Support for electron microscope

Abstract

1. A support for an electron microscope specimen, the support comprising a metal foil having one or more holes therethrough, the metal foil having a thickness of less than 50 nm and / or an average linear intercept grain size of 50 nm or less, the ratio of the diameter of each hole to the thickness of the metal foil being 15:1 or less, the metal foil being made of (a) one or more metals selected from transition metals, aluminum, and beryllium, or alloys thereof, or (b) the dopant element being 10 20 atoms / cm 3 a substrate comprising any of degenerately doped silicon selected from the group consisting of boron, aluminum, boron, and arsenic at a concentration of at least 1000 .mu.m;

Description

Technical Field

[0001] This application claims priority from GB2004272.7 filed on 24 March 2020, the content and elements of which are incorporated herein by reference for all purposes.

[0002] The present invention relates to an electron microscope (EM) sample support, a method of manufacturing an electron microscope sample support, an imaging method using an electron microscope sample support, and an apparatus capable of implementing the imaging method. The support is particularly useful in cryo-EM imaging.

Background Art

[0003] Samples can be imaged using electron microscope technology. According to such technology, a sample is "irradiated" with an electron beam. The presence of the sample in the electron beam causes a change to that beam. By examining the change in the beam induced by the sample, a magnified image of the sample can be created.

[0004] Since the sample is irradiated by an electron beam, it must be properly supported within that beam. In many cases, the electrons forming the electron beam have high energy, and bombarding an object, such as a sample for examination, together with a support that holds it in a predetermined position within the electron beam can cause physical, chemical, and / or electrical changes to the support and / or the sample. It will be understood that such changes can affect the results, including the resolution of the image obtained using electron microscope technology.

[0005] In this regard, there are at least two prominent problems in the art. First, the movement of the sample at the start of electron beam irradiation, in particular, degrades the image quality and removes information regarding non-damaged molecules from the structure. Thus, all current EM structure determination models have various degrees of radiation damage incorporated therein. Second, the throughput of modern synchrotron crystallography beamlines, at least in part due to current limitations in EM sample supports, far exceeds that of current state-of-the-art electron microscopes, which means that high-resolution structure determination for drug discovery and development is possible by EM technology, but the use of this technology is actually limited due to its low throughput.

[0006] Ermantraut 198 describes a carbon support foil ("Quantifoil" (registered trademark)) for cryo-EM and aims to minimize the total sample thickness to eliminate object distortion arising from interaction with the support structure. The foil has square holes that support an ice layer with a thickness up to 32 ± 2.3 nm. One carbon foil is said to be 15 nm thick and it is stated that a small pore diameter of about 500 nm is formed (not shown). This corresponds to a pore diameter to thickness ratio of 33.3:1.

[0007] Janbroers 2009 describes a carbon-free temperature-stable TEM grid. An 80% Au / 20% Pd metal film is supported on a standard mixed mesh Au TEM grid. The grid is formed by applying metal to a carbon-coated TEM grid and subsequently selectively removing the carbon using plasma cleaning. The circular holes shown in the figure have a diameter of about 1.5 μm or more. The thickness of the 80% Au / 20% Pd metal film was set to 7 nm, 10 nm, or 15 nm. This corresponds to a minimum pore diameter to thickness ratio of 100:1. The average particle size is 8.3 nm ± 2.0 nm and deposition occurs at room temperature.

[0008] Grant-Jacob 2016 describes a three-dimensional structured gold thin film with nanopores of defined periodic geometric shapes intended to provide spatially localized enhancement of the electric field by manipulating plasmons inside the nanopores. Since these films are not flat, they are not suitable for suspending samples for electron microscopy analysis. The substrate contains an array of inverted pyramids etched in a 4 mm × 4 mm square area on the surface, with inverted pyramids having a square of 1.5 μm × 1.5 μm, a pitch of 2 μm, a depth of 1 μm, and a thickness of 100 nm. Nanoscale holes of 50 nm squares are milled through the gold film at specific positions within the cavity to provide electric field control, which can then be used to enhance the fluorescence or Raman scattering of molecules within the nanopores. This corresponds to a ratio of pore diameter to thickness of 2:1 at the tip of the pyramid structure. However, the walls of the holes are not thick enough to support the sample film. The average particle size is ~40 nm, and deposition occurs at room temperature.

[0009] Jia 2019 describes a large-area free-standing gold nanomembrane with a thickness of 50 nm or more and nanoholes penetrating the membrane with a diameter of 250 nm. This corresponds to a pore diameter to thickness ratio of 5:1. However, since the gold nanomembrane is formed by room-temperature evaporation, it does not bring about as significant an improvement in image quality as the present invention.

[0010] Previous studies by the present inventors include Russo 2014, which shows a gold sample support that substantially eliminates movement of the substrate during irradiation. The support therein is a gold foil with a square pattern, having holes with a thickness of ~500 Å and a diameter of 1.2 μm. This corresponds to a pore diameter to thickness ratio of 24:1.

[0011] Also, Russo 2016 describes a fully gold support with a gold foil having holes with a thickness ranging from ~400 Å to 500 Å and a micrometer diameter. This corresponds to a pore diameter to thickness ratio of at least 20:1.

[0012] All of the above-known gold support foils have a thickness of at least 500 Å. This is because foils with a thickness of less than about 500 Å are not currently stable due to their polycrystalline grain structures, which typically have an average grain size of about 200 nm or more. When foils with a thickness of less than about 500 Å are formed, they suffer from structural defects such as gaps and cracks in the foil structure, which can cause a lack of structural rigidity and thus sample movement during any thermal expansion, for example, caused by electron beam heating. Furthermore, the holes through such membranes have significantly rough edges caused by individual particles, which is particularly prominent at the edges of very small holes. This roughness adversely affects the ease of imaging and stabilization. In addition, thin gold foils with a pore size of less than 0.5 μm, optimized for single-particle cryo-EM, cannot be manufactured by standard diffraction-limited photolithography.

[0013] It is desirable to provide a support that can address some of the problems of known sample supports, such as unwanted movement during imaging, for use in electron microscopes, particularly cryo-EM. The present disclosure has been made in view of the above problems.

Summary of the Invention

[0014] The inventors have found that unwanted movement during transmission electron microscopy imaging using current supports for electron microscopes is at least partially caused by the accumulation of tension in the sample membrane that can lead to buckling of the membrane when the force exceeds a certain threshold. For example, tension accumulates in the vitreous ice film used to immobilize the sample for cryo-EM, with a buckling threshold that directly depends on the shape of the sample membrane. For example, taking cryo-EM as an example, during cryo-plunging to freeze an aqueous sample, freezing occurs over a time interval of ≤ 10 -4 seconds. Within this time, the water density change is most rapid near the homogeneous nucleation temperature of 235 K. As water solidifies, the rapid cooling does not allow sufficient time for the structural rearrangement of water molecules, and as a result, compressive strain accumulates in the thin ice film. When the compression exceeds the critical value, the ice film buckles and the radial stress within the layer is instantaneously relaxed.

[0015] Buckling occurs only when the stress exceeds a critical point determined by the dimensions of the ice film, its modulus of elasticity, the specific volume change with respect to the support, and the constraints at the edges of the pores. As the vitreous ice film continues to be cooled to the temperature of the surrounding cryogen, typically liquid ethane at 90 - 93K, more stress can accumulate due to further relative density changes. This stress is stored indefinitely in the film when the film is completely cooled to 77K (liquid nitrogen temperature) and allowed to stand.

[0016] Even in situations where the stress accumulation within the ice layer does not exceed the threshold that causes buckling of that layer, local heating can at least partially relax the retained stress and cause movement of the sample when the sample is exposed to an electron beam in a transmission electron microscope (TEM).

[0017] Briefly, the inventors have found that the buckling threshold of the film depends on the shape and dimensions of the film and is determined by the shape of the pores in which the film is suspended. The present invention is designed to minimize or avoid the accumulation of unnecessary stress in the film, for example, to reduce or completely eliminate sample movement caused by buckling or stress relaxation during inspection. This significantly improves the image quality.

[0018] In a first aspect, there is provided a support for an electron microscope sample, the support comprising a metal foil having one or more through - holes, the thickness of the metal foil being less than 50 nm and / or the average linear section particle size being 50 nm or less, the ratio of the diameter of each hole to the thickness of the metal foil being 15:1 or less, and the metal foil being composed of (a) one or more metals selected from transition metals, aluminum, and beryllium, or alloys thereof, or (b) degenerate - doped silicon having a dopant element at a concentration of 10 20 atoms / cm 3 or more, selected from boron, aluminum, boron, and arsenic.

[0019] In particular, any alternative within the first aspect is the corresponding technical feature of these proposals. This is because these alternatives achieve the same technical effect of reducing sample movement and solve the same technical problem of improving imaging.

[0020] The transition metal is an element of Groups 3 to 11 of the periodic table.

[0021] In some cases, some or all of the one or more 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 alloys thereof. Preferably, the one or more transition metals are selected from noble metals ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, gold, or alloys thereof. Preferably, the one or more transition metals are selected from gold, palladium, and platinum, or alloys thereof. Preferably, the metal foil comprises or consists of gold or an alloy thereof. Preferably, the alloy is a binary alloy. Particularly preferred alloys include gold-silver alloy, gold-copper alloy, nickel-titanium alloy, gold-platinum alloy, and platinum-iridium alloy. The foil may not contain aluminum. The foil may not contain beryllium. The foil may not contain an alloy.

[0022] To avoid misunderstanding, the metal foil is not formed from an electrical insulator, a semiconductor material, or a carbon material such as amorphous or graphite carbon, although these may be present in the metal alloy as described herein. Semiconductor materials include materials whose conductivity decreases as the temperature decreases in the range of 300 to 80 K. This includes carbon (amorphous or graphite, but not diamond), silicon (with dopant elements such as boron, aluminum, phosphorus, and arsenic at less than 10 20 / cm 3 toppings), gallium arsenide, and other III-V or II-VI binary semiconductors.

[0023] In some cases, the ratio of the diameter of each hole to the thickness of the metal foil is 11:1 or less, 10:1 or less, 8:1 or less, 7:1 or less, or 5:1 or less. The ratio of the diameter of each hole to the thickness of the foil may be 11:1 to 1:1, 10:1 to 2:1, 9:1 to 3:1, or 8:1 to 4:1.

[0024] The advantage of using a specific ratio is to eliminate buckling of the sample film when the sample film, such as a suspended amorphous ice film used in cryo-EM, is formed within the holes. This enables accurate tracking of the foil during imaging using a high-speed detector and reduces the requirements for the accuracy and stability of the cryo-stage. Therefore, this support reduces the movement of particles to the limit set by pseudo-diffusion that is smaller than the resolution of the electron cryomicroscope. This enables the reconstruction of a complete map at a resolution of 1.9 Å with a fluence of less than 1 e - / Å 2 The sample film remains stable during irradiation and is under radial compression, and only diffusional movement limited to an RMS of less than 1 Å at 30 e - / Å 2 occurs. This movement-suppressing microscope sample support enables atomic structure determination at only 1 e - / Å 2 and extrapolation to the point before the destructive effect of the electron beam affects the reconstruction.

[0025] In some cases, the metal foil is substantially free of structural defects. The absence of structural defects means that the foil material is substantially uniform and continuous. That is, there are no visible gaps or cracks in the foil, and the surface roughness is reduced. For example, the edge roughness of each hole may be 20 nm or less, 15 nm or less, 10 nm or less, or 5 nm or less, measured as the root mean square deviation from the expected theoretical hole edge profile. The foil may have only one structural defect up to a diameter of 1 nm per 100 nm 2 of area.

[0026] When the thickness of the metal foil is less than 50 nm and there are no structural defects, the advantages are that the foil is structurally stable and can be used to generate images with much higher resolution by an electron microscope than was previously possible. Further, the thin foil thickness provides a thinner sample film within the pores with improved transmittance. Known metal foils with a thickness less than 50 nm provide inadequate images and often peel (i.e., they are not self - supporting or cannot be suspended across an EM grid square such as a 50 μm grid square without damage), and thus are not suitable for an electron microscope because they cannot properly suspend the sample film.

[0027] The advantage when the average linear section particle size is 50 nm or less is the absence of structural defects combined with a reduction in the roughness of the surface, particularly at the edges of the pores. As a result, the metal foil can be successfully used with a very high - resolution electron microscope. Foils of these particle sizes are known, but all have structural defects and non - uniformities and are particularly unsuitable for high - resolution electron microscopes. Such known foils are typically formed as a deposit on a support surface. In contrast, the present metal foil is sufficiently robust to be self - supporting when suspended across an opening, e.g., a 50 μm grid square within a TEM grid. Thus, the foil of the present disclosure is preferably not supported, i.e., not provided on a support layer. The defined particle size and lack of structural defects of the present metal foil enable the formation of more rounded and smoother nanoscale pores that provide a stable support for suspending the sample.

[0028] In some cases, the thickness of the metal foil is 49 nm or less, for example 45 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, 10 nm or less, or 5 nm or less. The thickness of the metal foil is preferably substantially constant throughout. Preferably, the foil thickness can vary by up to ±25 Å, ±10 Å, or ±5 Å throughout.

[0029] In some cases, the average linear slice particle size of the metal foil material may be 40 nm or less, for example, 30 nm or less, 20 nm or less, 10 nm or less, or 5 nm or less. A smaller particle size increases the stability of the foil at a lower thickness. To obtain an optimal balance of advantages, it is particularly preferred that the average linear slice particle size is 10 nm or less.

[0030] In some cases, the diameter of each pore is 750 nm or less, 700 nm or less, for example, 600 nm or less, 500 nm or less, 400 nm or less, 350 nm or less, 330 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 180 nm or less, 150 nm or less, 100 nm or less, or 75 nm or less. In some cases, the diameter of each pore is within a range selected from 750 nm to 75 nm, 500 nm to 100 nm, 400 nm to 150 nm, 300 nm to 150 nm, or 250 nm to 150 nm. The pores are preferably substantially circular. All the pores may have substantially the same diameter or different diameters. The diameter of each pore is preferably substantially the same throughout the depth of each pore. Each pore completely penetrates the metal foil. The spacing between the pores is preferably equal. The spacing between the pores is preferably equal to at least one pore diameter. The pore walls are preferably at an angle of 90 degrees with respect to the surface of the foil, optionally with an accuracy of ±2 degrees or better. For any pore diameter, the depth of the pore is preferably 500 nm or more. The side walls of the pores are preferably vertical or reverse (tapered). These two preferred configurations ensure that the pores formed in the foil are less likely to be blocked by the material deposited at the bottom / side walls of the wells in the later stage of the process. The metal foil may include other secondary pores that are larger than the above-defined diameter requirements and / or do not meet the above requirements for the diameter-to-thickness ratio. The secondary pores are extra because they do not meet the defined requirements. Alternatively, in some cases, the foil includes only pores that meet the defined requirements.

[0031] In some cases, the metal foil does not contain pores that do not meet the defined nanoscale diameter and / or diameter-to-thickness ratio requirements.

[0032] The advantages of nanoscale dimensions are that the movement of particles within the pores is isotropic (the same in the plane of the support and perpendicular thereto), spatially uncorrelated, and corresponds to the root of the incident electron fluence, similar to the case of purely random movement. In contrast, for larger pore diameters of known supports, there are sudden spatially correlated and undesirable particle displacements at the start of irradiation (e.g., at the first 4e - / Å 2 ). Subsequently, the decreasing correlated movement continues at a decreasing rate until the end of the exposure.

[0033] Another advantage is that pores of nanoscale dimensions provide plasmon resonance in the visible range, such that the support appears yellow when reflected with white light and blue when transmitted, and this property can be useful for characterizing the sample before imaging with electrons.

[0034] In some cases, the support has a maximum light wavelength transmittance of 650 - 800 nm, for example 700 - 750 nm or 714 nm. The foil support can have a minimum transmittance of 500 - 600, for example 625 - 675 or 645 nm.

[0035] In some cases, the purity of the metal foil material is 90% or more, preferably 99% or more, more preferably 99.999% or more (i.e., containing the relevant metal material of the stated %).

[0036] In some cases, the pores are arranged in a regular pattern on the metal foil, such as a hexagonal pattern or a square pattern. Preferably, the pores are arranged in a hexagonal pattern. The pattern is preferably regular. The hexagonal pattern provides a denser packing and improves structural rigidity. The hexagonal pattern also enables faster inspection of multiple pores by an automated system, as the denser packing of pores in the membrane results in a shorter distance between one pore and the next, speeding up the inspection time for multiple pores.

[0037] In some cases, the metal foil is provided on an EM support grid. The grid provides additional structural support to the foil. In some cases, the foil and the grid are an integral structure. The foil and the grid may be integrally formed. The foil and the grid may have the same elemental composition throughout. The foil and the grid may have different grain structures. Advantages provided by grids and foils having the same elemental composition include improved stability during imaging, which is because the two structures have the same coefficient of thermal expansion, or the difference in their mechanical behavior with respect to thermal changes (heating or cooling) is minimal or non-existent, or there is little or no relative movement between the two structures. Despite having the same elemental composition, the grain structure requirements for the grid material are not as strict as for the metal foil, so the foil and the grid may have different grain structures, for example, different particle sizes. The grid may be millimeter-sized, and such a circular grid may have a diameter of 5 mm, 4 mm, or most preferably 3 mm. The grid can include one or more support bars arranged to form a mesh capable of suspending the foil. In some cases, the mesh has an average pore size on the micrometer scale, for example, about 300 μm, about 200 μm, about 100 μm, or about 50 μm. The pores of the mesh may be hexagonal or square. Preferably, the pores of the mesh have a hexagonal shape. Preferably, the pores of the mesh are mosaic-like, that is, when the pores are square, the pores are arranged in a square array, and when the pores are hexagonal, the pores are arranged in a hexagonal array. The hexagonal pores and array provide a denser packing and improve structural rigidity. The pore pattern of the mesh may correspond to the foil pore pattern.

[0038] In some cases, the grid is formed from a material selected from the same options as those listed herein for the metal foil. In some cases, the grid comprises one or more metals selected from transition metals, aluminum, and beryllium, or alloys thereof. The one or more transition metals can be selected from one or more of the noble metals (ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, gold), copper, molybdenum, titanium, nickel, chromium, tungsten, hafnium, and tantalum, or alloys thereof. Preferably, the one or more transition metals are selected from the noble metals ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, gold, or alloys thereof. Preferably, the one or more transition metals are selected from gold, palladium, and platinum, or alloys thereof. Preferably, the grid comprises or consists of gold or an alloy thereof. Preferably, the alloy is a binary alloy. Particularly preferred alloys include gold-silver alloy, gold-copper alloy, nickel-titanium alloy, gold-platinum alloy, and platinum-iridium alloy. The grid may not contain aluminum. The grid may not contain beryllium. The grid may not contain an alloy.

[0039] In some cases, the grid is 10 20 atoms / cm 3 or more of a degenerate doped silicon having a dopant element selected from boron, aluminum, boron and arsenic.

[0040] The hexagonal arrangement of the holes in both the metal foil and the mesh increases the usable area by a factor of 10 relative to a standard cryo-EM grid, enabling the acquisition of over 800 images from a single stage position, for example, providing over 5000 individual holes within the holes of a single 25 μm-wide hexagonal mesh.

[0041] In some cases, the electron microscope is a transmission electron microscope, preferably a transmission electron cryo-microscope.

[0042] The grid or mesh that can exist as a support for the metal film can, in some cases, be placed within the support rim. The options for the material used for the support rim are the same as those for the grid described above. Preferably, the support rim is integral with the grid structure.

[0043] In a second aspect, there is provided the use of the support of the first aspect in an electron microscope, optionally a transmission electron microscope, preferably a transmission electron cryomicroscope.

[0044] In a third aspect, there is provided a method for manufacturing a metal foil for a support according to the first aspect, the method comprising: depositing a metal layer on a patterned substrate cooled to 200 K or less to form a layer having a thickness of 50 nm or less and having one or more through holes; removing the deposited metal layer; and forming a metal layer in a support for an electron microscope sample, wherein the metal foil consists of (a) one or more metals selected from transition metals, aluminum, and beryllium, or alloys thereof, or (b) degenerate doped silicon selected from boron, aluminum, boron, and arsenic having a dopant element concentration of 10 20 atoms / cm or more.

[0045] By cooling the substrate during the deposition process, a metal layer can be produced thereon that, for gold and other suitably described metals and materials in particular, not only has a smaller particle size compared to deposition at room temperature, but also has excellent uniformity and continuity at such particle sizes that were not previously known. This is achieved because a lower temperature reduces the thermal energy of the atoms faster than when the surface is not cooled, so that when the metal atoms are deposited on the substrate, the movement of the metal atoms on the deposition surface is reduced. Since the atoms are rapidly immobilized when deposited on the substrate, there is less movement of the atoms and they aggregate into smaller particles compared to when the surface is not cooled.

[0046] In some cases, the deposited foil is a metal foil as described herein.

[0047] Furthermore, for many small EM samples, the desired foil hole size is smaller in diameter than the diffraction limit of known photolithography techniques currently used to fabricate the sample support, and evaporated metal foils with a thickness of less than about 400 Å, particularly gold foils, are not stable due to their polycrystalline grain structure (e.g., they cannot be lifted from the deposition substrate without significant damage). The present method provides metal foils that do not have these defects.

[0048] In some cases, the patterned substrate is a silicon wafer, optionally having a diameter of 3 mm to 300 mm, such as 100 mm. The silicon wafer may be a degenerately doped silicon wafer having a resistivity of <0.02 ohm·cm. Alternatively, a silicon wafer having a higher resistivity of 1 to 30 ohm·cm can be used. The silicon wafer can be formed by Talbot displacement (phase coherent) lithography as described in Jefimovs 2017. The diameter of the holes in the silicon surface can be controlled using the template-substrate distance during phase coherent lithography. A regular template array can set the spacing between the holes patterned on the substrate. The silicon substrate is patterned to form recesses corresponding to the holes in the desired metal foil.

[0049] In some cases, the metal foil can be formed integrally with a grid bar support arrangement, such as a mesh. In such cases, if it is desired to set the foil a distance below the level of the upper surface of the grid bar, a mask having a grid bar pattern is applied to a photoresist-coated silicon template to expose only the grid bar regions after development. These are then etched, for example, by reactive ion etching (RIE), to form trenches of the desired depth. This step may be necessary to adapt the grid to the current sample plunging and blotting apparatus. If it is desired that the surface of the foil be at the same height as the upper surface of the grid bar, this step is not necessary.

[0050] In some cases, there is a step of Bosch etching the holes in the substrate. This controls their final depth and ensures that the hole walls are at an angle of 90 degrees with respect to the surface of the substrate with an accuracy of 2 degrees or better.

[0051] In some cases, there is a step of cleaning the patterned substrate before depositing the metal layer. The cleaning can be done, for example, by immersion in a piranha solution (3H2SO4:1H2O2, freshly mixed), oxygen plasma, or UV-ozone plasma. Cleaning is advantageous for removing contaminants that can lead to insufficient hole formation.

[0052] In some cases, the metal layer is deposited on the patterned substrate, and the substrate is at a temperature of 150 K or less, 125 K or less, 100 K or less, or 90 K or less. The temperature of the substrate can be set to 84 K - 92 K. Preferably, the substrate stage is maintained at 77 K (liquid nitrogen temperature) during evaporation so that the particle size of the metal foil can be reduced and more round and smooth through-holes can be formed. A higher deposition rate requires cooling to a lower temperature. The deposition can be by electron beam or thermal evaporation. For example, assuming surface-adsorbed atom diffusion with an activation energy of 0.5 eV, the temperature range for nucleating crystals of 10 nm or less at a deposition rate of 1 Å / s is 200 K or less. The substrate with the deposited metal layer can be slowly warmed (at about 50 K / hour or slower) to room temperature in a vacuum to prevent delamination of the foil.

[0053] Preferably, for the best growth of metal crystals, due to the high self-diffusion coefficient of metal materials such as gold, the optimal deposition temperature is lower. The purity of the material to be deposited is preferably 90% or more, more preferably 99% or more, and even more preferably 99.999% or more in order to form a stable continuous layer.

[0054] In some cases, the patterned substrate is provided with a first sacrificial layer applied to the patterned substrate on which the metal layer is to be deposited. Any layer that can be selectively etched with respect to the associated metal foil layer can be used for this layer. The sacrificial layer may be a metal, for example, it can be copper (particularly preferred for gold foil), etc. The sacrificial layer is preferably copper. The sacrificial layer can also be deposited on the substrate under the above-described cooling conditions. The deposition conditions are preferably the same as those for the metal foil layer for the sacrificial layer. This is preferred because undesirable incompleteness and roughness of the deposited sacrificial layer can otherwise be imparted to the side of the metal foil layer formed thereon. The thickness of the sacrificial layer is preferably at least the same as the specified particle size. For example, a sacrificial layer having a thickness of at least 10 nm, or at least 25 nm, or at least 50 nm can be used. The maximum thickness of the sacrificial layer is preferably less than the radius of the holes in the foil.

[0055] In some cases, there are one or more steps of transferring a metal foil onto an EM grid. The foil can be transferred onto a commercially available EM grid using a float process. Alternatively, the grid can be fabricated directly on a substrate carrying the foil to obtain an integrated foil and grid. The latter is a preferred option for large-scale manufacturing. Forming the foil integrally with at least the mesh of the grid bars, preferably the entire grid (the mesh of the grid bars and the thicker grid peripheral rim), is preferred because using the same metal for both the foil and the grid bars eliminates the difference in the coefficient of thermal expansion, and thus there is no movement of the substrate during heating (e.g., beam heating during exposure to an electron beam).

[0056] In some cases, the support is according to the first aspect.

[0057] Manufacture of the support by transferring a metal foil to a commercial grid To transfer a metal foil onto a commercial grid, there can be a process of lifting off the foil, a cleaning process, and a process of lowering it onto the grid. The foil can be coated with a 1 - 10 μm layer of negative photoresist before being lifted off. The lift-off process can include etching a sacrificial layer such as copper to peel the foil from the substrate. The cleaning process can be carried out by one or more HCl washings (e.g., 20%, 2%, 0.2% HCl, followed by at least 3 water rinses). The lowering process can include placing the grid on the bottom of a dish filled with water in which the foil is floating and slowly sucking up the water to lower the foil onto the clean grid. When using a support resist layer, the support resist layer can be removed by washing individual grids in a suitable solvent and treating them with low-energy plasma.

[0058] Optionally, as described in Naydenova 2019, after the foil is lowered onto the grid, polymer-assisted graphene transfer can be carried out.

[0059] Manufacture of a support by integrally forming a grid on a metal foil To manufacture a support having an integrated metal foil and grid, a photoresist is temporarily applied and excess foil between adjacent individual foils intended to be adjacent is etched away. A second temporary photoresist is applied so that the grid bars can be deposited by electroplating along with any additional features such as alignment marks, references, labels, unique identifiers, etc. The photoresist is applied, exposed under a mask, developed, and removed using standard techniques. After removing the second photoresist, the surface is cleaned with oxygen plasma or UV-ozone. A support plastic layer is rotated across the grid. Supports, each containing an integrated foil and grid and supported together on the same plastic layer, are then lifted off the substrate and cleaned, for example, by a series of 20 - 0.2% aqueous hydrochloric acid solutions and water washes.

[0060] The electroplating process can form a deposited metal layer with a total thickness of 10 - 15 μm on the grid bar and the rim. The metal layer can be deposited from an electrolyte solution, preferably a non - cyanide bath, such as a sulfite / thiosulfite. Such a solution typically has a metal material to be deposited at a concentration of 10 - 15 g / L and is used at 50 - 60 °C with an applied current density of 2 - 10 mA / cm 2 ². Both of these parameters are changed to control the residual stress in the electroplated metal layer. Under these conditions, for example, electroplated gold has a Young's modulus of at least 35 GPa and a hardness of at least 40 Vickers.

[0061] The lift - off process may typically involve chemically wet - etching a sacrificial layer, such as copper, in an etchant based on ferric chloride or ammonium persulfate. Lift - off is preferably performed within 20 minutes at room temperature to prevent non - selective etching of the metal foil layer by the copper etchant. When etching at a higher temperature, the time is approximately halved for every 10 - degree increase in temperature. The grid can preferably be detached from the silicon template by etching silicon in a stirred solution of KOH (30%) at a high temperature (80 °C). Lift - off occurs almost instantaneously when immersed in the solution and involves the removal of the photoresist by the same solution. The individual grids released by this process can then be transferred to a clean bath of KOH, followed by two de - ionized water baths, a clean bath of a piranha solution at room temperature (to remove any remaining copper), and then two more de - ionized water baths.

[0062] After the cleaning process, the integrated support is sufficiently stable on the plastic layer for packaging and transportation. The plastic layer can be removed by dissolving it in a suitable solvent immediately before using the integrated support. Any remaining slight traces of plastic can be removed by subsequent low - energy plasma treatment of the cleaned grid. The grid can be dropped onto a suitable support, such as filter paper, and dried for use.

[0063] Thus, the requirements for a suitable plastic layer are that (i) it can be easily removed with a solvent, (ii) it is insoluble in water, HCl, and a copper etchant (either ammonium persulfate or ferric chloride), and (iii) it has sufficient flexibility and structural rigidity to lift the substrate off and transfer it to a cleaning bath. Examples of suitable plastics include positive or negative photoresists, polystyrene, and collodion.

[0064] Optionally, as described in Naydenova 2019, polymer-assisted graphene transfer can be carried out immediately after the deposition of the metal foil to yield a graphene layer located between the foil and the grid bars. This is preferred for small-scale procedures. Alternatively, this can be carried out even after the grid is fully formed while it remains attached to the wafer. In the latter case, a plastic layer that aids graphene transfer can also be used as the plastic layer, or an additional plastic layer can be added as described above. When large-scale procedures (e.g., wafer scale) are used, the graphene can preferably be transferred onto the grid after releasing the grid from the wafer. This can be achieved, for example, by transferring the grid from the wafer onto another temporary support structure, such as a suitable polymer that can later be dissolved in an organic solvent.

[0065] It has been determined that excellent support quality can be ensured when one or more of the following parameters are met: (a) the percentage of clogged / malformed holes is less than 1% (caused by sub-optimal cleanliness of the substrate before evaporation); (b) the deviation from circularity of the holes is less than 10 nm (caused by an insufficiently small particle size due to the deposition rate during heating and / or evaporation, or by partial etching of the foil during lift-off); (c) the flatness of the hole edges is less than 10 nm (also caused by wear of the template as described above); (d) the grid bar defect area is less than 1% (caused by mask defects); (e) the adhesion of the foil to the grid bar is sufficient to withstand the stress due to rapid cooling from 277 K to 80 K (at least 106 K / s); and (f) the foil coverage rate is >99%.

[0066] These proposals also include EM supports formed by the methods described herein.

[0067] In a fourth aspect, there is provided an electron microscopy imaging method including the step of continuously imaging a sample suspended in a hole of the support described in the first aspect, wherein each image includes at least a part of the edge of the hole, and the electron beam includes the hole and the complete edge of the hole.

[0068] In some cases, at least a part of the edge of the hole in each image is compared with other images to remove any relative shift between consecutive images, and / or consecutive images of the sample in the hole are weighted taking into account damage to the sample.

Brief Description of the Drawings

[0069] To enable an understanding of the present invention and to further understand additional aspects and configurations of the present invention, embodiments illustrating the principles of the present invention will be described in more detail with reference to the accompanying drawings.

[0070]

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Mode for Carrying Out the Invention

[0071] The configurations disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, are expressed in terms of their specific forms, or means for performing the disclosed functions, or methods or processes for obtaining the disclosed results, and can be used, as required, separately or in any combination of such configurations, to implement the present invention in its various forms.

[0072] The present invention has been described in connection with the exemplary embodiments described above. Once the present disclosure is provided, many equivalent modifications and variations will be apparent to those skilled in the art. Therefore, the exemplary embodiments of the present invention described above are considered to be illustrative and not restrictive. Various changes can be made to the described embodiments without departing from the scope of the present invention.

[0073] To avoid misunderstanding, any theoretical explanations provided in this specification are provided for the purpose of improving the reader's understanding. The inventors are not bound by any of these theoretical explanations.

[0074] Any section headings used in this specification are for organizational purposes only and should not be construed as limiting the subject matter being described.

[0075] Throughout this specification including the following claims, unless the context requires otherwise, the words "have", "comprise", and "include", and variations such as "having", "comprises", "comprising", and "including", are meant 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.

[0076] It should be noted that in this specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. A range can be expressed herein as "about" one particular value and / or "about" another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation, it will be understood by using the antecedent "about" that a particular value forms another embodiment. The term "about" with respect to a numerical value is optional and means, for example, + / - 10%.

[0077] As used herein, the terms "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits in some situations. However, it should be understood that in the same or different situations, other embodiments may also be preferred. Thus, the description of one or more preferred embodiments is not meant or intended to imply that other embodiments are not useful, nor to exclude other embodiments from the scope of this disclosure or from the scope of the claims.

[0078] As used herein, the term "metal" is used to refer to a material or component (such as a foil) that exhibits the properties of a metal. In particular, they exhibit high electrical and thermal conductivity. Often, the conductivity in a metal material is higher than 10 4 S / m.

[0079] Electron microscope support A support for an electron microscope is a device that can transport a sample to be examined in an electron microscope both inside and outside the electron microscope. The support is provided with a certain degree of mechanical strength by a peripheral wall or rim, and typically a mesh of members (such as grid bars) is disposed inside it. The sample to be examined is placed on the support within an area defined by the periphery of the grid bars. In cryo-EM, the sample itself is suspended within a hole in a foil or within a membrane (such as a vitreous ice film) suspended within pores between the grid bars. The foil is generally also referred to as a support film.

[0080] The foil portion of the support typically has a mesh or "perforated film" structure. These foils are typically described in the art by two numbers, for example "2 / 1", which means a foil having pores of 2 μm at intervals of 1 μm. Similarly, a foil designated 2 / 4 has pores or holes of 2 μm at intervals of 4 micrometers. The term "support" includes cases where the foil is provided with and without a grid.

[0081] (Example 1) High-Resolution Cryo-EM Structure Determination of DNA Protection in the Starvation Protein (DPS) To demonstrate the use of a non-moving sample support for high-resolution cryo-EM, the structure of 220 kDa DNA protection in the starvation protein (DPS) was determined. DPS was plunge-frozen on a grid equipped with a 280 Å thick gold foil with 260 nm holes. The average resolution from an initial reconstruction from about 9 hours of automated data collection on a state-of-the-art 300 keV microscope easily reached <2 Å, and the total particle displacement was 35 e - / Å 2 of irradiation with an RMS of 0.86 Å. The absence of buckling also ensured no significant rotation of the particles during imaging. In contrast to all previous single-particle cryo-EM data sets to date, the maps reconstructed from each frame showed that the first frame (1 e - / Å 2 or 3 MGy) contained the most structural information, indicating that the quality (B factor) of consecutive frames decayed linearly with dose / fluence. The linear decay of the B factor with dose is predicted from studies of radiation damage in X-ray and electron crystallography but has not been observed to date for single-particle cryo-EM due to movement at the start of irradiation.

[0082] (Example 2) Gold Foil Property Evaluation The average linear section particle size of several gold foils fabricated as described herein was measured by TEM and found to be 100 ± 10 Å. This is approximately 20 times smaller than the particle size in gold foils fabricated under similar conditions but at room temperature. The small particle size enables both thinner foils and smoother hole edges. The typical edge roughness (deviation from circular) of holes of 200 - 300 nm is less than 10 nm.

[0083] (Comparative Example) Conventional Foil Is Unsuitable for the Purpose The following examples illustrate the improvement of this electron microscope support by detailing and comparing the defects in currently known supports.

[0084] (Defect Type 1) Poor Hole Formation Due to Increased Particle Size Caused by Increased Evaporation Rate The gold film shown in Fig. 11A was deposited at a rate of 6 Å / s on a patterned substrate maintained at approximately 90 K. A sacrificial copper layer (not shown) was evaporated at 27 Å / s. These evaporation rates resulted in poorly formed holes due to an increase in particle size. The pore diameters vary between 50 nm and 250 nm. Compare with the foil of Fig. 11D, which was produced by deposition at the same temperature but at a lower rate (1 Å / s for both copper and gold) on the same template and has the same thickness. In that foil, the typical deviation of the holes from circularity is 10 nm or less.

[0085] (Defect type 2) Poorly formed holes due to an increase in particle size caused by an increase in evaporation temperature are shown in Fig. 10A.

[0086] (Defect type 3) Poor hole formation due to overetching The gold foil shown in Fig. 11B was deposited on the same substrate as that from Fig. 11E in the same manner. The release of this film (etching of the sacrificial Cu layer in ferric chloride) was twice as slow as that of Fig. 11E (30 minutes vs. 15 minutes). As a result, the holes expanded irregularly (about 50 nm) due to the etching of gold by ferric chloride. The hole pitch is 600 nm.

[0087] (Defect type 4) Porosity The gold foil shown in Fig. 11C was fabricated by the method of Russo 2014, and gold deposition was carried out at room temperature. The foil thickness is 326 Å, and the holes have a diameter of 2 μm. This is thicker than this foil. Due to larger particle sizes of about 200 nm, the foil remains porous and unstable at this thickness. This was demonstrated using Russo 2014, which shows that this porosity and discontinuous metal foil make even a 397 Å thick foil unstable. Typical pore dimensions are 200 nm in length × 10 nm in width, and 30 - 40 / μm 2 and are.

[0088] In addition, the gold foils fabricated by the sputtering method of Janbroers et al. 2009 suffer from this porosity in addition to not being made of pure gold. In FIGS. 1C and 5, the pores are distinct. This is in contrast to the foils of the present invention that do not have such pores.

[0089] The data provided herein relates to gold, but similar improvements are expected to be seen in materials having similar structural and electrical properties, such as degenerately doped silicon having a second element selected from boron, aluminum, phosphorus, and arsenic at a concentration of 10 20 atoms / cm 3 or greater, of transition metals, aluminum, beryllium, and the like.

[0090] (Reference) 1.Ermantraut, E., Wohlfart, K. & Tichelaar, W. Perforated support foils with pre-defined hole size, shape and arrangement. Ultramicroscopy 74, 75-81 (1998). 2.Janbroers, S., de Kruijff, T. R., Xu, Q., Kooyman, P. J. & Zandbergen, H. W. Preparation of carbon-free TEM microgrids by metal sputtering. Ultramicroscopy 109, 1105-1109 (2009). 3.Russo, C. J. & Passmore, L. A. Ultrastable gold substrates for electron cryomicroscopy. Science 346, 1377-1380 (2014). 4. Russo, C. J. & Passmore, L. A. Ultrastable gold substrates: Properties of a support for high-resolution electron cryomicroscopy of biological specimens. Journal of Structural Biology 193, 33-44 (2016). 5. Grant-Jacob, J. A. et al. Design and fabrication of a 3D-structured gold film with nanopores for local electric field enhancement in the pore. Nanotechnology 27, 65302 (2015). 6. Jia, P. et al. Large-area freestanding gold nanomembranes with nanoholes. Materials Horizons 6, 1005-1012 (2019). 7. Naydenova, K., Peet, M. J. & Russo, C. J. Multifunctional graphene supports for electron cryomicroscopy. Proceedings of the National Academy of Sciences 201904766 (2019) doi:10.1073 / pnas.1904766116.

Claims

1. A support for an electron microscope sample, wherein the support includes a metal foil having one or more through holes, the thickness of the metal foil is less than 50 nm and / or its average linear section particle size is 50 nm or less, and the ratio of the diameter of each hole to the thickness of the metal foil is 15:1 or less, and the metal foil is made of one or more metals selected from transition metals, aluminum, and beryllium, or an alloy thereof.

2. The support according to claim 1, wherein the thickness of the metal foil is less than 50 nm and the average linear section particle size is 50 nm or less.

3. The support according to claim 1 or 2, wherein the edge roughness of each hole is 20 nm or less as measured by the root mean square deviation from the expected theoretical hole edge profile.

4. The support according to any one of claims 1 to 3, wherein the diameter of each hole is 750 nm or less.

5. The support according to any one of claims 1 to 4, wherein the support has a wavelength at which the light transmittance is maximum in the range of 650 to 800 nm.

6. The support according to any one of claims 1 to 5, wherein the holes are arranged in a hexagonal array or a square pattern array.

7. The support according to any one of claims 1 to 6, wherein the metal foil is suspended across the holes of an electron microscope grid.

8. The support according to claim 7, wherein the metal foil and the grid are integrally formed.

9. The support according to any one of claims 7 and 8, wherein the grid includes a mesh having an average hole size on the micrometer scale, and the holes of the mesh are mosaic hexagonal or mosaic square.

10. The metal foil consists of one or more of gold, palladium, and platinum, or an alloy thereof, and optionally, the metal foil consists of gold or an alloy thereof, the support according to any one of claims 1 to 9.

11. The support consists of one or more of gold, palladium, and platinum, or an alloy thereof, and optionally, the support consists of gold or an alloy thereof, the support according to any one of claims 1 to 10.

12. Use of the support according to any one of claims 1 to 11 in a transmission electron cryomicroscope.

13. A method for manufacturing a metal foil for a support according to any one of claims 1 to 11, the method comprising: depositing a metal layer on a patterned substrate cooled to 200 K or less to form a layer having a thickness of 50 nm or less and having one or more through holes; removing the deposited metal layer; and forming the metal layer in a support for an electron microscope sample, wherein the metal foil consists of one or more metals selected from transition metals, aluminum, and beryllium, or an alloy thereof.

14. A metal foil for a support formed by the method according to claim 13.

15. A method for electron microscope imaging, the method comprising continuously imaging a sample suspended in a hole of a support according to any one of claims 1 to 11, each image including at least a part of an edge of the hole, and an electron beam thereof including the hole and the complete edge of the hole.

16. Comparing at least a part of the edge of the hole in each image with other images to remove any relative shift between consecutive images, and / or weighting consecutive images of the sample in the hole in consideration of damage to the sample, the method for electron microscope imaging according to claim 15.

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