This research was primarily sponsored by the Laboratory Directed Research and Development Program of Oak&#160;Ridge National Laboratory (ORNL), managed by UT-Battelle LLC, for the U.S. Department of Energy (DOE). Part of the development to introduce water vapor into the&#160;in situ&#160;gas cell was sponsored by the U.S. DOE, Office of Energy Efficiency and Renewable Energy, Bio-Energy Technologies Office, under contract DE-AC05-00OR22725 (ORNL) with UT-Battle, LLC, and in collaboration with the Chemical Catalysis for Bioenergy (ChemCatBio) Consortium, a member of the Energy Materials Network (EMN). This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. DOE under Contract No. DE-AC36-08GO28308.&#160;Part of the microscopy was conducted at the Center for Nanophase Materials Sciences (CNMS), which is a DOE Office of Science User Facility. Early development of&#160;in situ&#160;STEM capabilities was sponsored by the Propulsion Materials Program, Vehicle Technologies Office, U.S. DOE. &#160;We thank Dr. John Damiano, Protochips Inc., for useful technical discussions. The authors thank Rosemary Walker and Kase Clapp, ORNL production team, for support with movie production. The views expressed in this article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.

1. E-chip preparation
<ol>
	<li>Direct powder deposition by drop-casting from a colloidal solution (Figure 2A).
	<ol>
		<li>Crush the powder if the powder particle aggregates are too large. Do this using a small mortar and pestle (crushed aggregates should be &#60;5 &#181;m in size). Mix a small amount (e.g., ~0.005 mg, amount determined by experience) of powder in 2 mL of the solvent (e.g., isopropanol or ethanol).</li>
		<li>Sonicate the mixture for around 5 min to create a colloidal suspension.</li>
		<li>Place the E-chip on the E-chip retaining fixture. Drop cast approximately 1 &#181;L of the suspension using a 0.5-2.5 &#181;L micro-pipette directly onto the E-chip.</li>
		<li>Clean the Au contacts to remove the suspension with an absorbent paper point while viewing through a stereo microscope.</li>
	</ol>
	</li>
	<li>Direct powder deposition through a mask (Figure 2B).
	<ol>
		<li>Crush the powder (e.g., Pt/TiO2) dry, if powder particles are too large (as in 1.1.1).</li>
		<li>Place a new clean E-chip on the E-chip retaining fixture (Figure 3D). Use a mask, which is another E-chip with the SixNy membrane removed (by breaking it with tweezers or compressed gas) and place it directly onto the E-chip within the fixture.</li>
		<li>Use the top plate to clamp a new clean E-chip and a mask together within the fixture.</li>
		<li>Deposit a small amount of the powder using a spatula directly on the silicon nitrile membrane in the mask.</li>
		<li>Gently vibrate the fixture to shake the particles down to the E-chip. This can either be done using a vacuum tweezer unit by holding the fixture to the top of the unit while it is running or using a sonication unit and placing the fixture in a dry beaker.</li>
		<li>Shake off the excess powder, disassemble the system and inspect the placement of dry powder on the E-chip using a stereo microscope.</li>
	</ol>
	</li>
	<li>Deposition method by either electron beam evaporation, ion, or magnetron sputtering. 
	NOTE: This method is used to create either a single-element system or model alloy specimens of known geometry and composition.
	<ol>
		<li>Create a pattern mask (Figure 3). 
		NOTE: Prepare the pattern mask in advance since it takes some time.</li>
		<li>Use a spacer chip with removed SixNy membrane. In this experiment, an E-chip commonly used in liquid-cell experiments was used after gently breaking out the SixNy membrane which resulted in 50 x 250 &#181;m opening. This spacer chip with removed SixNy membrane will be combined with another chip, having an array of holes (e.g., silicon nitride (SiN) Microporous TEM Window 33).</li>
		<li>Use cyanoacrylate (CA) glue (Table of Materials) to attach the SiN Microporous TEM Window face down (SiN pattern film away from the spacer chip) over the 50 x 250 &#181;m opening following the manufacturer&#39;s recommendation (Figure&#160;3B,C).</li>
		<li>Repeat the procedure to prepare as many pattern masks as needed, depending on the planned experiments.</li>
		<li>Place a new clean E-chip on the E-chip fixture (Figure 3D).</li>
		<li>Place the pattern mask on the E-chip (Figure&#160;3C,D).</li>
		<li>Cover with the top plate and clamp it (Figure 3D).</li>
		<li>Use either electron beam evaporation, ion sputtering or magnetron sputtering deposition techniques. These are the recommended methods used to sputter material of interest directly through the pattern mask. 
		NOTE: It may be important to purge the deposition system to remove residual oxygen prior to the deposition for higher purity material deposits33.</li>
		<li>Disassemble the system and inspect the E-chip with a stereo microscope to ensure good adherence of the deposited material on the E-chip&#39;s SixNy membrane.</li>
	</ol>
	</li>
	<li>Focused ion beam (FIB) milling (Figure 2C).
	<ol>
		<li>Prepare a standard TEM lamella using the FIB. Use low kV (e.g., 2-5 kV) for the final milling step to remove damage caused by FIB milling at high voltages (30-40 kV).</li>
		<li>Place the TEM lamella on the E-chip using standard FIB procedures. Do not damage the SixNy membrane when attaching the FIB-prepared TEM lamella to the E-chip. See Allard et al.34 and other publications30,35,36 for details of the variety of methods using Xe-PFIB and Ga-FIB instruments for lamella preparation.</li>
	</ol>
	</li>
</ol>
2. Preparation of the atmosphere (CCGR-TEM) holder
<ol>
	<li>Download the desired calibration file.</li>
	<li>Measure the resistance of the SiC heater to ensure that it is within the resistance range for that particular E-chip calibration as provided by the CCGR manufacturer.</li>
	<li>Remove the clamp from the CCGR-TEM holder.</li>
	<li>Clean the tip of the CCGR-TEM holder using absorbent paper points and/or compressed air, making sure no debris remains on the O-ring grooves. Then place the special double-gasket seal within the tip.</li>
	<li>Place the spacer chip into the CCGR-TEM holder.</li>
	<li>Place the E-chip containing the specimen that was prepared by one of the methods mentioned in section 1 with the heater contacts down onto the spacer chip, making a proper connection to the electrical contacts of the flex-cable within the holder.</li>
	<li>Position the holder clamp plate on the top of the E-chip using tweezers, place the screws into the designated location at the tip of the CCGR-TEM holder, then torque the set screws with a final torque to 0.2 lb-ft.</li>
	<li>Measure again, the resistance of the SiC heater after assembling the CCGR-TEM holder to ensure that it is within the resistance range for that particular E-chip calibration as provided by the CCGR manufacturer. 
	&#8203;NOTE: Here, a special adapter is used, which plugs directly into the holder&#39;s electrical connections. This allows for the resistance measurements to be made through the CCGR-TEM holder and paired microchip devices assembly while fully assembled into the holder.</li>
</ol>
3. Preparation of the experimental setup
<ol>
	<li>Bake and pump down the system (manifold, holder, gas tanks, and RGA chamber) overnight, either with or without the holder connected by pressing the Bake button in the gas-control software.</li>
	<li>Load the holder into the scanning transmission electron microscope and connect the gas tubing from the manifold to the CCGR-TEM holder.</li>
	<li>For the experiment, pump and purge the system with an inert gas (e.g., Ar or N2) twice from 100 Torr to 0.5 Torr.</li>
	<li>Perform a final pump and purge from 100 Torr to 0.001 Torr. This will ensure that the entire gas delivery system, from the gas manifold to the holder, is cleaned and flushed with inert gas.</li>
	<li>Residual gas analyzer - During the pump and purge procedure, turn on the RGA system to warm up the filament.</li>
</ol>
4. Preparing the water vapor delivery system (VDS)
NOTE: These instructions are for specific experiments that involve controlled delivery of gas in vapor form (e.g., water vapor). Gas delivery control is through the gas-control software provided by the manufacturer (Table of Materials).
<ol>
	<li>Attach the purge gas (e.g., N2) to the VDS, turn the lever knob to Exhaust, and then turn to the Park position.</li>
	<li>Purge the VDS (repeat 4.1) by flowing inert gas three times or until no more liquid is present.</li>
	<li>Turn the lever knob to the Park position and attach the VDS to the manifold.</li>
	<li>Turn the lever knob to the Fill position and remove the purge gas line.</li>
	<li>Set the vapor pressure to 18.7 Torr in the gas-control&#160;software.</li>
	<li>In the software, pump the VDS to vacuum (0.1 Torr) by selecting the input line and pressing the pump button.</li>
	<li>Fill the VDS with water (2 mL) via a syringe and tubing. 
	&#8203;NOTE: If higher purity vapor is needed, additional purging steps may be required.</li>
</ol>
5. Running the reaction
<ol>
	<li>Make sure all gases that are to be used in the experiments (e.g., N2, water vapor, and O2) are connected to the manifold.</li>
	<li>With the gas-control software under Naming, set the name(s) for the gas(es) required for the reaction and save the raw &#34;.csv&#34; file such that a running log file is generated for the experiment.</li>
	<li>Under the E-chip Setup, select the associated calibration file (i.e., as described in 2.5) for the E-chip being used and Run Calibration. As previously mentioned in the Introduction section, each E-chip is temperature calibrated using infrared radiation (IR) imaging from the manufacturer.</li>
	<li>Under Pump and Purge, see Preparation of Experimental Setup.</li>
	<li>Under Gas Control, select the desired gas name and its composition (e.g., select percentage for each gas) for the experiment.</li>
	<li>Under Temperature, select the desired heating rate and target temperature&#160;for the temperature of interest for the experiment and press the Start button.</li>
	<li>Start flowing the gas by pressing the Start button under the Gas Control section.</li>
</ol>
6. End of the experiment
<ol>
	<li>Once the reaction is complete, stop flowing the gas, turn off the temperature knob, and end the session using the Pump and Purge procedure (e.g., depending on the reaction that was performed, perform Pump and Purge procedure from 100 Torr to 0.1 Torr 2-3 times).</li>
	<li>Prior to removing the in situ CCGR-TEM holder from the electron microscope, ensure that that holder pressure is brought back up to atmospheric pressure.</li>
</ol>

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+Xe-plasma+FIB+for+preparing+aluminum+alloy+specimens+for+MEMS-based+in+situ+double-tilt+heating+experiments.">The utility of Xe-plasma FIB for preparing aluminum alloy specimens for MEMS-based in situ double-tilt heating experiments.</a> Microscopy and Microanalysis. 25 (2), 1442-1443 (2019).</li><li>Schilling, S., Janssen, A., Zaluzec, N. J., Burke, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+aspects+of+electrochemical+corrosion+measurements+during+in+situ+analytical+transmission+electron+microscopy+(TEM)+of+austenitic+stainless+steel+in+aqueous+media.">Practical aspects of electrochemical corrosion measurements during in situ analytical transmission electron microscopy (TEM) of austenitic stainless steel in aqueous media.</a> Microscopy and Microanalysis. 23 (4), 741-750 (2017).</li><li>Zhong, X. L., Schilling, S., Zaluzec, N. J., Burke, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preparation+methodologies+for+in+situ+liquid+and+gaseous+cell+analytical+transmission+electron+microscopy+of+electropolished+specimens.">Sample preparation methodologies for in situ liquid and gaseous cell analytical transmission electron microscopy of electropolished specimens.</a> Microscopy and Microanalysis. 22 (6), 1350-1359 (2016).</li><li>Duchamp, M., Xu, Q., Dunin-Borkowski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convenient+preparation+of+high-quality+specimens+for+annealing+experiments+in+the+transmission+electron+microscope.">Convenient preparation of high-quality specimens for annealing experiments in the transmission electron microscope.</a> Microscopy and Microanalysis. 20 (6), 1638-1645 (2014).</li><li>Unocic, K. A., Mills, M. J., Daehn, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+gallium+focused+ion+beam+milling+on+preparation+of+aluminum+thin+foils.">Effect of gallium focused ion beam milling on preparation of aluminum thin foils.</a> Journal of Microscopy. 240 (3), 227-238 (2010).</li><li>Unocic, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+battery+chemistry+with+liquid+cell+electron+energy+loss+spectroscopy.">Probing battery chemistry with liquid cell electron energy loss spectroscopy.</a> Chemical Communications. 51 (91), 16377-16380 (2015).</li></ol>

The authors declare no conflicts of interest.
This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).

In the present work, an approach to perform in situ STEM reactions with and without water vapor is demonstrated. The critical step within the protocol is E-chip preparation and maintaining its integrity during the loading procedure. The limitation of the technique is (a) the specimen size and its geometry to fit the nominal 5-&#181;m gap between paired (MEMS)-based silicon microchip devices as well as (b) a total pressure used in the experiments with water vapor since the highest total pressure depends on the quantity of water vapor6. The significance of this method with respect to existing methods is that we can perform operando experiments, i.e., we analyze the specimens under real conditions, enabled by an RGA system that confirms/monitors experimental conditions. Additionally, there are opportunities for future applications of the technique to diverse materials systems that may require different methods and procedures for sample deposition on E-chip heaters.
The E-chip preparation is shown in Figure 2, which highlights four different sample preparation methods; (1) direct powder deposition by drop cast from colloidal solution, (2) direct powder deposition through mask, (3) direct EBID/IBID or magnetron sputtering with patterned masks and (4) FIB milling. Powder deposition should include only powders with particles or aggregates less than 5 &#181;m thick to fit within the nominal 5 &#181;m gap between paired microchips in order to prevent damage to the SixNy viewing windows2,6. Researchers performing deposition through evaporation methods should adjust the parameters according to the elemental composition, temperature, and humidity and should minimize the oxygen level. Sample preparation using&#160;FIB milling requires users to be extremely careful to prevent damage to the SixNy membrane. Also, Ga implantation could alter alloy chemistry and affect surface diffusion. Regardless of what E-chip specimen method is selected, following sample deposition, examination of the E-chip using light optical microscopy and resistance measurements are required to verify the E-chip integrity before starting the in situ experiments.
This protocol for in situ CCGR-STEM studies enables new opportunities to visualize nanoscale gas reactions while they occur and under realistic conditions (temperature, pressure, and gas composition). Now, it is possible to reveal dynamic changes in the surface atoms and interfaces and to understand how the surface composition and structure may be controlled by external means7. For example, the structural changes in the Pt particle and rearrangement of its structure to expose {111} surfaces (Figure 6) were associated with minor shape changes (Figure 6A&#160;vs.&#160;Figure 6B&#160;vs.&#160;Figure 6C). Catalytic performance is determined by interfacial reactions that occur at site-specific catalyst interfaces, and in situ microscopy helped uncover gas-surface phenomena under water vapor in Pt/TiO2 catalysis research. Moreover, the experimental protocol presented here also contributes to an improved understanding of the in situ gas reaction process by monitoring the gas composition using an RGA. This is important because of the need to correlate the role of gas composition with structural and chemical changes that the material being studied undergoes as a direct effect of environmental exposure.
In summary, in situ CCGR-STEM studies can enable investigation of the deactivation or regeneration of catalyst materials via imaging and spectroscopy, and the investigation of chemical and morphological changes during gas reactions on bulk alloy materials. Such studies also allow for&#160;identification of the minimum temperature of initiation of e.g., the regeneration reaction and/or the maximum temperature for the reaction, as well as the nature of coarsening of supported metal particles from which kinetic information can be extracted. These studies provide a direct link to current computational models that predict the pathway of the reactions, but not time when it will happen, which is important&#160;for material optimization. The potential of this environmental closed-cell gas reaction protocol can be expanded to a number of different materials in conjunction with quantitative spectroscopy techniques such as electron energy-loss spectroscopy39 and energy-dispersive X-ray spectroscopy5,6 to identify chemical compositions and/or oxidation state changes. Moreover, this is just the beginning of a new capability that creates an advanced opportunity for materials characterization under a variety of realistic conditions.

There is a need to obtain detailed information on how a material undergoes structural and chemical changes under reactive gas exposure and at elevated temperatures. In situ closed-cell gas reaction (CCGR) scanning transmission electron microscopy (STEM) was developed specifically to study the dynamic changes occurring in a wide range of material systems (e.g., catalysts, structural materials, carbon nanotubes, etc.) when subjected to elevated temperatures, different gaseous environments, and pressures from vacuum to full atmospheric pressure1,2,3,4,5,6,7,8,9,10,11,12. This approach can be beneficial in several cases, e.g., in the accelerated development of next-generation catalysts that are important for a number of industrial conversion processes, such as the single-step conversion of ethanol to n-butenes over Ag-ZrO2/SiO213, catalysts for the oxygen reduction reaction&#160;and hydrogen evolution reaction in fuel cell applications14,15, catalytic CO2 hydrogenation16, methanol dehydrogenation to formaldehyde or dehydration to dimethyl ether that use either metal catalysts or multi-walled carbon nanotubes in a methanol conversion reaction in the presence of oxygen17. Recent applications of this in situ technique for catalysis research1,2,7,8,10,11,12,18,19,20,21,22 have provided new insight into catalyst dynamic shape changes10,11,23, faceting7, growth, and mobility8,20,24. Moreover, in situ CCGR-STEM can be used to investigate the high-temperature oxidation behavior of structural materials that are exposed to aggressive environments, from gas turbine engines to next-generation fission and fusion reactors, where not only strength, fracture toughness, weldability, or radiation are important but also high-temperature oxidation resistance25,26,27,28,29. Specific to structural alloys, in situ CCGR-STEM experiments allow for dynamic tracking of diffusion-induced grain boundary migration under reducing conditions9 and measurements of oxidation kinetics at high temperature5,6,30. For several decades prior to the recent development of CCGR technologies, in situ gas reaction studies were conducted using dedicated environmental TEMs (E-TEMs). A detailed comparison of E-TEM and CCGR-STEM has been previously addressed10; therefore, E-TEM capabilities are not discussed further in the present work.
In this work, a commercially available system (Table of Materials) comprising&#160;a computer-controlled manifold (gas delivery system) and a specially designed CCGR TEM holder that utilizes a pair of microelectromechanical (MEMS)-based silicon microchip devices (e.g., spacer chip and &#34;E-chip&#34; heater (Table of Materials)) were used. Each E-chip&#160;supports an amorphous, electron-transparent SixNy membrane. The spacer chip has a 50 nm thick SixNy membrane with a 300 x 300 &#181;m2 viewing area and 5 &#181;m thick epoxy-based photoresist (SU-8) &#34;spacer&#34; contacts that are microfabricated to provide a gas flow path and maintain a physical offset between the two paired microchips (Figure 1A). A portion of the E-chip is covered with a low conductivity ~100 nm SiC ceramic membrane; the membrane has a 3 x 2 array of 8 &#181;m-diameter etched holes overlapped by a ~30 nm thick amorphous SixNy membrane (SixNy viewing area) (Figure 1A and Figure 2D), through which images are recorded. The E-chip serves a dual role as both specimen support and heater6. Au contacts are microfabricated onto the E-chip to allow for resistive heating of the SiC membrane. Each E-chip is calibrated using&#160;infrared radiation (IR) imaging methods (Table of Materials)2 and has been shown to be accurate to within &#177;5%31. Temperature calibration is independent of the gas composition and pressure, thereby providing independent control over reaction temperatures under any chosen gas conditions. The benefit of a thin-film heater is that temperatures up to 1,000 &#176;C can be reached within milliseconds. In order to perform the reaction, the E-chip is placed on the top of the spacer chip, creating the closed-cell &#34;sandwich&#34; that isolates the environment around the specimen from the high vacuum of the TEM column. The advantage of this setup is that reactions can be performed from low pressures up to atmospheric pressure (760 Torr) with single or mixed gases and under static or flow conditions. The MEMS devices are secured with a clamp (Figure 1B) that allows the holder to be inserted within the mm-sized gap of the objective lens pole piece in an aberration-corrected S/TEM instrument (Table of Materials) (Figure 1C). Modern in situ S/TEM holders include integrated micro-fluidic tubing (capillaries) that are connected to the external stainless-steel tubing, which in turn is connected to the gas delivery system (manifold). An electronic control system permits the controlled delivery and flow of reactant gas through the gas cell. Gas flow and temperature are operated by a custom workflow-based software package provided by the manufacturer (Table of Materials)10,32. The software controls three gas input lines, two internal experimental-gas delivery tanks, and a receiving tank for gas flow returning from the cell during the experiment (Figure 1D).
Due to the variability of materials and their form factor, we first focus on several specimen deposition methods on the E-chip, then outline protocols for performing quantitative in situ/operando experiments with controlled temperature, gas mixing and flow.

<table><tbody><tr><td>Atmosphere Clarity Software</td><td>Protochips</td><td>6.5.14</td><td></td></tr><tr><td>Atmosphere Large Heating E-chips, 300 x 300 window, no spacer</td><td>Protochips</td><td>EAT-33AA-10</td><td>microchip device</td></tr><tr><td>Atmosphere Small E-chips, 300 x 300 micron window, 5 micron SU-8 spacer</td><td>Protochips</td><td>EAB-33W-10</td><td>microchip device</td></tr><tr><td>JEOL 2200FS</td><td>JEOL</td><td></td><td>microscope</td></tr><tr><td>M-bond 610</td><td>Electron Microscopy Sciences</td><td>50410-30</td><td>cyanoacrylate (CA) glue</td></tr><tr><td>Mikron M9103 IR camera</td><td>Micron</td><td></td><td>This is used by Protochips/ not available</td></tr><tr><td>Protochips &amp;ldquo;Fusion&amp;rdquo; E-chips</td><td>Protochips</td><td></td><td>spacer chip with removed Si&lt;sub&gt;x&lt;/sub&gt;N&lt;sub&gt;y&lt;/sub&gt; membrane</td></tr><tr><td>Protochips Atmosphere 200</td><td>Protochips</td><td>prototype</td><td>software</td></tr><tr><td>Residual Gas Analyzer R100 (RGA)</td><td>Stanford Research Systems</td><td>R100 SRS</td><td></td></tr></tbody></table>

performing in situ closed-cell gas reactions in the transmission electron microscope

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at length scales down to the atomic level. In situ closed-cell gas reaction (CCGR) studies performed using (scanning) transmission electron microscopy (STEM) can separate and identify localized dynamic reactions, which are extremely challenging to capture using other characterization techniques. For these experiments, we used a CCGR holder that utilizes microelectromechanical systems (MEMS)-based heating microchips (hereafter referred to as &#34;E-chips&#34;). The experimental protocol described here details the method for performing in situ gas reactions in dry and wet gases in an aberration-corrected STEM. This method finds relevance in many different materials systems, such as catalysis and high-temperature oxidation of structural materials at atmospheric pressure and in the presence of various gases with or without water vapor. Here, several sample preparation methods are described for various material form factors. During the reaction, mass spectra obtained with a residual gas analyzer (RGA) system with and without water vapor further validates gas exposure conditions during reactions. Integrating an RGA with an in situ CCGR-STEM system can, therefore, provide critical insight to correlate gas composition with the dynamic surface evolution of materials during reactions. In situ/operando studies using this approach allow&#160;for detailed investigation of the fundamental reaction mechanisms and kinetics that occur at specific environmental conditions (time, temperature, gas, pressure), in real-time, and at high spatial resolution.

Watch this Scientific Journal Video about Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope at JoVE.com

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Here, we present a protocol for performing in situ TEM closed-cell gas reaction experiments while detailing several commonly used sample preparation methods.

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at ...

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3.9K Views.  Oak Ridge National Laboratory. Tot voor kort analyseerden we materialen voor en na bepaalde testen. Er is echter behoefte aan gedetailleerde informatie over hoe materiaal structurele en chemische veranderingen ondergaat onder agressieve omgevingen bij verhoogde temperaturen. in situ gesloten-cel gasreactie, CCGR, scanning transmissie elektromicroscopie is speciaal hiervoor ontwikkeld.Het bestuderen van de dynamische veranderingen in real time bij verhoogde temperaturen in een gasomgeving, tot volledige atmosferische...

Video: Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.

We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted ...

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. The aggressive scaling of feature sizes, both on devices and interconnects, leads to serious challenges to ensure the required product reliability. Standard reliability tests and post-mortem failure analysis provide only limited information about the physics of failure mechanisms and degradation kinetics. Therefore it is necessary to develop new experimental approaches and procedures to study the TDDB failure mechanisms and degradation kinetics in particular. In this paper, an in situ experimental methodology in the transmission electron microscope (TEM) is demonstrated to investigate the TDDB degradation and failure mechanisms in Cu/ULK interconnect stacks. High quality imaging and chemical analysis are used to study the kinetic process. The in situ electrical test is integrated into the TEM to provide an elevated electrical field to the dielectrics. Electron tomography is utilized to characterize the directed Cu diffusion in the insulating dielectrics. This experimental procedure opens a possibility to study the failure mechanism in interconnect stacks of microelectronic products, and it could also be extended to other structures in active devices.

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. This paper demonstrates the procedure of an in situ TDDB experiment in the transmission electron microscope, which opens a possibility to study the failure mechanism in microelectronic products.

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Bioengineering

Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM. The microfluidic system consists of two silicon microchips supporting thin Silicon Nitride (SiN) membrane windows. This article describes the basic steps of sample loading and data acquisition. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. This protocol also includes a number of tests necessary to perform during sample loading in order to ensure correct assembly. Once the sample is loaded in the electron microscope, the liquid thickness needs to be measured. Incorrect assembly may result in a too-thick liquid, while a too-thin liquid may indicate the absence of liquid, such as when a bubble is formed. Finally, the protocol explains how images are taken and how dynamic processes can be studied. A sample containing AuNPs is imaged both in pure water and in saline.

This protocol describes the operation of a liquid flow specimen holder for scanning transmission electron microscopy of AuNPs in water, as used for the observation of nanoscale dynamic processes.

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Chemistry

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching

Graphene liquid cell electron microscopy provides the ability to observe nanoscale chemical transformations and dynamics as the reactions are occurring in liquid environments. This manuscript describes the process for making graphene liquid cells through the example of graphene liquid cell transmission electron microscopy (TEM) experiments of gold nanocrystal etching. The protocol for making graphene liquid cells involves coating gold, holey-carbon TEM grids with chemical vapor deposition graphene and then using those graphene-coated grids to encapsulate liquid between two graphene surfaces. These pockets of liquid, with the nanomaterial of interest, are imaged in the electron microscope to see the dynamics of the nanoscale process, in this case the oxidative etching of gold nanorods. By controlling the electron beam dose rate, which modulates the etching species in the liquid cell, the underlying mechanisms of how atoms are removed from nanocrystals to form different facets and shapes can be better understood. Graphene liquid cell TEM has the advantages of high spatial resolution, compatibility with traditional TEM holders, and low start-up costs for research groups. Current limitations include delicate sample preparation, lack of flow capability, and reliance on electron beam-generated radiolysis products to induce reactions. With further development and control, graphene liquid cell may become a ubiquitous technique in nanomaterials and biology, and is already being used to study mechanisms governing growth, etching, and self-assembly processes of nanomaterials in liquid on the single particle level.

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching

Graphene liquid cell electron microscopy can be used to observe nanocrystal dynamics in a liquid environment with greater spatial resolution than other liquid cell electron microscopy techniques. Etching premade nanocrystals and following their shape using graphene liquid cell Transmission Electron Microscopy can yield important mechanistic information about nanoparticle transformations.

Graphene liquid cell electron microscopy provides the ability to observe nanoscale chemical transformations and dynamics as the reactions are occurring in ...

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. The versatility of the GSMLCs is demonstrated in the context of a study about etching and growth dynamics of gold nanostructures from a HAuCl4 precursor solution. GSMLCs combine the advantages of conventional silicon- and graphene-based liquid cells by offering reproducible well depths together with facile cell manufacturing and handling of the specimen under investigation. The GSMLCs are fabricated on a single silicon substrate which drastically reduces the complexity of the manufacturing process compared to two-wafer-based liquid cell designs. Here, no bonding or alignment process steps are required. Furthermore, the enclosed liquid volume can be tailored to the respective experimental requirements by simply adjusting the thickness of a silicon nitride layer. This enables a significant reduction of window bulging in the electron microscope vacuum. Finally, a state-of-the-art quantitative evaluation of single particle tracking and dendrite formation in liquid cell experiments using only open source software is presented.

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics.

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. ...

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.
Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (&lt; -180&deg;C). For slender cells (up to ~500 nm in thickness3), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.1, 2
In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for ...

Biology

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass optical fiber inserted into the electron column above the objective lens polepiece, and a laser, an adjustable light source, was used to fabricate the device. After the illuminator has been calibrated using an external measuring system, it allows one to adjust the intensity of the lighting to the needs of the observed process. This lighting system was utilized to image antimicrobial photodynamic therapy phenomena, which are currently the subject of intense research. The sample was prepared by spotting a suspension of bacteria on a carbon, graphene, or silicon nitride substrate, blotting the excess solution, spotting the photosensitizer solution, blotting the excess liquid again, and then assembling the liquid cell with a second substrate or graphene film. The process of the imaging experiment itself includes choosing the right place for observation with the use of low magnification and a minimum dose of electrons, and then cyclical activation of the light source to capture subsequent images at specified intervals with the minimum amount of electrons necessary. The electron dose of each exposure and the time and intensity of lighting used need to be carefully recorded due to the complexity of the observed phenomena as, at the same time, the process is both light- and electron-driven. After the actual experiment is performed, additional control observations must be made, in which the same doses of electrons are used but without additional light influence and smaller doses of electrons are used for higher doses of light. This makes it possible to distinguish light-induced microstructural effects from those caused by electrons in both the fields of life and materials science.

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The present protocol describes transmission electron microscope (TEM) modifications with a light illumination system, the fabrication of liquid cells, and in situ TEM observations of light-induced interactions between bacterial cells and a photosensitizer. The sample preparation methods, electron beam damage, and imaging are also discussed.

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass ...

Fluid-cell Raman Spectroscopy for operando Studies of Reaction and Transport Phenomena during Silicate Glass Corrosion

Fluid-cell Raman Spectroscopy (FCRS) enables the real-time and space-resolved (operando) study of reaction mechanisms, kinetics, and their mutual interactions with transport processes during silicate glass corrosion at the micrometer scale and at elevated temperatures. This manuscript provides a detailed protocol for setting up an FCRS experiment, exemplified by a corrosion experiment with a ternary Na borosilicate glass and a 0.5 M NaHCO3 solution at a temperature of 86 &#177; 1 &#176;C. The protocol involves (i) sample preparation, (ii) assembly of the fluid cell, and (iii) setting of Raman measurement parameters for collecting Raman spectra across the sample/solution interface in regular time intervals. The results from the experiment show the formation of a water-rich zone between a silica-based surface alteration layer (SAL) and the pristine glass, which is an intrinsic feature of an interface-coupled dissolution-precipitation model for the formation of a SAL during silicate glass corrosion. The ability to track the reaction and transport processes during the corrosion of silicate glasses and potentially of other transparent materials, spatially resolved and in real-time, represents a unique strength of this technique, overcoming the disadvantages of conventional analysis of multi-step quenching experiments. The corrosion of the top side of the glass sample represents a current issue, reducing spatial resolution at depth due to precipitation within the laser pathway. This is caused by a solution-filled gap between the sapphire window of the fluid cell lid and the top side of the monolith, which is difficult to avoid during the experimental setup. This must be taken into account when choosing the depth at which the measurement should be made. In a few cases, the formation of air bubbles was observed, which disrupted or even led to the termination of the experiment. However, this can be avoided by carefully setting up the experiment, which requires little practice.

Fluid-cell Raman Spectroscopy (FCRS) enables in operando observations of reaction and transport phenomena during the aqueous corrosion of silicate glasses at a microscopic level, at elevated temperatures, and in real-time. Without interrupting ongoing processes, FCRS provides information about reaction mechanisms, kinetics, and transport processes.

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

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