Liquid sample grid for transmission electron microscope

Abstract

A grid for sampling a liquid standard includes meshes and a substrate. The mesh includes a plurality of mesh strips defining a plurality of openings. The plurality of grid strips form the periphery of each opening. The substrate is coupled to the mesh and the substrate includes a plurality of holes. A subset of the plurality of apertures is aligned with each of the plurality of openings. Each aperture in a subset is spaced apart from a periphery of a corresponding opening defined by the plurality of grid bars.

Description

Background Art

[0001] The present disclosure relates to a grid for sampling a liquid sample. The grid can be used to sample a liquid sample for transmission electron microscopy (TEM) analysis.

[0002] Samples for single-particle cryo-electron microscopy are typically prepared by manually pipetting a buffer solution containing the protein onto a glow-discharged TEM grid carrier. The grid is then blotted dry with filter paper and vitrified in liquid ethane. When using a TEM grid whose substrate has holes, the applied sample may wet the back side of the substrate by flowing through the micrometer-sized holes ("backside wetting"). Backside wetting can present several problems: (1) it complicates the removal of liquid from the grid; and (2) it complicates thinning the liquid on the grid. Both removing the liquid and thinning the liquid are essential to obtaining a sample film suitable for cryo-electron microscopy. Summary of the invention

[0003] In one embodiment, the present disclosure provides a grid for sampling a liquid sample. The grid includes a mesh and a substrate. The mesh includes a plurality of mesh bars defining a plurality of openings. The plurality of mesh bars form a periphery of each opening. The substrate is coupled to the mesh and includes a plurality of holes. A subset of the plurality of holes is aligned with each of the plurality of openings. Each hole in a subset is spaced apart from the periphery of a corresponding opening defined by the plurality of mesh bars.

[0004] In another embodiment, the present disclosure provides a grid for sampling a liquid sample. The grid includes a mesh and a substrate. The mesh includes a plurality of mesh bars defining a plurality of openings. The plurality of mesh bars form a perimeter of each opening. The substrate is coupled to the mesh and the substrate includes a plurality of holes. A subset of the plurality of holes is aligned with each of the plurality of openings. None of the holes in a subset of the holes at least partially overlaps with the plurality of mesh bars forming the perimeter of the corresponding opening.

[0005] In another specific implementation, the present disclosure provides a method for manufacturing a grid for sampling a liquid sample. The method includes providing a substrate having a plurality of holes and

[0006] A mesh is added to the substrate. The mesh includes a plurality of mesh bars defining a plurality of openings. The plurality of mesh bars form a perimeter of each opening. A subset of the plurality of holes is aligned with each of the plurality of openings. The method also includes spacing each hole in a subset from the perimeter of a corresponding opening defined by the plurality of mesh bars.

[0007] Other aspects of the disclosure will become apparent by consideration of the detailed description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 is a perspective view of a grid used to sample a liquid sample.

[0009] Figure 2A yes Figure 1 Schematic top view of a portion of a mesh of a grid.

[0010] Figure 2B yes Figure 1 Schematic top view of a portion of a grid.

[0011] Figure 3A yes Figure 1 Schematic cross-sectional view of a portion of a grid wherein a liquid sample is applied to a first side of the grid.

[0012] Figure 3B yes Figure 1 Schematic cross-sectional view of a portion of a grid wherein a liquid sample is applied to a second side of the grid.

[0013] Figure 4 is a schematic diagram of the electronic control system and microcontroller.

[0014] Figure 5 It is a description of a manufacturing Figure 1 Flowchart of the grid method. DETAILED DESCRIPTION

[0015] Before explaining any embodiments of the present disclosure in detail, it should be understood that the present disclosure is not limited in its application to the details of construction and arrangement of components set forth in the following description or shown in the following drawings. The present disclosure is capable of other embodiments and can be practiced or carried out in various ways.

[0016] The present disclosure relates to a sample preparation system 10 for sampling a liquid sample 14. The sample preparation system 10 is configured to prepare the liquid sample 14 for analysis by a charged particle microscope (e.g., a transmission electron microscope [TEM]). The application shown is just one example, and the sample preparation system 10 can be used in any sampling application, especially when sampling a liquid sample 14.

[0017] like Figure 1As shown, the sample preparation system 10 includes a pipette 16 for placing a liquid sample 14 and a grid 18 for holding the liquid sample 14. The pipette 16 is configured to place a droplet 19 of the liquid sample 14 onto the grid 18. In other embodiments, a different device (e.g., a printer, a syringe, etc.) can be used to place the liquid sample 14 onto the grid 18. The pipette 16 is configured to place the droplet 19 onto the second side 20 of the grid 18. In an alternative embodiment, the pipette 16 can place the droplet 19 onto the first side 21 of the grid 18.

[0018] The illustrated mesh 18 includes a mesh 22 and a substrate 26. The substrate 26 defines a first side 21 of the mesh 18. The mesh 22 defines a second side 20 of the mesh 18. The mesh 22 and the substrate 26 are substantially the same in shape and size. In the illustrated embodiment, both the mesh 22 and the substrate 26 are circular in shape. In alternative embodiments, the mesh 22 and the substrate 26 may be different shapes (e.g., square, rectangular, trapezoidal, etc.), or the shape of the mesh 22 may be different from the shape of the substrate 26 (e.g., the mesh 22 may be circular and the substrate 26 may be square).

[0019] The mesh 22 includes a plurality of mesh bars 46 defining a plurality of openings 50. Figure 2A and Figure 2B As shown, each of the openings 50 shown is generally square in shape. In alternative embodiments, each of the plurality of openings 50 may have other shapes (e.g., circular, rectangular, hexagonal, elliptical, etc.). The plurality of mesh bars 46 form an outer perimeter 54 of each of the plurality of openings 50. Figure 2A As shown, perimeter 54 corresponds to and overlaps with mesh bars 46. Each of the plurality of mesh bars 46 has a width 56. In this embodiment, width 56 is 25 microns. In other embodiments, mesh bars 46 may have other widths in the range of 10 microns to 100 microns.

[0020] In this embodiment, mesh 22 is hydrophilic and substrate 26 is hydrophilic. In alternative embodiments, mesh 22 may not be hydrophilic, or substrate 26 may not be hydrophilic, or both mesh 22 and substrate 26 may not be hydrophilic.

[0021] The substrate 26 includes a plurality of holes 58. In the illustrated embodiment, each hole in the plurality of holes 58 is generally circular. In alternative embodiments, the plurality of holes 58 may be of different shapes (e.g., square, triangular, elliptical, etc.). The plurality of subgroups 59 of the plurality of holes 58 are arranged into clusters 60. The clusters 60 are collections of holes 58 that are spaced apart from similar collections of holes 58. Each cluster 60 is aligned with one of the openings 50 defined by the mesh 22. The clusters 60 are separated from adjacent clusters 60 of the plurality of holes 58 by a distance 61. The distance 61 may be greater than the width 56 of one of the plurality of mesh bars 46. The mesh bars 46 may be domed in shape so that the contact length 62 between the mesh bars 46 and the substrate 26 may be smaller or greater than the mesh width 56. The contact length 62 between the mesh bars 46 and the substrate 26 may vary, depending on the tension of the substrate 26. The tension of the substrate 26 determines the extent to which the substrate 26 conforms to the curve of the mesh bars 46. The distance 61 may be greater than the contact length 62. In this embodiment, substrate 26 can be a plastic film. Amorphous carbon can be deposited on the plastic film, which can then be dissolved. Alternatively, the plastic film can be retained together with the amorphous carbon. In some embodiments, mesh 22 can be built on substrate 26 to form an integrated mesh / substrate structure, and substrate 26 can be made of a metal such as gold. In other embodiments, substrate 26 can be made of different materials (e.g., plastic, glass, etc.).

[0022] The rear portion 66 of the mesh 22 corresponds to and is in at least partial contact with the substrate 26. A subset 59 of the plurality of holes 58 on the substrate 26 is configured to align with one of the plurality of openings 50. Each hole 58 in a subset 59 is spaced apart from the periphery 54 of the corresponding opening 50. In this embodiment, none of the holes 58 in a subset 59 overlaps with a portion of the plurality of mesh bars 46 that form the periphery 54 of the corresponding opening 50. Instead, a solid portion 70 of the substrate 26 is in contact with the periphery 54. The solid portion 70 of the substrate 26 is continuous and does not include any of the plurality of holes 58. The solid portion 70 may extend along the entire contact length 62. The holes 58 are configured to align with the openings 50 to prevent backside wetting (i.e., when a portion of the liquid sample 14 penetrates through the holes 58 and contacts the second side 74 of the substrate 26).

[0023] Each of the plurality of holes 58 in a cluster 60 is spaced apart from one another by a cluster distance 57. The cluster distance 57 may be smaller than the distance 61 separating adjacent clusters 60 from one another. In alternative embodiments, such as when the amount of the plurality of holes 58 is low, the cluster distance 57 may be greater than the distance 61. By arranging the plurality of holes 58 at different distances from one another, the cluster distance 57 and the distance 61 distinguish different clusters 60 from one another. A cluster 60 may include any number of holes 58 between 1 and 1000. Figure 2BIn the example shown, one cluster 60 includes nine holes 58. In alternative embodiments, one cluster 60 may include fewer or more holes 58. The clusters 60 shown are arranged to form a square shape. The clusters 60 include three holes on each side of the square shape. In alternative embodiments, the clusters 60 may be arranged in any of a variety of different shapes (e.g., triangles, circles, rectangles, etc.).

[0024] In some embodiments, the holes 58 may be evenly spaced and distributed across the entire substrate 26. In such embodiments, the clusters 60 may be defined by the mesh bars 46 of the mesh 22. That is, the holes 58 within the perimeter 54 of one opening 50 may form a first cluster 60, while the holes 58 within the perimeter 54 of another opening 30 may form a second cluster 60. The holes 58 between adjacent clusters 60 (e.g., holes 58 that overlap with the mesh bars 46) may not be considered part of any cluster.

[0025] The mesh thickness of mesh 22 is 15 microns. The substrate thickness of substrate 26 is 10 nanometers. In alternative embodiments, mesh 22 and substrate 26 may have other thicknesses. For example, the nominal mesh thickness of mesh 22 may be in the range of 5 microns to 35 microns. The nominal substrate thickness of substrate 26 may be in the range of 5 nanometers to 50 nanometers.

[0026] Figure 3A The liquid sample 14 is shown applied to the first side 21 of the grid 18 such that the liquid sample 14 is supported by the substrate 26. As shown in the illustrated embodiment, the substrate 26 is closed about the grid bars 46. That is, none of the holes 80 in the substrate 26 partially overlaps one of the grid bars 46. In an alternative embodiment, none of the holes 80 in the substrate 26 partially or completely overlaps one of the grid bars 46. Instead, the first holes 80 in a subset 59 are

[0027] The holes 80 (i.e., the holes 80 closest to the grid bars 46) are formed at a hole distance 78 from the grid bars 46. The hole distance 78 may be between 5 microns and 20 microns. In other embodiments, the hole distance 78 may be larger or smaller. For example, the hole distance 78 may be in the range of 1 micron to 100 microns. By enclosing the substrate 26 adjacent to the grid bars 46, the liquid sample 14 is less likely to penetrate through a portion of the substrate 26 by adhering to the grid bars 46. Therefore, backside wetting may be reduced.

[0028] In alternative embodiments, the clusters 60 may be aligned only partially across the grid 18 (i.e., some clusters 60 may be perfectly aligned, while some clusters 60 may have holes 80 that overlap the grid bars 46). By applying the liquid sample 14 only to the perfectly aligned clusters 60, these grids 18 may still be usable. In addition, the user may accept that backside wetting of individual openings 50 may occur.

[0029] Figure 3B The liquid sample 14 is shown applied to the second side 20 of the grid 18 such that the liquid sample 14 is supported by the substrate 26 between the grid bars 46 of the mesh 22. Figure 3A In the scenario of FIG. 4 , the substrate 26 is closed near the grid bars 46. Therefore, it is less likely that the liquid sample 14 penetrates through a portion of the substrate 26 adjacent to the grid bars and causes backside wetting.

[0030] Figure 4 The sample preparation system 10 is shown to include a micromanipulator 88 to assist in aligning the substrate 26 with the mesh 22. In some embodiments, the micromanipulator 88 is configured to detect the position of the holes 58 and the position of the grid bars 46. The micromanipulator 88 then aligns the substrate 26 with the mesh 22 so that each cluster 60 of holes 58 is positioned within one of the openings 50. More specifically, the micromanipulator 88 aligns the substrate 26 with the mesh 22 so that none of the holes 58 overlaps with any of the grid bars 46. In other embodiments, the micromanipulator 88 may detect edges or other features of the mesh 22 and substrate 26 to properly align the substrate 26 with the mesh 22. The illustrated sample preparation system 10 also includes an electronic controller 90 configured to automatically or semi-automatically control the micromanipulator 88. The controller 90 may include a programmable processor 94 (e.g., a microprocessor, a microcontroller, or another suitable programmable device) and a memory 98, such as a non-transitory memory. Memory 98 may include, for example, a program storage area 102 and a data storage area 106. Program storage area 102 and data storage area 106 may include a combination of different types of memory 98, such as read-only memory ("ROM"), random access memory ("RAM") (e.g., dynamic RAM ["DRAM"], synchronous DRAM ["SDRAM"], etc.), electrically erasable programmable read-only memory ("EEPROM"), flash memory, a hard disk, an SD card, or other suitable magnetic memory device, optical memory device, physical memory device, electronic memory device, or other data structure.

[0031] Programming can be achieved through coding or through learning.

[0032] For example, the electronic controller 90 may be configured to control the rotation and movement of the micromanipulator 88. The electronic controller 90 may also be configured to control the position of the micromanipulator 88 in three-dimensional space, for example, to move or rotate the micromanipulator 88 to align the mesh 22 with the substrate 26, which may include moving or in any combination or orientation in the X direction, the Y direction, and / or the Z direction.

[0033] In some embodiments, the substrate 26 may initially be solid and not include a plurality of holes 58. In such embodiments, the rear portion 66 of the mesh 22 may be aligned with and attached to the substrate 26. Then, a plurality of holes 58 may be formed in the substrate 26 after the substrate 26 is coupled to the mesh 22. For example, a pattern forming device may form a pattern of a plurality of holes 58 on a portion of the substrate 26 corresponding to the opening 50. The pattern forming device may be controlled by an electronic controller 90. This may be achieved using wafer-based manufacturing (i.e., producing the grid 18 on a silicon wafer). Wafer-based manufacturing allows for the addition of customized fiducial marks and patterns that help locate and identify specific areas of several length scales of the grid 18 by eye, optical microscope, and electron microscope. These fiducial marks help form a pattern of a plurality of holes 58 on the substrate 26. In other embodiments, a micromanipulator 88 may align the template with the mesh 22 and substrate 26 that have been pre-aligned. The template may be configured to help form a pattern of a plurality of holes 58 on the substrate 26. Alternatively, the mesh 22 and substrate 26 may not be aligned, but the mesh may be built on the substrate to form a closed, one-piece mesh / substrate structure. The pattern forming device may then form a pattern of a plurality of holes 58 on a portion of the substrate corresponding to the opening 50. In an alternative embodiment, the substrate 26 may include a plurality of holes 58, and the micromanipulator 88 may be used to align the pre-made mesh 22 with the substrate. Alternatively, the substrate 26 may include a plurality of holes 58 and the mesh 22 may be built on the substrate 26 to form an integrally formed, one-piece mesh / substrate structure.

[0034] In other embodiments, the substrate 26 may have holes 58 throughout, and some of the holes 58 may be filled after the substrate 26 is attached to the mesh 22. For example, if the substrate 26 is positioned on the mesh 22 so that some of the holes 58 partially overlap the mesh bars 46, the holes 58 that partially overlap the mesh bars 46 may be filled with a suitable material or filler to close those holes 58. The substrate 26 may be attached to the mesh 22 using a micromanipulator 88, or the mesh 22 may be built onto the substrate 26 to form an integrated mesh / substrate structure.

[0035] Figure 51 is a flow chart depicting a method of making a grid 18 for sampling a liquid sample 14 for analysis in a charged particle microscope (e.g., a transmission electron microscope, a scanning electron microscope, etc.). Although the method includes specific steps, not all steps need to be performed or need to be performed in the order presented. In some embodiments, the method may include additional steps. At least some of the method may be performed by an electronic controller 90.

[0036] At step 501, a user provides a substrate 26. Step 501 may also include a user providing a mesh 22. The substrate 26 may include a plurality of holes 58. A subset 59 of the plurality of holes 58 is arranged in clusters 60. The clusters 60 are separated from adjacent clusters 60 of the plurality of holes 58 by a distance 61. Additionally, in this step 501, the substrate 26 may not include the plurality of holes 58.

[0037] At step 502, a mesh 22 is added to a substrate 26. This can be done by a user or a micromanipulator 88. The mesh 22 includes a plurality of mesh bars 46. The plurality of mesh bars 46 define a plurality of openings 50 and form a perimeter 54 of each opening 50. Each of the plurality of openings 50 is substantially square in shape. The mesh can be constructed on the substrate to form a closed, one-piece mesh / substrate structure. The clusters 60 are aligned with each of the plurality of openings 50 after the mesh 22 and substrate 26 are coupled. The mesh 22 can be added to the substrate 26 using any of a variety of different adhesives or bonding techniques. In some embodiments, no adhesive or bonding technique may be used.

[0038] At step 503, each hole 58 in a subset 59 is spaced apart from the perimeter 54 of the corresponding opening 50 defined by the plurality of mesh bars 46. In some embodiments of the method, step 503 may include aligning the substrate 26 with the mesh 22 so that none of the holes 58 in a subset 59 partially overlap the plurality of mesh bars 46. Step 503 may also include filling any holes 58 in a subset 59 that partially or completely overlap the plurality of mesh bars 46. In some embodiments, the patterning device may form a pattern of the plurality of holes 58 on a portion of the substrate 26 corresponding to the opening 50. The patterning device may not form a pattern of holes 58 on the perimeter 54. Additional method steps and intermediate method steps are apparent from the above description and the following description of operation.

[0039] In operation, the substrate 26 and the mesh 22 are aligned to form the grid 18 so that the holes 58 are aligned only with the openings 50. The holes 58 do not overlap with the plurality of grid bars 46. The liquid sample 14 is pipetted onto the grid 18, and the user blots the grid 18 with filter paper. The grid 18 is then inserted into, for example, liquid ethane to vitrify the liquid solution. The grid 18 may be immersed in a bath of liquid ethane, or a jet of liquid ethane may be focused on the grid 18. The grid 18 may then be analyzed by a microscope (e.g., TEM). It should be noted that other preparation techniques may be used. For example, a printer may spray print the liquid sample 14 onto the grid 18, and the user may not be required to blot the grid.

[0040] Aligning a subset of holes with each of the plurality of openings has various advantages. If the holes partially overlap the grid bars 46, backside wetting may occur due to the hydrophilicity of the grid bars 46. This may cause a backside meniscus to grow inward from the grid bars 46 toward the center of the opening 50. This may result in poor repeatability of the sample thickness 86 and may result in thick ice that is not suitable for cryo-EM acquisition of large areas of the grid 18. By keeping the substrate 26 solid near the grid bars 46, backside wetting is less likely to occur and the sample thickness 86 can be better controlled.

[0041] Various features and advantages of the invention are set forth in the following claims.

Claims

1. A grid for sampling a liquid sample, the grid comprising: a mesh comprising a plurality of mesh bars defining a plurality of openings, the plurality of mesh bars forming a perimeter of each opening; and A substrate is coupled to the mesh, the substrate comprising a plurality of holes, wherein a subset of the plurality of holes is aligned with each of the plurality of openings, and wherein each hole in the subset is spaced apart from a periphery of a corresponding opening defined by the plurality of mesh bars.

2. The grid according to claim 1, wherein: Each opening of the plurality of openings is substantially square in shape.

3. The grid according to claim 2, wherein: Each hole in the plurality of holes is substantially circular.

4. The grid according to claim 1, wherein: The substrate is a plastic film.

5. The grid according to claim 1, wherein: The mesh is hydrophilic.

6. The grid according to claim 1, wherein: Each subset of the plurality of holes is arranged into a cluster that is separated from an adjacent cluster of the plurality of holes by a distance greater than a width of one of the plurality of mesh bars.

7. The grid according to claim 1, wherein: The mesh thickness of the mesh is 5 microns to 35 microns.

8. The grid according to claim 1, wherein: The substrate has a thickness of 10 nanometers to 15 nanometers.

9. A grid for sampling a liquid sample, the grid comprising: a mesh comprising a plurality of mesh bars defining a plurality of openings, the plurality of mesh bars forming a perimeter of each opening; and A substrate is coupled to the mesh, the substrate comprising a plurality of holes, wherein a subset of the plurality of holes is aligned with each of the plurality of openings, and wherein no holes in the subset of holes at least partially overlap with the plurality of mesh bars forming a perimeter of the corresponding opening.

10. The grid according to claim 9, wherein: Each opening of the plurality of openings is substantially square in shape.

11. The grid according to claim 10, wherein: Each hole in the plurality of holes is substantially circular.

12. The grid according to claim 9, wherein: The substrate is a plastic film.

13. The grid according to claim 9, wherein: The mesh is hydrophilic.

14. The grid according to claim 9, wherein: Each subset of the plurality of holes is arranged into a cluster that is separated from an adjacent cluster of the plurality of holes by a distance greater than a width of one of the plurality of mesh bars.

15. The grid according to claim 9, wherein: The mesh thickness of the mesh is 5 microns to 35 microns.

16. The grid according to claim 9, wherein: The substrate has a thickness of 10 nanometers to 15 nanometers.

17. A method of manufacturing a grid for sampling a liquid sample, the method comprising: providing a substrate including a plurality of holes; adding a mesh to the substrate, the mesh comprising a plurality of mesh bars defining a plurality of openings, the plurality of mesh bars forming a perimeter of each opening, wherein a subset of the plurality of holes is aligned with each of the plurality of openings; as well as Each hole in the subset is spaced apart from a perimeter of the corresponding opening defined by the plurality of mesh bars.

18. The method according to claim 17, wherein: Spacing each hole in the subset from a perimeter of the corresponding opening includes aligning the substrate with the mesh such that none of the holes in the subset at least partially overlap the plurality of mesh bars.

19. The method according to claim 17, wherein: Spacing each hole in the subset from the periphery of the corresponding opening includes filling any hole in the subset that at least partially overlaps the plurality of mesh bars.

20. The method according to claim 17, wherein: Spacing each hole in the subset from a perimeter of the corresponding opening includes forming the plurality of holes in the substrate after coupling the substrate to the mesh.

Images