Liquid specimen grid for transmission electron microscope

Abstract

To provide a grid for sampling a liquid specimen.SOLUTION: The grid includes a mesh and a foil. The mesh includes a plurality of grid bars defining a plurality of openings. The plurality of grid bars form an outer perimeter of each opening. The foil is coupled to the mesh, and the foil includes a plurality of holes. A subset of each of the plurality of holes is aligned with each of the plurality of openings. Each hole of the subset is separated from the outer perimeter of the corresponding opening defined by the plurality of grid bars.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a grid for sampling a liquid specimen. The grid may be for sampling a liquid specimen for transmission electron microscopy (TEM) analysis. [Background technology]

[0002] Samples for single particle cryo-electron microscopy are traditionally prepared by manually pipetting a buffer solution containing the protein onto a glow-discharged TEM grid support. The grid is then blotted with filter paper and vitrified in liquid ethane. When using TEM grids with foils that have holes, the applied sample may wet the backside of the foil by flowing through the micrometer-sized holes ("backside wetting"). Backside wetting can present several problems: (1) complicating the removal of the liquid from the grid, (2) complicating the thinning of the liquid on the grid. Both liquid removal and liquid thinning are necessary to obtain a thin film of the sample suitable for cryo-electron microscopy. Summary of the Invention

[0003] In one implementation, the present disclosure provides a grid for sampling a liquid specimen. The grid includes a mesh and a foil. The mesh includes a plurality of grid bars that define a plurality of openings. The plurality of grid bars form a perimeter of each of the openings. The foil is coupled to the mesh, the foil including a plurality of holes. A subset of the plurality of holes is aligned with each of the plurality of openings. Each hole of the subset is spaced from a perimeter of a corresponding opening defined by the plurality of grid bars.

[0004] In another implementation, the present disclosure provides a grid for sampling a liquid specimen, the grid including a mesh and a foil, the mesh including a plurality of grid bars defining a plurality of openings, the plurality of grid bars forming a perimeter of each opening, the foil being coupled to the mesh, the foil including a plurality of holes, a subset of the plurality of holes being aligned with each of the plurality of openings, none of the subset of holes at least partially overlapping the plurality of grid bars forming the perimeter of a corresponding opening.

[0005] In another implementation, the present disclosure provides a method of manufacturing a grid for sampling a liquid specimen, the method comprising the steps of providing a foil having a plurality of holes; and adding a mesh to the foil, the mesh including a plurality of grid bars defining a plurality of openings, the plurality of grid bars forming a perimeter of each opening, a subset of the plurality of holes aligned with each of the plurality of openings, the method also including spacing each hole of the subset from a perimeter of a corresponding opening defined by the plurality of grid bars.

[0006] Other aspects of the disclosure will become apparent by consideration of the detailed description and accompanying drawings. [Brief description of the drawings]

[0007] [Figure 1] FIG. 2 is a perspective view of a grid for sampling a liquid specimen. [Figure 2A] FIG. 2 is a schematic top view of a portion of the mesh of the grid of FIG. 1. [Figure 2B] FIG. 2 is a schematic top view of a portion of the grid of FIG. 1. [Figure 3A] 2 is a cross-sectional schematic diagram of a portion of the grid of FIG. 1 with a liquid sample applied to a first side of the grid. [Figure 3B] 2 is a cross-sectional schematic view of a portion of the grid of FIG. 1 with a liquid sample applied to a second side of the grid. [Figure 4] FIG. 1 is a schematic diagram of the electronic control system and microcontroller. [Diagram 5] 2 is a flow diagram illustrating a method of manufacturing the grid of FIG. 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Before any implementations of the present disclosure are described in detail, it is to be understood that the disclosure is not limited in its application to the details of construction and the arrangements of components set forth in the following description or illustrated in the following drawings. The disclosure is capable of other implementations and of being practiced or carried out in various ways.

[0009] The present disclosure relates to a sample preparation system 10 for sampling a liquid specimen 14. The sample preparation system 10 is configured to prepare the liquid specimen 14 for analysis by a charged particle microscope (e.g., a transmission electron microscope [TEM]). The illustrated application is by way of example only, and the sample preparation system 10 may be utilized in any sampling application, particularly when sampling a liquid specimen 14.

[0010] 1 , the sample preparation system 10 includes a pipette 16 for depositing the liquid sample 14 and a grid 18 for holding the liquid sample 14. The pipette 16 is configured to deposit a droplet 19 of the liquid sample 14 on the grid 18. In other embodiments, a different device may be used to deposit the liquid sample 14 on the grid 18 (e.g., a printer, a syringe, etc.). The pipette 16 is configured to deposit the droplet 19 on a second side 20 of the grid 18. In an alternative embodiment, the pipette 16 may deposit the droplet 19 on a first side 21 of the grid 18.

[0011] The illustrated grid 18 includes a mesh 22 and a foil 26. The foil 26 defines a first side 21 of the grid 18. The mesh 22 defines a second side 20 of the grid 18. The mesh 22 and the foil 26 are substantially the same shape and size. In the illustrated embodiment, both the mesh 22 and the foil 26 are circular in shape. In alternative embodiments, the mesh 22 and the foil 26 may be different shapes (e.g., square, rectangular, trapezoidal, etc.) or the mesh 22 may have a different shape than the foil 26 (e.g., the mesh 22 may be circular and the foil 26 may be square).

[0012] The mesh 22 includes a plurality of grid bars 46 that define a plurality of openings 50. As shown in FIGS. 2A and 2B, each of the illustrated openings 50 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 grid bars 46 form a perimeter 54 of each of the plurality of openings 50. The perimeter 54 corresponds to and overlaps with the grid bar 46 as shown in FIG. 2A. Each of the plurality of grid bars 46 has a width 56. In this embodiment, the width 56 is 25 micrometers. In other embodiments, the grid bars 46 may have other widths ranging from 10 to 100 micrometers.

[0013] In this embodiment, the mesh 22 is hydrophilic and the foil 26 is hydrophilic. In alternative embodiments, the mesh 22 may not be hydrophilic, or the foil 26 may not be hydrophilic, or both the mesh 22 and the foil 26 may not be hydrophilic.

[0014] The foil 26 includes a plurality of holes 58. In the illustrated embodiment, each of the plurality of holes 58 is generally circular. In alternative embodiments, the plurality of holes 58 may be different shapes (e.g., square, triangular, oval, etc.). A subset 59 of the plurality of holes 58 is arranged in a cluster 60. A cluster 60 is a collection of holes 58 that is spaced apart from a similar collection of holes 58. Each cluster 60 is aligned with one of the openings 50 defined by the mesh 22. A cluster 60 is separated from an adjacent cluster 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 grid bars 46. The grid bar 46 may be dome-shaped such that a contact length 62 between the grid bar 46 and the foil 26 may be smaller or larger than the mesh width 56. The contact length 62 between the grid bar 46 and the foil 26 is variable depending on the tension of the foil 26. The tension in the foil 26 determines how well the foil 26 follows the curvature of the grid bar 46. The distance 61 may be greater than the contact length 62. In this embodiment, the foil 26 may be a plastic film. Amorphous carbon may be deposited on the plastic film, and then the plastic film may be dissolved. Alternatively, the plastic film may remain with the amorphous carbon. In some embodiments, the mesh 22 may be built on the foil 26 to form an integral mesh-foil structure, and the foil 26 may be made from a metal such as gold. In other embodiments, the foil 26 may be made from a different material (e.g., plastic, glass, etc.).

[0015] A rear portion 66 of the mesh 22 corresponds to and at least partially contacts the foil 26. A subset 59 of the plurality of holes 58 on the foil 26 is configured to align with one of the plurality of openings 50. Each hole 58 of the subset 59 is spaced from the perimeter 54 of the corresponding opening 50. In this embodiment, none of the holes 58 of the subset 59 overlaps with the plurality of grid bars 46 that form the perimeter 54 of the corresponding opening 50. Rather, a solid portion 70 of the foil 26 contacts the perimeter 54. The solid portion 70 of the foil 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 opening 50 to prevent backside wetting (i.e., when a portion of the liquid specimen 14 leaks through the holes 58 and contacts the second side 74 of the foil 26).

[0016] 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 less than the distance 61 by which adjacent clusters 60 are separated from one another. In alternative embodiments, such as when the total number of the plurality of holes 58 is small, the cluster distance 57 may be greater than the distance 61. The cluster distance 57 and the distance 61 distinguish the separate clusters 60 from one another by disposing the plurality of holes 58 at different distances from one another. A cluster 60 may include any number of holes 58, ranging from 1 to 1000. In the example shown in FIG. 2B, a cluster 60 includes nine holes 58. In alternative embodiments, a cluster 60 may include fewer or more holes 58. The illustrated cluster 60 is arranged to create a square. The cluster 60 includes three holes on each side of the square. In alternative embodiments, the cluster 60 may be arranged in any of a number of different shapes (e.g., triangle, circle, rectangle, etc.).

[0017] In some embodiments, the holes 58 may be evenly spaced and distributed throughout the foil 26. In such embodiments, the clusters 60 may be defined by the grid 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. Holes 58 between adjacent clusters 60 (e.g., holes 58 that overlap a grid bar 46) may not be considered part of either cluster.

[0018] Mesh 22 has a mesh thickness of 15 micrometers. Foil 26 has a foil thickness of 10 nanometers. In alternative embodiments, mesh 22 and foil 26 may have other thicknesses. For example, mesh 22 may have a nominal mesh thickness in the range of 5 to 35 micrometers. Foil 26 may have a nominal foil thickness in the range of 5 to 50 nanometers.

[0019] FIG. 3A illustrates the liquid specimen 14 applied onto the first side 21 of the grid 18 such that the liquid specimen 14 is supported by the foil 26. As shown in the illustrated embodiment, the foil 26 is closed adjacent to the grid bar 46. That is, none of the holes 80 in the foil 26 overlaps partially or completely with one of the grid bars 46. In an alternative embodiment, none of the holes 80 in the foil 26 overlaps partially or completely with one of the grid bars 46. Rather, the first holes 80 of the subset 59 (i.e., the holes 80 closest to the grid bar 46) are formed a hole distance 78 from the grid bar 46. The hole distance 78 may be between 5 and 20 micrometers. In other embodiments, the hole distance 78 may be greater or less. For example, the hole distance 78 may be in the range of 1 to 100 micrometers. By closing the foil 26 adjacent to the grid bar 46, the liquid specimen 14 is less likely to seep through a portion of the foil 26 by adhering to the grid bar 46. In this way, backside wetting can be reduced.

[0020] In alternative embodiments, the clusters 60 may only be partially aligned across the grid 18 (i.e., some clusters 60 may be perfectly aligned and some clusters 60 may have holes 80 that overlap the grid bars 46). These grids 18 may still be usable by applying liquid specimen 14 only to the clusters 60 that have perfect alignment. Alternatively, the user may accept backside wetting at the individual openings 50.

[0021] 3B illustrates the liquid specimen 14 applied onto the second side 20 of the grid 18 such that the liquid specimen 14 is supported by the foil 26 between the grid bars 46 of the mesh 22. Similar to the scenario of FIG. 3A, the foil 26 is closed near the grid bars 46. Therefore, the liquid specimen 14 is less likely to seep through the portions of the foil 26 adjacent to the grid bars and cause backside wetting.

[0022] FIG. 4 illustrates the sample preparation system 10 including a micromanipulator 88 that assists in aligning the foil 26 with the mesh 22. In some embodiments, the micromanipulator 88 is configured to detect the location of the holes 58 and the location of the grid bar 46. The micromanipulator 88 then aligns the foil 26 with the mesh 22 such that each cluster 60 of the holes 58 is positioned within one of the openings 50. More specifically, the micromanipulator 88 aligns the foil 26 with the mesh 22 such that none of the holes 58 overlaps any of the grid bars 46. In other embodiments, the micromanipulator 88 may detect edges or other features of the mesh 22 and the foil 26 to properly align the foil 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, microcontroller, or another suitable programmable device) and memory 98, such as non-transitory memory. The memory 98 may include, for example, a program storage area 102 and a data storage area 106. The program storage area 102 and the data storage area 106 may be any type of memory, including read only memory ("ROM"), random access memory (RANM), or the like. The programming may include a combination of different types of memory 98, such as dynamic access memory, "RAM" (e.g., dynamic RAM, "DRAM", synchronous DRAM, "SDRAM", etc.), electrically erasable programmable read-only memory (EEPROM), flash memory, hard disk, SD card, or other suitable magnetic, optical, physical, electronic memory device, or other data structure. The programming may be coded or learned.

[0023] As an example, the electronic controller 90 may be configured to control the rotation and translation 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 foil 26, which may include moving in the X, Y, and / or Z directions in any combination or orientation.

[0024] In some embodiments, the foil 26 may be initially solid and not include the holes 58. In such embodiments, the rear portion 66 of the mesh 22 may be aligned with and attached to the foil 26. The holes 58 may then be formed in the foil 26 after the foil 26 is bonded to the mesh 22. For example, a patterning device may pattern the holes 58 on the portions of the foil 26 that correspond to the openings 50. The patterning device may be controlled by an electronic controller 90. This may be done using wafer-based fabrication (i.e., the grid 18 is produced on a silicon wafer). Wafer-based fabrication allows for the addition of custom fiducial markers and patterns that help locate and identify specific areas of the grid 18 at several length scales by eye, in an optical microscope, and in an electron microscope. These fiducial markers aid in the patterning of the holes 58 on the foil 26. In other embodiments, the micromanipulator 88 may align a template to the pre-aligned mesh 22 and foil 26. The template may be configured to aid in the patterning of the plurality of holes 58 on the foil 26. Alternatively, the mesh 22 and the foil 26 may not be aligned, and instead the mesh may be built onto the foil to form an integrally closed mesh-foil structure. The patterning device may then pattern the plurality of holes 58 on the portions of the foil corresponding to the openings 50. In an alternative embodiment, the foil 26 may include the plurality of holes 58, and a prefabricated mesh 22 may be aligned with the foil using the micromanipulator 88. Alternatively, the foil 26 may include the plurality of holes 58, and the mesh 22 may be built onto the foil 26 to create an integrally formed integral mesh-foil structure.

[0025] In further embodiments, the foil 26 may have holes 58 throughout, some of which may be plugged after the foil 26 is attached to the mesh 22. For example, if the foil 26 is positioned on the mesh 22 such that some of the holes 58 overlap the grid bars 46, the holes 58 that overlap the grid bars 46 may be plugged with a suitable material or filler to close those holes 58. The foil 26 may be attached to the mesh 22 using the micromanipulator 88, or the mesh 22 may be constructed on the foil 26 to form an integral mesh-foil structure.

[0026] 5 is a flow diagram depicting a method of manufacturing a grid 18 for sampling a liquid specimen 14 for analysis in a charged particle microscope (e.g., a transmission electron microscope, a scanning electron microscope, etc.). The method includes certain steps, although not all of the steps need to be performed or in the order presented. In some embodiments, the method may include additional steps. At least a portion of the method may be performed by an electronic controller 90.

[0027] In step 501, a user provides a foil 26. Step 501 may also include a user providing a mesh 22. The foil 26 may include a plurality of holes 58. Subsets 59 of the plurality of holes 58 are arranged in clusters 60. The clusters 60 are separated from adjacent clusters 60 of the plurality of holes 58 by a distance 61. Alternatively, the foil 26 may not include a plurality of holes 58 in this step 501.

[0028] In step 502, the mesh 22 is added to the foil 26. This can be done by the user or by the micromanipulator 88. The mesh 22 includes a plurality of grid bars 46. The plurality of grid 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 generally square in shape. The mesh may be constructed on the foil to form an integral closed mesh-foil structure. The clusters 60 are aligned with each of the plurality of openings 50 after the mesh 22 and foil 26 are bonded. The mesh 22 may be added to the foil 26 using any of a number of different adhesives or bonding techniques. In some embodiments, no adhesives or bonding techniques may be used.

[0029] In step 503, each hole 58 of the subset 59 is spaced from the perimeter 54 of the corresponding opening 50 defined by the grid bars 46. In some embodiments of the method, step 503 may include aligning the foil 26 with the mesh 22 such that none of the holes 58 of the subset 59 overlaps the grid bars 46. Step 503 may further include blocking any holes 58 of the subset 59 that overlap partially or completely with the grid bars 46. In some embodiments, the patterning device may pattern the holes 58 on the portion of the foil 26 that corresponds to the opening 50. The patterning device may not pattern the holes 58 on the perimeter 54. Further and intermediate method steps will be apparent from the above description and the operational description below.

[0030] In operation, the foil 26 and mesh 22 are aligned to form the grid 18 such that the holes 58 are aligned only with the openings 50. The holes 58 do not overlap the plurality of grid bars 46. The liquid specimen 14 is pipetted onto the grid 18, and the user blots the grid 18 with filter paper. The grid 18 is then inserted, for example, into 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 onto the grid 18. The grid 18 may then be analyzed by a microscope (e.g., a TEM). It should be noted that other preparation techniques may be used. For example, a printer may print the liquid specimen 14 onto the grid 18, and the user may not need to blot the grid.

[0031] Aligning a subset of holes with each of the multiple openings provides an advantage. If the holes overlap the grid bar 46, backside wetting can occur due to the hydrophilic nature of the grid bar 46. This can cause the backside meniscus to grow inward from the grid bar 46 toward the center of the opening 50. This can result in a less reproducible sample thickness 86 and thicker ice that is not suitable for cryo-EM acquisition over large areas of the grid 18. By keeping the foil 26 solid near the grid bar 46, backside wetting is less likely to occur and the sample thickness 86 can be better controlled.

[0032] Various features and advantages of the invention are set forth in the following claims.

Claims

1. 1. A grid for sampling a liquid specimen, said grid comprising: a mesh including a plurality of grid bars defining a plurality of openings, the plurality of grid bars forming a perimeter of each opening; a foil coupled to the mesh, the foil including a plurality of holes, a subset of the plurality of holes being aligned with each of the plurality of openings, each hole of the subset being spaced from the perimeter of a corresponding opening defined by the plurality of grid bars.

2. The grid of claim 1 , wherein each of the plurality of openings is substantially square.

3. The grid of claim 2 , wherein each of the plurality of holes is generally circular.

4. 2. The grid of claim 1, wherein the foil is a plastic film.

5. The grid of claim 1 , wherein the mesh is hydrophilic.

6. 2. The grid of claim 1, wherein each subset of the plurality of holes is disposed in a cluster separated from an adjacent cluster of the plurality of holes by a distance greater than a width of one of the plurality of grid bars.

7. The grid of claim 1 , wherein the mesh has a mesh thickness of 5 to 35 micrometers.

8. The grid of claim 1 , wherein the foil has a foil thickness of 10 to 15 nanometers.

9. 1. A grid for sampling a liquid specimen, said grid comprising: a mesh including a plurality of grid bars defining a plurality of openings, the plurality of grid bars forming a perimeter of each opening; a foil coupled to the mesh, the foil including a plurality of holes, a subset of the plurality of holes being aligned with each of the plurality of openings, and none of the holes in the subset at least partially overlapping the plurality of grid bars that form the perimeter of a corresponding opening.

10. The grid of claim 9 , wherein each of the plurality of openings is generally square.

11. The grid of claim 10 , wherein each of the plurality of holes is generally circular.

12. 10. The grid of claim 9, wherein the foil is a plastic film.

13. 10. The grid of claim 9, wherein the mesh is hydrophilic.

14. 10. The grid of claim 9, wherein each subset of the plurality of holes is disposed in a cluster separated from an adjacent cluster of the plurality of holes by a distance greater than a width of one of the plurality of grid bars.

15. 10. The grid of claim 9, wherein the mesh has a mesh thickness of 5 to 35 micrometers.

16. 10. The grid of claim 9, wherein the foil has a foil thickness of 10 to 15 nanometers.

17. 1. A method of manufacturing a grid for sampling a liquid specimen, the method comprising the steps of: providing a foil including a plurality of holes; adding a mesh to the foil, the mesh including a plurality of grid bars defining a plurality of openings, the plurality of grid bars forming a perimeter of each opening, a subset of the plurality of holes aligned with each of the plurality of openings; and spaced each hole in the subset from the periphery of a corresponding opening defined by the plurality of grid bars.

18. 18. The method of claim 17, wherein spacing each hole of the subset from the perimeter of the corresponding opening comprises aligning the foil with the mesh such that none of the holes of the subset at least partially overlaps the plurality of grid bars.

19. 20. The method of claim 17, wherein spacing each hole of the subset from the perimeter of the corresponding opening comprises blocking any hole of the subset that at least partially overlaps the plurality of grid bars.

20. 20. The method of claim 17, wherein spacing each hole of the subset from the periphery of the corresponding opening comprises forming the holes in the foil after the foil is bonded to the mesh.

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