Method for functionalizing graphene, apparatus, and functionalized graphene product

Abstract

To provide a substrate holder and a substrate mounted thereon for use in electron microscopy, a method for functionalizing graphene or graphene oxide on the substrate, an apparatus for carrying out the method, and a product formed by the same.SOLUTION: A substrate having a graphene layer is held in a plasma treatment chamber and exposed to a plasma containing helium and at least one functionalizing compound for functionalization. Additionally, a mask is applied and removed to and from the substrate to form multiple functionalization zones.SELECTED DRAWING: Figure 12

Description

[Technical field]

[0001] The present invention relates to a method for functionalizing graphene, an apparatus for carrying out such functionalization, and products characterized by such functionalization, and in particular, but not exclusively, to their application in the field of electron microscopy, in particular electron cryomicroscopy (also called cryoelectron microscopy). [Background technology]

[0002] Thanks to recent technological advances, atomic resolution structures of purified biomolecular complexes can in principle be determined by single particle cryo-electron microscopy (cryo-EM). In practice, the biggest barrier to structure determination is the preparation of frozen samples suitable for high-resolution imaging.

[0003] High-resolution structural studies by cryo-EM require embedding biological samples in a thin film of amorphous water ice 1、2 During preparation and cryo-plunging, the specimen is exposed to the surface of this thin film of water and may be subject to deleterious interactions at the air-water interface immediately prior to vitrification. 1、3 These interactions can be improved with the help of a thin film covering the sample support. 4 , which provides an intimate contact surface for the sample, but is usually accompanied by background noise, movement during imaging, contamination, and the introduction of uncontrolled sample-surface forces.

[0004] Single-layer graphene has been proposed as a possible support membrane. 5、6、7、8 However, pure graphene is not a suitable substrate for preparing cryo-EM samples due to its hydrophobicity. Previous studies have demonstrated that partial hydrogenation using low-energy plasma can make graphene hydrophilic without destroying its lattice, making it suitable for cryo-EM. 8 . Summary of the Invention [Problem to be solved by the invention]

[0005] There is a need for improved supports and substrates for cryo-EM samples and suitable methods for their manufacture.

[0006] The present invention has been devised in light of the above considerations. [Means for solving the problem]

[0007] The present invention is based on the insight that by introducing functionalization into graphene or graphene oxide films the surface properties can be tailored for specific applications in the field of electron microscopy or cryo-EM. Furthermore, the inventors have discovered that the use of helium plasma as a carrier for functionalizing compounds provides relatively mild conditions that avoid damage to graphene during the plasma treatment / functionalization process.

[0008] Thus, a new multifunctional sample support for electron microscopy, e.g. cryo-EM, is presented. The support consists of, for example, large crystals of single-layer graphene or graphene oxide suspended on the surface of an ultrastable gold sample support. Using a low-energy plasma surface modification system, the surface of the support can be tailored to the sample by patterning a series of covalent functionalizations across the graphene or graphene oxide layers on a single grid. The design of the support reduces sample motion during imaging, improving image quality and enabling high-resolution structure determination with minimal material and data.

[0009] Accordingly, an apparatus for carrying out such functionalization, and a method therefor, are also presented.

[0010] In one aspect, there is provided a method of functionalizing graphene or graphene oxide on a substrate for use in electron microscopy, e.g., cryo-electron microscopy, the method comprising: (a) exposing a substrate having a layer of graphene or graphene oxide thereon to a plasma comprising helium and at least one functionalizing compound.

[0011] In a further aspect, there is provided a method of multi-functionalizing graphene or graphene oxide on a substrate for use in electron microscopy, e.g., cryo-electron microscopy, comprising: (a) masking regions of the substrate having a graphene or graphene oxide layer; (b) exposing unmasked regions to a plasma comprising helium and at least one functionalizing compound to form functionalized zones; and optionally (c) removing the mask from the substrate; and (d) repeating steps (a) through (c), using a different functionalizing compound in each repetition, to form a substrate having a graphene layer with multiple zones, each zone having a different functionalization.

[0012] Thus, the method of the invention may comprise functionalizing as defined herein and then using the functionalized substrate in electron microscopy, for example cryo-electron microscopy.

[0013] In such methods, a shutter fixed over the substrate and movable between an open position exposing the substrate and a closed position concealing the substrate may be used to control the exposure time of the substrate to the plasma, allowing the user greater control over factors such as exposure time and therefore the degree of functionalization.

[0014] In the method of the present invention, the functionalized compound or compounds have the following general formula (I): JPEG2025061648000002.jpg27109, where R 1 and R 2 Each of the groups may be H, halo (e.g., -F, -Cl, -Br), thiol (-SH), amino (-NH 2 ), carboxyl (-COOH) and hydroxyphenyl (-C 6 H 4 OH); R 1 and R 2 and n is an integer from 1 to 10.

[0015] In the method of the present invention, the or each functionalizing compound may be one or more selected from the group consisting of amylamine, hexanoic acid, 1-pentanethiol and 4-pentylphenol.

[0016] The methods described herein may further include exposing the substrate having the graphene or graphene oxide layer thereon to a hydrogen-containing plasma to hydrogenate the graphene or graphene oxide layer, or a portion of the graphene or graphene oxide layer, which may be performed before or after functionalization of the graphene or graphene oxide to provide or complement hydrophilicity of the graphene or graphene oxide layer.

[0017] In some embodiments, the substrate is a gold cryo-EM grid.

[0018] Apparatus is provided for carrying out the methods described herein.

[0019] In a further aspect, the invention provides an apparatus for functionalizing graphene or graphene oxide, optionally by a method described herein, comprising: a plasma generator, a process chamber connected to the plasma generator, a substrate holder positioned to hold a substrate for electron microscopy, e.g., cryo-electron microscopy, having a graphene or graphene oxide layer thereon within the process chamber and having a mounting area configured to mount the substrate therein, an injector for injecting a functionalizing compound into the process chamber, and a helium source for providing helium to the plasma generator, and optionally a shutter movable between an open position in which the mounting area of ​​the substrate holder is exposed within the process chamber and a closed position in which the mounting area of ​​the substrate holder is concealed.

[0020] The apparatus may be configured, in use, to supply helium to the plasma generator to generate a helium plasma, inject a functionalizing compound therein to form a mixture, and expose a substrate held on a substrate holder to the mixture in a process chamber.

[0021] In some embodiments, the apparatus further comprises a mask for masking areas of the substrate, which allows for more intricate patterning of the functionalized substrate.

[0022] The mask may be moveable, for example while a plasma is present in the process chamber, allowing for complex patterning, for example creating a "gradient" of functionalization by moving the mask in a given direction at a given speed.

[0023] The mask may be removable from the rest of the apparatus, so that it can be placed over the substrate outside the rest of the apparatus, for example in a larger environment.

[0024] The apparatus is preferably configured such that the mask does not contact the graphene or graphene oxide layer of the substrate when the substrate is held in the substrate holder, thereby eliminating the risk of the mask damaging the substrate. For example, the apparatus may be configured such that the mask is less than 200 μm from the surface of the substrate when the substrate is held in the substrate holder.

[0025] The apparatus may further comprise a hydrogen source for supplying hydrogen to the plasma generator, thereby allowing for partial hydrogenation as well as functionalization of graphene or graphene oxide in one apparatus.

[0026] The apparatus may further include a viewport opening into the processing chamber, and a UV-vis spectrometer configured to analyze radiation emitted from the processing chamber through the viewport.

[0027] The apparatus may further include one or more functionalizing compound sources each connected to the injector for supplying the functionalizing compound or compounds to the injector.

[0028] The apparatus may include multiple injectors, each of which may be connected to a respective source of functionalizing compound to provide the respective injector with the functionalizing compound.

[0029] The injector or injectors may be a gas injector or multiple gas injectors.

[0030] The invention also relates to a sample support for electron microscopy, such as cryo-electron microscopy, comprising a metal foil, such as a gold substrate, and a functionalized graphene or graphene oxide layer on the substrate, the graphene or graphene oxide having a plurality of different functionalizations. The graphene or graphene oxide may be partially hydrogenated.

[0031] The present invention includes combinations of the described aspects and configurations unless the combination is expressly not permitted or explicitly avoided. [Brief description of the drawings]

[0032] Next, embodiments and experiments illustrating the principles of the present invention will be described with reference to the accompanying drawings.

[0033] [Figure 1a] Figure 1a shows the effects of a residual air glow discharge and a helium plasma on a single layer of suspended graphene. [Figure 1b] Figure 1b shows the effects of a residual air glow discharge and a helium plasma on a single layer of suspended graphene. [Figure 1c] Figure 1c shows the effects of a residual air glow discharge and a helium plasma on a single layer of suspended graphene. [Figure 2a]Figure 2a shows experimental data on the wettability of functional graphene and the control of the orientational distribution of 20S proteasomes. [Figure 2b] Figure 2b shows experimental data on the wettability of functional graphene and the control of the orientational distribution of 20S proteasomes. [Figure 2c] Figure 2c shows experimental data on the wettability of functional graphene and the control of the orientational distribution of 20S proteasomes. [Figure 2d] Figure 2d shows experimental data on the wettability of functional graphene and the control of the orientational distribution of 20S proteasomes. [Figure 2e] Figure 2e shows experimental data on the wettability of functional graphene and the control of the orientational distribution of 20S proteasomes. [Figure 2f] Figure 2f shows experimental data on the wettability of functional graphene and the control of the orientational distribution of 20S proteasomes. [Diagram 3] Figure 3 shows a TEM analysis of multifunctional graphene on a gold support using amorphous carbon covered grid squares for electron microscope alignment. [Figure 4] Figure 4 shows Mollweide projection orientation distribution maps of the 30S ribosome on graphene functionalized with hexanoic acid (Fig. 4(a)), 1-pentanethiol (Fig. 4(b)), amylamine (Fig. 4(c)), 4-pentylphenol (Fig. 4(d)), all delivered by helium plasma, and preceded by partial hydrogenation, on partially hydrogenated graphene (Fig. 4(e)), and on graphene placed free in ice (Fig. 4(f)). [Diagram 5] Figure 5 shows (a) a diagram of a sample on graphene on a gold support and (b) a longitudinal section of a tomographic reconstruction of a functionalized graphene-covered hole containing a 30S ribosome in vitreous ice. The ice is 300 ± 10 Å thick, slightly larger than the size of the particles. All the particles are in one layer. [Figure 6] Figure 6 shows data on the improvement of efficiency and resolution by optimizing the orientation distribution of 30S ribosomal subunits on functionalized graphene. [Figure 7] Figure 7. High-resolution structure determination of apoferritin on graphene on a gold support. Figure 7(a) is a representative micrograph of an apoferritin particle in vitreous ice on graphene functionalized with amylamine. Scale bar is 500 Å. Figure 7(b) is the Fourier transform of the motion-corrected micrograph from Figure 7(a). [Figure 8] Figure 8 shows the RMS (root-mean-square) displacement during irradiation of apoferritin particles in unsupported ice on carbon grids and on graphene on gold. [Figure 9] An example of reduced movement of a biological sample on a graphene-coated all-gold grid is shown in Figure 9. The root-mean-square (RMS) displacement of a ribosome of MW 2 MDa is plotted against the electron fluence. [Figure 10a] Figure 10a shows the reduction of nanoparticle movement on a graphene-coated all-gold grid. [Figure 10b] Figure 10b shows the reduction in nanoparticle movement on the graphene-coated all-gold grid. [Figure 10c] Figure 10c shows the reduction of nanoparticle movement on the graphene-coated all-gold grid. [Figure 10d] Figure 10d shows the reduction in nanoparticle movement on the graphene-coated all-gold grid. [Figure 10e] Figure 10e shows the reduction of nanoparticle movement on the graphene-coated all-gold grid. [Figure 10f] Figure 10f shows the reduction of nanoparticle movement on the graphene-coated all-gold grid. [Figure 11]Figure 11 shows schematics of (a) a gold-on-gold substrate with a graphene layer formed on it, (b) a grid square covered with a suspended graphene film, and (c) a number of batch-produced graphene-coated all-gold grids. [Figure 12] Figure 12 shows (a) a schematic of the inductively coupled plasma apparatus design for covalently functionalizing graphene; (b) the slotted grid holder, and (c) the non-contact knife-edge mask design for patterning plasma-functionalized graphene grids. [Figure 12d] FIG. 12d shows the optical spectra for the pure helium carrier plasma (black) and for the introduction of the vapor of the chemical for functionalization. [Figure 12e] FIG. 12e shows the XPS spectrum of functionalized graphene indicating chemical modification. [Figure 13] Figure 13 shows (a)-(b) the orientation distribution of 30S ribosomal subunits on two different graphene functionalizations from one patterned grid plotted in the Mollweide projection, and (c)-(f) the surface of the 30S ribosomal subunit shaded with the Coulombic electrostatic potential. [Figure 14] Figure 14 shows (a) a contour density map of apoferritin showing the amino acid side chains in the structure, (b) Fourier shell correlation (FSC) of the apoferritin structure, (c) the mean square displacement of unsupported gold nanoparticles in ice (light circles) or gold nanoparticles on graphene on an all-gold support in ice (dark crosses), and (d) the calculated B-factor as a function of electron fluence for the apoferritin data set. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Aspects and embodiments of the present invention will now be described with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0035] (Graphene) Graphene as a material is well known in the art as are its methods of manufacture. The main known methods are "top-down" or "bottom-up" synthesis, which broadly involve synthesizing graphene structures by exfoliating layers from graphite or depositing carbon structures, e.g., by chemical vapor deposition (CVD).

[0036] When discussing graphene layers in this specification, it is assumed that either a single layer or several layers of graphene are included. For example, a graphene layer may be a single layer (mono) of graphene or may be composed of a graphene structure having 2 to 5 layers.

[0037] The graphene is preferably a single layer, which approaches invisible within the resolution range of interest for cryo-microscopes.

[0038] As described herein, the functionalized graphene layer is on a substrate that may itself have holes, for example, a gold foil with holes on a gold mesh or grid. In such a case, the graphene crystals in the layer are preferably each larger than one opening or square of the mesh / grid. For example, each graphene crystal may have a smallest two-dimensional dimension (not thickness) of at least about 50 μm. Also, each graphene crystal may have a hexagonal shape.

[0039] If the substrate has holes in it, the graphene, of course, does not "ride" directly "on" the substrate material in those holes. In such cases, the graphene can be said to be suspended; that is, if the substrate has holes, the graphene layer straddles the holes and is in contact with the substrate in some places and not in others.

[0040] Reducing the number of graphene grain boundaries in the graphene layers can be advantageous, for example, to improve the mechanical and chemical robustness of the graphene layers during functionalization processes (described herein) and during cryo-EM imaging.

[0041] The methods, apparatus, applications and products of the invention may alternatively utilize a graphene oxide layer instead of graphene, i.e., the methods of the invention may be applied to functionalize a graphene oxide layer on a substrate, the products of the invention may include a functionalized graphene oxide layer, etc. All of the preferred configurations and options discussed with respect to graphene may be applied to the graphene oxide embodiment, as appropriate.

[0042] (functionalization) The graphene layers described herein are functionalized, and the inventors have discovered a number of advantages associated with such functionalization.

[0043] Pure graphene is not a suitable substrate for preparing samples for cryo-EM due to its hydrophobicity. In previous studies, partial hydrogenation using low-energy plasma has been proposed as a way to make graphene hydrophilic without damaging its lattice, making it suitable for cryo-EM. However, we found that partially hydrogenated graphene only provides a single type of adhesive surface, which is not sufficient for all possible samples. By introducing and controlling the functionalization, we can support a variety of samples in different spatial orientations, which can greatly improve the efficiency of the cryo-EM analysis process.

[0044] Furthermore, knowing how purified proteins interact with other well-defined surfaces, such as those present in ion-exchange columns, can help guide appropriate functionalization and buffer conditions for a particular cryo-EM sample.Functionalized graphene not only represents a tunable surface for improving sample orientation in cryo-EM, but also offers a new way to map and quantify the interaction surfaces of specific biomolecules.

[0045] Functionalized graphene surfaces can be used to orient and disperse biomolecules within thin glassy ice films, and also reduce sample motion to improve image quality.

[0046] As used herein, "functionalized" graphene refers to graphene that has one or more chemical groups other than hydrogen covalently attached to its structure. The groups can be referred to as functionalized. Often, the groups are derived from hydrocarbon-based compounds that have had hydrogen atoms removed at the sites that attach / bond to the graphene structure. That is, the functionalized compound is generally one that has H- at the binding site of graphene, corresponding to the desired functionalization.

[0047] As described herein, the functionalizing compound and the corresponding functionalization are not particularly limited and can be selected depending on the desired effect of the functionalized graphene and the vapor pressure of the functionalizing compound.

[0048] When using the plasma functionalization technique described here, in principle, any small molecule compound can be functionalized as long as it has sufficient vapor pressure to be injected into the chamber. It is noted that the orientation and length of the functional groups are not specifically controlled, so that the surface may contain a mixture of functional fragments. That is, even if a single functionalization compound is used, the specific functionalization may not be uniform.

[0049] Possible examples are shown in Table 1 below, although variations on these are of course contemplated.

[0050] JPEG2025061648000003.jpg79146

[0051] In Table 1, the bond positions to graphene are Represented by JPEG2025061648000004.jpg3230.

[0052] For example, a functionalized compound is a hydrocarbon that is optionally substituted at one or more positions with a functional group, preferably a reactive functional group.

[0053] Functionalized compounds include, for example, halo (e.g., -F, -Cl, -Br), thiol (-SH), amino (-NH 2 ), carboxyl (-COOH) or hydroxyphenyl (-C 6 H 4 C optionally substituted with one or more groups selected from 1-10 Alkyl, preferably C 3-6 or C 5 The alkyl group may be an alkyl group or a compound having a vapor pressure at 25° C. of 0.01 to 50 Torr.

[0054] In practice, the vapor pressure of the compounds can be increased or decreased to a suitable level by heating or cooling the compounds and / or the injection system. A suitable range of temperatures to cool or heat them is from -60°C to +200°C.

[0055] In embodiments focused on cryo-EM, suitable functionalizing compounds include those that have a terminal first functional group with properties similar to amino acid side chains that biomolecules may encounter in vivo. Such functional groups include amines, thiols, carboxyls, phosphates, sugars, and other functional groups present in the native aqueous environment of cells. The compounds may also have a second functional group that can react with graphene to form an SP3 (covalent) bond. The second functional group may be less reactive with the first functional group. The second functional group may also be terminal.

[0056] These two functional groups, if both are present, may be separated by a hydrocarbon chain (preferably saturated), which separates the first functional group from the graphene surface but is otherwise unreactive. The length of the chain is limited by the vapor pressure. As the chain length increases, the vapor pressure decreases, eventually becoming so low that it cannot be injected into the chamber even with heating. Therefore, the optimal chain length is one that provides some separation from the graphene surface, but includes compounds whose vapor pressure is in a reasonable range for injection into the plasma chamber.

[0057] The functionalized compound or compounds have the following general formula (I): JPEG2025061648000005.jpg21121, wherein R 1 and R 2 Each of the groups may be H, halo (e.g., -F, -Cl, -Br), thiol (-SH), amino (-NH 2 ), carboxyl (-COOH) and hydroxyphenyl (-C 6 H 4 OH); R 1 and R 2 one of which is not H; and n is an integer from 1 to 10.

[0058] In some embodiments, n is an integer from 3 to 6, for example, 5.

[0059] In some embodiments, R 2 is H and R 1 are halo (e.g., -F, -Cl, -Br), thiol (-SH), amino (-NH 2 ), carboxyl (-COOH) and hydroxyphenyl (-C 6 H 4 OH).

[0060] In some embodiments, R 2 is H.

[0061] In some embodiments, R 1 is selected from carboxyl or thiol. In some embodiments, R 1 is a thiol.

[0062] In functionalized graphene, for example, 0.001 to 10% of the carbon atoms in the graphene structure are bonded to a functional group, i.e., the graphene may be 0.001 to 10% functionalized, for example, 0.1 to 1% functionalized.

[0063] Functionalization is performed on 1 nm of graphene. 2 The functionalization density may be about 0.5 to 5, for example 1 to 2 per molecule.

[0064] Functionalization with reactive group-containing compounds, such as thiol-containing compounds, may be preferred because the thiol groups can readily undergo further reactions to attach more complex structures to the functionalized graphene. For example, it may be desirable to use the short, flexible hydrocarbon chains discussed above to attach defined ligands or known binding groups to the graphene surface.

[0065] Graphene in which hydrogen is covalently bonded in the structure may be called hydrogenated graphene. In general, the level of hydrogenation is partial since not all sites are bonded with hydrogen. Partially hydrogenated graphene is known in the art. When partial hydrogenation of graphene is discussed here, preferably, about 0.001 to 10% of the carbon atoms in the graphene structure are bonded with hydrogen, i.e., graphene may be hydrogenated by 0.001 to 10%, for example, 0.1 to 1%.

[0066] In certain circumstances, partial hydrogenation is preferred. Partially hydrogenated graphene is more hydrophilic than pure graphene. Such hydrophilic behavior may be preferred. Thus, if graphene has at least one functionality that is not hydrophilic (i.e., is hydrophobic), it may be preferred to have partial hydrogenation.

[0067] For example, when graphene is functionalized with functional groups derived from carboxyls or thiols, it tends to be hydrophobic, so partial hydrogenation helps to make the functionalized graphene hydrophilic.

[0068] This type of hydrophilicity is particularly useful in the field of cryo-EM, where samples are analyzed in water.

[0069] Functionalized graphene may have one or more types of functionalization and may optionally be partially hydrogenated. Graphene with more than one type of functionalization may be referred to as multifunctional or multifunctionalized. Functionalized graphene may or may not be partially hydrogenated.

[0070] For example, the functionalized graphene may be functionalized with one or more (or exactly one), two or more (or exactly two), three or more (or exactly three), or four or more (or exactly four) functionalizations, which may be selected from the functionalizations shown in Table 1 above.

[0071] It may be preferred that the functionalized graphene is divided into several zones or regions, each zone or region having a different level, type or combination of functionalization. In this way, a single substrate with a functionalized graphene layer can be used to "hold" biological samples in various configurations, for example, by appropriately selecting the different functionalization and hydrogenation of the different zones.

[0072] For example, a substrate having a graphene layer can be divided into four zones or regions: a first zone having a first level and type of functionalization and (partial) hydrogenation, a second zone having a second level and type of functionalization and (partial) hydrogenation, a third zone having a third level and type of functionalization and (partial) hydrogenation, and a fourth zone having a fourth level and type of functionalization and (partial) hydrogenation.

[0073] It will be appreciated that in such a zone arrangement, all of the functionalization and hydrogenation options and possibilities discussed above apply both within each zone and to the functionalized graphene as a whole.

[0074] Such multi-functionalization, especially zonal functionalization, is advantageous as it means that grids in which every square has a different functionalization and surface properties or linker chemistry are possible, meaning for example that multiple conditions can be tested simultaneously on the same biological sample on a single grid.

[0075] (substrate) In one aspect, the present invention relates to a substrate having a functionalized graphene (or graphene oxide) layer thereon, such as a metal, particularly gold, substrate having such a layer. As described herein, the layer and its functionalization can be varied without particular restriction with respect to the substrate.

[0076] [Here, the term "support" is used to refer to the combination of substrate and graphene layer; in some embodiments, to the article on which a sample is placed for cryo-EM analysis].

[0077] In an embodiment of the invention, the substrate may be a metal article, such as a foil, for example of nickel, copper or gold. In particular, the substrate may be a gold-based substrate.

[0078] As described herein, substrates of the type known for supporting cryo-EM samples may be particularly preferred. Although several examples are known in the art, gold-based substrates are described here. Such substrates are known for their stability and 10 , structural integrity, and tensile strength, and also provides good electrical contact to graphene even at cryogenic temperatures.

[0079] Such substrates may be of the "gold-on-gold" or "all-gold" variety. Such substrates include gold meshes of any (but typically flat) shape, such as disks or square plates. Each such mesh may be, for example, a disk with a diameter of 1-5 mm, or a plate with sides of 1-5 mm. The mesh may have, for example, 100-500 lines per inch. This results in, for example, a continuous series of roughly square or hexagonal openings. This may also be referred to as a "grid."

[0080] On top of the gold mesh is a gold foil (hence the term gold-on-gold). This foil spans each opening in the mesh. The foil itself has holes in it, each hole having a size of 1 μm or less, for example 0.01-0.5 μm in diameter. The foil is thin, for example 100-1000 Å thick.

[0081] Such substrates are well known in the art. In the present invention, a functionalized graphene or functionalized graphene oxide layer can be added above or below the gold foil, on which the sample for cryo-EM analysis can be placed.

[0082] An example is shown in Figure 11. In Figure 11(a), an example of a gold-on-gold substrate is shown that includes a 3 mm disk-shaped 300 filaments / inch gold mesh ("Au grid"), with a gold foil of approximately 500 Å thickness ("Au foil") with a regular array of submicron-sized holes suspended over the square openings of the mesh. A graphene layer (here a monolayer) is deposited on top of the gold foil, covering all the holes ("holes"). As described herein, graphene can be covalently functionalized by exposure to a low-energy helium plasma containing hydrocarbon precursor molecules ("graphene", where the dots represent different functional groups). A sample (material for cryo-EM analysis) is applied to the graphene side of the grid and vitrified to form a continuous ice layer that can be imaged through the holes.

[0083] Part (b) of Figure 11 shows the coverage of the grid squares with a suspended graphene membrane (secondary electron micrograph). Scale bar is 10 μm. The insets show a fully covered hole (top) and a hole with destroyed graphene (bottom), both magnified 5 times (scale bar is 1 μm). As shown in Figure 11(c), one growth-and-transfer batch yields >150 all-gold grids with >95% graphene coverage (scale bar is 10 mm).

[0084] (Method and Apparatus for Functionalization) The inventors have developed a method for preparing a substrate having a functionalized graphene (or graphene oxide) layer thereon, and an apparatus for carrying out such a method. The substrate, graphene, and functionalization may, of course, be as described herein.

[0085] In particular, we find that the use of helium (inert, low atomic number) as the primary plasma gas in a remote plasma generator prevents sputter damage and other changes to the graphene while allowing chemical reactions between the graphene and chemical precursors (functionalizing compounds). This is in contrast to typical residual air glow discharge systems used in cryo-EM, which generate plasmas energetic enough to destroy suspended monolayer graphene even after brief exposures.

[0086] This is illustrated in Figure 1. Figure 1 shows the effect of residual air glow discharge and helium plasma on a single layer of suspended graphene. Scanning electron micrographs of graphene-coated all-gold grids after 45 seconds of exposure to a residual air glow discharge (PELCO easiGlow, negative bias, 15 mA, 0.3 Torr) (Figure 1(a)), after 45 seconds of exposure to a low-power helium plasma (Fischione 1070, forward power 9 W, 1 Torr) (Figure 1(b)), and after 4500 seconds of exposure to the same helium plasma (Figure 1(c)). Scale bars are 10 μm.

[0087] During exposure, the grid is covered with a contactless knife-edge half mask, with the approximate location of the mask edges indicated by dashed lines. The graphene exposed to the glow discharge is destroyed, and the uncovered holes in the gold foil appear black (see Fig. 1(a) right). The helium plasma does not affect the structural integrity of the graphene film, even when exposed for more than 100 times the typical time required for functionalization. The shadow of the mask edge is visible in the central square of Fig. 1(a), indicating that the patterning resolution of this masking method is less than 50 μm. The inset is a 5x magnification of the square area. The scale bar in the inset is 2 μm.

[0088] Therefore, the inventors have found that helium (He) is an excellent plasma carrier for the functionalization of graphene with functionalizing compounds, in particular for the functionalization of graphene layers on cryo-EM substrates, the plasma being relatively gentle so as not to damage or react with the graphene layers, and at the same time energetic enough to achieve functionalization with functionalizing compounds.

[0089] Plasmas of functionalizing compounds alone are too aggressive and may damage the integrity of the graphene layers, as well as fail to produce the desired functionalization. Furthermore, the use of such plasmas may result in the deposition of polymer by-products in the reaction chamber (depending on the functionalizing compounds present). Similarly, hydrogen plasmas are less suitable, as they react with the graphene layers, hydrogenating them.

[0090] Thus, in the functionalization processes described herein, helium is preferably the predominant plasma component, i.e., 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the atmosphere used to treat the graphene layer on the substrate may be helium plasma.

[0091] The power of the plasma used in this functionalization treatment may be, for example, 0.1 to 100 watts, such as 1 to 20 watts, or 5 to 15 watts.

[0092] The remaining portion may include, for example, a hydrogen plasma to affect partial hydrogenation, and one or more functionalizing compounds as described above. These compounds may be in the form of a vapor, gas, or plasma. In this example, the helium plasma effectively dilutes the other reactive components.

[0093] Treatment times and conditions can be appropriately selected depending on the desired functionalization level. For example, exposure times of 10 to 60 seconds to the functionalization plasma (He plasma + one or more functionalization compounds) can be appropriate.

[0094] For partial hydrogenation, a hydrogen plasma can be used.8 Hydrogen plasma may be used alone or in combination with helium plasma. In this case, the treatment time and conditions may be appropriately selected according to the desired hydrogenation level. For example, to hydrophilize the graphene layer, it may be appropriate to expose the graphene layer to hydrogen plasma for 30 to 300 seconds, for example, 150 to 210 seconds.

[0095] Typical plasma pressures are 0.1-10 Torr, e.g., 0.5-1 Torr. This applies to any plasma described herein, but the appropriate pressure can be selected depending on the plasma used, the functionalizing compounds added, the intended effect on the graphene layer, etc.

[0096] Such a hydrogenation treatment with a hydrogen-containing plasma can be carried out before or after a functionalization treatment with a helium plasma and one or more functionalizing compounds, and in some embodiments, it is carried out before the helium plasma treatment.

[0097] The inventors have found that by selectively masking and unmasking areas of the substrate, multiple functionalizations can be easily achieved and different zones of functionalization can be precisely controlled. By masking an area before a given treatment with plasma, the unmasked areas are affected. The mask can then be removed, i.e. removed from the substrate, and another masking can be performed, followed by another treatment with plasma. The masks can be the same or different. Alternatively, further masks can be added to the previous mask to perform different treatments with plasma, and different treatments can be performed depending on the order of the masks used.

[0098] Another possibility is that the mask is movable during the functionalization process. Depending on the design of the mask, it can allow a predefined functionalization in limited areas or patterns (for example, if the mask is essentially a moving hole on the substrate, then paths on the substrate can be selectively exposed to the functionalization). This allows one to "write" one or more paths or patterns of functionalization onto the substrate.

[0099] Other mask shapes that are moved over the substrate during functionalization can, for example, provide a gradual change in functionalization over the substrate, with areas initially uncovered by the mask being exposed to the functionalization treatment for longer than areas exposed for a shorter time, resulting in a gradient in the degree of functionalization. Many other variations are readily envisioned.

[0100] The movement of the mask may be continuous during the functionalization process, or alternatively, the mask may be moved in steps to give more distinct functionalization changes.

[0101] Thus, described herein is a method for functionalizing suspended graphene or graphene oxide on a substrate, the method comprising: (a) masking regions of the substrate having a graphene or graphene oxide layer, (b) exposing the unmasked regions to a plasma comprising helium and at least one functionalizing compound to form functionalized regions, and (c) modifying the masking of the substrate, for example by moving, adding, or replacing a mask. Step (c) may be performed during or after completion of step (b). This may be followed by a further step of repeating (b) with a different functionalizing compound.

[0102] This process can be repeated to build up a series of desired zones with varying functionalization and / or hydrogenation, using simple or complex masks as needed. Multiple exposures allow for multi-axis patterning and sequential patterning of multiple functionalizations in the same area.

[0103] This same patterning method can also be used to directly deposit, for example, amorphous carbon over a 100 μm diameter area in the center of the substrate, which is useful for stigmation and coma adjustment on multi-functional grids prior to high-resolution data collection.

[0104] The mask itself can have any shape selected for proper masking. For example, the mask can be a half mask that covers 50% of the substrate, such as one complete half. One mask can be used, removed, and then reapplied in a different orientation; alternatively, a new or differently shaped mask can be used.

[0105] The mask may have at least one "knife edge", i.e., a sharpened edge that tapers from its main thickness towards the edge. The knife edge is preferably at an edge that is intended to be on or across the substrate. An example is shown in Figure 12(c).

[0106] The mask may be placed slightly above the surface of the substrate, e.g., less than about 200 μm, e.g., about 20-100 μm, from the surface of the substrate. Since a larger distance results in less distinct boundaries of plasma treatment, the distance from the substrate to the mask may be increased to provide smoother surface patterning transitions or gradient functionalization, etc. Of course, the distance may be decreased if a more distinct boundary between treated and untreated regions is desired.

[0107] With a typical plasma process pressure of about 1 Torr and a mask-to-surface spacing of about 50 μm, the resolution of this patterning method is about 50 μm, or one grid square.

[0108] Preferably, when the mask is applied, it does not come into contact with the graphene layer on the substrate, thereby minimizing the risk of damage.

[0109] The mask itself may be a plate-like object that may have a series of holes or designs cut or patterned into it. These holes are positioned on the substrate being used to form the desired masking pattern. Some or each of these holes may have a knife edge.

[0110] For example, in FIGS. 12(b) and (c), it can be seen that there are masks with 10 half-moon (semicircular) holes in order to prepare 10 half masks for 10 disk-shaped substrates.

[0111] FIG. 12 shows aspects related to the functionalization and patterning of covalently bonded graphene by low-energy helium plasma. FIG. 12(a) shows a schematic of the design of an inductively coupled plasma apparatus for covalently functionalizing graphene. FIG. 12(b) shows a schematic of how a slotted grid holder reliably and precisely positions up to 10 grids under a single mask plate. Openable shutters are used to control the exposure time independent of establishing stable plasma reaction conditions in the chamber. FIG. 12(c) shows a non-contact knife-edge mask design for patterning graphene grids using plasma-assisted functionalization. Masks of any shape are possible. FIG. 12(d) shows the optical spectrum of a pure helium carrier plasma (lowest spectrum) and when a chemical vapor is introduced for functionalization. The He spectral line is saturated and other peaks corresponding to the atomic species of the precursor are visible. The Hα spectral line at 656 nm is used to monitor the decomposition of the hydrocarbon precursor in real time. FIG. 12(e) shows the XPS spectrum of functionalized graphene indicating chemical modification.

[0112] The preferred functionalization apparatus of Figure 12(a) can be further described. In its broadest scope, the apparatus comprises a plasma generator, a process chamber ("plasma chamber") connected to the plasma generator, a substrate holder ("grid holder") arranged to hold a substrate having a graphene layer in the process chamber and having a mounting area configured to mount the substrate therein, an injector ("gas injection manifold") for injecting a functionalization compound into the process chamber, and a helium source for supplying helium to the plasma generator. The invention also provides for the use of the apparatus (as described herein) in the functionalization of graphene (or graphene oxide), in particular in the functionalization of a graphene (or graphene oxide) layer on a substrate as described herein or a support as described herein.

[0113] Described herein is the use of an apparatus for functionalizing graphene, the apparatus comprising a plasma generator, a process chamber connected to the plasma generator, a substrate holder positioned to hold a substrate having a graphene layer in the process chamber and having a mounting area configured to mount the substrate therein, an injector for injecting a functionalizing compound into the process chamber, and a helium source for supplying helium to the plasma generator.

[0114] Such an apparatus is configured such that, in use, helium enters the plasma generator, a helium plasma is formed in the process chamber, a functionalizing compound is injected into the process chamber, and a graphene layer on a substrate held by the substrate holder is functionalized.

[0115] The apparatus may have various other configurations. For example, if desired, a hydrogen source may be included to supply hydrogen to the plasma generator. The hydrogen source may be coupled to the plasma generator so that hydrogen enters in the same way as helium from the helium source, or alternatively, a respective entry point may be provided. Each hydrogen or helium source may be controlled by a mass flow controller ("MFC").

[0116] The apparatus may optionally further comprise an oxygen source for supplying oxygen to the plasma generator, which may be used to generate an oxygen plasma for cleaning the process chamber and the apparatus between functionalization processes. Ideally, no substrate is present in the process chamber when oxygen is supplied to the plasma generator.

[0117] The oxygen source may be connected to the plasma generator such that the oxygen flows in the same path as the helium from the helium source and / or hydrogen from the hydrogen source, if present; alternatively, there may be separate entry points. Each oxygen source present may be controlled with a mass flow controller ("MFC").

[0118] The plasma generator may be, for example, an inductively coupled plasma generator of known type, for example, an RF plasma generator. The injector may be, for example, a gas injector or other injector suitable for the type of functionalization compound to be injected.

[0119] The apparatus may be configured to inject a number of different functionalization compounds. The apparatus may include a source of each functionalization compound. Each source of functionalization compound may use a common injector or each may have its own injector into the process chamber. If there is a common injector, each source of functionalization compound may have a control to adjust the flow of functionalization compound to the shared injector. This allows for good control over the flow of the various functionalization compounds and therefore the functionalization with the various functionalization compounds.

[0120] In the methods, apparatus and uses described herein, a suitable flow rate of each functionalizing compound into the process chamber or into the plasma may be, for example, from 1 to 1000 sccm, optionally from 10 to 100 sccm.

[0121] That is, under the conditions of 0° C. and 100 kPa, the flow rate of each functional compound is, for example, 1 to 1000 cm 3 / min, 10~100cm 3 / min, i.e. 0.1667 cm 3 / sec~16.667cm 3 / sec, arbitrarily 0.1667cm 3 / sec~1.667cm 3 / second.

[0122] The tool is equipped with a UV-vis spectrometer that can be used to view the process chamber through a viewport, e.g., a sapphire viewport, allowing the plasma conditions in the process chamber to be monitored.

[0123] The process chamber may also have known configurations such as gate valves and exhaust, e.g., pump-driven exhaust. The exhaust of the plasma chamber may be restricted, e.g., with a "butterfly valve" to limit the speed of the pump to control the pressure in the plasma (processing chamber) independent of the input gas flow rate and composition. The device and / or exhaust system may also include a real-time feedback loop for pressure, and may be controlled independent of the input flow rates. That is, depending on the flow rates of the plasma source (helium and / or hydrogen, or oxygen) and one or more functionalizing compound sources to the process chamber, the exhaust flow rate may be automatically increased or decreased to maintain the desired pressure in the process chamber.

[0124] The apparatus may also include a shutter that is movable relative to the substrate holder mounting area between an open position, in which the substrate holder mounting area is exposed to the process chamber, and a closed position, in which the substrate holder mounting area is concealed. An example of such a shutter is shown in Figure 12(b).

[0125] That is, the shutter itself may be moved to expose or hide the mounting area for the substrate holder, and the substrate holder itself may be moved to similarly expose or hide the mounting area for the substrate holder, and this movement of the shutter and / or substrate holder may be controlled from outside the process chamber.

[0126] The substrate holder itself may be configured to hold multiple substrates as described herein. In the example of FIG. 12(b), it holds, for example, 10 substrates. The substrate holder itself may also be comprised of a portion for receiving a substrate (mounting area), a portion for receiving a mask, and a portion for receiving a shutter mechanism. Of course, the substrate holder may be integrally formed with the shutter and / or mask as described herein.

[0127] When the shutter is closed to conceal the attachment area (and thus the substrate or a portion or all of the substrate), the concealed substrate or substrates are no longer subject to the plasma atmosphere. By opening the shutter in a controlled manner to expose the substrate or substrates (i.e., to partially or fully expose or uncover the attachment area), functionalization or hydrogenation can begin at the exposed portions as desired. This allows the time of functionalization or other plasma treatment to be carefully controlled. For example, some substrates can be exposed for a longer time than other substrates. This provides further options for rapidly creating a large number of substrates with different degrees or types of functionalization or hydrogenation.

[0128] The apparatus itself may include one or more pre-fabricated masks as described herein, in which case one need only place one or more substrates on the substrate holder or its mount to apply the masks and optional shutters of the apparatus to the substrates for use in the process chamber.

[0129] Where the apparatus includes a mask, it may further comprise means for moving the mask during or between functionalization processes.

[0130] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings are appropriately expressed in their specific form, or in terms of means for performing a disclosed function, or a method or process for obtaining a disclosed result, and such features can be utilized separately, or in any combination, to realize the invention in various of its forms.

[0131] While the present invention has been described in conjunction with the exemplary embodiments set forth above, many equivalent modifications and variations will be apparent to those skilled in the art upon receipt of this disclosure. Accordingly, the exemplary embodiments of the present invention set forth above are considered to be illustrative and not limiting. Various changes can be made to the described embodiments without departing from the spirit and scope of the invention.

[0132] For the avoidance of doubt, the theoretical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0133] All section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0134] Throughout this specification, including the claims which follow, unless the context otherwise requires, the words "comprise" and "include", as well as variations such as "comprises", "comprising", "including", etc., are understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of other integers or steps or groups of integers or steps.

[0135] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Ranges may be expressed herein from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" in connection with numerical values ​​is arbitrary and may mean, for example, ±10%. EXAMPLES

[0136] In the examples, a substrate with a graphene layer as described herein is used. For functionalization, an all-gold support or substrate (UltrAuFoil R 0.6 / 1, 300-mesh, Quantifoil) with a pore size of 800 nm is used, on which a graphene monolayer is provided. Such supports or substrates can be called grids.

[0137] For example, a support comprising a substrate having a graphene layer thereon can be prepared using techniques available in the art, such as by CVD growth of graphene on copper foil followed by transfer of the graphene layer to a gold substrate.

[0138] (Example 1) Furan-Functionalization (a) Covalent functionalization using low-energy helium carrier plasma Plasma treatment was performed in a modified commercial plasma cleaner (Fischione 1070) equipped with a custom grid and mask holder, a custom gas injection system, and a custom fiber-coupled sapphire viewport attached to a UV-vis spectrometer (Thorlabs). The apparatus thus modified was that of the present invention and is roughly as shown in FIG. 12(a).

[0139] The vacuum chamber is heated by a turbo pump for 10 -5The N6.0 grade helium carrier plasma was pumped to less than 5×10 Torr with a flow rate of 28 sccm. -1 It was produced at a standard pressure of 10 Torr. The 18 MHz RF coil was operated at 40% power, giving a typical forward power of 9 W and a reverse power of 0.6 W.

[0140] Vapors of chemicals used for functionalization were introduced through an evacuated five-channel manifold. Chemicals were stored in sealed evacuated 1 mL stainless steel vials, and vapors of the chemicals of interest were delivered to the plasma chamber via precision micrometer needle valves and shut-off valves. The effect of the introduced chemicals was observed by real-time spectra of the plasma. Spectra were acquired with an exposure time of 6 s, which allowed the helium lines to saturate and additional peaks from the precursors to become visible.

[0141] The most prominent feature in the optical spectrum due to the introduction of organic molecules was the Hα peak at 656 nm (Figure 12(d)). This is likely due to the high probability of terminal hydrogen being cleaved from the molecule. On the other hand, the cleavage of larger fragments or non-terminal moieties, such as atomic sulfur from thiol, requires the cleavage of two or more bonds, which is unlikely.

[0142] The actual amount of chemical present in the plasma depends on the vapor pressure of each compound and can be adjusted by controlling the temperature of the liquid container and the setting of the leak valve.

[0143] Once the desired plasma composition was established, the exposure time was controlled using a shutter covering the grid and mask holder. Such a shutter and mask are shown in Figure 12(b) and (c). Between uses of the different chemistries, the chamber was cleaned with pure He plasma at 70% power for 30-60 min until no traces of hydrogen were visible in the optical spectrum.

[0144] These manipulations were completed using amylamine, hexanoic acid, 1-pentanethiol, and 4-pentylphenol as functionalizing chemicals. Figure 12(e) shows the XPS spectrum of functionalized graphene indicating the chemical modification.

[0145] (b) XPS of functionalized graphene X-ray photoelectron spectroscopy (XPS) was used to study the covalent modification of graphene modified according to (a) above. The beam from an Al Kα source (1486.68 eV, ESCALAB 250 Xi) was focused onto the sample with a 900 μm diameter probe. For these measurements, graphene on copper (as grown) and graphene transferred to UltrAuFoil gold grids (according to example 2) were used and charge compensation was applied. Partially hydrogenated graphene and untreated graphene samples were used as controls to compare with the functionalized graphene. The pressure in the XPS chamber during data acquisition was 5 × 10 -9 It was mBar.

[0146] First, a spectral scan with 1 eV sampling was acquired in the range 136 eV to 1361 eV with a pass energy of 200 eV, then 30–50 scans with 0.1 eV sampling and pass energies of 30–50 eV were acquired around the region of interest and averaged to produce the spectrum plotted in Figure 12(e). These peaks were found in the NIST XPS database 27 Some of the observed peaks were attributed to signals from the metal substrate supporting the graphene.

[0147] (c) Contact angle measurement Contact angle measurements were performed on plasma-treated graphene suspended on an all-gold grid using a custom fixture. 28Briefly, a 1-2 μL drop of 18 MΩ deionized water was pipetted onto the center of the clamped grid and photographed parallel to the surface against a diffuse illumination background. Any images with a curved grid or asymmetric spreading of the drop were erased. The typical time between the end of the plasma treatment and the measurement was 1 min, with 3-5 repetitions for each data point. Contact angles were measured using ImageJ. 29 was quantified.

[0148] (d) Patterning of functionalized graphene To pattern grids with multiple functional groups on different regions of each grid, a knife-edge mask was precisely placed on top of each grid for each plasma treatment step (above (a)). This was done using a set of precision-machined masks that fit into defined positions on a grid alignment holder. This setup was capable of patterning 2 × 10 grids at a time.

[0149] The sharpness of the pattern depends on and can be controlled by varying the distance between the mask and the graphene surface, the sharpness of the mask edges, and the mean free path of the chemical species in the plasma. In the typical conditions used here (1 Torr, non-thermal remote plasma), the mean free path in the chamber is much larger than other relevant length scales. Therefore, the patterning resolution can be considered to be approximately equal to twice the distance between the grid and the mask, i.e., 50 μm. This can be seen in Figure 1(a) by the width of the transition region when the knife-edge mask is placed 20 μm from the surface of the foil.

[0150] (e) Deposition of amorphous carbon on the graphene-on-gold substrate. 10 -6Amorphous carbon was evaporated directly onto the graphene-coated gold grids using an Edwards 306A evaporator evacuated to mbar. The grids were placed in a custom slotted holder similar to that used for plasma treatment and covered with a mask plate. A single-slotted mask with a 100 μm hole diameter (EMS GA100-Au) was used as the mask, exposing only the center of the grid to the evaporated carbon. The distance between the grid and the mask was approximately 100 μm. Successful localized deposition of a continuous amorphous carbon film was confirmed by TEM observation (Figure 3).

[0151] Figure 3 shows a multifunctional graphene-on-gold support with grid squares covered with amorphous carbon for electron microscope alignment. Figure 3(a) is a diagram of the aperture mask used during carbon deposition to cover only about one grid square of the graphene-on-gold grid with a thin film of amorphous carbon. The deposition is performed before plasma treatment. Figure 3(b) is a low-magnification transmission electron micrograph of the modified multifunctional grid, showing the asymmetric center mark and the surrounding dark area covered with a ∼300 Å thick amorphous carbon film. The scale bar is 80 μm. Figure 3(c) shows that the Thon ring from the carbon-coated area (Fourier transform of a transmission electron micrograph at nominal magnification 155,000×, defocus 0.4 μm, and intentionally stigmatized by 700 Å) can be used to correct for astigmatism and coma. The lattice reflection of graphene at 2.13 Å is still visible. Clean monolayer graphene in the masked areas, in contrast, does not develop a Thon ring.

[0152] This patterning method involves the direct deposition of amorphous carbon onto a 100 μm diameter region in the center of a graphene-coated gold grid, allowing stigmation and coma alignment to be performed on the same multifunctional grid prior to high-resolution data collection.

[0153] (Example 2) Motion tracking in vitreous ice using gold nanoparticles (a) Grid preparation Samples were prepared by manual plunge freezing at 4 °C. All-gold supports (UltrAuFoil R 0.6 / 1, 300-mesh, Quantifoil) with pore diameter of 800 nm were used with or without graphene. Sample supports were plasma treated to make them sufficiently hydrophilic prior to vitrification as follows: all-gold grids were plasma treated with 9:1 Ar:O 2 The graphene-coated grid was exposed to hydrogen plasma for 60 s, then 3 μL of OD100 10 nm non-conjugated gold colloid solution (BBI Solutions) was applied to the foil side of the grid and blotted from the same side for 15 s before being immersed in liquid ethane held at 93 K. 30 .

[0154] (b) Imaging The graphene-supported ice data was obtained with a Titan Krios, and the graphene-unsupported ice data was obtained with a Tecnai Polara, both using a Falcon 3 detector in integral mode, under typical imaging conditions for single particle data collection (300 keV, 1e - The images were collected at 100 x 100 x 100 cm (1 / pix / frame, sample temperature 80 K). The illuminators were centered over each exposed hole, and the beam diameter was set to be slightly larger than the hole to minimize beam induced motion. 31 The magnification was chosen to include the edge of the hole in the micrograph for drift tracking. This corresponds to 1.74 Å / pix (nominal 47,000x) for Krios and 1.72 Å / pix (nominal 59,000x) for Polara. The movie was taken at a cumulative fluence of 60 e - / Å 2 The acquisition was performed for 5–8 s until the image was clear. Only holes from squares with no defects in the gold foil were imaged.

[0155] (c) Motion analysis The entire video of the micrographs was motion-corrected with MotionCorr. 20, stage drift was removed. The motion-corrected stacks were imaged at 1.5e per frame. - / Å 2 The particles were binned to a fluence of . 21 Each particle was manually selected and extracted from the motion-corrected stack in a 128 × 128 pixel frame. Each particle movie was then motion-corrected again with MotionCorr. Corrected particle movies were visually inspected. The first 20e - / Å 2 The trajectories of all particles during the irradiation of were used to calculate the mean square displacement and the root mean square displacement of the particles on the ensemble.

[0156] (Example 3) Cryo-EM of T. thermophilus 30S ribosomal subunit In this example, the 30S ribosomal subunit is used as a test specimen to demonstrate the potential of multifunctional graphene supports to optimize the orientation distribution of samples. 12,13 Graphene functionalized with amylamine, hexanoic acid, 1-pentanethiol, and 4-pentylphenol were prepared by applying these chemicals to half of a gold grid coated with partially hydrogenated graphene, respectively. The orientation distribution of 30S particles on each of these surfaces was analyzed (Figures 13(a)-(b), Figures 4(a)-(d)). These distributions are relative to the distribution on partially hydrogenated graphene and in unsupported ice (Figures 4(e)-(f)). A combination of functionalized surfaces was identified that increased the efficiency of the orientation distribution up to 0.8. 14 This allowed the calculation of a high-resolution reconstruction of only the 30S subunit (Figure 6). Clear differences in all observed distributions indicate that the particles interact with the functionalized graphene. This was confirmed by the tomographic reconstruction, which showed that the particles were in a monolayer on the graphene surface, in an ice layer that was slightly thicker than the particle diameter. 15 (Figure 5).

[0157] (a) Grid preparation Purified 30S ribosomal subunit of T. thermophilus (5 mM HEPES, 50 mM KCl, 10 mM NH 4 Cl, 10 mM Mg(OAc 2 ) was kindly provided (by the Ramakrishnan laboratory at the MRC Laboratory of Molecular Biology). Such samples are commercially available, for example from Sigma.

[0158] The concentration was adjusted to 8 mg / mL, except for the samples on partially hydrogenated graphene, which were 1.7 mg / mL. All graphene-coated grids were first hydrogenated with H 2 After 180 seconds of exposure to the plasma to render the entire surface hydrophilic, half of the grids were treated with He plasma carrying vapors of the corresponding functionalization chemicals (amylamine, hexanoic acid, 1-pentanethiol or 4-pentylphenol) for 30 seconds, and the other half was treated for an additional 30 seconds. Plain UltrAuFoil grids for control experiments without graphene were treated with a 9:1 Ar:O plasma. 2 The grids were treated with the plasma mixture for 60 seconds. The grids were plunged using an FEI Vitrobot equilibrated to 4 °C and 100% relative humidity, and the liquid ethane was kept at a fixed temperature of 93 K. 3–4 μL of the protein solution was pipetted onto the graphene-coated surface of the grid and double-blotted for 5 seconds before immediately immersing in ethane. The time from plasma treatment to vitrification was typically less than 10 minutes. The grids were stored in liquid nitrogen until they were transferred to the electron microscope for imaging.

[0159] (b) Imaging and data collection Micrographs of 30S ribosomes in ice on functionalized graphene were taken with a Tecnai Polara microscope operating at 300 kV with a Falcon 3 detector in integral mode with a nominal magnification of 93,000x, corresponding to 1.17 Å / pix (calibrated using the graphene reflection at 2.13 Å). Micrographs of 30S ribosomes in unsupported ice and in ice on partially hydrogenated graphene were acquired with a Titan Krios microscope operating at 300 kV with a Falcon 3 detector in integral mode with a nominal magnification of 59,000x, corresponding to 1.34 Å / pix.

[0160] (c) Single particle data analysis The functionalized sectors of the grid were identified using asymmetric center marks (see Figure 2(e)). Micrographs were motion corrected in Relion 3. 17 , fit the CTF with Gctf 22 Particles were manually picked in EMAN2 and 2D classification and 3D refinement were performed in Relion 3. All particles from the functionalized grids were processed together and the final orientation angles were classified into categories for each surface based on the name of the micrograph and the corresponding coordinates on the grid. Particles from the control experiment were processed separately due to different pixel sizes. The orientation distribution of the particles was analyzed by cryoEF. 14 , plotted in equal-area Mollweide projections. The shaded scale represents the local Gaussian kernel density (i.e., probability distribution function, PDF) of the distribution at all sampled orientations (see Fig. 13(a)-(b) and Fig. 6). The bandwidth of the kernel density was set to 5°. These plots also show the normalized probability that a particle interacts with the surface in a particular orientation, quantifying the strength of this interaction. Some variation in the assigned orientations could be due to local bending of the foil, wrinkling of the graphene, bulging of the ice, or bending of the mobile parts of the ribosomal subunits, all of which are less than 5°. The high-resolution crystal structure of the 30S ribosome (1J5E 12 ) converted to a surface and shaded with the surface Coulomb potential calculated with UCSF Chimera. 23We illustrated the dominant orientations of the particles on the graphene and analyzed the interaction interfaces (see Fig. 13(c)-(f)). We evaluated whether each orientation distribution could provide a uniform sampling of Fourier space that would enable high-resolution reconstruction using an efficiency index. 14 Efficiencies were calculated for each separate orientation distribution from approximately 10,000 particles. These efficiencies can be used to make rational decisions regarding the optimal surface or combination of surfaces for further data collection.

[0161] In this example, the surface chosen is graphene functionalized with amines and phenols, providing an overall efficiency of 0.8, thereby allowing the determination of near-atomic structures with approximately 100 times fewer particles compared to, for example, an efficiency of 0.3 for a data set of an unsupported 30S ribosome in ice. 14 (See Figure 6).

[0162] Figure 6 shows the improvement in efficiency and resolution by optimizing the orientation distribution of the 30S ribosomal subunit on functionalized graphene. Gold standard FSC plots are shown for 30S in unsupported ice (dashed line), which gave a resolution of 6.9 Å from 12,600 particles, and for 30S on amine / phenol functionalized graphene (solid line), which gave a resolution of 3.94 Å from 26,483 particles.

[0163] The combination of these functionalizations was predicted to maximize the efficiency of the orientation distribution. The difference in resolution is not due to a two-fold difference in particle number, as shown by the reconstruction from a subset of 12,600 particles of the high-efficiency data set (dotted line), which reached 4.68 Å. The inset shows the point spread function (PSF) corresponding to each orientation distribution, with the PSF extending in the weakest resolution direction.

[0164] (d) Tomography Single-axis bidirectional tilt series were collected on the same sample in 5° increments from -50° to +50° on a Titan Krios operating at 300 keV using a Falcon 3 direct electron detector in integral mode. Each image exposure was 2e over 1 s (40 frames) at a nominal magnification of 37,000x. - / Å 2 The video was motion corrected using MotionCorr. 20 ,The tomograms were aligned without fiducials and reconstructed using SIRT in Etomo (IMOD). 24 .

[0165] (e) Discussion These experiments reveal a functional interaction map of the surface of the 30S ribosomal subunit. This allows the creation of a physical model of particle-surface interactions that explains the observed orientation distribution. On carboxylated supports, the prevailing view is that the protein-rich outer side of the 30S faces the surface (Figure 13(d)). This face contains the Arg S2 226 , LysS10 80 , Lys S4 166,169Positively charged amino acid residues such as , , and , are exposed and appear to stabilize the contact with the surface. The same orientation is more favored on thiol-functionalized surfaces (Figure 4(b)), consistent with the lower pKa of the thiol groups. The next most common orientation on carboxylated graphene is with the 50S side of the 30S subunit facing the surface (Figure 13(c)). The interactions that cause this orientation are likely similar to those that occur at the 30S-50S interface in vivo. Putative anchoring points include the Arg / Lys-rich S13 chain near the 30S head and the 16S rRNA 5′ domain at the body surface. This orientation is highly favored on partially hydrogenated graphene (Figure 4(e)). On the other hand, this orientation is hardly seen on partially hydrogenated graphene treated with amylamine, consistent with the higher pKa of the amine groups. Of all the surfaces tested here, amylamine / hydrogen-functionalized graphene caused the least orientation bias for the samples tested. However, some acidic amino acid residues (Glu S6 24,31 , Glu S13 58 ) can be identified as putative interaction points of the 30S subunit contacting the amine-graphene surface (Figure 13(e)-(f)). Since the Debye screening length in the buffer used is about 10 Å, only electrostatic interactions with amino acid side chains that are in closer contact with the graphene surface are considered. The extent of the field of view around each preferred direction depends on the particle's shape and how it restricts rotation around fixed interaction points.

[0166] In Figure 13(a)-(b), the orientation distribution of 30S ribosomal subunits on two types of graphene functionalization ((a) graphene+hydrogen+carboxyl, i.e., partially hydrogenated graphene with carboxyl functionalization from hexanoic acid, and (b) graphene+hydrogen+amine, i.e., partially hydrogenated graphene with amine functionalization from amylamine) from a single patterned grid is plotted in Mollweide projection. Each point represents an observed particle, and the scale represents the calculated normalized probability density of observing a particle at a particular orientation. E represents the efficiency of each orientation distribution. 14 The two most probable view directions and their surrounding variations are labeled.

[0167] Figure 4 shows more completely the Mollweide projection orientation distribution plots of 30S ribosomes on graphene functionalized with (a) hexanoic acid, (b) 1-pentanethiol, (c) amylamine, and (d) 4-pentylphenol, all of which were delivered in helium plasma and preceded by partial hydrogenation; (e) the Mollweide projection orientation distribution plot of 30S ribosomes on partially hydrogenated graphene, and (f) the Mollweide projection orientation distribution plot of 30S ribosomes not supported on ice. Each point represents the orientation of a particle observed on the graphene surface, and the scale represents the calculated normalized probability distribution function (PDF) when the particle is observed in a field of view given by the Euler angles (φ, θ). All plots are displayed on the same scale as the peaks corresponding to the main field of view in the saturated state in (e)–(f). The efficiency E of each orientation distribution is shown.

[0168] (Example 4) Cryo-electron microscopy of human 20S proteasome (a) Grid preparation A sample of human 20S proteasome (Enzo) was buffer exchanged into TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA) at pH 8.3 and adjusted to a concentration of 0.8 mg / mL. Four-quadrant grids were prepared by exposing half of the grid to hydrogen plasma for 180 s, then rotating the mask by 90° and exposing half of the grid to amylamine under helium plasma for 30 s, rotating the mask by 180°, and exposing the remaining half of the grid to hexanoic acid under helium plasma for 30 s. Vitrification was performed in the same manner as above.

[0169] (b) Imaging and data collection The micrographs were taken on a Titan Krios microscope operating at 300 kV in integral mode with a Falcon 3 detector. The flux was 17e - / Å 2 The exposure time was 2 seconds, and a 70 μm objective aperture was used. The nominal magnification was 59,000x, corresponding to 1.34 Å / pix, and a 30 μm aperture was used. Anatase (TiO 2 ) The grid was calibrated using reflections from nanoparticles. The quadrants of the grid were identified with reference to the orientation of the asymmetric grid center mark as described above (see Figure 2).

[0170] Figure 2 illustrates our experimental efforts to control the wettability and orientational distribution of 20S proteasomes on functionalized graphene. Figure 2(a)-(d) are representative micrographs of 20S proteasomes on graphene in ice, taken from the four quadrants of a single patterned multifunctional graphene grid. The quadrant of the grid from which the micrographs were taken is highlighted in each inset. Hexanoic acid treatment (Figure 2(b)) renders the grid hydrophobic, leading to poor wetting of the graphene surface and partial drying of the proteins. This can be solved by pretreating the grid with hydrogen plasma (Figure 2(b)), which renders the surface hydrophilic and allows the formation of a uniform thin ice layer independent of subsequent functionalization. Thus, we demonstrate that an initial hydrogen plasma treatment can maintain the macroscopic surface wettability required to form a thin ice layer, even when the graphene surface is subsequently functionalized with compounds that may render it hydrophobic.

[0171] The axes dividing the grid into four different functionalized quadrants were defined by aligning the easily visible grid rim marks (Fig. 2(e), inset) parallel or perpendicular to the mask edges. The quadrants were also identified by locating the position and orientation of the asymmetric grid center marks (Fig. 2(e), inset) in the low magnification mode of the TEM. The inset in Fig. 2(e) is an optical micrograph of the true relative orientation of the rim and center marks. Scale bars are 500 Å in Fig. 2(a)-(d) and 50 μm in the inset in Fig. 2(e). Contact angle measurements of functionalized graphene can be utilized to monitor the extent of surface modification and to evaluate the hydrophilicity of the resulting surfaces, as shown in Fig. 2(f).

[0172] Based on previous experiments involving partial hydrogenation, a typical exposure would result in a 1 nm thick layer of graphene. 2It can be estimated that approximately one functional group is added per 100 nm. Functionalization with carboxyl and thiol groups tends to make the surface hydrophobic, as evidenced by the large contact angle, which can be problematic when freezing samples for cryo-EM. This can be avoided by pre-treatment with hydrogen plasma for 180 seconds (black data points) prior to the typical 30 second exposure to the functionalization chemistry.

[0173] This method of first partially hydrogenating the graphene prior to functionalization reliably produced a surface with sufficient wettability for preparing cryo-samples.

[0174] (Example 5) Cryo-electron microscopy of apoferritin In this example, the structure of horse spleen apoferritin is determined using amylamine-functionalized graphene on a gold support to demonstrate that this support is suitable for high-resolution structure determination.

[0175] (a) Grid preparation Horse (Equus caballus) spleen apoferritin (Sigma) was buffer-exchanged into 100 nM phosphate-buffered saline at pH 7.4 and adjusted to a concentration of 11.7 mg / mL. Graphene-coated grids treated with amylamine-carrying He plasma for 30 s were used. Vitrification was performed in the same manner as above.

[0176] (b) Imaging and data collection Movies were acquired in counting mode on a Titan Krios microscope operating at 300 kV with a Falcon 3 detector. The nominal magnification was 120,000x, pixel size 0.6495 Å / pix, and was calibrated using the 2.347 Å gold reflection in the Fourier transform of a micrograph of a gold substrate foil taken under the same conditions as data collection. A 100 μm objective aperture was used for data collection. Movies were acquired with an exposure time of 30.01 s and frames were compiled into 38 fractions (31 frames per fraction). The flux was 0.53e - / pix / sec, with a total fluence of 37e for each video. - / Å 2 It was.

[0177] Figure 7(a) shows a micrograph of apoferritin particles in vitreous ice on amylamine-functionalized graphene. When such electron micrographs are taken at appropriately high magnification, the graphene lattice is directly visible.

[0178] (c) Single particle data analysis and modeling Motion correction of micrographs was performed in Relion 3, CTF fitting in Gctf, particle pickup was performed manually in EMAN2, and 2D and 3D classification, 3D refinement, CTF refinement, particle polishing, and post-processing were all performed in Relion 3. As a result, the EM density map reached the gold standard FSC resolution of 2.14 Å, and the corresponding high-resolution crystal structure (2W0O 25 ) was matched.

[0179] JPEG2025061648000006.jpg105147

[0180] A 3D map of the fitted model was rendered in Chimera. The orientation distribution of the particles that contributed to the final reconstruction was analyzed with cryoEF, which showed an efficiency of 0.7. For the motion analysis, the displacement of each particle throughout the movie was calculated by subtracting the overall motion of the micrograph from the particle's motion calculated by Bayesian polishing in Relion. 26 This was necessary because the dominant component of motion was that the microscope stage was drifting at a rate of 0.6 Å / sec during the 30 sec exposure.

[0181] (d) Discussion Figure 14 shows details of high-resolution structure determination using multifunctional graphene-on-gold supports. Figure 14(a) shows a contour density map of apoferritin showing the amino acid side chains in the structure. Figure 14(b) shows the Fourier shell correlation plot (FSC) of the apoferritin structure. Figure 14(c) shows the mean square displacement of unsupported gold nanoparticles in ice on an all-gold support (light circles) or unsupported gold nanoparticles in ice on graphene (dark crosses). The addition of a graphene membrane reduces both the motion at the start of irradiation and the diffusive motion in subsequent frames by a factor of two. The dashed line is a linear fit to the data excluding the first frame. The addition of graphene to the all-gold sample support reduces the slope by 0.13 ± 0.1 Å. 2 / (e - / Å 2 ) to 0.048±0.005Å 2 / (e - / Å 2 ) for the apoferritin data set. Figure 14(d) shows the calculated B-factor as a function of electron fluence for the apoferritin data set. - / Å 2 The B factor at corresponds to the initial non-diffusive transfer stage. The B factor is 10-20e - / Å 2 7.7±0.8Å for a fluence in the range 2 / (e - / Å) (dashed line is linear fit).

[0182] Standard data collection and processing yielded a resolution of 2.1 Å (0.143 FSC) from 41,202 particles. 16 ) (see Table 2, Fig. 7, Fig. 14). The resolution of this reconstruction map is comparable to the highest resolution reported so far for this sample. 17 , the amount of data required is roughly half, as indicated by the clear density of side chains in the map (Figure 14(a)). This includes intact aspartic and glutamic acid residues, and density beyond Cβ is often not present in EM maps. 18The first 5-10e in this dataset - / Å 2 The decay rate of the high-resolution information as a function of fluence after irradiation (Figure 14(d)) is similar to the results of measurements of radiation damage in 2D protein crystals. 19 The particle motion on multifunctional graphene on a gold support was compared with previous measurements on several other supports. 4 Figure 8 shows the root-mean-square (RMS) displacement during radiation exposure of an apoferritin particle in unsupported ice on a carbon grid (top) and a particle on graphene-on-gold (bottom). The particle in unsupported ice is from a data set corresponding to the highest resolution structure published to date of commercially available horse spleen apoferritin. 17 Error bars are standard error of the mean. Functionalized graphene on a gold support reduced the initial motion of the particles during irradiation, the displacement rate, and the variance of the motion, compared to a porous amorphous carbon support. Figure 9 plots the root-mean-square (RMS) displacement of a ribosome of MW 2 MDa versus electron fluence. Figures 9(a)-(d) and (f) reproduce previously published work. 4 The motion on the graphene on gold support in Figure 9(e) was measured independently from the apoferritin data set obtained here, and for the purposes of this comparison, the effective diffusion constant was calculated as the molecular weight (MW 1 / 3 ) scaled by 9 The motion of the sample on the graphene-on-gold support is diffusive (mean square displacement is directly proportional to the fluence) except at the very beginning of the irradiation.

[0183] The addition of a graphene layer to the all-gold support reduced particle motion upon electron beam irradiation by a factor of two. Particle motion was reduced by a factor of three for graphene on a gold support compared to graphene on a carbon support. The reduction in particle motion was also confirmed by tracking the motion of individual gold nanoparticles on the all-gold support with and without the addition of a graphene layer (Figure 14(c) and Figure 10). Figure 10 shows the reduction in nanoparticle motion on a graphene-coated all-gold grid. Gold nanoparticles (10 nm diameter, 6 MDa mass) were photographed in unsupported vitreous ice on an all-gold grid (Figure 10(a)) and in vitreous ice on graphene on an all-gold grid (Figure 10(d)) at magnifications such that the field of view for drift tracking included the edge of the hole. Particle trajectories on the motion-corrected gross micrographs were plotted at 200x magnification (Figures 10(b) and 10(e)). The dashed boxes correspond to the areas in the micrographs, the empty circles are the initial particle positions, and the filled circles are the 20e - / Å 2 In Figure 10(c) and (f), the root-mean-square (RMS) displacement of the gold nanoparticles is plotted against the electron fluence for the shown micrographs (top panels) and for an average of n = 19 micrographs (gold) and n = 8 micrographs (graphene on gold) of the same type (bottom panels).

[0184] Importantly, in addition to suppressing random particle motion, the addition of the graphene layer suppresses motion during the first few hits of the electron beam, improving the quality of the initial frame (B-factor) with less molecular damage from the electron beam (Figure 14(c)-(d)). This two-fold reduction is likely due to the graphene layer restricting the bulk movement of the thin ice film within each pore, which occurs as stress is released at the beginning of the electron beam hit.

[0185] References In order to more fully describe and disclose the present invention and the state of the art to which it pertains, a number of publications are cited. Full citations for these publications are set forth below. Each of these publications is incorporated herein in its entirety. 1. J. Dubochet, et al., Q. Rev. Biophys. 21, 129 (1988). 2. RA Crowther, The Resolution Revolution: Recent Advances in Cryo-EM , vol. 579 of Methods in Enzymology (Elsevier, 2016). 3. RM Glaeser, Current Opinion in Colloid and Interface Science 34, 1 (2018). 4. C. Russo, LA Passmore, Current Opinion in Structural Biology 37, 81 (2016). 5. AK Geim, Science 324, 1530 (2009). 6. RS Pantelic, JC Meyer, U. Kaiser, W. Baumeister, JM Plitzko, J. Struct. Biol. 170, 152 (2010). 7. RS Pantelic, et al., J. Struct. Biol. 174, 234 (2011). 8. CJ Russo, LA Passmore, Nat. Methods 11, 649 (2014). 9. A. Einstein, Annalen der Physik 322, 549 (1905). 10. C. Russo, LA Passmore, Science 346, 1377 (2014). 11. T. E. Daubert, R. P. Danner, Physical and thermodynamic properties of pure chemicals: data compilation (Taylor & Francis, Washington DC, 1989). 12. B. Wimberly, et al., Nature 407, 327 (2000). 13. T. Hussain, J. L. Llacer, B. T. Wimberly, J. S. Kieft, V. Ramakrishnan, Cell 167, 133 (2016). 14. K. Naydenova, C. J. Russo, Nature Communications 8, 1 (2017). 15. A. J. Noble, et al., eLife 7, e34257 (2018). 16. P. B. Rosenthal, R. Henderson, J. Mol. Biol, 333, 721 (2003). 17. J. Zivanov, et al., eLife 7, e42166 (2018). 18. C. F. Hryc, et al., Proc Natl Acad Sci USA 114, 3103 (2017). 19. J. Hattne, et al., Structure 26, 759 (2018). 20. X. Li, et al., Nat. Methods 10, 584 (2013). 21. G. Tang, et al., Journal of Structural Biology 157, 38 (2007). 22. K. Zhang, Journal of Structural Biology 193, 1 (2016). 23. E. F. Pettersen, et al., J. Comput. Chem. 25, 1605 (2004). 24. D. N. Mastronarde, S. R. Held, Journal of Structural Biology 197, 102 (2017). 25. N. de Val, J.-P. Declercq, C. K. Lim, R. R. Crichton, J. Inorg. Biochem. 112, 77 (2012). 26. J. Zivanov, T. Nakane, S. H. W. Scheres, IUCrJ 6, 5 (2019). 27. National Institute of Standards and Technology, NIST X-ray Photoelectron Spectroscopy Database, vol. 20899 of NIST Standard Reference Database (Gaithersburg MD, 2000). 28. K. Naydenova, C. J. Russo, Microscopy and Microanalysis 24, 880 (2018). 29. C. A. Schneider, W. S. Rasband, K. W. Eliceiri, Nat. Methods 9, 671 (2012). 30. C. Russo, S. Scotcher, M. Kyte, Review of Scientific Instruments 87, 114302 (2016). 31. C. J. Russo, L. A. Passmore, Journal of Structural Biology 193, 33 (2016).

Claims

1. 1. An apparatus for functionalizing graphene or graphene oxide, comprising: Plasma generator; a process chamber connected to the plasma generator; a substrate holder arranged to hold in a process chamber a substrate having a graphene or graphene oxide layer thereon for electron microscopy, preferably cryo-electron microscopy, the substrate holder having a mounting area configured for mounting the substrate therein; a shutter movable between an open position exposing the mounting area of ​​the substrate holder to the processing chamber and a closed position concealing the mounting area of ​​the substrate holder; an injector for injecting a functionalizing compound into the processing chamber; and A helium source for supplying helium to the plasma generator. An apparatus comprising:

2. The apparatus of claim 1 , further comprising a mask for masking areas of the substrate.

3. The apparatus of claim 2 , wherein the mask is movable.

4. 4. The apparatus of claim 2 or claim 3, wherein the mask is removable from the remainder of the apparatus.

5. 5. The apparatus of claim 2, wherein the apparatus is configured such that the mask does not contact a graphene layer or a graphene oxide layer of the substrate when the substrate is held by the substrate holder.

6. The apparatus of claim 5 , wherein the apparatus is configured such that the mask is positioned less than 200 μm from a surface of the substrate when the substrate is held by the substrate holder.

7. The apparatus of any one of claims 1 to 6, further comprising a hydrogen source supplying hydrogen to the plasma generator.

8. 8. The apparatus of claim 1, further comprising a viewport opening into the processing chamber and a UV-vis spectrometer configured to analyze radiation emitted from the processing chamber through the viewport.

9. The apparatus of any one of claims 1 to 8, further comprising one or more functionalizing compound sources, each connected to said injector, for supplying said functionalizing compound or compounds to said injector.

10. The apparatus includes a plurality of injectors, each connected to a respective source of functionalizing compound for supplying the functionalizing compound to the respective injector. An apparatus according to any one of claims 1 to 9.

11. An apparatus according to any one of claims 1 to 10, wherein the injector or injectors are gas injectors or multiple gas injectors.

12. A sample support for cryo-electron microscopy A metal foil substrate; and A functionalized graphene layer or graphene oxide layer on said substrate having a plurality of different functionalizations.

1. A sample support for cryo-electron microscopy comprising:

13. 13. The cryo-electron microscopy sample support of claim 12, wherein the graphene or graphene oxide is also partially hydrogenated.

14. 14. The sample support for cryo-electron microscopy according to claim 12 or 13, wherein the substrate has holes in which the functionalized graphene or graphene oxide layer is suspended.

15. A method for functionalizing graphene or graphene oxide on a substrate for use in electron microscopy, preferably cryo-electron microscopy, comprising the steps of: (a) exposing a substrate having a graphene layer or a graphene oxide layer thereon to a plasma comprising helium and at least one functionalizing compound; The method includes:

16. 16. The method of claim 15, wherein prior to step (a), a mask is applied to the substrate bearing the graphene or graphene oxide layer to selectively expose only portions of the substrate.

17. The method of claim 16 , wherein the mask is moved during step (a).

18. 1. A method for multi-functionalizing graphene or graphene oxide on a substrate for use in electron microscopy, preferably cryo-electron microscopy, comprising the steps of: (a) masking an area of ​​a substrate having a graphene layer or a graphene oxide layer; (b) exposing the unmasked portion to a plasma comprising helium and at least one functionalizing compound to form a functionalized zone; (c) unmasking the substrate; (d) repeating steps (a) through (c) with different functionalizing compounds to form a substrate having a graphene layer with multiple zones, each zone having a different functionalization. The method includes:

19. 19. The method according to any one of claims 15 to 18, further comprising controlling the exposure time of the substrate to the plasma using a shutter fixed on the substrate and movable between an open position exposing the substrate and a closed position concealing the substrate.

20. The or each functionalizing compound has the following general formula (I): Formula (I) and wherein each of R1 and R2 is independently selected from the group consisting of H, halo (e.g., -F, -Cl, -Br), thiol (-SH), amino (-NH2), carboxyl (-COOH), and hydroxyphenyl (-CH4OH); one of R1 and R2 is not H; and n is an integer from 1 to 10 The method according to any one of claims 15 to 19.

21. A method according to any one of claims 15 to 20, wherein the or each functionalising compound is one or more selected from the group consisting of amylamine, hexanoic acid, 1-pentanethiol and 4-pentylphenol.

22. 22. The method of any one of claims 15 to 21, further comprising exposing the substrate having the graphene or graphene oxide layer thereon to a plasma comprising hydrogen to hydrogenate the graphene or graphene oxide layer, or a portion of the graphene or graphene oxide layer.

23. The method of any one of claims 15 to 22, wherein the substrate is a gold cryo-EM grid.

24. Apparatus for carrying out the method according to any one of claims 15 to 23.

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