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

Revestindo grades TEM com uma monocamada de grafeno

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.

Autor em destaque: Aprimorando a resolução do CryoEM usando grades revestidas de grafeno 

Fabricação de grades revestidas de grafeno monocamada para microscopia crioeletrônica

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

Revestimento de Folha de Grafeno-Cobre de Camada Única com MMA e Limpeza das Grades

Para começar, dentro de um exaustor, coloque uma tampa com a folha de grafeno colada em um revestidor de spin caseiro. Usando óculos de segurança, use uma pipeta de vidro para adicionar duas gotas de metacrilato de metila, ou MMA, na folha de grafeno e comece imediatamente a girar na velocidade máxima. Ao girar, adicione mais duas gotas no centro e gire por um minuto.Depois de limpar e secar três pratos cristalizantes, despeje 200 mililitros de clorofórmio, acetona e álcool isopropílico em cada prato. Coloque a base do bloco de fixação do suporte da grelha TEM no fundo da primeira placa contendo 200 mililitros de clorofórmio. Garantindo que o lado da película fenestrada esteja voltado para cima, transfira cada grade individualmente da caixa de grade para um poço no bloco de suporte de grade.Cubra o prato com papel alumínio e coloque-o em um agitador orbital para agitar suavemente por 30 minutos. Em seguida, coloque a tampa metálica no bloco de suporte da grade para fixar as grades para transferência para a próxima placa de cristalização. Usando um garfo dobrado e um tweezer longo, levante cuidadosamente o bloco do suporte da grade para fora do prato e coloque-o no fundo do segundo prato contendo 200 mililitros de acetona.Retire a tampa do bloco do suporte da grade e agite-a suavemente em um agitador orbital. Após 30 minutos, coloque a tampa no bloco do suporte da grade e transfira-a para uma placa contendo 200 mililitros de álcool isopropílico para remover o resíduo de acetona. Retire a tampa do bloco do suporte da grade e agite-a suavemente por 20 minutos.Transfira individualmente as grades com o lado da película fenestrada para cima do bloco do suporte da grade para uma placa de Petri de vidro coberta com papel mata-borrão.

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

500 visualizações.  Scripps Research. Para começar, dentro de um exaustor, coloque uma tampa com a folha de grafeno colada em um revestidor de spin caseiro. Usando óculos de segurança, use uma pipeta de vidro para adicionar duas gotas de metacrilato de metila, ou MMA, na folha de grafeno e comece imediatamente a girar na velocidade máxima. Ao girar, adicione mais duas gotas no centro e gire por um minuto.Depois de limpar e secar três pratos cristalizantes, despeje 200 mililitros de clorofórmio, acetona e álcool isop...

Vídeo: Revestimento de Folha de Grafeno-Cobre de Camada Única com MMA e Limpeza das Grades

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 ...

Biologia

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.