# In situ and operando transmission electron microscopy for the synthesis, fundamental understanding and catalytic applications of nanoalloys

Damien Alloyeau, Nathaly Ortiz Pena, Guillaume Wang, Christian Ricolleau, Jaysen Nelayah

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## To cite this version:

Damien Alloyeau, Nathaly Ortiz Pena, Guillaume Wang, Christian Ricolleau, Jaysen Nelayah. In situ and operando transmission electron microscopy for the synthesis, fundamental understanding and catalytic applications of nanoalloys. Advances in Physics: X, 2025, 10 (1), pp.2481277. 10.1080/23746149.2025.2481277. [hal-05348942](https://cnrs.hal.science/hal-05348942v1)

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# In situ and operando transmission electron microscopy for the synthesis, fundamental understanding and catalytic applications of nanoalloys

Damien Alloyeau, Nathaly Ortiz Pena, Guillaume Wang, Christian Ricolleau & Jaysen Nelayah

**To cite this article:** Damien Alloyeau, Nathaly Ortiz Pena, Guillaume Wang, Christian Ricolleau & Jaysen Nelayah (2025) In situ and operando transmission electron microscopy for the synthesis, fundamental understanding and catalytic applications of nanoalloys, Advances in Physics: X, 10:1, 2481277, DOI: 10.1080/23746149.2025.2481277

**To link to this article:** https://doi.org/10.1080/23746149.2025.2481277

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# In situ and operando transmission electron microscopy for the synthesis, fundamental understanding and catalytic applications of nanoalloys

Damien Alloyeau, Nathaly Ortiz Pena, Guillaume Wang, Christian Ricolleau and Jaysen Nelayah

Laboratoire Matériaux et Phénomènes Quantiques, Université Paris Cité - CNRS, Paris, France

## ABSTRACT

In line with the paradigm of nanoscience, developing functional nanoalloys for catalytic applications requires a thorough understanding of the correlations between their synthesis, structure, properties, and performance. Over the last two decades, the progresses of in situ and operando transmission electron microscopy (TEM) has revolutionized the way to meet this challenge by providing unparalleled observations of nanomaterial dynamics in their formation and application media. The atomic scale resolution and chemical sensitivity of TEM analyses in liquid or gaseous media of controlled composition and temperature are shedding new light on nanoalloys’ nucleation and growth mechanisms, the simultaneous kinetic and thermodynamic effects that dictate their structural evolution and their transformations under catalytic conditions. Through this review, we show the opportunities and challenges of in situ/operando TEM in rationalizing the synthesis

## ARTICLE HISTORY

Received 20 January 2025
Accepted 13 March 2025

## KEYWORDS

Nanoalloys; electron microscopy; fabrication; structural properties; catalysis

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and understanding the complex structural properties and as-
sociated performance of nanoalloys for catalysis and electro-
catalysis. We also discuss how future developments will extend
the impact of TEM analyses in optimizing the full life cycle of
functional nanoalloys.

GRAPHICAL ABSTRACT

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**1. Introduction**

Composed of two or more metals, alloy nanoparticles (NPs) also called
nanoalloys, exhibit unique catalytic, magnetic, or optical properties markedly
different from their bulk counterparts. The ability to tune these properties
by controlling their size, shape, atomic structure, and composition makes
nanoalloys highly valuable for various applications, including gas and liq-
uid phase catalysis, electronics, energy storage, sensing, and biomedicine
[1-3]. Transmission electron microscopy is the most complete characteri-
zation tool for studying multi-metallic alloy nanostructures because it gives
access to morphological, structural, and chemical information on individu-
ally analyzed NPs [4]. These unique opportunities allow for exploring the very
complex conformational space of nanoalloys commonly described in terms of
geometric structure (i.e. spatial arrangement of the atoms without reference
to their chemical species: octahedra, icosahedra, decahedra ...) and mixing
pattern (i.e. the arrangement of each metal within the geometric structure:
core-shell, Janus, chemical ordering ...) [2]. However, ex situ Transmission
Electron Microscopy analyses performed under vacuum remain limited to
fully understanding the structural properties of nanoalloys because, beyond
the influence of size, shape and composition, their atomic-scale conformation
is also governed by kinetics effects that can trap out-of-equilibrium architec-
tures and by chemical reactions that occur in real environments. Therefore,
understanding the structural behavior of nanoalloys directly in their forma-
tion and application media has become a major challenge for gaining control
over their fabrication, picturizing their full life cycle and better assessing their
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performances. In that regard, in situ and operando TEM have emerged as a crucial technique for exploring the dynamics of nanostructures in liquid and gaseous environments of controlled composition and temperatures, providing atomic-scale insights into their real-time structural evolution during their synthesis or under operational conditions [5–8]. This review provides a comprehensive overview of the applications of in situ and operando TEM techniques to nanoalloys, highlighting recent advancements, key methodologies, and prospects, with special emphasis on the liquid-phase synthesis, the thermodynamic properties and the applications of nanoalloys in catalysis and electrocatalysis.

## 2. In situ and operando TEM methods

In situ TEM covers a wide range of electron-based techniques. It refers to the real-time observation with high-energy electrons and recording of the structural and physicochemical properties of a material subjected to external stimuli in a TEM [9–11]. In situ TEM has over the recent decades developed into a pivotal ally to material scientists. With the constant development of novel in situ set-ups and advances in TEM instrumentation and methodologies, it is now possible to apply multiple external stimuli (light, heat, gas, liquid, mechanical stress, or electrical biasing) to nanomaterials in a controlled way and study materials in a TEM beyond the traditional ultra-high vacuum conditions.

Thermal TEM [12], liquid-phase [6] and gas-phase TEM [13] are key in situ TEM techniques that allow exploiting the performances of TEM imaging and spectroscopy to study the thermal behavior of nanomaterials and provide insights into solid-gas and solid-liquid interactions at their surface. Today, the introduction of liquids or gases into a TEM follows two approaches: employing state-of-the-art dedicated TEM equipped with a differential pumping (DP) system [14] or closed cell TEM holders incorporating nanoreactors based on micro-electro-mechanical systems (MEMS) [15]. The first approach is limited to pressurized gases or water vapor, confined in the sample region between the objective pole pieces. This is achieved by implementing inside the TEM column two or more differential pumping stages, separated by apertures, between the sample location and the gun, on one hand, and the sample and imaging detectors on the other (Figure 1a) [14,16]. DP gas TEM operates in mbar pressure domain to maintain a spatial resolution below 2 Å. The second approach is more versatile as it can be extended to liquids. It is implemented with pairs of silicon microchips with thin SiN electron-transparent windows that are vacuum-sealed on dedicated TEM holders and confine the gas or liquid environments within a volume around the specimen ranging from a few µm to a few tens of nanometers in thickness (Figure 1b) [15,18]. With the extreme confinement of the environment, it is possible to reach the
<figure><img src="image_19.png" /><figcaption>**Figure 1.** Technical approaches for the implementation of in situ liquid and gas TEM. (a) Open cell approach (also known as: dedicated environmental TEM or differential pumped TEM). This approach uses a dedicated TEM with an integrated differential pumping system that allows gas to be introduced and controlled in the sample chamber while maintaining the rest of the microscope column under vacuum. Adapted from [16], copyright (2012), with permission from Elsevier. (b) Closed cell approach. In this approach, the gaseous or liquid medium is confined around the sample using vacuum-seal nanoreactors based on silicon microchips. (c) Various designs of graphene liquid cells (GLC) in static and flow modes. Adapted from [17], copyright (2021), with permission from the American Chemical Society.</figcaption></figure>

atmospheric pressure regime while keeping atomic resolution in liquid and gas. The degradation of TEM resolution with the thickness of the in situ environment, especially in liquid [19], has motivated the microfabrication of various cell designs to minimize the bulging of the SiN membrane towards the vacuum to further reduce the cell thickness [20]. Interestingly, liquid and gas cells are compatible with a wide range of solvents and gases and the composition of the studied media is controlled by a microfluidic system that allows injecting chemical additives at the observation window. Furthermore, the use of graphene liquid cells with a liquid thickness below 100 nm drastically improves the signal-to-noise ratio of EDX spectra [17] and the resolution of TEM images which allows following dynamical processes with single atom sensitivity in aberration-corrected microscopes [21]. However, despite the development of graphene liquid cells with flow capabilities [17], the use of static graphene liquid cells is still the most common (Figure 1c).

With advances in microfabrication, MEMS-based chips also integrate heating membranes to tune the temperature of the sample up to 1200°C under vacuum or in gas, or heating resistances to control the temperature of liquid
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cells up to 100°C. The fabrication of electrochemical cells with three electrodes allows for the electrical biasing of the confined liquid volume. These key developments have expanded the possibilities of TEM in the understanding of the nucleation and growth of nanomaterials, their thermal behavior and their applications such as in (electro-)catalysis [22,23], gas sensing [24] and storage [25]. In the field of catalysis, in situ TEM becomes operating TEM when the composition of the liquid/gas environment is quantified by mass spectrometry or gas chromatography making it possible to detect (electro-)chemical reaction products and correlate the dynamics of catalysts with their catalytic performances [26,27]. Therefore, complementary imaging, diffraction, and spectroscopy techniques can be combined not only for material study under in situ conditions but also to understand the effects of solid-liquid and solid-gas interactions on the functional properties of materials.

# 3. Applications and findings

## 3.1. Liquid-phase synthesis of nanoalloys

Wet-chemical synthesis is a very versatile method for controlling the formation of nanoalloys [28]. Still, many aspects of the formation processes of multi-metallic nanostructures remain misunderstood because ex analyses provide an incomplete view of these dynamic processes simultaneously governed by kinetic and thermodynamic effects. By providing the unique opportunity to visualize the nucleation pathway of individual nanostructures and their subsequent growth mechanisms with atomic resolution and chemical sensitivity, liquid-phase TEM (LPTEM) has enabled colloidal chemists to take a fresh look at routinely used synthesis strategies to fabricate well-controlled nanoalloys in aqueous or organic solvents. The different reduction kinetics of metal precursors in solution is a long-lasting issue for controlling the structure and composition of nanoalloys. In that regard, directly observing the co-reduction of metal precursors driven by beam-induced radiolysis can provide crucial information. In this sense, Dachraoui and coworkers performed aberration-corrected STEM HAADF imaging and EDX spectroscopy in graphene liquid cells to reveal that PdPt nanoalloys formed by co-reduction of Pd and Pt precursors in water nucleate through a non-classical pathway with amorphous clusters as intermediate structures [21]. If amorphous intermediates bridging single atoms to nanocrystals were already identified in the formation of monometallic NPs [29–33], this work remarkably demonstrates that these ultra-small clusters are composed of Pd and Pt (Figure 2a), highlighting the homogenous reduction of both precursors. These primary alloy clusters grow by coalescence and monomer attachment but do not all crystallize at the same size leading to the coexistence of amorphous and crystalline nanoclusters in a size range between 1 and 5 nm. Then, mature PdPt
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<figure><img src="image_22.png" /><figcaption>**Figure 2.** Growth of PdPt NPs from aqueous solution under electron beam illumination in a graphene liquid cell. Time-lapse series of atomic resolution HAADF-STEM images show (a) the formation of Pt and Pd atoms in the illuminated area of the liquid and the growth of ultrasmall nanoclusters, (b) the growth of PdPt bimetallic NPs via cluster coalescence. Adapted from [21], copyright (2022) with permission from the American Chemical Society. Seed-mediated synthesis of Au@Ag rods. (c) Time-lapse STEM images showing the anisotropic deposition of silver on a gold bipyramid as well as homonucleation of Ag NPs. The scale bar is 50 nm on all images. (d) Contour plots of the Au@Ag rods extracted every second from image analysis in growth experiments with and without ascorbic acid. Adapted from [34], copyright (2020) with permission from the American Chemical Society.</figcaption></figure>

nanocrystals are formed mainly by nanocluster coalescence (Figure 2b). Still, the nanocluster structure affects the final shape of PdPt NPs since spherical nanoalloys are formed when at least one amorphous cluster is involved in the coalescence processes, while the coalescence of two crystalline nanoclusters generates faceted nanocrystals. Interestingly, the co-reduction of metal precursors can also be inhomogeneous and still leads to the formation of alloy NPs. Indeed, by studying the formation of NiPt NPs by co-reduction of acac precursors in a mixture of organic solvents (benzyl ether, oleylamine and oleic acid), the group of Haimei Zheng in Berkeley has shown that monodisperse and spherical alloy NPs form through a two-stage process, with the initial growth of Ni nanodendrites that are then consumed by the formation of NiPt NPs [35]. Further works of the same group demonstrated that the solute concentration and electron dose rate affect the formation mechanisms and resulting conformation of NiPt NPs [35,36]. The formation processes of nanoalloys become even more unpredictable when more than two metallic
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precursors are present in the reaction media. Recent in situ TEM studies have focused on high-entropy alloys (HEA) NPs formed by the co-reduction of five metals [37,38]. Both studies revealed strong composition disparities in the intermediate nanoclusters leading to the conclusion that the formation of HEA NPs with homogeneous composition requires controlling the aggregation and coalescence processes of these nanoclusters. It is worth noticing that high temperatures are commonly used in the bench-scale synthesis of nanoalloys, particularly to promote the homogeneous reduction of metal precursors [39,40]. Still, the role of this crucial synthesis parameter on the formation of nanoalloys by co-reduction remains unexplored, despite the possibility of tuning the temperature of the reactive media in the liquid cell [41–43].

Seed-mediated syntheses and face-blocking methods are commonly combined to control the shape of nanoalloys [28]. LPTEM has been deeply exploited to study the influence of seed structure and the role of organic ligands in overgrowth processes [34,44–47]. When metallic shells form over nanocrystals made of another metal, the shape of the resulting core-shell NPs depends on the ratio between the deposition and surface diffusion rates of metal atoms. If the atomic deposition is faster than the surface diffusion, the reaction is considered to be under kinetic control leading to branched morphology. On the contrary, the growth is under thermodynamic control if surface diffusion is faster than atomic deposition leading to well-faceted shells. Wu et al. studied Au adsorption and diffusion mechanisms at the surface of Pt icosahedral NPs [46]. By using low electron dose rate conditions to minimize the growth rate of the shell, they have shown that Au initially deposits on corner sites of the Pt icosahedra and then diffuses to the flat surface of the Pt core. The resulting shape of the core-shell NPs depends on the growth rate of Au islands at the corners of Pt icosahedra and the diffusion rate of gold atoms to the Pt facet leading to a conformal coating. Similarly, Aliyah et al. revealed that the deposition of Ag on pentagonal bipyramidal Au seeds starts on the (100) edges of the common base of the two pyramids, transforming the nanostructures into pentagonal nanorods with (100) side facets (Figure 2c) [34]. In this experiment, the pentagonal symmetry of the anisotropic core-shell NPs is conserved over a wide range of growth speeds because the (100) facets are stabilized by ascorbic acid, a molecule generally considered to merely act as a reducing agent (Figure 2d). This work demonstrates the potential of LPTEM in identifying the unexpected role of chemical additives in synthesis media. Interestingly, Tan et al. have shown that increasing the temperature up to 85°C boosts the diffusion of Pd at the surface of Au nanorods and favors growth processes governed by thermodynamics [44], leading to cuboidal shells. LCTEM has also been used to study galvanic replacement or selective etching reactions leading to the formation of hollow bimetallic NPs [48–52].
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More importantly, besides revealing nucleation and growth mechanisms that can sometimes be surprising, liquid phase TEM investigations should always be compared with in situ average measurements (SAXS, UV spectroscopy...) or at least with the intermediate and final products of bench-scale syntheses performed under similar chemical conditions to ensure that the observed atomic processes are not mainly due to confinement or electron beam effects. In most reaction media, there exist electron dose rate conditions in which the nanomaterials fabricated in the TEM are similar to the ones obtained at laboratory scale [34,53,54]. Such multi-scale comparisons are an essential prerequisite to make LPTEM an idea generator in nanochemistry.

## 3.2. Nanophase diagram and thermal behavior of nanoalloys

It is well established that physical and chemical routes produce nanoalloys with metastable structures that are stabilized by kinetic effects or specific deposition (or reduction) sequences during growth experiments. Thus, obtaining as-grown nanoalloys in the thermodynamically stable state is very rare. Heating experiments in a TEM is a powerful strategy to explore the nanophase diagram and thermal stability of nanomaterials from 25 to 1200°C. This approach was notably exploited to study the thermal behavior of Pt-based (NiPt [55–57], CuPt [58], PdPt [59] FePt [60–62] and CoPt [63]), Au-based (AgAu [64], AuFe [65,66]) and recently on high-entropy nanoalloys (CoCuFeNiPd [67] and AuCoCuNiPt [68]), often highlighting strong discrepancies in the phase diagram of bulk and finite-size alloys.

Pan et al [55] followed the thermally induced composition redistribution of NiPt octahedral NPs at a mild annealing temperature of 200–210°C. The as-grown NPs are formed by sandwiched nanostructures with Pt-rich regions in the core and at the surface separated by a sublayer with a NiPt composition in between. They showed a change in the composition and structure during the heating of the NPs. More precisely, they evidenced that the contrast changes in the *in situ* TEM image series are due to the dislocations formed by Ni atoms moving from the core of the particles to their surfaces whose composition becomes almost equiatomic (Ni₄₉Pt₅₁) after 1 h at 200°C. This mechanism gives a good insight into the origin of the enhanced oxidation/reduction reactions catalytic performance of NiPt nanoalloys. Similarly, Lu et al. [56] succeeded in stabilizing NiPt NPs in a solid solution form or in a core/shell Pt/Ni configuration by adjusting the growth kinetics. In situ heating TEM was used to demonstrate that these two configurations have different thermal stability. The solid solution is stable from room temperature to 800°C whereas the as-grown core/shell NPs start to evolve to the full mixing from 500°C by the migration of Ni atoms from the shell to the NP core. At 600°C, the NPs form a complete solid solution. This transformation was also associated with a morphology change implying the disappearance of dense facets ((111), (110) and (100)) at the expense of high index ones ((422) and (522)). In these two works,
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atomic diffusion tends to homogenize the composition of the whole NiPt NPs during annealing. The resulting Face-Centered Cubic (FCC) phase of the NPs is partially in agreement with the bulk phase diagram of NiPt in which im miscibility is not expected, but the L1₀ ordered structure was never observed in the NPs, although it is the thermodynamically stable phase below 650°C at equiatomic composition. As discussed later, chemical ordering is a kinetic process that requires staying long enough above the ordering temperature but below the order-disorder phase transition.

While bulk AuAg alloy forms a perfect solid solution over the whole com position range, it was shown by van der Hoeven [64] that Au/Ag core-shell nanorods can be obtained. By following *in situ* the atomic redistribution of atoms as a function of increasing temperature and time, they show that this redistribution is closely related to the relative Au/Ag composition and the vol ume of the NPs. For higher Ag content or for larger particle sizes, the diffusion is significantly lowered limiting the atomic redistribution within the NPs.

Kamp et al. [65] reported on the fabrication of Au₅₀Fe₅₀ and Au₂₀Fe₈₀ by pulsed laser ablation in liquid, forming core/shell and solid solution configu rations. These authors show that the thermodynamically stable configuration differs depending on the initial composition of the alloy. For the Au₂₀Fe₈₀ composition, whatever the initial configuration, the final stable structure at 700°C is composed of Janus or core shell configuration. For the Au₅₀Fe₅₀ composition, the final stable configuration at 700°C is composed of a homo geneous AuFe solid solution over the whole NPs (Figure 3). This last result is in complete contradiction with the bulk-phase diagram of AuFe since those two metals are not miscible over almost the entire composition range.

HEA NPs are a new class of nanostructures composed of at least 5 ele-
ments in equiatomic composition that crystalize in a solid solution. They have
promising mechanical and catalytic properties at high temperatures due to
the synergy effect between the constituting elements but the thermal stability
and the phase diagram of these nanomaterials are mostly unknown. Krouna
et al [68] have recently revealed by *in situ* heating TEM in vacuum, the for-
mation and melting of an AuCu layer at the surface of quinary AuCoCuNiPt
NPs at high temperatures. This phase separation appearing progressively with
increasing temperature is driven by enhanced atomic diffusion that is, unex-
pectedly, more active in the quinary system by comparison to monometallic
and bimetallic subsystems, ruling out the sluggish diffusion kinetics proposed
in HEA. As confirmed by molecular dynamic simulation, this phase separa-
tion driven by the low surface energy of Au and Cu indicates that the solid
phase solution of as-grown HEA NPs fabricated by Pulsed Laser Deposition
(PLD) is stabilized by kinetic effects rather than the entropy of mixing.

All these in situ TEM experiments illustrate that besides composition and
temperature that influence the thermodynamic equilibrium of bulk alloys,
the conformation of nanoalloys is also affected by size and surface effects.
Moreover, it also depends on the out-of-equilibrium structure obtained after
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<figure><img src="image_27.png" /><figcaption>**Figure 3.** HAADF STEM images of the structural evolution of gold-iron NPs as a function of increasing temperature (left side) and scheme of the temperature-dependent morphological transformation (right side, grey for Fe and yellow for Au) for (a) Au<sub>50</sub>Fe<sub>50</sub> and (b) Au<sub>20</sub>Fe<sub>80</sub> compositions. The scale bar is 25 nm. Adapted from [65], copyright (2018) with permission from the American Chemical Society.</figcaption></figure>

the synthesis. Beyond revealing conformational changes induced by temperature, in situ heating TEM experiments performed under vacuum are also important for the field of catalysis because they allow for the distinction of the effects of heat and gases on the dynamics of nanocatalysts.

Interestingly, in situ heating TEM also enables comparing the transformation kinetics of nanoalloys that are governed by various phenomena during annealing (phase transition, coalescence, Ostwald ripening ...). The fabrication of L1₀ chemically-ordered CoPt or FePt NPs below 10 nm in size opens up promising applications based on the ferromagnetic properties of these small nanoalloys. Nevertheless, low-temperature chemical and physical fabrication methods (below 600 °C) are mainly governed by kinetic effects leading to the stabilization of small FCC chemically disordered NPs in disagreement with the bulk phase diagram. Post-synthesis annealing is commonly used to obtain the L1₀ structure. Alloyeau et al. exploited in situ heating TEM to determine the activation temperature of chemical ordering and coalesce in CoPt NPs, with the view of rationalizing PLD protocols [59]. They have shown that coalescence induces a drastic growth of CoPt NPs above 700°C while ordering
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can be obtained within a few minutes at only 600°C. Therefore, they evidenced a temperature window between 600 and 700°C, in which small chemically ordered CoPt NPs can be directly fabricated by PLD without post-treatment annealing because the ordering time is then shorter than the fabrication time and the growth mechanisms remain poorly active. A similar ordering temperature was also seen in FePt NPs [60]. These works illustrate how in situ heating TEM experiments can be used as a guideline to better control kinetic effects on the synthesis of nanoalloys fabricated by vapor phase deposition. Note that size effects on the thermodynamic equilibrium of magnetic alloy NPs must also be considered since several experimental and theoretical investigations demonstrated the decrease of the FCC-L1₀ phase transition temperature by several hundred degrees in NPs smaller than 3 nm [69,70].

Furthermore, the possibility of maintaining the high-resolution performances of TEM at high temperatures provides a deeper mechanistic understanding of phase transitions in nanoalloys. Indeed, several in situ HRTEM studies revealed that chemically ordered phases (L1₀ and L1₂) nucleate at the surface of FCC-disordered FePt NPs and then propagate toward the core of the NPs (Figure 4) [60,61]. Interestingly, the ordering transformation pathway is driven by the counterdiffusion of Pt and Fe atoms between the surface and core regions that can lead to composition inhomogeneities in the resulting nanostructures [61]. Such in situ atomic-scale investigations open up many avenues for understanding the effects of crystal defects, NP size, and composition on the ordering mechanisms.

Finally, it is worth noting that high-temperature TEM analyses allow tracking the evaporation kinetics of nanoalloys at single particle level from which it is possible to determine the surface energy of nanoalloys in the liquid state. For instance, Chmielewski and coworkers have implemented such an approach to measure the surface energy in Cu-Au liquid NPs and showed that it follows a Vegard's rule-like dependence [71]. This work illustrates the potential of in situ TEM in experimentally determining key energetic parameters for the physics and chemistry of nanoalloys.

## 3.2. Reactivity and catalytic performances of nanoalloys

### 3.2.1. Gas-phase catalysis

Solid-gas interactions at the surface of metallic nanoalloys are key in many energy conversion and storage applications such as gas sensing, storage, and heterogenous catalysis in gas phase [2,72,73]. Over the recent years, in situ gas TEM has shown its ability to reveal material dynamics in gaseous environments with high temporal and spatial resolution. Most in situ studies on metallic nanoalloys have until now been directed towards understanding
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<figure><img src="image_30.png" /><figcaption>**Figure 4.** In situ STEM imaging of the ordering transformation in a Fe<sub>15</sub>Pt<sub>85</sub> NP at 700°C and in vacuum. (a–f) Time-sequence HAADF image snapshots showing the surface nucleation and growth of FePt<sub>3</sub> L<sub>1</sub><sub>2</sub> ordered domains (as marked by black dashed lines) that propagate into the bulk. The cyan lines in a, b, and e mark the formation of a Pt island via aggregation of Pt atoms rejected from the bulk. The white dashed circles in E and F mark the boundary between the Pt shell and the FePt<sub>3</sub> L<sub>1</sub><sub>2</sub> ordered core. The red dashed circles in E and F indicate the region showing the brighter image contrast due to the presence of a Pt island on the planar surface. (g and h) STEM EDS maps of Pt (green) and Fe (red) and (i) overlay of the Pt and Fe EDS maps of the well-annealed NP seen in F. (j) Schematic illustrating the nucleation and growth mechanisms of the ordering transformation in Fe<sub>15</sub>Pt<sub>85</sub> NPs. Adapted from [61], Copyright (2022), National Academy of Sciences.</figcaption></figure>

their exceptional catalytic properties, which are highly sensitive to their size and composition. Indeed, it has been shown that alloying of two or more metals in NPs can lead to less expensive catalysts (with a reduced noble metal
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content) with improved catalytic performance attributed to electronic (lig-
and) and/or geometrical (ensemble) effects associated with the arrangement
and bonding of neighboring atoms on the surface.

In situ gas TEM imaging and spectroscopies allow researchers to carry
out comprehensive studies to decouple ligand and electronic effects [74]. In
particular, there is growing interest in understanding structural and compo-
sitional dynamics in metallic nanoalloys and their relationship to catalytic
performances during reaction where variations in particle conformation
add layers of complexity to the understanding of catalytic processes. The
nanoscale direct imaging of NPs during catalytic reactions thus provides a
fundamental basis for the design and optimization of catalysts for different
classes of reactions.

In situ TEM studies on nanoalloy catalysts have mainly focused on sup-
ported bimetallic nanocatalysts mostly made of two noble metals (AuPd
[75-78] or AgAu [79]) and alloys of noble and non-noble metals (NiPt
[80-82], AuCu [83-85], NiRh [86], CuPd [87], CoPt [88,89], CoPd [90],
CuPt [91,92], PtPb [93] or AgIn [94]). These studies employ both differential
pumping system or closed cell approaches (see section 2), the latter becom-
ing increasingly popular because it is compatible with most TEMs and its
extension to operando conditions as discussed hereafter. These studies have
reported the complex restructuring dynamics of bimetallic nanocatalysts in
complex gas environments and external stimuli such as temperature, mimick-
ing the five stages of the catalyst life cycle: synthesis, activation, performance,
deactivation, and regeneration. With regard to the gas phase synthesis of
nanoalloy catalysts, the number of in situ TEM studies remains limited
[75,95]. Damsgaard et al. have for instance, using in situ HRTEM and EELS,
followed the formation of individual GaNi NPs from nitrate salt solution
heated in hydrogen flow at a temperature between 600 and 700°C. They could
reproduce in situ the formation of GaNi alloys within the same temperature
range as large-scale synthesis [95]. This study demonstrates the capability of
in situ TEM to examine nanoalloying processes in gas phase within a TEM.

Indeed, most in situ gas TEM studies on bimetallic nanocatalysts have
focused on the adsorbate-induced restructuring of particle conformation un-
der the activation/reaction conditions [77,78,83,85,88-90], the monitoring of
the active phases and their deactivation mechanisms [79,91-93]. It has been
shown that both the surface and core structures of bimetallic nanocatalysts
change continuously following different evolution pathways for different re-
action conditions [81-83,86,90,94,96]. For instance, in the investigation of
the origin of low-temperature activity of Ni – Rh NPs consisting of a Ni
core decorated with smaller Rh NPs in CO oxidation reaction, Gosh et al.
have revealed that the Ni core partially transforms into NiO under O₂-rich
conditions and the stable catalytic phase is of (Ni+NiO) – Rh hollow type
<figure><img src="image_32.png" /></figure>

[86]. In contrast, under O₂-poor conditions, the Rh NPs alloy with the surface of the Ni core to form NPs with a NiRh-alloy surface. Moreover, it has been observed that structural evolution and stable structure of bimetallic nanocatalysts depend on their initial architectures. For example, by comparing Pt – Ni rhombic dodecahedron NPs with segregated Pt frames and NiPt alloy NPs, Tan et al. have shown that their conformation during redox reactions and CO oxidation follows different pathways with different catalytic activity [80]. The NiPt rhombic dodecahedron evolve into a yolk – shell structure with Pt surrounded by NiO, whereas alloyed NiPt NPs tend to separate into a two-sided structure (Figure 5(a-c)). Oxidative and reducing redispersion of metals within the nanocatalysts are most often the main mechanisms through which particle conformation evolves. This is usually driven by the selective interaction of gas molecules with one element as evidenced recently in Cu-Au nanoalloys under H₂ by Wang et al. [84]. Using in situ gas STEM imaging, they showed an enhancement in the structural stability of Au NPs below 3–4 nm upon alloying with Cu. Whereas particle morphology evolved from facetted to rounded shapes, the observed AuCu NPs are found to keep their FCC symmetry under atmospheric hydrogen pressure (Figure 5d). Based upon ab initio molecular dynamics (AIMD) simulations, they assigned the enhanced structural stability to a Cu segregation process from the sub-surface toward the upper surface layer, and a reversed segregation of Au atoms from the surface towards the sub-surface sites driven by hydrogen-adsorption processes at the particle surface (Figure 5e).

The advent of operando transmission electron microscopy, which combines TEM imaging with mass spectrometry or gas chromatography [27,80,97–99] has constituted a major shift in the study of catalysis lately. This innovative approach combines the structural information of catalysts with real-time assessments of their catalytic activity closed to laboratory catalytic operating conditions (Figure 5(a-c)). Simultaneous access to nanocatalyst structure and activity opens new avenues for elucidating the often-missing and complex link in the structure-activity relationship in heterogeneous catalysis, including bimetallic nanaoalloys [80,86,87,96]. In the work by Gosh et al., the combination of structure observations and quantitative assessment of catalytic gas products have enabled to further investigate the low-temperature activity of the (Ni+NiO) – Rh hollow type NPs and NPs with a NiRh-alloy surface obtained under O₂-rich and O₂-poor conditions, respectively. Operando activity measurements using mass spectrometry have revealed that former conformation is the more active [86]. The enhanced low-temperature activity of the (Ni+NiO) – Rh hollow type NPs was attributed to the synergistic effect between the multiple Rh – NiO interfaces that are stabilized under reaction conditions.

HEA NPs have attracted much attention in the field of heterogeneous catalysis since the first report of their synthesis in 2018. Often described as
<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /><figcaption>**Figure 5.** (a,b) Operando closed-cell TEM monitoring of NiPt alloy NPs during CO oxidation with the assessment of catalytic activity based on the measurement of partial pressures of CO, $O_2$, and $CO_2$ as a function of time at different reaction temperatures using mass spectroscopy. (c) Elemental redispersion during CO oxidation at 650°C for 15 min evidenced by STEM imaging and EDX elemental mapping at single particle level CO oxidation over NiPt alloy NPs. Adapted from [80] copyright (2019) John Wiley and Sons. Atomic-scale visualization of the structural stability of a AuCu NP of size 2.3 nm supported on anatase $TiO_2$ in $H_2$ at atmospheric pressure. (d) STEM HAADF and BF STEM images of the NP acquired in situ at (A) 400°C under 40 Pa Ar, (B) 400 (C) 300 and (D and E) 200°C under $10^5$ Pa $H_2$, showing a continuous evolution of the morphology of AuCu NP under $H_2$ but with no change in crystal structure. FFT of the high-resolution BF STEM images showing a stable FCC NP structure between 400 and 200 °C. (e) Evolution of the shape and surface structure of Au and AuCu NPs after the adsorption of hydrogen atoms from DFT optimization and AIMD simulation. Blue, yellow and small white balls represent Cu, Au and H, respectively. Adapted from [84] Copyright (2023) Royal Chemical Society.</figcaption></figure>

the 'Swiss knife' of heterogeneous catalysis, the unique physical and chemical complexities of HEA NPs provide multiple active sites for oxidation and hydrogenation reactions within a single NP. Catalyzed by its inputs to the study of bimetallic nanocatalysts, in situ TEM holds much promise in understanding the catalytic properties of HEA NPs. Though the research in this area remains limited, in situ and in operando gas TEM will undoubtedly play a key role in the exploration of HEA nanocatalysts with regard to their growth mechanisms, and understanding of the origin of their superior catalytic properties. In two recent studies, Song et al. have investigated the oxidation of $Co_{0.21}Cu_{0.08}Fe_{0.28}Ni_{0.20}Pt_{0.23}$ NPs in air and the reduction of oxidized CuCuFeNiPt NPs under hydrogen environments successively using closed-cell gas TEM [100,101]. They demonstrated that the oxidation process is induced by Kirkendall effect but with a significant reduction in oxidation rate compared to binary alloys. During oxidation, Fe, Co, Ni, and Cu
<figure><img src="image_35.png" /></figure>

segregate at the surface while Pt remains in the core [100]. As for the re-
duction reaction, it involves only the external surface of the oxide layer on
the NPs with oxidized Cu fully reduced to metallic Cu, whereas Fe, Co, and
Ni stayed in their oxidized forms with porous structures stabilized during
reduction [101].

**3.2.2. Electrocatalysis**

Given the intrinsic properties theorized for nanoalloys, their role in the elec-
trocatalytic panorama comes along with major expectations. As discussed in
previous sections, the combination of the cocktail effect and the structural
defects make them excellent candidates to be high-performance electrocat-
alysts [102]. Nonetheless, the inherent complex character of multimetallic
nanocrystals makes it particularly difficult to deconvolute the effect of each
component in the link between structure-activity-stability. Thus, the design of
miniaturized electrochemical cells to directly monitor the catalyst dynamics
at the nanoscale under quasi-operational conditions allows a deeper insight
in the three main aspects of electrocatalytic applicability of nanoalloys: syn-
thetic pathways by electrodeposition, morphological transformation upon
electrocatalysis and degradation/deactivation processes. For electrocatalysis,
electrodeposition is an important fabrication method that improves the ad-
herence of the catalytic layer to the surface of the electrode leading to better
overall performance of a device. Therefore, electrochemical TEM has been
used by Parlinska-Wojtan and coworkers to understand the growth of NiPt
NPs electrodeposited on the surface of a glassy carbon electrode by cyclic
voltammetry [103]. This study demonstrated that the film growth rate is sig-
nificantly higher during the first four cycles. Such observations reveal that the
growth is limited by the reaction rate and not by the mass transport. Further-
more, they evidenced that during the anodic scan, partial dissolution takes
place but the subsequent deposition during the cathodic scan is higher than
the dissolution (Figure 6), an intermediate stage that would not be possible
to infer from post-mortem studies. Complementary in situ STXM analysis
permitted to show the oxidation state of Ni upon deposition.

Regarding the behavior of nanoalloys during electrocatalysis, most studies
focus on morphological transformations typically linked to activation/deac-
tivation processes and degradation. In particular for bimetallic noble/non-
noble NPs, intermediate stages of leaching or complete dissolution can be
discerned from in situ electrochemical analysis in conditions relevant in real
electrocatalytic devices. For instance, FePt NPs supported on carbon black
were studied in 0.1 M HClO₄ as electrolyte [104]. Thus, it was shown that
upon potential cycling, the particles over the matrix connected to the elec-
trode displayed coarsening. Additionally, they observed that the corrosion
<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /><figcaption>Figure 6. Time series of the nucleation and growth of three particles during the *in situ* electrodeposition of NiPt in LC-TEM, scanning the potential between 0.8 and –0.8 V vs Ag/AgCl with a scan rate of 80 mV/s. Adapted from [103] copyright (2024) the authors.</figcaption></figure>

of the carbon support is accelerated by a higher concentration of NPs em-
bedded in the matrix. Dealloying was also evidenced during slow potential
sweeping showing a slight size decrease at high potentials (~+1.2 V) attributed
to the Fe leaching. Later, similar degradation mechanisms were observed by
Beerman and workers in octahedral NiPt alloy NPs during 20 activation cy-
cles from 0 to 1 V at 100 mV/s, in which leaching of Ni was detected by the
change in intensity of the ADF signal, particularly for Ni-rich particles [105].
While this is the predominant process in Ni-rich particles, Pt-rich NPs tend
to aggregate. Further insights into the chemical and electrochemical etch-
ing processes, key in the shaping of bimetallic nanoalloys, were studied by
Tan and coworkers in NiPt rhombic dodecahedron NPs (Figure 7a) [52]. On
the one hand, during chemical etching localized pits in the surface of the al-
loys are the primary sources of dissolution generating a directional process
(Figure 7a). On the other hand, by electrochemical etching the dissolution
develops uniformly throughout the surface. Although both mechanisms lead
<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /><figcaption>**Figure 7.** (a) Comparison between chemical and electrochemical etching. (A) Time series of in situ TEM images showing the chemical etching of a NiPt rhombic dodecahedron NP with 5% (v/v) acetic acid solution inside a liquid cell at 90 °C. (B) Schematic of the chemical and electrochemical etching pathways. (C) Time series of in situ TEM images showing the electrochemical etching of a NiPt rhombic dodecahedron NP near the positively biased (+0.5 V) working electrode with 0.1 M H₂SO₄ as electrolyte. Modified from ref [52] copyright (2019) with permission from the American Chemical Society. (b) Electrochemical etching of NiPt NPs in 0.1 M H₂SO₄ during chronoamperometry at different temperatures. Time series of *in situ* TEM images showing electrochemical Ni dissolution in a liquid cell filled with 0.1 M H₂SO₄ electrolyte on application of continuous bias of (A) +150 and 160 mV for 60 s at *T* = 23°C and (B) +110 and 120 mV for 60 s at *T* = 50 °C. (C) Normalized area vs. time for NPs during reaction at *T* = 23°C (black line) and *T* = 50°C (red line). (D) Potential and current at *T* = 23°C and 50°C under the same chronoamperometry conditions. Adapted from [106] Copyright (2021) Royal Chemical Society.</figcaption></figure>

to Pt-rich tetradecapods, the fundamental differences observed in their intermediate states highlight the importance of in situ analysis for finely designing bimetallic structures.

Beyond the control of the electrochemical potential applied, temperature is an important parameter in the overall electrocatalytic process. Shifts in the dissolution potential can be tuned by increasing the temperature of the media. A clear example of such an effect was observed in rhombic dodecahedra NiPt NPs in 0.1 M aqueous H₂SO₄ where Ni dissolution was tracked at 500 mV of constant potential at 23 and 50°C (Figure 7b) [106]. At 50°C, the dissolution starts in half of the time compared to 23°C and the overall process goes 3 times faster at higher temperatures. Moreover, this dissolution exacerbation is also evidenced by the shift of the onset potential to lower values. More interestingly, this study allowed correlating the actual number of particles with the total charge passed (Q) demonstrating that this value increases with temperature, which is counterintuitive. Finally, they were able to deduct that secondary reactions like temperature-induced ligand reduction can have an impact over Q, fact that could only be recovered from in situ experiments.
<figure><img src="image_40.png" /></figure>

Thus, in situ electrochemical TEM is bringing knowledge gaps in the behav-
ior of electrocatalysts, especially bringing to light intermediate or transitory
stages during the formation and application of nanoalloys [23].

**4. Challenges and future directions**

The main limitation of in situ TEM remains the influence of the electron
beam on the sample's dynamic. Better understanding radiation damages in
liquid and gas cell TEM is a fundamental challenge to mitigate and some-
times exploit electron beam-driven effects. Liquid electron microscopy has
benefitted from decades of research into radiolysis in liquids to tackle this
problem. Radiation chemistry simulations can be used to evaluate the con-
centration of radiolysis products as a function of the electron dose rate in
simple solutions [107]. Beyond providing a way to study kinetics effects on the
formation or the degradation of nanostructures, LPTEM experiments moni-
toring the growth or the etching of nanostructures by using the electron dose
rate to control the kinetics of radiolytic reactions can be used to validate or
improve radiation chemistry simulations [50,107–111]. However, meaning-
ful models of complex liquid environments are more challenging because the
interplay between all the chemical species present and produced in the liq-
uid cell must be considered. Additionally, temperature and charging effects
at the interface between the liquid and the windows are also not fully un-
derstood either. Some review articles have undertaken the colossal task of
comparing LCTEM investigations to improve the understanding of beam ar-
tifacts, rationalize data interpretation and provide a good practice guide for
future experiments [112,113]. This fundamental work is less advanced for gas
phase TEM [114].

From a practical point of view, apart from experiments in which radiolysis
is used as the driving force of chemical reactions, any in situ or operando
TEM experiment requires determining the cumulative dose and dose rate
thresholds below which the electron beam does not dictate the sample dy-
namics. These dose-related constraints often limit spatial and time resolutions
and can even prevent in situ TEM observations. In that regard, compres-
sive STEM imaging that consists of acquiring sub-sampled images that are
then reconstructed using inpainting algorithms is a revolutionary solution
to improve the resolution-dose tradeoff. Moreover, this approach offers the
possibility to control the electron dose distribution, which can help mini-
mize beam-induced artifacts [115–117]. Developing machine learning-based
approaches to improve the signal-to-noise ratio and the detection limit of
nanomaterials is also a promising avenue. U-net architecture has emerged
as a robust method for the denoising and semantic segmentation of time-
series images, capturing the dynamic of NP assembly under environmental
conditions. More importantly, such video processing workflows allow the
<figure><img src="image_41.png" /></figure>

automated analysis of the large datasets generated by in situ TEM, leading to more accurate, insightful and quantitative interpretations. This deep learning approach has already been successfully applied to study the behavior of metal catalysts under gas reactions [118,119] or the motion, etching, growth and self-assembly of metal NPs in liquid [120,121]. Extending this approach to nanoalloys is not difficult but the specific contrast of the nanostructures (especially if core-shell NPs are studied) should be considered in the training data set, together with the particular noise of the used imaging conditions. Furthermore, improving the time resolution of EDX mapping without compromising the analysis of elemental distribution in NPs would also be a decisive advantage for studying the dynamic of nanoalloys in liquid or gaseous medium. Denoising approaches based on deep learning have considerably reduced the acquisition time and radiation dose for EDX tomography under vacuum, but the adaptation of this method to the noise and geometric constraints of close-cell environments remains to be done [122]. The quantitative analysis of temporally evolving nanostructures by combining STEM imaging and 4D STEM mapping in environmental conditions opens up a new way to study the rotational dynamic of nanocrystals or the role of nanostructural heterogeneity and strain on the reactivity of nanocrystals [123,124]. Combined with the very active development of direct-electron detectors that boost the time resolution of 4D STEM analysis, such simultaneous analyses of the real and reciprocal spaces could provide a deeper understanding of the thermally or chemically driven phase transformations in nanoalloys.

Besides pushing the microscope's functionalities and performances, the future progress of in situ TEM also requires optimizing the close-cell technology to reach better imaging conditions and to get as close as possible to real conditions. In that regard, graphene-based cells with flow capabilities [125,126] or amorphous windows with higher electron transparencies are essential features to improve imaging resolution while conserving the possibility of controlling the composition of the reactive media. Compared to gas cells that can go above 1000°C, the 100°C temperature limit of liquid cells mainly due to the mechanical instability of the holder has limited the application of LPTEM in the fields of nanomaterials synthesis and catalysis. However, the very recent possibility to reach 300°C in commercially available LPTEM holder opens many avenues to investigate the synthesis of nanoalloys in high boiling solvents or to study the behavior of electrocatalysts at high temperatures. Local temperature measurements based on in situ electron energy loss spectroscopy studies of the gas density or the plasmonic resonances of nanoparticles revealed that the local temperature in MEMS-based gas nanoreactors can deviate by several dozen degrees from the average heater temperature measured by ex situ calibration [127,128]. As accessing these discrepancies at the nanoscale is of paramount importance for the reliable
<figure><img src="image_42.png" /></figure>

interpretation of in situ TEM experiments, these EELS-based nanothermometry methods should also be applied to liquid cells. The development of liquid cells with true standard electrodes is also important to better interpret electrochemical results [129,130]. Also, increasing the pressure limit of gas cells (possibly above 10 atm) would bring a new level of operating conditions to TEM and would reduce the gap with real catalytic reactor conditions. Finally, correlative in situ/operando studies combining TEM with X-rays methods (SAXS, STXM, EXAFS ...), optical imaging (confocal and interference reflection microscopies ...), Spectroscopy (UV-Vis-IR, Raman ...) or near-field microscopy (AFM, Scanning electrochemical microscopy ...) constitute a logical step towards a more complete comprehension of nanoalloy dynamics at different length and time scales. If the interest and complementary of multi-modes in situ analysis in gaseous or liquid environments are now well demonstrated, the development of true correlative platforms enabling measurements performed in the same sample with identical cell geometries and exact conditions is still in its infancy [34,103,131–133]. Beyond all those technical developments, it is worth noting that comparing in situ TEM results with atomistic simulations (DFT, molecular dynamic, Monte Carlo ...) remains a strategy of choice for not only describing the atomic-scale behavior of nanoalloys but also explaining it by considering their energy landscape.

The present review only focuses on the in situ and operando studies of nanoalloy catalytic properties but alloy NPs have also promising optical and magnetic properties that can be very useful for sensing or biomedical applications. In that regard, studying the life cycle of nanoalloys in liquid or gaseous environments mimicking such application media provides a unique opportunity to correlate their degradation mechanisms with the evolution of their properties over time. Easily adaptable to nanoalloys, this strategy was notably employed to reveal the biodegradation mechanisms of a wide variety of nanomaterials in vivo, since LPTEM experiments can recapitulate in an accelerated way the oxidative-stress-driven transformation of nanomaterials inside macrophage cells [134–136].

## 5. Conclusion

Through real-time observations of the synthesis, thermal evolution, and transformations under catalytic conditions, in situ and operando TEM have remarkably advanced our understanding of the structural and physicochemical properties of nanoalloys. On the one hand, atomic-scale in situ insights into the nucleation and growth mechanisms or the thermal behavior of nanoalloys allow distinguishing the impacts of kinetic and thermodynamic effects on nanoalloy conformation leading to a better understanding of their structural properties and the rationalization of the synthesis protocols. On
<figure><img src="image_43.png" /></figure>

the other hand, in situ/operando TEM reveals the structural dynamics of
nanoalloys during catalytic or electrocatalytic cycles, including the formation,
activation, reaction, deactivation and regeneration phases, which provides the
unique opportunity to explain the catalytic performances because the inter-
mediate structures, the active sites of catalytic reactions and the degradation
processes can be identified. The on-going and future developments discussed
in the previous section aim at minimizing the artifacts mainly due to the
electron-beam irradiation and to mimic as closely as possible the real exper-
imental conditions used in the synthesis and catalytic testing of nanoalloys
while preserving the outstanding performances of TEM.

**Disclosure statement**

No potential conflict of interest was reported by the author(s).

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