Name: fabrication of micro-patterned chip with controlled thickness for high-throughput cryogenic electron microscopy
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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<...

<ol>
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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=Self-assembled+monolayers+improve+protein+distribution+on+holey+carbon+cryo-EM+supports.">Self-assembled monolayers improve protein distribution on holey carbon cryo-EM supports.</a> Scientific Reports. 4, (2014).</li><li>Palovcak, E., 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=A+simple+and+robust+procedure+for+preparing+graphene-oxide+cryo-EM+grids.">A simple and robust procedure for preparing graphene-oxide cryo-EM grids.</a> Journal of Structural Biology. 204 (1), 80-84 (2018).</li><li>Xu, B. 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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...

Watch this Scientific Journal Video about Fabrication of Micro-Patterned Chip with Controlled Thickness for High-Throughput Cryogenic Electron Microscopy at JoVE.com

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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