coating single-layer graphene-copper sheet with mma and cleaning the grids

TEM 그리드를 그래핀 단층으로 코팅

Single-particle cryogenic electron microscopy (cryoEM) is an increasingly applicable technology used to investigate the 3D structures of biomacromolecules. Technological advances in electron microscope optics, direct electron detection1, and computer algorithms2,3,4 over the past decade have enabled cryoEM users to determine the structures of biochemically stable macromolecular complexes to near-atomic resolution5,6,7,8. Despite these advances, there remain notable barriers to preserving samples for cryoEM imaging, which prevent the majority of biological specimens from being resolved to such high resolutions.
Sample preparation for high-resolution cryoEM analysis involves trapping macromolecules that are evenly distributed in a wide range of orientations within a thin layer of vitrified ice. The &#34;blot and plunge&#34; methods of freezing are the most widely used methods employed to generate thin films of biological samples on grids for cryoEM studies9,10. These methods involve applying a few microliters of sample solution to an EM grid containing a fenestrated film that has been made hydrophilic and subsequently blotting away the majority of the sample with filter paper before rapidly plunging the grid into a cryogen of liquid ethane or ethane-propane mixture9.
While this method has successfully been used to determine structures of a wide range of biological specimens, all commonly used cryoEM specimen preparation methods expose specimens to the hydrophobic air-water interface (AWI), which often introduces&#160;issues that limit high-resolution structure determination. It is established that biological specimens have a high propensity to denature when exposed to the AWI, which can lead to complex aggregation and disassembly11,12,13,14. Furthermore, hydrophobic patches on the surfaces of biological specimens cause particles to adopt preferred orientations in the ice12. In many scenarios, a single hydrophobic region of the sample forces all particles to adopt a singular orientation in the ice, thereby abolishing the ability to generate a reliable reconstruction. In addition to issues with the AWI, specimens may demonstrate an affinity for the surface of the fenestrated layer of film, limiting the number of particles suspended in the ice within the holes15.
Several methodological and technological solutions have been developed to diminish these issues arising from interactions with the AWI or the films16,17. One popular approach is to coat the fenestrated film of the EM grids with a thin (tens of nanometers) layer of amorphous carbon. This coating provides a continuous surface across the holes to which particles can adsorb, with the benefit of partially shielding the sample from interactions with the AWI15,18,19,20. However, the additional carbon layer elevates the amount of background signal in the imaged regions, introducing noise that can compromise attainable resolution, particularly for small (&#60;150 kDa) specimens. In recent years, the use of graphene oxide (GO) flakes to produce supporting films on cryoEM grids has been shown to have advantages over traditional amorphous carbon. GO flakes are produced through the oxidation of graphite layers, resulting in pseudo-crystalline sheets of monolayer graphite that are hydrophilic due to their substantial oxygen content in the form of carboxyl, hydroxyl, and epoxy groups on the surfaces and edges. Commercial GO flakes in aqueous suspensions are inexpensive, and there are numerous published methods for applying GO flakes to EM grids18,21. However, these methods often result in grids that are only partially covered with GO flakes, as well as regions that contain multiple layers of GO flakes. Further, GO flakes contribute a noticeable background signal to cryoEM images that is close to that observed with thin amorphous carbon22,23.
Pristine monolayer graphene, which consists of a single 2D crystalline array of carbon atoms, is distinct from GO in that it does not produce phase contrast in the electron microscope. Monolayer graphene thus can be used to generate an invisible support layer for imaging biological samples. Monolayer graphene is also stronger than GO and can be applied as a single monolayer on an EM grid, and recent advancements in the fabrication of graphene-coated EM grids have made it possible to prepare high-coverage monolayer graphene grids in-house24,25,26,27,28,29,30. However, despite the benefits of using graphene-coated grids for cryoEM structure determination, they are not widely used due to the prohibitive expense of commercial options and the complexity of in-house production. Here, we describe a step-by-step guide to effectively producing EM grids covered with a monolayer of graphene for cryoEM structure determination of biological specimens (Figure 1). By following this detailed protocol, cryoEM researchers can reproducibly prepare dozens of high-quality graphene support grids in a single day. The quality of the graphene-coated grids can be readily examined using a low-end transmission electron microscope (TEM) equipped with a LaB6 filament.

저자 스포트라이트: Enhancing CryoEM Resolution Using Graphene-Coated Grids 

Fabrication of Monolayer Graphene-Coated Grids for Cryoelectron Microscopy(초저온 전자 현미경을 위한 단층 그래핀 코팅 그리드 제작)

Currently optimally preserving biological samples is a major challenge in obtaining high resolution CryoEM structures. During the sample preparation process, specimens are exposed to hydrophobic air-water interface, which has denaturing effects on the samples and can cause stable samples to adopt a preferential orientation in the ice, which prevents high resolution structure determination. Coating CryoEM grids with a thin layer of carbon of like soft graphene oxide, has been commonly used to address issues related to interactions with air-water interface.However, this layers often increase background noise in our images, which compromise the resolutions of our structure. Coating the grid with a monolayer of graphene accomplishes similar protection without introducing significant background noise. Using graphene-coated CryoEM grids can help overcome issues related to interactions with air-water interface, however, commercial graphene grids are expensive and many labs consider graphene grids to be too complicated to prepare in-house.Researchers in the CryoEM field can now use this step-by-step protocol to reproduce fully produced dozens of high quality graphenes in one day.

Watch this Scientific Journal Video about Coating Single-Layer Graphene-Copper Sheet with MMA and Cleaning the Grids at JoVE.com

단층 그래핀-구리 시트를 MMA로 코팅하고 그리드를 청소합니다.

시작하려면 흄 후드 내부에 테이프로 감싼 그래핀 시트가 있는 커버 슬립을 수제 스핀 코터에 놓습니다. 보안경을 착용하고 유리 피펫을 사용하여 메틸 메타크릴레이트(MMA) 두 방울을 그래핀 시트에 떨어뜨리면 즉시 최대 속도로 회전을 시작합니다. 회전하는 동안 중앙에 두 방울을 더 넣고 1분 동안 회전합니다.3개의 결정화 접시를 세척하고 건조시킨 후 각 접시에 클로로포름, 아세톤 및 이소프로필 알코올 200mL를 붓습니다. 클램핑 TEM 그리드 홀더 블록의 베이스를 클로로포름 200밀리리터가 들어 있는 첫 번째 접시의 바닥에 놓습니다. 창공 필름 면이 위를 향하도록 하여 각 그리드를 그리드 상자에서 그리드 홀더 블록의 웰로 개별적으로 옮깁니다.접시를 알루미늄 호일로 덮고 오비탈 셰이커에 올려 30분 동안 부드럽게 흔듭니다. 다음으로, 그리드 홀더 블록에 금속 뚜껑을 놓아 다음 결정화 접시로 옮길 그리드를 고정합니다. 구부러진 포크와 긴 핀셋을 사용하여 접시에서 그리드 홀더 블록을 조심스럽게 들어 올려 200ml의 아세톤이 들어 있는 두 번째 접시 바닥에 놓습니다.그리드 홀더 블록에서 뚜껑을 제거하고 오비탈 셰이커에서 부드럽게 흔듭니다. 30분 후 그리드 홀더 블록에 뚜껑을 덮고 아세톤 잔여물을 제거하기 위해 200mL의 이소프로필 알코올이 들어 있는 접시에 옮깁니다. 그리드 홀더 블록에서 뚜껑을 제거하고 20분 동안 부드럽게 흔듭니다.창공 필름 면이 위로 향하게 하여 그리드 홀더 블록에서 압지로 덮인 유리 페트리 접시로 그리드를 개별적으로 옮깁니다.

coating-single-layer-graphene-copper-sheet-with-mma-cleaning

Research

JoVE Journal

Biology

Coating Single-Layer Graphene-Copper Sheet with MMA and Cleaning the Grids

504 조회수.  Scripps Research. 시작하려면 흄 후드 내부에 테이프로 감싼 그래핀 시트가 있는 커버 슬립을 수제 스핀 코터에 놓습니다. 보안경을 착용하고 유리 피펫을 사용하여 메틸 메타크릴레이트(MMA) 두 방울을 그래핀 시트에 떨어뜨리면 즉시 최대 속도로 회전을 시작합니다. 회전하는 동안 중앙에 두 방울을 더 넣고 1분 동안 회전합니다.3개의 결정화 접시를 세척하고 건조시킨 후 각 접시에 클?...

비디오: 단층 그래핀-구리 시트를 MMA로 코팅하고 그리드를 청소합니다.

Fabrication of Monolayer Graphene-Coated Grids for Cryoelectron Microscopy

Cryogenic electron microscopy (cryoEM) has emerged as a powerful technique for probing the atomic structure of macromolecular complexes. Sample preparation for cryoEM requires preserving specimens in a thin layer of vitreous ice, typically suspended within the holes of a fenestrated support film. However, all commonly used sample preparation approaches for cryoEM studies expose the specimen to the air-water interface, introducing a strong hydrophobic effect on the specimen that often results in denaturation, aggregation, and complex dissociation. Further, preferred hydrophobic interactions between regions of the specimen and the air-water interface impact the orientations adopted by the macromolecules, resulting in 3D reconstructions with anisotropic directional resolution. 
Adsorption of cryoEM specimens to a monolayer of graphene has been shown to help mitigate interactions with the air-water interface while minimizing the introduction of background noise. Graphene supports also offer the benefit of substantially lowering the required concentration of proteins required for cryoEM imaging. Despite the advantages of these supports, graphene-coated grids are not widely used by the cryoEM community due to the prohibitive expense of commercial options and the challenges associated with large-scale in-house production. This paper describes an efficient method for preparing batches of cryoEM grids that have nearly full coverage of monolayer graphene.

Here, we describe a protocol for applying a single monolayer of graphene to electron microscopy grids and how to prepare them for use in cryoEM structure determination.

Cryogenic electron microscopy (cryoEM) has emerged as a powerful technique for probing the atomic structure of macromolecular complexes. Sample ...

연구

생물학

To begin, inside a fume hood, place a cover slip with the taped graphene sheet on a homemade spin coater. Wearing safety goggles, use a glass pipette to add two drops of methyl methacrylate, or MMA, onto the graphene sheet, and immediately start spinning at maximum speed. While spinning, add two more drops in the center and spin for one minute.After cleaning and drying three crystallizing dishes, pour 200 milliliters of chloroform, acetone, and isopropyl alcohol into each dish. Place the base of the clamping TEM grid holder block at the bottom of the first dish containing 200 milliliters of chloroform. Ensuring the fenestrated film side is facing up, transfer each grid individually from the grid box to a well in the grid holder block.Cover the dish with aluminum foil and place it on an orbital shaker to gently shake for 30 minutes. Next, place the metal lid on the grid holder block to secure the grids for transfer to the next crystallizing dish. Using a bent fork and a long tweezer, carefully lift the grid holder block out of the dish and place it at the bottom of the second dish containing 200 milliliters of acetone.Remove the lid from the grid holder block and gently shake it on an orbital shaker. After 30 minutes, place the lid on the grid holder block and transfer it to a dish containing 200 milliliters of isopropyl alcohol to remove acetone residue. Remove the lid from the grid holder block and gently shake it for 20 minutes.Individually transfer the grids with the fenestrated film side up from the grid holder block to a glass Petri dish covered with blotting paper.

Transferring Grids to a Perforated Tray Under DI Water, Transferring Graphene to the Grids, Removing MMA, and Cleaning the Grids

Transfer the cleaned grids with the fenestrated film side facing up onto the wet blotting paper. Submerge the hydrophobic grids vertically into the water to prevent bending due to water tension. Arrange the submerged grids in a square array, keeping them close but not overlapping.Fill the grid coating trough with more deionized water, ensuring the water surface is at least five millimeters above the grids. To scoop out the MMA graphene film from the dish, slowly lower the cover slip into the dish at an angle, some distance away from the graphene film. Position the cover slip underneath the graphene MMA sheet and align its edges parallelly with the sheet.Then, lift the cover slip vertically from the water, bringing the MMA graphene film with it. Transfer the MMA graphene film to the trough by lowering the cover slip into the water at a 45-degree angle so that the film detaches from the cover slip and floats on the water surface. Position the MMA graphene film above the grids before the water level decreases.Using a Pasteur pipette whose tip has been melted closed, carefully manipulate the MMA graphene film's position. Using a syringe, slowly lower the water level such that the MMA graphene film perfectly lands on the grids and completely covers their surfaces. Using a pair of clean, dry tweezers, lift the blotting paper holding the grids to a clean, dust-free Petri dish.Place the base of the clamping TEM grid holder block at the bottom of the first crystallizing dish containing 200 milliliters of fresh acetone. Individually, transfer each grid with the MMA side facing up from the blotting paper to a well in the grid holder block. Cover the dish with foil, and gently shake on an orbital shaker for 30 minutes.Secure the grids by placing the metal lid on the grid holder block. Then, using a bent fork or long tweezers, carefully lift the block out of the dish and place it at the bottom of the second dish containing 200 milliliters of fresh acetone. After removing the lid from the block, gently shake it on an orbital shaker for 30 minutes.Place the lid back on the grid holder block and transfer it to the dish containing 200 milliliters of isopropyl alcohol. Remove the lid before gently shaking for 20 minutes. Transfer the grids to a small glass Petri dish covered with blotting paper and air dry the grids for 10 minutes.In TEM diffraction modes, EM grids covered with a monolayer of graphene show Bragg peaks corresponding to the hexagonal lattice of the graphene. Graphene grids have a concentrating effect on a sample as observed when comparing apoferritin applied to holy gold grids with and without the graphene support. A fast Fourier transform of the cryoEM micrograph of apoferritin on graphene-covered gold grids also showed Bragg peaks corresponding to the hexagonal graphene lattice.