Electron microscopy support

Abstract

The present invention relates to an electron microscopy sample support (20), and a method of manufacturing an electron microscopy sample support. The support is particularly useful in cryoEM imaging and sample preparation. The support comprises a metallic foil (21 ) having one or more holes therethrough and a support member (22), wherein the metallic foil is electrically insulated from the support member.

Description

[0001] ELECTRON MICROSCOPY SUPPORT

[0002] Related Applications

[0003] The present application is related to, and claims priority to and the benefit of GB 2311682.5 filed on 28 July 2023 (28.07.2023), the contents of which are incorporated by reference in their entirety.

[0004] Field of the Invention

[0005] The present invention relates to an electron microscopy (EM) sample support, and a method of manufacturing an electron microscopy sample support. The support is particularly useful in cryoEM imaging and sample preparation.

[0006] Background

[0007] Electron microscopy techniques can be used to image a specimen. According to such techniques, a beam of electrons is used to ‘illuminate’ a specimen. The presence of the specimen in the electron beam results in change to that beam. The changes to the beam induced by the sample can be examined to create a magnified image of the specimen.

[0008] In order to be illuminated by an electron beam, a specimen must be adequately supported in that beam. Often the electrons forming the electron beam have a high energy and it will be appreciated that bombarding an object, for example, a specimen for examination, together with the support holding the specimen in position within the electron beam, may result in physical, chemical and / or electrical changes to the support and / or specimen. Such changes may impact results, including resolution of image, obtained through use of electron microscopy techniques.

[0009] The use of metal grid supports in electron microscopy is known. Previous work by the present inventors includes Naydenova 2022 where an integrated foil-grid structure suitable for electron cryomicroscopy (cryoEM) specimen supports is disclosed. An all gold, HexAuFoil type design is described.

[0010] However, there remains a need to develop this technology, in particular to facilitate use of such grids in a wider variety of circumstances.

[0011] The present invention has been devised in light of the above considerations.

[0012] Summary of the Invention

[0013] In an all-gold support, the metal foil and the metal grid bars form an ohmic contact by design. That is to say, the contact between the metal foil and the grid bars acts as a non-rectifying junction, thus allowing for ease of movement of any free electrons between the foil and the grid. This minimises the build-up of charge when the grid is placed in a high-energy charged particle beam. However, this property may be undesirable if one wants to perform electrical measurements on the grid (or a part of the grid), or to apply a voltage bias between the grid and the foil. This may be the case when, for example, the specimen held by the grid is subject to thickness modification by plasma etching. In such a case, the thickness of the specimen can be monitored by analysis of electrical measurements taken from the grid.

[0014] A first aspect of the invention is a support for an electron microscopy sample, the support comprising a metallic foil having one or more holes therethrough and a support member, wherein the metallic foil is electrically insulated from the support member.

[0015] In this way, an electron microscopy sample support which enables electrical measurements to be performed on the support is provided. Without the present electrical insulation this is not possible, as explained above.

[0016] In some embodiments, the support comprises an insulating layer between the metallic foil and the support member. Examples of suitable insulating layers include silicon oxides, such as SiO, SiOz or non- stoichiometric silicon oxides SiOx (where x<2), aluminium oxide AI2O3 and silicon nitride SisN4, SixNx. Other examples include high-K dielectrics which have a dielectric constant greater than that of SiOz, such as hafnium silicate, zirconium silicate, hafnium dioxide and zirconium dioxide, typically deposited using atomic layer deposition. Preferably, the insulating layer comprises one or more silicon oxides, such as SiO or SiOz or non-stoichiometric silicon oxides SiOx (where x<2). The thickness of the insulating layer d which may be preferred to withstand a certain applied voltage l / can be estimated from the breakdown field E for the material using the definition V = Ed. Typical breakdown fields for the aforementioned oxides are E = 1 to 10 MV / cm. That is, for 1 kV maximal applied voltage, the thickness of the insulating layer d is 1 to 10 pm. Typically, the thickness of the insulating layer d is in the range 0.1 to 1 pm for providing at least 100 V breakdown voltage. If the support is to be used for fast electrical measurements or modulation, the capacitance C of the insulating layer also needs to be considered and minimised.

[0017] In some embodiments, the support further comprises an adhesion layer between the metallic foil and the insulating layer and / or between the insulating layer and the support member. The adhesion layer is useful to improve the adhesion between the metallic foil and insulating layer. For example, the adhesion of gold to silicon oxides is often poor. Preferably the adhesion layer is a thin metal adhesion layer. Suitable metals include titanium, aluminium, chromium and copper. Most preferably, the adhesion layer is a thin titanium layer. By ‘thin’, it is meant that the thickness of the adhesion layer is less than or equal to the thickness of the metallic foil, for example 5 to 10 nm.

[0018] In some embodiments, the diameter of each hole of the metallic foil is 5 pm or less, 2 pm or less, or 1 pm or less. In other embodiments, the diameter of each hole in the metallic foil is 750 nm or less, 700 nm or less, such as 600 nm or less, 500 nm or less, 400 nm or less, 350 nm or less, 330 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 180 nm or less, 150 nm or less, 100 nm or less, or 75 nm or less. In some cases the diameter of each hole is in a range selected from 750 nm to 75 nm, 500 nm to 100 nm, 400 nm to 150 nm, 300 nm to 150 nm, or 250 nm to 150 nm. The holes are preferably substantially circular. The holes may all be substantially the same diameter or different diameters. The diameter of each hole is preferably substantially the same throughout the depth of each hole. Each hole passes completely through the metallic foil. The spacing between the holes is preferably equal. The spacing between the holes is preferably equal to at least one hole diameter. The hole walls are preferably at a 90 degree angle to the surface of the foil, optionally with a precision of ±2 degrees or better. The metallic foil may comprise other secondary holes that are greater than the above specified diameter requirement and / or do not meet the above diameter to foil thickness requirement. Because secondary holes do not meet the specified requirements they are superfluous. Alternatively, in some cases, the metallic foil only comprises holes that meet the specified requirements.

[0019] In some embodiments, the metallic foil does not comprise any holes that do not meet the specified nanoscale diameter requirements.

[0020] An advantage of the nanoscale dimensions is that the movement of the particles in the holes is isotropic (the same in the plane of the support and perpendicular to it), spatially uncorrelated, and scales with the root of the incident electron fluence, as would be the case for purely random motion. By comparison, in the larger hole diameters of known supports there is an abrupt, spatially correlated unwanted displacement of the particles at the onset of irradiation (e.g. in the first 4 e7A2). This is followed by a decreasingly correlated movement that continues with reduced speed to the end of the exposure.

[0021] Another advantage is that nanoscale dimensioned holes provide plasmon resonances in the visible range, which causes the support to appear yellow on reflection with white light but blue on transmission, a property which may be useful for characterising a specimen before imaging with electrons.

[0022] In some embodiments, the holes are arranged in a regular pattern on the metallic foil, such as a hexagonal pattern or a square pattern. Preferably, the holes are arranged in a hexagonal pattern. The pattern is preferably regular. The hexagonal pattern provides closer packing and improves structural rigidity. The hexagonal pattern allows faster examination of multiple holes by automated systems because the closer packing of the holes in the film results in a shorter distance between one hole and the next which speeds up the time for examination of multiple holes.

[0023] In some embodiments, the thickness of the metallic foil is 50 nm or less. An advantage when the thickness of the metallic foil is less than 50 nm is that thinner specimen films are provided in the holes which have an improved transmission. Known metallic foils having a thickness of less than 50 nm are not suitable for electron microscopy because they provide poor images, often fall apart (i.e. they are not free standing or cannot be suspended across an EM grid square such as a 50 pm grid square, without damage) and cannot adequately suspend sample films.

[0024] In some embodiments, the metallic foil thickness is 49 nm or less, such as 45 nm or less, 40 nm or less, 30nm or less, 20 nm or less, 10 nm or less or 5 nm or less. The metallic foil thickness is preferably substantially constant throughout. Preferably, the metallic foil thickness may fluctuate by up to only ±25 A, ±10 A or ±5 A throughout.

[0025] In some embodiments, the metallic foil thickness is greater than 50 nm, up to 100 nm. In this way, the metallic foil has improved robustness to withstand high pressures during high pressure freezing. In some embodiments, the ratio of the diameter of each hole to the thickness of the metallic foil is 15:1 or less, 11 :1 or less, 10:1 or less, 8:1 or less, 7:1 or less, or 5:1 or less. The ratio of each hole diameter to the foil thickness may be between 11 :1 and 1 :1 , 10:1 and 2:1 , 9:1 and 3:1 or 8:1 and 4:1 .

[0026] An advantage of using the specified ratios is that it helps eliminate buckling of specimen films when such films are formed in the holes, such as a suspended amorphous ice film as used in cryoEM. This allows precise foil tracking during imaging with high-speed detectors, lessening demands on cryostage precision and stability. The present support therefore reduces particle movement to the limit set by pseudodiffusion, which is less than the resolution of the electron cryomicroscope. This allows reconstruction of a complete map at 1 .9 A resolution with a fluence of < 1 e7A2at 300 keV. The specimen films remain stable and under radial compression throughout irradiation, and only diffusive movement occurs which is limited to < 1 A RMS in 30 e7A2. The present movement-suppressing microscopy specimen support allows atomic structure determination at only 1 e7A2, and extrapolation back to the point before destructive effects of electron radiation affect the reconstruction.

[0027] In some embodiments the support member comprises a plurality of spaced support elements. The spaced support elements provide additional structural support to the foil. Preferably the plurality of spaced support elements are arranged to form a mesh. Accordingly, the support member may comprise an annular element which supports a grid-like structure. That grid-like structure may then support, between adjacent mesh elements, the metallic foil. In some embodiments, the porous region of the metallic foil is arranged to extend across a region of the mesh. In some embodiments, the mesh has a mean hole size that is on a micrometre scale, such as about 300 pm, about 200 pm about 100 pm, or about 50 pm. The mesh holes may be hexagonal or square. Preferably, the mesh holes have a hexagonal shape. Preferably the mesh holes tessellate, i.e. if the holes are square they are arranged in a square array and if they are hexagonal they are arranged in a hexagonal array. The hexagonal holes and array provides closer packing and improves structural rigidity; these may therefore be preferred. The mesh hole pattern may correspond to the metallic foil hole pattern.

[0028] In some embodiments, the metallic foil comprises one or more of gold, palladium and platinum or an alloy thereof. Preferably the metallic foil consists of gold or an alloy thereof.

[0029] In some embodiments, the support member comprises one or more of gold, palladium and platinum or an alloy thereof. Preferably the metallic foil consists of gold or an alloy thereof.

[0030] In some embodiments, the metallic foil and the support member have the same elemental composition. The advantages provided by the foil and the support member having the same elemental composition include increased stability during imaging because the two structures have the same thermal expansion coefficients, there is minimal, or no, difference in their mechanical behaviour on thermal change (heating or cooling) so little, or no, relative movement between the two structures.

[0031] In some embodiments, the plane of the metallic foil does not intersect the support member, providing an asymmetric support. That is, the support has a ‘foil side’ and a ‘grid bar side’. In some embodiments, the plane of the metallic foil intersects the support member. That is, the support member is present on both sides of the metallic foil. In this way, an electrically insulated symmetrical support is provided. This allows for access to both sides of the specimen on the support, facilitating, for example, two-sided etching of the specimen. The thickness of the support member on each side of the metallic foil (ti and ta) can suitably be controlled independently. Preferably the thickness is uniform across each side of the support member to within 0.1 pm accuracy. In some embodiments, ti = t2, preferably to within 0.1 pm accuracy, such that a perfectly symmetrical grid is provided.

[0032] A suitable total thickness of the support member (ti +t2) is in the range of 3 to 20 pm, preferably 5 to 7 pm.

[0033] A second aspect of the invention is the use of the support of the first aspect in electron microscopy, optionally in transmission electron microscopy, preferably in transmission electron cryo -microscopy.

[0034] A third aspect of the invention is a method of manufacturing a support for an electron microscope sample, comprising the sequential steps of i) providing a metallic foil having one or more holes therethrough, ii) defining the contours of a support member directly on the metallic foil using a positive photoresist layer, iii) depositing an insulating layer in the defined contours, iv) removing the photoresist layer of step ii) and reapplying a photoresist with the same pattern, and v) forming the support member directly on the deposited layer by electroplating.

[0035] Steps (i) and (ii) are as described in Naydenova and Russo 2022 for integrated wafer-scale manufacturing of cryoEM.

[0036] In some embodiments, the step of providing a metallic foil (step (i)) comprises depositing a metallic layer onto a patterned substrate.

[0037] In some cases, the patterned substrate is a silicon wafer, optionally between 3 mm and 300 mm in diameter, such as 100 mm. The silicon wafer may be a degenerately doped silicon wafer with a resistivity <0.02 Ohm cm. Alternatively, a silicon wafer with higher resistivity of 1 to 30 Ohm cm can be used. The silicon wafer may be formed by Talbot displacement (phase interference) lithography as described in Jefimovs 2017. The template-substrate distance during the phase interference lithography may be used to control the diameter of the holes in the silicon surface. A regular templating array can set the spacing between the holes to be patterned in the substrate. The silicon substrate is patterned so as to form recesses therein to correspond to the holes in the desired metallic foil.

[0038] In some cases, there is a step of cleaning the patterned substrate before deposition of the metallic layer.

[0039] The cleaning may be by immersion, for example in Piranha solution (3H2SO4 : I H2O2, freshly mixed), oxygen plasma or UV-Ozone plasma. Cleaning is advantageous to remove contaminants that can lead to poor hole formation.

[0040] In some embodiments, the metallic layer is deposited onto the patterned substrate wherein the substrate is at a temperature of 150K or less, 125K or less, 100K or less or 90K or less. The temperature of the substrate may be set from 80K to 90K. Preferably, to reduce the grain size of the metallic foil and allow for the formation of rounder, smoother holes therethrough, the substrate stage is kept at 77K (liquid nitrogen temperature) during the evaporation. Higher deposition rates require cooling to lower temperatures. The deposition may be by electron beam or thermal evaporation. For instance, the temperature range for nucleating 10 nm or smaller crystals at 1 A / s deposition rates is 200K or less, assuming surface adatom diffusion with activation energy of 0.5 eV. The substrate having the deposited metallic layer may be slowly (about 50K / hour or slower) warmed up to room temperature in vacuum to prevent the foil from delaminating.

[0041] Preferably, for the best growth of the metallic crystals, the optimal deposition temperature is lower, due to the high-self diffusion coefficient of metallic materials, such as gold. The purity of material to be deposited is preferably 90% or more in order to form a stable continuous layer, more preferably 99% or more; even more preferably 99.999% or more.

[0042] In some embodiments, the patterned substrate is provided with a first sacrificial layer applied to the patterned substrate onto which the metallic layer is deposited. Any layer which is selectively etchable with respect to the relevant metallic foil layer can be used for this layer. The sacrificial layer may be a metal, such as copper (which is particularly preferred for gold foils). It is preferred that the sacrificial layer is copper. The sacrificial layer may also be deposited on the substrate under the cooling conditions described above. The deposition conditions are preferably the same for the sacrificial layer as the metallic foil layer. This is preferred because unwanted imperfections and roughness of the deposited sacrificial layer may otherwise be imparted to the side of the metallic foil layer formed thereon. The thickness of the sacrificial layer is preferably at least the same as the specified grain size. For example a sacrificial layer having a thickness of at least 10 nm, or at least 25 nm, or at least 50 nm may be used. The maximal thickness of the sacrificial layer is preferably less than the radius of the holes in the foil.

[0043] In some embodiments, the insulating layer is deposited on the metallic foil by electron beam evaporation. Electron beam evaporation does not allow for precise control of the stoichiometry of the insulating layer. If the metallic foil is deposited on a holey silicon substrate, the insulating layer is required to be thick enough to fill the wells in the silicon substrate. For example, between 200 nm and 800 nm. Alternatively, the insulating layer may be deposited by thermal evaporation or by atomic layer deposition (ALD).

[0044] In some embodiments, there is a step of depositing an adhesion layer above and / or below the insulating layer. In this way the adhesion between the metallic foil and the insulating layer, and / or between the support member and the insulating layer, may be improved. In some embodiments, the adhesion layer is deposited onto the receiving layer (metallic foil or insulating layer) by electron beam evaporation. Alternatively, the adhesion layer may be deposited by thermal evaporation.

[0045] In some embodiments, a metallic layer is deposited on the insulating layer by electron beam evaporation. Alternatively the metallic layer may be deposited by thermal evaporation. If an adhesion layer is deposited above and below the insulating layer, the metallic layer is deposited on the adhesion layer coating the insulating layer. In this way, the surface is prepared for the subsequent electroplating to form the support member. Preferably the metallic layer is the same elemental composition as the support member.

[0046] In some embodiments, the positive photoresist layer is of a thickness of 7 pm or more. This allows for the resist layer to be lifted off after the insulating layer deposition by ensuring that the sidewalls of the resist remain exposed and uncoated. This is also facilitated by ensuring that the deposition rate is slow to prevent the resist from overheating. In some embodiments, the patterned substrate may be cooled during the deposition steps.

[0047] It is useful that the two photoresist layers are aligned. If the trenches for the electroplating (defined by the second photoresist layer) are offset from the deposited insulating layer, the support member may short the metallic foil. In order to mitigate this risk, it is preferable that the first photoresist layer defines wider contours of the same shape for the support member than the second photoresist layer to allow for more tolerance to offsets between the layers.

[0048] In this case, the foil layer is insulated from the grid bar layer. Consequently, there is no electrical connection useful for the electroplating step (step (v)). In some embodiments, the support member is connected via the metallic layer which was deposited to prepare the surface for electroplating. This can be achieved by modifying the electroplating mask with the addition of thin contact wires extending from each grid bar to its neighbours. Without such connections, it would not be possible to electroplate the support member. The insulation between the foils and the support member can be checked by contact resistance measurement between the foil and the support member.

[0049] In some embodiments, to separate the support from the patterned wafer, the silicon is dissolved in hot (80 °C) potassium hydroxide (KOH). In other embodiments, tetramethylammonium hydroxide (TMAH) may be used, which has an improved selectivity for silicon over SiOx compared with KOH.

[0050] In some embodiments, a sacrificial layer, such as copper, may be removed by etching. In some embodiments, a Piranha solution is used to remove the sacrificial layer.

[0051] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided. Summary of the Figures

[0052] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0053] Figure 1 shows a section view of a typical HexAuFoil grid.

[0054] Figure 2 shows a section view of an embodiment of the present electron microscopy support.

[0055] Figure 3A shows a schematic for a part of the process for manufacturing electron microscopy supports with an insulating layer between the grid and the metallic foil using lithography techniques.

[0056] Figure 3B shows a scanning electron micrograph of a silicon wafer after the deposition of the insulating layer and lift-off of the photoresist. This micrograph was acquired at 30° tilt, using 30 keV electrons and an Everhart-Thornley electron detector. In this example the insulating layer is only 0.4 pm thick, with additional overhangs due to deposition on the sidewalls of the resist.

[0057] Detailed Description of the Invention

[0058] Aspects and embodiments of the present invention will now be discussed 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.

[0059] Figure 1 shows a typical HexAuFoil support (10). The support has two different sides: the foil side (having the foil 11 at its surface) and the grid bar side (having the support member, in this illustration represented by grid bars 12, at its surface). In a typical support design, the grid bars and the foil are in electrical contact. This minimises the build-up of charge when the grid is placed in a high-energy charged particle beam. In this reference example, both the metallic foil and the support member comprising grid bars comprise gold.

[0060] In contrast to that, Figure 2 shows an embodiment of the electron microscopy support (20) of the present invention. The support (20) comprises a metallic foil (21 ) and a support member comprising grid bars (22). However, the support also comprises an extra insulating layer (23) between the grid bars (22) and the metallic foil (21 ). In this way, the metallic foil (21 ) is electrically insulated from the support member (22), which allows for electrical measurements to be carried out on the support. In this particular embodiment, the insulating layer (23) is an insulating oxide layer. In this particular embodiment, the insulating oxide layer is a non-stoichiometric silicon oxide (SiOx, where x<2). Preferably, the insulating oxide layer comprises stoichiometric silicon dioxide SiOz, which has a higher breakdown field.

[0061] In this particular embodiment, the plane of the metallic foil (21 ) does not intersect the support member (22), such that the support (20) has a foil side and a grid bar side. That is, the support is ‘asymmetrical’. Alternatively, the plane of the metallic foil may intersect the support member as to provide a symmetrical support. In this particular embodiment, the metallic foil (21 ) and the support member (22) both comprise gold. In this way, increased stability during imaging can be achieved since the two structures have the same thermal expansion coefficients.

[0062] Method

[0063] Figure 3A schematically shows part of an embodiment of a process for making supports of the present invention with an insulating layer between the grid and the metallic foil (30).

[0064] In the present invention a metallic foil is provided having one or more holes therethrough. The metallic foil may be deposited onto a patterned substrate. In this particular embodiment, the metallic foil (31 ) is deposited onto a silicon wafer (32) as the patterned substrate (Figure 3A upper left image) as disclosed in Naydenova and Russo 2022. The silicon wafer (32) is patterned so as to form recesses therein to correspond to the holes in the desired metallic foil. This may be achieved by phase interference (Talbot displacement) lithography with an ultraviolet source, or conventional photolithography.

[0065] The metallic foil may comprise one or more of gold, palladium and platinum or an alloy thereof. Preferably the metallic foil comprises or consists of gold. In this particular embodiment, the metallic foil consists of gold (31 ).

[0066] The metallic foil is deposited onto the substrate by cryogenic evaporation. Specifically, a thin sacrificial copper layer (33) is deposited onto the silicon wafer (32), followed by a gold layer (31 ). Both layers are deposited by electron beam evaporation, with the silicon wafer cooled using liquid nitrogen to 80-90 K.

[0067] Once the metallic foil (31 ) is deposited, the contours of the support member (39) are defined directly on the foil. This is achieved using a positive photoresist layer (34) as disclosed in Naydenova 2022. This is shown in the first image of the upper row of Figure 3A. This mask step is used to define the area of deposition of the insulating layer (35).

[0068] In the next step (Figure 3A upper row, second image from the left) the insulating layer (35) is deposited, for example by evaporation. That covers both the photoresist and the gold foil-coated Si wafer. In this particular embodiment, the insulating layer (35) is SiOx. Deposited both above and below the oxide layer is a titanium adhesion layer (36). In this way, the adhesion between the oxide insulating layer (34) and the gold foil (31 ) is improved. Alternatively, the insulating layer (35) may comprise silicon oxide SiO, silicon dioxide SiOx or silicon nitride SisN4 and the adhesion layer (36) may comprise chromium, aluminium or copper. On top of the titanium-coated oxide layer, a thin gold layer (37) is deposited to prepare the surface for the subsequent gold electroplating. Alternatively, the metallic layer (37) may comprise another metal as long as it is the same elemental composition as the support member. The adhesion layer (36) and the metallic layer (37) are both deposited by electron beam evaporation.

[0069] After deposition of the insulating, adhesion and preparatory metallic layers (35, 36, 37), the photoresist is removed, along with the residual layers deposited on it (Figure 3A upper row, second image from the right). An SEM image of one such embodiment is in Figure 3B. At this stage, a new photoresist (38) is applied with the same pattern as that which was removed, leaving the insulating layer present surrounded by the photoresist (Figure 3A upper right image).

[0070] Electroplating of the support member (39), in this case gold grid bars of 10 pm in thickness, follows (Figure 3A lower right image). Alternatively, the support member may comprise palladium or platinum, or an alloy thereof. Since the metallic foil (31 ) is insulated from the support member (39), there is no electrical connection useful for electroplating the support member. As such, the support member (39) is connected via the preparatory thin gold layer (37). This may be achieved by modifying the electroplating mask described in Naydenova 2022 to provide additional thin contact wires extending from each grid bar to its neighbours.

[0071] In a final step of the method, the formed supports are released from the substrate (32) and the sacrificial copper layer (33) is removed. The supports are separated from the substrate by dissolving the silicon in hot (80 °C) potassium hydroxide (KOH). KOH has a 100-fold selectivity for Si over SiOx, meaning that only the silicon substrate (32) is dissolved and not the oxide insulating layer (35). Alternatively, the selectivity for Si can be improved by up to a factor of 104by using tetramethylammonium hydroxide (TMAH). Any residual photoresist (38) is also removed by the KOH or TMAH.

[0072] The sacrificial copper layer (33) is removed from the released supports with a Piranha solution (3H2SO4 : 1 H2O2, freshly mixed). This step also eliminates any remaining organic surface contaminants from the photolithography steps.

[0073] The resulting supports can be kept in water or dried and stored in air for distribution and use.

[0074] Figure 3B shows a silicon wafer after deposition of the insulating layer and after lift-off of the photoresist (40). In this particular embodiment, the insulating layer (41 ) is an oxide insulating layer comprising SiOx. Alternatively, the insulating layer may comprise silicon oxide SiO, silicon dioxide SiO2, or silicon nitride SisN4.

[0075] In this particular embodiment the metallic foil (42) consists of gold. Alternatively, the metallic foil may comprise one or more of gold, palladium and platinum or an alloy thereof.

[0076] ***

[0077] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0078] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0079] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0080] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0081] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as 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 the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0082] References

[0083] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

[0084] K. Jefimovs, L. Romano, J. Vila-Comamala, M. Kagias, Z. Wang, L. Wang, C. Dais, H. Solak, M. Stampanoni, “High aspect ratio silicon structures by Displacement Talbot lithography and Bosch etching, Proc, of SPIE 10146 (2017).

[0085] K. Naydenova, C Russo, “Integrated wafer-scale manufacturing of electron cryomicroscopy specimen supports” Ultramicroscopy 232 (2022).

Claims

Claims:1 . A support for an electron microscope sample, the support comprising a metallic foil having one or more holes therethrough and a support member, wherein the metallic foil is electrically insulated from the support member.

2. The support according to claim 1 comprising an insulating layer located between the metallic foil and the support member.

3. The support according to claim 2 wherein the insulating layer comprises silicon oxides (SiOx).

4. The support according to claim 2 wherein the insulating layer comprises silicon nitrides (SiNx).

5. The support according to claims 2 to 4 further comprising an adhesion layer between the metallic foil and the insulating layer and / or between the insulating layer and the support member, optionally wherein the adhesion layer is a metal adhesion layer, preferably wherein the adhesion layer comprises titanium.

6. The support according to any of claims 1 to 5 wherein the diameter of each hole of the metallic foil is 5 pm or less.

7. The support according to any of claims 1 to 6 wherein the holes of the metallic foil are arranged in a hexagonal array or a square pattern array.

8. The support according to any of claims 1 to 7 wherein the metallic foil has a thickness of up to 100 nm, optionally wherein the thickness is less than 50 nm.

9. The support according to any of claims 1 to 8 wherein the ratio of the diameter of each hole to the thickness of the metallic foil is 15:1 or less.

10. The support according to any of claims 1 to 9 wherein the support member comprises a plurality of spaced support elements, optionally wherein the plurality of spaced support elements are arranged to form a mesh.11 . The support according to any of claims 1 to 10 wherein the metallic foil consists of one or more of gold, palladium and platinum or an alloy thereof, optionally wherein the metallic foil consists of gold or an alloy thereof; and / or wherein the support member consists of one or more of gold, palladium and platinum or an alloy thereof, optionally wherein the support consists of gold or an alloy thereof.

12. The support according to any of claims 1 to 11 wherein the metallic foil and the support member have the same elemental composition.

13. The support according to any of claims 1 to 12 wherein the plane of the metallic foil does not intersect the support member.

14. The support according to any of claims 1 to 12 wherein the plane of the metallic foil intersects the support member.

15. Use of the support according to any of claims 1 to 14 in transmission electron cryo-microscopy.

16. A method of manufacturing a support for an electron microscope sample comprising the sequential steps of i) providing a metallic foil having one or more holes therethrough, ii) defining the contours of a support member directly on the metallic foil using a positive photoresist layer, Hi) depositing an insulating layer in the defined contours, iv) removing the photoresist layer of step ii) and reapplying a photoresist with the same pattern, and v) forming the support member directly on the deposited layer by electroplating.

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