M.-H.K., S.K., M.L., and J.P. acknowledge the financial support from the Institute for Basic Science (Grant No. IBS-R006-D1). S.K., M.L., and J.P. acknowledge the financial support from Creative-Pioneering Researchers Program through Seoul National University (2021) and the NRF grant funded by the Korean government (MSIT; Grant Nos. NRF-2020R1A2C2101871, and NRF-2021M3A9I4022936). M.L. and J.P. acknowledge the financial support from the POSCO Science Fellowship of POSCO TJ Park Foundation and the NRF grant funded by the Korean government (MSIT; Grant No. NRF-2017R1A5A1015365). J.P. acknowledges the financial support from the NRF grant funded by the Korean government (MSIT; Grant No. NRF-2020R1A6C101A183), and the Interdisciplinary Research Initiatives Programs by College of Engineering and College of Medicine, Seoul National University (2021). M.-H.K. acknowledges the financial support from the NRF grant funded by the Korean government (MSIT; Grant No. NRF-2020R1I1A1A0107416612). The authors thank the staff and crew of the Seoul National University Center for Macromolecular and Cell Imaging (SNU CMCI) for their untiring efforts and perseverance with the cryo-EM experiments. The authors thank S. J. Kim of the National Center for Inter-university Research Facilities for assistance with the FIB-SEM experiments.

1. Fabrication of micro-patterned chip with GO windows (Figure 1)
<ol>
	<li>Deposit the silicon nitride.
	<ol>
		<li>Deposit low-stress silicon nitride (SixNy) on both sides of the Si wafer (4 inch diameter and 100 &#181;m thickness) using low-pressure chemical vapor deposition (LPCVD) at 830 &#176;C and a pressure of 150 mTorr, under a flow of 170 sccm dichlorosilane (SiH2Cl2, DCS) and 38 sccm ammonia (NH3).</li>
		<li>Using a deposition rate of ~30 &#197;/min, control the SixNy thickness to be within 25-100 nm by varying the deposition time. 
		NOTE: Extreme care should be taken when handling the Si wafer because the wafer is very thin and fragile. Take care not to bend the wafer during its handling or loading in the equipment.</li>
	</ol>
	</li>
	<li>Pattern the photoresist.
	<ol>
		<li>Apply a hexamethyldisilazane (HMDS) solution on the SixNy-deposited Si wafer with enough volume to cover the entire surface of the wafer, spin coat with a spin coater at 3,000 rpm for 30 s, and bake at 95&#176;C for 30 s on a hot plate to render the wafer surface hydrophobic and thus ensure a good coating performance with photoresist (PR).</li>
		<li>Apply positive PR (Table of Materials) with enough volume to cover the entire surface of the wafer, spin coat at 3,000 rpm for 30 s, and bake at 100 &#176;C for 90 s on a hot plate. Spin-coated PR has a thickness of 500 nm.</li>
		<li>Expose the PR-coated wafer with ultraviolet light (365 nm wavelength and 20 mW/cm2 intensity) for 5 s through a chromium mask (Figure 2A-D) using an aligner.</li>
		<li>Develop the PR for 1 min using a developer (Table of Materials) and rinse the wafer by immersing it in deionized (DI) water 2x. Fully dry the PR-patterned wafer by blowing N2 gas onto the wafer surface. 
		NOTE: Extreme care should be taken while blowing N2 gas onto the Si wafer because the wafer is very thin and fragile. Do not blow N2 gas with high pressure in a direction perpendicular to the wafer, as this may cause the wafer to fracture.</li>
	</ol>
	</li>
	<li>Pattern the SixNy.
	<ol>
		<li>Etch the exposed SixNy following the patterning of the PR using a lab-built reactive ion etcher (RIE), with 3 sccm sulfur hexafluoride (SF6) gas at a radiofrequency (RF) power of 50 W. The etching rate with these settings is ~6 &#197;/s. Set the etching time depending on the thickness of the SixNy layer deposited. 
		NOTE: The etching rate may vary and need in-lab optimization depending on the specifications of the RIE equipment used.</li>
		<li>Eliminate the PR by immersing the SixNy patterned wafer in acetone at room temperature for 30 min, followed by rinsing the wafer by immersing it in DI water 2x. Fully dry the wafer by blowing N2 gas onto the wafer surface. 
		NOTE: Extreme care should be taken while immersing or taking out the wafer from the solutions because the wafer can be fractured by the surface tension of the solution. Do not immerse or take out the wafer parallel to the surface of the solution. Use precision wafer handling tweezers with carbon fiber tips. Do not strongly grab the wafer with the tweezer; lift one side of the wafer until the wafer tilts to an angle, where it can be taken out from the solution. The wafer may fracture when it bends due to the firm grip during lifting.</li>
	</ol>
	</li>
	<li>Etch the Si.
	<ol>
		<li>Prepare a 1.5 M potassium hydroxide (KOH) solution by dissolving KOH powder in DI water at 80 &#176;C.</li>
		<li>Immerse the SixNy patterned wafer in KOH solution to etch the exposed Si. Leave the wafer in the solution with stirring until the free-standing SixNy windows can be observed at the opposite side of the patterned SixNy. 
		NOTE: The wet etching time may differ depending on the thickness of the Si; for a 100 &#181;m thick wafer, wet etching normally takes several hours. Do not set the stirring speed too high during Si etching because the free-standing SixNy windows are very thin and can be fractured by the flow of the fluid. In this experiment, the stirring rate was set to 250 rpm.</li>
		<li>Clean the etched wafer by dipping it several times in a DI water bath to eliminate etching residues. Dry the wafer in the air. 
		NOTE: Extreme care should be taken while immersing or taking out the Si patterned wafer from the solutions because the free-standing SixNy windows are very thin and fragile and can be fractured by the surface tension of the solution. The wafer should be immersed or taken out at an angle, such that the edge of the wafer enters and exits the solution first.</li>
	</ol>
	</li>
	<li>Eliminate the KOH etching residues.
	<ol>
		<li>Lightly press the boundaries of the chip array with a tweezer to obtain an array of chips that will be micro-patterned (Figure 1B).</li>
		<li>Prepare 1.5 M KOH solution at 80 &#176;C with stirring.</li>
		<li>Immerse the chip array in KOH solution for 30 s and rinse it by dipping it in DI water 2x. Fully dry the chips by blowing N2 gas. 
		NOTE: Extreme care should be taken while dipping the chips in solutions and blow-drying them with N2 gas because the free-standing SixNy windows are very thin and fragile. While the chip is immersed in KOH solution, stirring should be stopped. The chips should be dipped with their edges first in the direction perpendicular to the solution and blown with N2 gas in the parallel direction.</li>
		<li>Fully dry the chip array in the air for at least 1 h.</li>
	</ol>
	</li>
	<li>Pattern the PR.
	<ol>
		<li>Prepare a blank 525 &#181;m Si wafer as solid support. Spin coat the Si wafer with HMDS and positive PR, as described above, but attach the chip array (with the free-standing SixNy window side upward) on the Si wafer before baking the PR. The PR acts as an adhesive between the wafer and the chip array. Bake the Si wafer attached with the chip array at 100 &#176;C for 90 s on a hot plate.</li>
		<li>Spin coat the chip set with HMDS and positive PR, as described above.</li>
		<li>Expose the chip set with ultraviolet light (365 nm wavelength; 20 mW/cm2 intensity) for 5 s through a chromium mask (Figure 2E,F) using an aligner.</li>
		<li>Develop the PR using a developer for 15 s, rinse the chip set by dipping it in DI water 2x, and fully dry the PR patterned chip set by blowing N2 gas.</li>
	</ol>
	</li>
	<li>Prepare the micro-patterned SixNy.
	<ol style="margin-left: 40px;">
		<li>Etch SixNy following the PR patterning using a lab-built RIE, with 3 sccm SF6 gas at RF power of 50 W. Control the etching time depending on the thickness of the SixNy layer.</li>
	</ol>
	</li>
	<li>Eliminate the PR.
	<ol>
		<li>Eliminate the PR by immersing the patterned chip set in 1-methyl-2-pyrrolidinone (NMP) solution at 60 &#176;C and leaving it overnight. Rinse the chip set by dipping it in DI water 2x, and fully dry the patterned chip set by blowing N2 gas.</li>
		<li>Eliminate the PR residues with an O2 plasma process using 100 sccm O2 gas at RF power of 150 W for 1 min with the lab-built RIE.</li>
	</ol>
	</li>
	<li>Rinse the micro-patterned chip.
	<ol>
		<li>Prepare 1.5 M KOH solution at 80 &#176;C.</li>
		<li>Immerse the micro-patterned chips in KOH solution for 30 s to fully eliminate the PR residues and rinse the chips by immersing them in DI water 2x. Fully dry the chips by blowing N2 gas.</li>
		<li>Fully dry the chips in the air for at least 1 h.</li>
	</ol>
	</li>
	<li>Transfer graphene oxide (GO) by the drop-casting method.
	<ol>
		<li>Dilute GO solution (2 mg/mL) to 0.2 mg/L with DI water and sonicate for 10 min to break up aggregates of GO sheets. Centrifuge the diluted GO solution at 300 x g for 30 s.</li>
		<li>Glow discharge the Si-etched side of the micro-patterned chip to render the chip surface with positive charge using a glow discharger (Table of Materials) at 15 mA for 1 min.</li>
		<li>Drop 3 &#181;L of the GO solution onto the glow discharged side of the micro-patterned chip and leave the drop on the chip for 1 min. After 1 min, blot away the excess GO solution on the chip with filter paper.</li>
		<li>Wash the GO-transferred chip with DI water droplets prepared on paraffin film and blot away the DI water on the chip with filter paper. Repeat this procedure 2x on the GO transferred side and 1x on the opposite side. Dry the GO-transferred chip at room temperature overnight.</li>
		<li>Wash the micro-patterned chip with GO windows by immersing it in the DI water and blow-dry the chip with N2 gas.</li>
	</ol>
	</li>
</ol>
2. Cryo-EM imaging
<ol>
	<li>Prepare the cryo-sample.
	<ol>
		<li>Prepare the cryo-sample using a mechanical cryo-plunging machine (Table of Materials), which controls the temperature, humidity, blotting time, and force. After loading the blotting pad onto the blotters, ensure that the humidity and the temperature in the chamber are maintained at 100% and 15 &#176;C, respectively.</li>
		<li>Pick up the micro-patterned chip with a typical cryo-tweezer and load the tweezer to the cryo-plunging machine. Pipet 3 &#181;L of sample solution onto the micro-patterned chip at the hole-patterned side, with GO windows on the bottom. Control the blotting time and force depending on the sample solution. 
		NOTE: Here, biological specimens, namely human immunodeficiency virus (HIV-1), ferritin, proteasome 26S, groEL, apoferritin protein particles, and tau filament proteins were used for cryo-EM imaging. In addition, diverse types of inorganic materials, such as Fe2O3 nanoparticles (NP), Au nanoparticles, Au nanorods, and silica nanoparticles, were used for cryo-EM imaging. The desired blotting time and force were set on the cryo plunger for different types of samples.</li>
		<li>After the blotting process, plunge-freeze the sample-loaded chip immediately in liquid ethane. Transfer the chip to the grid box in liquid nitrogen (LN2) and store it in LN2 before cryo-EM imaging.</li>
	</ol>
	</li>
	<li>Carry out cryo-EM imaging.
	<ol>
		<li>Load the cryo-sample to a cryo-EM holder with the temperature maintained at -180 &#176;C.</li>
		<li>Load the cryo-EM holder into a TEM and observe the samples with the minimum dose system (MDS) mode.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest.

The microfabrication processes for producing micro-patterned chips with GO windows are introduced here. The fabricated micro-patterned chip is designed to regulate the thickness of the vitreous ice layer by controlling the depth of the micro-hole with GO windows depending on the size of the material to be analyzed. A micro-patterned chip with GO windows was fabricated using a series of MEMS techniques and a 2D nanosheet transfer method (Figure 1). The major advantage of using the MEMS fabric...

Cryogenic electron microscopy (cryo-EM) has been developed to resolve the three-dimensional (3D) structure of proteins in their native state1,2,3,4. The technique involves fixing proteins in a thin layer (10-100 nm) of vitreous ice and acquiring projection images of randomly oriented proteins using a transmission electron microscope (TEM), with the sample maintained at liquid nitrogen temperature. Thousands to millions of projection images are acquired and used to reconstruct a 3D structure of the protein by computational algorithms5,6. For successful analysis with cryo-EM, cryo-sample preparation has been automated by plunge-freezing the equipment that controls the blotting conditions, humidity, and temperature. The sample solution is loaded onto a TEM grid with a holey carbon membrane, successively blotted to remove the excess solution, and then plunge-frozen with liquid ethane to produce thin, vitreous ice1,5,6. With the advances in cryo-EM and the automation of sample preparation7, cryo-EM has been increasingly used to solve the structure of proteins, including envelope proteins for viruses and ion channel proteins in the cell membrane8,9,10. The structure of envelope proteins of pathogenic viral particles is important for understanding viral infection pathology, as well as developing the diagnosis system and vaccines e.g., SARS-CoV-211, which caused the COVID-19 pandemic. Moreover, cryo-EM techniques have recently been applied to material sciences, such as for imaging beam-sensitive materials used in battery12,13,14 and catalytic systems14,15 and analyzing the structure of inorganic materials in solution-state16.
Despite noticeable developments in cryo-EM and relevant techniques, limitations exist in cryo-sample preparation, hindering high-throughput 3D structure analysis. Preparing a vitreous ice film with optimal thickness is especially important for obtaining the 3D structure of biological materials with atomic resolution. The ice must be thin enough to minimize background noise from electrons scattered by the ice and to prohibit overlaps of biomolecules along the electron beam path1,17. However, if the ice is too thin, it can cause protein molecules to align in preferred orientations or denature18,19,20. Therefore, the thickness of vitreous ice should be optimized depending on the size of the material of interest. Moreover, extensive effort is typically needed for the sample preparation and manual screening of ice and protein integrity on the prepared TEM grids. This process is extremely time-consuming, which hinders its efficiency for high-throughput 3D structure analysis. Therefore, improvements in the reliability and reproducibility of cryo-EM sample preparation would enhance the utilization of cryo-EM in structural biology and commercial drug discovery, as well as for material science.
Herein, we introduce microfabrication processes for making a micro-patterned chip with graphene oxide (GO) windows designed for high-throughput cryo-EM with controlled ice thickness21. The micro-patterned chip was fabricated using microelectromechanical system (MEMS) techniques, which can manipulate the structure and dimensions of the chip depending on the imaging purposes. The micro-patterned chip with GO windows has a microwell structure that can be filled with the sample solution, and the depth of the microwell can be regulated to control the thickness of the vitreous ice. The strong affinity of GO for biomolecules enhances the concentration of biomolecules for visualization, improving the efficiency of the structure analysis. Furthermore, the micro-patterned chip is composed of an Si frame, which provides high mechanical stability for the grid19, making it ideal for handling the chip during sample preparation procedures and cryo-EM imaging. Therefore, a micro-patterned chip with GO windows fabricated by MEMS techniques provides reliability and reproducibility of cryo-EM sample preparation, which can enable efficient and high-throughput structure analysis based on cryo-EM.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-methyl-2-pyrrolidinone (NMP)</td>
			<td>Sigma Aldrich, USA</td>
			<td>443778</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AFM</td>
			<td>Park Systems, South Korea</td>
			<td>NX-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Aligner</td>
			<td>Midas System, South Korea</td>
			<td>MDA-600S</td>
			<td></td>
		</tr>
		<tr>
			<td>AZ 300 MIF developer</td>
			<td>AZ Electronic Materials USA Corp., USA</td>
			<td>184411</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo-EM holder</td>
			<td>Gatan, USA</td>
			<td>626 single tilt cryo-EM holder</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo-plunging machine</td>
			<td>Thermo Fisher SCIENTIFIC, USA</td>
			<td>Vitrobot Mark IV</td>
			<td></td>
		</tr>
		<tr>
			<td>Focused ion beam-scanning electron microscopy (FIB-SEM)</td>
			<td>FEI Company, USA</td>
			<td>Helios NanoLab 650</td>
			<td></td>
		</tr>
		<tr>
			<td>Glow discharger</td>
			<td>Ted Pella Inc., USA</td>
			<td>PELCO easiGlow</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphene oxide (GO) solution</td>
			<td>Sigma Aldrich, USA</td>
			<td>763705</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisizazne (HMDS), 98+%</td>
			<td>Alfa Aesar, USA</td>
			<td>10226590</td>
			<td></td>
		</tr>
		<tr>
			<td>Low pressure chemical vapor deposition (LPCVD)</td>
			<td>Centrotherm, Germany</td>
			<td>LPCVD E1200</td>
			<td></td>
		</tr>
		<tr>
			<td>maP1205 positive PR</td>
			<td>Micro resist technology, Germany</td>
			<td>A15139</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide (KOH), flake</td>
			<td>DAEJUNG CHEMICALS &#38; METALS Co. LTD., South Korea</td>
			<td>6597-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>Raman Spectrometer</td>
			<td>NOST, South Korea</td>
			<td>Confocal Micro Raman System HEDA</td>
			<td></td>
		</tr>
		<tr>
			<td>Reactive ion etcher (RIE)</td>
			<td>Scientific Engineering, South Korea</td>
			<td>Lab-built</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM</td>
			<td>Carl Zeiss, Germany</td>
			<td>SUPRA 55VP</td>
			<td></td>
		</tr>
		<tr>
			<td>Si wafer</td>
			<td>JP COMMERCE, South Korea</td>
			<td></td>
			<td>4&#34; Silicon wafer, P(B)type, (100), 1-30ohm.c m, DSP, T:100um</td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Dong Ah Trade Corp., South Korea</td>
			<td>ACE-200</td>
			<td></td>
		</tr>
		<tr>
			<td>TEM</td>
			<td>JEOL, Japan</td>
			<td>JEM-2100F</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of micro-patterned chip with controlled thickness for high-throughput cryogenic electron microscopy

A major limitation for the efficient and high-throughput structure analysis of biomolecules using cryogenic electron microscopy (cryo-EM) is the difficulty of preparing cryo-EM samples with controlled ice thickness at the nanoscale. The silicon (Si)-based chip, which has a regular array of micro-holes with graphene oxide (GO) window patterned on a thickness-controlled silicon nitride (SixNy) film, has been developed by applying microelectromechanical system (MEMS) techniques. UV photolithography, chemical vapor deposition, wet and dry etching of the thin film, and drop-casting of 2D nanosheet materials were used for mass-production of the micro-patterned chips with GO windows. The depth of the micro-holes is regulated to control the ice thickness on-demand, depending on the size of the specimen for cryo-EM analysis. The favorable affinity of GO toward biomolecules concentrates the biomolecules of interest within the micro-hole during cryo-EM sample preparation. The micro-patterned chip with GO windows enables high-throughput cryo-EM imaging of various biological molecules, as well as inorganic nanomaterials.

A micro-patterned chip with GO windows was fabricated by MEMS fabrication and 2D GO nanosheet transfer. Chips for micro-patterning were mass-produced, with about 500 chips produced from one 4 in wafer (Figure 1B and Figure 2A,B). The designs of the micro-patterned chips can be manipulated using different designs of the chromium mask (Figure 2) during the photolithography procedure. The fabricated micro-patterned chi...

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Fabrication of Micro-Patterned Chip with Controlled Thickness for High-Throughput Cryogenic Electron Microscopy

A newly developed micro-patterned chip with graphene oxide windows is fabricated by applying microelectromechanical system techniques, enabling efficient and high-throughput cryogenic electron microscopy imaging of various biomolecules and nanomaterials.

A major limitation for the efficient and high-throughput structure analysis of biomolecules using cryogenic electron microscopy (cryo-EM) is the ...

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Research

JoVE Journal

Engineering

2.5K Views.  The Catholic University of Korea. A newly developed micro-patterned chip with graphene oxide windows is fabricated by applying microelectromechanical system techniques, enabling efficient and high-throughput cryogenic electron microscopy imaging of various biomolecules and nanomaterials.

Video: Fabrication of Micro-Patterned Chip with Controlled Thickness for High-Throughput Cryogenic Electron Microscopy

Fabrication of Gate-tunable Graphene Devices for Scanning Tunneling Microscopy Studies with Coulomb Impurities

Owing to its relativistic low-energy charge carriers, the interaction between graphene and various impurities leads to a wealth of new physics and degrees of freedom to control electronic devices. In particular, the behavior of graphene&#8217;s charge carriers in response to potentials from charged Coulomb impurities is predicted to differ significantly from that of most materials. Scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS) can provide detailed information on both the spatial and energy dependence of graphene&#39;s electronic structure in the presence of a charged impurity.&#160;The design of a hybrid impurity-graphene device, fabricated using controlled deposition of impurities onto a back-gated graphene surface, has enabled several novel methods for controllably tuning graphene&#8217;s electronic properties.1-8 Electrostatic gating enables control of the charge carrier density in graphene and the ability to reversibly tune the charge2 and/or molecular5 states of an impurity. This paper outlines the process of fabricating a gate-tunable graphene device decorated with individual Coulomb impurities for combined STM/STS studies.2-5 These studies provide valuable insights into the underlying physics, as well as signposts for designing hybrid graphene devices.

This paper details the fabrication process of a gate-tunable graphene device, decorated with Coulomb impurities for scanning tunneling microscopy studies. Mapping the spatially dependent electronic structure of graphene in the presence of charged impurities unveils the unique behavior of its relativistic charge carriers in response to a local Coulomb potential.

Owing to its relativistic low-energy charge carriers, the interaction between graphene and various impurities leads to a wealth of new physics and degrees ...

Preparation of Liquid-exfoliated Transition Metal Dichalcogenide Nanosheets with Controlled Size and Thickness: A State of the Art Protocol

We summarize recent advances in the production of liquid-exfoliated transition metal dichalcogenide (TMD) nanosheets with controlled size and thickness. Layered crystals of molybdenum disulphide (MoS2) and tungsten disulphide (WS2) are exfoliated in aqueous surfactant solution by sonication. This yields highly polydisperse mixtures containing nanosheets with broad size and thickness distributions. However, for most purposes, specific sizes (in terms of both lateral dimension and thickness) are required. For example, large and thin nanosheets are desired for (opto) electronic applications, while laterally small nanosheets are interesting for catalytic applications. Therefore, post-exfoliation size selection is an important step that we address here. We provide a detailed protocol on the efficient size selection in large quantities by liquid cascade centrifugation and the size and thickness quantification by statistical microscopic analysis (atomic force microscopy and transmission electron microscopy). The comparison of MoS2 and WS2 shows that both materials are size-selected in a similar way by the same procedure. Importantly, the dispersions of size-selected nanosheets show systematic changes in their optical extinction spectra with size due to edge and confinement effects. We show how these optical changes are related quantitatively to the nanosheets dimensions and describe how mean nanosheets length and layer number can be extracted reliably from the extinction spectra. The exfoliation and size selection protocol can be applied to a broad range of layered crystals as we have previously demonstrated for graphene, gallium sulphide (GaS) and black phosphorus.

A protocol for the liquid exfoliation of layered materials to nanosheets, their size selection and size measurement by microscopic and spectroscopic techniques is presented.

We summarize recent advances in the production of liquid-exfoliated transition metal dichalcogenide (TMD) nanosheets with controlled size and thickness. ...

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 ...

Enhanced Electron Injection and Exciton Confinement for Pure Blue Quantum-Dot Light-Emitting Diodes by Introducing Partially Oxidized Aluminum Cathode

Stable and efficient red (R), green (G), and blue (B) light sources based on solution-processed quantum dots (QDs) play important roles in next-generation displays and solid-state lighting technologies. The brightness and efficiency of blue QDs-based light-emitting diodes (LEDs) remain inferior to their red and green counterparts, due to the inherently unfavorable energy levels of different colors of light. To solve these problems, a device structure should be designed to balance the injection holes and electrons into the emissive QD layer. Herein, through a simple autoxidation strategy, pure blue QD-LEDs which are highly bright and efficient are demonstrated, with a structure of ITO/PEDOT:PSS/Poly-TPD/QDs/Al:Al2O3. The autoxidized Al:Al2O3 cathode can effectively balance the injected charges and enhance radiative recombination without introducing an additional electron transport layer (ETL). As a result, high color-saturated blue QD-LEDs are achieved with a maximum luminance over 13,000 cd m-2, and a maximum current efficiency of 1.15 cd A-1. The easily controlled autoxidation procedure paves the way for achieving high-performance blue QD-LEDs.

A protocol is presented for fabricating high-performance, pure blue ZnCdS/ZnS-based quantum dots light-emitting diodes by employing an autoxidized aluminum cathode.

Stable and efficient red (R), green (G), and blue (B) light sources based on solution-processed quantum dots (QDs) play important roles in next-generation ...

Single-Digit Nanometer Electron-Beam Lithography with an Aberration-Corrected Scanning Transmission Electron Microscope

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by employing an aberration-corrected scanning transmission electron microscope as the exposure tool. Here, we present results of single-digit nanometer patterning of two widely used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. The method achieves sub-5 nanometer features in poly (methyl methacrylate) and sub-10 nanometer resolution in hydrogen silsesquioxane. High-fidelity transfer of these patterns into target materials of choice can be performed using metal lift-off, plasma etch, and resist infiltration with organometallics.

We use an aberration-corrected scanning transmission electron microscope to define single-digit nanometer patterns in two widely-used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. Resist patterns can be replicated in target materials of choice with single-digit nanometer fidelity using liftoff, plasma etching, and resist infiltration by organometallics.

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by ...

Graphene-Assisted Quasi-van der Waals Epitaxy of AlN Film on Nano-Patterned Sapphire Substrate for Ultraviolet Light Emitting Diodes

This protocol demonstrates a method for graphene-assisted quick growth and coalescence of AlN on nano-pattened sapphire substrate (NPSS). Graphene layers are directly grown on NPSS using catalyst-free atmospheric-pressure chemical vapor deposition (APCVD). By applying nitrogen reactive ion etching (RIE) plasma treatment, defects are introduced into the graphene film to enhance chemical reactivity. During metal-organic chemical vapor deposition (MOCVD) growth of AlN, this N-plasma treated graphene buffer enables AlN quick growth, and coalescence on NPSS is confirmed by cross-sectional scanning electron microscopy (SEM). The high quality of AlN on graphene-NPSS is then evaluated by X-ray rocking curves (XRCs) with narrow (0002) and (10-12) full width at half-maximum (FWHM) as 267.2 arcsec and 503.4 arcsec, respectively. Compared to bare NPSS, AlN growth on graphene-NPSS shows significant reduction of residual stress from 0.87 GPa to 0.25 Gpa, based on Raman measurements. Followed by AlGaN multiple quantum wells (MQWS) growth on graphene-NPSS, AlGaN-based deep ultraviolet light-emitting-diodes (DUV LEDs) are fabricated. The fabricated DUV-LEDs also demonstrate obvious, enhanced luminescence performance. This work provides a new solution for the growth of high quality AlN and fabrication of high performance DUV-LEDs using a shorter process and less costs.

A protocol for graphene-assisted growth of high-quality AlN films on nano-patterned sapphire substrate is presented.

This protocol demonstrates a method for graphene-assisted quick growth and coalescence of AlN on nano-pattened sapphire substrate (NPSS). Graphene layers ...

High Throughput Analysis of Liquid Droplet Impacts

Experimental studies of liquid drop impacts on surfaces are often restricted in their scope due to the large range of possible experimental parameters such as material properties, impact conditions, and experimental configurations. Compounding this, drop impacts are often studied using data-rich high-speed photography, so that it is difficult to analyze many experiments in a detailed and timely manner. The purpose of this method is to enable efficient study of droplet impacts with high-speed photography by using a systematic approach. Equipment is aligned and calibrated to produce videos that can be accurately processed by a custom image processing code. Moreover, the file structure setup and workflow described here ensure efficiency and clear organization of data processing, which is carried out while the researcher is still in the lab. The image processing method extracts the digitized outline of the impacting droplet in each frame of the video, and processed data are stored for further analysis as required. The protocol assumes that a droplet is released vertically under gravity, and impact is recorded by a camera viewing from side-on with the drop illuminated using shadowgraphy. Many similar experiments involving image analysis of high-speed events could be addressed with minor adjustment to the protocol and equipment used.

This protocol enables efficient collection of experimental high-speed images of liquid drop impacts, and speedy analysis of those data in batches. To streamline these processes, the method describes how to calibrate and set up apparatus, generate an appropriate data structure, and deploy an image analysis script.

Experimental studies of liquid drop impacts on surfaces are often restricted in their scope due to the large range of possible experimental parameters ...

Fabrication and Characterization of Thickness Mode Piezoelectric Devices for Atomization and Acoustofluidics

We present a technique to fabricate simple thickness mode piezoelectric devices using lithium niobate (LN). Such devices have been shown to atomize liquid more efficiently, in terms of &#64258;ow rate per power input, than those that rely on Rayleigh waves and other modes of vibration in LN or lead zirconate titanate (PZT). The complete device is composed of a transducer, a transducer holder, and a &#64258;uid supply system. The fundamentals of acoustic liquid atomization are not well known, so techniques to characterize the devices and to study the phenomena are also described. Laser Doppler vibrometry (LDV) provides vibration information essential in comparing acoustic transducers and, in this case, indicates whether a device will perform well in thickness vibration. It can also be used to &#64257;nd the resonance frequency of the device, though this information is obtained more quickly via impedance analysis. Continuous &#64258;uid atomization, as an example application, requires careful &#64258;uid flow control, and we present such a method with high-speed imaging and droplet size distribution measurements via laser scattering.

Fabrication of piezoelectric thickness mode transducers via direct current sputtering of plate electrodes on lithium niobate is described. Additionally, reliable operation is achieved with a transducer holder and &#64258;uid supply system and characterization is demonstrated via impedance analysis, laser doppler vibrometry, high-speed imaging, and droplet size distribution using laser scattering.

We present a technique to fabricate simple thickness mode piezoelectric devices using lithium niobate (LN). Such devices have been shown to atomize liquid ...

In Situ Transmission Electron Microscopy with Biasing and Fabrication of Asymmetric Crossbars Based on Mixed-Phased a-VOx

Resistive switching crossbar architecture is highly desired in the field of digital memories due to low cost and high-density benefits. Different materials show variability in resistive switching properties due to the intrinsic nature of the material used, leading to discrepancies in the field because of underlying operation mechanisms. This highlights a need for a reliable technique to understand mechanisms using nanostructural observations. This protocol explains a detailed process and methodology of in&#160;situ nanostructural analysis as a result of electrical biasing using transmission electron microscopy (TEM). It provides visual and reliable evidence of underlying nanostructural changes in real time memory operations. Also included is the methodology of fabrication and electrical characterizations for asymmetric crossbar structures incorporating amorphous vanadium oxide. The protocol explained here for vanadium oxide films can be easily extended to any other materials in a metal-dielectric-metal sandwiched structure. Resistive switching crossbars are predicted to serve the programmable logic and neuromorphic circuits for next-generation memory devices, given the understanding of the operation mechanisms. This protocol reveals the switching mechanism in a reliable, timely, and cost-effective way in any type of resistive switching materials, and thereby predicts the device&#39;s applicability.

In Situ Transmission Electron Microscopy with Biasing and Fabrication of Asymmetric Crossbars Based on Mixed-Phased a-VOx

Presented here is a protocol for analyzing nanostructural changes during in&#160;situ biasing with transmission electron microscopy (TEM) for a stacked metal-insulator-metal structure. It has significant applications in resistive switching crossbars for the next generation of programmable logic circuits and neuromimicking hardware, to reveal their underlying operation mechanisms and practical applicability.

Resistive switching crossbar architecture is highly desired in the field of digital memories due to low cost and high-density benefits. Different ...

Cryogenic Liquid Jets for High Repetition Rate Discovery Science

This protocol presents a detailed procedure for the operation of continuous, micron-sized cryogenic cylindrical and planar liquid jets. When operated as described here, the jet exhibits high laminarity and stability for centimeters. Successful operation of a cryogenic liquid jet in the Rayleigh regime requires a basic understanding of fluid dynamics and thermodynamics at cryogenic temperatures. Theoretical calculations and typical empirical values are provided as a guide to design a comparable system. This report identifies the importance of both cleanliness during cryogenic source assembly and stability of the cryogenic source temperature once liquefied. The system can be used for high repetition rate laser-driven proton acceleration, with an envisioned application in proton therapy. Other applications include laboratory astrophysics, materials science, and next-generation particle accelerators.

This protocol presents the operation and principles of micron-scale cylindrical and planar cryogenic liquid jets. Until now, this system has been used as a high repetition rate target in laser-plasma experiments. Anticipated cross-disciplinary applications range from laboratory astrophysics to material science, and eventually next-generation particle accelerators.

This protocol presents a detailed procedure for the operation of continuous, micron-sized cryogenic cylindrical and planar liquid jets. When operated as ...