A sample holder for cryo-em imaging

Abstract

The application discloses a sample table for cryo-EM imaging, which comprises a base and a detachable filling component, wherein the base is in a discoid structure, a convex edge is arranged on the base, the convex edge and the base form a cavity for accommodating a sample, and the filling component can fill a step structure formed by the outer side wall of the convex edge and the base during high-pressure freezing and be removed in a pre-thinning process. The application can accommodate a high-end cryo-TEM sample ring, can be stably fixed between the sample ring and a sample base, can be gripped by a mechanical hand in an electron microscope Autoloader, and can be placed into a lens barrel for imaging. Therefore, the sample table provided by the application can be applied to the cryo-EM of the Autoloader.

Description

Technical Field

[0001] This invention relates to the field of biological sample detection, and in particular to a sample stage for cryo-electron microscopy imaging. Background Technology

[0002] In recent years, thanks to the development of cryo-electron tomography (cryo-ET), in-situ structural biology research has progressed rapidly. The research subjects are now in states closer to physiological conditions, and the resolution of structural analyses is constantly improving. Its ultimate goal is to resolve the structures of biological macromolecules and molecular machines in situ, thereby elucidating the essence of life activities at different levels, including molecules, organelles, and cells.

[0003] Cryo-electron tomography (cryo-electron tomography) generally requires sample thicknesses below 500 nm; otherwise, excessive inelastic scattering electrons during imaging severely degrade the signal-to-noise ratio, making subsequent image processing difficult. Since most cell and tissue samples are much thicker than 500 nm, the technical approach for these samples is cryo-fixation – cryo-focused ion beam (Cryo-FIB) thinning – cryo-electron tomography. Typically, small cells are rapidly frozen and fixed onto a special electron microscope grid, then thinned using cryo-FIB before cryo-electron microscopy imaging. However, rapid freezing only achieves a freezing depth of a few micrometers. For tissues with thicknesses ranging from tens to hundreds of micrometers, and for large-scale cells, rapid freezing alone cannot provide adequate fixation. Therefore, high-pressure freezing is necessary. The resulting sample block after high-pressure freezing is trimmed using a cryostat, then thinned to a few hundred nanometers using cryo-FIB, and finally sent to the cryo-electron microscope for imaging.

[0004] To improve imaging resolution, cryo-electron microscopes with autoloaders, such as the FEI Titan Kros, are used. These microscopes offer high resolution and can obtain more realistic and clear in-situ structures. However, the sample support tool used in electron microscopes is a grid, while the sample support tool used in autoloading cryo-electron microscopes is a stage. Autoloaders cannot mount sample stages, therefore, existing sample stages cannot be used on cryo-electron microscopes with autoloaders. Summary of the Invention

[0005] The purpose of this invention is to provide a sample stage for cryo-electron microscopy imaging, so as to solve the problem that existing sample stages cannot be applied to the cryo-electron microscope of Autoloader.

[0006] According to an embodiment of the present invention, a sample stage for cryo-electron microscopy imaging is provided, including a base and a detachable filling component. The base has a disc-shaped structure and a raised edge. The raised edge and the base form a cavity for accommodating the sample. The filling component can fill the stepped structure formed by the outer wall of the raised edge and the base during high-pressure freezing and can be removed during the pre-thinning process.

[0007] Specifically, both the protruding edge and the filling component are annular structures, and the filling component can be fitted onto the outer wall of the protruding edge.

[0008] Specifically, the center of the protruding edge coincides with the center of the base.

[0009] Specifically, the height of the filling component is the same as the height of the outer wall of the protrusion.

[0010] Specifically, the base and filling component are made of thermally conductive metal material.

[0011] This invention provides a sample stage for cryo-electron microscopy imaging. It can accommodate a loading ring for a high-end cryo-transmission electron microscope and is stably fixed between the loading ring and the loading base. It can be grasped by a robotic arm in an autoloader and placed into the microscope tube for imaging. Therefore, the sample stage provided by this invention can be applied to cryo-electron microscopes with autoloaders. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 This is a structural diagram of a sample stage for cryo-electron microscopy imaging provided in an embodiment of the present invention;

[0014] Figure 2 This is a structural diagram of the base;

[0015] Figure 3 This is a structural diagram of the filling component;

[0016] Figure 4 This is a diagram illustrating the usage state of a sample stage for cryo-electron microscopy imaging, provided in an embodiment of the present invention.

[0017] Among them, 1-base, 2-filling component, 3-protruding edge, 4-cavity, 5-sample loading base, 6-sample loading ring. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] According to an embodiment of the present invention, see Figures 1 to 3 A sample stage for cryo-electron microscopy imaging is provided, including a base 1 and a detachable filling component 2. The base 1 has a disc-shaped structure and a protrusion 3 on the base 1. The protrusion 3 and the base 1 form a cavity 4 for accommodating the sample. The filling component 2 can fill the stepped structure formed by the outer wall of the protrusion 3 and the base 1 during high-pressure freezing and can be removed during the pre-thinning process.

[0020] Both the raised edge 3 and the filling component 2 are annular structures. The filling component 2 can be fitted onto the outer wall of the raised edge 3, facilitating installation and removal by operators. The center of the raised edge 3 coincides with the center of the base 1. The height of the filling component 2 is the same as the height of the outer wall of the raised edge 3. The base 1 and the filling component 2 are made of thermally conductive metal material, which can be pure copper / silver / gold-plated copper, etc., but is not limited to these materials; this embodiment does not impose any limitation. The size of the sample stage can be set according to actual needs; this embodiment does not impose any limitation.

[0021] The sample preparation process is as follows: First, in the high-pressure freezing section, the filling component 2 is fitted onto the outer wall of the protruding edge 3, so that the filling component 2 fills the stepped structure formed by the outer wall of the protruding edge 3 and the base 1. The biological sample to be frozen is placed in the cavity 4, and a cryoprotectant is added to fill the cavity 4. Then, a sapphire sheet is placed on top to form a whole, which is then placed in the high-pressure freezer for freezing and fixation. The filling component 2 fills the space formed between the sapphire sheet and the base 1, improving the freezing effect and freezing speed.

[0022] Next, the frozen sample is transferred to a cryo-microtome in liquid nitrogen. After removing the sapphire slide, the assembly of base 1 and the collar is pre-thinned, trimming off approximately one-third to one-half, i.e., from a circle to a semi-circle / large semi-circle, exposing the surface of the frozen biological sample and thinning it from a scale of approximately hundreds of micrometers to a scale of tens of micrometers. After being trimmed into a semi-circle, the filling component 2 can be easily removed without affecting the subsequent use of the sample stage. The purpose of this step is to expose the sample and remove the collar to facilitate subsequent FIB cutting and electron microscopy imaging.

[0023] Then, as Figure 4As shown, the sample stage base 1 after pre-thinning is loaded with a sample loading ring 6 (c-clip) and a sample loading base 5 (AutoGrid). The sample stage is stably fixed between the two, forming a whole sample loading ring 6-sample stage-sample loading base 5, and is then transferred in liquid nitrogen to the scanning electron microscope cryo-FIB chamber for cryo-focused ion beam (cryo-FIB) thinning. The thinned sample thickness is approximately 200 nm, which meets the requirements for transmission electron microscopy imaging.

[0024] Finally, the entire process of loading ring 6, sample stage, and loading base 5 is grasped by the robotic arm in the electron microscope's Autoloader and placed into the microscope tube for cryo-transmission electron microscopy imaging and data collection.

[0025] This invention provides a sample stage for cryo-electron microscopy imaging. It can accommodate a loading ring 6 for a high-end cryo-transmission electron microscope and is stably fixed between the loading ring 6 and the loading base 5. The stage can be grasped by a robotic arm in the electron microscope's autoloader and placed into the microscope tube for imaging. Therefore, the sample stage provided by this invention can be applied to cryo-electron microscopes with autoloaders.

[0026] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the following claims.

[0027] It should be understood that the present invention is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The scope of the invention is limited only by the appended claims.

Claims

1. A sample stage for cryo-electron microscopy imaging, characterized in that, Includes a base (1) and a detachable filling component (2). The base (1) has a disc-shaped structure and a raised edge (3) on it. The raised edge (3) and the base (1) form a cavity (4) for accommodating the sample. The filling component (2) can fill the stepped structure formed by the outer wall of the raised edge (3) and the base (1) during high-pressure freezing and can be removed during the pre-thinning process. Both the protruding edge (3) and the filling component (2) are annular structures. The filling component (2) can be fitted onto the outer wall of the protruding edge (3). The annular structure is circular before the pre-thinning and semi-circular or semi-circular after the pre-thinning, exposing the surface of the frozen sample.

2. The sample stage for cryo-electron microscopy imaging according to claim 1, characterized in that, The center of the protruding edge (3) coincides with the center of the base (1).

3. The sample stage for cryo-electron microscopy imaging according to claim 1, characterized in that, The height of the filling component (2) is the same as the height of the outer wall of the protrusion (3).

4. The sample stage for cryo-electron microscopy imaging according to claim 1, characterized in that, The base (1) and filling component (2) are made of thermally conductive metal material.

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