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><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 &amp;amp; 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&quot; 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

2.6K Views.  The Catholic University of Korea. Dit protocol is belangrijk voor het reguleren van de dikte van het beeldformaat op nanoschaal om eiwitten en nanomaterialen van verschillende groottes met succes te observeren met behulp van cryo EM. De fabricagetechniek van de Mem maakt de massaproductie van de microchip mogelijk. Het maakt ook de selectie van diepten en ontwerpen van de micropatroonputten mogelijk. Afhankelijk van de experimentele doeleinden.De techniek kan bijdragen aan het verbeteren van de efficiëntie van 3D-stru...

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

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 ...

Atomically Traceable Nanostructure Fabrication

Reducing the scale of etched nanostructures below the 10 nm range eventually will require an atomic scale understanding of the entire fabrication process being used in order to maintain exquisite control over both feature size and feature density. Here, we demonstrate a method for tracking atomically resolved and controlled structures from initial template definition through final nanostructure metrology, opening up a pathway for top-down atomic control over nanofabrication. Hydrogen depassivation lithography is the first step of the nanoscale fabrication process followed by selective atomic layer deposition of up to 2.8 nm of titania to make a nanoscale etch mask. Contrast with the background is shown, indicating different mechanisms for growth on the desired patterns and on the H passivated background. The patterns are then transferred into the bulk using reactive ion etching to form 20 nm tall nanostructures with linewidths down to ~6 nm. To illustrate the limitations of this process, arrays of holes and lines are fabricated. The various nanofabrication process steps are performed at disparate locations, so process integration is discussed. Related issues are discussed including using fiducial marks for finding nanostructures on a macroscopic sample and protecting the chemically reactive patterned Si(100)-H surface against degradation due to atmospheric exposure.

We report a protocol for combining the atomic metrology of the Scanning Tunneling Microscope for surface patterning with selective Atomic Layer Deposition and Reactive Ion Etching. Using a robust process involving numerous atmospheric exposures and transport, 3D nanostructures with atomic metrology are fabricated.

Reducing the scale of etched nanostructures below the 10 nm range eventually will require an atomic scale understanding of the entire fabrication process ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Biology

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

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Biochemistry

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

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.

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.

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

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

Volume is an important parameter regarding physiological and pathological characteristics of cells. We describe a fluorescent exclusion method allowing full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution required for the analysis of neurites and dynamic structures implied in neuronal growth.

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique ...

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

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