could not be stripped during delithiation. These ‘dead’ Li parts
become detached from the electrode during delithiation, shown as the residual pieces after ‘Li dissolution’
in figure 5b. Despite the difference in the reported SEI morphology and location, one thing in common in
all three reports was that the SEI does not change much during the stripping process. Another
observation reported by Mehdi et al. was that cracking could be found in the Pt electrode after cycling,
which can be seen in figure 5b. Such cracking is probably due to the strain from the Pt-Li alloying process
due to lithiation. Such cracking was also reported in Au electrode by Zeng et al. [124].
Additives are commonly introduced into electrolytes in commercial battery to stabilize SEI formation
and to supress dendrite formation [125]. Using Zn deposition as a model, Park et al. [126] successfully
showed that the addition of Bi into the electrolyte can greatly mitigate the formation of Zn dendrites
and gives a much more uniform and smooth coverage over the electrode. The additive effect on Li
dendrite formation was studied in situ in 2015 by Mehdi et al. [122,127]. By adding trace amount of
water, HF was formed and studied as an additive. This is shown in figure 5d. With more HF as the
(a) + 14
(i) (ii) (iii) (iv) (ix) a0
0
(v) (vi) (vii) (viii) (x) b0
1 mm 1 mm
–
(b) cyclic voltammetry Li deposition Li dissolution
100
80 1st cycle BF STEM <4 S 4:20 min 7:00 min
60
40
J/mA cm–2
20 Li dissolution
0
–20
–40 Li deposition
–60 1 mm

–80
–100
100
80 2nd cycle BF STEM <4 S 3.52 min 7.00 min
60
40 Li dissolution
J/mA cm–2
20
0
–20
–40 Li deposition
–60 2 mm
–80
–100
100
80 3rd cycle BF STEM <4 S 5:00 min 7:00 min
60
40
J/mA cm–2
20 Li dissolution
0
–20
–40 Li deposition
–60
–80
2 mm
–100
–4 –3 –2 –1 0
E/V versus Pt
(c)
(i) (ii) (iii) 1.29 mm (iv) 1.93 mm
920 nm
120 nm 536 nm 714 nm
280 nm 440 nm
571 nm
400 nm 464 nm
2 mm 2 mm 2 mm 2 mm
(d) 18
normalized volume of
80 (i) 50 ppm of H2O

10 ppm of H2O
16 (ii) 50 ppm of H2O (iii) 10 ppm (iv) 50 ppm
14 10 ppm of H2O
lithium/mm3
J/mA cm–2
40 12
stripping of Li
10
0 8
deposition of Li
6
–40
4
2 5 mm
–80 5 mm
0
–4.0 –3.5 –3.0 –2.5 –2.0 –1.5 –1.0 –0.5 0 200 250 300 350 400
E versus Pt/V time (s)
(e) (i) time (s)

400 600 800
voltage (V)
0
(ii) (iii) (iv) (v) (vi) (vii) (viii)
Figure 5. (a) In situ study of lead dendrite plating and stripping from lead ions in aqueous solution [120]. Sequential dendrite plating
at −1.3 V (i–iv) and stripping at +0.3 V (iv–vii). (viii) Specimen surface after two cycles of plating and stripping. (ix–x) The change in Pb2+
concentration in the liquid visualized by subtracting two consecutive frames from the in situ movie, lighter or darker regions indicate where
the intensity increases or decreases. (b) In situ Li plating and stripping on Pt working electrode in LiPF6/PC [29]. The first, second and third
rows correspond to the first, second and third charge/discharge cycles, showing the cyclic voltammogram, electrode before plating, after
plating and after stripping for each cycle. (c) The interface between the Pt working electrode and the LiPF6/PC electrolyte after cycles 5–8,
the red line shows the outside of the bright contrast SEI layer while the blue line shows the outline of the Pt electrode [29]. (d) Effect of HF
formed from water as an additive to the Li plating and stripping process in LiPF6/PC electrolyte [122]. (i) Cyclic voltammograms of electrolyte
with 10 and 50 ppm water. (ii) Quantified total area of Li deposited and stripped for the electrolyte with 10 and 50 ppm of water. (iii) Li
deposited on Pt working electrode in electrolyte with 10 ppm water. (iv) Li deposited on Pt working electrode in electrolyte with 50 ppm
water. (e) Sequential evolution of a LiFePO4/FePO4 cluster during one cycle [123]. (i) The charge/discharge voltage profile. (ii–viii) 5 eV
EFTEM images, brighter particles indicated by the arrows are FePO4 particles formed from delithiation of LiFePO4 during charging. Scale bar
200 nm.
additive, rather than the typical dendrite morphology, a smooth and dense layer was deposited. In 15
addition, more Li was deposited and stripped during the cycle and a better coulombic efficiency was
achieved. This is due to the increased LiF concentration within the SEI, which helped sustain faster Li+
cation diffusion through the highly conductive LiF channels. The effect of LiNO3 as an additive was
also studied [128], which has been shown to induce distinctive difference on the dendrite morphology.
Other battery systems beyond Li-ion can also be tested with liquid cell TEM. Mg plating and SEI
formation were visualized by Singh et al. [129] in 2018. The non-dendrite growth and the formation of
the highly functional SEI, which allows continuous deposition and dissolution of Mg metal, have
shown Mg as a promising candidate for future batteries. Liquid TEM provides us a means for the
direct visualization of time-resolved dendrite morphology change [130], allowing such processes to be
quantified [131] and for the growth mechanism to be better understood. This can help us find good
ways for morphology control to mitigate the dendrite problem in batteries, via tuning of the
electrolyte and electrode chemistry and electrode topography.

5.2.2. Electrode materials lithiation/delithiation
As mentioned, lithiation/delithiation are the incorporation/removal process of lithium from an electrode
in a lithium ion battery. The volume and structural change during lithiation and delithiation is one of the
key electrode degradation mechanisms which leads to battery capacity loss and failure. Therefore,
understanding these processes within electrode materials is critical.
Lithiation has been performed on various materials with ionic liquids using open-cell TEM. However, ionic
liquid is rarely used in actual batteries so the representativeness of such experiments compared with real
lithiation processes in batteries is limited. The development of the TEM liquid cell has allowed lithiation
experiments to be performed using commonly used Li-battery electrolytes. Gu et al. [132] showed the in situ
lithiation/delithiation process of Si nanowire within a liquid cell. This experiment showed different dynamic
structural evolution to the case for the open cell. This set-up allowed more insight into understanding of the
lithiation process in common electrolytes, for example, the formation and evolution of the SEI.
Another way of studying electrode materials is depositing these materials onto the Pt or Au
electrodes of commercialized liquid cells, as performed by Holtz et al. [123], and shown in figure 5e.
LiFePO4, one of the most commonly used cathode materials, was deposited onto the cell electrode
using an inkjet printer capable of precise localized deposition of picolitres of LiFePO4 nanoparticle
suspension. By using energy-filtered TEM (EFTEM), the transition between the lithiated LiFePO4 state
and the delithiated FePO4 state can be clearly distinguished during charging and discharging. In 2015,
Zeng et al. [133] performed lithiation and delithiation of MoS2 nanosheets, showing the irreversible
decomposition process of nanosheets into nanoparticles upon lithiation. By using commonly used
commercialized electrolyte, these experiments are more representative of the actual degradation
process of the electrodes in commonly used Li batteries. Other than batteries, the liquid cell can also
be used to test other energy materials, including for fuel cells [134] and photocatalysts [135].
One aspect that is important to control for in these experiments is the effect of the electron beam on
the electrolyte. As mentioned, the electron beam can lead to the formation of radiolysis species like
hydrated electrons, e h , a reducing agent, which may lead to the degradation of electrolyte, depositing
various species without electrical biasing. Abellan et al. [136] tested the electron beam’s effect on
several common Li electrolytes, and showed strong beam-induced artefacts especially for LiAsF6
solutions. LiPF6 salt electrolytes were found to exhibit fewer artefacts under the beam. For in situ TEM
results to be representative, it is always a good idea to check that such beam effects are not too severe
by performing control experiments.
5.3. Biological cells

Applying in situ liquid cell TEM toward the study of biological specimens has emerged as an alternative
to the more commonly used cryogenic TEM technique. In 2008, Liu et al. [56] demonstrated that a closed
cell can be used to image biological specimens. Two types of bacteria and their in situ tellurite reduction
process were imaged. Liu et al. also showed that neither the sealing nor the TEM beam does significant
damage to the studied bacteria. Within the same year, de Jonge et al. [8] demonstrated for the first time
that a liquid cell can be used to image a whole eukaryote cell (fibroblast) of several micrometre size fully
immersed in liquid.
Visualizing cells without significant damage requires imaging with a low dose. Combining this
limitation with the presence of a thick liquid layer makes it difficult to achieve reasonable resolution
or contrast [137]. However, de Jonge showed that labelling with Au-tagged epidermal growth factor 16
(EGF) molecules, a resolution down to 4 nm could be achieved despite having a liquid thickness of
7 ± 1 µm. Since then, combining Au-tagging and phase contrast in STEM mode became a popular
technique to identify and trace the in situ changes of specific proteins within live biological cells in
liquid state [138]. For example, in 2011, using such a technique, Peckys et al. [11] were able to identify
nanoparticles around vesicles which helped trace and quantify vesicles’ accumulation process.
Another advantage of using liquid cell for biological specimen imaging is that cells can be easily
grown onto the SiN membrane. And by labelling certain proteins using both affibody and quantum
dots, the same cell can be imaged with light microscopy [139], SEM and STEM [140] using a
technique called correlative fluorescence and electron microscopy [139,141,142]. This is demonstrated
in figure 6. HER2 within a SKBR3 cell were labelled with quantum dots, which can emit bright
fluorescence signals under conventional light microscopy. Quantum dots’ electron-dense core can also
be easily distinguished under STEM. In this case, the STEM was performed with an environmental
SEM with saturated water vapour atmosphere. However, the same chip is also compatible with

imaging under liquid cell STEM, as shown in figure 6b(iii).
This labelling technique, combined with high-resolution TEM, can be used for quantitative study of
the distribution of the proteins within the cell [143]. For example, from the inter-label distances, single-
labelled protein monomer and double-labelled protein pairs can be distinguished, and from their
distribution, Peckys et al. [144] revealed the dimeric conformation of the TMEM16A protein. This
technique is an efficient way to elucidate the stoichiometry distribution and any possible change of
various proteins on the cell membrane [47,145,146]. And not only proteins, it has been shown that
nanoparticle labelling can also be done on DNA [49].
Apart from imaging proteins, in situ interaction studies of cells can also be performed. For example,
Pohlmann et al. [147] monitored the interaction of glioblastoma (brain tumour) stem cells with
nanoparticles. Such visualization can help the understanding and improvement of nanoparticle-based
therapy to treat diseases like glioblastoma.
5.4. Other research works based on liquid cell TEM

Apart from nanomaterial synthesis and manipulation, battery cycling and biological cells, liquid cell
TEM has also been used in a wide range of other fields. Examples include localized corrosion kinetics
[16,148,149], biological mineralization processes [150–152], and in situ patterning [153] and electron
beam lithography [154]. And with better resolution and more customized functionalities, more
research fields will be explored using liquid cell TEM in the future [155].
6. Challenges and possible future improvements

There are three main challenges in performing liquid cell TEM: resolution, electron beam damage and
representativeness.
6.1. Resolution
Liquid cell TEM suffers from poor resolution mainly due to electron scattering from the SiN window and
the liquid layer. Liquid thicknesses of up to several micrometres can be imaged, but resolution will be
heavily compromised [8,11]. A quantitative study showing the correlation between the resolution and
the liquid layer thickness was done by de Jonge et al. [58].
Unnecessarily thick liquid can be formed due to bowing of the membrane, leading to reduced
resolution [11,57]. Such bowing is caused by the pressure difference between the inside and outside
the liquid cell. There are several possible ways to avoid bowing. Increasing the membrane thickness
can reduce bowing and make the window more robust, but resolution will decrease due to the
electron scattering with the membrane material itself. Another way is to incorporate micrometre-sized
pillars pinning the top and bottom window together [156]. However, this requires the manufacture of
top and bottom chips as a single piece, which is more expensive and prone to breaking. The most
popular way to reduce bowing is to simply reduce the size of the window.
A good way to mitigate electron scattering by the window is to use alternative membrane materials.
For example, graphene-membrane cells achieve better resolution than SiN [47–52]. Recently, MoS2 [157]
was also successfully used as the membrane material to see Pt nanoparticle growth. However, as
17
(a)
nm
HER2-monomer
(extracellular domains)
4
~1
streptavidin-QDs
4 nm
biotinylated anti-HER2 affibody
~8 nm
(b)
(i) imaging buffer (ii) 3°C, 720–740 Pa saturated H2O vapour
electron
beam

SiN membrane microchip (30 kV) liquid H2O film
cell
nucleus
glass QDs on ErbB2
63× oil
immersion scattered electrons (towards
objective dark-field STEM detector)
(iii)
silicon
microchip electron
beam
cell liquid
flow
SiN membrane
scattered
electrons
(c)
(i) (ii) (iii) (iv)
(v) (vi)
Figure 6. (a) Schematics of HER2 labelling with affibody-quantum dots [141]. (b) Schematics showing correlative microscopy of
whole cells in liquid cell. (i) Fluorescence imaging [141]. (ii) For STEM imaging within environmental SEM [141]. (iii) For STEM
imaging inside TEM [139]. (c) Correlative light and electron microscopic images of HER2-labelled SKBR3 cell [141].
(i) Fluorescence overview image showing several dozen cells. (ii) Higher-resolution fluorescence image from marked solid line
rectangle in (i). (iii) Even higher-resolution fluorescence image from selected area in (ii). (iv) STEM image of the same area as
(iii). (v) STEM image of the boxed region shown in (iii) and (iv). (vi) Automatically detected labels outlined in light green.
Scale bars, 100 µm (i), 20 µm (ii); 2 µm (iii and iv); 200 nm (v and vi).
Table 2. Different types of electron beam damage and their effects on liquid cell TEM (dotted lines indicate relatively 18
insignificant contribution).
electron beam damage
electron irradiation damage
scattering elastic scattering inelastic scattering

types
atom electrostatic heating radiolysis hydrocarbon

beam damage displacement and charging contamination
types sputtering

effects on unwanted particle biological cell bubble unwanted relatively
relatively
liquid cell movement damage formation reactions insignificant
insignificant
- low-energy beam - high-energy beam - liquid flow - cooling - cooling - cooling

- low e– dose - low e– dose - extra cooling - low e– dose - low e– dose - low e– dose
possible - change membrane - low e– dose - high-energy - high-energy - high-energy
solutions - high-energy beam beam beam
beam - radical - radical - radical
- use correlative scavengers scavengers scavengers
microscopy - specimen
preparation
mentioned, these membrane materials can only encapsulate a very small amount of liquid, and they are
not compatible with microfabrication and therefore functions like electrical biasing and liquid flow.
Using correlative techniques like AFM, traction force microscopy (TFM) [158] can also help obtain
more information and potentially better resolution.
6.2. Electron beam damage

The electron beam can induce many artefacts during the imaging process within a liquid cell [159]. Here
we will cover the origin of these artefacts and discuss with respect to the five types of beam damage that
commonly occur in electron microscopy, as summarized by Egerton et al. [160] and shown in table 2. Both
elastic and inelastic electron scattering can lead to beam damage. Elastic scattering results from the
beam’s interaction with the atomic nucleus, with no energy change of the scattered electron. Inelastic
scattering results from the beam’s interaction with the electron shells of atoms, with accompanying
energy loss that results in secondary electrons, X-ray emission etc.
Atom displacement and sputtering is a type of beam damage due to high-energy, high-angle elastic
electron scattering, which displaces atomic nuclei to interstitial positions, or sputters surface atoms away
from the specimen. Since liquid cell TEM contains mainly liquid phase specimens, such damage is not as
significant as the other types, except potentially when imaging crystalline formation within liquid. A low
electron dose rate or lower beam energy can be used to mitigate its effects.
Electrostatic charging can be due to both elastic and inelastic scattering, although inelastic scattering
has a dominant effect due to the generation of secondary electrons. Within a liquid cell, charge can build
up on the insulating SiN membranes. And such surface charge is worsened by a local enhancement of
electron dose formed at the solid–liquid interface which acts as secondary electron source due to
scattering [100]. Such surface charge not only interferes with the incoming beam, it can also induce
unwanted particle movement within liquid due to repulsion [114]. One of the proposed solutions is to
use a very low-energy beam that does not have enough energy to generate secondary electrons.
However, such energy limit is usually 50–150 eV [160], which is way too low for any electrons to
transmit through the specimen and therefore is not a plausible method. A better solution for TEM is,
instead, to use very high energy that has a bigger mean free path for scattering, accompanied by a
thin specimen, so most of the beam will transmit through the specimen without generating secondary
electrons. Reducing the electron dose rate can also reduce the total charging effect. Another potential
solution is to use a different membrane material. For example, using graphene instead of SiN was 19
reported to reduce such beam damage, allowing higher dose by one magnitude for imaging [161].
Heating is another type of beam damage due to inelastic scattering of electrons where part of their
kinetic energy was converted to thermal energy. The relatively large liquid volume within the liquid
cell and possible liquid flow system allows good heat exchange to prevent localized heating, which
makes heating a relatively insignificant beam damage mechanism in the liquid cell. However, this can
still contribute to the degradation of biological cells that are generally very sensitive to localized
heating and are normally characterized within an enclosed cell without liquid flow.
A bigger contributor to biological cell degradation is radiolysis due to the electron beam, which
destroys the cells [12,138] and induces structural change to biological materials [54]. Keeping a low
electron dose is critical to limit the cell damage during TEM imaging. A systematic damage study on
the cell by beam dose was performed by Hermannsdörfer et al. [162] in 2016. They found a beam
dose limit of around 102 electrons nm−2 should not be exceeded during imaging [59,162]. It is almost
impossible to have good contrast or resolution of organic biological features at such low dose [137].

Therefore, the best way is to use nanoparticle labelling protein [8,12] and use correlative light and
electron microscopy [139,141], which should give enough contrast at very low dose.
Radiolysis is the most critical beam damage mechanism in most liquid cell TEM experiments.
Radiolysis can lead to bubble formation and expansion, and other unwanted reactions. It has been
shown that intensive illumination can lead to bubble formation [163] due to hydrolysis of water and
lead to the growth of existing bubbles [164]. Systematic studies of beam-induced gas formation within
liquid cell were performed [165,166]. It is well known that the electron beam can induce reactions,
which can be beneficial as a way to control the reactions we want to observe [3,6]. However,
unwanted reduction of the ions within the solution can lead to crystallization of unwanted
nanoparticles [167] which is a common artefact. Other artefacts include particle shape change [108],
gelation of ionic liquid [34] etc. These arise from the radical species and aqueous electrons formed
within the liquid due to beam irradiation. As mentioned before, these radicals and aqueous electrons
are strong reducing species that will react with aqueous electronegative ions and induce or accelerate
the reaction [86]. Apart from using a lower dose, radiolysis can also be reduced by reducing
temperature and adding scavenger molecules to reduce the radicals.
Hydrocarbon contamination due to inelastic scattering is not well understood within a liquid cell, since
it is hard to distinguish its effects from those due to radiolysis, especially on the inside surface exposed to
liquid. However, better cell preparation including plasma cleaning, heating and electron beam shower can
help get rid of the hydrocarbon contamination on the SiN outside surface exposed to vacuum.
Overall, electron beam damage from inelastic scattering is responsible for most of the artefacts
observed during TEM imaging of liquid phase samples. The best way to mitigate their effects is to use
a high beam energy (to increase the mean free path and reduce total scattering events, and therefore
the generation of secondary electrons) and a low electron dose rate (to reduce total scattering events).
It is also important to perform appropriate control experiments where possible, so that beam-induced
effects can be distinguished from the experiment of interest.
Thanks to the recent development in (S)TEM, better image contrast can be achieved at a much lower
dose. For example, sub-sampling approaches [168,169] allow for high-resolution imaging with very low
dose. Acquisition optimization techniques like feature adaptive sampling [170] can also reduce beam
damage by reducing unnecessary beam scans to have a lower total beam dose. Further development
of low-dose optimized electron microscopy techniques will be essential to realize the objective of in
situ liquid TEM studies without beam-induced artefacts, and thus an accurate representation of the
actual processes that occur in biological cells, batteries and nanostructure synthesis.
6.3. Representativeness
Due to the limited solution volume and confined space, in addition to the beam damage mechanisms
discussed above, liquid cell TEM results’ representativeness is always in doubt. For example, studies of
battery electrode materials using liquid cell are currently limited. The electrode materials were either
assembled onto the microchips by picolitre drop casting [123] or by flowing a suspension [133]. Not only
do these methods have a low success rate, the amount of material deposited onto the chip is too small,
which makes such systems far from being representative of their real battery counterparts. To improve its
representativeness, a larger amount of material with better attachment to the electrode should be achieved
by either nanoscale thin-film patterning [153,154,171] or by focused ion beam (FIB) and nanoscale
welding [172]. Membrane materials can also cause problems, making liquid cell TEM less representative.
Donev et al. [173] showed that for electron-beam-induced deposition, the membrane material can have a great 20
influence on the morphology and size of the nanoparticles formed. For the Cu deposition and stripping
experiment [1], the cyclic voltammogram in liquid cell is quite different from that performed in bulk
solution. This is due to the hindered diffusion of liquid and particle within the liquid cell. Such hindered
diffusion can also contribute to the unexpected size and number distribution of the deposited material
[99]. Nanoparticle diffusion in liquid cell was reported to be up to seven to nine orders slower than
expected for a bulk solution [155]. This slowing is due to increase in liquid viscosity (by five orders of
magnitude) [110] caused by the formation of an ordered liquid layer due to the surface charge [174] of the
silicon nitride windows. A solution for this problem is to either find a way to cancel the charge on the SiN
membrane or to use a different membrane material that does not interact or charge as easily.
6.4. Other problems

Apart from the three main challenges mentioned above, there are other common issues like trapped

bubbles [4,56] that can be solved with better cell design, and sample movement during imaging
which can lead to compromised resolution and requires additional post-experiment processing to
track individual particles [84]. In this case, immobilization by functionalizing SiN [175,176] can be
used to fix material to improve the image stability and resolution. A final problem that is worth
noting is the technically demanding nature of performing in situ liquid cell TEM. However, the
competitive commercial environment for liquid cell TEM holders has led to increasingly user-friendly
systems with each generation.
So far, compared with imaging solid specimens using TEM, imaging liquid is still quite limited. There
is still great potential to improve the technique. For example, most studies only perform bright/dark field
STEM. More information can be potentially extracted if the liquid cell experiments can be further
optimized for functions like diffraction, EELS and tomography. The cell design could also be
improved so that the final electrode products from the in situ experiment could be non-destructively
removed from the sample chip, such that they could be analysed using other complementary
characterization techniques ex situ. Finally, developing TEM holders that allow the sample-holding tip
to be detached from the holder would allow complementary experiments to TEM to be performed
more easily, as the tip could be loaded in the appropriate instrument.
Data accessibility. This article has no additional data.
Authors’ contributions. S.P. and C.G. both wrote the initial review drafts. A.W.R. set the scope, advised on references and
finalized the manuscript.
Competing interests. We declare we have no competing interests.
Funding. A.W.R. is funded by a Royal Society University Research Fellowship.
Acknowledgements. A.W.R. would like to thank the support of the Royal Society.
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Liquid cell transmission

This article has been edited by the Royal Society 1. Introduction

R. Soc. open sci. 7: 191204

2. Different TEM configurations for imaging liquid samples

low-vapour- TEM with differential microchip graphene liquid

R. Soc. open sci. 7: 191204

advantages: advantages: advantages: advantages:

disadvantages: disadvantages: disadvantages: disadvantages:

deposit SiN layer assemble with top chip

R. Soc. open sci. 7: 191204

3. Liquid cell design and application

R. Soc. open sci. 7: 191204

dSTEM / T 1=2 : ð3:2Þ

dTEM,STEM / D1=4 : ð3:3Þ

4. Liquid research with conventional TEM and open environmental TEM

R. Soc. open sci. 7: 191204

R. Soc. open sci. 7: 191204

5. Research with liquid cell

materials observation author methods importance

R. Soc. open sci. 7: 191204 royalsocietypublishing.org/journal/rsos

materials observation author methods importance

R. Soc. open sci. 7: 191204 royalsocietypublishing.org/journal/rsos

R. Soc. open sci. 7: 191204

(v) (vi) (vii) (viii)

(d) 001 view along [011]

(ii) 0:00 4:44 6:13 9:39 12:06 16:06

R. Soc. open sci. 7: 191204

5.1.2. The growth of more complex structures

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5.1.4. Nanoparticle movement and interactions within the liquid cell

5.2. Battery research

R. Soc. open sci. 7: 191204

R. Soc. open sci. 7: 191204

80 (i) 50 ppm of H2O

(e) (i) time (s)

R. Soc. open sci. 7: 191204

5.3. Biological cells

R. Soc. open sci. 7: 191204

5.4. Other research works based on liquid cell TEM

6. Challenges and possible future improvements

biotinylated anti-HER2 affibody

R. Soc. open sci. 7: 191204

electron irradiation damage

scattering elastic scattering inelastic scattering

atom electrostatic heating radiolysis hydrocarbon

R. Soc. open sci. 7: 191204

- low-energy beam - high-energy beam - liquid flow - cooling - cooling - cooling

6.2. Electron beam damage

R. Soc. open sci. 7: 191204

6.4. Other problems

R. Soc. open sci. 7: 191204

R. Soc. open sci. 7: 191204

R. Soc. open sci. 7: 191204

R. Soc. open sci. 7: 191204

R. Soc. open sci. 7: 191204

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