Abdelali Khelfa

Jaysen Nelayah

Guillaume Wang

Christian Ricolleau

Damien Alloyeau
email: damien.alloyeau@univ-paris-diderot.fr

Studying the Effects of Temperature on the Nucleation and Growth of Nanoparticles by Liquid-Cell Transmission Electron Microscopy

Keywords: exfoliated vs CVD, number of layers and cleaning

Quantitative in situ visualization of thermal effects on the formation of gold nanocrystals in solution 4 Selective shortening of gold nanorods: when surface functionalization dictates the reactivity of nanostructures ii Contents iii 5 Structural analysis of single nanoparticles in liquid by low-dose STEM nanodiffraction 6 Fabrication and preliminary testing of flow capable graphene liquid cells for enhanced imaging conditions and composition control 6.1 Advantages of graphene liquid cells . . . . . . . . . . . . .

Résumé

La microscopie électronique à transmission en cellule liquide (LCTEM) est une technique in situ puissante pour révéler la dynamique des nanomatériaux en solution avec une résolution atomique. Cependant, la LCTEM peut encore être améliorée, notamment en ce qui concerne l'optimisation du rapport signal/bruit et la résolution des images TEM acquises dans un liquide, la compréhension et la minimisation des effets du faisceau et le contrôle de l'environnement réactif (température et composition). L'objectif principal de ce travail était de relever les défis techniques sur l'optimisation du LCTEM et les défis scientifiques sur la dynamique des nanoparticules d'or dans leur milieu de synthèse. Outre un article vidéo décrivant le protocole de nos expériences de LCTEM à température contrôlée [1], quatre résultats principaux émergent de ce travail : (i) Pour la première fois, nous avons étudié les effets de la température sur la nucléation et la croissance des nanoparticules d'or dans l'eau par LCTEM et nous avons révélé de nouvelles informations in situ sur les processus de mûrissement d'Ostwald et de facettage des nanostructures [2]. (ii) En collaboration avec Hakim Amara (Laboratoire d'études de microstructure -ONERA -Châtillon) et Hazar Guesmi (Institut Charles Gerhardt -Montpellier), nous avons combiné des expériences TEM en cellule liquide avec des simulations atomistiques pour étudier les mécanismes de dissolution des nanorods d'or et déterminer si leurs processus de dégradation sont dictés par leur structure atomique ou leur fonctionnalisation de surface [3]. (iii) Nous avons montré que la nanodiffraction STEM peut être exploitée dans des analyses en milieu liquide pour surmonter les deux principales limitations de l'analyse structurale HRTEM : la résolution limitée et le flux de dose d'électrons très élevé. En effet, nous mettons en évidence que la nanodiffraction STEM permet une analyse rapide de la structure de nanoparticules d'or uniques quelle que soit leur orientation et nous montrons également que cette technique peut être utilisée dans des conditions de très faible dose (réduite de près de quatre ordres de grandeur par rapport à l'analyse HRTEM conventionnelle) [4]. (iv) Enfin, en collaboration avec Kristian Mølhave (DTU Nanolab -Technical University of Denmark), nous avons fabriqué et testé de nouvelles cellules liquides en graphène avec des capacités de flow combinant des conditions d'imagerie améliorées et un contrôle de la composition de la solution.

Chapter 1

General introduction 1.1 General context

Humanity has always tried to adapt its environment to its needs and desires. For this purpose, humankind have developed tools to shape the world and understand it. With time, knowledge of physics and chemistry allowed humanity to control the environment and even shape or create new materials. In the beginning of the 20 th century, X-ray diffractions [5] and later the transmission electron microscope (TEM) [6] were developed allowing scientists to determine the structural and morphological characteristics of materials at the atomic scale. These important tools opened up new ways of analyzing materials which allowed understanding the origin of their properties and therefore enhanced our ability to fabricate functional materials.

Following the introduction of "nanotechnology" by Richard Feynman during his lecture "There's plenty of room at the bottom" in 1959 [7], nanomaterials have induced unprecedented research effort because of their astonishing physico-chemical properties and their various applications in optical sensing [8], medicine [9], catalysis [10,11] or energy development [12] just to name a few. However, by reducing the size of materials one must take into account many parameters that were ignored with bulk materials. Indeed, besides the composition and atomic structure that impact the properties of bulk materials, the size, 3D shape and interface with the surrounding environment are essential parameters to understand and control the properties of nanostructures. Exploiting these new parameters for designing materials with enhanced properties has emerged as one of the most exciting challenge in materials science.

The main objective of nanomaterial synthesis is to develop reliable ways to obtain nanoobjects with well controlled size, shape, chemical composition and atomic structure. For decades, wet chemical synthesis has made it possible to fabricate a broad variety of nanomaterials in liquid media [13]. However, a deeper understanding of the nucleation, growth and etching processes that occur in solution is crucial to rationalize the synthesis strategies in colloidal chemistry. It is now well established that ex situ characterization techniques, with which nanomaterials are studied at the atomic scale but outside their synthesis environment, only provide a very partial comprehension of the dynamic of nanomaterials in solution and the influence of the synthesis parameters. This issue has motivated the development of many in situ approaches to probe in real time and at the atomic level the behavior of nanomaterials in liquid media. Nowadays, these in situ techniques can be classified in two categories.

On the one hand, global analysis techniques, such as X-ray scattering methods and UV-Vis spectroscopy, allow real-time averaged measurements of the size, shape, density, and volume fraction of nano-objects in liquid of controlled composition and temperature [14][15][16]. Such global measurements in large volume of solution have been fantastic tools to measure the collective behavior of colloids during their formation. However, given the impossibility to follow the different behaviors of individual nanostructures and the necessity to develop models to retrieve the structural information, these in situ approaches provide an incomplete and indirect overview of the processes implied in wet-chemical synthesis. On the other hand, in situ microscopy approaches have been developed on both near-field microscopes (AFM and STM) [17] and electron microscopes (SEM and TEM) [18] with the view to directly observe chemical reactions that occur at the interface between solid and liquid with nanometer or even atomic resolution. In this context, Liquid Cell Transmission Electron Microscopy (LCTEM) has rapidly emerged as a very powerful method to reveal the nucleation, growth and etching mechanisms at the single nanoparticle level [19]. The goal of my thesis is to use this in situ TEM technique to explore new frontiers in the synthesis of gold nanoparticles and to develop new liquid cells and TEM techniques that improve the image resolution, the structural analysis, and the control over the synthesis media.

In this introduction, I will first discuss various strategies employed in colloidal chemistry to control the shape of gold nanoparticles and the remaining grey areas of the atomic-scale processes involved in these synthesis protocols that could be cleared with in situ studies.

Then, I will describe the technical history of LCTEM and its main contributions in the mechanistic understanding of metal nanoparticles synthesis. Finally, I will present in details the goals of my PhD.

Metal nanoparticles synthesis using solution-based methods

By reducing gold chloride with phosphorous in water [20], Michael Faraday prepared for the first time gold colloids in 1857 and studied their interactions with light. For the last 150 years, many techniques were developed to synthesize metal nanoparticles. The objective of the development of these synthesis protocols is to have a fast and reliable way to obtain the desired nanoparticles. Depending on the application, one can choose a specific protocol that allows a control of a set of characteristics. In general two main methods of fabrication can be defined: the top-down and the bottom-up approaches. The principle of the topdown approach is to carve, mold and slice a bulk material giving us the image of a sculptor working with marble. Whereas with the bottom-up approach we build step by step, atom by atom, molecule by molecule the nanoparticles. Both of these approaches have their own advantages and disadvantages.

In my thesis, we focused on the bottom-up approach and specifically the chemical reduction method.

The chemical reduction method [21] is the most commonly used process to synthesize noble metal nanoparticles in solution. Reducing agents accompanied with stabilizing agents are used to provoke a reduction of molecular metal precursors and thus the nucleation and growth of nanoparticles. Although in principle very simple, the physics behind the synthesis is complex. Indeed, this method is very sensitive to various parameters such as the ionic salt concentration, temperature, the nature and concentration of the stabilizing agents etc. and it is difficult to study the exact role of each parameters without using specialized tools.

While the chemical reactions involved in the metal nanoparticles synthesis are fairly simple, the growth mechanisms are not. Indeed, for a long time the synthesis of metal nanoparticles has been developed empirically. As with all bottom-up synthesis, there is a competition between kinetic and thermodynamic effects, between the flow of matter inside the liquid and the stability of nanomaterials. The key of the research on metal nanoparticles growth is therefore to control the growth speed and/or the equilibrium of the nanostructures with respect to their environment. Moreover, many physical and chemical parameters play a role in the growth and the complex phenomenons taking place inside the liquid environment are at the atomic scale. Therefore, the advent of atomic scale characterization tools like the electron microscopy, near field microscopy and synchrotron techniques allowed scientists to better understand all these phenomenons at play in the liquid media.

In a typical synthesis of metal nanoparticles, metal precursors are mixed up with a reducing agent in a solvent. The metal precursors are in a higher oxydation state than the reducing agent in the solvent solution, thus a redox reaction takes place where zero-valent atoms will be created. As described by LaMer [22] in the 1950's (see figure 1.1), the concentration of atoms increases through the reduction of metal salt until the concentration reaches a point called the minimum supersaturation level. After reaching this point, the atoms start to aggregate to form small clusters called nuclei via self-nucleation. These nuclei then grow by capturing nearby atoms in the liquid media, thus decreasing rapidly the concentration of atoms. Nuclei continue to appear inside the solution until the concentration drops below the minimum supersaturation level. Then, no more new nuclei appear and the remaining nuclei continue to grow larger. When the cluster reaches a well-defined structure, then it becomes a seed. A seed can take a single crystal structure, a singly twinned crystal structure, a multiply twinned crystal or a plate with stacking faults. All these structures can coexist inside the solution. After that, adatoms diffuse on the surface of the seeds until they find an appropriate site for them to stay inducing a growth of the seeds. This growth is dependent on the competition between a decrease in bulk energy (that promotes growth) and an increase in surface energy (that promotes dissolution). At one point, the bulk energy and the surface energy reach an equilibrium or the metallic atoms in the solution are all consumed and the growth will stop. As the nucleation and growth processes depend on many parameters, chemical reduction is a very versatile method that allows scientist to obtain various nanoparticle shapes. atoms, nucleation and subsequent growth [22].

Strategies to control the synthesis

Different strategies have been investigated to control the synthesis and obtain a homogeneous set of nanoparticles with a given shape. In this section, we will describe some synthesis methods with the view to present the context and the questions that motivate the in situ TEM experiments presented in the result chapters.

Seed-mediated synthesis and face-blocking methods

The shape of the seeds can determine the final shape of the nanoparticles, although there is not only one final shape possible from one seed. We can use that to our advantage in seed-mediated growth: we insert a homogeneous set of seed that will grow into the desired nanoparticles through epitaxial growth. It is also possible to use seeds that are chemically different of the atoms in the solution. However, heteroepitaxial growth requires that the interatomic distance of the seed and the growing materials must be close. The seed selection methods usually employ oxydative reactions that enable the dissolution of small clusters with specific atomic structures on specific crystal defects. These selective etching processes will be described in the subsection 1.3.4. Many shapes and forms can be obtained using this seed-mediated technique: gold spheres [23], gold nanorods [24], palladium nanocubes [25],

silver nanoshells on silica spheres [26], gold-silver core-shell nanorods [27]...

The final shape of the nanoparticles depends tremendously on the shape of the seeds. However, seeds are never used alone to obtain a specific shape of nanoparticle. Indeed, surface capping agents or ligands can be used to cover specific facets of a nanocrystal and thus promote the growth of a given nanostructure. The capping agent makes these facets thermodynamically more favorable by reducing their interfacial free energies through chemisorption.

The capping agent can be a byproduct of the synthesis or it can be added into the synthesis. Indeed, let us give the example of the work of Hubert et al. [28] where gold nanorods were produced using cetyltrimethylammonium bromide silver bromide complex (CTASB) as a capping agent without seeds. In this study, the authors have mixed up tetrachloroauric acid, cetyltrimethylammonium bromide (CTAB), silver nitrate, ascorbic acid and sodium borohydride and obtained gold nanorods at the end of the synthesis. Silver nitrate and CTAB interact and form CTASB. According to the authors, CTASB then deposit itself on the surface of the nucleating gold seeds in the solution. We can see the obtained gold nanorods in the figure 1.2. However, this study lacks a critical information: where does CTASB deposit itself on the surface of gold seeds ? Without this information, it is not possible to explain the real atomistic mechanisms taking place during the growth of the nanorods.

Despite the fact that in this study the nanoparticles growth was without initial seeds, most experiments and protocols of nanoparticles growth use a combination of seed-mediated growth and face-blocking methods. In this case, how can we distinguish between the effects of the atomic structure and defects of the seeds and the ligands that cover specific facets of the growing nanoparticles ?

Kinetically-controlled synthesis

Kinetically-controlled synthesis consists in tuning the reaction speed to favor the formation of a given shape [29]. For example, by reducing the rate of creation of metal atoms in the solution sufficiently, multiply twinned seeds tend to expand their {111} facets and achieve the lowest total free energy. This also negates the creation of single crystal seeds. One can even produce plate-like seeds that are not possible to obtain if the growth is too fast.

The final shape of the nanoparticle is the result of an intimate interaction between kinetic effects and thermodynamic effects. Indeed, the final shape is often very thermodynamically stable meaning that the nanoparticles are very facetted. Different methods have been made

to achieve kinetically-controlled synthesis: using a weak reducing agent [30], coupling the reduction to an oxidation process [31] or taking advantage of Ostwald ripening [32].

As an example, we can describe the work from Xiong et al. [33] where palladium nanobars and nanorods were synthesized. In the synthesis, Na 2 PdCl 4 (the metal precursor) was coreduced by ethylene glycol (EG) and poly(vinyl pyrrolidone) (PVP) in an aqueous solution containing potassium bromide (KBr). According to the authors, higher reducing agent concentration resulted in faster reduction rate for promoting directional growth of nanorods and the aspect ratios increased with the increase of reducing agent concentration (see figure 1.3). The effects of growth speed are dynamic by nature and they are very difficult to understand without the possibility to visualize the growth processes in real time. These effects are also present directly at the atomic scale. Moreover, the speed of the growth depends on various parameters and in the usual protocols of synthesis, many of these parameters are tuned at the same time. Then it is difficult to distinguish the exact effect of each individual parameter. Therefore, there is a need to have new in situ tools of investigation with high time and spatial resolution.

Temperature-controlled synthesis

Temperature is an important parameter in the synthesis of nanocrystals. However, its role is very difficult to study and understand because of its multiple interacting effects on the nucleation and growth. Indeed, on one hand, it modifies the reaction rate and increases the diffusion of precursor in the solution. On the other hand, it also influences the surface diffusion mechanisms of the adatoms on the growing nanoparticles and affects their nanophase diagrams [34]. All these effects simultaneously change the size and shape of the nanoparticles.

To illustrate a way to control the synthesis using temperature, we describe the polyol synthesis of metal nanoparticles [35]. The polyol processes use poly-alcohol or polyol such as ethylene glycol as a solvent and reducing agent at the same time. The presence of several OH group induces a high boiling point and a high viscosity compared to water. The synthesis can therefore be done at higher temperature than in water meaning that nanoparticles with higher cristallinity will be obtained. The high viscosity helps obtaining nanoparticles separated from one another and minimizes coalescence. It also favors a kinetically-controlled growth. The synthesis follows the same general steps than a "classical" solution-based synthesis: (1) mix up the metal precursors with the polyol solution at a given temperature, (2) reduction of the metal precursors by the polyol solution and nucleation of metal atoms, (

formation of seeds by clustering of metal atoms and (4) growth of the seeds into nanoparticles. The shape of the nanoparticles depend on the nature of the polyol solution, the capping agents used and the temperature of the solution.

One interesting example of polyol synthesis is the one done by Kim et al. [36]. In this work, silver nitrate was prepared as the precursor for silver nanoparticles, ethylene glycol was used as the polyol and polyvinylpyrrolidone (PVP) was used as a capping agent to avoid the agglomeration of the nanoparticles. They compared two protocols of synthesis: first, mixing up silver nitrate, ethylene glycol and PVP and then heating up the mixed solution step by step, second, mixing up ethylene glycol and PVP and heating up this solution to a given temperature and only then injecting the silver nitrate in the heated solution. For the first protocol, silver nitrate is reduced slowly meaning that the nucleation lasts a long time.

Thus, seeds continue to appear while other nanoparticles are already large meaning that they obtained at the end of the synthesis, when all metal precursors are consumed, a very large size dispersion of nanoparticles. The figure 1.4 shows that the faster the heating rate, the narrower the size distribution of nanoparticles. On the contrary for the hot injection method (second protocol), silver atoms are formed very rapidly and the nucleation phase is very short. Therefore, nanoparticles with a narrow size distribution are obtained. Another interesting point of this study is that depending on the temperature of the solution in the hot injection method, we can observe different average size and size distribution of nanoparticles.

Indeed, in the figure 1.5 we can see that the mean size is smaller and the size distribution is narrower at lower temperature. Temperature is influencing both kinetic and thermodynamic aspects of nanoparticle formation. Moreover, temperature is often combined with the effects resulting from other parameters (in the polyol synthesis, capping agents and seeds are often used). It is therefore necessary to develop in situ tools that can monitor the growth of the nanoparticles in real time and in real space at the nanoscale so that we can distinguish the exact effects of temperature. The chapter 3 focuses on these important questions.

Selective etching

The synthesis of metal nanoparticles is often driven by a reduction of metal precursors.

However, with the presence of coordination ligands, the zero-valent metal can be oxidized back into metal ions. This phenomenon of oxydative etching [37] can happen during the nucleation/growth of nanoparticles. Although oxydative etching is opposite to the reduction of metal precursors, it can be useful and even desired. Indeed, the oxydative etching is a phenomenon that have specific targets like defect sites on nanocrystals rather than singlecrystal. Therefore, various efforts have been made to pilot the oxydative etching in order to obtain the desired nanoparticles. Selective etching have various applications: (1) Tuning the distribution of seeds and manipulate the ratio of multiply twinned to single-crystal seeds [31]. Thus the final shape of the nanoparticles generated can be controlled. (2) Slowdown the growth and therefore change the growth mode. (3) The oxydative etching dissolves some of the seeds into precursor ions and then these ions get reduced again into metal atoms. The metal atoms then tend to either form new seeds or be absorbed by the growing nanoparticles.

By doing this multiple times, it is possible to increase the size of the final nanoparticles [31].

(4) Oxydative etching have a tendency to attack preferentially defects or sharp features such as corners. This can be used to transform the nanoparticles shape (for example from cubes to spheres) [38]. (5) Apart from defect sites selectivity, it has been shown that oxydative etching can attack different facets of a nanocrystal depending on the the difference of surface energy between them. This can also be used to form anisotropic nanoparticles [39]. (6) Hollow metal nanostructures are nanoparticles with hollow body meaning that only their facets remain of the original nanostructure. These hollow nanoparticles have an increased surface area and a reduced density. These characteristics induce powerful optical, electronic and catalystic properties. One simple way to create hollow nanoparticles is to use oxydative etching to remove the interior of existing nanoparticles [40]. (7) The oxydation can happen simultaneously to the reduction of metal ions. This can be useful when metal ions are introduced while chemically different nanoparticles are already present in the solution. Indeed, by doing that, nanoalloys can be formed from the two metal components in the solution.

This process is known as galvanic replacement [START_REF] Chen | Facile synthesis of gold-silver nanocages with controllable pores on the surface[END_REF].

Let us give one interesting example of selective etching by Chen et al. [31]. In this work, triangular gold nanoplates have been synthesized using oxydative etching as a strategy to increase the yield without using additional seeds. A scheme explaining the reaction taking place in the solution is shown at the figure 1.6. Hexadecyltrimethylammonium chloride (CTAC) used as a surfactant for the growth is mixed up with potassium iodide. This solution is mixed up with chloroauric acid, the gold precursor. Ascorbic acid and NaOH is then added to the solution. This leads to the formation of many gold atoms and seeds.

These seeds can take many of the forms we described before but due to the presence of tri-iodide ions only planar twined seeds are preserved, the other seeds are oxidized back into gold ions. Tri-iodide ions also bind on gold {111} facets and thus facilitate the growth of nanoplates. Although, selective etching is already in use, the exact process of oxydative etching is still not well understood. Indeed, many questions remain unanswered when we analyze the end products of synthesis: what are the mechanisms of dissolution of nanoparticles ? How can we observe and confirm that oxydative agents attack preferentially one facet and not the others ? How does the presence of ligands around the nanoparticles influences the oxydative etching ? The chapter 4 focuses on these important questions.

Technical history and applications of in situ liquid

TEM

As mentioned before, ex situ tools provide only a partial view of nanoparticle synthesis because, most of the time, only the final morphology of the nanoparticles is revealed. Although this structural information can be very accurate, the atomic scale mechanisms are only guesses. The effects of seeds, the effects of ligands, the interaction between the kinetic and thermodynamic effects, all these phenomenons are still not well understood because their individual consequences on the growth are not observed. What needs to be done is to distinguish between all these parameters and effects. Moreover, many theoretical studies cannot be confirmed or refuted if there are no direct view into the synthesis. All these issues require the development of in situ tools that capture in real time and in real liquid media the nanoparticles growth.

In this section, we will describe the technical history of liquid cell transmission electron microscopy (LCTEM) and some crucial answers that this in situ technique brings to the field of metal nanoparticles synthesis.

Technical history of LCTEM

The first "modern" liquid cell was built by Williamson et al. in 2003 [42]. It consisted of two silicon (Si) wafers glued face to face with silicon nitride (SiN) windows transparent to electrons. The liquid cell was sealed and static (no liquid flow). Gold wires inserted inside the liquid cell allowed electrochemical experiments.

However, because of the radiolysis effects granted by the electron beam, there was a need to rethink about a way to renew the solution inside the liquid cell. First developed in 2009 by de Jonge et al. [START_REF] De Jonge | Electron microscopy of whole cells in liquid with nanometer resolution[END_REF], flow capable liquid cells consist of two microfabricated Si chips

(called e-chips) with a SiN window transparent to electrons in each chips. The liquid cell is assembled by squeezing the two e-chips in a dedicated TEM holder that allows to flow a liquid during the experiment in the TEM. Indeed, this so called flow capability is possible due to the presence of microfluidic canals inside the liquid cell holder that can transport a liquid in and out of the liquid cell. The e-chips are also equipped with spacers, small pillars that allow the liquid to not be completely encapsulated between the two e-chips. O-rings allow the liquid to flow from the holder to the inside of the liquid cell without risking a leak of the liquid inside the TEM. An additional feature available in liquid cells is the presence of electrodes that can connect to the holder in order to perform electrochemical measurements.

This design was then used to make commercially available liquid cells by Protochips [START_REF]Tem liquid microscopy cell: Poseidon select -porotochips[END_REF] and Hummingbird Scientific [START_REF]Liquid flow holder for in-situ tem -hummingbird scientific[END_REF]. For our experiments, we used the e-chips and liquid cell holder made by Protochips (see figure 1.7). The size of the chips and the windows can vary but typically the dimensions of the smaller one is 2 x 2 mm while the larger one is 4.5 x 6 mm. The thickness of the window can also vary but it is typically around 50 nm. Protochips also developed an e-chip with an embedded electrode inside the larger e-chip that allows to heat up the whole liquid cell uniformly. We used it for our heating experiments. Note that DENSolutions, the third company that develops TEM holders, made a liquid cell holder [START_REF]Stream in situ tem liquid -densolution[END_REF] with a microchannel where the liquid is guided over the sample by a defined channel whereas in other liquid cell holders, the liquid enters the liquid cell by diffusion from "liquid wells" (the liquid cell is surrounded by liquid that diffuses to the window). Additionally, thanks to a pressure-based liquid pump, they are able to control the speed, pressure and direction of the liquid inside the liquid cell. Interestingly, it has been shown that it is possible to control the thickness of the liquid layer with the pressure-based liquid pump [START_REF] Keskin | Liquid-phase electron microscopy with controllable liquid thickness[END_REF].

Furthermore, several academic laboratories have also developed homemade liquid cells with specific designs, such as the liquid cell with thinner SiN membranes developed by the group of Haimei Zheng [START_REF] Liao | Facet development during platinum nanocube growth[END_REF][START_REF] Liang | In situ study of spinel ferrite nanocrystal growth using liquid cell transmission electron microscopy[END_REF], the multi-nanochannels designed by the group of Kristian Mølhave at DTU Nanolab [START_REF] Murat Nulati Yesibolati | Electron inelastic mean free path in water[END_REF] and of course graphene liquid cells [START_REF] Frances | Liquid Cell Electron Microscopy[END_REF]. As this is one of the challenges of this thesis, we will present a state of the art of graphene liquid cell development at the beginning of the chapter 6 to introduce the motivation of our work.

Seed-mediated growth and face-blocking method

In the work of Wu et al. [START_REF] Wu | Growth of au on pt icosahedral nanoparticles revealed by low-dose in situ tem[END_REF], LCTEM has been used for the study of the growth of gold (Au) on platinum (Pt) seeds. In order to have realistic reaction conditions, the solution was injected directly inside the liquid cell while the TEM was used at low electron dose. Thus, the continuous flow of liquid means that the same irradiated liquid is not kept during the experiment and the effects of the irradiation itself are minimized. Pt icosahedral seeds were first introduced into the liquid cell. Then, the aqueous mixture of chloroauric acid (the Au precursor, HAuCl 4 ) and citric acid as a reducing agent are injected. They observed that Au grew first at the corners of the Pt seeds forming large protrusions and then the gold atoms diffuse from these protrusions on the surface of the Pt facets (see figure 1.8). To better observe the diffusion of Au on the surface and slow down the growth, they turned off the flow after a few minutes. From the images, the growth kinetics were analyzed quantitatively.

It was shown that the growth depends on the competition between the deposition of Au in the corners of the Pt seed into protrusions or islands and the diffusion of the Au atoms on the surface of the seed. In the first phase of the growth, the former effect of formation of protrusion is prevalent but after some time (and after the protrusions reach a critical size)

the diffusion process starts to take over and the size of the island decreases because of the diffusion of its atoms. While this study confirms some assumptions on the fact that metal grows preferentially on defect sites and edges, a more complicated effect happening at the surface of the seeds is discovered: the competition between growth and diffusion. Other studies on anisotropic Au/Ag nanoparticles [START_REF] Aliyah | Real-time in situ observations reveal a double role for ascorbic acid in the anisotropic growth of silver on gold[END_REF], spherical Au/Pd nanoparticles [START_REF] Sutter | Determination of redox reaction rates and orders by in situ liquid cell electron microscopy of pd and au solution growth[END_REF],

icosahedron Au/Ag nanoparticles [START_REF] Mark | Stepwise evolution of spherical seeds into 20-fold twinned icosahedra[END_REF] have been performed.

Another study made by the MEANS group at MPQ [START_REF] Ahmad | Exploring the formation of symmetric gold nanostars by liquid-cell transmission electron microscopy[END_REF] was on the formation of symmetric gold nanostars by LCTEM. In this work, gold seeds of various shapes were synthesized by radiolysis of a mixture of chloroauric acid (HAuCl 4 ) with methanol. Indeed, the radiolysis of the methanol by electron beam induces the production of solvated electrons that play the role of the reducing agent in the growth of the gold seeds. The synthesis is done at a low electron dose. Therefore, the nanoparticle growth is thermodynamically driven because nanoparticles have enough time to reach energetically stable shapes (2D plates, 3D icosahedral and decahedral mainly, see figure 1.9). The icosahedral nanoparticles were used as seeds for the formation of nanostars. This study therefore shows us an example of a very complex dynamic taking place during nanoparticles growth: the nanostars growth is a combination of seed-mediated growth, kinetically-driven growth and the effect of the capping agents. In the work of Liao et al. [START_REF] Liao | Facet development during platinum nanocube growth[END_REF], the facet development of platinum (Pt) nanoparticles was studied. Indeed, Pt(acetylacetonate) 2 (metal precursor) was dissolved in a mixture of oleylamine, oleic acid, and pentadecane. After putting this solution inside the liquid cell, the electron beam induces the growth of Pt nanoparticles by radiolysis of the solution. The growth kinetics were studied for each crystal direction. However, the growth along the [START_REF] Koo | Live cell electron microscopy using graphene veils[END_REF] direction stops very early compared to the (101) and [START_REF] Daniel | Nanometer resolution elemental mapping in graphene-based tem liquid cells[END_REF] directions leading to the formation of nanocubes. The authors explained this by the presence of the ligand, the capping agent covering the facets of the growing nanoparticles. This ligand is used traditionally to drive the growth using the face-blocking method. According to ab initio calculations performed to determine the binding energies of the ligands on the different facets of the Pt nanocubes, the ligands could move out of the [START_REF] Daniel | Nanometer resolution elemental mapping in graphene-based tem liquid cells[END_REF] facets keeping it exposed to the solution and allowing it to continuously grow. On the contrary, the ligands have low mobility on (100) facets meaning that the ligands don't move and effectively block the growth along the (100) direction. This phenomenon happens however only when the nanocube is sufficiently large and when it is small, all the facets grow at the same speed.

The objective of the seed-mediated synthesis is to create specific reactive sites that will allow the formation of nanocrystals into a specific shape. Playing with the ligands in the faceblocking method has more or less the same objective: pilot the growth to obtain a specific shape. In these two methods, two main questions remain: what are the reaction sites and why are they more reactive than others ? What is the respective weight of the seeds and the ligands and their attraction to these reaction sites ?

Kinetically-controlled synthesis

In the work of Woehl et al. [START_REF] Woehl | Direct in situ determination of the mechanisms controlling nanoparticle nucleation and growth[END_REF], the mechanisms of nucleation and growth by radiolysis of silver nanoparticles were revealed. Silver nitrate (metal precursor) diluted in water was injected in the liquid cell using flow during the experiment. Various reaction products resulting from the radiolysis of water like solvated electrons played the role of the reducing agents. The authors performed a parametric study of the effects of the electron beam current, the pixel dwell time and the magnification on the the nucleation induction time in order to establish the induction threshold dose necessary for nucleation and growth of silver nanocrystals. Surprisingly, they discovered that these parameters do not influence the nucleation induction dose but it changes the number of nanoparticles and their distribution through time.

They also tried to compare the dose-dependant growth mechanisms of the silver nanoparticles with the Lifshitz-Slyozov-Wagner growth model (LSW). The model doesn't predict the final shape of the nanoparticles and considers only spherical nanoparticles. According to LSW, two types of growth can occur as a function of the amount of reducing agent in the solution. The first type of growth is the reaction limited growths when the amount of reducing agent is so small that the atoms have enough time to diffuse on the surface of the existing nanoparticles and find the lowest energy facet they can find. The model predicts that the radius of a spherical nanoparticle will grow with time as r ≈ t 1/2 . The second type of growth is the diffusion limited growth that occurs when the amount of reducing agents is high. In this case, the high concentration of reducing agents induces a depletion of metal precursor in the region of reaction. Then, the metal precursor in the adjacent regions need to diffuse in the zone where the reducing agents are in order to continue the growth. Here, the model predicts that the radius will grow as r ≈ t 1/3 . In the experiment (see figure 1.11), they could choose between these two growth modes by simply changing the beam current: when it is "low" they obtained a reaction limited growth and when it is "high" they obtained a diffusion limited growth. For the low beam current condition, the nanoparticles radius followed the r ≈ t 1/2 law predicted by LSW. They found highly crystalline nanoparticles with singly twinned seeds that can form anisotropic plates or bipyramids. In the high beam current condition, they found multiply twinned seeds that became polyhedral or spherical shapes. However, the power law followed by the radius of the nanoparticles is r ≈ t In another work made by my team [START_REF] Alloyeau | Unravelling kinetic and thermodynamic effects on the growth of gold nanoplates by liquid transmission electron microscopy[END_REF], the kinetic and thermodynamic effects were revealed in the growth of gold nanoparticles. Gold nanopartircles were synthesized by radiolysis of an aqueous solution containing chloroauric acid (HAuCl 4 ). Similarly to the results obtained by Woehl et al., 2D nanoplates and well-faceted 3D gold nanoparticles were obtained at low electron dose which corresponds to the reaction limited growth where the thermodynamic effects are prevalent (see figure 1.12). At high electron dose, dendritic nanoparticles were obtained corresponding to a diffusion limited growth where kinetic effects are prevalent.

Remarkably the growth of gold nanoplates do not require to introduce capping agents in the solution, showing us that the slow growth kinetics play a major role in the formation of anisotropic nanoparticles. The growth is however guided by the formation of planar defects (stacking faults or twin planes) that breaks the cubic symmetry of gold lattice during the growth. In short, this study shows us the great capability of LCTEM to reveal the growth mechanisms of nanoparticles and the nanophase diagram of nanostructures as a function of the growth kinetics and the chemical environment.

Temperature-controlled synthesis and selective etching

In the study made by Xin and Zheng [START_REF] Huolin | In situ observation of oscillatory growth of bismuth nanoparticles[END_REF], a mechanism of oscillatory growth of bismute (Bi) nanoparticles by Ostwald ripening induced by temperature is shown. Oswald ripening is a growth phenomenon where larger nanoparticles consume smaller nanoparticles around them without any coalescence phenomena. Bi nanoparticles were directly put inside the liquid cell along with a solvent mixture of oleylamine (surfactant) and dichlorobenzene. In this study, the authors used a static liquid cell and a conventional heating TEM holder made by Gatan. This design allows observing the effect of temperature. However, the temperature control of the liquid media is poorly calibrated. Bi precursors were present in the solution after dissolving some of the Bi nanoparticles. This was done by increasing the temperature of the solution to 180 • C (corresponding to the boiling temperature of dichlorobenzene). Surprisingly, an oscillating regime of growth and dissolution was observed at this temperature: Bi nanoparticles grew while others dissolved back and forth. Indeed, on the figure 1.13 one nanoparticle grow while the other is dissolved and once it is dissolved completely, the process is inverted and the particle dissolved starts to grow while the bigger nanoparticles starts to dissolve. They also confirmed the presence of a depletion zone around the nanoparticles: when nanoparticles are within the depletion zone of each other, they tend to have this oscillatory growth that we described. In the work made by Niu et al. [START_REF] Niu | Revealing bismuth oxide hollow nanoparticle formation by the kirkendall effect[END_REF], the growth of bismude (Bi) oxide hollow nanoparticles by in situ heating and using the Kirkendall effect has been demonstrated. Indeed, Bi 2-ethylhexanoate was dissolved in a mixture of oleylamine and dichlorobenzene and put into the liquid cell. After reaching a temperature of 180 • C, the growth of Bi spherical nanoparticles was observed. The Bi nanoparticles are then oxidized with a thin bismuth oxide layer on the nanoparticles surface. The continuous oxidation of Bi leads to the formation of void surfaces inside the nanoparticle and then to a hollow nanoparticle by the Kirkendall effect.

The different pathways to obtain these hollow nanoparticles were studied: the first one is dominated by nonuniform diffusion of Bi through void surfaces and the second one is dominated by diffusion of Bi directly through the shell.

It should be noted that commercially available holders that can control precisely the temperature of a liquid cell were developed only very recently. Temperature control in LCTEM is still in its infancy and thus, to our knowledge, there have been no systematic study of the effects of temperature on the growth of nanoparticles in liquid. This is the goal of our study described in the chapter 3. Nevertheless, last generation temperature-controlled liquid cell TEM holder have been used to investigate nanoscale processes that occur at high temperature [START_REF] Shu Fen Tan | In situ kinetic and thermodynamic growth control of au-pd core-shell nanoparticles[END_REF][START_REF] Ambrožič | Controlling the radical-induced redox chemistry inside a liquid-cell tem[END_REF].

For example, in the work made by Tan et al. [START_REF] Shu Fen Tan | Intermediate structures of pt-ni nanoparticles during selective chemical and electrochemical etching[END_REF], nickel (Ni) was dissolved in Pt-Ni bimetallic nanoparticles using chemical oxydative etching and eletrochemical etching (see figure 1.14 for the results). Pt-Ni nanoparticles were first put in a liquid cell. For the selective etching, an aqueous solution containing acetic acid was introduced inside the liquid cell while it was heated to 90 • C. The liquid cell was heated during the experiment using a liquid cell holder with flow and heating capability (by Hummingbird Scientific). This induced the selective etching of some of the facets of the Pt-Ni nanoparticles. The etching started on the surface of the Ni by the formation of pits. These pits then expanded into the Ni until all the Ni was gone and only the Pt skeleton remained. On the contrary, for the electrochemical dissolution, the etching was isotrope and no pits appeared on the surface of the Ni. One clear advantage of the electrochemical method is the ability to control the final shape of the nanoparticles by interrupting the dissolution at intermediate structures. If the dissolution is complete, the resulting Pt skeleton obtained is similar to the one obtained using the chemical etching.

Using DFT calculations, they suggested that a surface layer of nickel oxide appears and this layer is unstable compared to the Ni facets. This leads to a localized dissolution on these nickel oxide facets whereas for the electrochemical method, there is no formation of these nickel oxide facets and so the dissolution is isotrope. 1.5 Goals and outline of this thesis

As we have just seen, direct TEM observations of nanoscale processes in liquid environments can allow for new insights into nanomaterials formation. Nevertheless, there is still a lot of room for improvement in LCTEM techniques in particular on the optimization of the signal to noise ratio and resolution of TEM images acquired in liquid, the understanding and minimization of the beam effects and the control over the reactive environment (temperature and composition). Therefore, the main objective of my thesis is to face the technical challenges on the optimization of the LCTEM and the scientific challenges on the dynamic of gold nanoparticles in their synthesis media. The results of my thesis are described in the next 5 chapters. Chapters 2, 3, 4 and 5 are peer reviewed publications and their supplementary materials.

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Chapter 2

Experimental chapter and methods

Electron-matter interactions

The TEM uses high speed (and thus high energy) electrons as a probe of matter. However, the absorption of electrons by matter is very high; the electron-matter interactions are about 10 4 to 10 5 higher than X-ray and neutron interactions. This implies that the TEM requires thin samples that can be penetrated by the electrons.

Electrons can have multiple interactions with matter that can give us information about the sample. Some of these interactions are depicted in the figure 2.1. We can subdivide two types of electron-matter interactions: elastic scattering where the electrons conserve their energy and inelastic scattering where electron energy loss occurs.

Elastic scattering

The electrons can go through the sample without changing direction. Thus, they are parallel to the incoming electron beam or they can be scattered in another direction. This incident electron. While the unscattered electrons are used for contrast imaging, the scattered electrons form a diffraction pattern that reveals crystallographic information about the sample. This diffraction pattern is often compared to tables of diffraction or to computer simulations to identify the nature of the crystal and retrieve other useful information.

Inelastic scattering

Some of the electrons lose energy while interacting with the sample. This energy is then used to generate other species that can be analyzed using a detector.

A typical product of this interaction is an X-ray photon. An incoming high energy electron can give part of its energy to a core electron of an atom. This core electron is then knocked out of its electron shell and leaves a hole behind. Following this process, an electron of an outer shell can then fill up this hole and generate a photon. This X-ray photon has a precise energy that corresponds to the energy difference between the electronic levels of the transition. This characteristic X-rays is very interesting because its energy is specific to the nature of the atom. Using this phenomenon, we can quantitatively analyze the atomic composition of the sample. This analysis is called energy dispersive X-ray spectroscopy (EDX).

Radiolysis

The electron microscope uses electrons to image the sample studied but in liquid phase experiments, the energy of the electrons is transferred to the liquid medium. This increased energy induces an excitation of the orbital electrons in the molecules of the solvent which can then escape the molecules to generate heat, radicals and molecular species. The products of this irradiation then react further with the species inside the medium thus creating a cascade of chemical reactions and additional species. Radiolysis is the initial reaction taking place when the incoming electrons interact with the liquid media. Radiolysis is the subject of a lot of studies since the beginning of the nuclear era. Some studies such as the work of Schneider et al. [START_REF] Nicholas | Electron-water interactions and implications for liquid cell electron microscopy[END_REF] have tried to predict the chemistry taking place inside a liquid cell filled with water or a metal precursor solution diluted in water [START_REF] Ambrožič | Controlling the radical-induced redox chemistry inside a liquid-cell tem[END_REF][START_REF] Hun | Control of electron beam-induced au nanocrystal growth kinetics through solution chemistry[END_REF]. However, if all the radiolysis reactions are known in such simple solutions, their models require very strong approximations notably because the electron dose rate in TEM is very high compared to the conventional radiation chemistry studies and they become very inaccurate for real experiments where other species are present in the solution (nanoparticles, ligands, salt, NaCl and HCl...).

Nevertheless, let's take the case where the solvent is pure water. When the electrons interact with water molecules thus exciting orbital electrons, the water molecules first decompose themselves into hydrated (solvated) electrons e - h , hydrogen radicals H • , hydroxyl radicals OH • and dihydrogene atoms H 2 . This initial yield is nonuniformly concentrated on the incident electrons path less than 10 ps after the first penetration of the electrons. Less than 1 µs after the energy transfer, primary products start to form in the liquid: all the molecules of the initial yield plus hydrogen peroxide H 2 O 2 , hydronium H 3 O + and hydroperoxyl HO • 2 . 1 µs after the energy transfer, the primary products are uniformly dispersed in the irradiated region. The primary products then participate to additional reactions that result in the production of dioxygene O 2 .

Radiolysis has multiple side-effects that can be detrimental to liquid experiments. For example, if the electron dose rate is sufficiently high (above 10 3 Gy/s) and the initial pH of water is sufficiently high (7 or above), Schneider et al. [START_REF] Nicholas | Electron-water interactions and implications for liquid cell electron microscopy[END_REF] have shown that the pH will decrease, thus having a drastic impact on the chemical pathways inside the liquid cell. Other side effects such as the formation of gas bubble (H 2 mainly) can also disturb the experiment [START_REF] Joseph M Grogan | Bubble and pattern formation in liquid induced by an electron beam[END_REF].

There are two strategies to deal with the radiolysis effects. On the one hand, because it is known that the concentration in radiolysis products has a power law relationship with the electron dose rate, the first strategy consists in reducing to a minimum the concentration in radiolysis species by keeping a low electron dose rate during the experiment. For example, this strategy can be used to study the nanoscale electrochemical mechanisms of charge or discharge of a battery [START_REF] Holtz | Nanoscale imaging of lithium ion distribution during in situ operation of battery electrode and electrolyte[END_REF][START_REF] Mehdi | Observation and quantification of nanoscale processes in lithium batteries by operando electrochemical (s)tem[END_REF]. Here, the challenge is to keep the electron dose rate low enough to avoid the degradation of the electrolyte because of beam induced growth processes. On the other hand, the effects of the radiolysis of water can be exploited to pilot a specific chemical reaction pathway. Indeed, in our case, most of our experiments are driven by radiolysis we can use the electron dose rate to control the reaction kinetics. An example of such reaction will be described in the chapter 3.

TEM modes and techniques in use for LCTEM

Conventional TEM Imaging

What we call contrast can be simply defined as: C = I 1 -I 0 I 0 = ∆I I 0 where ∆I is the difference of intensity between the object and the image background. When passing through the sample, the electron wave has its amplitude and phase modified. This modification can then be compared to the original amplitude and phase of the electron wave. This gives rise to the amplitude contrast and the phase contrast. Although both contrasts are present in an image, we tend to change conditions so that one dominates the other. We can classify two types of amplitude contrasts: mass-thickness contrast and diffraction contrast. Amplitude contrast can be observed in two kind of images: bright-field (BF) and dark-field (DF) (see figure 2.2). In BF, we select the unscattered electrons that went through the sample while in DF we select the scattered electrons. We can switch between these two types of images by changing the position of the objective aperture on the diffraction plane.

Aberration-Corrected High Resolution Transmission Electron Microscopy (AC-HRTEM)

While in conventional imaging a small objective aperture allows us to select either unscattered or scattered electrons, high resolution imaging uses a large or no objective aperture in order to retrieve unscattered and scattered electrons at the same time. This allows the diffracted beams to interfere. The resulting image depends on the relative phase between the interfering beams. Although HRTEM is wildly used as a tool of investigation for the structural analysis of nanoparticles, lens aberration and energy dispersion impose a physical limit to the microscopes. The resolution was limited to the range of 0.18-0.17 nm for 200 kV uncorrected microscopes. Low order aberrations like defocus and the two-fold astigmatisms can be directly measured on the FFT of a single image of amorphous materials and thus corrected.

However, high order aberrations remained difficult to correct, especially spherical aberrations. Thanks to the development of corrector technology for magnetic electron lenses, high end electron microscopes can effectively compensate spherical aberrations of the objective lens. The ARM 200F is an aberration corrected microscope that is also equipped with a CEOS corrector that negates other high order aberrations (axial coma, three-fold astigmatism, star aberration and four-fold astigmatism). Coupled with cold FEG electron source, these aberration-corrected optics are able to achieve a point resolution of 0.75 Å under vacuum at 200 kV. The probe correction, installed in 2020 on this microscope, allows to reach similar resolution levels in STEM mode.

Scanning Transmission Electron Microscopy (STEM)

STEM combines the principles of the TEM and the scanning electron microscope (SEM). A series of condenser lenses allows the electron beam to be focused on one point on the sample.

By adjusting the scan coils in the microscope, we can make the beam scan an area of the sample. The signal is then detected as depicted in the figure 2.4.

In STEM high angle annular dark field (HAADF), only the electrons with high scattering angle are detected by an annular disk with inner radius in a range of few tens of milliradians to an outer radius of few hundred milliradians. One of the main advantages of the STEM HAADF mode is that image intensity is directly proportional to ρ.t.Z α where α is a constant between 1.5 and 2 while ρ, t and Z are density, thickness and atomic number of matter respectively. This means that STEM HAADF is very sensitive to the chemical composition of the sample which is correlated with different levels of contrast in the image. This is why STEM HAADF is also called Z contrast imaging. Moreover, the contrast of the images also depends on the thickness of the nano-objects. Finally, STEM HAADF is relatively good at imaging thick samples compared to other TEM modes and techniques. Thanks to this, STEM HAADF is the main technique of investigation for LCTEM where the liquid cell is very thick (more than 150 nm).

TEM holders

Depending on the experiment and the nature of the sample studied, the choice of TEM holder is important. Two TEM holders were used:

• Simple tilt TEM holder. This holder is used for the alignment of TEM and ex situ experiments.

• Protochips Poseidon Select LCTEM holder with heating and flow capability. This holder is used for all the LCTEM experiments. It is possible to inject a solution inside the liquid cell using a syringe pump and the microfluidic canals inside the holder. The holder can also be plugged in a temperature controller so that the liquid cell can be heated. The holder can heat up the liquid cell up to 100 °C.

Calculation of electron dose rates

The radiolysis of the solution is an inevitable phenomenon in LCTEM experiments. Many chemical reactions then take place and all of them depend on the amount of electrons interacting with the solution. Thus, it is understandable that the electron dose rate ḋ has a profound effect on the reaction kinetics. ḋ can be expressed with this equation:

ḋ (electrons/s. Å2 ) = Beam current (C/s) Elementary charge (C/electron) × Irradiated area ( Å2 )
ḋ varies differently depending on the TEM mode in use (TEM or STEM).

In conventional TEM mode, an image of the beam on the CCD device without any detail through a vacuum is taken. The number of counts detected per Å2 is measured on the image. By combining this measure with the conversion efficiency of the detector (number of photons generated per electron, 52.37 counts/electron at 200kV for our JEOL ARM microscope) and with the exposure time of the image, we can deduce directly the electron dose rate ḋ. Remarkably, changing the brightness of the electron beam changes ḋ because it changes the irradiated area but changing the magnification has no impact on ḋ as this simply changes the lens current of the projection lenses.

In STEM mode, the electron dose rate varies with the magnification because when the magnification is increased the irradiated area (scan area) is smaller. In fact, ḋ is inversely proportional to the square of the magnification. To calculate ḋ, we take directly an image of the STEM probe on the CCD camera and we retrieve the beam current from it. The irradiated area is known for each magnification meaning that it is possible to calculate ḋ for each of the magnification. Furthermore, ḋ is in electrons/s. Å2 and can be converted to Gys -1 (J.kg -1 .s -1 ), the unit used for radiation chemistry, by multiplying ḋ by the density normalized stopping power of the solvent at the applied acceleration voltage and finally by the electron charge (1.6 x 10 -19 C).

The figures 2.5, 2.6, 2.7 and 2.8 show the calculated electron dose rate for STEM imaging and its variation with the magnification, aperture sizes and probe sizes for each microscope used.

Standard liquid-cell preparation, sample preparation and an example of liquid-phase TEM experiment are described extensively in the section 2.5.

Introduction

Metal nanoparticles (NPs) have promising physico-chemical properties that can be used in various domains such as optical sensing 1 , medicine 2 or energy 3 . Wet-chemical synthesis is a very versatile method to fabricate metal NPs with well-defined size and shape. Over the last decades, many strategies have been developed to gain control over NPs synthesis: seed-mediated growth 4 , face-blocking method 5 , kineticallycontrolled synthesis 6 , selective etching 7 or temperature-controlled synthesis 8 . However, while the chemical reactions driving the synthesis are fairly simple, the nucleation and growth mechanisms are not, because many parameters play a role in the formation processes and their individual influence are difficult to retrieve from ex situ snapshots of the resulting nanomaterials extracted from their formation medium at given time points of the synthesis. To truly understand the nucleation and growth processes and establish ways to control them, we must employ in situ tools that allow their real time observation in finely controlled liquid environment.

In that regards, Liquid-Cell Transmission Electron Microscopy (LCTEM) has been a very powerful method for shedding new light on the synthesis of metallic nanoparticles 9 , 10 , 11 , 12 , 13 .

By imaging the dynamics of individual nanostructures directly in their liquid formation media, this technique has provided a deeper understanding of nucleation and growth mechanisms, notably the role of crystal defects, seed morphology and organic ligands that allow driving directional growth or etching processes and obtaining nanomaterials with specific shapes (nanorods, nanostars, nanoplates, nanoshells) 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 . When the electron beam of a TEM interacts with liquids, radiolysis processes produce strong reducing and oxidizing species that modifies the solution chemistry in the irradiated area and can be used to drive growth or etching processes. Interestingly, the concentration of radiolytic products is known to increase with the electron dose rate, a parameter that can be finely tuned in an electron microscope 20 . Therefore, this dose-rate dependency of radiolysis has been exploited to control the reaction speed and reveal kinetic effects on the formation processes and final morphology of nanostructures 11 , 15 , 20 .

Although the temperature is a crucial parameter in nanomaterials synthesis, its effects have so far not been carefully investigated by LCTEM, because commercial liquidcells with reliable temperature control have only recently been developed. Yet, such in situ studies are indispensable to unravel the complex kinetics and thermodynamic effects induce by temperature changes. Indeed, on the one hand increasing the temperature has drastic impacts on the faceting processes during the growth, speeds up atomic and molecular diffusion in liquid and modifies the reaction rates.

On the other hand, the nano-phase diagram of nanostructures is also very sensitive to temperature. In this article, we exploit recently developed heating liquid cells to follow the radiolytic growth of gold nanoparticles in water with a temperature control between room temperature and 100 °C.

This methodology combining STEM imaging and diffraction in an environment that is getting closer and closer to the real synthesis conditions reduces the gap between in situ TEM observations and bench-scale syntheses.

Protocol 1. Align the transmission electron microscope for STEM HAADF imaging

1. Follow manufacturer instructions for microscope alignment.

2. Use a conventional dried sample to align the microscope.

Do not use a liquid sample.

3. To minimize the growth speed, minimize the electron dose rate (see discussion section) which implies using small condenser aperture and small spot size to reduce the beam current.

E-chip handling

NOTE: Commercial liquid-holders fit on almost all TEM but use the holder that is specifically design for the microscope brand and pole piece. A liquid cell is made of two MEMSbased silicon chips called E-chips, both of which are silicon substrates with a 500 x 50 µm window covered by a 50 nm thick amorphous silicon nitride (SiN) film that is electron transparent (Figure 1A). These two e-chips have different sizes. The small one is 2 x 2 mm with gold spacers that fix the distance between the two E-chips (150 nm here) and the liquid thickness. The large one is 4 x 6 mm and it has a resistance embedded inside the silicon substrate that allows a uniform heating of the liquid sample (Figure 1B). Because of the way they are fabricated in clean rooms, the E-chips have two different sides: one where the window looks small (here after called the front side) and the other where the window is large with a sink shape (here after called the back side).

1. When handling the E-chips, never touch the window with tweezers and grip the chips by the sides. To avoid scratching the surface of the silicon substrate use carbon-tipped tweezers.

2. If placing an E-chip on a surface, make sure that the back side is in contact with the surface because the SiN film is fragile and it is deposited on the front side.

Cleaning of the liquid cell holder (before the experiment)

1 4. Dry up the tip of the liquid-cell holder using an air pistol.

Preparation of the liquid cell (E-chips)

1. Cleaning of the liquid cells.

1. Fill a glass Petri dish with acetone.

2. Fill a glass Petri dish with methanol.

CAUTION: Due to the toxicity of methanol, the Petri dish with methanol must be put under a fume hood.

Methanol should be handled with the adequate protective gear (gloves).

3. Put one small and one large E-chip in the Petri dish with acetone and wait for 2 minutes.

NOTE: The E-chips are coated with a protective layer that needs to be removed before the experiment. Acetone will remove the photoresist and clean up the E-chips of debris. To enhance the cleaning, the solution can be gently agitated. 2. Put the small E-chip inside liquid cell holder (Figure 3C). To reduce the bowing of the SiN films towards the vacuum of the microscope, place the windows of the liquid cell in a crossed configuration. Therefore, the window of the small E-chip must be parallel to the length of the holder and the front face up. Make sure that the small E-chip is well inserted inside the gasket.

3. Prepare the liquid sample (here, 1 mM of HAuCl 4 in water).

4. Drop ≈2 µL of the liquid sample on the small E-chip using a micropipette (Figure 3D). If the small E-chip has been properly plasma cleaned, the aqueous liquid sample will spread evenly across the surface of the chip.

5.

Remove the extra liquid with a filter paper. With a sharply cut piece of filter paper, reduce the thickness of the liquid layer on the small E-chip until it forms a flat dome.

6. Put the big E-chip inside the liquid cell holder (Figure 3E). Place the large E-chip on the small one with its front face down (the front sides of the two chips must face each other). The electrodes on the large E-chip must be in contact with the electrode pad on the holder.

7. Slide the lid back on the liquid cell holder. Gradually tighten each screw (Figure 3F).

8. Dry up the eventual liquid coming out of the E-chips using small cut-out filter paper. Verify that there is no liquid coming out on both sides of the liquid cells by rotating the liquid cell holder around its axis.

Use the liquid holder in flow mode

1. Fill up 2 syringes with a few milliliters of the solution to be injected (1 mM of HAuCl 4 in water in our case). 3. Inject the solution with a flow rate of 5 µL/min in each inlet.

Heating of the liquid environment

STEM imaging of nanoparticles growth

1. Use the microscope in STEM mode using the HAADF detector. Go to a pristine area of the sample, near a corner of the observation window where the liquid thickness is minimum. Acquire videos of nanoparticle growth for different temperatures of the liquid (Figure 5).

NOTE: Gold nanoparticles immediately appear and grow

in the scanned area. Video recording with a frame rate of one image per second is a good compromise to observe the growth processes with good signal to noise ratio and a good time resolution.

STEM nanodiffraction of single nanoparticles

1. Acquire a STEM HAADF image of several nano-objects.

Acquire the diffraction pattern of individual nanoparticles selected on the image using the STEMx software (Figure 6). Overgrowth experiments in a different solvent can then be performed. 23 2. Inject 5 mL of distilled water in the inlet and outlet tubing of the liquid cell holder.

3. Clean the tip of the liquid cell holder using an ultrasonic bath for 20 minutes. The contact pad can be immersed in the bath. Only immerse the part that is covered with the lid. Do not immerse the vent holes in liquid.

4. Dry up the liquid cell holder using an air pistol.

5. Put back the gasket used with the dummy liquid cells. Put back the dummy liquid cells and the lid.

6. Store the sample holder in a vacuum station.

Post-experiment analysis using Fiji (ImageJ)

NOTE: It is recommended to split each frame of the video taken into single images. The purpose of this post-experiment analysis step is to transform the original videos of the nanoparticles into binary videos that can be analyzed by Fiji.

A median filter is used in order to enhance the contrast of the nanoparticles on the background (Figures 7B &7E). This is essential to facilitate the binarization of the video. Save the image sequence in tiff format.

2. Crop out all the undesired artifacts from the video (for example the scale bar or the edge of the liquid cell window).

Click on

Representative Results

Figure 5

Discussion

The described protocol enables following the nucleation and growth of gold nanoparticles driven by radiolysis in a temperature-controlled liquid media. Combined with automated video processing, it allows measuring the effect of the temperature on key parameters of nanoparticle synthesis such as the density, the size, the shape and the atomic structure of nanoparticles. These valuable inputs allow evaluating the effect of the temperature on the nucleation and growth rates, detecting possible phase transitions and visualizing the faceting processes that dictate the final outcome of colloidal solutions. Together with the possibility to control the composition of the reactive media, temperaturecontrolled liquid cell TEM is another step towards the direct observation of the nucleation and growth processes of various nanostructures in realistic synthesis conditions. The interpretation of the results presented in this article and their comparison with nucleation and growth models will be discussed elsewhere. Here, we want to highlight several methodological aspects that must be considered to conduct relevant in situ TEM experiments.

First of all, it is crucial to identify the electron beam effects in the reaction media because they can drastically influence the results of the experiment. Here, as water radiolysis is the driving force of nanoparticle formation, the growth speed increases rapidly with the electron dose rate which will impacts the final shape of nano-objects 11 , 15 . Therefore, to study the effects of the temperature on the nucleation and growth of nanoparticles, it is necessary to compare growth experiments acquired with the same electron dose rate. In STEM mode, the electron dose rate corresponds to the beam current (in electron per second) divided by the image size (in nm 2 ). Therefore, a constant electron dose rate implies to maintain the same beam current (i.e., same condenser aperture and same spot size) and the same magnification for each experiment. Quantifying the beam current of the imaging conditions using a CCD camera or a Faraday cup is important to interpret and reproduce the data. The magnification and resulting dose rate should be selected according to whether one wishes to visualize the growth of a large assembly of nanoparticles to extract statistically relevant results on the growth kinetics (Figure 5) or the growth mechanisms at the single nanoparticle scale to identify the preferential adsorption sites on the nanoparticle surfaces (Figure 10). If the nucleation and growth processes are too fast, particularly at high magnification, small condenser aperture and small spot size should be selected to minimize the dose rate. The nucleation and growth of nanoparticles can also slow down by reducing the concentration of metal precursor in the analyzed solution but note that the concentration of radiolytic products will increase with temperature. In a general manner, it is also important to take into account the electron irradiation history of the whole sample. Here, for example, if several growth experiments are rapidly performed in areas close to each other, the density of nanoparticles will decrease over time because the concentration of gold precursors in the studied area decreases. This effect can be minimized by separating the growth experiments both in space and time and by using the liquid-holder in flow mode.

Interface-tracking algorithms are extremely helpful to automatize the analysis of videos and extract quantitative results on the nucleation and growth of large nanoparticles assemblies. However, it is worth noting that the imagebinarization step is always data specific, meaning that the filters and the data processing that must be applied on images to optimize the detection of the nanoparticle/liquid interface will vary from an experiment to another. Moreover, it is essential to compare the results of these automated analyses with manual measurements performed on a few images to optimize the image-processing workflow and to know its limitations. Here, for example, multiple scattering events in the increasingly thick 3D nanoparticles formed at high temperature induces a contrast inversion of their core after 30 seconds of observation because the angular broadening of scattered electrons results in a decrease of the collected signal in the angular range of the annular detector. To keep measuring the true surface area of these nanoparticles, we used a "fill holes" data process after the binarization of the image that fills the inner circle of ring shape contrasts (Figure 7F,G). However, we had to use a small dilatation of the objects to make sure that these ring shape contrasts are always fully connected. This latter step leads to a slight overvaluation of the mean surface area of nanoparticles in the automated measurements (Figure 9). Similarly, for the detection of nanoparticles, we have to define a minimal size of detected objects (S mim ) to avoid detecting the noise, but this parameter affects the measured nucleation rate. to noise ratio is a common challenge in liquid-phase TEM that can be further improved using other segmentation and denoising methods 24 or machine learning approaches 25 .

As seen in

Last but not least, the preparation of the liquid cell and the cleaning of the liquid holder must be very carefully performed to avoid contaminations of the reaction media.

In general, controlling the temperature of the sample during LCTEM analyses provides the opportunity to investigate thermal effects on chemical reactions that occur at the interface between solids and liquids. Therefore, we hope that the present method paves the way to other in situ TEM experiments designed to reveal the dynamics of hard, soft or biological materials in temperature-controlled liquid media.

Chapter 3

Quantitative in situ visualization of thermal effects on the formation of gold nanocrystals in solution

This chapter is a scientific paper from Advanced Materials (Wiley) [2]. Because of the embargo on this paper, I am not allowed to reuse it online so I encourage readers to look for the paper and its supplementary material with this link: https://doi.org/10.1002/adma.202102514.

Introduction

Oxidative etching plays a key role in the dynamics of metallic nanostructures in their formation or application media. On the one hand, the use of oxidizing agents and coordination ligands for the metal ions is a well-known strategy to shape metal nanoparticles during liquid-phase synthesis. 1 On the other hand, oxidative degradation is an integral part of the life cycle of nanomaterials, especially when used in fuel cells 2,3 and biological media. 4-8 Therefore, understanding the atomicscale mechanisms of nanomaterial dissolution in liquid electrolyte is of primary importance to design more efficient nanotechnologies. In that regards, liquid cell transmission electron microscopy (LCTEM) has become a method of choice to observe the oxidative etching of individual nanostructures such as metallic nanoparticles 9-17 and nanoalloys 18-21 and it has provided a unique observation window on the intermediate nanostructures formed during these dynamic processes.

Like the nucleation and growth of nanoparticles, 22,23 the dissolution mechanisms are simultaneously driven by kinetic and thermodynamic effects that depend on the reaction speed and the structural stability of nanomaterials, respectively. Therefore, LCTEM allowed highlighting the effects of oxidation potential, nanoparticle faceting and crystal defects on selective-etching processes. 11-13,20 However, the influence of surface functionalization on the corrosion dynamics has always been disregarded, although the nature and spatial distribution of organic shells are known to affect the stability and reactivity of nanostructures. 4,6,24,25 From theoretical point of view, different approaches have been actively developed to understand the role of surfactants on the structure of metal nanoparticles. 26,27 Generally, methods of quantum chemical calculations enable a detailed study of a system at the atomic level, but the computational cost for such methods is high. To overcome these difficulties, classical molecular dynamic (MD) approaches involving force fields with different level of accuracy were developed to investigate the evolution of large molecular assemblies for long periods. Moreover, many MD works focused on the interaction between surfactants and metal surfaces have been performed to explain the growth of anisotropic nanostructures coated with ligands such as citrate, piptide, CTAB …. 28-31 However, to our knowledge such theoretical approaches have never been used to reveal the effect of surface functionalization on the dissolution processes of nanoparticles.

In the present study, we combine experimental and theoretical investigations to demonstrate at the atomic scale the key roles of degradation kinetics and capping agents in the selective shortening of gold nanorods (NRs) in oxidative media.

Experimental section and methods

Nanorods and chemicals

Citrate-coated gold NRs were purchased from Nanocomposix Inc. According to the supplier, the gold NRs were first fabricated by a conventional wet-chemical method assisted by cetyl trimethylammonium bromide (CTAB). This very common surfactant is then replaced by sodium citrate molecules. The NRs are highly purified to remove all residual reactants following synthesis and dispersed in Milli-Q water. Other chemicals used for liquid-cell TEM were purchased from Sigma Aldrich Inc., with ACS Reagent grade.

TEM experiments and data processing

All TEM experiments were performed on a JEOL ARM 200F microscope equipped with a CEOS aberration corrector for the objective lens and a cold field emission electron source. 32 We used a 200 kV acceleration voltage.

Liquid STEM imaging was performed using a liquid-cell TEM holder commercialized by Protochips Inc. A colloidal suspension of citrate-coated gold NRs dispersed in an acidic saline solution containing 1 M of NaCl and 10 -2 M of HCl was encapsulated in the liquid-cell by using the conventional loading process. 22,23 The Au spacers of the liquid cell were 150 nm thick and the experiments were performed in the corners of the observation window, where the liquid thickness is minimum. The acidic saline solution was continuously flowing into the liquid-cell during the experiment with a flow rate of 5 µL min -1 . STEM HAADF imaging was performed with a pixel dwell time of 5 μs and a probe current of 1.1 10 8 electron per s. The electron dose rate was then tuned with the STEM magnification. The dose rate given in electron per s per Å 2 , can be calculated by dividing the probe current by the irradiated area (i.e. the image area in STEM mode). Videos of NRs etching were acquired with a frame rate of one image per second using Digital Micrograph software. The length and width evolution of NRs during the etching processes was extracted with an automated video processing using Fiji and a homemade Python code.

Aberration-corrected HRTEM imaging was exploited to study the spatial distribution of citrate coating on NRs deposited on a thin carbon film. Using the CEOS aberration corrector, the first order aberrations (A1 and C1 (i.e. the focus)), the second-order aberrations (A2, B2) and the third order aberrations (C3, A3 and S3) were tuned below 2 nm, 20 nm and 1 µm, respectively. These optical conditions optimize the contrast of organic molecules adsorbed on gold nanocrystals because they minimize the noise arising from the carbon substrate and they avoid the contrast delocalization of the gold surfaces. We note that the citrate layer is more easily detected on NRs flatly deposited on the carbon film than on NRs laying at the edge of carbon film which highlights the influence of the local environment of NRs on the conformation the citrate layer.

The following digital processing was applied to HRTEM images to better visualize the citrate layers. We used a low-pass filter with cutoff frequency at 3 nm -1 to suppress the atomic contrast of the NR and to make the citrate layer the area of the images with the highest local intensities. Then, the color thresholding method implemented in Digital micrograph software allows us coloring in hot colors the high signal of the citrate layer.

To take into account the high variability in thickness of the citrate coating, the average thickness of citrate layer on the body, the tips and the corners of the NRs was calculated by measuring the average thickness on 5 nm long surface sections. These measurements were performed over 15 NRs (corresponding to 1000 nm of analyzed projected surface).

Grand canonical monte carlo (GCMC) simulations

At first, the atomic position of a 13 nm long and 5.8 nm wide NR was calculated with the Nanofabric software. 33 Then, the Au-Au interaction is modeled by a tight-binding semi-empirical potential within the second moment approximation. Such model is particularly well suited to model transition and noble metals where the cohesion is governed by the d-electron band. 34,35 The parameters for gold, fitted on the bulk cohesive energy, lattice parameter and elastic constants, can be found in Chmielewski et al. 36 This model, both fast to compute and accurate, is then implemented in a MC code using GC algorithm with fixed temperature and Au chemical potential (µ Au ). The GC algorithm used consists in a series of MC steps. Each step randomly alternates displacement moves but also attempts to incorporate and remove Au atoms in a previously defined active zone. In order to mimic the etching process from the surface, the active zone for extracting/inserting Au atoms is defined as a region ± 5 Å from the surface of the NR. It is important to specify that in order to respect the detailed balance and thus ensure that the Markov chain is satisfied, both events (extraction and incorporation of Au atoms) are to be tried. 37 Although studying etching, one cannot simply extract only the Au atoms which would bias the simulation. In the present work, we typically performed up to 2000-8000 MC steps to get a complete degradation of the NP depending on µ Au . Within each MC step, we systematically performed 2000 attempted displacement trials per atom. To extract or incorporate Au atoms, 500 attempts were made and ended as soon as one successful event has occurred. We then performed GCMC simulations at T = 500 K and different µ Au ranging from -3.75 to -3.95 eV. The Au chemical potential is referred to a fictitious ideal monoatomic gas, explaining its values that are of the order of the cohesive energies of Au in FCC bulk (e.g. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

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µ Au (at a given T ), one would tend to favor the adsorption or extraction of Au atoms. In our case, we worked in a chemical potential range below -3.70 eV to observe the dissolution of the NRs.

In order to analyze the etching mechanisms in detail, we calculated a desorption rate through the tip and body of the NR defined as follows: desorption rate = n Au removed /A where A is the surface of the tip or body of the NR and n Au removed is the number atoms removed during the MC simulation on each surfaces. Consequently, the calculation of the desorption rate implies determining the surface of the tip and the body of the NR during the simulation. However, the surface of the NP is always evolving. At the atomic scale, it is therefore difficult to represent it as a multifaceted NR as can be seen in its initial state. A simple way to approximate the surface of the object is to assimilate it to a cylinder closed by two half-spheres. By doing so, the cylinder corresponds to the body of the NR and the two half-spheres to the tip (Fig. S1 †).

DFT calculations

Theoretical calculations were performed within plane-wave pseudopotential method based on density functional theory as implemented in the Vienna Ab initio Simulation Package (VASP). 38,39 Ions are described by employing PAW pseudopotential for atomic core region. 40,41 The exchange-correlation energy was calculated within the Perdew, Burke, and Ernzerhof formulation of the generalized-gradient approximation (GGA-PBE). 42,43 After the extensive test calculations for the total energy convergence, the kinetic energy cut-off for plane wave expansion was set to 400 eV. Six Au surface orientations [START_REF] Daniel | Nanometer resolution elemental mapping in graphene-based tem liquid cells[END_REF], ( 100), [START_REF] Noh | Lithographically patterned well-type graphene liquid cells with rational designs[END_REF] (for low index surfaces) and ( 221), (311), (331) (for high index surfaces) were considered. The Au surfaces were simulated by the (4 × 4) slab models containing 6 atom layers, in which the 2 bottom layers are fixed to mimic the bulk state. The calculated equilibrium lattice constant of the bulk was found to be of 4.17 Å, which is in good agreement with the experimental value of 4.08 Å. 44 The slab models are separated with a 15 Å vacuum space along the Z-axis and the K points is set as (2 × 2 × 1). The geometry of the acetate and citrate molecules in a gas phase is optimized in a 15 Å × 15 Å × 15 Å cubic box; the k-point sampling is set as (1 × 1 × 1). The convergence criterion for the electronic self-consistent cycle was fixed to 10 -6 eV per supercell. Atomic positions and lattice parameters were both relaxed using the conjugate gradient algorithm until all the forces are below 0.02 eV Å -1 . The surface energies of Au [START_REF] Daniel | Nanometer resolution elemental mapping in graphene-based tem liquid cells[END_REF], Au(100) and Au(110) are calculated to be 0.64 J m -2 , 0.80 J m -2 and 0.91 J m -2 , respectively. Even underestimated compared with the experimental value of 1.5 J m -2 , these DFT predicted surface energies are in good agreement with the literature. 45 More theoretical details and comparisons with other DFT functional could be found in our previous works. 46,47 The electronic structure of the adsorption systems was analyzed through Bader charge density analysis 48 with the implementation of Henkelman and co-workers 49 which allows analyzing the density issued from VASP code. From a theoretical point of view, classic DFT cannot accurately capture weak interactions such as van der Waals forces and it is always difficult to ascertain the existence of a physisorbed molecular state on the gold surfaces. In order to evaluate the effect of the dispersion correction term in the DFT calculated adsorption energies of citrate molecules, we employed the dispersion term DFT-D3 proposed by Grimme 50 and implemented in the VASP code. However, although we have adopted the dispersion correction in the above calculations, the DFT-GGA method tends to underestimate the energies (surface energies and adsorption energies), which is a well-known artifact. 51 3. Results and discussion The ROS-induced degradation of gold nanostructures that also occurs in a slower way in cellular media, 8 can be summarized as followed:

Au 0 þ 4Cl À ! ½AuCl 4 À þ 3e À 3e À þ 3OH • ! 3OH À 3OH À þ 3H þ ! 3H 2 O H 2 O 2 þ 2H þ þ 2e À ! 2H 2 O
In these conditions, with an electron dose rate of 3.3 electrons per s per Å 2 , the dissolution time of NRs varies from 100 seconds (Fig. S3 †) to 180 seconds (Fig. 1a) but the etching process is always selective. Indeed, the length of the NRs decreases linearly with time while their width remains constant until spherical nanostructures remain (Fig. 1b). Then, the etching rate accelerates leading to a quick disappearance of the nanocrystals. Consequently, the aspect ratio of the nanostructures (i.e. length/width) decreases linearly with time from 3.8 to 1 (Fig. 1c andS3 †). As the concentration of ROS increases with the electron dose rate, 52 View Article Online possibility to modulate the etching kinetics and visualize its impacts on the degradation mechanisms. As illustrated in Fig. 1d ande, with a dose rate of 20.5 electrons per s per Å 2 the dissolution of NRs takes only about 30 seconds and both the length and width decrease over time. Throughout this fast dissolution process, the width and length loss percentages have the same time dependence (Fig. S4b †) resulting in a constant aspect ratio of the nanostructures, except for the very last seconds in which their shape anisotropy decreases (Fig. 1f and S4 †). These speed-controlled experiments reveal a transition between a low driving force regime leading to the slow selective shortening of NRs and a high-driving force regime in which fast and more isotropic dissolution are observed. These real-time in situ observations confirm in a straightforward manner many studies carried out by UV-Vis spectroscopy on the colloidal stability of NRs in oxidative media with various concentration of etching agent. 13,53-56 To summarize, in the high driving force regime, the dissolution kinetics is domi- This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

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nated by the high etching power of the solution which depends on the concentrations of ROS and Cl -, whereas the low driving force regime allows visualizing the difference in reactivity between the body and the two tips of NRs and the observed selective etching is then driven by the intrinsic thermodynamic properties of nanostructures.

3.2. Why the ends of nanorods are more reactive?

The preferential etching of NR's ends can either be explained by the surface structure of nanocrystals (i.e. defects and faceting) that modifies the local reactivity or by an inhomogeneous passivation layer that dictates surface accessibility for etching agents. 12,17,25,54,55 In the following, we combined aberrationcorrected HRTEM imaging and atomistic simulations to evaluate the respective impacts of the crystal surface and the citrate coating on the anisotropic etching of NRs. At first, we performed atomic-scale TEM investigations under vacuum to determine the faceting of nanocrystals. The HRTEM images of nanostructures along the [START_REF] Koo | Live cell electron microscopy using graphene veils[END_REF], [START_REF] Noh | Lithographically patterned well-type graphene liquid cells with rational designs[END_REF] and [001] zone axes (Fig. 2a-c) clearly reveal that the body of the rods exhibits four {100} and four {110} faces, whereas the tips consist of four {110} and four {111} faces (herein referred to as the corners of the NRs) together with a {100} face at the apex. If small terraces and steps are occasionally observed at the interface between faces, NRs are monocrystalline and do not present classical defects of FCC metals, such as stacking faults or twin planes. These results is consistent with the 3D shape of NRs obtained by seed-mediated growth in aqueous solution, assisted by the surfactant cetyltrimethylammonium bromide (CTAB), 57 and it confirms that the surface exchange protocol used to replace the CTAB with citrate capping agents does not affect the faceting of nanostructures (see section 2.1).

To determine if the selective shortening of NRs origins from their atomic-scale faceting, Grand Canonical Monte Carlo (GCMC) simulations were performed using a specific N-body potential derived from the second moment approximation of the tight-binding scheme. 34,35 The chemical reactions involve in the gold etching, is translated in the GCMC algorithm into a change of chemical potential of Au atoms (µ Au ). 58 As required in GCMC simulations that take the system to thermodynamic equilibrium, long atomic relaxations are performed after each accepted Au removal, as well as attempts to incorporate Au atoms in the structure. 37 Simulation details are provided in section 2.3. Starting from a 13 nm by 5.8 nm NR containing about 19 000 atoms with the same crystal faceting than the NRs studied experimentally (Fig. 2d), we simulated atomic-desorption processes with various chemical potentials at 500 K until the NR completely dissolved (Fig. 3a). As expected, the stronger (i.e. the more negative) the chemical potential, the faster the dissolution of the NR (Fig. S5a †), but regardless of the chemical potential, the number of extracted atoms per MC step is constant until 6000 atoms remain within the nanostructures, then the dissolution process speeds up. This size effect on the desorption processes is explained by the decrease in the cohesive energy of nanocrystals when their dimensions are reduced to a few nanometers (Fig. S5b †). 36,59 It is worth noting that this effect occurs in a size range (width < 3.5 nm) which prevents its experimental observation because of the resolution limit of our LCTEM experiments and the very short life time of degraded NRs with a size below 5 nm. The coordination-number distribution of extracted atoms shows that strong chemical potential favors the extraction of atoms with higher coordination numbers (Fig. S5c †), which reflects the possibility to extract atoms with higher desorption energies (Fig. S5d †). More importantly, GCMC simulations never show a selective shortening of NRs but rather isotropic dissolution processes. As illustrated in Fig. 3 and S6 † for the weaker and stronger chemical potentials, respectively, the reduction in length and width of the NRs follow very similar trends (Fig. 3b and S6b †) and we measure similar desorption rates on the body and at the tips of NRs (Fig. 3c andS6c †) throughout the simulations. Consequently, the aspect ratio of NRs increases when the dissolution process speeds up because the loss percentage is more important along the width than along the length (Fig. 3d andS6d †). Fig. 3a and S6a † also show that the initial crystal facets of NRs rapidly transform into rough surfaces which likely favors the isotropic degradation of the NRs. It also indicates that atomic desorption goes faster than surface diffusion that should maintain the facetted morphology of nanocrystals. Ultimately, the disagreements between the LCTEM observations and the GCMC simulations reveal that crystal-faceting alone does not explain the directional etching of gold NRs observed experimentally in the low driving force regime. The isotropic etching observed in the GCMC simulations that does not take into account the effects of the ligands on the thermodynamic properties of nanostructures, simply results from the very similar surface structure of the body and ends of NRs. Indeed, they both exhibit {100} and {110} faces and the {111} faces that are only observed on the tips are known to be the most stable gold surface. 60 Since surface functionalization reduces access to gold nanocrystals for the chelating agent and stabilizes crystal surface, we evaluate the role of citrate coating in the chemical etching of NRs by studying both experimentally and theoretically the citrate/Au interfaces. The absence of delocalization contrast at the edges of nanocrystals and the low noise level arising from the amorphous substrate are two characteristic features of aberration-corrected HRTEM images acquired close to zero focus 61 that allow revealing the inhomogeneous distribution of the citrate layer over the NRs (Fig. 4a and S7 †). 4 Obviously, these HRTEM images provide only a partial view of these soft/ hard material interfaces since the citrate layer can be detected only if it is not superimposed with the gold crystal. Moreover, the conformation of the organic layer around the NRs is very likely influenced by the local environment of the NRs during the deposition and drying processes. Nevertheless, in general, we observe much more citrate on the body of the NRs than at the extremities. Local-thickness measurements of the citrate layer illustrated in Fig. 4a have resulted in an average thickness (T ˉcit ) of 4.5 ± 3.0 nm and 1.9 ± 0.7 nm on the body and the tips of the NRs, respectively (see details in section 2. View Article Online cisely, the corners of the NRs are the less covered surfaces with a T ˉcit of only 1.1 ± 0.5 nm with a minimum for the {111} corners (T ˉ111 cit = 0.8 ± 0.2). These atomic scale observations of dried nanostructures highlight the lower density of citrate layers on the (111) facets, and more generally on the tips of NRs. Note that, it would be very interesting, but still challenging, to confirm these results by LCTEM to minimize experimental artefacts.

At first attempt to explain theoretically this inhomogeneous citrate coating, DFT calculations were performed to determine the binding modes and adsorption energies of citrate mole-cules, which contain three carboxylate groups (Fig. S8 †), on {100}, {110} and {111} gold surfaces. We considered the dissociative adsorption via carboxylate O-H terminal where H atom was assumed to be dissociated in the solution and to diffuse onto the Au surface (Fig. S9 †). In agreement with the recent results of Al-Johani et al., 62 we found that carboxylate groups tend to bridge two Au surface atoms, with each carboxylate oxygen interacting with one Au atom. On {100}, {110} and {111} gold surfaces, carboxylate-group adsorption is always stronger when bridging two first neighbor Au atoms (see details in ESI, Fig. S10 and Table S1 †). We found that citrate This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

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anions have two stable geometries on gold surfaces: the C 2 conformation, where one terminal carboxylate group links to the gold surface (Fig. 4b) and the C 4 configuration, where one terminal and the central carboxylate groups interact with the substrate (Fig. 4c). Interestingly, the calculated adsorption energies (E ad ) show endothermic adsorption over {111} surfaces for both C 2 (E ad = 0.05 eV) and C 4 (E ad = 0.79 eV) configurations, whereas exothermic adsorptions are found for the In order to go further in the comprehension of the reactivity of the different facets of gold NRs toward citrate molecules, we performed additional calculations on high index surfaces such View Article Online as {221}, {311} and {331}. These latter could be representative of step atoms at the edge of small terraces that are sometimes observed at the interfaces between faces on the tips of NRs (Fig. S12 †). DFT results show that these sites are also exothermic adsorption points for citrate molecules with competitive adsorption energies (E ad < -0.30 eV) (see details in ESI, Fig. S13 and Table S2 †). All these theoretical calculations emphasis that the {111} surfaces are the least prone to citrate adsorption. In line with HRTEM observations, this indicates that the {111} corners of NRs are less protected by the citrate layer which favors their degradation. Therefore, in the low driving force regime, these easily accessible surfaces are very likely the most, if not the only, reactive site for oxidative etching which drives the selective shortening of NRs. The more isotropic etching observed in the high driving force regime is due to the higher etching power of the solution that either becomes too strong for the citrate-covered surfaces or results in the degradation of the citrate shield itself. Interestingly, the identified distribution of citrates over gold NR facets and their role in the anisotropic dissolution is very similar to the identified distribution of surfactants and their role in the anisotropic growth phenomena. Indeed, using This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

View Article Online molecular dynamic simulations, Sulpizi et al. 28 have identified the role of surfactant layer that preferentially covers the ( 100) and ( 110) facets, leaving the [START_REF] Daniel | Nanometer resolution elemental mapping in graphene-based tem liquid cells[END_REF] facets unprotected. This inhomogenous surfactant layer promotes anisotropic growth with the less protected tips growing faster, which explains the shape of NRs. Facet-specific coverage has been also reported by Heinz and coworkers for peptides on Pt nanoparticles. 31 Moreover, these authors have introduced the concept of soft epitaxial binding mechanism, which control the facet recognition and binding strength in solution. 63 It is worth noting that our DFT calculations of single adsorbed citrate molecule interacting with gold surfaces are an initial proof of the effect of ligands on the protection of NRs from degradation and that further analyses should be undertaken. Indeed, it is actually well known that for charged molecules, such as citrate, the contribution of the polarization effect to the adsorption energy should be taken into account. 64,65 The development of an accurate force field including polarization effects and the possible influence of the solvent and the charged species generated by its radiolysis to implement in molecular dynamic simulations is an essential future step to reproduce the observed dissolution process.

Conclusion

Real-time monitoring of reaction processes is crucial for understanding the reaction mechanisms. So far, UV-Vis spectroscopy has been the standard method to characterize in real time the corrosion of gold NRs and reveal selective-shortening processes because the resulting blue shift of the longitudinal surface plasmon resonance of NRs allows tracking the decrease of their aspect-ratio. 13,53-55 In that regards, this impact visible to the naked eye on the optical response of the solution opens up new perspectives for sensing applications. 56 In this letter, we exploited LCTEM to directly image and control the oxidative etching gold NRs. For the first time, we found experimental conditions that allows visualizing the selective shortening of gold NRs at the nanoscale. These in situ investigations confirmed that this directional etching process requires reducing the etching power of the solution to make the tips of the NRs the only reaction site. Then, we combined HRTEM and atomistic simulations to establish the link between the anisotropic dissolution of NRs and their structural properties. We demonstrated that the higher reactivity of NR tips is not due to their intrinsic crystal structure but results from a less abundant citrate coating. These results highlight the key role of surface accessibility in corrosion processes and the necessity to consider the structure of organic/inorganic interfaces to understand the degradation of nanostructures.

Detailed analysis of citrate adsorption

The computed adsorption configurations of citrate on Au surfaces through one terminal carboxylate group (C2 configuration) are shown in Figure S10. Here as well, we have tested several possible adsorption sites to check the effects of the different surface sites. After optimizations, the results show that the most stable configuration is when the two oxygen atoms are combined to one Au surface atom and its first nearest neighbor Au atom (Table S1). Over the (110) surface, the configuration in which oxygen atoms of carboxylate group interact with two second Au atom neighbors is found to be less stable than the one engaging two Au first neighbors by about 0.08 eV.

The adsorption of citrate on high index surfaces (Au(221), Au(311) and Au(331))

As the tips of NRs present some surface defects (i.e. steps and small terraces) at the interface between ( 111), ( 110) and ( 100 The electron dose irradiating the analyzed nanostructures during data acquisition can be reduced by almost four orders of magnitude compared to conventional HRTEM analysis. Finally, dynamical analyses in reciprocal space are used to provide new insights into the shape-dependent rotation of nanocrystals in the liquid-cell.

Introduction

Liquid-cell transmission electron microscopy (LCTEM) has opened up exciting opportunities in materials, live and Earth sciences in which nanoscale processes at liquid/solid interfaces are key determinants [START_REF] Ross | Opportunities and challenges in liquid cell electron microscopy[END_REF]. The dream of imaging nanostructure dynamics with atomic resolution in liquid has motivated the development of optimized liquid-cells with reduced liquid-layer thickness and windows with better electron transparency [START_REF] Ross | Liquid Cell Electron Microscopy[END_REF]. Therefore, graphene liquid STEM nanodiffraction was developed to obtain the image and the diffraction pattern of single nanostructures without switching between the image and diffraction modes (J.M., 1999). As described in figure 1, this technique consists in acquiring a STEM-HAADF survey image (fig. 1a) on which the beam can then be positioned on any pixel to acquire the diffraction pattern of single nano-objects on a CCD camera (fig. 1b-e). Firstly, the structural analysis of nanomaterials by STEM nanodiffraction requires using a nano-sized electron probe to acquire diffraction patterns of single nano-objects. Secondly, the convergence angle of the probe must be small to avoid the overlapping of diffraction spots in the diffraction patterns. Over the last two decades, many alignment methods have been proposed to meet this two prerequisites and nearly parallel STEM probe (convergence angle below 1 mrad) with a size around 1 nm can now be achieved [START_REF] Alloyeau | STEM nanodiffraction technique for structural analysis of CoPt nanoparticles[END_REF][START_REF] Ganesh | D-STEM: A Parallel Electron Diffraction Technique Applied to Nanomaterials[END_REF]. Thus, STEM nanodiffraction has been exploited on various nano-objects studied under vacuum, such as nanotubes [START_REF] Cowley | Applications of electron nanodiffraction[END_REF], ferritins [START_REF] Cowley | The Structure of Ferritin Cores Determined by Electron Nanodiffraction[END_REF], nanoparticles [START_REF] Ganesh | D-STEM: A Parallel Electron Diffraction Technique Applied to Nanomaterials[END_REF], nanoalloys [START_REF] Alloyeau | STEM nanodiffraction technique for structural analysis of CoPt nanoparticles[END_REF])… The key advantages of this technique is the fast and possibly automated acquisition process of nanodiffractions and the possibility for complete structural analysis of nanomaterials with a much better information limit than with direct imaging, because the level of structural detail in reciprocal space is only limited by the observable range of electron scattering. In practice, STEM nanodiffraction allows determining the individual structure of much more randomly-oriented nano-objects in an assembly. Such statistically-significant structural analyses were notably crucial to reveal size and shape effects on the structural properties of nanostructures [START_REF] Alloyeau | Size and shape effects on the order-disorder phase transition in CoPt nanoparticles[END_REF].

Here, we show for the first time that STEM nanodiffraction can be exploited in LCTEM and opens up new opportunities to characterize nanomaterial structure and dynamics in liquids. Most importantly, the use of high speed acquisition process allows conducting low-dose structural analysis of single nano-objects. By comparing our results with aberration-corrected HRTEM analyses, we reveal the advantages and drawbacks of these reciprocal-space investigations with respect to direct imaging.

Methods

Sample preparation

Liquid cell experiments were carried out on a JEOL ARM 200F microscope equipped with cold

STEM nanodiffraction acquisition

STEM images were acquired on a JEOL high angle annular dark field detector (STEM-HAADF), while nano-diffraction patterns were acquired with a high-speed CCD (Gatan, OneView) by selecting the position of the electron probe directly on the STEM-HAADF image (spot analysis of the DigiScan system). The convergence angle of the electron probe (α) can be easily determined by comparing the diameter of the diffraction spots (D) and the distance (Rhkl) between the spots (000) and (hkl). It is then given by: 𝛼 = 𝐷 * 𝜆 𝑅 !"# * 𝑑 !"# with λ the electron wavelength (2.51 pm at 200 kV) and dhkl the lattice spacing (hkl) planes. Here, the convergence angle was reduced to 7.4 mrad only by using the smallest condenser aperture available (diameter = 10µm). The convergence angle can be further reduced by modifying the alignment of the illumination system, more precisely by powering on the condenser mini lens that is switch off in conventional STEM mode [START_REF] Alloyeau | STEM nanodiffraction technique for structural analysis of CoPt nanoparticles[END_REF][START_REF] Ganesh | D-STEM: A Parallel Electron Diffraction Technique Applied to Nanomaterials[END_REF].

Nevertheless, modifying the alignment of the STEM probe was not necessary for the structural analysis of gold nanoparticles, because a convergence angle of 7.4 mrad is low enough to avoid diffraction-spot overlapping. Thus, this simple alignment process is a very good compromise to obtain clear STEM images and analyzable diffraction patterns on which the atomic structure and orientation of metal nanoparticles can be determined. Pixel dwell time for image acquisition was fixed to 5 µs and two camera lengths of 8 cm (with the HAADF detector inserted) and 12 cm (with the HAADF detector retracted) were used.

The beam current (ie) for each probe size was measured prior inserting the sample by acquiring the image of the probe on the CCD camera. ie varies from 5.5*10 6 to 1.2*10 8 electron/s when the probe size increases from 10c to 5c. Note that using the smallest condenser aperture provides also the advantage to reduce ie. HRTEM images were acquired in a separated experiment, using the same sample preparation method. The beam current was also measured prior inserting the sample for similar illumination conditions.

Results and discussions

Studying the structure of single metal nanoparticles without spatial-resolution limitation

We have conducted aberration-corrected HRTEM and diffraction STEM analyses of gold NRs in water by using LCTEM. Both experiments were performed after the formation of a gas bubble in the liquid-cell to reduce the liquid thickness (see methods). In these conditions, the atomic contrast of gold NRs can be observed on HRTEM images (Fig. 2a). Nevertheless, as illustrated in figure 2b, only the [START_REF] Daniel | Nanometer resolution elemental mapping in graphene-based tem liquid cells[END_REF] and (002) lattice spacings of gold can be imaged. This restricted resolution drastically reduces the possibility of HRTEM for analyzing the atomic structure of randomly oriented gold nanoparticles, because it can only be visualized when the nanocrystals are oriented along the <001> and <101> directions.

On the contrary, STEM nanodiffraction allows analyzing the atomic structure of individual NRs without zone-axis orientation restriction. In fact, the better information limit of reciprocal space analyses is a decisive advantage because NRs are rarely in, or close to, low-index zone axis orientation in the liquid-cell. Indeed, only 7 % of the 75 studied NRs were found close to the <001> or <101> orientations (fig. 1b). The great majority of NRs were found out of zone axis (fig. 1c) or they were oriented along high-index directions, in which diffraction spots with subangstrom lattice spacing can be observed (fig. 1c &1d). Structural information are detected up to 0.05 nm on these diffraction patterns obtained with probe current of 1.2*10 8 electrons/s and an acquisition time of 79.8 ms. As HRTEM, STEM nanodiffraction allows correlating the size, shape and atomic structure of single nanocrystals, but over the same period of time, it allows analyzing substantially more nano-objects. This new possibility is an important step forward because the lack of statistical results is a major drawback of in situ TEM studies. It is worth noting that STEM nanodiffraction is here used to study monocrystalline nanoparticles, but it is obviously more challenging to study the full structure of nanostructures with crystal defects or immiscible elements. In that regards, HRTEM remains the best option to study the local atomic arrangements at the solid/solid or solid/liquid interfaces.

Low-dose structural investigations

Studying dynamic processes at the atomic scale while minimizing the electron dose is a major challenge in LCTEM. In that regards, we want to stress that STEM nanodiffraction provides the opportunity to probe the atomic structure of nanomaterials with an electron-dose level way below the one of HRTEM. As the position and the geometry of the electron beam are different in direct imaging (i.e. stationary and parallel beam) and in STEM nanodiffraction (i.e. scanning and then stationary focused beam) analyses, the dose in electrons.nm -2 or the dose rate in electrons.nm -2 .s - 1 are not the best parameters to compare the beam irradiation of these two techniques. Therefore, we have compared the number of electrons that irradiate a single NR during the acquisition of HRTEM (𝑁 $% ) and STEM nanodiffraction (𝑁 &' ) data.

In high resolution imaging, 𝑁 $% is given by:

𝑁 $% = 𝑑 ̇ * 𝑡 ( * 𝑆
with 𝑑 ̇ the dose rate, ti the acquisition time of the image and S the nanoparticle surface (here considered equal to 517 nm 2 for all the NRs). Calculating 𝑁 &' requires to sum the number of electrons necessary to acquire the survey image (Ns) and the diffraction pattern (Nd) that are given by:

𝑁 ) = 𝑖 * * 𝑡 + * 𝑆 𝑆 + 𝑁 , = 𝑖 * * 𝑡 ,
With ie the beam current (in electron.s -1 ), tp the pixel dwell-time, Sp the pixel surface and td the acquisition time of the diffraction pattern.

The high resolution image in figure 2a, was acquired with a dose rate of 2.95*10 5 electrons.nm - 2 .s -1 and an acquisition time of one second. Consequently, the NR has been irradiated by 1.5*10 8

electrons to obtain this image (𝑁 $% ). This very high dose could possibly be reduced by using higher frame rate or lower beam current, but the noise level of images acquired in liquid would rapidly hinder the detection of crystal planes. Remarkably, diffraction signal remains detectable even if the frame rate of the CCD camera and the beam current of the STEM probe are set to their maximum and minimum values, respectively. Indeed, the figure 3d shows the diffraction pattern of single NR that is a few degrees away from the <001> zone axis orientation. This image was obtained with an acquisition time of 3.3 ms and beam current of 5.5*10 6 electrons.s -1 by using the smallest probe size available. Consequently, only 1.8*10 4 electrons was necessary to acquire this diffraction pattern (Nd). Given the low magnification (250k) and the short pixel dwell-time (5 µs), the same STEM probe was focused on the NR only for 730 µs during the acquisition of the survey image (fig. 3a). Therefore, this image adds a small contribution (Ns = 0.4 *10 mode, but other synchronization between the beam position and the diffraction acquisition can be employed to further reduce the period of immobility of the electron-beam that may generate harmful radiolytic effects.

Studying nanoparticle rotation in liquid

Studying nanoparticles motion in liquid has been one of the many opportunities provided by liquid has been investigated with nano-scale TEM or STEM imaging which gives access only to translation motion of nano-objects. To take a fresh look at the motion of gold nanoparticles in the liquid cell, we have exploited STEM nanodiffraction to investigate the beam-induced spinning of nanocrystals as a function of their shape. As illustrated in Figure 4, probing the motion of single nanoparticles in reciprocal space reveals that during their period of immobility, nanoparticles have a stationary position but they keep rotating under the electron beam. When low electron dose are used, the diffraction pattern of nanocrystals can be followed for several seconds before that they jump to a non-irradiated area. These observations show that the spinning motion of nanoparticles is governed by their shape and their local environment. Indeed, spherical nanoparticles tend to rotate very rapidly in a random fashion. This result is illustrated in figure 4a in which several crystallographic orientation are identified on the diffraction pattern of a spherical nanoparticle over 1.2 s of observation. This dynamic analysis of diffraction signals shows that spherical nanoparticles can rotate by several tens of degrees in a few tenth of second. For example, the jump from the <323> to the <100> orientations observed in figure 4a occurs in 0.4s and it implies a rotation of 50.14 or 64.46 degrees depending on the rotation direction. We have also analysed the rotation of gold nanoprisms, consisting in 2D nanostructures with large (111) triangular facets.

These nanostructures are most of the time observed along the <111> zone axis orientation and they continuously rotate around this <111> direction (fig. 4b). This spinning motion is consistent with the direct imaging of these planar nanostructures (fig. 5) showing that the triangular projected shape of nanoprisms is always clearly observed but presents different orientation from frame to frame. For their parts, NRs are less prone to rotation under the STEM probe. Most of the time they are observed along the same orientation for several seconds before jumping out of the irradiated area. The local environment of NRs could contribute to their higher stability. Unlike spherical or 2D nanoparticles, NRs frequently form aggregates with up to several tens of nanoobjects (fig. 1) most likely stabilized by their citrate ligands. Although nanocrystals are not in direct contact within these aggregates, the high cohesion of these nano-assemblies is evidenced by the diffusion behaviour of NRs that always have collective motions rather than individual trajectories (fig. S1c in supporting information). We can easily assume that the strong interactions between NRs in such aggregates prevent their individual rotation. Nevertheless, isolated NRs are also more stable than spherical or flat NPs. Sometime they randomly rotate by a few degree but they never radically change of orientation. For example, by comparing experimental and simulated diffraction patterns calculated with JEMS software, we measured that the NR in figure 4c rotates in an angular range of 7° around the <101> zone axis orientation during observation times (fig. S2 in supporting information). All these results illustrate the impact of nanoparticle dimensionality on their spinning motion. In a general manner 0D nanoparticles rotate more rapidly than anisotropic nanostructures. The 2D shape of nano-platelets dictates their spinning direction that tends to maximize their surface contact with the membrane, while the 1D shape of NRs provides them a high stability that is probably reinforced by inter-connections with other NRs.

Conclusion and perspectives

We have shown that STEM nanodiffraction can be exploited in LCTEM experiments to reveal the atomic structure of single gold nanoparticles in liquid. The fast acquisition process and the possibility to determine the structure of nanoparticles without zone-axis orientation restriction are two decisive advantages of STEM nanodiffraction as compared to HRTEM investigations.

Consequently, STEM nanodiffraction allows analyzing much more nanoparticles in the liquidcell in which colloids are always randomly oriented. More importantly, we have demonstrated that this structural analysis of single nano-objects can be performed in very low dose conditions.

Indeed, the use of high-speed camera allows a significant decrease of the electron dose seen by the analyzed nanostructures during data acquisition. This latter is reduced by almost four orders of magnitude with respect to conventional HRTEM analysis. This high frame rate analysis of the nanocrystal reciprocal space was also exploited to reveal the effect of nanoparticle shape on their spinning motion.

Interestingly, STEM nanodiffraction in liquid media could greatly benefit from the many electron optic and electronic developments that have emerged over the last decades. Notably, precession methods could be applied to obtain quasi-kinematical diffraction patterns of nanostructures that would be less sensitive to the slight misorientation of the zone axis from the optical axis of the microscope (Eggeman and Midgley, 2012; Vincent and Midgley, 1994). More importantly, the use of very short camera length (8 cm) allows the acquisition of diffraction patterns without removing the HAADF detector (figure 4b-c). Therefore, this simple microscope alignment is then The race to make the next generation of liquid cells to replace standard SiN chips have begun recently. Indeed, static graphene liquid cells (GLC) [START_REF] Frances | Opportunities and challenges in liquid cell electron microscopy[END_REF][START_REF] Mohammadreza Ghodsi | Advances in graphene-based liquid cell electron microscopy: Working principles, opportunities, and challenges[END_REF][START_REF] Park | Graphene liquid cell electron microscopy: Progress, applications, and perspectives[END_REF] have been used to enhance the quality of images during LCTEM experiments. Although they are useful for some experiments, there is still a need to control the volume and nature of the reaction media. This is why flow capable GLCs are interesting: they combine the enhanced image quality of static GLCs with the additional chemical control of the standard flow capable SiN liquid cells.

In this chapter, we will explain the interest and objective of GLCs by reminding the limitations of standard SiN liquid cells. Then, we will describe the state-of-the-art GLC research including static GLCs and flow capable GLCs. Finally, we will describe how we modified existing SiN liquid cells to obtain flow capable GLCs. We will also show preliminary TEM experiments realized with the latter that will allow us to discuss how to improve on what we have done. 106

6.1 Advantages of graphene liquid cells

A necessary mechanical stability and impermeability

The SiN window of the standard e-chips have its dimensions constrained by the very high vacuum of the TEM (reducing the size of the window will reduce the bulging effect and thus the mechanical straining on the window). Moreover, while it is transparent to electrons, SiN doesn't let liquid or molecules go through it. While the commercially available SiN based e-chips have these properties, the homemade liquid cells using 2D materials must have them too to replace SiN.

Thankfully, graphene exhibits a very high mechanical stability [START_REF] Lee | Measurement of the elastic properties and intrinsic strength of monolayer graphene[END_REF][START_REF] Koenig | Ultrastrong adhesion of graphene membranes[END_REF] while retaining a good flexibility [START_REF] Xu | Flexible graphene films via the filtration of water-soluble noncovalent functionalized graphene sheets[END_REF] and can therefore sustain the high pressures of the microscope. Increasing the number of layers of graphene improves on its mechanical stability and flexibility. Mono-layer graphene also has a very dense electron cloud that doesn't let molecules such as neon or argon to penetrate its surface [START_REF] Hu | Proton transport through one-atomthick crystals[END_REF][START_REF] Sun | Limits on gas impermeability of graphene[END_REF]. However, graphene can have defects that can compromise its impermeability. In fact, it has been shown that mono-layer graphene needs to lose 6 carbon atoms in order to lose this essential property although losing less than 6 atoms isn't critical [START_REF] Leenaerts | Graphene: A perfect nanoballoon[END_REF]. The appearance of these defects is dependent on the graphene synthesis method (synthesized CVD graphene exhibits more defects than exfoliated graphene for example) or can be beam-induced (the energy threshold of atomic-displacement for single-layer graphene is of 82 keV [START_REF] Banhart | Irradiation effects in carbon nanostructures[END_REF][START_REF] Jannik | Accurate measurement of electron beam induced displacement cross sections for single-layer graphene[END_REF]). Operating at 80 keV or below is therefore highly recommended when working with GLCs but pre-existing defects on graphene lower the energy threshold of atomic displacement and favor the formation and growth of holes under radiation [START_REF] Mouhoub | Probing structural and electronic properties of h-BN by HRTEM and STM[END_REF][START_REF] Jannik | Accurate measurement of electron beam induced displacement cross sections for single-layer graphene[END_REF].

Reducing the electron dose or using multilayer graphene will help to limit beam-induced damage.

An increase of the spatial resolution in LPTEM

The first advantage of LPTEM compared to other tools of observation like optical microscopy is its high spatial resolution (the second advantage being its high temporal resolution) [START_REF] De Jonge | Resolution and aberration correction in liquid cell transmission electron microscopy[END_REF]. In order to truly understand the impact of GLCs on the imaging conditions, we need to explain what parameters must be taken into account when we calculate the spatial resolution.

In a traditional TEM experiment, the sample is prepared so that it is as thin as possible.

Thus, in this case, the first limiting factor is the intrinsic resolution of the electron optics.

However, in liquid phase TEM experiments, the thickness of the sample is very high, thus limiting the spatial resolution. Indeed, the electron scattering effects of the liquid expand the energy dispersion of the electron beam. Moreover, as we already discussed before, radiolysis effects must be controlled and minimized in most cases. The electron dose must then be reduced but by reducing it we also reduce the signal-to-noise ratio. Therefore, the reduction of the electron dose is the first factor of limitation of the spatial resolution in liquid experiments. In short, three additional parameters must be taken into account when considering spatial resolution in LPTEM: the electron dose, the liquid thickness (without forgetting the liquid cell bulging inducing an increased liquid thickness) and the position of the studied object in the liquid cell.

The position of the studied object inside the liquid cell is very important and will determine the optimal mode to use for imaging [START_REF] De Jonge | Electron microscopy of specimens in liquid[END_REF]. In TEM mode, the object should be positioned on the bottom membrane. The most limiting effect is the electron "beam broadening" which is more a blurring of the image of the object on the camera due to the layer of liquid below the object. On the contrary, in STEM mode, the object should be positioned on the top membrane of the liquid cell in order to maximize the resolution. Otherwise, beam broadening effects due to the scattering of electrons in contact with the liquid will decrease the resolution. Thankfully, in our liquid experiments, the dynamic of the gold nanoparticles occurred close to the "top" membrane of the liquid cell window (at the entry point of the electron beam). Based on the optical principle of reciprocity, a bright field STEM image with a point detector is equivalent to an image taken in TEM mode. In any case, we will write the beam broadening/blurry effect with the term d blur .

It is possible to define a criterion in which a pixel surrounded by a background noise can be detected if the signal-to-noise ratio (SNR) is above a threshold of between 3 and 5: this is called the Rose criterion [START_REF] Colliex | Unconventional modes for stem imaging of biological structures[END_REF][START_REF] Rose | Television pickup tubes and the problem of vision[END_REF][START_REF] Rose | Vision: human and electronic[END_REF]. We will define d SN R the smallest size of an object that can be detected in a thick water layer inside a liquid cell when only considering the SNR.

d SN R is itself limited by the electron dose.

We also have to take into account the fact that the spatial resolution can be limited by diffraction effects. Indeed, in optical microscopy, two objects appearing close to each other in an optical device can only be distinguished if there is a minimal distance between them. This minimal distance called the angular resolution is obtained when the diffraction maximum of the Airy disk (diffraction pattern with a bright central region due to uniform illumination of a circular aperture) of one image corresponds to the first minimum of the Airy disk of the other image. This can be translated into the Raleigh criterion in the form of the equation:

θ = 1.22 λ D
where θ is the angular resolution, λ the wavelength of light and D the diameter of the lens aperture. In STEM mode, the Raleigh criterion is important because the probe size and the pixel size need to be adjusted to satisfy it (the dip of the signal between two images must reach at least 20 %). Therefore, the beam diameter must be adjusted to satisfy the Raleigh criterion where the angular resolution is represented by the term 2d 50 (d 50 is the probe size diameter containing 50 % of the current in STEM mode).

To sum it up, the spatial resolution in a STEM mode experiment is given by [START_REF] De | Theory of the spatial resolution of (scanning) transmission electron microscopy in liquid water or ice layers[END_REF]:

d ST EM = (2d 50 ) 2 + d 2 SN R + d 2 blur (6.1)
In a LCTEM experiment, the high thickness of the liquid layer and the SiN membrane can modify the energy and angular distributions of incident electrons and thus limit the spatial resolution. Standard SiN windows have spacers that can adjust the space between the echips but due to the high pressure differences between the interior and the exterior of the liquid cell, the membrane extends itself and bulges toward the exterior thus increasing the thickness of the liquid layer up to a few micrometers. Moreover, the membrane itself has a thickness of 50 nm which in itself isn't much but is still not negligible considering that a few tens of nanometers can change considerably the resolution. Reducing the thickness of the membrane is possible but because of the loss of mechanical stability, it is required to reduce the size of the window. It is therefore crucial to find other strategies to limit the thickness of the liquid layer and the membrane.

Replacing the SiN membrane with graphene could be a solution to this problem. Indeed, graphene consists of a single layer of carbon atoms [START_REF] Geim | The rise of graphene[END_REF] which has a thickness of about 3.4 Å, way thinner than the existing SiN membrane for standard liquid cells. The low atomic number of graphene (6 for carbon) plays also an important role in reducing the energy lost by the electron when penetrating the window [START_REF] Radosav | Graphene: Substrate preparation and introduction[END_REF]. The SNR obtained with graphene is often higher than on other types of membranes [START_REF] Meyer | Imaging and dynamics of light atoms and molecules on graphene[END_REF].

A reduction of the electron beam effects

In LCTEM experiments, radiolysis is the most important beam effect [START_REF] Nicholas | Electron-water interactions and implications for liquid cell electron microscopy[END_REF][START_REF] Wang | Precise in situ modulation of local liquid chemistry via electron irradiation in nanoreactors based on graphene liquid cells[END_REF][START_REF] Le | Water radiolysis: Influence of oxide surfaces on h2 production under ionizing radiation[END_REF]. It results in the breaking of some of the bonds between atoms or molecules (see subsection 2.1.3).

Various experiments cannot be carried out because of undesired chemical reactions that occur in the liquid media (formation of bubble of hydrogen gas that can interact with the sample, destruction of bio/soft materials such as DNA or the death of live cells due to hydroxyl radicals...). Moreover, the complex chemistry that takes place inside the liquid cell [START_REF] Nicholas | Electron-water interactions and implications for liquid cell electron microscopy[END_REF] can make an experiment unpredictable or unrepresentative of the real chemical conditions. One of the most well-known strategies to limit the impact of radiolysis is again to lower the electron dose. But lowering the electron dose has its limits because the higher the flux of electrons, the better the quality of images taken.

Remarkably, graphene also happens to mitigate the effects of radiolysis in the liquid media [START_REF] Cho | The use of graphene and its derivatives for liquid-phase transmission electron microscopy of radiation-sensitive specimens[END_REF][START_REF] Keskin | Reduced radiation damage in transmission electron microscopy of proteins in graphene liquid cells[END_REF][START_REF] Koo | Live cell electron microscopy using graphene veils[END_REF]. Interestingly, Cho et al. have first suggested that graphene is a radical scavenger, a material that can consume the products of the radiolysis of water thus protecting sensitive materials such as DNA strands. Keskin and de Jonge have shown that this protection allows scientists to observe biomaterials at higher electron dose than the recommended limit. Wang et al. [START_REF] Wang | Longer-lasting electron-based microscopy of single molecules in aqueous medium[END_REF] demonstrated also that it is possible to combine the radical scavenging capabilities of other agents such as deuterium water with that of graphene in order to further limit the diffusion of radicals and slow down the generation of bubbles of hydrogen.

6.2 State-of-the-art of graphene liquid cells The same design gave birth to the first generation of GLCs [START_REF] Min | High-resolution em of colloidal nanocrystal growth using graphene liquid cells[END_REF] which consists of a liquid pocket containing the sample studied encapsulated between two graphene sheets (see figure 6.2). In the work of Yuk et al., the growth of colloidal platinum nanoparticles was studied at the highest possible resolution at the time. The GLCs were prepared by transferring graphene to standard holey amorphous carbon-coated TEM grids. A droplet of liquid containing the nanoparticles was then put on one of the graphene sheets and enclosed with the other graphene sheet. Although the results are impressive compared to standard SiN liquid cells, the first generation of GLCs have strong limitations. Indeed, the formation of the liquid pockets is random and inhomogeneous resulting in random number of liquid pockets dispersed in the liquid cell with a high variety of volume. Sometimes, the amount of pockets and their volume are so small that it can be difficult to argue that this is a real liquid environment (especially if there is no movement of the nano-objects). Various fabrication techniques have been invented to counteract this problem [START_REF] Textor | Strategies for preparing graphene liquid cells for transmission electron microscopy[END_REF][START_REF] Chang | Preparation of graphene liquid cells for the observation of lithium-ion battery material[END_REF][START_REF] Mohammadreza Ghodsi | Advances in graphene-based liquid cell electron microscopy: Working principles, opportunities, and challenges[END_REF][START_REF] Xin | Simplified fabrication strategy of graphene liquid cells for the in situ transmission electron microscopy study of au nanoparticles[END_REF][START_REF] Pauline | Graphene liquid cells assembled through loop-assisted transfer method and located with correlated light-electron microscopy[END_REF].

Well or channel-type graphene liquid cells

Controlling the volume of liquid and the amount of "liquid pockets" is a key feature of the second generation of GLCs. This generation of liquid cell consists of a thick substrate (generally a few hundreds nanometers of SiN) with wells or channels in its structure. These wells/channels are covered with graphene sheets on both sides so that the liquid and the sample is trapped in the wells/channels. Various techniques exist to obtain the wells/channels desired such as focused ion beam (FIB) [START_REF] Rasool | Graphene-sealed si/sin cavities for high-resolution in situ electron microscopy of nano-confined solutions[END_REF], photolithography [START_REF] Noh | Lithographically patterned well-type graphene liquid cells with rational designs[END_REF] or electron beam lithography [START_REF] Daniel | Nanometer resolution elemental mapping in graphene-based tem liquid cells[END_REF].

In the work from Rasool et al. [START_REF] Rasool | Graphene-sealed si/sin cavities for high-resolution in situ electron microscopy of nano-confined solutions[END_REF], two different designs of GLCs were made (see figure 6.4). One well-type where holes were formed by milling into an SiN membrane using a FIB until reaching a given depth into the substrate. The other is a channel-type design where the holes are made in the same manner but the substrate is milled form side to side. In both cases the holes have a diameter of 500 nm and are covered with CVD graphene after putting a droplet of the liquid sample consisting of 10 nm diameter spherical gold nanoparticles in a phosphate buffer solution (PBS). It should be noted that 80 % of the wells successfully encapsulated the sample while only 40 % of the channels were successful. The authors successfully demonstrated the capability to image moving nanoparticles (which was not possible in the first generation of GLCs) as well as obtaining atomic-scale resolution (figure 6.5) in these GLCs.

While these GLCs were stable, the fabrication process is less accessible because it requires additional machines that are not available to everyone (in the case of the paper we described, a FIB is required). The low success rate of capturing the liquid sample inside the channels can be explained by the size of the holes. Indeed, when the holes have a diameter of a few hundreds of nanometers, the liquid has a hard time entering the holes whereas when the holes are bigger with a diameter of a few micrometers, the liquid enters the hole more easily but it tends to stay only on the edges due to capillarity effects [START_REF] Noh | Lithographically patterned well-type graphene liquid cells with rational designs[END_REF]. By reducing the diameter of the holes, it is possible to avoid a collapse of the graphene sheets (figure 6.6). holes). 30 nm platinum spacers are placed on the bottom e-chip. The entire e-chips are then covered with CVD graphene using a wet transfer. Mono-layer graphene is transferred multiple times in order to have 3-5 layers of graphene that can enhance the stability of the window. The thickness of the window and the number of layers of graphene are specifically chosen in order to achieve a liquid thickness below 100 nm. The relatively high thickness of the SiN window ensures that the bulging effect in vacuum is minimized. According to the calculations made by the authors, above this target the liquid thickness starts to be the main limitation to spatial resolution. Various tests have been made with these e-chips to verify their stability and their limits. Indeed, the pressure limit (interior pressure above which the window breaks) was determined and compared with 50 nm thick SiN standard liquid cells, and the flow rate limit (liquid flow rate above which the window breaks) was evaluated (see figure 6.10).

Gold nanoparticles were first used to show that the signal-to-noise ratio and the beam blur are significantly improved with these liquid cells compared to standard SiN liquid cells.

Moreover, they showed that they achieved a maximum resolution of 1.47 Å, which is a performance close to that of the static GLCs (the first generation in particular). Additionally, the Brownian motion of gold nanoparticles was observed and linked to the injection rate of the solution. After that, they further demonstrated the capability of the liquid cell by injecting a solution of 0.1 M HAuCl 4 and 0.1 M KCl inside the liquid cell, thus inducing an oxidative etching of the gold nanoparticles (see figure 6.11). According to the authors, the etching process occurs preferentially in the corner and after all the faces have a similar coordination, the dissolution starts to be isotropic. A variety of soft materials such as liposomes or E. coli bacteria were also imaged using these liquid cells. Interestingly, the authors suggest that this GLC could be used to study the conformational dynamics of soft materials at the molecular scale such as DNA hybridization or virus infection.

Graphene liquid cell preparation

Here, we will focus on describing the preparation for the flow capable GLCs that I made during the first six months of my PhD at DTU in Denmark under the supervision of Kristian

Mølhave.

The main idea behind our GLCs is to take the standard Protochips liquid cell and use a Focused Ion Beam (FIB) to etch holes in the SiN window. The holes are covered with multilayer graphene and the liquid cell is then ready to be used in the Poseidon Select holder that we exploit at the MPQ lab in France.

We made the choice to modify the existing SiN e-chips because, at first, it seemed to us to be the easiest way to rapidly obtain and test flow capable GLCs. After this first step, it was planned that I make a special e-chip adapted to the graphene transfer that will also reduce the liquid thickness in the cleanroom of DTU. However, due to the Covid-19 pandemic, I couldn't return to DTU to continue this work and so we redirected our work on the heating experiments that we described in the chapter 3.

Two different transfer techniques were used to put graphene on the holes of the modified e-chips. The first graphene transfer technique was used in a lab at DTU. We will call this graphene transfer technique the wet transfer technique. The second graphene transfer technique was used in the cleanroom of MPQ at the Université de Paris. We will call this graphene transfer technique the hot pick-up transfer technique. Although the principles of the techniques are similar, the tools and the temperatures used are different.

Modification of existing flow capable standard SiN liquid cells

Before making holes in the FIB, the e-chips must be cleaned in acetone and methanol for 2 minutes in each solvent in order to remove the protection layer that covers the e-chips and that could interact with the FIB.

The etched holes are circular with a diameter varying between 1 µm and 8 µm. We varied the diameter of the holes in order to test the stability of the SiN window. Two holes were etched on both the small and large e-chips in the central region of the SiN window as indicated in the figure 6.12. By choosing this configuration, it is possible to align the holes of the small e-chips and the holes of the large e-chips when assembling the liquid cell in cross configuration in the TEM holder.

Wet graphene transfer 6.3.2.1 Material

• Natural graphite crystals. Extraction of graphene from the graphite flakes.

• Clean standard tape. Used for the graphene exfoliation.

• Oxygen plasma cleaner. Used to enhance the adherence of graphene on the SiO 2 wafers.

• Cut and marked SiO 2 wafers. The wafers should have markings that allow us to easily recall the position of a good graphene flake on the wafer.

• Optical microscope with a large movable plane. • Reverse action anti-capillary tweezers. Used to pickup graphene+CAB.

• Cellulose acetate butyrate (CAB) polymer.

• Spin coater.

• Scalpel knife.

• Distilled water.

• Syringe with thin needle.

• Hot plate that goes up to 120 °C.

• Acetone.

• Isopropanol.

• Standard liquid flow e-chip (top or bottom) with holes in the SiN membrane.

Protocol

The first step is to put graphene on SiO 2 . For this, we exfoliate by hand natural graphite crystals with a standard clean tape. Then, we put pre-cut and marked SiO 2 wafer pieces in a plasma cleaner and treat it in oxygen plasma (300 mbar O 2 , 120 W). The loaded tape must immediately be applied on the SiO 2 wafer after the end of the plasma cleaning. The tape must be pressed strongly on the SiO 2 wafer to make sure that the graphene is transferred on the wafer. The tape have to be removed slowly and carefully so as to not pickup the graphene again from the SiO 2 wafer. Using an optical microscope, the presence of graphene should be verified and its position marked on a notebook using the markings on the SiO 2 wafer. We can recognize a good graphene flake by its color and size: it should be purple/pink and large enough to cover all the holes in the SiN membrane of the e-chip. Very thick layers of graphene appear to be white and reflect light. The lighter the color, the thinner the graphene flake with transparent violet flakes being bilayer or monolayer.

Afterwards, CAB is spun on top of the SiO 2 wafers using the spin coater at 1500 rpm for 40 seconds. These CAB coated wafer pieces are then heated up to 80 °C for 5 minutes to make sure that the graphene is attached to the CAB.

Using the optical microscope, we find the marked zone on the CAB coated wafer pieces and cut a square of CAB using a scalpel knife around the graphene. The CAB square must be big enough so as to be easily manipulated but smaller than the e-chip. Using a syringe, water is injected between the CAB and the SiO 2 wafer piece. Then, the square cut CAB is picked up carefully using reverse action anti-capillary tweezers. The CAB can be very tricky to pickup depending on various random parameters (closeness to the edge of the wafer piece, how much water is injected...) and it requires a lot of training. After picking up the CAB, it must be put on the holes of the SiN window. As long as the CAB is hydrated, it can be moved around to position it better. The graphene flake must be positioned directly on the holes, covering them. This step is quite difficult and one can try again to move the CAB if it starts to be dehydrated by adding water using the syringe. After positioning the graphene over the holes, we must wait for the water to evaporate from the CAB and the e-chip (about 5 minutes).

If the graphene flake is still above the holes, then the e-chip is heated using the hot plate to 120 °C. At 120 °C, the CAB starts to melt and the graphene flake sticks to the SiN window.

Before removing the CAB, we need to make sure that the graphene didn't move using the optical microscope. After this verification step, the e-chip is cleaned in acetone and then in isopropanol for 10 minutes each. Finally, the presence of graphene above the holes must be verified using the optical microscope. If the graphene is very thin, it can be difficult to see it. I noticed that using a green filter can help to find the graphene flake.

Hot pick-up graphene transfer

This graphene transfer technique was developed by Jacko Rastikian and Salvatore Timpa in the clean room of the MPQ lab [START_REF] Jacko | Experimental investigation of charge and spin transport in WSe 2 connected with palladium contacts[END_REF][START_REF] Rastikian | Magnetic properties of devicelike cobalt/2d materials interfaces[END_REF][START_REF] Timpa | Strongly reduced thermal conductivity of supported multilayer-graphene nanowires[END_REF]. It follows the same principles of the other graphene transfer technique with a few changes. The protocol of this technique is heavily inspired from the work of Pizzocchero et al. [START_REF] Pizzocchero | The hot pick-up technique for batch assembly of van der waals heterostructures[END_REF]. It is extensively explained in the PHD report of Jacko Rastikian.

Material

• Natural graphite crystals. Extraction of graphene from the graphite flakes.

• Clean standard tape. Used for the graphene exfoliation.

• Oxygen plasma cleaner. Used to enhance the adherence of graphene on the SiO 2 wafers.

• SiO 2 wafers. These wafers do not need to be marked or cut.

• Optical microscope with a large movable plane. A heater must be installed on the movable plane. The heater can go up to 80 °C.

• Glass slide with a polypropylene carbonate (PPC) coated Polydimethylsiloxane (PDMS)

drop. PPC has the property to be "glassy" at room temperature, rubbery and viscous at 40 °C and liquid at 70 °C.

• Metallic micro-manipulator for the glass slide. The manipulator can move to the x, y and z directions to the few nearest microns.

• Weakly adhesive tape so that the wafer stays attached to the heater during the pickup.

The adhesive tape must not be too strong or else the wafer could break after trying to remove it from the heater.

• Standard liquid flow e-chip (top or bottom) with holes in the SiN membrane. I personally let the e-chip in its corresponding box that contains a gel. The gel will allow the e-chip to stick to the box and not be picked up by the PPC+PDMS drop when the lift up is done.

Protocol

The graphene exfoliation and the preparation of the SiO 2 wafers are the same as in the other wet transfer.

Once prepared, the SiO 2 wafer is sticked to the heating plate which is placed on the moving plane of the optical microscope. Then, using the optical microscope, a good graphene flake is found. The glass slide with the PPC+PDMS drop is attached to the manipulator with the drop facing the hot plate. The glass slide is moved so as to center the drop above the graphene flake. The drop is then carefully and slowly brought down while regularly verifying that its center is still aligned with the graphene flake by changing the focus of the optical microscope. At one point the contact is made between the drop and the wafer. If the center of the drop is not aligned on the graphene flake when the contact is made, it still possible to bring up the glass slide and try again the process.

If the center of the drop is successfully in contact with the graphene flake, the hot plate must be heated up to 54 °C. This will allow the graphene flake to stick to the drop. After waiting 3 minutes after reaching the temperature, the drop is rapidly brought up. By changing the focus, one can verify the presence of the graphene flake at the center the drop.

Then, the plane of the optical microscope is moved around so that the e-chip is positioned below the center of the drop and the holes in the SiN window must be positioned directly below the graphene flake. After that, the drop is lowered slowly while verifying the position of the graphene flake and the holes. If it is not well aligned, the plane must be moved and not the glass slide. The drop will then enter in contact with the SiN window. The drop mustn't be pressed too hard on the window or else the membrane might break under the pressure. If the graphene is not above the holes, it is still possible to correct the mistake by heating the hot plate to 54 °C and then lifting the glass slide again. Once the graphene is in the holes, the temperature can be raised to 76 °C to allow the graphene to stay on the SiN window. After waiting 3 minutes, the micromanipulator can be lifted slowly at a constant speed in order to avoid breaking of the drop.

If we compare the practical aspects of these two transfer methods, it is worth noting that the hot pick-up transfer is easier to complete than the wet transfer. Indeed, the micromanipulator allow a precise control over the glass slide whereas the tweezers in the wet transfer technique were controlled by hand. Also, the amount of movement is highly reduced because everything is done under the optical microscope, thus reducing the chances of mistake. It is also easier to correct errors when they occur. This technique is also easier to learn and faster to complete. As we will see in the next section, the success/failure ratio of this hot pick-up graphene transfer technique is also higher than in the wet transfer technique. I also did the hot pick-up transfer in a clean room, thus I had a cleaner graphene transfer with less impurities. The only notable disadvantage of the hot pick-up transfer is that it requires a specific setup that needs to be prepared in advance. But once the optical microscope and the PPC+PDMS drop are prepared, the graphene transfer is quite fast to do. As shown in table 6.13, the wet transfer was largely ineffective with a success rate of only 20% of graphene transfer whereas the hot pick-up transfer is more effective with 86% successful graphene transfer. The figure 6.14 (a-b) shows an example of the result of a successful graphene transfer where the holes are covered by a graphene flake. We tested the window stability by loading the GLCs filled with aqueous solutions in the TEM. We can see that the GLCs made using the hot pick-up transfer were more stable with 100% of them withstanding the vacuum compared to only 50% with the GLCs using the wet transfer. However, in the case of the vacuum test, the most probable reason why the GLCs made with the hot pickup transfer were more stable is because of the reduced size of the holes made in the SiN membrane (1 µm for the ones made for the hot pick-up transfer and 8 µm for the ones made for the wet transfer). Most of the GLCs that we used with flow were stable. The flow of liquid in the cell was confirmed by the observation of droplets at the outlet tubing of the sample holder. Moreover, the observation of gold nanoparticles growth only after the injection of metal precursors confirmed that molecules can diffuse up to the graphene windows (see the experimental subsection described below). In short, we conclude from this table that the hot pick-up transfer is more suited to GLCs with a total success rate of flow-capable GLCs of 64%. We will discuss in the section section 6.5 some of the solutions that we can consider to increase the stability of the GLCs in vacuum.

Results

We also performed ex situ TEM observations of graphene layers on single e-chips. The figure 6.14 (c-d) represents high magnification images of a hole covered with graphene.

Interestingly, we can see that the hole is fully covered by a multilayer graphene patch with large single domain area. However, we observed a lot of polymer residues on the graphene.

By using the image 6.14 (c), we can extract a measurement of the signal-to-noise ratio (SNR) [START_REF] Lee | Electron dose dependence of signal-to-noise ratio, atom contrast and resolution in transmission electron microscope images[END_REF] in and outside the hole as defined in the equation: SN R = I 0 σ where I 0 is the mean intensity of the pixels in a target area and σ is the standard deviation of the intensity of the pixels in the same area. We find a SNR of 42 inside the hole and 28 outside the hole. In other words, the SNR is 1.5 times better in graphene alone than in the SiN+graphene layer without liquid sample. It's worth noting that even when the GLCs sustain the vacuum of the TEM and the flow of liquid, polymer residues from the graphene transfer can still disturb the experiment and obstruct the area of observation. This problem can be observed in the figure 6.15 which is a TEM image of an extreme case where a lot of polymer residues covered a large part of the observation area. We tried to use the flow of liquid to remove the polymer or use the beam to destroy it but we only moved the polymer around the window without really removing it. In the section 6.5, we will explain some of the options we have to avoid or minimize the presence of polymer residues on graphene. Finally, it's worth noting that we didn't succeed in overlapping the holes on both e-chips when assembling the liquid cells. Thus, all images we will show in this section were obtained with the beam passing through one graphene window and one SiN window.

LCTEM experiments with graphene liquid cells

We will show here two experiments that we carried out with our GLCs.

Signal-to-noise ratio and resolution limit in TEM mode

In a first experiment, a droplet of water containing gold nanoparticles was deposited on the small graphene e-chip and we closed the liquid cell with another standard SiN e-chip. The graphene was on the bottom e-chip and thus we performed our experiment in TEM mode.

We intentionally created a bubble of hydrogen by radiolysis of water with the electron beam in order to reduce the thickness of the liquid layer and thus enhance the imaging conditions. To counteract the effects of radiolysis and to avoid the drying of the liquid cell, water was continuously injected during the TEM observations. We confirmed that the graphene window was still covered by a thin layer of liquid by observing the random motion of nanoparticles.

In the figure 6.16, we can observe gold nanoparticles and a copper pollution that originates from the water we used in this experiment: the electron beam induced the copper that was present in water to grow inside the liquid cell in the form of aggregates. It should be noted that the hole shown here is located close to the central region of the observation area.

Moreover, we found a SNR of 36 inside the hole and 24 outside hole. Note that illumination conditions are different in images 6.16 and 6.14 (c) so their SNR values cannot be compared. In these conditions, HRTEM images of gold nanoparticles could be acquired as shown in the figure 6.17. In the FFT of this HRTEM image, we observed a diffraction spot demonstrating that we achieved a resolution of 1.8 Å.

In situ gold nanoparticles growth in STEM mode

The second experiment that we show here is a simple in situ STEM experiment of growth of gold nanoparticles by radiolysis. We first deposited a droplet of water on a standard small SiN e-chip and we closed the liquid cell with a graphene equipped large e-chip. Because the graphene is located on the membrane of the top e-chip, STEM mode is more suited to this experiment. No hydrogene bubbles were formed in this experiment. We first observed the We calculated once again the SNR inside and outside hole but didn't find a significant difference between the two (we obtained a SNR of 27 for both cases). This confirms that when the thickness of the liquid layer and the bulging of the liquid cell are too high, the advantages of graphene for the imaging conditions are nullified. Interestingly, we can clearly see in the figure 6.18 that gold tend to grow preferentially on graphene and particularly on the edges. It is probable that the radiolysis of water changed the chemical composition of the outer layer of graphene thus becoming a graphene oxide layer. Oxygen functionalities at the surface of graphene have already been shown to provide reactive sites for the nucleation and growth of gold nanoparticles [START_REF] Goncalves | Surface modification of graphene nanosheets with gold nanoparticles: The role of oxygen moieties at graphene surface on gold nucleation and growth[END_REF].

We also observed the growth of gold aggregates at higher magnification on the graphene window (see figure 6.19). We can remark that the growth is driven more by aggregation and coalescence than in standard SiN liquid cells in which monomer attachment is the main growth mechanism. We can conclude that graphene patches have a strong influence on the nucleation and growth mechanisms of gold nanoparticles which confirms the key role of the substrate topology and surface chemistry in the nanoparticles formation processes observed by LCTEM [START_REF] Woehl | The mechanisms for nanoparticle surface diffusion and chain self-assembly determined from real-time nanoscale kinetics in liquid[END_REF]. We choose to use exfoliated graphene for our GLCs because we thought that it would be easier to use than CVD graphene. However, exfoliated graphene has the big disadvantage of having a size way smaller than CVD graphene (exfoliated graphene flakes had the size of about 50 µm whereas CVD graphene could cover the whole liquid cell). Nevertheless, CVD graphene also has more defects than exfoliated graphene. Therefore, the stability of CVD graphene must be carefully tested to confirm that it can be a more practical solution to make GLC windows.

Besides, the number of graphene layers used for GLCs should be well controlled. Using exfoliated graphene, the number of layer can be linked to the color of the graphene flake.

But the approximate number of layer is difficult to state (it is possible to differentiate 1-2 layers from 5-6 layers but more difficult to differentiate 3 layers from 6 layers). CVD graphene offers the advantage of a controllable number of layer of graphene thus resolving this issue. Moreover, the number of layers ensuring safe experiments with the best imaging conditions should be determined. Although this choice is highly dependent on the design of the liquid cell, Koo et al. [START_REF] Koo | Liquid-flowing graphene chip-based high-resolution electron microscopy[END_REF] have shown that a number of 3-5 layers is ideal to combine the advantages of graphene for liquid phase experiments while retaining a certain mechanical stability and to counteract the eventual defects that are randomly present in CVD graphene.

Moreover, our experiments showed that the graphene flakes were not very clean which is sometimes dramatic for the transparency of the graphene window. Beside the copper contamination that was coming from the water we used in the first experiment, the other contamination observed on graphene probably directly came from the residues of the polymer (whether CAB in the wet transfer or PMMA in the hot pick-up transfer) used. Acetone was used in both transfer methods to clean up residues of polymer but it seems to be not as effective as expected. An additional annealing step could be added to clean up the graphene.

We should note that standard SiN e-chips are cleaned with acetone and methanol before plasma cleaning them with argon and oxygen. This plasma cleaning recipe is very effective in removing carbon atoms and it is thus dangerous to graphene. Remarkably, Koo et al.

[114] used a low-energy hydrogen plasma for cleaning the surface of graphene after the transfer. We should add that plasma cleaning also allows the surface to be hydrophilic, a crucial property for liquid experiments where graphene is a hydrophobic material. Therefore, using a hydrogen plasma seems to be a good solution that should be tried in further experiments.

The design of the flow capable graphene liquid cell

The first generation of GLCs were unreliable with their random volume of liquid and disposition of the sample. The well-type and flow-type cells tend to be more reliable. The design of flow capable GLCs must allow the reproducibility of liquid experiments.

First, we recommend to use 8 µm holes because 1 µm holes are too small for experiments although the SiN window seems to be less stable with bigger holes. Indeed, we observed that a few GLCs with 8 µm holes didn't sustain the vacuum in the TEM whereas GLCs with 1 µm holes always sustained the vacuum. However, it must be noted that 8 µm holes were used with the wet transfer whereas 1 µm holes were used with the hot pick-up transfer.

Moreover, it is certainly possible to multiply the holes on SiN window in order to maximize the area of observation. Further work is necessary to determine the number of holes that can be etched in the window while preserving the stability of the liquid cell. Finally, we never succeeded in overlapping the holes of both e-chips so increasing the number of holes will also solve this issue. These holes should have a minimum distance of 5 µm but shouldn't be too far from each other so that the majority of the holes are in the observation window after the alignment of the two e-chips in the liquid cell holder and also so that the graphene flake can cover all the holes at the same time.

The thickness of the SiN liquid cell window is crucial to the design. As shown in the state-ofthe-art as described in the section 6.2, other teams demonstrated that thicker SiN windows allow to multiply the number of holes and thus increase the graphene observation areas and at the same time facilitate the alignment of holes between the two e-chips. 300 nm thick SiN windows were used by Koo et al. [START_REF] Koo | Liquid-flowing graphene chip-based high-resolution electron microscopy[END_REF] making it mechanically stable in vacuum despite having dozens of holes in it. The size of the holes are also important because as we have seen in our experiments, holes of 1 µm are way too small to use them in a real liquid phase experiment. 8 µm holes seem to be effective and large enough to be used. Koo et al. used this size in some of their GLCs successfully.

The final dilemma is to choose between modifying existing liquid cells as we did or fabricating from scratch GLCs in a clean room. As seen in our experiments, decreasing the liquid thickness is crucial to benefit from the enhanced SNR of GLCs. It is thus essential to design a GLC with a very thin liquid layer, a challenge that can only be met by fabricating new e-chips. In that regard, nanochannels are promising designs [START_REF] Murat Nulati Yesibolati | Electron inelastic mean free path in water[END_REF][START_REF] Murat Nulati Yesibolati | Mean inner potential of liquid water[END_REF]. Moreover, at this time, no graphene transfer technique can be upscaled to the industrial level and this step must be made by hand in the lab. Therefore, if a team has the equipment and the resources to build their own flow capable GLCs from scratch, it seems to me to be the best solution.

Maybe in the future if GLCs gain more popularity, we can think of commercially available SiN liquid cells equipped with holes and directly ready for a graphene transfer.

Chapter 7

General conclusion

LCTEM is fantastic tool to investigate the dynamics of gold nanoparticles in water. Its ability to image nanomaterials formation and degradation processes in real time and in realistic of liquid media of controlled composition and temperature opens up a lot of opportunities. In our work, we demonstrated how we overcame technical and scientific challenges to improve this already effective tool.

For the first time, we conducted a heating experiment demonstrating that the drastic changes in nucleation and growth of gold nanoparticles originate from the increase of the molecular diffusion and gold solubility with temperature. We optimized and explained the protocol to conduct such an experiment smoothly. We showed that the ability of LCTEM to image single nanoparticle dynamics allowed unraveling that beyond the ideal view of Ostwald ripening, nanomaterial growth is not only driven by size effects and other parameters should be taken into account. We revealed how the competition between the deposition rate of atoms and the diffusion rate of atoms on the surface of nanoparticles controls the shape of nanomaterials in water. LCTEM is therefore a complementary tool to investigate the effects of temperature on the nanochemistry of nanomaterials along with UV-Vis spectroscopy and X-ray scattering methods.

In a second study, we determined that the anisotropic dissolution of gold nanorods is highly dependent on the reduction of the etching power of the solution and the inhomogeneous distribution of the citrate coating around the nanorods. LCTEM studies complemented with atomistic calculations can evaluate the respective role of numerous complex parameters influencing the reactivity of nanoparticles such as organic/inorganic interfaces.

Besides these in situ investigations into the behavior of gold nanoparticles in solution, we also carried out technical works to improve TEM analysis in liquid media. At first, we showed 131 that STEM nanodiffraction is a simple and fast technique that can extract the diffraction pattern of any nanoparticle regardless of its orientation with an electron dose reduced of as much as four order of magnitudes compared to HRTEM techniques.

Finally, we fabricated flow capable graphene liquid cells by modifying existing standard SiN liquid cells and performed the first tests of this promising technology. These preliminary results allow drawing the roadmap to optimize the success rate, the TEM imaging conditions and the environment control with the next generation of graphene liquid cells.

This thesis is merely a step in the TEM research of the MEANS group to understand the dynamic of metal nanoparticles and some future experiments can already be drawn on the board. Indeed, the next generation of liquid cell holder will probably be able to heat up the solution above 100 °C and this could open up new opportunities to study the synthesis of gold at higher temperatures using well established methods such as polyol synthesis. More complex nanosystems, such as high entropy nanoalloys that require high synthesis temperatures could also be studied with these future liquid cell holders. For graphene liquid cells, there is still a lot of room for improvement for the design we proposed (increasing the number of holes, increasing the thickness of the SiN, using CVD graphene...). Moreover, an interesting experiment to do is to use the heating capable SiN e-chips for graphene liquid cells:

the thermal properties of graphene could bring unexpected results to a metal nanoparticle nucleation and growth experiment. On the topic of data handling, the recently installed AXON software developed by Protochips allows the sample to stay in place when doing a heating experiment that can induce a drift of the observation area. AXON also facilitates data handling by increasing the visibility of anomalous events during an experiment or by allowing data plotting directly without using any additional software/program. Finally, machine learning algorithms have been shown to be very powerful to extract interesting data from very noisy images and videos [START_REF] Yao | Machine learning to reveal nanoparticle dynamics from liquid-phase tem videos[END_REF]. Machine learning could also allow an automatic classification of nano-objects that is useful for automatic data analysis with a large dataset.

The MEANS group has recently obtained the support of the ANR to develop new machine learning approaches to study nanomaterials synthesis and reactivity.

Chapter 8

Abstract of the PhD report in French

A summary written in French is required by my graduate school (EDPIF) for a PhD report written in English.

Introduction générale

Figure 1 . 1 :

 11 Figure 1.1: Plot of atomic concentration against time, illustrating the generation of atoms, nucleation and subsequent growth [22].

Figure 1 . 2 :

 12 Figure 1.2: TEM image of nanorods obtained with [CTASB] = 0.55 mM. Right inset: aspect ratio distribution. Left inset: rod fraction (percentage in number of objects) as a function of the CTASB initial concentration [28].

Figure 1 . 3 :

 13 Figure 1.3: TEM images of palladium nanoparticles obtained from co-reduction of Na 2 PdCl 4 by PVP) and EG, with varying volume percent of EG: (A) 0%, (B) 9.1%, (C) 45.5%, and (D) 72.7%. The effects of the type and concentration of reducing agent onthe formation of 1-D nanoparticles is evident here.[33]

Figure 1 . 4 :

 14 Figure 1.4: SEM images of the synthesized silver nanoparticles by heating the dissolved solution (first protocol) at different heating rates of (a) 1 • C.min -1 , (b) 5 • C.min -1 and (c) 7.5 • C.min -1 [36].

Figure 1 . 5 :

 15 Figure 1.5: SEM images of the synthesized silver nanoparticles using the hot injection method (second protocol) at different temperatures of (a) 100 • C, (b) 120 • C and (c) 150• C[36].

Figure 1 . 6 :

 16 Figure 1.6: Scheme of the proposed growth pathway of gold nanoplates through oxidative etching [31].

Figure 1 . 7 :

 17 Figure 1.7: In situ liquid cell setup with flow capability. (A) Tip of liquid cell holder with provisions for o-rings and e-chips. This holder can transport liquid in and out of the two e-chips. (B) Small e-chip with spacers and big e-chip. SiN membrane is etched in the centre of the two e-chips. (C) Syringe pump for precise liquid flow during the experiment. (D) Schematic cross section of a liquid cell with flow and heating capability.

Figure 1 . 8 :

 18 Figure 1.8: TEM images showing the shape evolution of the Pt@Au nanoparticles during the nucleation and growth stages: (a-c) with a flow of HAuCl 4 and citric acid solution,and (d-f) at the stage for surface diffusion when the flow was turned off. (g) Illustration of the shape evolution of a Pt@Au nanoparticle during the process as measured from the TEM images[START_REF] Wu | Growth of au on pt icosahedral nanoparticles revealed by low-dose in situ tem[END_REF].

Figure 1 . 9 :

 19 Figure 1.9: Nucleation and growth of Au seeds in methanol (a) Time series of STEM HAADF image series in the corner of the liquid cell showing the nucleation and growth of 2D and 3D nanostructures in methanol. The irradiation time of the observed area is indicated in the bottom right corner of each image. (b) Ex situ TEM image of a nanoprism formed in the liquid cell. (c) Ex situ STEM HAADF image of a decahedral nanoparticle. (d) Ex situ HRTEM of an icosahedral nanoparticle. The FFT of the HRTEM image and a 3D model of the icosahedral nanoparticle are in the bottom and top right inserts, respectively. Such icosahedral nanoparticles served as seeds for the formation of symmetric nanostars. [56]

Figure 1 . 10 :

 110 Figure 1.10: Growth mechanisms of Au nanostars. LCTEM image series showing the stellation process transforming an icosahedral seed into a well symmetric Au NS with twenty sharp arms (10 arms are visible because of 2D projection). As illustrated in the model bellow, the growth of pyramidal arms on each of the 20 triangular facets of the seed occurs when DMA is injected in the liquid-cell [56].

Figure 1 . 11 :

 111 Figure 1.11: Summary of the synthesis and the growth mechanisms of silver nanoparticles in water [58].

Figure 1 . 12 :

 112 Figure 1.12: Growth of gold nanoparticles by radiolysis observed by LCTEM. (a) Fast growth process induced by high electron dose rate in TEM mode [61]. (b) Slow growth process induced by low electron dose rate in STEM mode. (c) Schematic illustrating the defect-mediated growth of multi-twinned 3D nanoparticles and 2D nanoplates [60].

Figure 1 . 13 :

 113 Figure 1.13: TEM images of the in situ growth of Bi nanoparticles and the corresponding color gradient maps showing oscillatory growth of a pair of nanoparticles. A few nanoparticles with significant changes of volume are marked by arrows in image (III) as a comparison with image (II). [62]

Figure 1 . 14 :

 114 Figure 1.14: Comparison between chemical and electrochemical etching. (A) Time series of in situ TEM images showing the chemical etching of a Pt-Ni nanoparticle using acetic acid inside a liquid cell at 90 • C. Red arrows indicate the formation of pits on the nanoparticle surface. (B) Schematic of the chemical and electrochemical etching pathways. (C) Time series of in situ TEM images showing the electrochemical etching of a Pt-Ni nanoparticle near the positively biased working electrode of an electrochemical cell containing H 2 SO 4 electrolyte solution. [66]

  scattering effect is dependent on the Couloumb interactions between the incident electrons and the atoms and electrons constituting the sample. If the sample is crystalline, the elastic scattering generates strong interference patterns in determined directions. This gives rise to a diffraction pattern called Bragg's diffraction. The angle between the scattered electrons and the unscattered beam of electrons is equal to 2θ B where θ B is the Bragg angle. We can relate the Bragg angle, a Bragg diffraction spot and the incident beam in the equation 2dsinθ B = nλ where d is the lattice spacing of the sample and λ the wavelength of the

Figure 2 . 1 :

 21 Figure 2.1: Signals generated when a high-energy beam of electrons interacts with a thin specimen [67].

  The liquid cell experiments were done on two microscopes, both of them installed at the University of Paris: the JEOL 2100F and the JEOL ARM 200F. They both operate at a voltage of 80 or 200 kV. The ARM 200F is equipped with a CEOS hexapole type spherical aberration (C S ) image corrector and a very fast camera OneView allowing us to have a high spatial and time resolution. An Ascor probe corrector (CEOS) was added on the JEOL ARM 200F during my PhD.

Figure 2 . 2 :

 22 Figure 2.2: Comparison of the use of an objective aperture in TEM to select (A) the direct or (B) the scattered electrons forming BF and DF images, respectively. [67]

Figure 2 . 3 :

 23 Figure 2.3: Image formation process and associated ray-diagram in HRTEM.

Figure 2 . 4 :

 24 Figure 2.4: STEM diagrams to select (A) the direct or (B) the scattered electrons forming BF and DF images, respectively. [67]

Figure 2 . 5 :

 25 Figure 2.5: JEOL ARM 200F: Electron dose rates in STEM mode calculated as a function of magnification, aperture size and probe size.

Figure 2 . 6 :

 26 Figure 2.6: JEOL 2100 plus (part 1): Electron dose rates in STEM mode calculated as a function of magnification, aperture size and probe size.

Figure 2 . 7 :

 27 Figure 2.7: JEOL 2100 plus (part 2): Electron dose rates in STEM mode calculated as a function of magnification, aperture size and probe size.

Figure 2 . 8 :

 28 Figure 2.8: JEOL 2100 plus (part 3): Electron dose rates in STEM mode calculated as a function of magnification, aperture size and probe size.

2. 5

 5 Description of a new method to study the thermal behavior of metal colloids in solution with the TEM This section is a scientific paper reused from the Journal of Visualized Experiments (Jove Methods) with the permission from MyJove Corporation[1]. JOVE being a video article, I encourage the readers to visualize the seven-minute movie associated with this publication (link: https://www.jove.com/v/62225/studying-effects-temperature-on-nucleation-growth-nanoparticles)

2 . 3 .

 23 . Remove the lid covering the tip of the holder. Remove the dummy liquid cells using tweezers. Remove the gasket used with the dummy liquid cells. NOTE: Dummy liquid cells are liquid cells without the SiN window and only silicon. They are used for storing the liquid cell holder in the vacuum pump. Pay attention to the state of brass screws because they crumble easily over time. Particularly, if the screw heads are damaged, the screws must be changed. Otherwise, it may be difficult to unscrew them after the experiment and small debris may also disrupt sample loading. Manually inject 2 mL of distilled water inside the holder using syringes and the external PEEK tubing to connect to the back of the holder. NOTE: There are 3 microfluidic tunnels inside the holder. All three must be cleaned up with water. Pay attention to the water coming out of front of the holder: if the water is colored because of a previous experiment, continue to put water inside the holder until the liquid is uncolored. If injecting a solution in the liquid cell during the experiment (1 mM of HAuCl 4 in water in our case), fill the tubing of the sample holder with this solution.

4 . 5 . 6 . 7 . 1 .

 45671 Put both E-chips in the Petri dish with methanol and wait for 2 minutes. The methanol will clean up the Echips from the acetone and the rest of the debris.CAUTION:The transfer of the E-chips between the acetone and the methanol must be done as fast as possible in order to not let the E-chips dry up in the air. Dry up the liquid cells using an air pistol. Hold the E-chip using tweezers while using the air pistol. Be careful to not press too much on the air pistol trigger otherwise the E-chip can drop out of the tweezers. If they drop out, restart the cleaning with acetone and methanol. Verify the integrity of the silicon nitride window using a binocular magnifier or an optical microscope (Figure 2). NOTE: Make sure that the windows of both E-chips are clean and not broken. If the E-chips do not seem clean, try to put them back in acetone and methanol again. If the dirt is still on the window or if the window is broken, the E-chips must be changed with new ones. Plasma clean the E-chips with a mixture of argon and oxygen gas for 2 minutes. Plasma cleaning the E-chips allows them to be hydrophilic. Here are the details of plasma cleaning set up: argon gas flow = 35 sccm, oxygen gas flow = 11.5 sccm, gas flow timeout = 20 s, Forward RF target = 50 W, Forward RF Range = 5 W, Maximum reflected RF = 5 W.2. Loading the liquid cells in the TEM holder (Figure3). Load the gasket O-rings inside the liquid cell holder (Figure3B). Verify that the gasket used is clean. If not, clean it rapidly with distilled water. Dry it up using a clean filter paper. To remove debris and fibers on the gasket, press it between two sheets of parafilm multiple times.

3 .

 3 Test the vacuum sealing of the liquid cell in a pumping station. If the vacuum level of the pump reaches 5 x 10 -2 Pa then continue the protocol. If not, check the integrity of the window (it is most likely broken) and start the protocol from the beginning with a new set of E-chips. 4. Verify one last time the integrity of the silicon nitride window using a binocular magnifier or an optical microscope. Sometimes, the liquid cell will be able to sustain the vacuum of the pumping station even if the window is broken. This is because when the window breaks and the liquid spills out, it can form aggregates of salt on the broken part of the window thus covering the hole. If it happens, prepare a new set of E-chips. 5. Load the liquid cell holder in the TEM and check the vacuum level. Even if the liquid cell sustained the vacuum of pumping station and there is no visible problem with the window, micro-leak of the liquid-cell can prevent to reach the vacuum level required to operate the TEM. If the microscope cannot reach the required vacuum level to operate (2-5 x 10 -5 Pa), remove the sample holder and prepare a new set of E-chips.

2 .

 2 Connect 2 external PEEK tubes to the syringes. Place the 2 syringes on the syringe pumps. Insert the external PEEK tubes in the 2 entries of the liquid cell holder. Insert one additional external PEEK tube for the output of the liquid cell holder.

1 .

 1 Connect the power supply to the holder. Connect the power supply to the computer on which the heating software is installed. 2. Power up the computer and open the Heating software. Power up the power supply.3. Click on the device check button. If the software indicates "passed" then the experiment can continue. Otherwise, the large E-chip might have a problem (incorrect loading of the E-chip, broken electrodes…). 4. Click on the Experiment tab. Click on Manual to activate the manual mode of heating. 5. Select the targeted temperature and change accordingly the temperature rate. Press apply to heat up the E-chips to the targeted temperature (Figure 4). NOTE: The E-chips can be heated up to 100 °C. If an aqueous solution is used for the experiment (as in our case), avoid heating the E-chips above 90 °C. Otherwise, the liquid sample can dry up. When heating up the liquid, the temperature can temporarily rise above the targeted temperature and then fall back to the desired temperature. Use low heating rate to minimize such overshoots (1 °C/s is fine).6. Click on Ambient to go back to the ambient temperature (25 °C). Click on Stop to stop the heating abruptly. Click on the End session tab to end the heating experiment.

  NOTE: STEM nanodiffraction is a technique that allowsacquiring the diffraction pattern of single nanoparticles in liquid during growth experiments22 .2. After the acquisition of a STEM HAADF image, select several nano-objects on the image and the STEMx software automatically synchronizes the position of the probe and the CCD camera to acquire the diffraction pattern at each position of the probe. To avoid the overlapping of the diffraction spots, use a small convergence angle of the STEM probe (7.4 mrad in our case) by using small condenser aperture (10 µm in our case).9. Cleaning of the liquid cell holder (after the experiment)NOTE: Here we describe a standard cleaning procedure for the liquid cell holder. If this cleaning is not efficient enough, it is possible to use diluted nitric acid and methanol to flush out the eventual nanoparticle aggregates in the liquid cell holder. The chemical compatibility documentation of the liquid cell holder should be consulted before. In any case, always finish the cleaning with the injection of distilled water.1. Remove the lid. Remove the used E-chips. Remove the internal gasket.NOTE:The used E-chips can be stored in an adapted box. It is then possible to perform ex situ TEM or SEM analyses of the nano-objects that remained attached to the SiN windows after unsealing the liquid-cell15 . It is not advisable to reuse the E-chips for another in situ experiment, but it is still possible if the SiN film has not been broken during the unsealing of the liquid cell.

1 .

 1 Open the file directory containing the images of the video on Fiji by clicking on File | Import | Image sequence.The sequence options window will pop-up. Select the appropriate starting image (if the beginning of the video is to be discarded). Enter the increment number for the image sequence (it corresponds to the number of frames it takes for the STEM scan to reach the bottom of the image). Check the box for Convert to 8-bit grayscale.

8 ,

 8 Figure 5 shows two STEM HAADF image series of gold nanoparticle formation acquired over 80 seconds at 25 °C and 85 °C. In all these experiments the nucleation and growth of nanoparticles is driven by the radiolysis of water. Among the chemical species generated by this electron-beam induced phenomena, strong reducing agents (i.e., aqueous electrons and hydrogen radicals) can reduce the tetrachloroauric acid leading to the formation of gold nanocrystal at the interface between the SiN windows and the liquid. These two in situ observations performed with the same electron dose rate confirm that the present method allows visualizing the drastic impact of temperature on the formation of nanoparticles in

Figure 6

 6 Figure 6 represents a typical STEM image and the diffraction pattern of two gold nanoparticles that have been selected directly on the image (indicated by red arrows on Figure 6A).Here, we can identify the face-centered cubic (FCC) structure of gold oriented along the [001] (Figure6B) and[START_REF] Utkur | Imaging protein structure in water at 2.7 nm resolution by transmission electron microscopy[END_REF] (Figure6C) zone axes.

Figure 1 :

 1 Figure 1: Schematic of the E-chips and the tip of the liquid cell holder. (A) The large e-chip with the resistance used to heat the liquid cell (top) and the small E-chip (bottom). (B) Both E-chips are loaded in the liquid cell holder. The electrodes of the large E-chip are in contact with electrode pads of the liquid cell holder. The resistance of the large E-chip can heat up the liquid cell. Please click here to view a larger version of this figure.

Figure 2 :

 2 Figure 2: Optical microscope pictures of E-chips illustrating: (A) An intact SiN window that is necessary for the experiment. (B) A damaged silicon wafer at the edge of the E-Chip. This type of E-chips can be used if the damaged area is outside the wet area once the liquid cell is sealed (i.e., if the damage is outside the area defined by the O-rings). (C) Residues on the E-chip surface. If such residues do not leave after repeating the cleaning processes (see section 4.1), do not use the E-chip. (D to F) Damaged SiN windows (unusable E-chips). Please click here to view a larger version of this figure.

Figure 3 :

 3 Figure 3: Photos of the step-by-step process of the loading of the liquid cell in the TEM holder. (A) Sample holder alone. (B) Put the gasket O-ring in the cavity. (C) Insert the small E-chip in gasket O-rings. (D) Put a drop of solution on the small E-chips. (E) Put the large E-chip over the small one. (F) Seal the whole liquid-cell by screwing the lid. Please click here to view a larger version of this figure.

Figure 4 :

 4 Figure 4: Screenshot of the heating software controlling the temperature of the liquid cell. Please click here to view a larger version of this figure.

Figure 5 :

 5 Figure 5: Low-magnification STEM HAADF image series of the growth of gold nanoparticles. (A) At 25 °C. (B) At 85 °C. The corresponding time is indicated in the bottom left corner of each image. The 2D nanostructures are indicated by red arrows. All images are acquired with the same electron dose rate of 3.4 electron•s -1 •nm -2 . Please click here to view a larger version of this figure.

Figure 6 :

 6 Figure 6: STEM nanodiffraction of single nanoparticles. (A) STEM image used to select the diffracting nanoparticles (the positions of the probe during diffraction acquisitions are indicated by red arrows). (B,C) Diffraction pattern of the two selected nanoparticles. Please click here to view a larger version of this figure.

Figure 7 :

 7 Figure 7: Data processing and analysis of STEM HAADF images using Fiji. The images were acquired 40 seconds after the beginning of the growth. (A to C) Image acquired at 25 °C. (D to G) Image acquired at 85 °C. (A,D) Raw STEM image. (B,E) Processed image (median filter). (C,F) Binary image. (G) A dilatation of the pixels is applied two times and the "Fill holes" process is then applied. Please click here to view a larger version of this figure.

Figure 8 :

 8 Figure 8: Graph representing the number of gold nanoparticles as a function of time at 25 °C and 85 °C. The two curves at 25°C are automatically measured with a minimal detection size (S min ) of 20 (red) and 50 (blue) pixels 2 . The green dots measured after 12 and 60 seconds of acquisition represent the number of nanoparticles counted manually on the video acquired at 25 °C. Please click here to view a larger version of this figure.

Figure 9 :

 9 Figure 9: Graphs representing the average surface area of gold nanoparticles as a function of time for 25 °C and 85 °C. The green dots represent manual measurements of the average area of nanoparticles at given time points of the video acquired at 85° C. Please click here to view a larger version of this figure.

Figure 10 :

 10 Figure 10: High-magnification STEM HAADF image series of the growth of single gold nanocube at 85 °C. This image series was acquired with an electron dose rate of 83.6 electron.s -1 .nm -2 . Please click here to view a larger version of this figure.

Figure 8 ,

 8 the number of detected nanoparticles increase at the beginning of the experiment to reach a plateau. When S min is large (50 pixels 2 corresponding to 1543 nm 2 ), automatic and manual measurements agreed on the level of this plateau (835 nanoparticles after 60 seconds) but the detection of nanoparticles is delayed in the automatic analysis since 835 nanoparticles are manually counted after only 12 s, but not automatically detected until later. This extended detection time leads to an undervaluation of the nucleation rate. Reducing S min down to 20 pixel 2 (i.e., 617 nm 2 ) reduces the error on the nucleation time of the nanoparticle assembly but it leads to an overvaluation of the nanoparticle density particularly at the early stage of the experiments (Figure8) which also affects the nucleation rate. The detection and the size and shape measurements of nano-objects with a very dynamical behavior and a low signal

- 3 .

 3 81 eV per at. in our TB model). Depending on the value of Nanoscale Paper This journal is © The Royal Society of Chemistry 2020 Nanoscale, 2020, 12, 22658-22667 | 22659 Open Access Article. Published on 02 November 2020. Downloaded on 2/1/2021 1:44:59 PM.

3. 1 .

 1 In situ imaging of selective-etching processesThe dissolution of 50 nm long and 13 nm wide gold NRs coated with citrate molecules was followed at the nanoscale by LCTEM in an acidic saline solution containing 1 M of NaCl and 10 -2 M of HCl (Fig.1). As the corrosion of NRs only occurs under electron beam irradiation (see ex situ control experiment in ESI, Fig.S2 †), the reactive oxygen species (ROS) produced by water radiolysis, particularly hydroxyl radicals (OH • ) and hydrogen peroxide (H 2 O 2 ), are obviously implied in the oxidizing reactions. By studying the colloidal stability of gold nanoparticles under electron-beam irradiation in various saline solutions, Hermannsdörfer and coworkers demonstrated that the etching of gold nanoparticles requires, as in our experiment, high NaCl concentration and low pH.10 They concluded that the strong oxidizing power of OH • and H 2 O 2 drives this corrosion processes but the presence of chlorine ions (Cl-) is necessary to lower the redox potential of the reaction and stabilize tetrachloro gold complexes ([AuCl 4 ] -) . The reactivity of ROS increases at low pH, because a high concentration of oxonium ions favors the formation of H 2 O.

  LCTEM also offers the Paper Nanoscale 22660 | Nanoscale, 2020, 12, 22658-22667 This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 02 November 2020. Downloaded on 2/1/2021 1:44:59 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

Fig. 1

 1 Fig. 1 Reaction-kinetics effects on the etching processes of gold NRs in an acidic saline solution. (a) STEM HAADF image series acquired with an electron dose rate of 3.3 electron per s per Å 2 . The acquisition time is indicated in the top right corner of each image. See Video 1 in ESI. † Time evolution of the NR (b) length, width, and (c) aspect ratio. (d) STEM HAADF image series acquired with an electron dose rate of 20.5 electron per s per Å 2 . The acquisition time is indicated in the top right corner of each image. See Video 2 in ESI. † Time evolution of the NR (e) length, width, and (f ) aspect ratio. The analyzed NR is indicated by arrows in (d). The shape evolution of the other NR is quantified in Fig. S4. †

  2). More pre-Paper Nanoscale 22662 | Nanoscale, 2020, 12, 22658-22667 This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 02 November 2020. Downloaded on 2/1/2021 1:44:59 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

Fig. 2

 2 Fig. 2 Atomic-scale faceting of nanorods. Aberration-corrected TEM images of NRs along the (a) [100], (b) [110] and (c) [001] zone-axis orientations. The white, blue and pink dashed lines indicate the {100}, {110} and {111} edge-on facets, respectively. In image (c) the NR is seen along its anisotropy axis. (d) Scheme of the atomic scale faceting of NRs deduced from HRTEM images. This 3D nanostructure is used as a starting point of Monte Carlo simulations.

C 2

 2 configuration over the {100} surface (E ad = -0.21 eV) and for both the C 2 (E ad = -0.49 eV) and C 4 (E ad = -0.45 eV) over the {110} surface. The strong adsorption of citrates on {110} surfaces is due to stronger charge transfers from the under-coordinated gold surface to the molecules (Fig. S11 †).

Fig. 3

 3 Fig. 3 Monte Carlo (MC) simulations of the dissolution of a citrate-free NR using a weak chemical potential (µ Au = -3.75 eV). (a) Snapshot images of the simulated NR seen along its transversal (left) and longitudinal (right) axes at different MC steps. (b) Length and width of the NR as function of MC step. (c) Rate of desorption of gold atoms on the tips and the body of the NR as function of MC step. (d) Aspect ratio of the NR as function of MC step. see Video 4 in ESI † and calculation details section 2.3. †

Fig. 4

 4 Fig. 4 Spatial distribution of citrate functionalization over gold NRs. (a) Low-pass filtered HRTEM image of a NR acquired close to zero focus. A color thresholding process was applied to display the citrate layer around the NR in hot colors (see details in section 2.2). On the right, signal to noise ratio profile measured along the arrow seen on the image, on which we can measure the local thickness of citrate (T cit ) on each side of the NR's body. Optimized C2 (b) and C4 (c) configurations of citrate on {100}, {110} and {111} gold surfaces and their corresponding adsorption energies obtained by DFT calculations (see details in section 2.3 and in ESI †). Au, C, O and H atoms are represented in yellow, black, red and white, respectively.

Figure S2 .

 S2 Figure S2. Ex situ control experiment (without electron beam). TEM image series showing the stability of NRs after an immersion time in the acidic saline solution (1M of NaCl and 10 -2 M of HCl). After depositing the NRs on the carbon lacey TEM grid, the same area of the sample was imaged by TEM after each immersion step (the time indicated in the top-right corner of each image corresponds to the cumulative immersion time after each step). No degradation of the NRs was observed after 75 minutes in the solution revealing the key role of the electron beam in the etching processes observed by LCTEM.

Figure S3 .

 S3 Figure S3. Selective shortening of gold NRs observed by LCTEM at low electron dose rate in the acidic saline solution. STEM HAADF image series acquired with an electron dose rate of 3.3 electron.s -1 .Å -2 . The acquisition time is indicated in the top right corner of each image. The time evolution of aspect ratio of the two observed NRs is seen in the botom right corner of the figure (the NRs corresponding to the black and red data points are indicated on the first image). See Video 3 in SI.

Figure S4 .

 S4 Figure S4. Isotropic etching observed by LCTEM at high electron dose rate in the acidic saline solution. (a) STEM HAADF image series acquired with an electron dose rate of 20.5 electron.s -1 .Å -2 . The acquisition time is indicated in the top right corner of each image. Time evolution of (b) the normalized length and width and (c) the aspect ratio of the NR indicated with an arrow in (a). See Video 2 in SI.

Figure S5 .

 S5 Figure S5. Grand Canonical Monte Carlo (GCMC) simulations of the dissolution of a citratefree NR for three different chemical potentials. (a) Number of gold atoms in the NR as a function of MC step. (b) Cohesive energy per atom in the NR as a function of MC step (c) Coordinationnumber distribution of extracted atoms. (d) Desorption-energy distribution of extracted atoms.

Figure S6 .

 S6 Figure S6. Monte Carlo (MC) simulations of the dissolution of a citrate-free NR using a strong chemical potential (µAu = -3.95 eV). (a) Snapshot images of the simulated NR seen along its transversal (left) and longitudinal (right) axes at different MC steps. (b) Length and width of the NR as function of MC step. (c) Rate of desorption of gold atoms on the tips and the body of the NR as function of MC step. (d) Aspect ratio of the NR as function of MC step (see video 5 in SI and the calculation details in the methods section).

Figure S7 .

 S7 Figure S7. Spatial distribution of citrate functionalization over 8 gold NRs. On the left, original aberration-corrected HRTEM image acquired close to zero focus. On the right, same image on which we applied a low-pass filter and a color thresholding process to better visualize the citrate layer displayed in hot colors. Such images were used the measured the average thickness of the citrate layer on the body, the tips and the corners of NRs (see materials and methods).

Figure S8 .

 S8 Figure S8. The optimized configurations and the structural formula of citrate in the gas phase.

Figure S9 :

 S9 Figure S9: Illustration of the considered dissociative adsorption of the molecule. Au, C, O and H atoms are represented in yellow, black, red and white, respectively.

Figure S10 .

 S10 Figure S10.The optimized configuration of citrate on Au(100), Au[START_REF] Noh | Lithographically patterned well-type graphene liquid cells with rational designs[END_REF] and Au[START_REF] Daniel | Nanometer resolution elemental mapping in graphene-based tem liquid cells[END_REF] surfaces though one terminal carboxylate group.

  understand the superior reactivity of the Au(110) surface toward the citrate and to compare it to the Au(100) and the Au(111) surfaces (-0.49 eV Vs. -0.21 eV and 0.05 eV), we calculated the Bader charges and the charge transfers between the different surfaces and the molecule. The Figure S11, shows the calculated Bader charges of the carboxylate atoms connected to two Au first neighboring surface atoms for each surface and the corresponding charge density difference represented by isosurfaces. The results show strong charge transfer from the surface to the citrate molecule adsorbed on Au[START_REF] Noh | Lithographically patterned well-type graphene liquid cells with rational designs[END_REF]. Interestingly, the surface charge depletion seems to arise from upper as well as from deeper surface atoms.

Figure S11 .

 S11 Figure S11. The Bader charges and the charge density difference of citrate adsorbed on the Au(100), Au(110), and Au(111) surfaces with the terminal COO -group. The isosurface of the charge density is 0.005 e/Bohr 3 .

Figure S13 .

 S13 Figure S13. The optimized configurations of citrate on (a) Au(221) (b) Au(311) and (c) Au(331) surfaces though one terminal COO -group.

  cells have been exploited to acquire high-resolution TEM images of nanostructures in static liquid, but the lack control over the composition of the solution remains a strong limitation for many studies (Textor and de Jonge, 2018;[START_REF] Yuk | High-Resolution EM of Colloidal Nanocrystal Growth Using Graphene Liquid Cells[END_REF]. With commercially-available liquid TEM holder, the formation of a large hydrogen bubble within the SiN liquid cell is a well-known trick to reduce the liquid thickness to a few tens of nanometers, giving access to atomic resolution(Ahmad et al., 2018;[START_REF] Zhu | Atomic resolution liquid-cell transmission electron microscopy investigations of the dynamics of nanoparticles in ultrathin[END_REF]. These strategies have notably provided unseen atomicscale insights into the growth and etching mechanisms of nanostructures[START_REF] Ngo | Toward Ending the Guessing Game: Study of the Formation of Nanostructures Using In Situ Liquid Transmission Electron Microscopy[END_REF].Nevertheless, the use of high resolution TEM in liquid presents two major drawbacks. At first, as liquids and amorphous materials broaden both the energy dispersion of electrons (reducing TEM resolution) and the electron probe size (reducing STEM resolution), image resolution is limited between 2 to 3 Å.(de Jonge, 2018a, b; de Jonge and Ross, 2011)Although such a resolution allows observing the atomic structure of metals, oxides and semiconductors, it drastically limit the number of zone-axis orientations along which structural information can be obtained. More importantly, high resolution imaging requires very high dose rate leading to drastic changes in the local chemistry of the liquid sample(Schneider et al., 2014;[START_REF] Woehl | Defining the radiation chemistry during liquid cell electron microscopy to enable visualization of nanomaterial growth and degradation dynamics[END_REF]. Beam-induced effects are well-known issues in LCTEM that have generated intense theoretical and experimental works to understand the radiolytic processes occurring in the liquid-cell[START_REF] Ross | Liquid Cell Electron Microscopy[END_REF]. Furthermore, low-dose imaging strategies have been proposed and exploited to minimize the beam-induced phenomena that can influence in situ TEM experiments(Buban et al., 2010; Evans et al., 2008; Helveg et al., 2015; Mehdi et al., 2017; Stevens et al., 2018; Vendelbo et al., 2014). However, the structural analysis of individual nanomaterials in low dose conditions remains challenging in liquid media.

FEG

  and CEOS image corrector[START_REF] Alloyeau | Following Ostwald ripening in nanoalloys by high-resolution imaging with single-atom chemical sensitivity[END_REF][START_REF] Ricolleau | Performances of an 80-200 kV microscope employing a cold-FEG and an aberration-corrected objective lens[END_REF]. Accelerating voltage in all experiments was maintained at 200 kV. We used a Poseidon select liquid-cell holder designed by Protochips. Two types of citrate-coated gold nanoparticles purchased from Nanocomposix were used for this study: 20 nm spherical nanoparticles and nanorods (NRs) with average dimensions of 11 nm and 47 nm. This two types of nanoparticles were firstly deposited by drop casting on the window of the top E-chip. Then, a droplet of water was sandwiched between two silicon E-chips separated by 150 nm gold spacers. The sealed liquid-cell provide a 50 µm by 50 µm field of view in which the liquid is encapsulated between two electrontransparent SiN films. Water was continuously flowing through the liquid-cell during the experiment with a flow rate of 5 µL/min. A gas bubble was intentionally formed in the liquid-cell prior data acquisition in order to reduce the thickness of the liquid layer. Bubble formation was obtained by irradiating the liquid cell in TEM mode for one or two minutes with a dose rate above 3000 electrons.nm -2 .s -1 . As soon as the bubble is formed, a characteristic liquid cell configuration is observed with a very thin liquid film in the middle of the observation window and thicker liquid areas at each corners (fig.S1a-bin supporting information). The presence of water near the windows was confirmed by the diffusion of the nanoparticles under electron irradiation (fig.S1cin supporting information). Note that using the holder in flow mode slowly refills liquid cell with water and avoids the full drying of the observation area during STEM analysis. Gold nanoprisms were synthesized directly in the liquid-cell by injecting an aqueous solution of HAuCL4, (1 mM).As previously reported, such faceted nanostructures grow by radiolysis in presence of gold precursor when low electron dose rate are used to drive the reduction process(Ahmad et al., 2017; Alloyeau et al., 2015).

  cell microscopy. Many studies have demonstrated that the electron beam drives a sub-diffusive motion of metal nanoparticles in the liquid-cell (Lu et al., 2014; Piffoux et al., 2017; Verch et al., 2015; White et al., 2012; Woehl and Prozorov, 2015). Under the beam, nanoparticles move via sporadic jumps on the windows separated by period of immobility due to their strong electrostatic interactions with the positively-charged SiN film. Confinement effects imposed the liquid-cell geometry also influence the motion behaviour of nano-objects which cannot be described as bulk mediated surface diffusion. (Parent et al., 2018; Parent et al., 2017) So far, nanoparticle motion

FiguresFig. 1 .

 1 Figures

Fig. 2 .

 2 Fig. 2. (a) HRTEM image of a gold NR in water obtained with a dose rate of 2.95*10 5 electrons.nm -2 .s -1 and an exposure time of 1 second. Atomic contrasts are observed in the zoomed

Fig. 3 .

 3 Fig. 3. Low-dose STEM nanodiffraction analyses of gold NRs with a beam current ie = 5.5*10 6

Fig. 4 .

 4 Fig. 4. STEM nanodiffraction analysis of nanoparticle rotation in water with a beam current ie = 1.9*10 7 electrons/s (probe size 8c). After acquiring a STEM HAADF image (left) the beam was

Fig. 5 .

 5 Fig. 5. Image series of gold nanoparticles (beam current ie = 1.9*10 7 electrons/s). The zoomed

Figure S1 :

 S1 Figure S1: Liquid-cell configurations. (a) Liquid-cell fully filled with liquid before the formation of a gas bubble. From left to right: schematic cross section view of the liquid cell; Low-mag TEM image of the observation window; STEM-HAADF image of the window's corner. Image contrast reveals thin-liquid area at the corners and thick-liquid region at the center of the liquid cell. (b) Liquid-cell after the formation of the gas bubble. From left to right: schematic cross section view of the liquid cell; Low-mag TEM image of the observation window; Bright field TEM image of the window's corner. Image contrast reveals a thin liquid layer at the center and thicker liquid areas at the corners of the liquid cell (c) STEM-HAADF image series showing the diffusion of gold NRs in the central area of the liquid cell after the formation of the gas bubble, confirming the presence of liquid layers over and under the bubble.

Figure S2 . 6 Fabrication

 S26 Figure S2. NR misorientation with respect to zone axis orientation. Comparison of experimental (left) and simulated (right) diffraction patterns allows estimating the tilt angle with respect to the <101> zone axis orientation. Experimental diffractions are the same than in figure 4c (time series on a gold nanorod). (a) Tilt angle = 2.7°. (b) Tilt angle = 7.3°. (c) Tilt angle = 7.4°.

6. 2 . 1

 21 Static pockets type of graphene liquid cellsThe initial work of Yuk et al.[START_REF] Min | Graphene veils and sandwiches[END_REF] corresponds to an in situ heating experiment where nanoparticles were preserved from evaporation when inside what they called a "graphene veil superstructure" which was a precursor of the GLCs (see figure6.1).

Figure 6 . 1 :

 61 Figure 6.1: Schematic of a graphene veil superstructure[START_REF] Min | Graphene veils and sandwiches[END_REF]. While it is not represented in this schematic, the bottom layer of graphene rests on a substrate such as silicon and the top graphene layer is transferred to standard carbon-coated TEM grid. The two layers of graphene encapsulate gold nanoparticles.

Figure 6 . 2 :

 62 Figure 6.2: Schematic of a first generation GLC [103]. The TEM grids are not represented.

Figure 6 . 3 :

 63 Figure 6.3: STEM HAADF image sequence of Pt nanoparticle growth via coalescence [103]. White arrow: small nanoparticle that coalesces into the bigger one. Red dot line: twin boundary. On the right: FFT of the final image taken. Scale bar = 2 nm. Z.A. = zone axis.

Figure 6 . 4 :

 64 Figure 6.4: Schematic of the two types of GLCs made by Rasool et al. [109]. Left: well-type GLC. Right: channel-type GLC.

Figure 6 . 5 :

 65 Figure 6.5: In situ TEM image of a gold nanoparticle with atomic resolution using a well/channel-type GLC [109].

Figure 6 . 6 :

 66 Figure 6.6: Schematic of a channel filled with liquid[START_REF] Noh | Lithographically patterned well-type graphene liquid cells with rational designs[END_REF]. Top: a bigger hole induces a collapse of both graphene sheets unto themselves and the liquid tends to be on the edges of the channel. Bottom: a smaller hole allows to avoid a collapse of the graphene sheets.

Figure 6 . 7 :

 67 Figure 6.7: Schematic representation of the top and bottom graphene e-chips [113]. The bottom e-chip is at the exit the e-beam. It consists of a stack of SiN (thickness of 7 nm, blue)/SiO 2 (75 nm, light blue) / SiN (14 nm, not represented) on top of a Si chip (green). The nanochannel patterned by ebeam and SiO 2 is selectively etched away using a BHF etch. The holes have a diameter of 200 nm although the cavities themselves have a diameter of 2 µm. The integration of the channel into a single e-chip and the limitation of the channel width to about 2 µm allow to avoid the bowing/bulging effects seen in standard SiN liquid cells.

Figure 6 . 8 :

 68 Figure 6.8: TEM image series highlighting the motion of gold nanoparticles in a phosphate buffer solution [113]. (a) Yellow circle = rotating nanoparticle. Red circles = moving individual nanoparticle with the red arrow indicating the direction. (b) Red circle = pair of nanoparticles getting closer to each other. Blue circles = moving pair of nanoparticles with the blue arrow indicating the direction. A false background color (blue) has been applied to enhance the visualization conditions.

Figure 6 . 9 :

 69 Figure 6.9: Schematic of the flow capable GLC made by Koo et al. [114]. Apart from the graphene, these e-chips design are similar to the commercial standard SiN e-chips made by Protochips.

Figure 6 . 10 :

 610 Figure 6.10: Some of the physical properties of the flow capable GLC made by Koo et al. [114]. (a) Pressure limit before rupture of the membrane between the flow capable GLC and a standard SiN liquid cell. (b) Flow rate limit as a function of the number of layers of graphene for the flow capable GLC and standard SiN liquid cell.

Figure 6 . 11 :

 611 Figure 6.11: Oxidative etching of an icosahedral gold nanoparticle [114]. The inset shows the diffraction pattern of the initial nanoparticle. Yellow arrows = position of flattening. White arrows = position of etching. The electron dose was reduced to minimize the effects of the electron beam on the etching.

Figure 6 . 12 :

 612 Figure 6.12: Drawing representing the two holes dispositions on the SiN window. The SiN window is in orange with a dimension of 500 x 50 µm. The holes have a diameter varying between 1 µm and 8 µm. (a) Hole disposition for small e-chips. The distance between one hole and the closest edge of the SiN window is of about 5 µm. (b) Hole disposition for large e-chips. The distance between the two holes is of about 40 µm.

6. 4 . 1

 41 Graphene liquid cell fabrication success rate and general remarks Before we dive into the results of our experiments, we need to first examine the success rates of our fabrication methods and give some general remarks about their use.

Figure 6 . 13 :

 613 Figure 6.13:Table of the different success rates of GLCs as a function of the two graphene transfer techniques.

Figure 6 . 14 :

 614 Figure 6.14: (a-b) Optical microscope images of a graphene flake covering holes in the SiN membrane. (a) Bright field image. (b) Dark field image. The red arrows indicate the holes. The graphene flake is more visible in dark field. (c-d) Ex situ TEM images of a hole and graphene. (c) The red squares are the areas used to calculate the signal-to-noise ratio in (1) and outside (2) the hole. The green square is the area corresponding to the zoomed in HRTEM image (d). (d) HRTEM image of graphene with polymer residues on it. Inset corresponds to the FFT of the image.

Figure 6 . 15 :

 615 Figure 6.15: TEM image of polymer residues from the graphene transfer in water.

Figure 6 . 16 :

 616 Figure 6.16: In situ TEM image of gold nanoparticles in a thin layer of water and a hole. The blue arrow indicate a gold circular nanoparticle that appears black/dark grey. The orange arrow indicate copper pollution that appears light grey. The red squares are the areas used to calculate the signal-to-noise ratio in (1) and outside (2) the hole.

Figure 6 . 17 :

 617 Figure 6.17: In situ HRTEM image of gold nanoparticles in a thin layer of water. (a) HRTEM image. The gold nanoparticle is at the center while copper grew around it. (b) FFT of the HRTEM image. The spot circled in red demonstrates that we reach a resolution of 1.8 Å.

Figure 6 . 18 :

 618 Figure 6.18: In situ STEM image series of a hole covered by multilayer graphene. Between each image, we made various higher magnification images and videos at different positions of the observation window. The red squares are the areas used to calculate the signal-to-noise ratio in (1) and outside (2) the hole. The blue arrows indicate a step in the multi-layer graphene patch.

Figure 6 . 19 :

 619 Figure 6.19: High magnification in situ STEM image series of the growth of gold aggregates. The red arrows indicate small aggregates that agglomerate with larger aggregates.

  Les nanoparticules métalliques ont des propriétés physico-chimique qui en ont fait des candidats idéals pour une très grande variété d'applications en optique, médecine et dans le domaine de l'énergie. Pour adapter au mieux les nanoparticules métalliques aux différentes applications, la recherche actuelle tente de comprendre quelles sont les paramètres qui contrôlent leurs propriétés et ainsi obtenir de manière fiable et non empirique des nanoparticules fonctionnelles. De plus, l'objectif principal de la synthèse des nanoparticules métalliques est de comprendre comment obtenir des nano-objets dont la taille, la forme, la composition chimique et la structure atomique sont bien contrôlées. Depuis des décennies, la synthèse chimique en solution a permis de fabriquer une très grande variété de nanomatériaux en milieu liquide[13]. Cependant, une compréhension plus approfondie des processus de nucléation, de croissance et de dissolution qui se produisent en solution est cruciale pour rationaliser les stratégies de synthèse en chimie colloïdale. Pour cela différents outils d'investigation sont utilisés. Les outils ex situ comme le diffractomètre à rayons X, le microscope électronique à balayage ou le microscope électronique à transmission (TEM) nous donnent de nombreuses informations sur la structure finale des nanoparticules métalliques[14][15][16]. Cependant, il est maintenant bien établi que ces techniques de caractérisation ex situ, avec lesquelles les nanomatériaux sont étudiés à l'échelle atomique mais en dehors de leur environnement de synthèse, ne fournissent qu'une compréhension très partielle de la dynamique des nanomatériaux en solution et de l'influence des paramètres de synthèse.133Cette problématique a motivé le développement de nombreuses approches in situ pour sonder en temps réel et à l'échelle atomique le comportement des nanomatériaux en milieu liquide. Récemment, des cellules liquide nano-fabriquées ont été développées et permettent d'observer des nanoparticules directement dans leur environnement de nucléation et de croissance à l'aide du TEM. Le design des cellules liquide que nous avons utilisées (cellules Protochips) consiste en deux chips de silicium avec une fenêtre en nitrure de silicium transparente aux électrons. Ces deux e-chips sont superposées dans un porte-objet que l'on insère dans le TEM et l'étanchéité de la cellule dans le vide du microscope est assurée par des joints circulaires (voir figure8.1 (a)). Le porte-objet est muni de canaux micro fluidique qui permettent l'injection d'une solution directement dans la cellule liquide et aussi de contrôler la composition du milieu réactionnel. La microscopie électronique à transmission en cellules liquides (LCTEM) s'est révélée être un puissant outil d'investigation des différentes stratégies de contrôle des synthèses des nanoparticules métalliques comme les synthèses basées sur des germes ou des blocage de facettes[START_REF] Ahmad | Exploring the formation of symmetric gold nanostars by liquid-cell transmission electron microscopy[END_REF], les synthèses par contrôle de la cinétique de croissance[START_REF] Woehl | Direct in situ determination of the mechanisms controlling nanoparticle nucleation and growth[END_REF] ou les synthèses par contrôle de la température[START_REF] Huolin | In situ observation of oscillatory growth of bismuth nanoparticles[END_REF][START_REF] Niu | Revealing bismuth oxide hollow nanoparticle formation by the kirkendall effect[END_REF]. Cependant, beaucoup d'améliorations sont envisagées pour cette technique de TEM in situ en particulier pour l'optimisation du rapport signal/bruit et de la résolution des images acquises dans le liquide, la compréhension et la minimisation des effets du faisceau et le contrôle de l'environnement réactif (température et composition). L'objectif principal de ce travail était de relever les défis techniques de l'optimisation du LCTEM et les défis scientifiques de la dynamique des nanoparticules d'or dans leur milieu de synthèse. Les résultats de ma thèse ont été décrit dans les chapitres 2, 3, 4, 5 et 6 avec les chapitres 2 à 5 correspondants à des articles publiés dans des journaux scientifiques. Nous allons ici résumer l'ensemble de ces chapitres. 8.2 Description d'une nouvelle méthode pour étudier le comportement thermique des colloides métalliques en solution avec le TEM Le contrôle de la température est un développement récent qui offre un degré de liberté supplémentaire pour étudier la nanochimie par microscopie électronique à transmission en cellule liquide. Dans un article vidéo [1], nous décrivons comment préparer une expérience in situ pour étudier l'effet de la température sur la formation de nanoparticules d'or induite par radiolyse de l'eau. Le protocole de l'expérience est assez simple et implique une cellule liquide spéciale avec des capacités de chauffage uniforme jusqu'à 100 °C (voir figure 8.1),

Figure 8 . 1 :

 81 Figure 8.1: (a) Coupe transversale schématique d'une cellule liquide intégrée dans le porte-objet avec capacité de flow et de chauffage. (b) Schéma des chips utilisées pour les expériences de LCTEM chauffantes produites par Protochips. La grande chip est munie d'une électrode intégrée dans le silicium qui permet un chauffage intégral et uniforme de la cellule liquide. (c) Schéma du porte-objet Poseidon Select produit par Protochips. Le porte-objet combine les capacité de flow de liquide avec une possibilité d'augmenter la température jusqu'à 100 °C.

8. 3 Figure 8 . 2 : 8 . 4 Figure 8 . 3 :Figure 8 . 4 :

 382848384 Figure 8.2: Images STEM HAADF in situ de la croissance de nanoparticules d'or à différentes températures et un graphique représentant la variation du logarithme du rayon moyen des nanoparticules en fonction du logarithme du temps et des températures utilisées. Les courbes sont linéaires avec une pente d'environ 0.33 ce qui signifie (d'après la théorie LSW) que les croissances observées sont limitées par la diffusion des atomes d'or en solution.

Figure 8 . 5 :

 85 Figure 8.5: Schéma d'une petite chip modifiée avec des trous et avec un patch de graphène. Les carrés jaunes sont les "spacers" en or qui permettent de maintenir un espace entre les e-chips après assemblage dans le porte-objet. Le schéma n'est pas à l'échelle.

Table S1 .

 S1 The adsorption energies of citrate on the Au surfaces through one terminal carboxylate group.

	Au atoms	Au(100)	Au(110)	Au(111)
	first neighbors -0.21	-0.49	0.05
	2 nd neighbors	unstable	-0.41

Table S2 .

 S2 The adsorption energies of citrate on high index Au surfaces.

	Citrate on Au(221) surface
	Configuration	1	2	3	4
	Ead (eV)	-0.17	-0.33	0.03	0.20
	Citrate on Au(311) surface
	Configuration	1	2
	Ead (eV)	-0.08	-0.31
	Citrate on Au(331) surface
	Configuration	1	2	3
	Ead (eV)	-0.33	-0.43	-0.06

It is worth noting that on these three high-index surfaces, step atoms at the edge of terraces are stable adsorption sites for citrate with competitive adsorption energies (Ead<-0.3 eV). They are even the most stable sites on Au(221) and Au(311) surfaces. The DFT-D3 optimizations show the Au(331) to be the most reactive gold surface for citrate adsorption. Over this latter surface, the computed adsorption energies show two competitive adsorption sites (E=0.1 eV), on the edge Au sites with Ead predicted to be of -0.33 eV and over the Au terrace sites with Ead=-0.43 eV. The adsorption energy of citrate on the Au(331) terrace sites is similar to the calculated adsorption energy on the (110) surface.

  4 electrons) to the total amount of electron seen by the NR during the acquisition of the STEM nanodiffraction data (𝑁 &' ) that reaches 2.2*10 4 electrons. Thus, the number of electrons necessary to determine the atomic structure of metal nanoparticles immersed in liquid is more than 6800 times smaller in

STEM nanodiffraction than in HRTEM. Interestingly, the information limit on these low-dose nanodiffractions remains below 0.1 nm. Although strategies have been developed to reduce the electron dose of high-resolution (S)TEM imaging,

(Buban et

al., 2010; Evans et al., 2008; Helveg et al., 2015; Mehdi et al., 2017; Stevens et al., 2018) this huge gap makes of STEM nanodiffraction a unique technique to extract structural information on single nanomaterials in beam-sensitive liquid environments. Note that here nanodiffractions are acquired in spots-analysis

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Acknowledgments

We gratefully acknowledge the financial support of the Region Ile-de-France (convention SESAME E1845 for the JEOL ARM 200 F electron microscope installed at the University of Paris), the Labex SEAM (GLOIRE Project) and the CNRS (Defi Nano Program). We thank Madeline Dukes and Daniel Franck for sharing the schematics and the optical pictures of the liquid cells seen in figures 1 and 2.

Acknowledgements

We gratefully acknowledge the help of Yann Le Bouar for the use of the Nanofabric Software Developed for the geometrical construction of free or supported crystalline nanoclusters. We also acknowledge the financial support of the Region Ile-de-France (convention SESAME E1845 for the JEOL ARM 200F electron microscope installed at the University of Paris), the Labex SEAM, the CNRS and the ANR (CYCLYS project).

Acknowledgments

We gratefully acknowledge the financial support of the Region Ile-de-France (convention SESAME E1845 for the JEOL ARM 200F electron microscope installed at the Paris Diderot University), the Labex SEAM and the CNRS.

The authors have no competing interests to declare capacité de flow tout en augmentant le signal sur bruit des images grâce au graphène. Ceci permet d'obtenir une résolution maximale de 1.8 Å en HRTEM. Enfin, nous remarquons que la présence du graphène modifie fortement les mécanismes de nucléation et croissance des nanoparticules d'or. Ces travaux préliminaires sont riches de leçons techniques pour de futurs designs de cellules liquide en graphène.

Bibliography

Disclosures

The authors have nothing to disclose.

Chapter 4

Selective shortening of gold nanorods: when surface functionalization dictates the reactivity of nanostructures This chapter is a scientific paper reused from Nanoscale with the permission from the Royal Society of Chemistry [3].

Conflicts of interest

There are no conflicts of interest to declare.

View Article Online

Supporting information

Selective shortening of gold nanorods: when surface functionalization dictates the reactivity of nanostructures Abdelali Khelfa, 1 Jun Meng, 2.3.4 Caroline Byun, 1 Guillaume Wang, 1 Jaysen Nelayah, 1 Christian Ricolleau, 1 Hakim Amara, 1, 5 Hazar Guesmi, 2 Damien Alloyeau For each surface, different positions of the citrate bonding to the Au atoms are tested. The corresponding configurations which mostly correspond to C2 conformations are summarized in Figure S13 and the adsorption energies are depicted in Table S2. In addition to the described configurations, we also studied the configurations of citrate bonded to the Au(221), (311) and the (331) facets through one terminal carboxylate and the central carboxylate (C4 conformation).

However, the citrate became very unstable and messed up during the optimizations.

Associated Videos

There are 5 video files in SI: Video 5 corresponds to the GCMC Simulations shown in figure 3 Chapter 5

Structural analysis of single nanoparticles in liquid by low-dose STEM nanodiffraction

This chapter is a scientific paper accepted in Micron and reused with the permission from Elsevier [4]. The readers can visualize the paper in the published format with this link: https://doi.org/10.1016/j.micron.2018.09.008.

Structural analysis of single nanoparticles in liquid by low-dose STEM nanodiffraction

Abdelali Khelfa, a,# Caroline Byun, a,# Jaysen Nelayah, a Guillaume Wang, a Christian Ricolleau, a Here we show that STEM nanodiffraction can be exploited in liquidcell TEM experiments to overcome these two limitations. We evidence that this technique allows quick analysis the structure of single gold nanoparticles whatever their zone axis orientation, which substantially increases the percentage of analysable nanostructures with respect to HRTEM investigations. Moreover, STEM nanodiffraction can also be used in very low dose conditions.

Supporting information

Structural analysis of single nanoparticles in liquid by low-dose STEM nanodiffraction

Abdelali Khelfa, a,# Caroline Byun, a,# Jaysen Nelayah, a Guillaume Wang, a Christian Ricolleau, a Damien Alloyeau

Flow capable graphene liquid cells

Liquid flow, meaning the capacity to inject a liquid inside a liquid cell, is an indirect solution of some of the problems we described in the other GLCs. Indeed, the precursor concentration can be controlled, the chemical nature of the liquid can be changed through targeted reactions and the dynamics of nanomaterials are closer than ever to the real life conditions. Liquid flow could even facilitate the diffusion of the products of the radiolysis of water and thus reduce the chance of beam induced damage to the sample [START_REF] Utkur | Imaging protein structure in water at 2.7 nm resolution by transmission electron microscopy[END_REF].

Very few attempts at making flow capable GLCs were successful because of multiple difficulties in various aspects of the experiment such as the design, the resources necessary to achieve the design, the graphene transfer and the experiment itself. However, two very recent papers reported successful flow capable GLCs.

Dunn et al. [START_REF] Gabriel Dunn | Graphenesealed flow cells for in situ transmission electron microscopy of liquid samples[END_REF] described the fabrication process of liquid flow graphene e-chips. The essential part of the bottom e-chip consists of a stack of SiN/SiO 2 /SiN with cavities in it (see figure 6.7). These cavities are connected to each other thus forming a nanochannel.

Multiple nanochannels are made in parallel in one e-chip. Thus if one channel is broken or blocked, other channels can be used for the experiment. Gold electrodes are deposited on the bottom e-chip to allow electrical biasing and electrochemistry. Graphene is deposited using a wet transfer technique over the holes of the nanochannel. It should be noted that the window aperture is smaller than on standard SiN liquid cells so that it spans only the holes covered with graphene and not the full channel.

The authors demonstrated that these GLCs are working effectively by studying the movement of gold nanoparticles in a phosphate buffer solution (see figure 6.8). The nanoparticles are seen rotating and moving by translation individually or in pair. Although the authors successfully imaged these phenomenons as well as another experiment involving a uranyl acetate crystallization process, it should be noted that atomic resolution was not reached (or at least the authors never stated that they reached it). This limited resolution by comparison to the performance of static GLCs is explained by the authors with the fact that the flow capable GLCs they made are thicker and that they used a JEOL 2010 TEM operating at 80 keV. Koo et al. [START_REF] Koo | Liquid-flowing graphene chip-based high-resolution electron microscopy[END_REF] made a flow capable GLC with another design. This design consists of two clean room fabricated standard Si e-chips with 300 nm thick SiN windows (see figure 6.9). The SiN windows are perforated with a holey pattern with many holes so that it easier to align the holes on both e-chips when assembling them in the liquid cell holder. Different holey patterns have been tested and used (for example a holey pattern of 8 µm

Abstract

Liquid Cell Transmission Electron Microscopy (LCTEM) is a powerful in situ technique to reliably observe the dynamics of nanomaterials in solution with sub-nanometer resolution.

However, there is still room for improvement for LCTEM in particular on the optimization of the signal-to-noise ratio and resolution of TEM images acquired in liquid, the understanding and minimization of the beam effects and the control over the reactive environment (temperature and composition). The main objective of this work was to face the technical challenges on the optimization of the LCTEM and the scientific challenges on the dynamic of gold nanoparticles in their synthesis media. Besides a video article describing the protocol of our temperature-controlled LCTEM experiments [1], four main results emerge from this work: (i) For the first time, we investigated the effects of temperature on the nucleation and growth of gold nanoparticles in water by LCTEM and revealed new in situ insights into the Ostwald ripening and faceting processes [2]. (ii) In collaboration with Hakim Amara (Laboratoire d'études de microstructure -ONERA -Châtillon) and Hazar Guesmi (Institut Charles Gerhardt -Montpellier), we combined liquid cell TEM experiments with atomistic simulations to study the etching mechanisms of gold nanorods and determine if their degradation processes are dictated by their atomic structure or their surface functionalization [3].

(iii) We showed that STEM nanodiffraction can be exploited in liquid cell TEM experiments to overcome the two main limitations of HRTEM structural analysis in liquid media: the limited resolution and the very high electron dose rate. Indeed, we evidence that STEM nanodiffraction allows quick analysis of the structure of single gold nanoparticles whatever their zone axis orientation and we also show that this technique can also be used in very low dose conditions (reduced by almost four orders of magnitude compared to conventional HRTEM analysis) [4]. (iv) Finally, in collaboration with Kristian Mølhave (DTU Nanolab -Technical University of Denmark) we have fabricated and tested new graphene liquid cells with flow capabilities combining enhanced imaging conditions and solution composition control.