Sample support for cryo-electron microscopy

Abstract

Sample support foils and grids for cryo-electron microscopy (cryo-EM) systems, methods for making / using the disclosed cryo-EM foils, grids, devices, and / or systems, and cryo-EM systems equipped with the disclosed foils, grids, and / or devices are presented. The sample support grid assembly may include a grid body having opposing first and second grid surfaces and a plurality of grid holes extending through the grid body from the first grid surface to the second grid surface. A foil sheet is seated against the first surface and covers at least a subset of the grid holes in the grid body. The foil sheet includes a plurality of foil regions filled with the foil holes and separated from one another by a plurality of elongated slots. These elongated slots allow the foil regions to move relative to one another when the grid assembly is under load. The slots may be arranged in an auxetic pattern.

Description

Technical Field

[0001] Priority Claim and Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 197,077, filed on June 4, 2021.

[0002] Approval of Government Support This research is supported by the National Institutes of Health, General Medical Sciences, under Award Number R43 - GM137720 - 01.

[0003] The present disclosure generally relates to the field of biotechnology. More specifically, aspects of the present disclosure relate to sample supports and sample cooling systems for cryo - electron microscopy.

Background Art

[0004] Single - particle cryo - electron microscopy (cryo - EM) is a powerful approach for obtaining structures approaching atomic resolution of large biomolecular complexes, membrane proteins, and other targets important in science, pharmacy, and biotechnology. Dramatic increases in achievable resolution and throughput have been achieved through the development of high - efficiency, high - frame - rate direct electron detectors, algorithms for correcting “movies” acquired against electron - beam - induced motion, and computational tools for classifying and averaging 10 5 ~10 6 molecular images. Substantial investment in new cryo - EM facilities and the development of user - friendly software have greatly expanded access, especially to non - specialists. Unlike X - ray crystallography, cryo - EM requires only small amounts of biomolecular samples dispersed in solution. It enables the structural study of systems that are difficult to handle for crystallization and is emerging as a forward method for initial attempts in structure determination.

[0005] Similar to the case of X-ray cryo-crystallography, an important issue in single-particle cryo-EM is related to the preparation and handling of samples. The basic principles and methods currently in use were developed in the 1980s, and recent developments in sample preparation techniques are firmly rooted in the ideas and methods developed at that time. Historically, biomolecular samples are expressed, isolated, and purified. A cryoprotectant-free buffer containing the biomolecule of interest at about 0.3 mg / mL is applied to a glow-discharged, clean, and charged carbon or metal (often gold) film 10 - 50 nm thick on a 200 - 400 mesh, 10 - 25 mesh supported "foil", a metal (usually copper or gold) grid 3 mm in diameter and 0.1 - 0.25 mm thick. Excess sample is removed by blotting and evaporation targeting a thickness of several times that of the biomolecule, i.e., about 10 - 50 nm, to maximize image quality while limiting the proportion of biomolecules preferentially oriented by interaction with the interface. To vitrify the buffer for optimal imaging, the foil + grid containing the sample is plunged into liquid ethane (generated by cooling the gas in a liquid nitrogen-cooled cup) at about 90 K at 1 - 2 m / sec. The sample is transferred from ethane to liquid nitrogen (LN2), placed in a grid box, transferred to an additional container, and then to a storage dewar. The sample is removed from the storage dewar flask and grid box and then loaded onto the cryo-microscope stage or "clipped" and loaded into a cryo-sample cassette, and then the stage or cassette is loaded into the microscope.

[0006] These complex procedures are challenging. Grids, especially foils, are routinely bent, torn, and otherwise damaged in each step of the many manual operations. Sample dispensing, blotting, and evaporation are inaccurate. The final film thickness of the sample is poorly controlled. Biomolecules accumulate at interfaces that may have a preferential orientation or undergo denaturation. Plunge-cooled samples often generate significant crystalline ice and are contaminated by ice formed on other cryogenic surfaces exposed to ethane, nitrogen, and moisture. When first irradiated by the electron beam during imaging, the vitrified sample undergoes rapid movement that blurs the image and causes a large loss of information.

SUMMARY OF THE INVENTION

[0007] Aspects of the present disclosure relate to systems, methods, and apparatuses that use a sample support and a sample cooling device for cryo-electron microscopy. Described herein are, for example, innovations in sample support design and sample cooling devices. These innovations should help reduce electron beam-induced sample motion, which is a significant factor limiting the achievable resolution in cryo-EM, and increase the achievable sample cooling rate. The disclosed features also help facilitate more controlled sample preparation and cooling, as well as automatic storage of the grid after plunge cooling within the grid box.

[0008] At least some of the disclosed sample supports for cryo-EM consist of a metal grid having a diameter of about 2.5 to about 3.5 mm, or in some configurations about 3.05 mm, and a thickness of about 10 micrometers (μm) to about 25 μm, and can be covered on the top surface by a significantly thin (e.g., about 10 to about 50 nanometers) sample support film / foil of carbon or metal. In at least some configurations, the grid may have a mesh pattern with about 200 to 400 mesh corresponding to a pitch / repeat period of about 50 to 150 μm, or in at least some implementations about 62 to 125 μm, and through holes with a diameter / width of about 20 to 130 μm, or in at least some implementations about 37 to 106 μm. The grid may also have a solid outer edge region without openings. The foil may typically have a pattern of much smaller through holes of about 0.2 to about 2 micrometers. Conventional designs are handled with metal tweezers having sharp tips and are often damaged during handling.

[0009] Described herein are a series of innovations for grids and foils that help improve functionality and useful throughput.

[0010] According to an aspect of the disclosed concept, the grid under the sample support film provides an area that can safely grip the grid on both sides using tweezers, rather than at a single point, and may have a substantial area that together includes at least 10% and less than 50% of the grid area on two diametrically opposed sides that are solid or substantially solid.

[0011] According to an aspect of the disclosed concept, the sample support film or foil may be sized and shaped so as not to substantially overlap the solid gripping portions of the grid configuration having solid gripping portions on both sides, whereby the grid can be gripped on the solid area without contacting the foil or damaging the foil.

[0012] According to an aspect of the disclosed concept, the grid may have one or more internal regions that are mechanically isolated from the surrounding grid region, for example, to reduce stress and deformation in the internal region due to the difference in shrinkage between the internal region and the surrounding region during cooling, and these differences in shrinkage may result from differences in temperature during cooling.

[0013] According to an aspect of the disclosed concept, in at least one internal region of the grid, the width and / or thickness of the grid bars is reduced and / or the grid opening area ratio is increased, for example, to reduce the total thermal mass per unit area of the grid and increase its cooling rate.

[0014] According to an aspect of the disclosed concept, at least one internal region of the grid may be connected to the surrounding region by bars that contact the periphery of the internal region and are connected to the surrounding region only at their ends, whereby, for example, when the internal region and the surrounding region are at different temperatures and shrink by different amounts, these bars deflect laterally at their contact points with the internal region.

[0015] According to an aspect of the disclosed concept, the internal region of the grid may be connected to each tangent bar via a small tab that extends perpendicular to the axis of the bar.

[0016] According to aspects of the disclosed concept, the interior region of the grid may be connected to the surrounding region via a zigzag or wavy spring-shaped metal member that deforms with respect to the interior and surrounding regions, for example, when the grid is placed under stress.

[0017] Aspects of the present disclosure further relate to foils present on the grid and improvements to those foils, for example, to reduce beam-induced sample motion.

[0018] According to aspects of the disclosed concept, the foil may be placed on the grid and may have one or more interior regions that are mechanically isolated from the surrounding grid region to reduce stress and deformation in the interior region when the outer region is under stress, for example, as may occur when the grid bar to which the outer region is attached cools more slowly than the foil in the inner region.

[0019] According to aspects of the disclosed concept, one or more foil interior regions may be connected to the surrounding region by bars that contact the perimeter of the interior region and are connected to the surrounding region only at their ends, such that, for example, when the interior and surrounding regions are at different temperatures and shrink by different amounts, these bars deflect laterally at their points of contact with the interior region.

[0020] According to aspects of the disclosed concept, the interior region may be connected to each tangent bar via a small tab that extends perpendicular to the axis of the bar.

[0021] According to aspects of the disclosed concept, the interior region of the foil is connected to the surrounding region via a zigzag or corrugated spring-shaped metal tab that preferentially deforms with respect to the interior and surrounding regions when the grid is placed under stress.

[0022] According to an aspect of the disclosed concept, the foil placed on the grid may have an internal region having a pattern of slits or openings that, for example, allow for the release of stress when the foil is placed under tensile stress. As a result, some regions of the foil within these internal regions remain substantially stress-free, while the stress concentrates in other regions.

[0023] According to an aspect of the disclosed concept, the slits on the foil may be arranged in a pattern that defines rectangular regions, and these slits may open when the foil is placed under tensile stress and may be designed to reduce the stress in the internal regions bounded by the slits.

[0024] According to an aspect of the disclosed concept, the slits on the foil may be arranged in a pattern that defines rectangular regions. When the foil is placed under tensile stress, the slits open, and the pattern of their openings is such that the rectangular regions rotate while maintaining a substantially stress-free state, with stress concentration occurring in the "hinge" regions between the slits.

[0025] According to an aspect of the disclosed concept, the slits in the foil may be arranged to define other aesthetic patterns including rotated parallelograms, rotated squares of different sizes, and rotated triangles, each of which can expand when placed under tensile stress in such a way as to involve rotation of substantially stress-free elements and stress concentration in the links between the elements.

[0026] According to an aspect of the disclosed concept, the regions of the foil having stress-relieving features may form a checkerboard pattern across the regions of the grid, whereby some regions of the foil have a standard hole pattern and retain full mechanical strength, while other regions have stress-relieving features, thereby making the foil more robust during handling and grid assembly.

[0027] According to an aspect of the disclosed concept, the foil covering the grid may have regions having at least two different thicknesses, and one or more of these regions may have an array of through-holes.

[0028] According to aspects of the disclosed concept, these regions of two different thicknesses can be produced by depositing metal onto a master, then spraying particles of a narrow size range thereon, then depositing again, and then removing the particles. The particles are much larger than the hole spacing but smaller than several grid squares.

[0029] Aspects of the present disclosure further disclose the design of tools, such as tweezers, for holding a cryo-EM grid within a plunger cooling device.

[0030] According to aspects of the disclosed concept, the tool may include or consist essentially of a wand having a push-button actuator at one end and a pair of grid-gripping jaws actuated by a push button at the other end. According to aspects of the disclosed concept, the jaws may have protrusions that squeeze together to grip opposite edges of the grid while leaving most of the grid accessible for sample deposition and uptake.

[0031] According to aspects of the disclosed concept, the jaws of the tool can facilitate uptake by leaving large free regions above and below the grid that are not obstructed by the grippers.

[0032] According to aspects of the disclosed concept, the jaws may be made of a low thermal conductivity material such as stainless steel or a polymer such as Teflon®. In one example, the jaws are made as thin and narrow as possible to minimize the total thermal mass in contact with the grid. According to aspects of the disclosed concept, the jaws may have a textured surface to minimize the area of direct contact with the grid and improve grid gripping.

[0033] Aspects of the present disclosure also present systems, devices, and means for automatically capturing and storing cryo-EM grids after plunge cooling within a grid box. In one example, a motor-driven mechanical stage within a main liquid nitrogen chamber receives standard cryo-EM sample holder storage boxes / cassettes and automatically positions them to align with a sample plunge path defined by a vertical translation stage such that, for example, each cryo-sample can be deposited within a separate compartment within each holder by a combination of only vertical movement of the vertical translation stage and only linear or circular movement in the horizontal direction of the mechanical stage on which the sample holder storage box is placed.

[0034] According to aspects of the disclosed concepts, a representative system, device, or means can comprise a vertical linear sample translation stage that can push a sample into liquid nitrogen at a speed of 1 - 10 m / s and then translate it downward along the same axis. A grid gripping mechanism is attached to this stage, capable of gripping a cryo-EM grid, holding its plane precisely perpendicular to the surface of the liquid nitrogen, and releasing the grid after cooling. Further, a dewar or insulated container is used to contain a cryogenic fluid such as liquid nitrogen. A stage having one degree of freedom, which can be either translational or rotational, operates within the liquid cryogen within the insulated container. A platform is attached to this stage and can hold one or more grid boxes in a clearly defined orientation. A motor and control system automatically translate / rotate the stage to align successive grid slots within each grid box with the axis of the vertical translation stage such that the grid can be released therein.

[0035] According to aspects of the disclosed concepts, a funnel device is positioned at the end of the plunge path of the vertical translation stage within the liquid nitrogen and just above the top of the grid box platform to guide the grid into the grid box.

[0036] According to aspects of the disclosed concept, the gripping mechanism can automatically release the cryo-EM grid into the storage container after the grid has been plunge cooled.

[0037] The above summary does not represent all embodiments or all aspects of the present disclosure. Rather, the foregoing summary merely provides some examples of the novel concepts and features described herein. The above features and advantages of the present disclosure, as well as other features and attendant advantages, will become readily apparent from the following detailed description of the exemplary embodiments and representative forms for practicing the present disclosure when considered in connection with the accompanying drawings and the appended claims. Further, the present disclosure explicitly includes any and all combinations and sub-combinations of the elements and features presented above and below.

Brief Description of the Drawings

[0038]

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[0039] The present disclosure is suitable for various modified and alternative forms, and some representative embodiments are shown by way of example in the drawings and described in detail herein. However, it should be understood that the novel aspects of the present disclosure are not limited to the specific forms shown in the drawings listed above. Rather, the present disclosure encompasses all modifications, equivalents, combinations, sub-combinations, substitutions, groupings, and alternatives that fall within the scope of the present disclosure, as for example encompassed by the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present disclosure can accept embodiments in many different forms. Representative embodiments of the present disclosure are shown in the drawings and are described in detail herein with the understanding that these embodiments are not limitations of the broad aspects of the present disclosure, but are provided as examples of the disclosed principles. In that regard, elements and limitations described, for example, in the summary, introduction, abstract, and detailed description sections, but not explicitly recited in the claims, should not be incorporated into the claims, either individually or collectively, by implication, inference, or otherwise.

[0041] For purposes of the detailed description of the present invention, unless otherwise specified, the singular includes the plural and vice versa, the words "and" and "or" are both conjunctive and disjunctive, the words "any" and "all" mean "any and all", and the words "comprising", "containing", "including", "having", etc. each mean "including but not limited to". Further, approximating words such as "about", "substantially", "generally", "approximately", etc. can each be used herein in the sense of, for example, "at, near, approximately", or "within 0-5% of", or "within acceptable manufacturing tolerances", or any logical combination thereof.

[0042] At least some of the goals of the disclosed concepts include, by way of some non-limiting examples, developing sample supports, sample handling tools, and tools including cooling equipment that enable reliable vitrification of cryo-EM samples, reduce the damage rate of grids and foils, and facilitate automated sample tracking.

[0043] Scientific motivation Current generation cryo-EM sample supports and cooling devices are mostly based on research and principles developed in the 1980s. Some of the important problems addressed by this disclosure are described below.

[0044] The method of holding the grid during plunge cooling is inadequate. The grid is typically held using tweezers that grip the upper center of the grid and is never supported from the back. During liquid adhesion and uptake, the grid can easily deflect, making accurate uptake more difficult and potentially causing it to bend permanently. When the grid plunges into the liquid cryogen at high speed, a slight deviation from vertical can cause the grid to bend dramatically as it enters and moves through the liquid cryogen. This can negatively affect grid cooling and result in damage to the foil and grid.

[0045] Beam-induced motion is an important factor limiting the achievable resolution in single-particle cryo-EM, most likely arising from the large temperature difference between the grid bar and the sample + foil during cooling. The motion of the sample during irradiation is the main factor limiting the resolution in single-particle cryo-EM. The motion occurs even during low-dose irradiation, is most rapid (dose-based) at the start of irradiation where radiation damage is minimal and highest-resolution structural information is available. If a sufficient signal-to-noise ratio is available, the “movie” of the sample can be analyzed to correct for this motion and improve the final resolution. Bare sample foils undergo substantial beam-induced motion (BIM) due to stress and buckling associated with the differential shrinkage during cooling of the foil and grid materials, and this motion can be minimized by using the same material (e.g., Au) for both. For biomolecule-containing samples within the holes of the foil, particle-tracking experiments suggest that the primary mode of motion corresponds to “doming” (like a drumhead) of the sample film. For holes with a diameter of 1.2 mm, the radiation-induced displacement of the sample perpendicular to the plane of the foil was about 150 Å and the radius of curvature was 25 μm. This doming motion indicates that the sample within the hole is under substantial compressive stress.

[0046] The observed attenuation of sample motion with increasing electron dose indicates that the motion mainly arises from radiation-induced creep driven by sample stress, and as the creep progresses, the driving stress is relieved and thus the creep rate decreases. In recent experiments, it has been found that BIM decreases by a factor of four when the sample is briefly warmed to about 150–170 K, which is higher than the devitrification temperature that allows diffusive water motion to relieve the sample stress. However, where does the initial sample stress come from?

[0047] The sample stress can be generated by the difference in shrinkage between the sample being cooled and the foil support. Between room temperature and the glass transition temperature of water, Tg ~ 136 K, the sample undergoes a net volume expansion of ~8%, but since the sample remains liquid (assuming no crystallization), this expansion is essentially decoupled from the shrinkage of the support foil (in contrast to the claims of reference 37). Below Tg, the vitrified water has a positive coefficient of thermal expansion (comparable to that of hexagonal ice), but the shrinkage of the sample is strongly coupled to the shrinkage of the support foil. The sample stress induced by cooling can be reduced by matching the expansion coefficients of the vitrified ice and the foil between Tg and the final storage temperature of 77 K. Amorphous carbon, Cu, and Au foils all shrink less than vitrified ice in this temperature range (Au is the best fit). All are likely to generate tensile stress in the sample. However, radiation-induced creep in the presence of tensile sample stress does not cause the observed doming.

[0048] The main cause of the stress driving the radiation-induced sample motion has been shown to be related to the substantial transient temperature difference between the grid bars and the sample + foil that occurs during cooling. The foil is much thinner and mechanically less rigid than the grid bars, so the contraction of its length (and area) is mainly determined by the contraction of the grid bars. The grid bars are cooled and thus contract more slowly than the foil between them, so the foil generates a transient tension (even if it is of the same material as the grid bars). The sample then vitrifies (and adheres firmly to this foil) on the tensioned foil. As the grid bars are cooled towards the final temperature of the foil + sample, their separation decreases and the tension in the foil is released. However, this then places the sample under compressive stress here. In the holes in the foil where the sample is not constrained by the foil in its relaxed state, irradiation-induced creep generates a "doming" to relieve this stress. Calculations have shown that the observed "doming" of height ~150 Å in 1.2 mm foil holes can be generated by a temperature difference between the grid bars and the foil of about 20 K during sample vitrification. Recent experiments have confirmed the prediction that BIM scales linearly with the diameter of the foil holes. Molecular diffusion and devitrification that occur when warming to 150 - 170 K relieve this stress, and even when recooled to 77 K, most of the sample remains solid and the stress does not recover because the magnitude of the transient temperature and thermal contraction differences between the foil and the grid are much smaller.

[0049] Two other causes of sample stress related to the grid contribute to the non-doming components of the beam-induced sample motion. First, damaged or improperly "pinched" grid / foil can undergo significant bending when placed in ethane, and if the bending radius is small enough, it generates stress in the vitrified sample film. Second, about 10 5 K / s to 10 6At a cooling rate of K / s and an entry velocity of 2 m / s, the cooling of the grid and the foil occurs in zones approximately 3 or 0.3 mm along the entry direction when they enter the liquid cryogen. Within this zone, the grid and the foil have a large transient temperature gradient, which creates a transient gradient across the grid spacing and transient stresses in the foil. The relaxation of these transient grid deformations after the sample has vitrified creates a directional stress in the foil + sample

[0050] Description of the example shown FIG. 1 shows a cryo-EM grid (10) having two solid gripping regions (20) without openings on its opposing left and right sides for gripping, for example, with parallel "jaws". The grid gripped in this way is rigid, for example, during sample deposition and uptake and does not bend or flex during plunge cooling. The gripping regions can have a width of about 200 to about 600 micrometers in the radial direction and, in at least some mounting configurations, about 400 micrometers. Generally speaking, the gripping regions may be wide enough for sufficient gripping, but not so wide as to interfere with access to the rest of the grid and overly reduce the cooling of the rest of the grid. The exemplary design of FIG. 1 also has a third solid gripping region (30) along its side connecting the left and right sides of the grid (1). This third gripping region (30) may be used for gripping using the jaws of a plunge cooler. The marked pattern (40) can uniquely identify each grid, and a thin graphene or graphite foil (50) can cover one or more of the marked patterns for microscope calibration.

[0051] Figure 2 shows several grid designs that help mechanically and thermally isolate the inner region of the grid (e.g., the radially centered region of a circle or square) from the surrounding region (e.g., the annular radially outer region). The inner region may be thinner than the surrounding region, have grid bars that are narrower than those of the surrounding region, a different grid bar spacing from the surrounding region, etc. The designs of FIGS. 2A, 2B, and 2C are shown with "tangent beam" bars that extend in contact with the inner region and connect to the outer region at their ends. For example, if the inner region is cooler than the surrounding region, the inner region is more likely to contract than the surrounding region. The "tangent beam" has limited rigidity against bending perpendicular to its axis and thus allows this contraction to occur while reducing the stress that would otherwise occur in the inner region. If the inner region cools faster than the surrounding region during plunger cooling, this reduces the transient stress generated and reduces the associated beam-induced motion.

[0052] In FIG. 2(A), the bar / beam (60) is a linear structure that contacts and directly adheres to the outer periphery of the circular inner region (70). The bar (60) of FIG. 2(A) may extend continuously around the inner region (70) to form an integral structure surrounding the inner region (70), forming a hexagonal structure as shown. In FIG. 2(B), the beam (80) similarly connects tangentially to the rectangular inner region (90) and can surround it. These beams (80) may be bent in a wavy shape, for example, like a leaf spring, and may be attached to the inner region (90) such that when the beam (80) bends, the inner region (90) can move laterally and axially with respect to the diameter center of the grid (10).

[0053] In FIG. 2(C), the beam (100) is in contact with, but spaced from, the outer periphery of the circular inner region and is attached to the inner region via short tabs (110) extending radially between the inner and outer regions. These tabs (110) can serve to reduce the contact area with the inner region. The width and / or length of the tabs (110) may be adjusted, for example, to ensure that the tabs do not break under the stress applied to the tabs during cooling. In FIG. 2(D), the inner region is isolated from the outer region using a plurality (four) of separate beams (120), each of which may be shaped like a compression - extension type zigzag spring member. Each beam (120) may be circumferentially spaced from its adjacent beam, extend radially between the inner and outer regions, and join the innermost peripheral edge of the outer region to the outermost peripheral edge of the inner region. In at least some configurations, all components of the grid are made of the same material using a method such as electroforming. Each grid may be formed as a single monolithic structure. Alternatively, the grid may be formed as a multi - piece (e.g., two - piece or three - piece) structure in which the components are physically joined.

[0054] Other available options include that the configuration of the through - holes on the inner region (70) of the grid may be different from (e.g., in shape, size, arrangement, etc.) or substantially the same as the configuration of the through - holes on the outer region. For example, in FIG. 2(A), both the inner and outer regions include square through - holes arranged in a square array of rows and columns. However, the through - holes in the inner region (70) are two to three times the size (e.g., area, width, diameter, etc.) of the through - holes in the outer region. For this purpose, one or both regions may include through - holes having other shapes, as shown by the circular holes in FIG. 4, and may be arranged in similar or different patterns such as a grid structure, a hexagonal structure, or a staggered distribution. The inner and outer perimeters of the outer region may be defined, for example, by rigid frames without holes, as seen in the outer perimeter (13) of FIG. 2(A). Comparing FIG. 2(A) and FIG. 2(B), it can be seen that only the outer perimeter (and / or the outer perimeter of the inner region) of the outer region can be surrounded by a rigid frame without holes.

[0055] For example, the same features and options shown in FIG. 2 for mechanically separating the inner region of a grid from its outer region may be used to separate the inner portion of a perforated foil from the outer portion of the foil. For example, FIG. 3 shows a small portion of a "perforated" foil (200) that may have a square or other polygonal planar profile. The inner portion (220) of the foil (200) is isolated from the outer portion via a plurality (three) of tangential beams (230) and a plurality (three) of radial tabs (240). It may be desirable for the total size of the inner portion (220) and the accompanying support beams (230) to be smaller than the total size of the openings in the underlying grid (e.g., approximately 60 micrometers in a 300 mesh grid with a 85 micrometer repeat and 25 micrometer grid bars). During cooling, the foil near the center of the grid bar openings cools much faster than the grid bars, such that, since the foil is firmly attached to the grid bars, tension is applied to the foil between the grid bars. The structure shown in FIG. 3 may serve to reduce the resulting tension in the inner region. When the grid finally reaches the final temperature of the foil and the liquid cryogen, the tensile stress in the foil is released, causing beam-induced motion except in the inner region where the tensile stress is minimized.

[0056] FIG. 4 shows two representative configurations that can be used to reduce transient tensile stress in the foil between grid bars, for example. In FIG. 4(A), for example, a square array of elongated slits (250) is disposed in the foil (200) in rows parallel to each other and columns perpendicular to the rows parallel to each other. A single through-hole may be located at each node junction between four intersecting slits (250). When the foil (200) is placed under tensile stress, these slits (250) expand, thereby reducing the stress in the foil (200). In FIG. 4(B), the pattern of slits (260) takes an azeotropic configuration and can be adjusted to more effectively reduce the tensile stress in the regions bounded by the slits. These slits (260) are also arranged in rows and columns, but unlike FIG. 4(A), in FIG. 4(B), the slits (260) can be arranged alternately such that the opposing ends of each slit (260) face and terminate at spaced-apart relationships in the most central section of the adjacent slit (260).

[0057] Figure 5(A) shows a portion of a representative foil (270) disposed within a single grid opening defined by a grid bar (280). The pattern of slits (290) as in Figure 4(B) may each be continuous slits (such as in Figures 4(A) and 4(B)), or may be a series of linearly aligned mini-slits that act as fragile "weak" links connecting the structural sides of the slits (as shown), and are arranged in an aesthetic pattern. Figure 5(B) is an inset showing an enlarged view of a selected section of the foil (210) to better view the through-holes of the foil (270) used for sample imaging. Figure 5(C) shows how the foil (270) of Figure 5(A) having the slit pattern of Figure 4(A) can respond when it is at a lower temperature than the surrounding grid bars and is thus under a tensile stress that acts to stretch the foil. The slit (290) opens and the rectangular region (300) defined by the slit rotates, such that the rectangular region (300) remains substantially stress-free and the stress concentrates in the "hinge" region (310). The total area filled by the (expanded and rotated) foil within each grid opening can remain equal to the area of the grid opening even if the actual solid area of the foil decreases due to thermal contraction. During cooling, this allows the sample to be vitrified on the foil (270) maintaining a stress-free state within the rectangular region (300), even if there can be a large temperature difference between the grid and the foil that creates a stress acting to stretch the foil within each grid opening. The actual transient strain can be <0.1% or <0.1 mm. Only a small "opening" within the grid opening and rotation that does not affect step-and-repeat imaging in TEM may be required. These patterns can be limited to the TEM-accessible regions of the foil to maintain the robustness of the foil during grid assembly. Alternatively, the pattern can be limited to a smaller region encompassing a few to several grid squares that may be arranged in a "checkerboard" pattern with foil regions without slits to maintain the robustness of the foil, for example, during lift-off and assembly onto the grid.Smaller regions may be comparable to or somewhat smaller than a single grid square (85 micrometers for a 300 mesh grid), or they may encompass several grid squares.

[0058] Figure 6 is another rendering of another exemplary foil design having a slit (290) arrangement similar to that of FIGS. 4(B) and 5, showing how the foil can respond to tensile stress via the opening of the slit (290) and the rotation of the rectangular region (300).

[0059] According to the aesthetical theory (see, e.g., Grima and Evans, J. Mat. Sci. Lett. 19, 1563 - 1564 (2000)), it is envisioned that a number of other slit patterns can be implemented to relieve stress in the planar film by allowing rotation of elements with near - zero stress and stress concentration in small connecting regions. Any of these can be used for stress relief in cryo - EM foils (or cryo - EM grids having regions of different thickness, grid bar width, and / or grid bar density). These include slit patterns that define parallelograms, two different - sized squares, triangles, etc.

[0060] Figure 7 shows an alternative foil design in which at least a portion of the foil has a triangular pattern of slits (320). In this case, the triangular region (330) defined by the slits (320) rotates when tensile stress is applied to the foil, maintaining a near - stress - free state, and the stress re - concentrates in the hinge region (340) that interconnects the adjacent regions (330).

[0061] Figure 8 shows an example of how the slit / stress relaxation portion of the foil (350) (indicated by the dashed square) can be laid out across the area of the foil (360) used in a standard 3.05 mm cryo-EM grid (370). Figure 8 is a view from the grid side of the grid-foil assembly. The presence of the slit weakens the foil and can make it more prone to tearing during lift-off from the master, floating onto the grid, and plunge cooling. Since only a small area of the grid / foil is needed to obtain a complete single-particle cryo-EM dataset, the stress relaxation pattern is limited to a small area encompassing a few grid squares and can be surrounded, for example, by a "slit-free" foil (i.e., a foil structure without slits, through-holes, voids, etc.) to increase the overall robustness of the foil.

[0062] Variable-thickness foils: The thickness of the foil, along with the size of the holes in the foil, is an important geometric parameter of the grid + foil in determining the thickness of the final sample film and thus whether imaging is achievable. Ideally, absorption and evaporation remove almost all of the liquid except that within the pores. Here, the feasibility and performance of foils with variable thickness across the TEM-accessible grid area are investigated. In at least some implementations, the simplest approach is to evaporate a base foil layer and then use a mechanical mask (e.g., made from a Ni or polyimide sheet) to cover portions of the foil that are small enough so as not to compromise the mechanical properties of the overall foil during a second evaporation step.

[0063] The foil covering the grid can be manufactured to have regions with two or more different thicknesses, one or more of these regions having an array of through-holes. The thickness of the holes can determine the thickness of the ice when the sample is properly blotted. Having two thicknesses on the same foil can help increase the chance of obtaining an optimal sample thickness for imaging. The outer perimeter of the foil can be made substantially thicker than the rest to facilitate handling and placement of the foil on the grid. A foil having a thickness that increases stepwise as the radius increases can be manufactured by using a disk-shaped shadow mask with an increasing radius during the continuous deposition of metal or carbon. In this case, the innermost foil region is the thinnest and the outer perimeter is the thickest. The shadow mask can be manufactured using standard photolithography and etching processes from, for example, a 1-mil-thick Cu foil or a light-exposable polymer such as SU-8 or polyimide. The disks of the foil array may be connected by fine wires. The shadowing by these wires can be reduced by using different wire positions on the continuous mask or by placing the mask in near contact and depositing metal or carbon at an angle.

[0064] Tool for handling the grid: Figure 9 shows an example of a sample wand (400) having a push-button actuation (410) for gripping the grid (420) during plunge cooling and holding it on a vertical translation stage. Figure 9(A) shows a gripping jaw (430) that holds the grid at its edge. The angle between the two jaws is exaggerated in the figure and should be small, preferably less than about 15 degrees, to minimize the impact cross-section of the jaws when they enter the liquid cryogen. The surface of the jaw that contacts the grid can be textured to reduce the area of physical and thermal contact with the grid. The jaw can be made of any rigid material including stainless steel. Figures 9(B) and 9(C) show views of the sample wand. The jaw is opened using a push button via a rod that moves downward to push the jaws apart.

[0065] Automatic storage of the grid after plunger cooling. All plunger coolers have a vertical translation stage (driven or gravity drop) that translates the grid into the liquid cryogen. Figure 10 shows a part of the design of a plunger cooler that includes a system for automatically storing the cold grid after plunging. The grid is held by a wand as shown in Figure 9 attached to a vertical movement stage and is first moved rapidly into the liquid cryogen and then slowly downward to the grid release point. The grid is released by a mechanism that presses a push button on the wand and falls into the grid capture system. The grid capture system, which is present within a thermally insulated container of liquid nitrogen or other liquid cryogen (not shown), consists of a fixed "funnel" (500) that helps guide the grid into the storage slot, a storage platform (510) on which the grid is placed, and an electric stage (520) on which the platform is mounted and that rotates or translates the platform with a single degree of freedom to position the successive grid storage slots under the funnel. In Figure 10, the rotation of the stage is driven by a shaft 530 connected to a motor. Figure 10(A) shows the platform and stage in a position for capturing the grid, and Figure 10(B) shows the platform and stage rotated to allow removal and storage of the platform containing the grid. Figure 10(C) shows an enlarged view of the funnel, the platform with grid slots, and the electric stage.

[0066] Figure 11 shows an alternative platform (540) that accepts a cryo-EM grid box (550) in a standard format, and its receptacle (560) positions and orients the grid box to enable automated grid storage with a single degree of freedom stage. The form factor of the grid box is standardized in the industry, but the orientation of the grid slots (570) within the box varies among manufacturers and can be adapted as shown to facilitate loading with a single degree of freedom.

[0067] The stress-reducing foils described herein, including those shown in FIGS. 3-8, may each be manufactured as a single sheet of foil from gold (Au), chromium (Cr), and combinations and alloys thereof, and may each have a thickness of about 20 to about 60 nm. These foil sheets may be manufactured by vapor deposition on a master covered with a release layer. The foil is then floated off the master by dissolving the release layer and can then be floated onto a 200-300 mesh Cu or Au low temperature EM grid having square and hexagonal grid patterns. The foil has a hole size of about 0.3 to about 2.0 micrometers and has selected regions that constitute about 20% of the foil area covered with stress relaxation features.

[0068] A foil having the stress relaxation features of FIG. 3 in a given foil area may be made from Au or an Au / Cr alloy and may be placed on a 300 mesh Cu or Au grid. For example, FIG. 12 shows an optical micrograph of a foil (200) having a grid that employs the pattern of FIG. 3. The diameter of the separation region (220) was selected to ensure that a portion of the region is completely contained within a single grid opening that is about 55-65 micrometers wide. In FIG. 12(A), the foil (200) was placed on a 300 mesh square grid having "mushroom" profile grid bars created by filling and overfilling the master during electroforming. The foil (200) shows visible deformation around the underlying grid bars (280), which can make the position of the grid bars more visible. This is a characteristic deformation pattern of the foil on a commercially available grid.

[0069] Within each grid square, the foil appears to be unstrained. However, for grid openings where the foil (200) has the structure shown in FIG. 3, the tangent bars (230) connecting the isolation regions (220) to the surrounding foil can be deformed, indicating that the foil between the grid bars was / were under tensile stress. The deformation of the tangent bars can also indicate that the isolation regions (220) are at least partially detached from that stress. The amount of deformation of the tangent bars may vary across the foil, providing a direct optical readout of the stress distribution within the foil. This direct optical measurement of foil stress was used to optimize the process of placing the foil onto the grid. As shown in FIG. 12(B), this optimization enabled the production of the foil on the grid, where the foil was essentially stress-free.

[0070] Foils having an “aesthetic” stress relaxation feature in a given foil region, as shown in FIGS. 4B and 6, may also be made from Au or Au / Cr alloy and placed on a 300 mesh Cu or Au grid. For example, FIG. 13 shows an optical micrograph of a foil (200) having an “aesthetic” pattern as shown in FIGS. 4(B) and 6, consisting of slits (260) arranged at right angles on the grid. In this example, the area covered by the slit region may be larger than the size of a single grid opening, but the square region (300) defined by adjacent slits may be smaller than the grid opening. The slit width may be from about 2 to about 5 micrometers, and the slit length may be from about 20 to about 25 micrometers. The hinge region between the slits may have a length of from about 1 to about 5 micrometers, and the square region bounded by adjacent slits may have dimensions of from about 12 to about 25 micrometers. In this example, the width of the square region was selected to ensure that part of the region was completely contained within a single grid opening having a width of from about 55 to about 65 micrometers.

[0071] In FIG. 13(A), a foil having a region with a selected aesthetic pattern can be placed on a 300-mesh square grid having a “mushroom”-shaped grid bar. The foil can show a visible deformation around the underlying grid bar, which makes the position of the grid bar more clearly visible. This is a characteristic deformation pattern of the foil on a commercially available grid. The large residual stress in the foil is revealed by the slit openings and the rotation of the squares between the slits. By using a grid with a flat upper shape and by improving the process by which the foil is placed on the grid, an essentially stress-free foil can be obtained, as shown by the lack of square rotation and slit opening (FIG. 13(B)). During cooling, this aesthetic foil pattern should allow the sample to vitrify on a stress-free foil, even if the grid can cool and shrink much more slowly than the foil. Tiling the foil having an aesthetic region surrounded by a normal foil as in FIG. 8 preserves the robustness of the foil during release and placement.

[0072] Imaging of foil stress at room temperature and cryogenic temperatures: Foil stress and deformation are important measurement criteria that can affect particle imaging performance. Starting with a completely flat and stress-free foil, as in FIGS. 12(B) and 13(B), subsequent deformation of the foil upon cooling can be optically detected by the deviation from specular reflection. When the foil has the aesthetic pattern of FIG. 4(B), even more sensitive visualization of the stress state of the foil is possible. The relationship between the total strain (expansion of the foil) e = ΔL / L of the aesthetic / slit region and the square rotation angle q is q = 2 arccos(1 / (1 + e)). 10 -5 、10 -4 、10 -3 、and 10 -2Regarding the strain, the rotation angles are 0.5°, 1.6°, 5.1° and 16.1°. It is easy to detect a small rotation of about 0.5°. Whether the foil is initially under tensile stress (Figure 13(A)) or stress-free (Figure 13(B)) determines the range of compressive and tensile strains that can be observed. These strains may be related to the stress underlying the foil. Thus, the foil patterns of Figures 3(B) and 4 not only enable mechanical separation of the foil regions so that the foil regions remain substantially stress-free, but also provide a highly sensitive method for optically visualizing and quantifying the stress within the foil.

[0073] The features of the present disclosure have been described in detail with reference to the illustrated examples, but those skilled in the art will recognize that many modifications can be made thereto without departing from the scope of the present disclosure. The present disclosure is not limited to the exact configurations and compositions disclosed herein, and any and all modifications, changes, and variations apparent from the foregoing description are within the scope of the present disclosure as defined by the appended claims. Further, this concept explicitly includes any and all combinations and sub-combinations of the foregoing elements and features.

[0074] Additional features and options may be defined by the following clauses.

[0075] Clause 1: A metal cryo-EM grid for use in single-particle cryo-electron microscopy, having a diameter of about 3.05 mm and a thickness of about 10 - 25 micrometers, the upper surface of which is covered by a foil having a thickness of about 10 - 50 nm, made of one form of metal or carbon, and may have an array of through-holes sized about 0.2 - 2 micrometers, the metal cryo-EM grid.

[0076] Clause 2: One or more internal regions that are mechanically enclosed and spaced apart from the surrounding region, and that reduce the stress in the foil in these internal regions below the stress that could occur due to the difference in shrinkage of the foil and the grid during the assembly / manufacture of the foil + grid, and more specifically due to the slower cooling of the grid compared to the foil during cooling to low temperature, structured to have one or more internal regions, the foil according to clause 1.

[0077] Clause 3: One or more of the internal regions of the foil are connected to the surrounding region by bars / strips / beams that are in contact with the perimeter of the internal regions and are connected to the surrounding region only at their ends, such that these bars deflect laterally at the contact points with the internal regions when the foil is under tensile stress, the foil according to clause 1 or 2.

[0078] Clause 4: The internal regions are connected to each tangent bar via small tabs that extend perpendicular to the axis of the bar, the foil according to any one of clauses 1 to 3.

[0079] Clause 5: The internal regions of the foil defined by tangent bars / beams are smaller than the opening regions of the underlying metal grid, the foil according to any one of clauses 1 to 4.

[0080] Clause 6: The internal regions of the foil have width and length dimensions smaller than about 60 micrometers, the foil according to any one of clauses 1 to 5.

[0081] Clause 7: Each of the tangent beams has a length that is at least 10 times (e.g., 14 times) greater than its width, the foil according to any one of clauses 1 to 6.

[0082] Clause 8: The foil has internal regions with a pattern of slits or openings that allow for stress relief when the foil is placed under tensile stress, such that some regions of the foil within these internal regions remain substantially stress - free while the stress can be concentrated in other regions, the foil according to any one of clauses 1 to 7.

[0083] Clause 9: Each of the slits has a length that is at least 10 times (e.g., 17 times) greater than its width, and the foil described in any one of Clauses 1 to 8.

[0084] Clause 10: The foil regions surrounded by adjacent slits contain at least 16 holes for sample imaging, and the foil described in any one of Clauses 1 to 9.

[0085] Clause 11: The slits of the foil are arranged in a pattern that defines a rectangular region, and these slits open when the foil is placed under tensile stress, reducing the stress in the internal region bounded by the slits, and the foil described in any one of Clauses 1 to 10.

[0086] Clause 12: The slits of the foil are arranged such that their major axes are along one of two orthogonal axes in the plane of the foil, each end of each slit is closest to another slit only at its ends, and adjacent parallel slits are collinear, and the foil described in any one of Clauses 1 to 11.

[0087] Clause 13: The slits of the foil are arranged such that their major axes are along one of two orthogonal axes in the plane of the foil, the slits are oriented at right angles at their closest points, and a given slit is adjacent to two collinear and orthogonally oriented slits at its midpoint, such that when the foil is placed under tensile stress, the slits open and the rectangular region bounded by the slits rotates with substantially no stress, and the stress concentrates in the hinge regions connecting each rectangular region to the surrounding foil, and the foil described in any one of Clauses 1 to 12.

[0088] Clause 14: The pattern of the slits of the foil includes a slit pattern that defines rotating triangles and parallelograms, and defines other aesthetic patterns that allow the foil to expand when placed under tensile stress while keeping most of the foil in the patterned region stress - free, and the foil described in any one of Clauses 1 to 13.

[0089] Clause 15: The foil according to any one of Clauses 1 to 14, wherein the inner region of a square or rectangular foil defined by a slit is smaller than the opening region of the underlying metal grid.

[0090] Clause 16: The foil according to any one of Clauses 1 to 15, wherein the inner region of the foil has a width dimension and a length dimension each smaller than 60 micrometers.

[0091] Clause 17: The patterning of the stress relaxation features within the foil is limited to specific regions of the foil that form a proportion of less than 1 of the total area of the foil, whereby some regions of the foil retain full mechanical strength to make the foil more robust during handling, grid assembly, and use. The foil according to any one of Clauses 1 to 16.

[0092] Clause 18: A system or device for automatically capturing and storing a cryo-EM grid within a grid box after plunge cooling, comprising a container for containing a liquid cryogen, a grid translation stage for translating the grid into the liquid cryogen and then to a release point within the liquid cryogen, a grid gripping mechanism attached to this stage for gripping the grid and holding its plane perpendicular to the surface of the liquid cryogen and then releasing the grid at the release point within the liquid cryogen after cooling, a funnel device disposed within the liquid cryogen with its upper opening aligned perpendicular to the release point, and a grid box having a series of receptacles for storing the grid, wherein one of the receptacles is aligned perpendicular to the lower opening of the funnel device such that when the grid is pushed into the liquid cryogen, translated to the release point within the liquid cryogen, and released, the grid falls through the funnel under the influence of gravity and then enters a receptacle within the grid box.

[0093] Clause 19: The system / apparatus according to Clause 18, further comprising: a stage within the liquid coolant, the stage having at least one degree of freedom that enables movement in a plane perpendicular to the vertical line at the end of the plunger path; and a platform placed on the stage having a receptacle for the grid, wherein the platform can be translated or rotated so that each grid receptacle can be positioned directly below the bottom outlet of the funnel device.

[0094] Clause 20: The system / apparatus according to Clause 18 or 19, wherein the platform has a receptacle for holding the grid box, and the stage enables positioning each receptacle within each grid box directly below the bottom outlet of the funnel device.

[0095] Clause 21: A system or apparatus for optically measuring the stress in a thin film, comprising: a thin film; a pattern of slits in a portion of the thin film that enables the slit region to exhibit an optical behavior when the thin film is placed under tensile stress, the pattern of slits being arranged such that the major axis of the slit is along one of two orthogonal axes in the plane of the foil, the slits being oriented at right angles at the closest proximity points, and a given slit being adjacent at its midpoint to two collinear and orthogonally oriented slits; an optical microscope as other means for imaging the thin film; wherein the relative rotation angle of adjacent square regions bounded by the slits is determined by the stress in the surrounding thin film and can be used to directly estimate the stress.

[0096] Clause 22: A method for measuring the stress of a thin film, comprising: fabricating a thin film having an optical pattern of slits in a portion of the region; observing the film using a microscope; and measuring the relative rotation angle of the major axes of adjacent squares bounded by the slits.

[0097] Clause 23: A method or computer-readable medium for storing instructions for manufacturing and / or using any of the foils, grids, grid + foil assemblies, and / or cryo-EM systems described herein.

[0098] Clause 24: A cryo-EM system or cryo-EM device comprising any of the foils, grids, and / or grid + foil assemblies described herein.

[0099] Clause 25: A grid body having opposing first and second grid surfaces and a plurality of grid holes extending through the grid body from the first grid surface to the second grid surface, and a foil sheet seated on the first surface and covering a subset of the grid holes of the grid body, the foil sheet having a plurality of foil holes and including a plurality of foil regions separated by a plurality of elongated slots, the elongated slots being configured to allow the foil regions to expand and / or move relative to each other, a grid assembly for sample support for a cryo-electron microscope system.

[0100] Clause 26: The grid assembly according to clause 25, wherein the grid body has a diameter or width of from about 2.5 millimeters (mm) to about 3.5 mm.

[0101] Clause 27: The grid assembly according to clause 25, wherein the grid body has a thickness of from about 10 micrometers (μm) to about 25 μm.

[0102] Clause 28: The grid assembly according to clause 25, wherein the grid body is a substantially flat single-piece structure.

[0103] Clause 29: The grid assembly according to clause 25, wherein the grid body is formed from a rigid metallic material.

[0104] Clause 30: The grid assembly according to clause 25, wherein each of the grid holes in the grid body has a width or diameter of from about 20 micrometers (μm) to about 130 μm.

[0105] Clause 31: The foil sheet has a thickness of from about 10 nanometers (nm) to about 50 nm and is the grid assembly described in Clause 25.

[0106] Clause 32: The foil sheet has a substantially flat single-piece structure and is the grid assembly described in Clause 25.

[0107] Clause 33: The foil sheet is flexible and is formed from a metallic material or a carbon material and is the grid assembly described in Clause 25.

[0108] Clause 34: Each of the foil holes in the foil sheet has a width or diameter of from about 0.2 micrometers (μm) to about 2.0 μm and is the grid assembly described in Clause 25.

[0109] Clause 35: The foil region of the foil sheet includes an inner foil region and an outer foil region surrounding the inner foil region, and the elongated slots are interposed between the inner foil region and the outer foil region and separate the inner foil region from the outer foil region, and is the grid assembly described in Clause 25.

[0110] Clause 36: The foil sheet further includes a plurality of beams connecting the inner foil region to the outer foil region and is the grid assembly described in Clause 35.

[0111] Clause 37: The beams are in a tangential direction with respect to the outer periphery of the inner foil region and is the grid assembly described in Clause 36.

[0112] Clause 38: The inner foil region has a circular or polygonal planar profile and is the grid assembly described in Clause 37.

[0113] Clause 39: The beams are spaced apart from the inner foil region and are connected to the inner foil region by a plurality of radially elongated tabs and is the grid assembly described in Clause 37.

[0114] Clause 40: Each of the beams is joined to the outer foil region at both ends thereof and is joined to the inner foil region at the central portion thereof and is the grid assembly described in Clause 37.

[0115] Clause 41: Each of the beams has a beam width and a beam length that is approximately 7 to 10 times greater than the beam width, and is the grid assembly described in Clause 37.

[0116] Clause 42: Each of the beams has a straight or corrugated shape, and is the grid assembly described in Clause 37.

[0117] Clause 43: The foil area of the foil sheet includes a grid of polygonal foil areas, and the elongated slots include multiple rows and columns of straight slots that are interposed between and separate adjacent ones of the polygonal foil areas, and is the grid assembly described in Clause 25.

[0118] Clause 44: The rows of straight slots are parallel to each other, the columns of straight slots are parallel to each other, and the rows are orthogonal to the columns, and is the grid assembly described in Clause 43.

[0119] Clause 45: Each opposite end of each straight slot terminates at an adjacent opposite end of a plurality of adjacent slots among the straight slots, and is the grid assembly described in Clause 43.

[0120] Clause 46: Each opposite end of each straight slot terminates adjacent to a central region of one adjacent straight slot among the straight slots, and is the grid assembly described in Clause 43.

[0121] Clause 47: Each of the polygonal foil areas has a square shape in plan view and includes an array of foil holes, and is the grid assembly described in Clause 43.

[0122] Clause 48: Each of the straight slots includes a series of linearly aligned slits or a single continuous slit, and is the grid assembly described in Clause 43.

[0123] Clause 49: Each of the straight slots has a slot width and a slot length that is approximately 7 to 10 times greater than the slot width, and is the grid assembly described in Clause 43.

Claims

1. A grid assembly for supporting a sample in a cryo-electron microscope system, comprising a grid body having opposing first and second grid surfaces and a plurality of grid holes extending through the grid body from the first grid surface to the second grid surface; a foil sheet seated on the first grid surface and covering a subset of the grid holes of the grid body, the foil sheet having a plurality of foil holes and including a plurality of foil regions separated by a plurality of elongated slots, the elongated slots being configured to allow the foil regions to expand and / or move relative to each other.

2. The grid assembly according to claim 1, wherein the grid body has a diameter or width of 2.5 millimeters (mm) to 3.5 mm.

3. The grid assembly according to claim 1, wherein the grid body has a thickness of 10 micrometers (μm) to 25 μm.

4. The grid assembly according to claim 1, wherein the grid body has a flat single-piece structure.

5. The grid assembly according to claim 1, wherein the grid body is formed from a rigid metal material.

6. The grid assembly according to claim 1, wherein each of the grid holes of the grid body has a width or diameter of 30 micrometers (μm) to 110 μm.

7. The grid assembly according to claim 1, wherein the foil sheet has a thickness of 10 nanometers (nm) to 50 nm.

8. The grid assembly according to claim 1, wherein the foil sheet has a flat single-piece structure.

9. The grid assembly according to claim 1, wherein the foil sheet is flexible and formed from a metal material or a carbon material.

10. The grid assembly according to claim 1, wherein each of the foil holes of the foil sheet has a width or diameter of 0.2 micrometers (μm) to 2.0 μm.

11. The grid assembly according to claim 1, wherein the foil regions of the foil sheet include an inner foil region and an outer foil region surrounding the inner foil region, the elongated slots being interposed between the inner foil region and the outer foil region and separating the outer foil region from the inner foil region.

12. The grid assembly according to claim 11, wherein the foil sheet further includes a plurality of beams connecting the inner foil region to the outer foil region.

13. The grid assembly according to claim 12, wherein the beams are in a tangential direction with respect to the outer periphery of the inner foil region.

14. The grid assembly according to claim 13, wherein the inner foil region has a circular or polygonal planar profile.

15. The grid assembly according to claim 13, wherein the beams are spaced apart from the inner foil region and are connected to the inner foil region by a plurality of radially elongated tabs.

16. The grid assembly according to claim 13, wherein each of the beams is joined to the outer foil region at its opposing ends and to the inner foil region at its central portion.

17. The grid assembly according to claim 13, wherein each of the beams has a beam width and a beam length that is 7 to 10 times longer than the beam width.

18. The grid assembly according to claim 13, wherein the beams have a straight or corrugated shape.

19. The foil region of the foil sheet includes a grid of polygonal foil regions, and the elongated slots include a plurality of rows and columns of straight slots intervening between and separating adjacent said polygonal foil regions, the grid assembly according to claim 1.

20. The grid assembly according to claim 19, wherein the rows of the straight slots are parallel to each other, the columns of the straight slots are parallel to each other, and the rows are orthogonal to the columns.

21. The grid assembly according to claim 19, wherein opposing ends of each of the straight slots terminate at adjacent opposing ends of a plurality of adjacent slots among the straight slots.

22. The grid assembly according to claim 19, wherein opposing ends of each of the straight slots terminate adjacent to a central region of an adjacent slot among the straight slots.

23. The grid assembly according to claim 19, wherein each of the polygonal foil regions has a square planar profile and includes an array of foil holes.

24. The grid assembly according to claim 19, wherein each of the straight slots includes a series of linearly aligned slits or a single continuous slit.

25. The grid assembly according to claim 19, wherein each of the linear slots has a slot width and a slot length that is 7 to 10 times longer than the slot width.

Images