Electron microscopy sample support

Abstract

The present disclosure provides an electron microscopy sample support comprising a metallic support, the metallic support comprising a plurality of wells, wherein each well comprises a solid, continuous base and the ratio of the depth to cross-sectional diameter or longest cross-sectional dimension of each well is in the range of 15:1 to 1:2. A processed electron microscopy sample support is also described comprising: a metallic support, the metallic support comprising a support region surrounding an imaging region, wherein the support region is thicker than the imaging region, and the support region and the imaging region are integral, wherein the plane of the surface of the support region is at an angle to the plane of the surface of the imaging region, wherein the imaging region comprises a plurality of through-holes in the metallic support, and wherein each through-hole optionally houses a section of a sample suspended in frozen liquid. Also described are methods of manufacturing an electron microscopy sample support, and supports obtained by such methods, the method comprising the steps of: providing a substrate comprising a plurality of protrusions; depositing a layer of metallic material on the substrate covering and incorporating a plurality of the protrusions; and separating the layer of metallic material from the substrate to provide an electron microscopy sample support comprising a plurality of wells complementary to the protrusions of the substrate. Methods of preparing a sample for electron microscopy are also described.

Description

[0001] ELECTRON MICROSCOPY SAMPLE SUPPORT Field of the invention The present invention relates to an electron microscopy (EM) sample support; a method of manufacturing such an electron microscopy sample support; a method of preparing a sample for electron microscopy; and a processed electron microscopy sample support. The support is particularly useful in cryo-EM imaging. Background Electron microscopy techniques can be used to image a specimen. In such techniques, a beam of electrons is used to “illuminate” a specimen. The presence of the specimen in the electron beam results in changes to that beam. The changes to the beam induced by the sample can be examined to create a magnified image of the specimen. In order to be illuminated by an electron beam, a specimen must be adequately supported in that beam. 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 the results, including resolution of image, obtained through use of electron microscopy techniques. Cryo-electron microscopy (cryo-EM) involves studying specimens at cryogenic temperatures. The thickness of the specimen is a key parameter affecting signal-to-noise ratio in the images collected due to the limited mean free path length of electrons in frozen biological materials. Other key challenges in electron microscopy and cryo-EM are associated with sample preparation and handling. It is known to use grid-based sample supports with an overlaying thin foil or film having a porous region which receives the sample. In cryo-EM the support grid provides mechanical stability to the sample. A sample dispersed in a liquid medium is provided to the pores of the film and then flash-frozen to immobilise the sample in vitrified ice. Among other problems, upon imaging such samples, the electron flux of the illuminating beam may heat and / or electrically charge the sample and grid support elements. Differential thermal expansion and / or electronic interactions can result in unwanted motion of the sample in the beam which can impair the image obtained. As an attempt to improve image quality by producing thinner specimens, to account for the limited path length of electrons, and to reduce motion, it is known to prepare samples for cryo-EM by preparing a sample supported in vitrified ice within a relatively large grid aperture and using cryo focussed ion beam (FIB) milling to etch away the vitrified ice. This forms thin lamellae of ice containing the sample which can be used for cryo-EM and electron cryotomography (cryo-ET). However, due to the relatively large size of the grid apertures (and similarly in cases with relatively large sample sizes), to prepare the vitrified ice, straightforward plunge freezing is not suitable. This is because the fluid (usually water) suspending the sample does not freeze fast enough or uniformly, which can result in ice crystals surrounding and within the sample, i.e., the ice is not vitrified ice. Such crystals can damage specimens and degrade image quality due to electron diffraction. This effect also limits the thickness of samples that can be prepared by this route – samples freeze from the outside progressively inwards and in thick samples this does not occur quickly enough to form vitrified ice so, again, ice crystals are formed with the associated problems noted above. As an attempt to overcome this, high pressure freezing equipment may be used at considerable additional cost and complication. Even then, the thickness is still limited by the rate of cooling of the material in conjunction with the rate it is compressed. Furthermore, due to the relatively large amount of water in the grid apertures, the sample has a high degree of freedom of motion prior to freezing – specimens may exhibit preferred or random orientations in relation to the grid, preventing certain aspects of specimens from being imaged in the vitrified ice due to undesirable orientations, or may deposit in random locations across the grid, making directed imaging challenging. Other techniques include preparing a sample supported by vitrified ice on top of a grid with blotting and plunge freezing techniques. However, this can produce inconsistent results between batches of grids, and any part of a specimen not fully embedded in the ice may be subject to additional radiation damage. The problems remain that a sample may exhibit undesirable preferred orientations or locations on the grid, or these may be random, making directed imaging challenging. Even when FIB-milling is used there may still be a large amount of vitrified ice on the grid which can contribute to increased motion and poorer image quality. It is therefore desired to provide a specimen support, for use in electron microscopy, which may address some of the features of known specimen supports. Summary of invention Accordingly, a first aspect of the present disclosure provides an electron microscopy sample support comprising a metallic support, the metallic support comprising a plurality of wells, wherein each well comprises a solid, continuous base and the ratio of the depth to cross- sectional diameter or longest cross-sectional dimension of each well is in the range of 15:1 to 1:2. The metallic support provides structural strength to the electron microscopy sample support. In contrast to conventional grid-based supports the metallic support may be more mechanically stable as a greater proportion of the electron microscopy sample support may be substantially thicker than the sample for imaging. The metallic nature of the support material also ensures that the support has high thermal conductivity to maximise cooling rates and minimise formation of crystalline ice during freezing (because the water in the wells freezes very rapidly on cooling, e.g., plunge freezing), and minimise charging (due to dissipation of beam-induced charges before they accumulate). A second aspect provides a method of manufacturing an electron microscopy sample support, the method comprising the steps of: (i) providing a substrate comprising a plurality of protrusions, (ii) depositing a layer of metallic material on the substrate covering and incorporating a plurality of the protrusions, (iii) separating the layer of metallic material from the substrate to provide an electron microscopy sample support comprising a plurality of wells complementary to the protrusions of the substrate. A third aspect provides a method of preparing a sample for electron microscopy comprising the steps of: (i) providing an electron microscopy sample support according to the first aspect, (ii) loading a plurality of wells in the electron microscopy sample support with a sample suspended in a liquid, (iii) freezing the liquid, (iv) processing a portion of the electron microscopy sample support by removing the material defining the base of a plurality of the wells. A fourth aspect provides a processed electron microscopy sample support comprising: a metallic support, the metallic support comprising a support region surrounding an imaging region, wherein the support region is thicker than the imaging region and the support region and the imaging region are integral, wherein the plane of the surface of the support region is at an angle to the plane of the surface of the imaging region, wherein the imaging region comprises a plurality of through-holes in the metallic support, and wherein each through-hole optionally houses a section of a sample suspended in frozen liquid. Brief description of the figures So that the invention may be understood, and so that further aspects and features thereof may be appreciated, embodiments illustrating the principles of the invention will now be discussed in further detail with reference to the accompanying figures, in which: Figure 1 shows a schematic illustration of steps in a method of preparing a sample for electron microscopy according to the present invention. Figure 2 shows scanning electron microscopy (SEM) images of an electron microscopy sample support according to the present invention. Figure 3 shows an illustration of how the wells of an electron microscopy sample support according to the present invention may enable imaging of particular cell orientations. Figure 4 shows a schematic illustration of a method of manufacturing an electron microscopy sample support according to the present invention. Figure 5 shows a scanning electron microscopy (SEM) image of a substrate for use in a method of manufacturing an electron microscopy sample support according to the present invention (left) and an image of electron microscopy sample supports manufactured according to the present invention (right). Figure 6 shows a lithography mask design at increasing zoom factors for use in a method of manufacturing an electron microscopy sample support according to the present invention. Figure 7 shows SEM (a), TEM (b), and cryo-confocal microscopy (c) images of a processed electron microscopy sample support according to the present invention with TEM (d) and cryo-fluorescence (e) images of a cell slice in the support. Figure 8 shows a violin plot comparing mean motion per frame of tilt series collecting from a processed electron microscopy support according to the present invention with that of conventional standard lamellae. Figure 9 shows electron cryo-tomography (cryo-ET) images of E. coli bacteria cells obtained with an electron microscopy sample support according to the present invention. Figure 10 shows electron cryo-tomography (cryo-ET) images and tomogram models of E.coli cells showing FtsZ filaments obtained with an electron microscopy sample supportaccording to the present invention. Figure 11 shows sub-tomogram average maps of a 70S ribosome obtained using an electron microscopy sample support according to the present invention and a Fourier shell correlation plot of half-maps generated during refinement. Figure 12 shows a diagram of the sub-tomogram averaging workflow used to map the E. coli 70S ribosome with data collected according to the present invention. Detailed description While the invention has been described in conjunction with exemplary embodiments, many equivalent modifications and variations falling within the scope of the claims 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 scope of the invention as defined by the claims. 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 are not bound by any of these theoretical explanations. 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 words “have”, “comprise”, and “include”, and variations such as “having”, “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. The words "preferred" and "preferably" are used herein refer to embodiments of the invention that may provide certain benefits under some circumstances. However, it is to be appreciated that other embodiments may also be preferred under the same or different circumstances. The recitation of one or more preferred embodiments therefore does not mean or imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure, or from the scope of the claims. As used herein, the term “metallic” is used to refer to a material or component displaying properties of a metal. In particular they display high electrical and thermal conductivity. In many cases electrical conductivity in metallic materials is higher than 104S / m. As used herein, the term “well” is used to refer to a cavity recessed into a surface of a substrate that does not penetrate through the substrate in a thickness direction. A well has one or more sides and a base. The base of a well is a solid, continuous layer that does not comprise any perforations or holes penetrating through the substrate. As used herein, the term “through-hole” is used to refer to a hole or aperture in a surface of a substrate which penetrates through the substrate in a thickness direction. This is as opposed to a recess or well in the surface of the substrate. As used herein, the term “sample” or “specimen” may refer to any material that it is desirable to image with electron microscopy. For example, a sample may comprise biological material such as cells or proteins. A sample may be in aqueous solution. A sample may comprise single molecules. A sample may be suspended in a liquid. Typically, a sample is suspended in water. References herein to an “electron microscopy sample support” relate to the sample support of the present invention. They do not necessarily imply that the support is suitable for transmission electron microscopy prior to processing, e.g., to remove the at least one of the well bases. References herein to a “processed electron microscopy sample support” refer to an “electron microscopy sample support” defined herein that has been processed to remove the base of at least one of the wells to provide a through-hole. References herein to electron microscopy refer to any type of electron microscopy but may preferentially refer to transmission electron microscopy, in particular transmission electron cryo-microscopy. The features, options, and preferences that are described herein in relation to one aspect or embodiment may be equally applicable to other aspects or embodiments insofar as they are compatible and combinable. In the usual way, values disclosed as endpoints of a range are disclosed independently and are combinable with other endpoint values to form a different range. For example, any lower endpoint may be combined with any upper endpoint, or with any other lower endpoint, to form a different range. Similarly with upper endpoints which may be combined with any lower endpoint or any other upper endpoint. Electron microscopy sample support A first aspect of the present disclosure provides an electron microscopy sample support comprising a metallic support, the metallic support comprising a plurality of wells, wherein each well comprises a solid, continuous base and the ratio of depth to cross-sectional diameter or longest cross-sectional dimension of each well is in the range of 15:1 to 1:2. The metallic support provides structural strength to the electron microscopy sample support. In contrast to conventional grid-based supports the metallic support may be more mechanically stable as a greater proportion of the electron microscopy sample support may be substantially thicker than the sample for imaging. The metallic nature of the support material also ensures that the support has high thermal conductivity to maximise cooling rates and minimise formation of crystalline ice during freezing (because the water in the wells freezes very rapidly on cooling, e.g., plunge freezing), and minimise charging (due to dissipation of beam-induced charges before they accumulate). The wells do not penetrate through the metallic support. The presence of wells enables the metallic support to receive a sample as the wells act as containers for the sample. In some cases, the plurality of wells accepts a plurality of samples, e.g., with each well containing only one or a few samples. The dimensions and / or aspect ratio of the wells ensure that, in some cases, each well can bias a sample into a given, preferred, orientation. For example, an elongate sample may be biased into an orientation in which the longest axis of the sample is orientated substantially aligned with the long axis of a well. For example, a sample such as non-spherical bacteria can be biased into an orientation with its long axis along the long axis of the well accommodating it, typically with one bacterial cell per well. Specimens may thus be prevented from adopting undesirable orientations and biased into orientations that are preferential for imaging. This provides more control over the orientations that can be imaged by electron microscopy once the electron microscopy sample support has been processed and prepared for imaging. In some cases, the electron microscopy is transmission electron microscopy, preferably transmission electron cryo-microscopy (cryo-EM) or electron cryo-tomography (cryo-ET). Exemplary electron microscopy sample supports (100) are depicted in Fig.1, Fig.2 (left), and Fig.5 (right). Metallic support In some examples, the metallic support is an integral object providing a unitary body of material defining the plurality of wells. This provides a structurally robust electron microscopy sample support, typically more robust than known supports which use a separate grid and foil that may break during handling. It allows the entire electron microscopy sample support to be manufactured in one process. Furthermore, this avoids the presence of parts or portions of the electron microscopy sample support with different thermal expansion coefficients or joints between different elements of the support grid (e.g., grid bars and foil), so reduces stress, strain, relative movement and / or damage such as cracking during changes in temperature or electrical charging phenomena, such as on freezing or during electron illumination. In some examples, the metallic support provides a unitary body of material at least 1 µm thick. This provides structural strength and support for a sample. The electron microscopy sample support may be handled more easily, and a sample may be added with less risk of damaging the electron microscopy sample support. Methods such as centrifuging may be used to introduce a sample into the wells, which a conventional grid and foil support could not withstand. In some examples the metallic support is between 1 and 50 µm thick. The thickness may be at least 1 µm, at least 2 µm, or at least 5 µm. The thickness may be up to 50 µm, up to 30 µm, up to 20 µm, or up to 10 µm. Some exemplary thickness ranges may be 1-10 µm, 2-10 µm, 5-10 µm, 2-10 µm, 2-20 µm, 5-20 µm, 1-50 µm, 2-50 µm, and 5-50 µm. In some examples the metallic support comprises an edge portion and a sample receiving portion, wherein the wells are located in the sample receiving portion. In some cases, wells are not located in the edge portion. In some examples, the metallic support has regions of different thicknesses. The edge portion may be thicker than the sample receiving portion. The ratio of the thickness of the edge portion to the sample receiving portion may be at most 10:1. The edge portion provides structural stability and a robust area for handling of the electron microscopy sample support. In some examples, the sample receiving portion does not comprise any through-holes. This allows a sample to be loaded into the wells of the electron microscopy sample support without any leakage and increases the structural stability of the support. In some examples, there are a plurality of edge portions and / or sample receiving portions in one electron microscopy support. In some examples, the edge portion is a circumferential region of the metallic support and the sample receiving portion is a central region. This allows easy handling of the electron microscopy sample support and provides some structural protection for the sample receiving portion. In some examples, the metallic support is a disc shape. In some examples, the disc has a diameter of between about 1 and 5 mm, such as between about 2 and 3.5 mm, preferably about 3.05 mm. A 3.05 mm diameter disc provides compatibility with conventional electron microscopes and cryo-EM imaging devices. An exemplary electron microscopy sample support (100) is depicted in Fig.1(a) with an edge portion of the metallic support (110) around the circumference of a sample receiving portion of the metallic support (120) which contains a plurality of wells. The exemplary electron microscopy sample support of Fig.1 also shows orientation markings in white. Fig.2 (left) shows a corresponding SEM image of an exemplary electron microscopy sample support. An edge portion of the metallic support (110) is visible around the circumference of a sample receiving portion of the metallic support (120) which contains a plurality of wells. Orientation markings can be seen located in the edge portion of the metallic support. The scale bar represents 500 μm. Metallic support material In some examples, the metallic support comprises or consists of, a metal or an alloy. In some examples, the metallic support comprises or consists of a metal. Metals are used to minimise charging, chemical and / or other similar motion-inducing processes, which may occur on exposure of the electron microscopy sample support to a high energy electron beam. Without being bound by any theory, it is thought that a metallic material can conduct electrons away from the sample during exposure so dissipating charge before it builds up. Metallic materials also have high thermal conductivity, so are chosen to maximise the dissipation of heat away from a sample on the electron microscopy sample support and minimise formation of crystalline ice during freezing and further storage or use and to minimise sample heating due to electron flux in the imaging beam. Similarly, metallic materials may be able to minimise heating during FIB milling due to their high thermal conductivity and ability to conduct charge away from the sample, so higher currents may be used in the milling process. In some examples, the metallic support comprises, or consists of, one or more metals selected from transition metals, aluminium and beryllium, or an alloy thereof. Transition metals are elements in groups 3 to 11 of the periodic table of elements. In some cases, the transition metals are selected from one or more of noble metals (ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, gold), copper, molybdenum, titanium, nickel, chromium, tungsten, hafnium, and tantalum or an alloy thereof. In some cases, the one or more transition metals are selected from the noble metals ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, gold or an alloy thereof. In some examples, the metallic support comprises, or consists of, a metal selected from the group of gold, platinum, tungsten, tantalum, or palladium or an alloy comprising a metal from this group. The metals of this group are relatively inert and non-toxic to cells, avoiding damage to samples contacted with them, and can be milled using focussed ion beam (FIB) milling. In some examples, the metallic support consists essentially of one or more of the materials listed above. In some cases, the metallic support consists of only one of the materials listed above. This may ensure that the support has a consistent thermal expansion coefficient. In some cases, the metallic support is gold or an alloy thereof. In some cases, the alloy is a binary alloy, such as an alloy selected from gold-silver alloy, gold-copper alloy, nickel- titanium alloy, gold-platinum alloy and platinum-iridium alloy. It may be that the metallic support does not comprise aluminium. It may be that the metallic support does not comprise beryllium. It may be that the metallic support does not comprise an alloy. Gold is particularly stable under irradiation by an electron beam and results in less motion during data collection than other materials. Without being bound by any theory, this is thought to be because of much reduced charging. For the avoidance of doubt, the metallic support is not formed from an electrical insulator, a semi-conductor material or a carbon material such as amorphous or graphitic carbon, although these may be present in a metallic alloy as described herein. Semi-conductor materials include materials whose conductivity drops with decreasing temperature in the 300 to 80 K range. This includes carbon, silicon (below 1020 / cm3doping with a dopant element such as boron, aluminium, phosphorus and arsenic), gallium arsenide and other III-V or II-VI binary semiconductors. In some cases, the purity of the metallic support material is 90% or more, preferably 99% or more; even more preferably 99.999% or more. (i.e., contains the stated % of the relevant metallic material). Wells in the metallic support In some examples, the base of a well is made from the same material as the side(s). In some examples, the wells are completely defined by the metallic support. The side(s) and base of each well are formed from the metallic support material. In some examples, the base and side(s) of a well are integral. In some examples, the wells act as containers for a sample. Individual samples e.g., cells, or parts of a sample, may sit inside each well. This is different from previous cryo-EM techniques where the sample typically sits on top of a grid or is suspended in the pores of a foil and is not contained in the same way. Containing the sample in the wells in this way allows for better control during imaging and may lead to improved resolution. In some examples, the shape of the wells is chosen or manufactured to be compatible with the shape and dimensions of the sample they are to receive, with regards to the limitations of the electron microscopy sample support manufacturing process described below. In some examples, the wells are cylindrical in shape with one open and one closed end. This allows them to act as containers for a sample and is particularly suited to biological samples such as cells where each well can act as a container for an individual cell. A cylindrical shape reduces the amount of excess solution and thus ice around a sample, especially if, as in some cases, the dimensions of each well are compatible with (e.g. slightly larger than) the dimensions of the sample. This minimises the formation of crystalline ice during freezing and improves the resulting image quality by reducing the total amount of ice around samples. Excess ice may contribute to movement, charging and contamination of samples during the imaging process. When the electron microscopy sample support is processed (e.g., to remove the closed end, as described below), the wells enable the formation of through-holes for imaging sample cross-sections. In some examples, the thickness of the metallic support below each of the wells is at least 100 nm, i.e., the base or ‘floor’ of the wells is at least 100 nm thick. In some examples this thickness is at least 150 nm, or at least 200 nm, or at least 300 nm, or at least 400 nm, or at least 500 nm, or at least 750 nm. This contributes to robustness of the well formation in the sample support. In some examples, the ratio of the thickness of the metallic support below each of the wells, i.e., the base or ‘floor’ of the wells, to the depth of the wells is in the range 1:150 to 1:1. The lower limit of this ratio range may be 1:1, or 1:2, or 1:5, or 1:10, or 1:20. The upper limit of this range may be 1:150, or 1:100, or 1:75, or 1:50, or 1:20. In some examples, the sides of each well are substantially orthogonal to the surface of the metallic support, i.e., the sides of each well are parallel to the normal of the plane of the surrounding surface (e.g., the surface of the sample receiving portion). In this case, if material is removed from the electron microscopy sample support along a plane at an acute angle to the plane of the surface during processing / sample preparation then the processed electron microscopy sample support may comprise cross-sections of the wells taken at different heights through different wells. The resulting processed electron microscopy sample support may have through-holes with sides (previously the sides of the wells) at an angle of between >0° to 20°, for example 1° to 15°, or for example 5° to 10°, to the normal of the plane of the surrounding processed surface (e.g., the surface that has been formed by FIB milling, also referred to as the surface of the imaging region). This has the effect of removing a layer of the metallic support at an acute angle to the original surface to cut through different heights of different individual wells. Thus, in an electron microscopy sample support, cross-sections from throughout the length / height of different wells, and therefore at different heights through a series of samples in the plurality of wells, may be obtained and imaged. In some examples, the wells may recess into the surface of the metallic support such that the sides of each well are at an angle to the normal of the plane of the surface of the metallic support into which they are recessed (e.g., the surface of the sample receiving portion of the electron microscopy sample support). The angle may be complementary to the angle at which material is removed from the electron microscopy sample support during processing / sample preparation such that after sample support processing, the sides of the through-holes (which were previously the sides of the wells), may be parallel to the normal of the plane of the processed surface (e.g., the FIB-milled surface or surface of the imaging region), i.e., the sides may be perpendicular to the plane of the processed surface. The angle at which the wells recess into the surface may be from >0° to 20°, for example 1° to 15°, such as 5° to 10°. The resulting processed electron microscopy sample support may have through-holes with sides substantially orthogonal to the surrounding surfaces, i.e., sides parallel to the normal of the plane of the surrounding surfaces. In some examples, each well has a cross-sectional diameter (or longest cross-sectional dimension) of less than 4 μm. This ensures that any water in the wells forms vitreous ice relatively easily on freezing, e.g., plunge freezing, and minimises the formation of crystalline ice. Without being bound by any theory, it is thought that diameters larger than this will always result in the formation of some crystalline ice due to the limitations of the thermal conductivity of water. This is explained, for example, in Dubochet et al., 1988. In some examples, each well has a cross-sectional diameter (or longest cross-sectional dimension) between 0.2 and 3 μm. In some examples each well has a cross-sectional diameter (or longest cross-sectional dimension) between 1 and 2 μm. The size may be chosen to be compatible with (e.g., slightly larger than) a dimension, e.g., the width, of the sample being imaged. In some examples, the wells all have approximately the same cross-sectional diameter (or longest cross-sectional dimension). In some examples, each well has a cross-sectional diameter (or longest cross-sectional dimension) of approximately 1.5 μm. This dimension is particularly useful for cells such as non-spherical bacteria as it may bias the bacteria towards a particular orientation, e.g., with long axis aligned with the long axis of the well. In some examples, each well has a depth between 1 and 15 μm, such as between 1 and 10 μm, such as between 1 and 6 μm, such as between 1.5 and 5 μm, such as between 2 and 4 μm. In some examples, each well has a depth of approximately 3 μm. In some examples, the wells all have approximately the same depth. The ratio of the depth to cross-sectional diameter or longest cross-sectional dimension of each well is in the range of 15:1 to 1:2. In some examples, the wells each have a depth to cross-sectional diameter ratio of between 4:1 and 1:2, such as between 3:1 and 1:1, such as between 3:1 and 2:1. In some examples, the wells are deeper than their cross-sectional diameter (or longest cross-sectional dimension). In some examples, the depth to cross- sectional diameter ratio of the wells is at most 10:1 (the depth is at most 10 times the cross- sectional diameter). In some examples, the depth to cross-sectional diameter ratio of the wells is at least 1:1 (the depth is at least the value of the cross-sectional diameter). In some examples, no internal linear dimension of a well is greater than 10 times any other internal linear dimension of the well. In some examples, the wells are arranged in a regular array. The array may be a hexagonal pattern or a square repeat pattern. Preferably, the wells are arranged in a hexagonal pattern. The hexagonal pattern provides closer packing and improves structural rigidity of the metallic support. The hexagonal pattern also 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. In some examples the distance between the external perimeters of any two wells is at least half the cross-sectional diameter of the wells. In some examples, the distance between the external perimeters of any two wells is at least the cross-sectional diameter of the wells. In some examples the distance between the external perimeters of any two neighbouring wells is at most 10 times the cross-sectional diameter of the wells. In some cases, it is desirable that the spacing of the wells is such that the material in between the wells has a great enough thermal mass to take in heat from a sample in the wells e.g., so that they cool quickly to minimise the formation of crystalline ice. In some cases, it is desirable that the material surrounding the wells (i.e., the material of the metallic support) has a high thermal conductivity to maximise the rate at which the metallic support cools. It may be desirable that the metallic support cools quickly itself and that the metallic support can conduct heat away from a sample in the wells quickly. In some examples the electron microscopy sample support comprises approximately 5000 to 300,000 wells in total. The electron microscopy sample support may comprise up to 300,000, up to 200,000, up to 150,000, or up to 100,000 wells in total. In some examples, the electron microscopy sample support comprises at least 5000 wells. In some examples, the electron microscopy sample support may only comprise wells recessed into a single surface. In some cases, the wells have a circular or polygonal cross section, preferably circular. In some cases, the wells have a uniform cross-section throughout their depth. In some examples, the plurality of wells contains an array of aligned samples. An exemplary depiction of the surface of the sample receiving portion of the metallic support (120) comprising a plurality of wells (130) is depicted in Fig.1(b) and a cross-sectional side view of the same sample receiving portion of the metallic support (120) is shown in Fig.1(c). The wells have a cylindrical shape and are arranged in a regular array or honeycomb pattern. A sample (131) such as an elongate cell is shown housed in each well, with the long axis of the cell biased to align with the long axis of the well. In some examples, the sample in each well comprises a liquid which fills the well and may be frozen for cryo-EM imaging. Fig.2 (right) is a corresponding SEM image of an exemplary electron microscopy sample support showing the top surface of the sample receiving portion of the metallic support (120). The openings of the wells (130) in the metallic support can be seen arranged in a regular array or honeycomb patten. The scale bar represents 1 μm. Manufacture of an electron microscopy sample support A second aspect provides a method of manufacturing an electron microscopy sample support, the method comprising the steps of: (i) providing a substrate comprising a plurality of protrusions, (ii) depositing a layer of metallic material on the substrate covering and incorporating a plurality of the protrusions, (iii) separating the layer of metallic material from the substrate to provide an electron microscopy sample support comprising a plurality of wells complementary to the protrusions of the substrate. In some examples, the substrate comprises a plurality of aligned protrusions. In some examples, the protrusions are in a regular array. In some examples, the protrusions extend from a flat surface of the substrate. As the wells in the metallic support are formed complementary to the protrusions, the options and preferences stated above regarding the morphology, dimensions, orientation, and distribution of the wells apply equally to the protrusions. In some examples, the heights and diameters of the protrusions correspond to the depths and diameters of the wells detailed above for the electron microscopy sample support. In some examples, the substrate is provided by patterning a wafer with protrusions, e.g., columns, such as cylindrical columns, of polymeric material. For example, the polymeric material may be photoresist. The wafer may be any suitable substrate material, such as a silicon wafer. In some examples, the wafer may be a silicon wafer with a thickness of 200 μm to 1 mm, for example, 500 μm. In some examples, the wafer is a

[0100] -oriented Si single crystal wafer. One side of the wafer may be polished flat. In some cases, the substrate is provided with a first sacrificial layer applied to the substrate onto which the metallic material is deposited. Any material which is selectively etchable with respect to the relevant metallic material can be used for this layer. The sacrificial layer material may be a metal, such as copper (which is particularly preferred for gold supports). It is preferred that the sacrificial layer is copper. The layer may be evaporated onto the wafer, for example, using an electron beam evaporator to form a film. The layer may be approximately 20 to 100 nm thick. Preferably, the layer is 40 to 50 nm thick. This layer of metal may act as an electrode in an electroplating step. This layer may also be used as a release layer to separate the electron microscopy sample support from the wafer as it is etchable, so can be removed, for example, with a "piranha solution” (3:1 conc H2SO4:30% H2O2). The first sacrificial layer may be applied before or after the protrusions of the substrate are formed. In some examples, the wafer is patterned with columns of polymeric material using UV lithography techniques. The wafer may be patterned with a lithography mask. Lithography masks can be designed to produce a desired 2D pattern. The depth of the polymeric material layer can be altered by changing the particular polymeric material used or altering the coating parameters. The pattern and depth of the polymeric material layer are chosen to provide the desired pattern of wells in the resulting electron microscopy sample support. The pattern shape and dimensions are therefore chosen based on the pattern and dimensions of the wells discussed above. In some examples, orientation marks and / or larger features, such as outlines, may also be patterned with a polymeric material. In some examples, the layer of metallic material is deposited on the substrate by electroplating. The conditions of electroplating may be chosen to maximise the purity and / or conformity of the material. The current may be monitored during the electroplating process to ensure the quality of the layer. In some examples, the plating conditions are chosen to minimise toxicity of the chemicals used. In some examples, multiple layers of metallic material are deposited on the substrate. 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. In some examples, multiple rounds of lithography and plating are used to build up the metallic material in multiple layers. In this way, electron microscopy sample supports with more complex well geometries may be manufactured, e.g., wherein the plurality of wells are non-uniform. In some examples, the layer of metallic material is separated from the substrate by etching away the polymeric material (e.g., photoresist) and optionally the film of etchable material on the wafer. This leaves the layer of metallic material which has been formed into an electron microscopy sample support. In some examples, a piranha solution is used for etching. For example, a mixture of sulphuric acid and hydrogen peroxide. The piranha solution may be 3:1 concentrated sulphuric acid: 30% hydrogen peroxide. In some examples, etching is performed for at least 12 hours. In some examples, individual electron microscopy supports are separated by cutting any joints between them. This may be done with a scalpel. In some examples, a plurality of electron microscopy sample supports may be formed in a singular process with one substrate. The plurality of electron microscopy sample supports may be separated into groups by cutting the substrate before the step of releasing the metallic material. In some examples, at least 300 electron microscopy sample supports may be formed in a singular process on one substrate. The metallic material may be any of the materials discussed for the metallic support above. Accordingly, a method of manufacturing an electron microscopy sample support may comprise the steps of: (i) patterning a wafer with columns of polymeric material, (ii) electroplating the wafer, (iii) etching away the polymeric material to release an electron microscopy sample support comprising a plurality of wells complementary to the columns of polymeric material. In some cases, the electron microscopy sample support is according to the first aspect. These proposals also include an electron microscopy sample support as formed by a method described herein. Fig.4 shows a schematic illustration of an exemplary method of manufacturing an electron microscopy sample support. A substrate (340), e.g., a silicon wafer, is provided comprising a plurality of protrusions (330), e.g., columns of polymeric material such as photoresist. In this example, a first sacrificial layer (350), e.g., of copper, is also provided as part of the substrate. The first sacrificial layer may be formed before or after the protrusions, in this example it can be seen underneath the protrusions. A layer of metallic material (320) is deposited on the substrate (340) covering and incorporating a plurality of the protrusions (330) to form the metallic support on release from the substrate. The sample receiving area of the metallic support (120) can be seen after release from the substrate below, with wells (130) formed complementarily to the protrusions (330) of the substrate. Fig.5 (left) shows an SEM image of an exemplary substrate (340) provided with a plurality of protrusions (330). The scale bar represents 5 μm. Fig.5 (right) shows an exemplary set of electron microscopy sample supports (100), formed on a single substrate, after release from the substrate. Each disc is one electron microscopy sample support. Fig.6(a) shows the design of an example mask used to pattern a wafer to form a substrate. The filled areas would be clear on the printed mask, while all white areas are opaque. The black square and circular outlines represent the edge of the mask and approximate position of the wafer for patterning, respectively. Figures 6(b)-(d) show the same mask at increasing zoom factors. Fig.6(b) shows the mask design for one whole electron microscopy sample support (surrounded by several others), with the edge portion of the metallic support in white and the sample receiving portion in dark grey. Fig.6(c) shows an area of the mask design for the sample receiving portion in more detail. A plurality of grey circles can be seen for patterning the protrusions of polymeric material and thus determining the position and shape of the wells in the resulting electron microscopy sample support. Fig.6(d) shows a smaller area within Fig.6(c). Method of preparing a sample for electron microscopy A third aspect provides a method of preparing a sample for electron microscopy comprising the steps of: (i) providing an electron microscopy sample support according to the first aspect, (ii) loading a plurality of wells in the electron microscopy sample support with a sample suspended in a liquid, (iii) freezing the liquid, (iv) processing a portion of the electron microscopy sample support by removing the material defining the base of a plurality of the wells. In some examples, a sample is loaded into the wells of the electron microscopy sample support by adding the sample to the surface of the electron microscopy sample support into which the wells are recessed and performing centrifugation to urge the samples into the wells. In some examples, multiple rounds of centrifugation may be used. In some examples, the sample is pipetted across the relevant surface of the electron microscopy sample support. This can help to dislodge any sample, e.g., cells, blocking well entrances. In some examples, the proportion of wells occupied by a sample after the loading step is at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%. The sample-containing electron microscopy sample support may be incubated before freezing. In some examples, excess fluid may be removed from the surface of the electron microscopy sample support after loading and prior to freezing, e.g., by contacting with absorbent material. In some examples, dyes or other markers may be included with the sample or loaded independently, for example, to identify or stain certain features in the sample. In some examples, the electron microscopy sample support is plunge frozen to freeze the liquid. The electron microscopy sample support may be frozen in liquid ethane. A manual plunger or an automated system may be used. The present methods and sample supports are particularly suitable for plunge freezing and typically do not require the additional experimental complication of high-pressure freezing methods. However, the electron microscopy sample support may be used with high-pressure freezing methods, for example, if required for a particular specimen. In some examples, any water present when the electron microscopy sample support is frozen forms vitrified ice. Preferably, at least 90%, such as at least 95%, or at least 99% of water present in or on the electron microscopy sample support forms vitreous ice rather than crystalline ice. In some examples, the electron microscopy sample support is processed by focussed ion beam (FIB) milling. FIB milling is a known technique for precise removal / ablation of material. FIB milling may be used to remove a layer of material from the electron microscopy sample support such that the bases of the wells are removed to leave through-holes. Further material may be removed to control the thickness of this region of the processed electron microscopy support. A layer of material may also be removed from the top surface of the electron microscopy sample support at the openings of the wells, e.g., to thin the region containing the wells. Thus, a thin lamella portion or slice of the electron microscopy sample support is produced comprising through-holes formed from cross-sections of the wells. This area of the processed electron microscopy sample support from which material has been removed may be referred to as the imaging region. A portion of a sample in a well may be removed simultaneously to leave cross-sections through the sample in the resulting through-holes. Therefore, thin (suitably electron beam transparent) cross-sections of the sample may be obtained and imaged, for example with transmission electron microscopy (TEM) through the through-holes. Due to geometrical constraints, FIB milling may be performed at an angle (e.g., an acute angle) to the plane of the surface of the electron microscopy sample support (e.g., the surface of the sample receiving portion). As a result, cross-sections are taken through the wells at an angle and at different heights for different wells. This provides a selection of cross-sections through samples in different wells. The angle may be 0 to 20°, typically greater than 0 to 20°. The angle may be 1 to 15°, such as 5° to 10°. The angle may be chosen to ensure that cross-sections are taken through all or a plurality of the wells. In some examples, the axis of the wells (i.e., the sides of the wells) may be formed to be angled relative to the normal of the plane of the surface of the electron microscopy sample support and milling may be performed at a complementary angle. In this case, the cross- sections of the wells may be taken substantially orthogonally to the sides of the well. Advantageously, this may prevent the edges of the resulting through-holes from obscuring part of the sample during imaging as the sides of the through-holes would be substantially orthogonal to the plane of the surrounding surfaces (the sides of the through-holes would be substantially parallel to the electron beam). The removal of the material defining the base of the wells may be performed across any of the wells. Since each portion where material has been removed (e.g., each imaging region) may contain many wells there may be no requirement for particular targeting. In comparison, a grid-based electron microscopy sample support requires targeting for the sample- containing portions and to avoid the grid bars. In some examples, the wells may be pre-screened with light microscopy to identify well occupancy, ice thickness, or specific biological features. A portion of the electron microscopy sample support may be processed until it is at most 500 nm thick, or 300 nm, or 200 nm, or 150 nm, or 100 nm, or 50 nm, or 30 nm, or 20 nm, or 10 nm thick. Preferably the electron microscopy sample support is processed until a portion is at most 200 nm thick, more preferably 150 nm. Without being bound by any theory, at this thickness enough electrons may penetrate the sample (which may be processed in the wells simultaneously with the processing of the support) to produce a good image. This is explained in Dickerson et al., 2022. The processing step may be performed manually, or it may be automated. Plasma FIB milling (pFIB milling) may be used. This may increase the speed of the process. In some examples, the electron microscopy sample support may additionally be processed to introduce expansion joints. The expansion joints may be introduced using the same method as the other processing steps. The expansion joints may be FIB milled. In some examples, an autoloader system may be used throughout the process. This reduces handling steps and minimises ice contamination. An exemplary schematic illustration of the method is shown in Fig.1. Fig.1(c) shows a cross-sectional view of a sample receiving portion of the metallic support (120) before processing. Wells (130) can be seen, each loaded with a sample (131). During processing material is removed from the electron microscopy sample support along the plane indicated by the dashed line in Fig.1(c) and the FIB milling arrow in Fig.1(d). As shown by the dashed lines, material is removed from the top and bottom of the support, i.e., from around and including the base of a plurality of the wells and the opening of the wells. Thus, a slice of the metallic support is taken through a plurality of wells leaving a thin imaging region or lamella, as depicted in Fig.1(d). Fig.1(d) shows that the imaging region of the metallic support (220) in the processed electron microscopy sample support comprises through-holes (230), each of which may contain a cross-section of a sample (231). It can be seen from Fig.1(d) how removal of material or FIB milling along an angled plane can result in through-holes containing cross-sections through samples at different heights, thus enabling imaging of different features of a sample. It can also be seen that the processed regions or imaging regions will have a surface which is at an angle to the plane of the surrounding unprocessed or un-milled surface (i.e., the support regions). Processed electron microscopy support A fourth aspect provides a processed electron microscopy sample support comprising: a metallic support, the metallic support comprising a support region surrounding an imaging region, wherein the support region is thicker than the imaging region and the support region and the imaging region are integral, wherein the plane of the surface of the support region is at an angle to the plane of the surface of the imaging region, wherein the imaging region comprises a plurality of through-holes in the metallic support, and wherein each through-hole optionally houses a section of a sample suspended in frozen liquid. A processed electron microscopy sample support according to this aspect enables the imaging of sample cross-sections which would be difficult or impossible to achieve with known sample supports. It may provide better support for the sample than a conventional grid-based sample support as fragile foils are not required and a larger proportion of the support may have a thickness of, for example, more than 3 µm. Issues with non-uniform layers of ice are avoided as the sample is added before processing of the electron microscopy sample support. Additionally, less ice may be present around the sample than in alternative methods, and less crystalline ice may be present. This improves mechanical stability and reduces charging and movement during imaging, thus improving image quality. The imaging region and support region may both be within the area of the original sample receiving portion of the electron microscopy sample support before processing. An area that has been processed by removal of material defining the base of a plurality of wells during manufacture of the processed electron microscopy sample support may form the imaging region while the surrounding unprocessed areas form the support region. Hence the support region is thicker than the imaging region. In a single electron microscopy support, there may be multiple imaging regions. In a single electron microscopy support, there may be multiple support regions. In a single support region, there may be multiple imaging regions. The imaging region comprises a plurality of through-holes. In some examples, the support region may comprise one or more wells. The angle between the plane of the surface of the support region and the plane of the surface of the imaging region means that different through-holes may house cross-sections through different heights of a sample, such as bacteria. The angle corresponds to the angle of processing during manufacture of the processed electron microscopy sample support. The processed electron microscopy sample support may have been formed with FIB milling at an angle. The resulting angle between the plane of the surface of the support region and that of the imaging region may be >0° to 20°. The angle may be 1 to 15°, such as 5° to 10°. As a result of the angled removal during processing, if the wells in the electron microscopy sample support had sides substantially orthogonal to the surrounding surface before processing, then the processed electron microscopy samples support may have through- holes with sides that are at an angle to the normal of the plane of the processed surface (the sides may not be parallel to the normal of the plane of the processed surface, i.e., the surface of the imaging region). The angle of the sides of the through-holes to the normal of the plane of the processed surface may correspond to the angle of processing. The angle may be >0° to 20°. The angle may be 1 to 15°, such as 5° to 10°. In some examples, the imaging regions are up to 35 μm wide. In some examples, the imaging regions are at least 25 μm wide. This increased width compared to known lamellae is enabled by the improved mechanical stability. The processed electron microscopy sample support thus has a greater ratio of useable to unusable area than known sample supports as adjacent thicker material prevents the collection of tilt series during transmission electron microscopy from the edges of any kind of lamella. In some examples, the imaging region has a thickness of at most 500 nm, or 300 nm, or 200 nm, or 150 nm, or 100 nm, or 50 nm, or 30 nm, or 20 nm, or 10 nm. This ensures that a cross-section of a sample housed in the through-holes of the imaging region is thin enough for a high proportion of electrons to penetrate and produce a good image during exposure to an electron beam during, for example, TEM imaging. In some examples, each through-hole has a cross-sectional diameter corresponding to those of the wells in the electron microscopy sample support before processing as described above. In some examples, the ratio of the cross-sectional diameter of each through-hole to the depth or thickness of the through-hole (which corresponds to the thickness of the imaging region) is at least 1:3. The ratio may be at least 1:1, or at least 2:1, or at least 10:1. In some examples, the ratio of the cross-sectional diameter of each through-hole to the thickness of the through-hole is at most 200:1. The ratio may be at most 100:1, or at most 50:1, or at most 30:1, or at most 20:1. In some examples, the support region has a thickness of at least 2 μm, or 5 μm, or 10 μm, or 50 μm, or 100 μm. In some examples, the ratio of the thickness of the imaging region to the thickness of the support region is at least 1:1000, or at least 1:500, or at least 1:100, or at least 1:50. In some examples, the ratio is at most 1:4, or at most 1:10. In some examples, the processed electron microscopy sample support comprises expansion joints. On rare occasions ice formed in the wells can crack due to expansion on formation. Expansion joints allow the wells and supporting structures to expand, minimising damage to samples. In some cases, the processed electron microscopy sample support is prepared according to the methods of the third aspect. These proposals also include a processed electron microscopy sample support as formed by a method described herein. Fig.7 shows SEM (a), TEM (b), and cryo-confocal microscopy (c) images of an exemplary imaging region from a processed electron microscopy sample support with the same through-hole indicated by the grey box. Method of imaging a sample In some examples, a method of imaging a sample comprises the steps of: (i) arranging a processed electron microscopy sample support as described herein in an electron beam of a microscope, and (ii) collecting image data for analysis. The processed electron microscopy sample support may be according to the fourth aspect. The processed electron microscopy sample support may be prepared according to the method of preparing a sample for electron microscopy described above. Image data may be collected with a transmission electron microscope (TEM). In particular, electron cryo-microscopy (cryo-EM) or electron cryo-tomography (cryo-ET) may be performed. The image data may be collected by imaging (in the case of cryo-ET by sequentially imaging) a sample suspended in a through-hole of a processed electron microscopy samplesupport according to the fourth aspect, wherein in some cases each image encompass atleast a part of the edge of the through-hole and the electron beam encompasses the through-hole and the complete edge of the through-hole. In some cases, at least a part of the edge of the through-hole in each image is compared to the other images to remove any relative shift between sequential images and / or wherein the sequential images of the specimen in the through-hole are weighted to account for damage to the specimen. Bacterial Imaging A specific application of the EM sample support described herein is in the imaging of elongate samples, such as non-spherical bacteria, particularly those exhibiting preferential orientations on a known EM sample grid. When used with such samples, an electron microscopy sample support according to the first aspect sections through cells at multiple heights, including the division plane, which is normally perpendicular to the imaging plane, so that it may be imaged. Thus, the electron microscopy sample support provides imaging access to sections of samples that are not accessible using known supports. Fig.3 is a diagram showing a comparative illustration of the images that could be obtained of an elongate sample using a conventional grid for TEM and an electron microscopy sample support according to the present invention. With a conventional grid (left) the sample is located on the surface of the support and thus adopts a preferred horizontal orientation relative to the electron beam. With the electron microscopy sample support according to the present invention (right) the sample is housed in a well which enables the long axis of the sample to be approximately aligned with the electron beam so that a cross-section of the long axis of the sample may be imaged. For example, an average of the prokaryotic 70S ribosome was reconstructed by sub- tomogram averaging to demonstrate the high-resolution capabilities of this method of imaging. The vast majority of archaea and bacteria utilise the protein FtsZ to organise cell division (Barrows and Goley, 2021; Du and Lutkenhaus, 2019). FtsZ is a tubulin homologue that forms filaments around the circumference of the cell at the division site, making a structure known as the Z-ring (Bi and Lutkenhaus, 1991; Li et al., 2007; Wang and Lutkenhaus, 1993). It has not previously been possible to image intact rings via cryo-ET. This is a technical limitation of the method and is due to the aforementioned issue of preferred sample orientation in combination with the known missing wedge limitation of cryo-ET. When rod- shaped cells are plunge frozen on EM grids, they orient with the plane of the grid. As the Z ring runs round the circumference of the cell cross-section, this results in images where the Z-ring is mostly visible in cross section and FtsZ appears as dots under the membrane (Fig. 3, left). It would be necessary for the cell to be oriented vertically in order to visualise the intact ring (Fig.3, right). This has not previously been possible in cryo-EM. While the use of tomography allows the reconstruction of 3D volumes, limitations of sample thickness and most microscope hardware means that sample tilting is restricted to ± 60-70°. This results in a missing wedge of information, making it impossible to visualise the entire ring, unless the sample is tilted closer to ± 90° (Palmer and Löwe, 2014), which is not typically possible in EM apparatus. The electron microscopy sample supports and methods described herein are able to vertically trap and section rod-shaped bacteria. When combined with FIB milling, the supports and methods allow the generation of lamellae (processed electron microscopy sample supports) containing cross sections through the bacteria at different heights (Fig. 1(a) - (d)). Features and parameters that are described herein independently of other features and parameters may be combined in some aspects insofar as they are compatible. Furthermore, features and parameters that are described in the context of one aspect of the invention are applicable to other aspects described herein insofar as they are compatible. In the usual way, in numerical ranges and ratios defined herein, upper and lower limits are individually disclosed and may be combined with other upper or lower limits to form alternative ranges, each of which is envisaged herein. EXAMPLES As noted above, the following examples are illustrative and do not limit the scope of the invention which remains defined by the claims. Example 1: Manufacture of an electron microscopy sample support Electron microscopy sample supports were produced using a combination of UV lithography and electroplating (Fig.4). They were manufactured using a 4-inch silicon wafer as a base, yielding approximately 400 electron microscopy sample supports per wafer. Firstly, a thin (a25pprox.40-50 nm thick) film of copper was evaporated onto the wafer. This acted as both an electrode for the plating step and also as a release layer for the completed electron microscopy sample supports, but formed no part in the final electron microscopy sample supports. Then, using a lithography mask (Fig.6), the wafer was patterned with columns of photoresist 1.5 µm in diameter and protruding 3 µm from the wafer surface (Fig.5, left), plus orientation marks and larger features to make the electron microscopy sample support outline. Following this, the wafer was electroplated with gold, with the photoresist acting as a substrate (Fig.4). Finally, piranha solution (3:1 conc. H2SO4:30% H2O2) was used to etch away the copper and the photoresist, releasing the gold film from the wafer and leaving nothing but the plated gold film remaining (Fig.4). The resulting electron microscopy sample supports had a plurality of wells approximately 1.5 µm in diameter and 3 µm in depth (Fig.4 and Fig.5, right). This process is now described in more detail. Electron microscopy sample supports were manufactured using 4-inch <100> silicon wafers (University Wafer) as a base. First a 40-50 nm layer of copper was evaporated onto the wafer using an electron beam evaporator (Moorfield Minilab 080). Immediately after removal from the vacuum chamber of the evaporator, the wafer was transferred to a spin coater (Cee Apergee 450). Approximately 4 mL of Omnicoat (Kayaku Advanced Materials) was static dispensed onto the wafer before spin coating using a 2-step program (spread step: 500 rpm, acceleration 100 rpm / s, 5 s; coat step: 3000 rpm, acceleration 300 rpm / s, 30 s). The wafer was incubated on a hotplate at 200 °C for 60 s to soft bake the Omnicoat, removed and allowed to cool to room temperature before spin coating with SU-82005 (Kayaku Advanced Materials). Approximately 4 mL of resist was statically dispensed onto the wafer and spin coated using a 2-step program (spread step: 500 rpm, acceleration 100 rpm / s, 6 s; coat step: 4000 rpm, acceleration 300 rpm / s, 40 s). The wafer was incubated on a hotplate at 95°C for 2 mins to soft bake the SU-8, then left to cool at room temperature for 2 min before transfer to a mask aligner (Neutronix-Quintel Q4000). The wafer was raised to vacuum contact with a lithography mask (Fig.6, ordered from Compugraphics) and UV exposed for 15 seconds at a dose rate of 9.0 mW / cm². This was followed by a post-exposure bake at 95°C for 2 min and the wafer was allowed to cool to room temperature. The SU-8 was developed by immersion in propylene glycol monomethyl ether acetate for 2 min with gentle agitation, washed by immersion in isopropanol, and dried with a stream of dry nitrogen gas. Finally, the Omnicoat was developed by immersion into Microposit MF 319 developer (Kayaku Advanced Materials) for 15 s with very gentle agitation, then washed by immersion in 3 successive water baths. The wafer was dried with a stream of dry nitrogen gas, left in a laminar flow hood for 30 min to allow any remaining moisture to dry and hard baked in an oven at 70°C for 10 mins. The patterned wafer was electroplated with gold using a Yamamoto 4-inch silicon wafer plating set with MetGold ECF33B cyanide-free plating solution (Metalor Advanced Coatings). The wafer was cleaned using a UVO cleaner (Jelight, model 42 series) for 4 minutes, assembled into the cathode holder, pre-heated to 60°C in a dry oven for a minimum of 20 min and the gold solution pre-heated to the plating temperature of 54°C. The wafer was plated for 15 minutes with the voltage continuously monitored and adjusted to give a current of 0.28 A, to provide a nominal depth of plating of 4 μm for the design used. The wafer was transferred to a water bath at 54°C and gradually cooled to room temperature by the addition of cold water. The cathode holder was disassembled and the wafer further washed by sequential immersion in 2 water baths and dried with a stream of dry nitrogen gas. Before any further processing, the wafer was cut into 13 separate chips, each chip containing 40 electron microscopy sample supports. The film was loosened around the edges of the chips and the film was released from the wafer by an overnight piranha etch (3:1 conc. Sulphuric acid: 30% hydrogen peroxide). To ensure all copper and photoresist was removed, the released film was subjected to a second piranha etch in fresh solution for a further 4 hours. This was followed by 3 sequential water washes and the films allowed to air dry. Finally, the electron microscopy sample supports were inspected by SEM to check for defects and confirm the diameter of the wells, before separation by cutting the joints holding supports together manually with a scalpel. Example 2: Bacterial culture and electron microscopy sample support preparation For FtsZ(D212A) overexpression, C41(DE3) E. coli cells were transformed with plasmid PMZ120 (Szwedziak et al., 2014) and cultured in M9 minimal media supplemented with 0.4% glycerol at 37°C until approximately OD6000.4.10 mL of culture were centrifuged and washed in PBS before a 10-minute incubation with 200 µL of PBS premixed with 1 μL CellBrite Fix 488 (Biotium). The dye was washed out by centrifugation and the pellet mixed with 200 µL of M9 supplemented with 0.1% arabinose. The electron microscopy sample supports were glow discharged at 30 mA for 2 min on each side.30 µL of the concentrated culture was added to the surface of each electron microscopy sample support to be vitrified and centrifuged for 1 min in a custom-made plate holder in a table-top swinging bucket centrifuge at 3000 rpm (~1800 g). A further 10 μL was added to the electron microscopy sample support, pipetting across the top surface to dislodge cells blocking entrance to the wells, and the centrifugation step repeated. A final 10 μL was added as above and the electron microscopy sample supports subjected to a third and final centrifugation step. The surface of the electron microscopy sample support was washed with arabinose supplemented M9, a 50 μL drop of fresh media added to the surface and the electron microscopy sample supports were incubated inside a humid chamber at 37°C for approximately 1 hour. The electron microscopy sample supports were removed from the incubator, front (hole side) blotted and plunge frozen in liquid ethane using a manual plunger. For tomography of native FtsZ, C41(DE3) E. coli cells were cultured in LB at 37°C until OD 0.5-0.6, washed into PBS and incubated with membrane dye as above. Following the membrane staining step, they were returned to LB and centrifuged into the electron microscopy sample supports as above. Following centrifugation, the electron microscopy sample supports were directly washed and plunge frozen as above. Example 3: FIB milling Lamellae (imaging regions) were milled on either a Scios Dual Beam FIB-SEM (Thermo Fisher Scientific) or a Crossbeam 550 (Zeiss), both equipped with a PP3010 cryo-stage and loading system (Quorum Technologies). Prior to milling, the electron microscopy sample supports were coated with an organo-platinum layer for 30-45 s using the Gas Injection System (GIS). Bulk milling and expansion joint milling were performed at 30 keV, 5-15 nA using rectangular milling patterns and all subsequent steps were performed using cross- sectional milling. Lamellae were milled at a shallow angle to the grid (typically 8-10º) and therefore sampled through different heights in the wells from the top of the wells at the front of the lamella to the bottom of the wells at the back of the lamella (Fig.1(d), 7(a), (b)). The milling currents were gradually reduced as the lamellae were thinned till the final polishing step at 30-100 pA. Once the lamellae were below ~1 μm thickness, milling was performed with an under / over tilt of 0.5-1° until the final polish, which was performed at the original milling angle. The lamellae correspond to the imaging region of a processed electron microscopy sample support described herein. Example 4: Cryo fluorescence microscopy (CLEM) Light microscopy was used to identify specific targets in the sections using a non-specific fluorescent membrane dye that highlights the presence of cell membranes in sections of cells in each well. The fluorescence signal, in the form of rings, correlated with the wells where there are visible cells, both in the SEM imaging from the milling and in the medium magnification montage from the TEM (Fig.7(a) – (e)). Milled lamellae (processed electron microscopy sample supports) were fluorescently imaged on a LSM900 Axio Imager Z2 Airyscan confocal microscope (Zeiss), equipped with a cryo- stage for imaging grids at liquid nitrogen temperature (Linkam CMS196V3). Z stacks were collected using a 100x air objective (LD EC Epiplan-Nerofluar 100x / 0.75 DIC) using airyscan, with a pixel sampling of 0.079 µm x 0.079 μm x 0.210 μm and a pixel time of 27.3 µs. The raw stacks were 3D processed with auto-filtering. Z stacks were imported into Fiji software (Schindelin et al., 2012) and a maximum intensity Z-projection generated. In Fig.7 milled lamellae were imaged using SEM (a), TEM (b) and cryo-confocal microscopy (c). The cryo-confocal image is a projection of the acquired Z stack; the fluorophore is the non-specific membrane dye cellBrite-488. The same cell / well has been indicated in all 3 images (a, b, c). The tomogram in Fig.10(g) is from this cell and the cell is shown as seen in the TEM (d) and as a single slice from the cryo-fluorescence Z-stack (e). Scale bars (a-c): 5 μm; (d, e): 200 nm. Example 5: Electron microscopy Lamellae (imaging regions of processed electron microscopy sample supports) FIB-milled through the wells produce cross-sections that run approximately perpendicular to the long axis of the elongated bacteria (Fig.1(c), (d) and Fig.9(a), (d), (e), (f)). Electron cryo- tomography (cryo-ET) was performed on these lamellae in a transmission electron microscope (TEM), operated at 300 keV. The TEM was a Titan Krios electron microscope (Thermo Fisher Scientific) equipped with a Gatan imaging filter and K3 direct electron detector (Gatan). Tilt series were collected using SerialEM (Mastronarde, 2005), either using the SerialEM batch tomography set up or PACE-tomo (Eisenstein et al., 2023). Tilt series were collected with a magnified pixel size of 2.1 or 2.7 Å, depending on data set, and was chosen so that the entire cross-section of a cell fits in the field of view on a ~4k x 4k image. Tomograms were collected from a start tilt of -10° (to flatten the lamella, which are milled at an angle of approximately 10° and loaded with a rotation of approximately -90° between the milling orientation and the Krios autoloader) in a dose symmetric manner (Hagen et al., 2017), with an increment of 2° and a tilt range of +50-60° from the start tilt. Each tilt series had a total fluence of approximately 150 e- / Ų, distributed evenly across the tilt series and each tilt was collected in 5-6 frames. Fig.8 is a violin plot showing mean motion per frame of tilt series collecting from lamellae milled from the custom support, as compared to conventional lamellae. Means: conventional grids 1.136, supports according to the invention 0.444. Fig.9 shows (a, d, e, f) tomogram slices showing cross sections through cells, most likely not at a division plane. The dark regions in the corners are the edges of the gold wells. (b) and (c) are sections of the tomogram in (a) depicting the membrane region shown with and without annotation. The inner (IM) and outer (OM) membranes and peptidoglycan (PG) are visible as well as various protein densities in the periplasm, including those bridging the peptidogylcan to the outer membrane, which could be the very abundant Braun’s lipoprotein (Lpp). Scale bars (a, d, e, f): 250 nm; (b, c): 25 nm. Example 6: Tomogram reconstruction Frames were aligned using IMOD (version 4.10.25) alignframes (Kremer et al., 1996). Tilt series were aligned using patch tracking and reconstructed with back projection, both with etomo, part of the IMOD package (Kremer et al., 1996). Tomograms were denoised using cryoCARE (Buchholz et al., 2019). For training of the cryoCARE noise2noise model, tilt series were produced using odd / even aligned frames and then reconstructed using the alignments generated for all frames. For each tomogram to be denoised, the model was trained separately on just the odd / even frame generated tomograms of the tomogram in question. Segmentation and modelling were done manually in IMOD. Membranes were modelled by drawing contours every 20 slices. Instead of directly tracing individual filaments of FtsZ, contours were drawn on filaments on every tomogram slice where there were filaments and the resulting contours then resolved into filaments in the 3D model. While it is difficult to measure the resolution of the resulting tomograms, their quality can be readily estimated from their resolving power of known features of bacterial cells. In the tomograms it is possible to resolve the two leaflets of the membrane bilayers, the peptidoglycan layer, and some protein densities in the periplasm (Fig.9(b), (c)). In particular, there were a number of stick-like densities bridging between the peptidoglycan and the outer membrane. One of the most highly expressed proteins in E. coli is Braun’s lipoprotein (Lpp), a small trimeric coiled coil-containing protein that anchors the peptidoglycan to the outer membrane [reviewed in (Asmar and Collet, 2018)] and is a likely candidate for the bridging densities observed in the tomograms. Fig.10 shows (a) a slice from tomogram collected from lamella of E. coli cells over- expressing a GTPase mutant of FtsZ (FtsZ[D212A]). There is an extensive single-layered ring or band of filaments under the inner membrane, as modelled in (b). A close-up of the tomogram is shown in (c). (d – f) are FtsZ(D212A) filaments in the overexpression system, this time at a constriction site. This is a cell that sits tilted in the support well. The models in (e) and (f) show top down and tilted side views, respectively. Additionally, the lamella’s top and bottom boundaries have been modelled in (f) for clarity. (g – i) show partial, native Z-ring (no FtsZ mutant or overexpression). Here the plane of the lamella and the plane of the Z-ring ring are not perfectly aligned so that only part of the ring is present in the tomogram. Scale bars (c) and (i): 50 nm, all others 100 nm. OM = outer membrane, PG = peptidoglycan, IM = inner membrane. Note that for the modelling of FtsZ filaments, contours have been drawn on every section containing filaments, rather than tracing individual filaments. Example 7: Sub-tomogram averaging In order to demonstrate that these gold supports enable high-resolution structure determination, especially given the gold-redeposition described above, an average of the prokaryotic 70S ribosome was reconstructed by sub-tomogram averaging (STA), Ribosomes are the most tractable target for STA: they are large (2.5 Mda) and abundant (thousands per cell). In the following process (shown in Figure 12) 14,458 sub-tomograms from 21 tomograms were averaged to generate a structure of the 70S ribosome, at 6.6 Å nominal resolution (Fig. 11 (a), (b), (h)). At this resolution, protein α-helices are clearly visible as tubular densities and RNA is well resolved (Fig.11 (c)-(f)). Unsurprisingly, local resolution estimation indicates that peripheral parts of the rRNA are not as well resolved as the core (Fig.11(g)). For instance, the L1 stalk displays local resolution worse than 10 Å, likely owing to its flexibility. These results show clearly that the lamellae (i.e., the imaging regions of processed electron microscopy sample supports) generated by FIB milling electron microscopy sample supports can be effectively interrogated for sub-nanometer-resolution structural information. 143 tilt series (collected at 2.7 Å / px) were automatically reconstructed at bin 5 using a custom script based around IMOD programs (Kremer et al., 1996), using alignframes for frame alignment, cross-correlation based tilt series alignment and reconstruction by back projection. The resulting tomograms were inspected to determine lamellae thickness and 83 with a thickness above 200 nm were discarded. The remaining 60 tomograms (thicknesses ranging from 60 to 200 nm) were selected, and the tilt series were manually realigned by patch tracking in etomo, part of IMOD (Kremer et al., 1996). Defocus estimations were calculated in Warp 1.0.9 (Tegunov and Cramer, 2019). Tomograms for particle picking were reconstructed in RELION 4.0 (Zivanov et al., 2022) at 8x binning (21.3 Å / px) with CTF demodulation. For further processing 23 tomograms were selected out of an initial shortlist of 60 whose alignments were visually were superior and whose reconstructions captured the greatest biological detail: membranes and ribosomes were clearly visible. Tomograms in which there was cracked ice or heavy gold or ice contamination were discarded. Any tomograms that were collected from horizontally oriented cells in the ice layer on the top of the supports were also discarded. On a previous dataset (collected at 2.13 A / px).1,280 suspected ribosomes were picked by hand from 33 representative slices across 8 tomograms. These particles were used to train a crYOLO model (Wagner et al., 2019). This model was then allowed to pick on the 23 tomograms, producing -43,500 picks. These particles were inspected in the tomograms that they originated from, and those that were obviously not ribosomes (because, for instance, they lay outside any cell) were manually discarded. This left ~39,000 particles. The particle coordinates were supplied to Warp for sub-tomogram generation, using starfile (Burt et al., 2021). Sub-tomograms were first extracted at 4x binning (10.6 A / px). These sub- tomograms were classified in RELION, with the aim to filter out particles that were not ribosomes. It appeared that almost all particles were in fact ribosomes, and they averaged to a resolution of 21 Å (Nyquist sampling limit at 4x binning), so the same coordinates were used to generate sub-tomograms at 2x binning (5.3 Å). Classifying these sub-tomograms into 8 classes with alignment in RELION yielded 3 sensible classes, comprising 40% of the particles (15,500). Refining the poses of these 15,500 particles produced an 11 Å-resolution map (Nyquist at 2x binning). Finally, un-binned sub-tomograms were extracted for these 15,500 particles. Refinement generated a 9.3 Å map. From here, M (Tegunov and Cramer, 2019) was used to refine geometric parameters and defocus. For unknown technical reasons, it was necessary to discard 2 of the 23 tilt series, reducing the particle set to 14,500. M produced a 6.6 Å-resolution map, clearly resolving protein secondary structure. Fig.11 shows (a) the final map, shaded by ribosomal subunit: large subunit, small subunit, and E-site tRNA. (b) The same map, shaded by chemistry: protein and nucleic acid. (c–f) Close-up views of the map rendered semi-transparently over PDB model 6H4N, shaded by ribosomal subunit: (c) 50S ribosomal proteins L5 and L31, (d) 50S ribosomal proteins L20 and L21, (e) 30S ribosomal protein S13, (f) 30S ribosomal proteins S2 and S5. (g) The map shaded according to local resolution. Higher resolution (< 8 Å) is apparent in the protein components of the large subunit, lower resolution (> 10 Å) in the L1 stalk. Images rendered by UCSF ChimeraX (Goddard et al., 2018). (h) Fourier shell correlation (FSC) plots of half- maps generated during refinement by M. The corrected FSC curve crosses 0.143 at 6.6 Å. References Asmar, A.T., Collet, J.-F., 2018. Lpp, the Braun lipoprotein, turns 50-major achievements and remaining issues. FEMS Microbiol Lett 365, fny199. https: / / doi.org / 10.1093 / femsle / fny199 Barrows, J.M., Goley, E.D., 2021. FtsZ dynamics in bacterial division: What, how, and why? Curr Opin Cell Biol 68, 163-172. https: / / doi.org / 10.1016 / J.CEB.2020.10.013 Bi, E., Lutkenhaus, J., 1991. FtsZ ring structure associated with division in Escherichia coli. Nature 354, 161-164. https: / / doi.org / 10.1038 / 354161a0 Bisson-Filho, A.W., Hsu, Y.-P., Squyres, G.R., Kuru, E., Wu, F., Jukes, C., Sun, Y., Dekker, C., Holden, S., Van Nieuwenhze, M.S., Brun, Y. V, Garner, E.C., 2017. Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division. Science (1979) 355, 739 LP-743. Buchholz, T.O., Krull, A., Shahidi, R., Pigino, G., Jékely, G., Jug, F., 2019. Content-aware image restoration for electron microscopy. Methods Cell Biol 152, 277-289. https: / / doi.org / 10.1016 / BS.MCB.2019.05.001 Burt, A., Gaifas, L., Dendooven, T., Gutsche, I., 2021. A flexible framework for multi-particle refinement in cryo-electron tomography. PLoS Biol 19, e3001319-. Dickerson, J.L., Lu, P.-H., Hristov, D., Dunin-Borkowski, R.E., Russo, C.J., 2022. Imaging biological macromolecules in thick specimens: The role of inelastic scattering in cryoEM. Ultramicroscopy 237, 113510. https: / / doi.org / https: / / doi.org / 10.1016 / j.ultramic.2022.113510 Du, S., Lutkenhaus, J., 2019. At the Heart of Bacterial Cytokinesis: The Z Ring. Trends Microbiol 27, 781-791. https: / / doi.org / 10.1016 / J.TIM.2019.04.011 Dubochet, J., Adrian, M., Chang, J.-J., Homo, J.-C., Lepault, J., Mcdowall, A.W., Schultz, P., 1988. Cryo-electron microscopy of vitrified specimens. Quarterly Review of Biophysics 21, 129-228. https: / / doi.org / 10.1017 / S0033583500004297 Eisenstein, F., Yanagisawa, H., Kashihara, H., Kikkawa, M., Tsukita, S., Danev, R., 2023. Parallel cryo electron tomography on in situ lamellae. Nature Methods | 20, 131-138. https: / / doi.org / 10.1038 / s41592-022-01690-1 Goddard, T.D., Huang, C.C., Meng, E.C., Pettersen, E.F., Couch, G.S., Morris, J.H., Ferrin, T.E., 2018. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Science 27, 14-25. https: / / doi.org / https: / / doi.org / 10.1002 / pro.3235 Hagen, W.J.H., Wan, W., Briggs, J.A.G., 2017. Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J Struct Biol 197, 191-198. https: / / doi.org / 10.1016 / J.JSB.2016.06.007 Hayles, M.F., de Winter, D. a M., Schneijdenberg, C.T.W.M., Meeldijk, J.D., Luecken, U., Persoon, H., de Water, J., de Jong, F., Humbel, B.M., Verkleij, A.J., 2010. The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy. J Struct Biol 172, 180-90. https: / / doi.org / 10.1016 / j.jsb.2010.07.004 Hoffmann, P.C., Kreysing, J.P., Khusainov, I., Tuijtel, M.W., Welsch, S., Beck, M., 2022. Structures of the eukaryotic ribosome and its translational states in situ. Nat Commun 13, 7435. https: / / doi.org / 10.1038 / s41467-022-34997-w Kelley, K., Raczkowski, A.M., Klykov, O., Jaroenlak, P., Bobe, D., Kopylov, M., Eng, E.T., Bhabha, G., Potter, C.S., Carragher, B., Noble, A.J., 2022. Waffle Method: A general and flexible approach for improving throughput in FIB-milling. Nat Commun 13, 1857. https: / / doi.org / 10.1038 / s41467-022-29501-3 Khanna, K., Lopez-Garrido, J., Sugie, J., Pogliano, K., Villa, E., 2021. Asymmetric localization of the cell division machinery during Bacillus subtilis sporulation. Elife 10, e62204. https: / / doi.org / 10.7554 / eLife.62204 Klykov, O., Bobe, D., Paraan, M., Johnston, J.D., Potter, C.S., Carragher, B., Kopylov, M., Noble, A.J., 2022. In situ cryo-FIB / SEM Specimen Preparation Using the Waffle Method. Bio Protoc 12, e4544. https: / / doi.org / 10.21769 / BioProtoc.4544 Kremer, J.R., Mastronarde, D.N., Mcintosh, J.R., 1996. Computer visualization of three- dimensional image data using IMOD. J Struct Biol 116, 71-6. https: / / doi.org / 10.1006 / jsbi.1996.0013 Li, Z., Trimble, M.J., Brun, Y. V, Jensen, G.J., 2007. The structure of FtsZ filaments in vivo suggests a force-generating role in cell division. EMBO J 26, 4694 LP-4708. Mahamid, J., Schampers, R., Persoon, H., Hyman, A.A., Baumeister, W., Plitzko, J.M., 2015. A focused ion beam milling and lift-out approach for site-specific preparation of frozen- hydrated lamellas from multicellular organisms. J Struct Biol 192, 262-269. https: / / doi.org / https: / / doi.org / 10.1016 / j.jsb.2015.07.012 Marko, M., Hsieh, C., Schalek, R., Frank, J., Mannella, C., 2007. Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy. Nat Meth 4, 215- 217. Mastronarde, D.N., 2005. Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152, 36-51. https: / / doi.org / 10.1016 / j.jsb.2005.07.007 Mercogliano, C.P., DeRosier, D.J., 2007. Concatenated metallothionein as a clonable gold label for electron microscopy. J Struct Biol 160, 70-82. https: / / doi.org / 10.1016 / J.JSB.2007.06.010 Palmer, C.M., Löwe, J., 2014. A cylindrical specimen holder for electron cryo-tomography. Ultramicroscopy 137, 20-29. https: / / doi.org / 10.1016 / j.ultramic.2013.10.016 Redick, S.D., Stricker, J., Briscoe, G., Erickson, H.P., 2005. Mutants of FtsZ Targeting the Protofilament Interface: Effects on Cell Division and GTPase Activity. J Bacteriol 187, 2727-2736. https: / / doi.org / 10.1128 / jb.187.8.2727-2736.2005 Rigort, A., Bäuerlein, F.J.B., Villa, E., Eibauer, M., Laugks, T., Baumeister, W., Plitzko, J.M., 2012. Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc Natl Acad Sci USA 109, 4449-54. https: / / doi.org / 10.1073 / pnas.1201333109 Rubino, S., Akhtar, S., Melin, P., Searle, A., Spellward, P., Leifer, K., 2012. A site-specific focused-ion-beam lift-out method for cryo Transmission Electron Microscopy. J Struct Biol 180, 572-6. https: / / doi.org / 10.1016 / j.jsb.2012.08.012 Schaffer, M., Pfeffer, S., Mahamid, J., Kleindiek, S., Laugks, T., Albert, S., Engel, B.D., Rummel, A., Smith, A.J., Baumeister, W., Plitzko, J.M., 2019. A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue. Nat Methods 16, 757-762. https: / / doi.org / 10.1038 / s41592-019-0497-5 Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D.J., Hartenstein, V., Eliceiri, K., Tomancak, P., Cardona, A., 2012. Fiji: an open-source platform for biological-image analysis. Nat Meth 9, 676-682. https: / / doi.org / 10.1038 / nmeth.2019 Silvester, E., Vollmer, B., Pražák, V., Vasishtan, D., Machala, E.A., Whittle, C., Black, S., Bath, J., Turberfield, A.J., Grünewald, K., Baker, L.A., 2021. DNA origami signposts for identifying proteins on cell membranes by electron cryotomography. Cell 184, 1110-1121.e16. https: / / doi.org / 10.1016 / J.CELL.2021.01.033 Stricker, J., Erickson, P.H., 2003. In Vivo Characterization of Escherichia coli ftsZ Mutants: Effects on Z-Ring Structure and Function. J Bacteriol 185, 4796-4805. https: / / doi.org / 10.1128 / jb.185.16.4796-4805.2003 Szwedziak, P., Wang, Q., Bharat, T.A.M., Tsim, M., Löwe, J., 2014. Architecture of the ring formed by the tubulin homologue FtsZ in bacterial cell division. Elife 3, e04601. https: / / doi.org / 10.7554 / eLife.04601 Tegunov, D., Cramer, P., 2019. Real-time cryo-electron microscopy data preprocessing with Warp. Nat Methods 16, 1146-1152. https: / / doi.org / 10.1038 / s41592-019-0580-y Wagner, T., Merino, F., Stabrin, M., Moriya, T., Antoni, C., Apelbaum, A., Hagel, P., Sitsel, O., Raisch, T., Prumbaum, D., Quentin, D., Roderer, D., Tacke, S., Siebolds, B., Schubert, E., Shaikh, T.R., Lill, P., Gatsogiannis, C., Raunser, S., 2019. SPHIRE- crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun Biol 2, 218. https: / / doi.org / 10.1038 / s42003-019-0437-z Wang, K., Strunk, K., Zhao, G., Gray, J.L., Zhang, P., 2012.3D structure determination of native mammalian cells using cryo- FIB and cryo-electron tomography. J Struct Biol 180, 318-326. https: / / doi.org / https: / / doi.org / 10.1016 / j.jsb.2012.07.003 Wang, Q., Mercogliano, C.P., Löwe, J., 2011. A Ferritin-Based Label for Cellular Electron Cryotomography. Structure 19, 147-154. https: / / doi.org / 10.1016 / J.STR.2010.12.002 Wang, X., Lutkenhaus, J., 1993. The FtsZ protein of Bacillus subtilis is localized at the division site and has GTPase activity that is dependent upon FtsZ concentration. Mol Microbiol 9, 435-442. https: / / doi.org / https: / / doi.org / 10.1111 / j.1365- 2958.1993.tb01705.x Wang, Z., Grange, M., Wagner, T., Kho, A.L., Gautel, M., Raunser, S., 2021. The molecular basis for sarcomere organization in vertebrate skeletal muscle. Cell 184, 2135- 2150.e13. https: / / doi.org / 10.1016 / J.CELL.2021.02.047 Whitley, K.D., Jukes, C., Tregidgo, N., Karinou, E., Almada, P., Cesbron, Y., Henriques, R., Dekker, C., Holden, S., 2021. FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in Bacillus subtilis cell division. Nat Commun 12, 2448. https: / / doi.org / 10.1038 / s41467-021-22526-0 Whitley, K.D., Middlemiss, S., Jukes, C., Dekker, C., Holden, S., 2022. High-resolution imaging of bacterial spatial organization with vertical cell imaging by nanostructured immobilization (VerCINI). Nat Protoc 17, 847-869. https: / / doi.org / 10.1038 / s41596- 021- 00668-1 Wolff, G., Limpens, R.W.A.L., Zheng, S., Snijder, E.J., Agard, D.A., Koster, A.J., Bárcena, M., 2019. Mind the gap: micro-expansion joints drastically decrease the bending of FIB- milled cryo-lamellae. J Struct Biol. https: / / doi.org / 10.1016 / J.JSB.2019.09.006 Yang, X., Lyu, Z., Miguel, A., McQuillen, R., Huang, K.C., Xiao, J., 2017. GTPase activity- coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis. Science (1979) 355, 744-747. https: / / doi.org / 10.1126 / science.aak9995 Zivanov, J., Otón, J., Ke, Z., von Kügelgen, A., Pyle, E., Qu, K., Morado, D., Castaño-Díez, D., Zanetti, G., Bharat, T.A.M., Briggs, J.A.G., Scheres, S.H.W., 2022. A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0. Elife 11, e83724. https: / / doi.org / 10.7554 / eLife.83724

Claims

Claims 1. An electron microscopy sample support comprising a metallic support, the metallic support comprising a plurality of wells, wherein each well comprises a solid, continuous base and the ratio of the depth to cross-sectional diameter or longest cross-sectional dimension of each well is in the range of 15:1 to 1:

2.

2. The electron microscopy support of claim 1, wherein each well comprises a base and side(s) and the base and side(s) of each well are formed from the same material.

3. The electron microscopy support of claim 1 or 2, wherein the metallic support is an integral object comprising the wells.

4. The electron microscopy sample support of any of the preceding claims, wherein the metallic support comprises a metal selected from the group of gold, platinum, tungsten, tantalum, or palladium or an alloy comprising a metal from this group.

5. The electron microscopy sample support of any of the preceding claims, wherein the wells are arranged in a regular array.

6. The electron microscopy support of any of the preceding claims, wherein the plurality of wells contains an array of aligned samples.

7. The electron microscopy sample support of any of the preceding claims, wherein the wells are cylindrical in shape.

8. The electron microscopy sample support of any of the preceding claims, wherein the wells have a cross-sectional diameter or longest cross-sectional dimension of less than 4 μm and / or a depth of 6 μm or less.

9. A method of manufacturing an electron microscopy sample support, the method comprising the steps of: (i) providing a substrate comprising a plurality of protrusions, (ii) depositing a layer of metallic material on the substrate covering and incorporating a plurality of the protrusions, (iii) separating the layer of metallic material from the substrate to provide an electron microscopy sample support comprising a plurality of wells complementary to the protrusions of the substrate.

10. The method of manufacturing an electron microscopy sample support according to claim 9, wherein the substrate of step (i) comprises a plurality of aligned protrusions.

11. The method of manufacturing an electron microscopy sample support according to claims 9 or 10, comprising a step of patterning a wafer with columns of polymeric material to form the substrate for step (i).

12. The method of manufacturing an electron microscopy sample support according to any of claims 9 to 11, wherein step (ii) comprises electroplating the substrate.

13. The method of manufacturing an electron microscopy sample support according to any one of claims 9 to 12, wherein step (iii) comprises etching removal of the substrate to separate the layer of metallic material.

14. A method of preparing a sample for electron microscopy comprising the steps of: (i) providing an electron microscopy sample support according to claim 1, (ii) loading a plurality of wells in the electron microscopy sample support with a sample suspended in a liquid, (iii) freezing the liquid, (iv) processing a portion of the electron microscopy sample support by removing the material defining the base of a plurality of wells.

15. The method of preparing a sample for electron microscopy according to claim 14 wherein step (ii) comprises centrifuging the electron microscopy sample support with a sample.

16. The method of preparing a sample for electron microscopy according to claims 14 or 15, wherein step (iv) comprises focussed ion beam (FIB) milling a portion of the electron microscopy sample support.

17. A processed electron microscopy sample support comprising: a metallic support, the metallic support comprising a support region surrounding an imaging region, wherein the support region is thicker than the imaging region and the support region and the imaging region are integral, wherein the plane of the surface of the support region is at an angle to the plane of the surface of the imaging region, wherein the imaging region comprises a plurality of through-holes in the metallic support, and wherein each through-hole optionally houses a section of a sample suspended in frozen liquid.

18. The processed electron microscopy sample support of claim 17, wherein the imaging region has a thickness of less than 500 nm.

19. The processed electron microscopy sample support of claims 17 or 18, wherein the sides of the through-holes are at an angle to the normal of the plane of the surface of the imaging region.

Images