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 plane of the metallic foil intersects 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 2311684.1 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 and Russo 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] Existing grids generally have a ‘grid bar side’ and a ‘foil side’. That is, the plane of the metallic foil does not intersect the support member, providing an asymmetric support. This means that a specimen held by the support can generally only be addressed, for example if it is to be etched or patterned, from a single side. In some circumstances it may be preferable to have access to both sides of the specimen. For example, if the specimen is to be processed by plasma etching, etching on or from both sides may increase efficacy and efficiency of the process.

[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 plane of the metallic foil intersects the support member and wherein the ratio of the thickness of the support member on one side of the plane of the metallic foil ti to the thickness of the support member on the opposite side of the plane of the metallic foil t2 (ti / t2) is 1 to 10.

[0015] In this way, a symmetrical support is provided. This allows for access to both sides of the specimen in the support, facilitating, for example, two-sided etching, and preparation of two-sided vitreous specimens by high-pressure freezing.

[0016] The thickness of the support member on one side of the plane of the metallic foil ti may be not equal to the thickness of the support member on the opposite side of the plane of the metallic foil t2. Preferably the thickness is uniform across each side of the support member to within 0.1 pm accuracy.

[0017] In some embodiments, the ratio ti / t2 is 1 to 5.

[0018] In some embodiments, the ratio ti / t2 is 1 . That is, the thickness of the support member on either side of the plane of the metallic foil is substantially the same (ti = t2), preferably to within 0.1 pm accuracy.

[0019] A suitable total thickness of the support member (ti +t2) is in the range of 3 to 20 pm, preferably 5 to 7 pm.

[0020] 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.

[0021] In some embodiments, the metallic foil does not comprise any holes that do not meet the specified nanoscale diameter requirements.

[0022] 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 e_ / A2). This is followed by a decreasingly correlated movement that continues with reduced speed to the end of the exposure.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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 .

[0029] An advantage of using the specified ratios is that it helps to eliminate buckling of specimen films when such films are formed in the holes, such as a suspended amorphous ice film that are 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 pseudo-diffusion, 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 e / A2at 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 e / A2. The present movement-suppressing microscopy specimen support allows atomic structure determination at only 1 e / A2, and extrapolation back to the point before destructive effects of electron radiation affect the reconstruction,

[0030] 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 provide closer packing and improves structural rigidity; these may therefore be preferred. The mesh hole pattern may correspond to the metallic foil hole pattern.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] In some embodiments, the metallic foil is electrically insulated from the support member. In this way, an electron microscopy sample support which enables electrical measurements to be performed on the support is provided.

[0035] 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 or SiO2 or non- stoichiometric silicon oxides SiOx (where x<2), aluminium oxide AI2O3, or silicon nitride SisN4, SixNx.

[0036] Other examples include high-K dielectrics which have a dielectric constant greater than that of SiO2, 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 silicon dioxide SiO, SiO2 or non-stoichiometric silicon oxides SiOx (where x<2). The thickness of the insulating layer c / 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 required 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.

[0037] 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, 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.

[0038] 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.

[0039] 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 on a patterned substrate, ii) defining the contours of a support member directly on the metallic foil using a positive photoresist layer, iii) etching the metallic foil present in the defined contours, iv) etching the exposed patterned substrate to form trenches in the defined shape of the support member, v) forming the support member in the trenched by electroplating, and vi) releasing the support from the patterned substrate.

[0040] Steps (i), (ii), (v) and (vi) are as described in Naydenova and Russo 2022 for integrated wafer-scale manufacturing of cryoEM.

[0041] 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.

[0042] In some cases, there is a step of cleaning the patterned substrate before deposition of the metallic layer.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] In some embodiments, the contours of the support member are outlined using a wet etchant. Examples of suitable wet etchants include etchants based on iodine and sodium or potassium iodide, or alkaline cyanide etchants. Preferably the wet etchant is an iodine-based gold TFA etchant. It is also possible to use a dry etchant, for example reactive plasma etching based on fluorine, chlorine or bromine chemistry. Examples include Ch, HBr, BCh, CF4 and CCk.ln some embodiments, the same wet etchant used to define the contours of the support member is used for etching the metallic foil. Preferably, the wet etchant is an iodine-based gold TFA etchant.

[0048] In some embodiments, plasma etching is used to etch the patterned substrate. In this way, the open patterned substrate surface is made accessible for etching to make trenches which outline the support member shape. The photoresist-covered metallic foil can act as a mask for the etching of the patterned substrate, removing the need for an additional masking layer. In the case where the patterned substrate is a silicon wafer, preferably SFe plasma is used. In this way, a nearly isotropic etch profile can be achieved. This is suitable for features, such as the support member, where the sidewall shape is not critical. Alternatively, a Bosch-type process (deep reactive ion etching) as disclosed in Jefimovs 2017 may be used to provide vertical sidewalls and less undercutting. The plasma etching time of the silicon determines the thickness of the support member on the substrate side of the metallic foil (ti). The longer the etching time the greater ti. The etching rate depends on the total exposed area of patterned substrate in the chamber, and so the flow-rate and the plasma time are preferably re-optimised for any given substrate.

[0049] The total electroplating time determines the total thickness of the support member (ti + t2), provided that the photoresist layer is sufficiently thick. In this configuration, the deposition during electroplating states at the mid-plane of the support member, on the edge of the metallic foil. Advantageously, this ensures that the metallic foil remains connected to the support member, even if the metallic foil has been overetched.

[0050] In some embodiments, to release the support from the patterned wafer, the silicon is dissolved in hot (80 °C) potassium hydroxide (KOH).

[0051] 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.

[0052] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0053] Summary of the Figures

[0054] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0055] Figure 1 shows a section view of a typical HexAuFoil grid.

[0056] Figure 2 shows a section view of an embodiment of the present electron microscopy support.

[0057] Figure 3A shows a schematic of part of the process for manufacturing electron microscopy supports wherein the plane of the metallic foil intersects the support member.

[0058] Figure 3B shows a scanning electron micrograph of the resist-coated wafer after etching the grid bar trenches in SFe. This micrograph was acquired at 45° tilt, using 30 keV electrons and an Everhart- Thornley electron detector. The inset is a magnified view of the boxed area, acquired at the same imaging condition, but displayed with a different contrast to highlight the roughness of the etched silicon.

[0059] Figure 3C shows a scanning electron micrograph of an electron microscopy support after release from the wafer, acquired at 52° tilt, using 30 keV electrons and an Everhart-Thornley detector. A position where the foil is broken was specifically selected for the imaging in order to reveal the grid bars on both sides of the foil. In this particular example, the grid bars were plated to a height over that of the resist, leading to a lateral overgrowth on the top side. The contrast between the grid bar on side 1 and side 2 is due to the different roughness of the templating surfaces (etched Si versus photoresist).

[0060] Detailed Description of the Invention

[0061] 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.

[0062] 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 (11) and the support member comprising grid bars (12) comprise gold.

[0063] In contrast to that, Figure 2 shows an embodiment of the electron microscopy support (20) of the present invention. The support comprises a metallic foil (21) and a support member comprising grid bars (22). The plane of the metallic foil (21) intersects the grid bars (22). In this particular embodiment the metallic foil (21) and the grid bars (22) both comprise gold. In this way, increased stability during imaging can be achieved since the two structures have the same thermal expansion coefficients. The thickness of the grid bar on each side of the plane of the metallic foil (ti an t2) can be controlled independently by controlling the etching time and the electroplating time, respectively. In the illustrated embodiment ti = t2.

[0064] Method

[0065] Figure 3A schematically shows part of an embodiment of a process for manufacturing electron microscopy sample supports of the present invention, wherein the plane of the metallic foil intersects the support member.

[0066] 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 degenerately doped silicon wafer (32) as the patterned substrate as described in Naydenova and Russo 2022. The doped silicon wafer 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.

[0067] 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).

[0068] It is preferable that the metallic foil and the support member have the same elemental composition. The metallic foil is deposited onto the substrate by cryogenic evaporation. The doped silicon wafer (32) is first provided with a sacrificial copper layer (33) before the gold foil layer (31) is deposited. Both layers are deposited by electron beam evaporation, with the doped silicon wafer (32) cooled using liquid nitrogen to 80-90 K.

[0069] Once the metallic foil (31) is deposited, the contours of the support member (35) 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 Figure 3A. This mask step is used to remove nearly all the metallic foil from the regions in between the support member outlined by wet etching. Typically, the wet etchant used is an iodine-based gold TFA etchant. Alternative etchants may be other wet etchants such as those based on iodine and sodium or potassium iodide, or alkaline cyanide etchants, or dry etchants based on reactive plasma etching.

[0070] In the next step, the doped silicon wafer (32), exposed after etching of the metallic foil (Figure 3A second image from the left), is isotropically etched using SFe plasma etching (Figure 3A second image from the right). The photoresist-covered metallic foil acts as a mask for etching of the silicon wafer, removing the need for an additional masking layer. An SEM image of such a stage is shown in Figure 3B. The silicon etching is carried out in a Henniker-100 reactor operated at 100 W, with a gas flow of 5 seem. After exposure to 40 minutes of the SFe plasma, 4 pm deep trenches are etched on all exposed areas of the 4 inch wafer, while the photoresist and the underlying foil remain intact (Figure 3A second image from the right). Such an etching method provides a nearly isotropic etch profile suitable for features such as grid bars where the sidewall shape is not critical. A different etch process (for example, a Bosch-type process) can alternatively be used to provide vertical sidewalls and less undercutting, if required. The plasma etching time of the substrate determines the thickness of the support member on the substrate side of the metallic foil (ti). The longer the etching time the greater ti.

[0071] In the trenches formed in this manner, the support member (35) can be formed by, for example, electroplating (Figure 3A right image). In this particular embodiment, the support member (35) comprises gold grid bars of 10 pm in thickness. Alternatively, the support member (35) may comprise palladium or platinum, or an alloy thereof. Gold can be electroplated directly onto the silicon trenches, especially with the use of degenerately-doped silicon wafers with a low resistivity. It is also possible to directly electroplate onto lightly-doped wafers despite the higher resistivity of such wafers. In this particular embodiment, the gold deposition by electroplating starts at the mid-plane of the grid bar of the support member (35) on the edge of the metallic foil (31). This is beneficial as even if the metallic foil is previously over-etched, the electroplating will ensure that the metallic foil remains connected to the support member. The electroplating time determines the total thickness of the support member (ti + t2). The longer the plating time, the thicker the support member.

[0072] 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). The supports lifts off the substrate (32) almost instantly on immersion into the KOH solution, which also removes the residual photoresist (34). The sacrificial copper layer (33) is removed from the released supports with a Piranha solution (3H2SO4 : I 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] An SEM image of such a product is shown in Figure 3C.

[0075] In an alternative to this type of method, the electron microscope support may be formed by aligning and bonding a bare grid to the foil-side of a foil-coated grid; that is, by forming a grid according to known methods (of the type shown in Figure 1) and then attaching an additional support member such as a grid onto the foil surface on the foil side. However, the method partly depicted in Figure 3A is advantageously more easily applied to the wafer scale and is therefore preferable.

[0076] Figure 3B shows a micrograph of the resist-coated silicon wafer (41). In this particular embodiment, the silicon wafer has been etched with SFe plasma. In this particular embodiment, the photoresist (42) has a hexagonal pattern.

[0077] Figure 3C shows a micrograph of an electron microscopy support after release from the wafer. In this particular embodiment the metallic foil and the support member comprising grid bars both comprise gold. In this way, increased stability during imaging can be achieved since the two structures have the same thermal expansion coefficients.

[0078] In this particular embodiment, the electron microscopy support is not perfectly symmetric (ti = 7.2 pm and t2 = 4.1 pm). The thickness of the support member either side of the plane of the metallic foil can be independently controlled by controlling the etching time and electroplating time, respectively.

[0079] ***

[0080] 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.

[0081] 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.

[0082] 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.

[0083] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. 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.

[0084] 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%.

[0085] References

[0086] 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.

[0087] 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).

[0088] 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 plane of the metallic foil intersects the support member wherein the ratio of the thickness of the support member on one side of the plane of the metallic foil ti to the thickness of the support member on the opposite side of the plane of the metallic foil t2 (ti / t2) is 1 to 10.

2. The support according to claim 1 wherein the ratio ti / t2 is 1 to 5.

3. The support according to claim 1 or 2 wherein the ratio ti / t2 is 1 .

4. The support according to any of claims 1 to 3 wherein the diameter of each hole of the metallic foil is 5 pm or less.

5. The support according to any of claims 1 to 4 wherein the holes of the metallic foil are arranged in a hexagonal array or a square pattern array.

6. The support according to any of claims 1 to 5 wherein the metallic foil has a thickness of up to 100 nm, optionally wherein the thickness is less than 50 nm.

7. The support according to any of claims 1 to 6 wherein the ratio of the diameter of each hole to the thickness of the metallic foil is 15:1 or less.

8. The support according to any of claims 1 to 7 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.

9. The support according to any of claims 1 to 8 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.

10. The support according to any of claims 1 to 9 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.

11. The support according to any of claims 1 to 10 wherein the metallic foil and the support member have the same elemental composition.

12. The support according to any of claims 1 to 11 wherein the metallic foil is electrically insulated from the support member.

13. The support according to any of claims 1 to 12 wherein the metallic foil and the grid are integrally formed.

14. Use of the support according to any of claims 1 to 13 in transmission electron cryo-microscopy.

15. 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 on a patterned substrate, ii) defining the contours of a support member directly on the metallic foil using a positive photoresist layer, iii) etching the metallic foil present in the defined contours, iv) etching the exposed patterned substrate to form trenches in the defined shape of the support member, v) forming the support member in the trenches by electroplating, and vi) releasing the support from the patterned substrate.

Images