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 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.
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.
1. Fabrication of micro-patterned chip with GO windows (Figure 1)
2. Cryo-EM imaging
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 chips had controlled numbers and dimensions of free-standing SixNy membranes. The numbers of the free-standing SixNy membranes were controlled from 48 (6 x 8) to 50 (5 x 10) and the dimensions from 50 x 40 μm2 to 250 x 40 μm2 (Figure 3A,B,F,G). Each free-standing SixNy membrane can have tens to hundreds of micro-holes with customizable diameters ranging from 2-3 μm with different hole spacing. Fabricated micro-patterned chips have up to ~25,000 GO-suspended holes, while the number of the holes is also controllable (Figure 3B–D and Figure 3G–I). The existence of the thin GO layer across the hole was confirmed by Raman spectroscopy and electron diffraction. The Raman spectrum at the GO window showed representative peaks of GO, namely D and G bands at 1360 cm-1 and 1590 cm-1, respectively22 (Figure 3E). The multiply-oriented hexagonal diffraction patterns indicate that the windows consist of multilayer GO (Figure 3J).
The micro-patterned chip with GO windows was fabricated in three representative target depths (25 nm, 50 nm, and 100 nm) by controlling the deposition thickness of the SixNy on the Si wafer during the LPCVD process to confirm the feasibility of regulating the depth of the micro-holes. To evaluate the structure and the thickness of the micro-holes with GO windows, 40°–tilted and cross-sectional scanning electron microscope (SEM) images and atomic force microscopy (AFM) images of the micro-patterned chip with GO windows were obtained. Well-type structure of the micro-hole with a GO window was clearly observed, with the depth of the micro-hole corresponding to the targeted depth (Figure 4). The results confirm that controlling the number and the design of the micro-patterned chip with GO windows is possible.
To demonstrate the use of the micro-patterned chip for cryo-EM imaging, various cryo-samples of biomolecules and inorganic NPs were prepared using the micro-patterned chip. For biological specimens, HIV-1, ferritin, proteasome 26S, groEL, apoferritin protein particles, and tau filament proteins were imaged with cryo-EM using the micro-patterned chip with GO windows (Figure 5A–F). Besides biomolecules, inorganic materials such as Fe2O3 NPs, Au NPs, Au nanorods, and silica NPs were also observed by cryo-EM using micro-patterned chips (Figure 5G–J).
Figure 1: Schematics and images of the fabrication procedure of the newly developed micro-patterned chip with GO windows for cryo-EM. (A) Schematics of the fabrication process and cross-sections of the micro-patterned chip with GO windows during the fabrication process. (B) Images of the fabrication products at each fabrication step. Please click here to view a larger version of this figure.
Figure 2: Brief illustration of the chromium masks used for the photolithography process. (A,B) Mask design for mass production of chips for a 4 in Si wafer (24 x 24 array of chips), (C,D) designs of 2 x 2 array of chips, and (E,F) designs of micro-hole patterns. Please click here to view a larger version of this figure.
Figure 3: Structure of the micro-patterned chips with GO windows. (A,F) Optical microscopy images of whole micro-patterned chips, (B,G) SEM images of single micro-patterned SixNy membranes, (C,H) SEM images of micro-patterns, and (D,I) SEM images of single micro-holes with GO windows. (E,J) Confirmation of GO at the micro-hole through (E) the Raman spectrum and (J) the selected area electron diffraction (SAED) pattern of the GO window. Please click here to view a larger version of this figure.
Figure 4: Well-structure and depth of the micro-hole with GO windows. (A–C) 40° tilted SEM images of a single micro-hole with a GO window, and (D–F) cross-sectional SEM image of the micro-patterned chip with GO windows in different depths (25 nm, 50 nm, and 100 nm). (G) Atomic force microscopy (AFM) 3D rendering image, (H) AFM deflection image, and (I) line profile along the red line in (H) showing the depth of the micro-patterned chip with GO windows fabricated with 100 nm SixNy membrane. Please click here to view a larger version of this figure.
Figure 5: Cryo-EM images of various sized biomaterials and inorganic nanomaterials using the micro-patterned chip with GO windows. (A) HIV-1 virus particle, (B) ferritin, (C) proteasome 26S, (D) groEL, (E) apoferritin, (F) tau protein (arrows indicating fibrillized tau protein), (G) Fe2O3 NP, (H) Au NP, (I) Au nanorod, and (J) silica NP. Please click here to view a larger version of this figure.
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 fabrication technique is its capability for mass production and the feasibility of manipulating the structure and dimensions of the microchip by using different designs of the chromium mask during photolithography (Figure 2). The LPCVD-deposited SixNy layer with low stress ensures the stability of the tens of nanometers thick free-standing SixNy23,24,25,26. However, the nanometer-scale free-standing SixNy layer is still vulnerable to forces in the perpendicular direction27. Therefore, extreme caution is needed while handling the micro-patterned chip, such as when dipping in solution or blow-drying. In addition, the fabrication process for the micro-patterned chip uses the 100 μm Si wafer, which ensures compatibility with most cryo-EM specimen holders and autoloaders. However, caution is needed during the fabrication processes to prevent the fragile wafer from fracturing.
The micron-scale regular array of well-type structures with GO windows was confirmed with an optical microscope and an SEM (Figure 3 and Figure 4). Besides, the drop casting method for transferring GO enables GO deposition with high flatness and without noticeable wrinkles (Figure 3D,E,I,J). The micro-patterned chip is suitable for loading in the cryo-EM autoloader, and tens of thousands of micron-scale holes in a regular array allow the automated collection of large image data for single particle analysis. Moreover, the number and the morphology of SixNy membranes and GO-supported micro-holes can be manipulated easily in the MEMS fabrication process, allowing for high-throughput single particle analysis and other cryo-EM imaging experiments depending on the research purposes. Furthermore, extended applications of micro-patterned chips with controlled thickness can be facilitated by the fabrication of chips that have holes patterned at the nanometer scale. Nano-patterning techniques developed in the semiconductor industry can be adopted in the fabrication of those chips28,29,30.
The capability of regulating the depth of the micro-holes has been demonstrated here by fabricating micro-patterned chips with GO windows in three representative target depths: 25 nm, 50 nm, and 100 nm. Different depths of the microwell structure were achieved by controlling the deposition time of the SixNy layer on the Si wafer (Figure 4). For evaluating the morphology and the thickness of the micro-patterned chip with GO windows, cross-sections of the devices obtained from focused ion beam (FIB) sectioning were observed with SEM, and the depth profile was measured with AFM (Figure 4). The well-type structure of the micro-hole with GO window was clearly shown in the SEM and AFM images, confirming successful control of the depth of the SixNy micro-hole and transfer of the GO window. The use of the customizable micro-patterned chip with GO windows is likely to ensure a high success rate in producing regions of optimal ice thickness for cryo-EM imaging.
Since the materials to be observed with cryo-EM have different sizes, producing vitreous ice with an appropriate thickness can ensure enhanced contrast resolution, a wide orientation coverage, and reduced denaturation of the structure during cryo-EM imaging. To demonstrate the use of the cryo-EM imaging process for biological applications, various biological samples of different sizes, including HIV-1, ferritin, proteasome 26S, groEL, apoferritin, and tau protein, were imaged using the micro-patterned chip with GO windows. The biomolecules were clearly observed using the micro-patterned chip with GO windows (Figure 5A–F). Besides biomolecules, diverse types of inorganic nanomaterials, such as Fe2O3 NPs, Au NPs, Au nanorods, and silica NPs, were also observed using the micro-patterned chip with GO windows (Figure 5G–J). The micro-patterned chip and fabrication method show compatibility for the cryo-imaging of various materials. Thus, the newly developed micro-patterned chip with GO windows provides a reliable and reproducible sample preparation strategy for efficient and high-throughput structure analysis with cryo-EM.
The authors have nothing to disclose.
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-methyl-2-pyrrolidinone (NMP) | Sigma Aldrich, USA | 443778 | |
Acetone | |||
AFM | Park Systems, South Korea | NX-10 | |
Aligner | Midas System, South Korea | MDA-600S | |
AZ 300 MIF developer | AZ Electronic Materials USA Corp., USA | 184411 | |
Cryo-EM holder | Gatan, USA | 626 single tilt cryo-EM holder | |
Cryo-plunging machine | Thermo Fisher SCIENTIFIC, USA | Vitrobot Mark IV | |
Focused ion beam-scanning electron microscopy (FIB-SEM) | FEI Company, USA | Helios NanoLab 650 | |
Glow discharger | Ted Pella Inc., USA | PELCO easiGlow | |
Graphene oxide (GO) solution | Sigma Aldrich, USA | 763705 | |
Hexamethyldisizazne (HMDS), 98+% | Alfa Aesar, USA | 10226590 | |
Low pressure chemical vapor deposition (LPCVD) | Centrotherm, Germany | LPCVD E1200 | |
maP1205 positive PR | Micro resist technology, Germany | A15139 | |
Potassium hydroxide (KOH), flake | DAEJUNG CHEMICALS & METALS Co. LTD., South Korea | 6597-4400 | |
Raman Spectrometer | NOST, South Korea | Confocal Micro Raman System HEDA | |
Reactive ion etcher (RIE) | Scientific Engineering, South Korea | Lab-built | |
SEM | Carl Zeiss, Germany | SUPRA 55VP | |
Si wafer | JP COMMERCE, South Korea | 4" Silicon wafer, P(B)type, (100), 1-30ohm.c m, DSP, T:100um | |
Spin coater | Dong Ah Trade Corp., South Korea | ACE-200 | |
TEM | JEOL, Japan | JEM-2100F |