The differentially pumped system approach relies on a modified TEM setup to perform environmental studies. In this case, the environment cell (E‐Cell), generally intended as the experimental chamber where the specimen comes in contact with user‐introduced gaseous species, is constituted by the portion of TEM column around the sample that is limited by a double series of additional apertures with small holes, which are positioned in correspondence with the objective lens pole pieces to confine the gaseous phases. Two additional sets of apertures along the column combined to a differential pumping system create zones of decreasing pressure between the high pressure zone of the E‐Cell and the high vacuum of the other parts of the TEM column, thus protecting the electron gun from any gas coming from the E‐Cell (**Figure 6A**).

This architecture allows the separate vacuum control of the E‐Cell from the rest of the TEM column so that all the volume around the sample delimited by the objective lens can be filled with gases at low pressure and evacuated independently. The introduction of external gases implies that the E‐Cell needs to be connected to an external gas manifold and flow controller system equipped with gas reservoir tanks, unreactive pipelines, and pressure gauges for any

tuning and monitoring procedures [71] but no modification is necessary with regard to the holder and any type of tilting and heating TEM holders can be used to perform *in situ* experiments.

The main advantage of the differential pumping is that it is an *open* system, meaning that the electron scattering comes only from the sample and the gas molecules, while no containment window is needed, although it is also worth noting that this architecture can reach only limited pressure values (around 20 Torr) without jeopardizing the TEM and electron gun [81]. However, this solution comes with a cost, i.e., the blocking of electrons scattered at high angle by the lower series of differential apertures below the sample [67]. This constraint imposes a strong limitation to the capabilities of different analytical techniques, e.g., STEM imaging by annular dark field (ADF) and high angle annular dark field (HAADF) detectors, electron diffraction of patterns collected at high angle [82, 83], and EELS, since the range of collection semiangle of the spectrometer cannot exceed the angular value of the lower E‐Cell and differentially pumped apertures. Moreover, the introduction of a differentially pumped system results in a more complex and more expensive system.

In addition, the imaging resolution is not only affected by the presence of gases, but it is also influenced by additional electro‐optical and microscope‐related parameters with opposite effects, i.e., the electron dose and the primary electron energy. In fact, under the same condi‐ tions of E‐Cell gas pressure and primary high tension, reducing the electron‐dose increases

**Figure 6.** (A) Schema of a differentially pumped TEM system after [71]; (B) sequence of HRTEM images and the corre‐ sponding diffractograms of an Si 011 crystal lattice measured under 4 mbar of N2 gas pressure and different electron doses. Adapted with permission from [84]; (C) HRTEM and the corresponding power spectrum of an Au film on car‐ bon film, showing the pressure dependence of the transmitted information limit under different conditions of accelera‐ tion tension and N2 gas pressure. The white circle corresponds to 10 nm−1. Adapted with permission from [85].

the spatial resolution (**Figure 6B**) [84], while reducing the high tension worsens the spatial resolution (**Figure 6C**) [85]. These effects can be explained thinking that a low electron dose will lower the electron density of the beam and the overall number of *extrinsic scattering* events, while, on the other hand, a lower tension will induce a stronger scattering effect between the electron beam and the atoms of the sample and the gas molecules, the latter resulting in a worsening of the signal‐to‐noise ratio [86]. Therefore, a proper evaluation of the best setting of the microscope according to the type of material, gas phase, pressure, and chemical reaction to be studied is mandatory before starting any experiment of environmental (S)TEM.

tuning and monitoring procedures [71] but no modification is necessary with regard to the holder and any type of tilting and heating TEM holders can be used to perform *in situ*

The main advantage of the differential pumping is that it is an *open* system, meaning that the electron scattering comes only from the sample and the gas molecules, while no containment window is needed, although it is also worth noting that this architecture can reach only limited pressure values (around 20 Torr) without jeopardizing the TEM and electron gun [81]. However, this solution comes with a cost, i.e., the blocking of electrons scattered at high angle by the lower series of differential apertures below the sample [67]. This constraint imposes a strong limitation to the capabilities of different analytical techniques, e.g., STEM imaging by annular dark field (ADF) and high angle annular dark field (HAADF) detectors, electron diffraction of patterns collected at high angle [82, 83], and EELS, since the range of collection semiangle of the spectrometer cannot exceed the angular value of the lower E‐Cell and differentially pumped apertures. Moreover, the introduction of a differentially pumped

In addition, the imaging resolution is not only affected by the presence of gases, but it is also influenced by additional electro‐optical and microscope‐related parameters with opposite effects, i.e., the electron dose and the primary electron energy. In fact, under the same condi‐ tions of E‐Cell gas pressure and primary high tension, reducing the electron‐dose increases

**Figure 6.** (A) Schema of a differentially pumped TEM system after [71]; (B) sequence of HRTEM images and the corre‐ sponding diffractograms of an Si 011 crystal lattice measured under 4 mbar of N2 gas pressure and different electron doses. Adapted with permission from [84]; (C) HRTEM and the corresponding power spectrum of an Au film on car‐ bon film, showing the pressure dependence of the transmitted information limit under different conditions of accelera‐ tion tension and N2 gas pressure. The white circle corresponds to 10 nm−1. Adapted with permission from [85].

system results in a more complex and more expensive system.

experiments.

240 Microscopy and Analysis

In general, the combination of *open* architecture and limited *extrinsic scattering* contributions due to the use of low working pressures make the open E‐Cell suitable for chemical investi‐ gations of materials through EELS analysis. For these reasons, many recent basic and appli‐ cative studies of catalytic materials took advantage of this combination to study the structural and textural modifications of NPs exposed to reactive gas, following their change of valence state via EELS near‐edge fine structure analysis.

In particular, studying the modifications occurring to catalysts in different temperature and pressure regimes is a focal point for assessing the best working conditions and comprehending variations in catalytic performance. This is the case of cuprous oxide (Cu2O) NPs, which represents an active photocatalyst for hydrogen production by water splitting through direct irradiation with visible light (*λ* > 400 nm) [87]. Studying the variations in the oxidation state of copper on Cu2O nanocubes under water vapor (5 mbar) and light exposure (*λ* = 405 nm) (**Figure 7A**) by EELS near‐edge structure analysis of the Cu L2,3 edge highlighted a variation in the near‐edge features throughout the photocatalytic reaction. In fact, the two strong L2 and L3 ionization edges—"white lines"—showed by oxidized copper (Cu+ , Cu2+) at 951 and 931 eV, respectively, were substituted by one step‐like abrupt onset of L3 edge followed by weak features, which is typical of metallic copper (Cu0 ) and revealed that the photocatalytic reaction also led to a self‐degradation process of the NPs to metallic Cu. Another example is offered by the study on the evolution of Ni NPs during oxidation [88]. In fact, Ni is a viable catalyst for a wide range of applications, including energy production in solid fuel cells, but its oxidation leads to a lessening of its catalytic performance. Therefore, analyzing in detail by *in situ* E‐TEM the structural and chemical transformations induced by oxidation to crystallites represents a first step in understanding and, possibly, control this process. In particular, the comparison of structural and chemical data showed that the oxidation process progressed as the pervasive formation of a thin NiO layer covering the Ni NPs, and subsequently growing at the expense of the Ni component by increasing the temperature, until the final evolution into porous, polycrystalline NiO structures, which are responsible for the worsened catalytic performance. Xin used a similar approach to monitor the transformation of nanoporous CoOx/silica nano‐ composites under H2 flux at different temperatures [89]. In fact, while the chemical analysis of the Co L2,3 edge of EELS signal exhibits a clear fingerprint of reduction of Co valence from Co2+ to Co0 during the exposure to H2 at high temperatures, the *in situ* TEM showed a coars‐ ening of the composites, reflected by an increase in size of the Co component at the expense of the composites porosity. This evidence translated in the quantification of a structural trend from a porous nanosized network to solid NPs, which can be used to tailor the catalyst features in its actual working conditions (**Figure 7B**).

**Figure 7.** (A) HAADF STEM images of a Cu2O nanocube before (left) and 30 min after (right) the photocatalytic reac‐ tion. The corresponding EEL spectra of the peripheral region (below) show the coexistence of metallic Cu portions (marker 1) in the Cu2O nanocube (Marker 2). Adapted with permission from [87]; (B) *in situ* observation of structural changes of Co/CoO*x* NPs under different reduction conditions and temperatures. Adapted with permission from [89]; (C) representative TEM image of Ru/SiO2 NPs (left) and background subtracted EEL spectra acquired at different tem‐ peratures during the oxidation reaction of CO (right). Adapted with permission from [91].

On the other hand, E‐TEM is also a powerful tool for direct observation in more applicative‐ inclined studies, devoted to test and observe the performance and limits of materials with more immediate practical repercussions. Such is the case of the study on yttria‐stabilized zirconia (YSZ) NPs as a treatment device for soot exhaust products by their oxygen channeling capability [90]. *In situ* E‐TEM experiments, conducted under high temperature conditions with a 3 mbar pressure of O2 on a tight mix of YSZ and soot, offered direct evidence of the role of YSZ. In fact, the direct observation showed how the oxidation front of soot was indeed at the surface of the NPs, where the O2− is made available, while soot particles not in contact with YSZ remained unaffected despite being surrounded by gaseous oxygen, thus confirming how the oxidation of carbon soot to CO2 was due to the capability of YSZ of conducting O2− ions toward the NPs surface and making them available for oxidation. Similarly, Chenna et Crozier [91] were able to detect and quantify the catalytic process in the E‐TEM by EELS, showing the feasibility of an *Operando TEM* approach for simultaneous structural and reactivity studies. To achieve this goal, they investigated the oxidation effects of Ru/SiO2 nanocomposites on CO gas. Taking advantage of the high catalytic performance of Ru/SiO2 and of the variation of C π\* peak, going from 286.4 eV for CO to 289.7 eV for CO2, they heated the sample and collected the EELS spectra during the oxidation of CO to CO2. By monitoring the appearance and growth of the C π\* peak of CO2 and the variation of its intensity with respect to the C π\* peak of CO, it was possible to confirm that the oxidation reaction was taking place to observe temperature‐ dependent variations in the oxidation (**Figure 7C**), thus showing the feasibility and power of the *Operando* approach in E‐TEM.

#### **3.2. The window‐closed E‐cell**

from a porous nanosized network to solid NPs, which can be used to tailor the catalyst features

**Figure 7.** (A) HAADF STEM images of a Cu2O nanocube before (left) and 30 min after (right) the photocatalytic reac‐ tion. The corresponding EEL spectra of the peripheral region (below) show the coexistence of metallic Cu portions (marker 1) in the Cu2O nanocube (Marker 2). Adapted with permission from [87]; (B) *in situ* observation of structural changes of Co/CoO*x* NPs under different reduction conditions and temperatures. Adapted with permission from [89]; (C) representative TEM image of Ru/SiO2 NPs (left) and background subtracted EEL spectra acquired at different tem‐

On the other hand, E‐TEM is also a powerful tool for direct observation in more applicative‐ inclined studies, devoted to test and observe the performance and limits of materials with more immediate practical repercussions. Such is the case of the study on yttria‐stabilized zirconia (YSZ) NPs as a treatment device for soot exhaust products by their oxygen channeling capability [90]. *In situ* E‐TEM experiments, conducted under high temperature conditions with a 3 mbar pressure of O2 on a tight mix of YSZ and soot, offered direct evidence of the role of YSZ. In fact, the direct observation showed how the oxidation front of soot was indeed at the surface of the NPs, where the O2− is made available, while soot particles not in contact with YSZ remained unaffected despite being surrounded by gaseous oxygen, thus confirming how the oxidation of carbon soot to CO2 was due to the capability of YSZ of conducting O2− ions toward the NPs surface and making them available for oxidation. Similarly, Chenna et Crozier [91] were able to detect and quantify the catalytic process in the E‐TEM by EELS, showing the feasibility of an *Operando TEM* approach for simultaneous structural and reactivity studies. To achieve this goal, they investigated the oxidation effects of Ru/SiO2 nanocomposites on CO gas. Taking advantage of the high catalytic performance of Ru/SiO2 and of the variation of C π\* peak, going from 286.4 eV for CO to 289.7 eV for CO2, they heated the sample and collected the EELS spectra during the oxidation of CO to CO2. By monitoring the appearance and growth of the C π\* peak of CO2 and the variation of its intensity with respect to the C π\* peak of CO, it was possible to confirm that the oxidation reaction was taking place to observe temperature‐ dependent variations in the oxidation (**Figure 7C**), thus showing the feasibility and power of

peratures during the oxidation reaction of CO (right). Adapted with permission from [91].

in its actual working conditions (**Figure 7B**).

242 Microscopy and Analysis

the *Operando* approach in E‐TEM.

The window‐closed cell is the main alternative to the differentially pumped system for environmental TEM analysis. In this case, the E‐cell is incorporated into the tip of a dedicated TEM holder and uses two parallel electron‐transparent windows to confine the sample and the gases inside a tiny‐volume nanoreactor (**Figure 8A**), while no modification to the TEM column is required. By reducing the volume to a small, closed layer around the specimen, the gaseous phases can reach pressures above one atmosphere [92–94], while the nanoreactor can be safely inserted in the TEM column. The main advantage, other than the higher working pressures, is that this setup can be installed in different TEMs without any modification to the column and vacuum system with a direct saving of costs of purchasing and maintenance.

The confinement diaphragms of the window‐closed cell must have some fundamental prerequisites. Namely, they must (i) be electron-transparent, (ii) be strong enough to confine the pressurized gas, and (iii) provide a low diffraction contribution in order to reduce any effects of diffraction contrast and limit the superposition of additional periodic patterns to the "proper" signals coming from the sample [95]. These are the reasons why the diaphragms are made of thin and amorphous materials with low average atomic number (e.g., carbon, silicon nitride), thus also minimizing their *extrinsic scattering* contribution [95]. However, the addi‐ tional contributions of diaphragms to the *extrinsic scattering* can increase the energy spread of primary electrons, resulting in a worsening of spatial resolution of both the microscope [75] and EELS capabilities because of a stronger damping effect on contrast transfer function and a worse energy spread of the beam entering the specimen, respectively. In a similar fashion, the escape of any X‐ray signals generated during the scattering events between primary electrons and sample atoms will be partially or totally absorbed by the diaphragms [92]. The "closed" nature of this setup does not require any additional apertures in the lower part of objective lens and column, thus making available to the detectors the forwarded signals coming from electrons scattered at high angle and removing some of the limitations encoun‐ tered in the differentially pumped systems. However, the gas path length between the windows is still a crucial parameter: the spatial resolution displays an overall worsening under high‐pressure conditions (1 atm) in nanoreactors with thin windows but longer gas paths (more than 1 mm), due to the dramatic increase in the number of *extrinsic scattering* events [96], akin to the differentially pumped systems.

Also, in this configuration the gas pressure and the temperature inside the cell can be moni‐ tored and varied continuously, up to the instrumental limit. The holder is generally connected to an external gas line equipped with gas reservoir tanks, unreactive pipelines, and manometer pressure gauges capable to control the pressure from few mbars to few bars, while a dedicated vacuum pump is used for purging operations [93].

In this regard, the introduction of micromechanical system (MEMS) technology led to impor‐ tant improvements in the mechanical characteristics, design, and performance of window materials and in the engineering of holders with respect to previously available setups (**Figure 8B**) [92, 94, 97]. The last generations of closed cell nanoreactors, obtained via MEMS technology, integrate electron‐transparent diaphragms made of 10 nm thick amorphous silicon nitride, flow gas pipes of a few tens of micrometer wide, micropressure controlling gauges, and microheaters [98, 99]. Usually, the heater system is a metallic spiral strip (e.g., underdoped Pt or Pt‐based alloys) embedded on the lower thin window and the gas path between the windows is in the order of some tens of micrometers, which allows a reduction in the volume of the cell. Moreover, the spiral shape of the heater helps to minimize any spurious magnetic fields interfering with those of the electromagnetic lenses [98] and any thermal gradient upon the sample. The combination of these parameters makes MEMS‐based nanoreactors suitable for HRTEM and HRSTEM imaging in Cs‐corrected microscopes even at atomic scale, as demonstrated, e.g., by the study of Allard et al. [93] on catalytic nanomaterials, which exhibited atomic columns resolution under vacuum in Au NPs grown on Fe2O3 and in Rh nanocrystals on CaTiO3 under pressures up to 1 atm. Ultimately, the improvement in performance over a wide range of working pressures and temperatures and the adaptability to different (S)TEMs without any modification of columns and vacuum systems provide the window‐closed cells a high experimental flexibility, which makes them valuable in a variety of scientific studies devoted to the *in situ* observation of chemical reactions. Obviously, given the novelty of the most recent developments of the E‐TEM technique, different technical approaches are being explored. While third‐party solutions are already available in the market, many groups are also developing their own custom‐built setups to perform environmental TEM experiments.

**Figure 8.** (A) Schema of TEM holder with window‐closed cell; (B) MEMS‐based nanoreactor. Top: Schematic lateral cross section with the electrical contact for the heater; middle: schematic 3D view of the MEMS nanoreactor; bottom: active area, featuring the microheater spiral (in white) with ovaloid electron‐transparent windows and circular spacers. Adapted with permission from [92].

In this context, Sun et al. adapted a wet cell they previously developed into a closed‐cell setup for *in situ* E‐TEM experiments devoted to observe the oxidation of Ag NPs in air [100]. Due to its ionizing effects on molecular oxygen, the electron beam was used to obtain atomic and ionic oxygen inside the E‐Cell, which acted as ionizing agents for Ag. Therefore, variations in the electron beam current intensity were reflected in the oxidation reactions products: at first increases in current density were revealed by the progressive decrease in Ag NPs size accompanied by the nucleation of new Ag and silver oxide grains; further increases in current density resulted in the progressive predominance of oxide over metallic silver with the heating effects of the electron beam having an increasingly prominent role. The melting of crystalline silver oxide due to beam heating led to the formation of a noncrystalline, highly dynamic vapor phase, which swept through the E‐Cell either aligning to nucleate clusters of Ag NPs or dissolving NPs (**Figure 9A**).

and microheaters [98, 99]. Usually, the heater system is a metallic spiral strip (e.g., underdoped Pt or Pt‐based alloys) embedded on the lower thin window and the gas path between the windows is in the order of some tens of micrometers, which allows a reduction in the volume of the cell. Moreover, the spiral shape of the heater helps to minimize any spurious magnetic fields interfering with those of the electromagnetic lenses [98] and any thermal gradient upon the sample. The combination of these parameters makes MEMS‐based nanoreactors suitable for HRTEM and HRSTEM imaging in Cs‐corrected microscopes even at atomic scale, as demonstrated, e.g., by the study of Allard et al. [93] on catalytic nanomaterials, which exhibited atomic columns resolution under vacuum in Au NPs grown on Fe2O3 and in Rh nanocrystals on CaTiO3 under pressures up to 1 atm. Ultimately, the improvement in performance over a wide range of working pressures and temperatures and the adaptability to different (S)TEMs without any modification of columns and vacuum systems provide the window‐closed cells a high experimental flexibility, which makes them valuable in a variety of scientific studies devoted to the *in situ* observation of chemical reactions. Obviously, given the novelty of the most recent developments of the E‐TEM technique, different technical approaches are being explored. While third‐party solutions are already available in the market, many groups are also developing their own custom‐built setups to perform environmental TEM experiments.

**Figure 8.** (A) Schema of TEM holder with window‐closed cell; (B) MEMS‐based nanoreactor. Top: Schematic lateral cross section with the electrical contact for the heater; middle: schematic 3D view of the MEMS nanoreactor; bottom: active area, featuring the microheater spiral (in white) with ovaloid electron‐transparent windows and circular spacers.

In this context, Sun et al. adapted a wet cell they previously developed into a closed‐cell setup for *in situ* E‐TEM experiments devoted to observe the oxidation of Ag NPs in air [100]. Due to its ionizing effects on molecular oxygen, the electron beam was used to obtain atomic and ionic oxygen inside the E‐Cell, which acted as ionizing agents for Ag. Therefore, variations in the electron beam current intensity were reflected in the oxidation reactions products: at first increases in current density were revealed by the progressive decrease in Ag NPs size

Adapted with permission from [92].

244 Microscopy and Analysis

More recently, Vendelbo et al. [97] used a MEMS‐based nanoreactor to study *in situ* the oscillatory variations in the structure of catalytic Pt NPs during CO oxidation. In fact, while the oscillatory behavior of catalytic reactions under stable conditions was already known, the role of nanosized catalysts was still unclear. Experimental data were acquired while 1.0 bar of CO/O2/He was flowing through the E‐Cell containing Pt NPs at a steady temperature of 659 K. The catalytic performance of the NPs was verified by measuring variations in mass spectrometry of the CO and CO2 phases exiting the nanoreactor, while time‐resolved HRTEM imaging recorded variations in the structure of the Pt NPs. Finally, calorimetric information about the chemical reaction was extracted by recording fluctuations in the electrical power consumption of the MEMS nanoreactor due to the heat released by the CO oxidation. By correlating the reaction data with the variations in morphology observed on the catalytic NPs, it was observed that a periodic and reversible faceting of Pt between a spherical and a facetted shape is indeed the cause of the periodic oscillations in these catalytic reactions (**Figure 9B**). In a similar fashion, the *in situ* study performed by Zhang et al. [101] on the evolution of Si‐ supported Pd@CeO2 nanocatalysts revealed the presence of dynamical processes that altered drastically the structure of the nanocomposites and that could only be observed by closed cell *in situ* E‐TEM. In fact, while the performance of Pd@CeO2 was usually attributed to the formation of a core‐shell structure, *in situ* calcination at 500°C in a 150 Torr O2 atmosphere

**Figure 9.** (A) Metallic evolution of Ag NPs in an air‐filled cell after exposure. Top, from left to right: after 0 s with 0.18 A cm2 current density; after 20 s with 0.18 A cm2 current density (a new grain is indicated by a white arrow); after 0 s with 0.44 A cm2 current density; after 20 s with 0.44 A cm2 current density. Bottom, from left to right: after 0 s with 0.66 A cm2 current density; after 20 s with 0.66 A cm2 current density; after 20 s with 0.72 A cm2 current density; after 20 s with 0.8 A cm2 current density (the black arrow marks the vertical alignment of the AgO vapor phase). Adapted with permission from [100]; (B) time‐resolved TEM images of a Pt nanoparticle at the gas exit of the reaction zone. The elapsed time is indicated in the top left corner. Adapted with permission from [97]; (C) (top) disappearance of an atom cloud and growth of a nearby particle, with subsequent coalescence at 650°C in 150 Torr O2. The atomic cloud is delim‐ ited by a dashed line; (bottom) appearance of subnanometric features. The elapsed time indicated in minutes is at the top right corner of each image. Adapted with permission from [101].

revealed the dissociation of the Pd and CeO2 particles to atomic clouds. Subsequent calcina‐ tions lead to re‐organization of the atomic species into bigger sized ceria and Pd NPs or to the nucleation of a new structure featuring very small, highly dispersed silica, Pd, and ceria (**Figure 9C**). Assuming a conventional catalytic behavior of the bigger particles, the exceptional catalytical performance of Pd@CeO2 above 800°C was attributed to this new structure.

## **4.** *In situ* **(S)TEM imaging of liquid specimens**

As mentioned in the Section 1, since the invention of the electron microscope, one of the main scientific challenges has always been the imaging of liquid or wet samples. Even if this is usually possible by using light/fluorescence/confocal microscopy, and even taking into account the most recent and dramatic resolution improvements achieved due to the super‐ resolution approaches [102], its resolution (currently less than 100 nm) is still far from that of any electron microscope and represents a strong limit for nanoscale analyses of liquid samples. That being said, this section deals with the methods and devices that allow to achieve much higher resolution of liquid/wet samples performing *in situ* TEM instead of light‐microscopy‐ based imaging. Analogously to what described in the previous section, also here the term "E‐ Cell" will be used to simply define the volume in which the interaction between the liquid environment and the specimen of interest occurs, irrespective of the device used to allow this interaction to happen.

A brief historical overview looks helpful in understanding what were, since the first decades of electron microscopy, the main limitations related to the possibility to perform *in situ* TEM of specimens kept in a liquid environment. First, the liquid has to be isolated from the rest of the column, which being under vacuum conditions would give rise to an almost immediate liquid evaporation, unless it would have a very low vapor pressure, and to the concomitant loss of the high vacuum conditions. The first trials to realize an *in situ* specimen holder capable to locally isolate the liquid in an E‐Cell was proposed in the works of Abrams and McBain [103, 104]. It can be basically described as a platinum cell containing the liquid sample and consti‐ tuted by two parts that were glued together, each provided with a very small hole where a window was attached, the latter thin enough to allow the electron beam to pass through them and the internal part that contained the liquid. This kind of E‐Cell was just put in a normal TEM holder instead of the grid. The material used to fabricate the 50 nm thin electron‐ transparent windows was called collodion, and it was basically constituted by nitrocellulose. In none of the two papers of Abrams and McBain is reported any EM image of a liquid sample (gold particles in water) imaged by using this *in situ* specimen holder, and the authors affirmed that this was due to the particles' too rapid‐free Brownian movement in a liquid as fluid as water. It was needed waiting almost 30 years to read a further paper [105] in which Ernest Fullam showed a novel way to fabricate a wet E‐Cell for TEM. The design of this latter cell basically followed that of Abrams and McBain, but Fullam indicated that one of the most important failure of the colleagues' previous cell was due to the material chosen to fabricate the thin windows: the nitrocellulose films were invariably broken by the pressure difference between inside and outside the E‐Cell, once it was inserted in the TEM column. In fact, the most important novelty shown by Fullam was the manner followed to fabricate the thin and electron‐transparent windows, which were now constituted by a triple‐layer film: Formvar and nitrocellulose separately cast from fresh solutions and deposited on 400 mesh copper TEM grids. Fullam estimated this configuration having a total thickness ranging from 30 to 40 nm and showed that it did not suddenly ruptured, allowing to image chromium hydroxide spheres in water suspension even if, despite the low magnification (5k) used, still in such a case the loss in spatial resolution due to the water electron scattering as well as once again to the Brownian movement of the particles suspended in water is clearly reported. To limit both these effects, only a possible solution was indicated: making the liquid thickness contained by the E‐Cell as low as possible.

revealed the dissociation of the Pd and CeO2 particles to atomic clouds. Subsequent calcina‐ tions lead to re‐organization of the atomic species into bigger sized ceria and Pd NPs or to the nucleation of a new structure featuring very small, highly dispersed silica, Pd, and ceria (**Figure 9C**). Assuming a conventional catalytic behavior of the bigger particles, the exceptional catalytical performance of Pd@CeO2 above 800°C was attributed to this new structure.

As mentioned in the Section 1, since the invention of the electron microscope, one of the main scientific challenges has always been the imaging of liquid or wet samples. Even if this is usually possible by using light/fluorescence/confocal microscopy, and even taking into account the most recent and dramatic resolution improvements achieved due to the super‐ resolution approaches [102], its resolution (currently less than 100 nm) is still far from that of any electron microscope and represents a strong limit for nanoscale analyses of liquid samples. That being said, this section deals with the methods and devices that allow to achieve much higher resolution of liquid/wet samples performing *in situ* TEM instead of light‐microscopy‐ based imaging. Analogously to what described in the previous section, also here the term "E‐ Cell" will be used to simply define the volume in which the interaction between the liquid environment and the specimen of interest occurs, irrespective of the device used to allow this

A brief historical overview looks helpful in understanding what were, since the first decades of electron microscopy, the main limitations related to the possibility to perform *in situ* TEM of specimens kept in a liquid environment. First, the liquid has to be isolated from the rest of the column, which being under vacuum conditions would give rise to an almost immediate liquid evaporation, unless it would have a very low vapor pressure, and to the concomitant loss of the high vacuum conditions. The first trials to realize an *in situ* specimen holder capable to locally isolate the liquid in an E‐Cell was proposed in the works of Abrams and McBain [103, 104]. It can be basically described as a platinum cell containing the liquid sample and consti‐ tuted by two parts that were glued together, each provided with a very small hole where a window was attached, the latter thin enough to allow the electron beam to pass through them and the internal part that contained the liquid. This kind of E‐Cell was just put in a normal TEM holder instead of the grid. The material used to fabricate the 50 nm thin electron‐ transparent windows was called collodion, and it was basically constituted by nitrocellulose. In none of the two papers of Abrams and McBain is reported any EM image of a liquid sample (gold particles in water) imaged by using this *in situ* specimen holder, and the authors affirmed that this was due to the particles' too rapid‐free Brownian movement in a liquid as fluid as water. It was needed waiting almost 30 years to read a further paper [105] in which Ernest Fullam showed a novel way to fabricate a wet E‐Cell for TEM. The design of this latter cell basically followed that of Abrams and McBain, but Fullam indicated that one of the most important failure of the colleagues' previous cell was due to the material chosen to fabricate the thin windows: the nitrocellulose films were invariably broken by the pressure difference between inside and outside the E‐Cell, once it was inserted in the TEM column. In fact, the

**4.** *In situ* **(S)TEM imaging of liquid specimens**

interaction to happen.

246 Microscopy and Analysis

A further and strong novelty was then proposed in the work of Parsons [66], who described a new kind of experimental design to perform *in situ* TEM of liquid samples. It is the birth of the so‐called environmental TEM (E‐TEM) that consisted, and still consists, in a completely different type of E‐Cell inside the column, the details of which have been already described in the previous section. Basically, and to remind its working principle, this kind of E‐Cell is no longer physically limited by two thin electron‐transparent windows and then just inserted into the TEM column as a part of a special specimen holder, but it is itself a part of the TEM column, where the ability to kept it under low gaseous pressure conditions is given by a system constituted by two differentially pumped and apertures' limited volumes put just above and below the E‐TEM cell. Parson indicated that in the E‐TEM cell he proposed a water vapor pressure of around 0.03 bar was enough to establish and keep a wet compartment. However, by using such a configuration he also mentioned that a maximum resolution of about 4.0 nm in the presence of water vapor pressure could be attainable, with the microscope operating at an acceleration voltage of 200 kV, and showed an E‐TEM image of unstained and unfixed coliform bacteria and intracytoplasmic organelles, even if not highly resolved.

A variation of E‐TEM is that shown by Gai in [106], where both environmental and high‐ resolution TEM (EHRTEM) were shown with a double possible configuration: first, a specific microreactor inserted inside the E‐Cell of the EHRTEM column enables studies of thermally driven gas‐solid reactions, while keeping an atomic resolution. Similarly, a way to develop *in situ* TEM studies of wet samples that could be even heated was indicated. It consisted in inserting a dedicated specimen holder in the E‐Cell, capable to inject liquids onto the sample, located in the furnace of the holder.

However, what was mainly developed in the following years and so far to perform *in situ* TEM imaging is mainly based on sample holders with the isolating double thin window systems. This is not only due to the not secondary point that purchasing an E‐TEM is obviously much more expensive than acquiring a dedicated sample holder to be inserted into a conventional S(TEM). However, it was actually shown that *in situ* E‐TEM imaging of liquid/wet specimen unavoidably brings to work with a liquid film thickness not less than 1 μm and whose fine control is very hardy achievable as a consequence of a high and systematic loss in the final point resolution.

More recently, double window‐based E‐Cells for liquids were then and again used for *in situ* specimen holders' fabrication, being adaptable to any standard. The sample of interest can be sealed inside the double thin‐window E‐Cell together with gases or liquids, the latter possibly also let to flow. Although the E‐Cell thin windows' presence unavoidably makes a little worse the final resolution, by such a way the sample can be surrounded by a thin gaseous or liquid environmental layer, with a thickness that can be *a priori* chosen within a large range, providing more or less resolution depending on both the chosen TEM imaging geometries and the total thickness of both sealing windows and interposed sample [107, 108].

The major improvement achieved for the development of specimen holders capable to allow *in situ* (S)TEM imaging of liquid samples was the fabrication of devices with robust but thin windows for the relevant E‐Cells. These were realized producing thick microchips of amor‐ phous silicon nitride (SiN) with a central zone having a thin or a very thin thickness (20–100 nm). In addition, further specific advances in the microchip fabrication allowed the integration of electrodes and microfluidic circuits to mimic physiological and/or dynamic conditions in the E‐Cell for more specific *in situ* (S)TEM imaging such as, just for instance, the one of proteins or NPs interactions with whole cells [107, 109, 110], electrochemical reactions [111, 112], nanocrystals' growth in liquid solution [113, 114].

In the following, we first deal with the equations needed to determine the final point resolution when an *in situ* sample holder containing a water layer of given thickness is used in both TEM and STEM geometries since each of them brings to the optimal way to prepare the specimen that should be looked at. Second, it will continue and end with some examples of *in situ* (S)TEM imaging for both materials and biological science studies.

#### **4.1. The point resolution when using an** *in situ* **sample holder for liquids**

#### *4.1.1. TEM*

To determine how to calculate the final point resolution when using an *in situ* sample holder for liquids, a first approximation could be adopted. It consists in considering the material constituting the E‐Cell windows with a scattering factor negligible for typical beam energies (200–300 keV), with respect to that of the liquid layer contained in the E‐Cell, which is valid for most of the cases. Under such a condition, the main resolution's limiting factor is the chromatic aberration due to the inelastic scattering of electrons by the liquid. Taking then into account the most diffused and simple case of water as liquid medium, the electron energy broadening due to the beam‐water inelastic scattering and the fact that chromatic aberration is the one effectively governing the final resolution, then *d*TEM could be expressed by the following equation:

$$d\_{\rm TEM} = 6 \times 10^{12} \left(\frac{\alpha C\_{\rm C} T}{E^2}\right) \tag{2}$$

where *CC* is the spherical aberration of the objective lens, usually ranging between 0.5 and 2 mm depending on the microscope used, *α* is the objective semi angle, *E* is the beam energy, and *T* is the whole thickness of the cell filled by water. The water features (density, mean atomic number, and atomic weight) have been taken into account in the numerical coefficient appearing in the equation. Just for instance, with *E* = 200 keV, *α* = 10 mrad, *CC* = 2mm, and *T* =1 μm, a final resolution *d*TEM of 4 nm is calculated [108].

Thus, taking into account what indicated by Eq. (2) to look at any liquid sample (i.e., at a solid sample surrounded by a liquid environment) by TEM using the parallel electron beam geometry, the highest attainable resolution is obtained for solid objects surrounded by liquid in a configuration where electrons have to pass the lowest possible liquid volume after having crossed the solid objects, in order to minimize the energy broadening that they suffer in scattering with the liquid's molecules. For this reason, the solid part of the sample should be put onto the internal side of that E‐Cell windows from which the electrons definitely exit from the E‐Cell or, analogously, if the solid objects are homogeneously distributed all over the liquid the best choice to image them by TEM is that to focalize the ones closest to the lower E‐Cell's window. In other words, to obtain the best resolution, the objective lens focal plane should be placed as near as possible to the internal side of the lower E‐Cell's window. **Figure 10A** shows this experimental configuration.

**Figure 10.** Different possible configurations for electron microscopy in liquid. (A) TEM imaging of nanoparticles in a liquid fully enclosed between two electron‐transparent windows. Adapted with permission from [108]. (B) STEM imaging in a fully enclosed light liquid, used to image nanoparticle labels on whole biological cells. Adapted with per‐ mission from [108].

#### *4.1.2. STEM*

sealed inside the double thin‐window E‐Cell together with gases or liquids, the latter possibly also let to flow. Although the E‐Cell thin windows' presence unavoidably makes a little worse the final resolution, by such a way the sample can be surrounded by a thin gaseous or liquid environmental layer, with a thickness that can be *a priori* chosen within a large range, providing more or less resolution depending on both the chosen TEM imaging geometries and the total

The major improvement achieved for the development of specimen holders capable to allow *in situ* (S)TEM imaging of liquid samples was the fabrication of devices with robust but thin windows for the relevant E‐Cells. These were realized producing thick microchips of amor‐ phous silicon nitride (SiN) with a central zone having a thin or a very thin thickness (20–100 nm). In addition, further specific advances in the microchip fabrication allowed the integration of electrodes and microfluidic circuits to mimic physiological and/or dynamic conditions in the E‐Cell for more specific *in situ* (S)TEM imaging such as, just for instance, the one of proteins or NPs interactions with whole cells [107, 109, 110], electrochemical reactions [111, 112],

In the following, we first deal with the equations needed to determine the final point resolution when an *in situ* sample holder containing a water layer of given thickness is used in both TEM and STEM geometries since each of them brings to the optimal way to prepare the specimen that should be looked at. Second, it will continue and end with some examples of *in situ* (S)TEM

To determine how to calculate the final point resolution when using an *in situ* sample holder for liquids, a first approximation could be adopted. It consists in considering the material constituting the E‐Cell windows with a scattering factor negligible for typical beam energies (200–300 keV), with respect to that of the liquid layer contained in the E‐Cell, which is valid for most of the cases. Under such a condition, the main resolution's limiting factor is the chromatic aberration due to the inelastic scattering of electrons by the liquid. Taking then into account the most diffused and simple case of water as liquid medium, the electron energy broadening due to the beam‐water inelastic scattering and the fact that chromatic aberration is the one effectively governing the final resolution, then *d*TEM could be expressed by the

12 C

where *CC* is the spherical aberration of the objective lens, usually ranging between 0.5 and 2 mm depending on the microscope used, *α* is the objective semi angle, *E* is the beam energy, and *T* is the whole thickness of the cell filled by water. The water features (density, mean atomic

*E* æ ö a= ´ ç ÷

è ø (2)

TEM 2 6 10 *C T d*

thickness of both sealing windows and interposed sample [107, 108].

nanocrystals' growth in liquid solution [113, 114].

*4.1.1. TEM*

248 Microscopy and Analysis

following equation:

imaging for both materials and biological science studies.

**4.1. The point resolution when using an** *in situ* **sample holder for liquids**

Differently from the TEM geometry, in which a parallel electron beam is used to illuminate whole areas of the sample, in the STEM the imaging is performed by a convergent beam that scans the sample point by point and where the contrast is then obtained by collecting the electron signal produced by each irradiated point with an appropriate detector. Even consid‐ ering that the transmitted beam is the simplest way to form an image (the so‐called Bright field mode), to image the contrast due to small but atomically heavy objects (like NPs) dispersed in a lighter liquid medium, the signal emitted with an high angle of divergence is usually the one collected to form the STEM image, being this STEM detection's geometry called HAADF. In such a case, the contrast is due to the fact that the detected signal's intensity is roughly proportional to *Z*2 , with *Z* being the atomic number of the material irradiated by the electron beam. Under these experimental conditions, the STEM resolution (*d*STEM) achievable for a NPs' sample surrounded by a liquid environment is usually defined as the diameter of the smallest NP visible above the background noise as given by the following equation:

$$d\_{\rm STEM} = \mathfrak{S} l\_{\rm nano} \sqrt{\frac{T}{N\_0 l\_{\rm liquid}}} \tag{3}$$

where *l*nanop and *l*liquid are the mean free path length for elastic scattering of NP and surrounding liquid, respectively, *N*0 is the number of incident electrons, and *T* is the whole thickness of the liquid cell [107, 108].

Thus, the highest resolution for the STEM imaging of an object within a liquid is achieved when the object is at the entrance side of the sample. Moreover, keeping the specimen holder filled with the same water volume, the NP STEM imaging will be performed with a resolution in principle quite higher than the one obtainable by using a TEM imaging.

According to Eq. (3), a gold NP (marker) with a 1.4 nm in diameter can be resolved on a water layer of *T* = 5 μm. For NPs positioned deeper in the liquid, imaging takes place with a blurred probe, but a resolution better than 10 nm is still achieved in the top 1 μm layer of the liquid. As previously illustrated for the TEM, **Figure 10B** shows the experimental configuration corresponding to the STEM geometry with an *in situ* sealed E‐Cell for liquid specimens.

#### **4.2.** *In situ* **liquid TEM for materials science studies**

Just as short overview of the different fields of application of *in situ* (S)TEM of liquid sample and devices, in the following some quite known examples are reported. One of the important papers that started to show the terrific possibilities offered by this technique was that of Williamson et al. [111], while a quite similar study was also published by Radisic et al. [112]. Both works deal with electrochemical processes monitored over time by providing a sealed electrochemical E‐Cell well fitted for both vacuum environment and restricted volume available in the TEM column. The liquid E‐Cell is filled with a water‐based solution (usually electrolytes) and also contains electrodes as shown in **Figure 11A**. Both papers showed time‐ resolved imaging of electrochemically driven process of nucleation and following growth of individual Cu clusters onto an electron‐transparent Au electrode patterned over one of the windows of an E‐TEM liquid cell, with simultaneous measurement of voltage and current involved. It should be noticed that in both papers the TEM operating mode and the corre‐ sponding beam geometry was chosen, and in fact the Cu nanoclusters' formation is observed when they reach a size of several tens of nanometers (**Figure 11B** and **C**).

a lighter liquid medium, the signal emitted with an high angle of divergence is usually the one collected to form the STEM image, being this STEM detection's geometry called HAADF. In such a case, the contrast is due to the fact that the detected signal's intensity is roughly

beam. Under these experimental conditions, the STEM resolution (*d*STEM) achievable for a NPs' sample surrounded by a liquid environment is usually defined as the diameter of the smallest

where *l*nanop and *l*liquid are the mean free path length for elastic scattering of NP and surrounding liquid, respectively, *N*0 is the number of incident electrons, and *T* is the whole thickness of the

Thus, the highest resolution for the STEM imaging of an object within a liquid is achieved when the object is at the entrance side of the sample. Moreover, keeping the specimen holder filled with the same water volume, the NP STEM imaging will be performed with a resolution

According to Eq. (3), a gold NP (marker) with a 1.4 nm in diameter can be resolved on a water layer of *T* = 5 μm. For NPs positioned deeper in the liquid, imaging takes place with a blurred probe, but a resolution better than 10 nm is still achieved in the top 1 μm layer of the liquid. As previously illustrated for the TEM, **Figure 10B** shows the experimental configuration corresponding to the STEM geometry with an *in situ* sealed E‐Cell for liquid specimens.

Just as short overview of the different fields of application of *in situ* (S)TEM of liquid sample and devices, in the following some quite known examples are reported. One of the important papers that started to show the terrific possibilities offered by this technique was that of Williamson et al. [111], while a quite similar study was also published by Radisic et al. [112]. Both works deal with electrochemical processes monitored over time by providing a sealed electrochemical E‐Cell well fitted for both vacuum environment and restricted volume available in the TEM column. The liquid E‐Cell is filled with a water‐based solution (usually electrolytes) and also contains electrodes as shown in **Figure 11A**. Both papers showed time‐ resolved imaging of electrochemically driven process of nucleation and following growth of individual Cu clusters onto an electron‐transparent Au electrode patterned over one of the windows of an E‐TEM liquid cell, with simultaneous measurement of voltage and current involved. It should be noticed that in both papers the TEM operating mode and the corre‐ sponding beam geometry was chosen, and in fact the Cu nanoclusters' formation is observed

0 liquid

NP visible above the background noise as given by the following equation:

STEM nanop

in principle quite higher than the one obtainable by using a TEM imaging.

when they reach a size of several tens of nanometers (**Figure 11B** and **C**).

**4.2.** *In situ* **liquid TEM for materials science studies**

5 *T d l*

, with *Z* being the atomic number of the material irradiated by the electron

*N l* = (3)

proportional to *Z*2

250 Microscopy and Analysis

liquid cell [107, 108].

**Figure 11.** (A) Sketch of the liquid cells' components. Adapted with permission from [111]. (B) The center‐of‐mass posi‐ tions of Cu clusters during four deposition experiments (different colors) on the same area of the electrode. Inset is the micrograph that corresponds to the black circles. Adapted with permission from [111]. (C) Small regions extracted from a video recorded during deposition at 5 mA cm‐2. Images taken at the times shown after current flow began. Adapted with permission from [111].

In 2009, Zheng et al. again used TEM geometry and a double SiN window *in situ* E‐Cell for liquid specimens to image Pt nanocrystals' growth and coalescence, obtaining a final resolution well lower than 1 nm, mainly due to the low spherical aberration of the high‐resolution objective lens but also to the 200–300 kV accelerating voltage used [113]. For the particle synthesis, a stock solution was prepared by dissolving Pt(acetylacetonate)2 in a mixture of *o*‐ dichlorobenzene and oleylamine. About 100 nanoliters of such a growth solution were put into

**Figure 12.** Growth and coalescence of Pt nanocrystals. (A) Top images: video frames acquired at different time points of exposure to the electron beam are reported, with specific particles labeled by arrows. Bottom image: the number of particles (left axis) and the number of coalescence events (Nc, right axis) vs. time, as measured with a time rate of 2.0 s. Adapted with permission from [113]. (B) Video images showing nanoparticle's growth occurred by monomer addition (left column, a) or growth by means of coalescence (left column, b). Particles are selected from the same field of view. Right columns (a) and (b): color images of the left ones, enlarged 1.5 times. The yellow arrows are indicating recrystal‐ lization observed in the coalesced particle. Adapted with permission from [113]. (C) Top images: histogram of particle size distribution at different time points, with black curves corresponding to Gaussian fittings. Bottom images: particle size vs. growth time of particles highlighted in panel (A). Inset shows two types of growth trajectories: simple growth and growth with coalescence. A relaxation time (*τ*) was observed after a coalescence event. Adapted with permission from [113].

the E‐Cell, where the SiN windows and the liquid layer had a thickness of 25 and 200 nm, respectively. It is crucial to highlight here that the driving force used to promote the nucleation, growth, and coalescence of the Pt NPs was the sole electron beam irradiation. The main results published in the paper of Zheng et al. are summarized in **Figure 12A−C**. They consisted in showing not only the nucleation and growth of the Pt nanoclusters over time in the sealed E‐ Cell, but also in showing the fundamental role played by the coalescence of the larger NPs with the smaller ones during the growth process.

Similarly to what cited above, Evans et al. synthesized PbS NPs using as electrolyte a solution containing lead acetate, poly(vinyl alcohol), isopropyl alcohol, and thioacetamide [114]. The selective decomposition of thioacetamide was again promoted using the illumination with high‐energy electron beam as driving force, and it gave rise to the production of free sulfur ions in solution. Surrounding lead ions then reacted with the free sulfur ions to allow the PbS NPs to nucleate and grow, while the poly(vinyl alcohol) acted as a stabilizing agent. However, two quite important improvements were shown in the work of Evans et al. and consisted in (a) using a continuous flow of reactants, drawn in and out from the E‐Cell by appropriate microfluidic lines and (b) monitoring the processes occurring inside the E‐Cell by using a spherical aberration corrected STEM, with higher resolution than the one attainable using a conventional TEM, for the reasons explained above.

#### **4.3.** *In situ* **liquid TEM for biological sciences studies**

For biological purposes, both E‐TEM and sealed E‐Cells for liquid specimen holders have been used, with a large predominance of the sealed ones. However, a premise is crucial before showing some examples. When using any EM imaging technique, either TEM or SEM based, no living specimen could be directly looked at without destroying it. That is due to the intrinsic conditions needed for any electron microscope to work (high vacuum and radia‐ tion damage due to the electrons‐matter interactions) that are incompatible with the high water content, low density, and very low hardness of any living organism. To look at any living matter *post mortem*, so far a very large series of experimental protocol have been then developed, which allow making it rigid, dehydrated or frozen, and possibly with enough contrast if seen by a transmission geometry. In any case, all preparation protocols unavoid‐ ably require to kill either the cells or the micro‐organisms prior to inserting them in any EM. The *in situ* liquid sample holders are then the only devices that are currently able to allow looking at living matter without killing it, and keeping it into a physiological aqueous environment, even if some effects of the radiation damage have to be taken into account. First, the small temperature rise can alter diffusion and reaction rates via convective motions within the liquid volume. Second, nonthermal effects may be more critical and more complicated to deal with: (a) high‐energy electrons in water give rise to the pretty long‐ lived species' production such as OH radicals and hydrated electrons, even if charged species could quench radicals resulting in the concomitant reduction of their reactivity in solution; (b) substrate chemistry also plays a nonnegligible role since reactive species can be generat‐ ed from interactions between the beam and the substrate, i.e., the thin windows that seal the E‐Cell; (c) organic specimens are known to be intensely affected by a small enough electron dose, which means that for pristine biological materials, structural damage begins to happen at the subnanometer scale when the low electron dose of ∼102 electrons per nm2 is exceed‐ ed. This effect could be limited: (a) considering that the electron dose usually scales quad‐ ratically with the resolution, and then the latter could be appropriately chosen in order to have still good images but low radiation damage; (b) making use of an ultrasensitive CMOS camera, which allows to work using very low electron doses. If higher electron doses are required, the main way to preserve the cell/organism ultrastructure is stabilizing it by a chemical or cryofixation.

the E‐Cell, where the SiN windows and the liquid layer had a thickness of 25 and 200 nm, respectively. It is crucial to highlight here that the driving force used to promote the nucleation, growth, and coalescence of the Pt NPs was the sole electron beam irradiation. The main results published in the paper of Zheng et al. are summarized in **Figure 12A−C**. They consisted in showing not only the nucleation and growth of the Pt nanoclusters over time in the sealed E‐ Cell, but also in showing the fundamental role played by the coalescence of the larger NPs

Similarly to what cited above, Evans et al. synthesized PbS NPs using as electrolyte a solution containing lead acetate, poly(vinyl alcohol), isopropyl alcohol, and thioacetamide [114]. The selective decomposition of thioacetamide was again promoted using the illumination with high‐energy electron beam as driving force, and it gave rise to the production of free sulfur ions in solution. Surrounding lead ions then reacted with the free sulfur ions to allow the PbS NPs to nucleate and grow, while the poly(vinyl alcohol) acted as a stabilizing agent. However, two quite important improvements were shown in the work of Evans et al. and consisted in (a) using a continuous flow of reactants, drawn in and out from the E‐Cell by appropriate microfluidic lines and (b) monitoring the processes occurring inside the E‐Cell by using a spherical aberration corrected STEM, with higher resolution than the one attainable using a

For biological purposes, both E‐TEM and sealed E‐Cells for liquid specimen holders have been used, with a large predominance of the sealed ones. However, a premise is crucial before showing some examples. When using any EM imaging technique, either TEM or SEM based, no living specimen could be directly looked at without destroying it. That is due to the intrinsic conditions needed for any electron microscope to work (high vacuum and radia‐ tion damage due to the electrons‐matter interactions) that are incompatible with the high water content, low density, and very low hardness of any living organism. To look at any living matter *post mortem*, so far a very large series of experimental protocol have been then developed, which allow making it rigid, dehydrated or frozen, and possibly with enough contrast if seen by a transmission geometry. In any case, all preparation protocols unavoid‐ ably require to kill either the cells or the micro‐organisms prior to inserting them in any EM. The *in situ* liquid sample holders are then the only devices that are currently able to allow looking at living matter without killing it, and keeping it into a physiological aqueous environment, even if some effects of the radiation damage have to be taken into account. First, the small temperature rise can alter diffusion and reaction rates via convective motions within the liquid volume. Second, nonthermal effects may be more critical and more complicated to deal with: (a) high‐energy electrons in water give rise to the pretty long‐ lived species' production such as OH radicals and hydrated electrons, even if charged species could quench radicals resulting in the concomitant reduction of their reactivity in solution; (b) substrate chemistry also plays a nonnegligible role since reactive species can be generat‐ ed from interactions between the beam and the substrate, i.e., the thin windows that seal the E‐Cell; (c) organic specimens are known to be intensely affected by a small enough electron

with the smaller ones during the growth process.

252 Microscopy and Analysis

conventional TEM, for the reasons explained above.

**4.3.** *In situ* **liquid TEM for biological sciences studies**

**Figure 13.** (A) Top image: single Si microchip with an electron‐transparent SiN window. The sizes of the microchip are 2.0 × 2.3 × 0.3 117. Bottom image: sketch of a microfluidic chamber constituted by two Si microchips such as the ones presented in the top image and showing the liquid flow direction. Adapted with permission from [107, 108]. (B) Liquid STEM images of COS7 fibroblast cells labeled EGF‐Au. Left image: edge of a fixed COS7 cell after 5 min incubation with EGF‐Au. The labels are visible as bright spots and the cellular material is shown as light‐gray matter on a dark‐ gray background (pixel size: 5.7 nm). Central image: COS7 cell incubated with EGF‐Au for 10 min and then in buffer (without EGF‐Au) for additional 15 min. Circular cluster of labeled EGF receptors are visible. Arrow 1 indicates sharp‐ edged gold labels in the cluster, whereas the labels in the cluster at arrow 2 appear blurred and cannot be distinguish‐ ed as individual entities, indicating that cluster 2 is out of the vertical plane of focus. The observation of circular clusters of labels at different vertical positions in the cell indicates that the gold nanoparticles labels were internalized (pixel size: 5.7 nm). Right image: sample shown in left image, but imaged after the flow cell opening and sample dry‐ ing in air. The dried sample displays much stronger contrast on the cellular material: the white large zones in the im‐ age correspond to the edge of a dried cell, while the gold nanoparticles labels are still visible as bright spots (pixel size: 8.9 nm). Adapted with permission from [107]. (C) Left image: COS7 cell edge labeled with EGF‐Au, where the bright spots indicate the labels (pixel size: 2.9 nm). Central image: magnification of the area defined by the white square in left image showing the individual labels. Right top line profile: HAADF signal as a function of the electron probe posi‐ tion over an individual gold nanoparticle, corresponding to the line labeled "C" in the central image. Right bottom line profile: HAADF signal as detected on a 10 nm gold nanoparticle at the bottom of 1.3 μm thick liquid layer (10% PBS buffer in water) in a test sample without cells (pixel size was 0.91 nm). Adapted with permission from [107].

As first example of study of biological living matter by *in situ* E‐Cell for liquid specimens, we report here what Liu et al. showed in their paper [115]. It reports the detailed fabrication using microelectromechanical system (MEMS) techniques to obtain a novel microchip constituted by two SiO2 films used as a substrate and the 9 nm thin windows for *in situ* imaging of living organisms in aqueous environment using a conventional TEM geometry. Such a kind of microchips—with a gap between them ranging from 2 to 5 μm—allowed the effective TEM observation of living bacteria *Escherichia coli* and the tellurite reduction process in *Klebsiella pneumonia,* the height of which were estimated ranging between 0.5 and 1μm. The most important results of this paper were (a) the capability of bacterium *K. pneumoniae* and the yeast *Saccharomyces cerevisiae* to stay alive onto the microchip substrate under continuous electron beam irradiation needed for the TEM imaging for up to 14 and 42 s, respectively; (b) the possibility to follow over time, even if with not very high resolution as expected from Eq. (2), biological processes such as the tellurite reduction by *K. pneumonia*.

As already mentioned and shown by Eq. (3), the use of STEM allowed to achieve a dramatically increased resolution and to image by using HAADF geometry even very small heavy particles dispersed in a light liquid medium. This was the case of Au‐labeled eukaryotic fibroblast cells (COS7) immersed in a several micrometer‐thick liquid layer [107, 109]. In both the papers a flow liquid E‐Cell was used, allowing the aqueous solution to pour through the E‐Cell and around the whole fibroblast cells. The experimental configuration is reported in **Figure 13A**. The cells were grown, then labeled, and finally fixed prior to be inserted in the flow E‐Cell, on the internal side of the silicon nitride film that was finally placed to seal the upper part of the E‐Cell, in order to maximize the STEM resolution according to Eq. (3). The cells' immunolab‐ eling was realized by using gold NPs sizing 10 nm, in order to target the epidermal growth factor (EGF) molecules bound to the cellular EGF receptors. Thus, using a water layer 10 μm thick, the theoretical resolution limit determined by using Eq. (3) was found to be equal to 1.9 nm. In agreement with that, the 10 nm sized labeling gold NPs were detected with a spatial resolution of about 4 nm, as shown in **Figure 13B** and **C**, using the STEM in HAADF geometries, and performed with a beam accelerated by a voltage of 200 kV and a dwell time of 20 μs. If it is not weird that the electron beam did not perturb the fixed cells, it is however remarkable that for the dose used (7 × 104 electrons per nm2 ) and within the flowing liquid no visible damage consequences were observed on the spatial distribution of the labeling NPs on the cells.

As last and most recent example the results achieved by Pohlmann *et al.* are shown [110]. In their paper they showed an *in situ* TEM imaging, time resolved, of the uptake of typical drug delivery vehicles—in such a case gold nanorods (GNR) encapsulated in polyvinylpyridine (PVP)—from glioblastoma stem cells (GSCs), which actually is one of the most aggressive cancers among brain tumors. This kind of study is quite interesting, allowing to image by TEM the evolution over time of NP uptake from living cells, a phenomenon so far investigated just by light/confocal microscopy. To perform it, the authors first prepared a multiwell E‐Cell with a maximum height of 2 μm where the GSCs were forced to be tethered on the lower side, thanks to a surface functionalization that involved their NOTCH1 receptors, as shown in **Fig‐ ure 14A**. Then, the GSCs contained in the multiwell E‐Cell were incubated together with the PVP‐GNR NRs for 20 and 60 min, respectively, prior to inserting the sealed E‐Cell into the TEM. The evolution of the PVP‐GNRs within the GSCs was then followed over a total time of 30 s, using the TEM classical geometry performed at medium magnification, and with a high contrast objective lens and an electron beam acceleration voltage of 120 kV. Even under these conditions, the resolution was enough to study the PVP‐GNRs uptake evolution over time. As shown in **Figure 14B**, the movement of the PVP‐GNRs inside the GSCs is similar for both the incubation times: a local displacement of the PVP‐GNRs and the solution surrounding them were observed. The main difference between the GSCs incubated for 20 and 60 min with the PVP‐GNRs consisted in the number of those uptaken from the GSCs: higher the incubation time, higher the amount of PVP‐GNRs internalized from the tumor cells.

**Figure 14.** (A) First row, left images: sketches of microchip with integrated microwells (10 μm in diameter) functional‐ ized to specifically capture GS9‐6/NOTCH+ glioblastoma stem cells; right images: comparison between the functional‐ ized microwells (called "capture") and the negative control, i.e., identical microwells not treated for functionalization and then not able to tether GSCs. Second row: sketch showing a cross‐sectional view of the microfluidic system con‐ taining GSC positioned in the TEM column. Adapted with permission from [110]. (B) Gold nanorods penetrated in a GSCs after two different times of incubation. Top images show the capability of unlabeled gold nanorods to perforate the cells within 20 min incubation period; moreover, the liquid dispersed around the nanorods displaces their position within a common region. These movements are represented in a contour map generated at the same threshold level and within the same region of interest at the initial imaging stage (0 s) and final stage (30 s). Regions exhibiting the greatest density (included both nanorods and their surrounding liquid) were contoured at the darker colored thresh‐ old while regions showing less density were represented using increasingly lighter gradients of color. Bottom images: cell incubated for 60 min, where more NPs inside the cell than in the solution are observed, if compared to the sample incubated for 20 min. Adapted with permission from [110].

## **5. Conclusions**

As first example of study of biological living matter by *in situ* E‐Cell for liquid specimens, we report here what Liu et al. showed in their paper [115]. It reports the detailed fabrication using microelectromechanical system (MEMS) techniques to obtain a novel microchip constituted by two SiO2 films used as a substrate and the 9 nm thin windows for *in situ* imaging of living organisms in aqueous environment using a conventional TEM geometry. Such a kind of microchips—with a gap between them ranging from 2 to 5 μm—allowed the effective TEM observation of living bacteria *Escherichia coli* and the tellurite reduction process in *Klebsiella pneumonia,* the height of which were estimated ranging between 0.5 and 1μm. The most important results of this paper were (a) the capability of bacterium *K. pneumoniae* and the yeast *Saccharomyces cerevisiae* to stay alive onto the microchip substrate under continuous electron beam irradiation needed for the TEM imaging for up to 14 and 42 s, respectively; (b) the possibility to follow over time, even if with not very high resolution as expected from Eq. (2),

As already mentioned and shown by Eq. (3), the use of STEM allowed to achieve a dramatically increased resolution and to image by using HAADF geometry even very small heavy particles dispersed in a light liquid medium. This was the case of Au‐labeled eukaryotic fibroblast cells (COS7) immersed in a several micrometer‐thick liquid layer [107, 109]. In both the papers a flow liquid E‐Cell was used, allowing the aqueous solution to pour through the E‐Cell and around the whole fibroblast cells. The experimental configuration is reported in **Figure 13A**. The cells were grown, then labeled, and finally fixed prior to be inserted in the flow E‐Cell, on the internal side of the silicon nitride film that was finally placed to seal the upper part of the E‐Cell, in order to maximize the STEM resolution according to Eq. (3). The cells' immunolab‐ eling was realized by using gold NPs sizing 10 nm, in order to target the epidermal growth factor (EGF) molecules bound to the cellular EGF receptors. Thus, using a water layer 10 μm thick, the theoretical resolution limit determined by using Eq. (3) was found to be equal to 1.9 nm. In agreement with that, the 10 nm sized labeling gold NPs were detected with a spatial resolution of about 4 nm, as shown in **Figure 13B** and **C**, using the STEM in HAADF geometries, and performed with a beam accelerated by a voltage of 200 kV and a dwell time of 20 μs. If it is not weird that the electron beam did not perturb the fixed cells, it is however remarkable

electrons per nm2

damage consequences were observed on the spatial distribution of the labeling NPs on the

As last and most recent example the results achieved by Pohlmann *et al.* are shown [110]. In their paper they showed an *in situ* TEM imaging, time resolved, of the uptake of typical drug delivery vehicles—in such a case gold nanorods (GNR) encapsulated in polyvinylpyridine (PVP)—from glioblastoma stem cells (GSCs), which actually is one of the most aggressive cancers among brain tumors. This kind of study is quite interesting, allowing to image by TEM the evolution over time of NP uptake from living cells, a phenomenon so far investigated just by light/confocal microscopy. To perform it, the authors first prepared a multiwell E‐Cell with a maximum height of 2 μm where the GSCs were forced to be tethered on the lower side, thanks to a surface functionalization that involved their NOTCH1 receptors, as shown in **Fig‐ ure 14A**. Then, the GSCs contained in the multiwell E‐Cell were incubated together with the

) and within the flowing liquid no visible

biological processes such as the tellurite reduction by *K. pneumonia*.

that for the dose used (7 × 104

cells.

254 Microscopy and Analysis

In this chapter, some of the most recent developments for the *in situ* TEM and STEM imaging techniques have been shown. The chapter aimed at showing the impact on the *in situ* TEM/ STEM imaging of the progress achieved in the last 10–15 years in terms of novel microscope architecture developments, fabrication and commercial availability of novel and much more performing dedicated specimen holders. This in turn has allowed achieving unprecedented results in terms of sample stability and diversity of setups, variation of the local sample environment, and possible external *stimuli*. Due to the vastness of the topic, the three sections of this chapter have been dedicated to describe three particular applications of the *in situ* TEM/ STEM that witness how this technique evolved in the last decade: the *in situ* sample heating under high vacuum condition, the *in situ* sample heating in a controlled gaseous atmosphere, and, finally, the *in situ* imaging of samples immersed in a liquid layer of user‐controlled thickness. The importance of these improvements lies in the widening of their possible application not only to inorganic but also to biological materials, even in living conditions, which paves the way to novel, so far unattainable studies.

## **Author details**

Alberto Casu, Elisa Sogne, Alessandro Genovese, Cristiano Di Benedetto, Sergio Lentijo Mozo, Efisio Zuddas, Francesca Pagliari and Andrea Falqui\*

\*Address all correspondence to<EMAIL_ADDRESS>
King Abdullah University of Science and Technology (KAUST), Biological and Environmental Sciences and Engineering (BESE) Division, Thuwal, Kingdom of Saudi Arabia

## **References**

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results in terms of sample stability and diversity of setups, variation of the local sample environment, and possible external *stimuli*. Due to the vastness of the topic, the three sections of this chapter have been dedicated to describe three particular applications of the *in situ* TEM/ STEM that witness how this technique evolved in the last decade: the *in situ* sample heating under high vacuum condition, the *in situ* sample heating in a controlled gaseous atmosphere, and, finally, the *in situ* imaging of samples immersed in a liquid layer of user‐controlled thickness. The importance of these improvements lies in the widening of their possible application not only to inorganic but also to biological materials, even in living conditions,

Alberto Casu, Elisa Sogne, Alessandro Genovese, Cristiano Di Benedetto, Sergio Lentijo Mozo,

King Abdullah University of Science and Technology (KAUST), Biological and Environmental

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**Author details**

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**Abstract**

The widespread utilization of nonaqueous redox flow batteries is hindered by the low performance. Including some kinds of additives in electrolyte is a possible and facile solution. In this chapter, the effects of carbon dioxide gas, EC/DMC, and antimony ions on the electrochemical performance of nonaqueous redox flow batteries are disclosed. The results show that the ohmic resistance of the deep eutectic solvent (DES) electrolyte reduces significantly when adding carbon dioxide gas and EC/DMC, the percentage of reduction increases with the volume percentage of EC/ DMC in electrolyte, and the reaction kinetics almost keeps unchanged for carbon dioxide gas and EC/DMC additives. For the additive of antimony ions, the electrochemical reaction kinetics of active redox couple is enhanced, the diffusion coefficient of active ions also increases, and the charge transfer resistance decreases. The antimony ions electrodeposited on the surface of graphite felt contribute a catalytic effect on the electrochemical reaction so as to improve the performance. However, due to the trade-off between the enhanced kinetics and reduced active surface area, the optimum concentration of antimony ions is found to be 15 mM. In addition, the flow battery assembled with negative electrolyte containing antimony ions exhibits 31.2% higher power density than that of pristine DES electrolyte.

**Keywords:** redox flow batteries, deep eutectic solvent (DES), electrolyte additives, carbon dioxide, EC/DMC, antimony ions