CN114486432A - Novel high-flux half-moon-shaped carrier net for freezing double-beam extraction of transmission electron microscope sample - Google Patents

Abstract

The invention provides a novel high-flux half-moon-shaped grid for freezing double-beam extraction of a transmission electron microscope sample, which comprises a half-moon-shaped gold, copper or molybdenum grid, wherein four teeth are arranged at the chord position of the grid, and the four teeth are vertical to the chord; gaps are reserved between adjacent teeth; the teeth are also provided with gaps vertical to the strings; or a plurality of gaps are formed at the chord position of the grid, the gaps are perpendicular to the chord, and long teeth are formed between adjacent gaps. The invention provides a novel grid which is simple in structure, convenient to process and convenient to use, and the grid can be combined with a frozen focused ion beam to realize the preparation of a high-quality and high-flux frozen transmission electron microscope sample.

Description

Novel high-throughput meniscus grid for cryo-dual-beam extraction of TEM samples

technical field

The invention provides a novel high-throughput half-moon-shaped carrier net for freezing double-beam extraction transmission electron microscope samples, belonging to the technical field of cryo-electron tomography.

Background technique

Cryo-electron tomography is a high-resolution, cross-scale in situ cryo-electron microscopy technique, which can obtain in situ three-dimensional high-resolution ultrastructure of cell and tissue samples, in situ structural information of biological macromolecules, and in situ interaction of protein machinery. Action information. Compared with the traditional cryo-electron microscopy method, the raw data of cryo-electron tomography adds accurate Z-axis information, so the requirements for sample purity and assembly rigor are lower, and there is no need to remove the sample from the original environment during the preparation of the sample. The structure is more physiological. As it becomes more refined, gaps in structural biology will be filled, from viruses to bacteria, cells and even tissue dimensions. However, this technique requires that the thickness of the sample must be below 300 nm, and to obtain high-resolution information, a thinner sample of 150 nm or less is required. However, in order to study the structure of biological tissue samples in situ, it is usually necessary to use high-pressure freezing and other techniques for biological tissue samples. Freeze fixed. The thickness of fixed samples is generally about 100 μm. How to prepare high-quality biological tissue sample slices suitable for cryo-electron tomography research is an important technical problem in the field of in situ structural biology. The nanomechanical extraction technology in the latest frozen focused ion beam can realize slice extraction of biological tissue samples under the condition of full freezing, which solves the difficulty of preparing thick biological tissue samples.

The general steps for preparing cryo-TEM samples by cryo-focused ion beam nanomechanical extraction are: 1. Deposit a platinum (Pt) protective layer on the surface of the biological sample; 2. Etch the region of interest with an ion beam to form a Cut the block; 3. Etch the bottom and both sides of the block with an ion beam, leaving only a few microns on one side to connect to the block as a support; 4. Place the tip of the manipulator (usually a tungsten needle tip) close to the block, using Pt Deposition or reverse deposition method to stick the flake on the tip of the tungsten needle; 5. The side left in step 3 is etched away with an ion beam to disengage the dicing block from the sample; 6. The dicing block is close to a half-moon shape by a mechanical control system 7. Use the reverse deposition method to stick the cut pieces on the focused ion beam carrier grid; 8. Use the focused ion beam to cut and separate the tungsten needle and the slice to complete the sample extraction; 9. Use the focused ion beam to remove the slice Further thinning to below 200 nm completes the preparation of frozen TEM samples.

It can be seen from the sample preparation steps that, different from conventional frozen focused ion beam etching, the extraction of frozen TEM samples involves not only sample etching and thinning, but also the transfer and bonding of frozen cut pieces. It is particularly important, which is closely related to the quality and efficiency of frozen sample extraction, and directly determines whether the experiment can be carried out. The carrier grid of common commercial focused ion beams is generally half-moon-shaped, and there are four teeth in the middle of the half-moon, so the extracted cut can only be fixed to one side of the teeth. Only one end of the sample is fixed, which makes the thin sheet extremely vulnerable to bending and twisting during the thinning process, which affects the quality of the thin sheet and ultimately the quality of TEM data collection. On the other hand, due to the limitation of the needle entry angle, only 4 samples can be adhered at the same time at one time, which is inefficient and cannot meet the preparation requirements of a large number of samples. Therefore, a new carrier grid is urgently needed to meet the preparation of high-quality cryosections, and to maximize the throughput when conditions permit.

As shown in the schematic diagram of Fig. 1, the existing commercial half-moon-shaped net carrier has a four-tooth structure, the width of the teeth is about 0.16mm, the length of the teeth is about 0.8mm, and the spacing between the teeth is about 0.12mm. As shown in Figure 2, after the frozen sample is extracted, it is bonded to the side of the tooth to realize the transfer of the sample.

Because the spacing between the teeth of the existing commercial carrier mesh is too large, usually greater than 0.16mm (the cutting area is usually 3-40μm), it cannot be mounted on both sides, and the frozen sample can only be bonded to one side of the teeth, so only one end can support the sample , so the bending and curling of the sample will occur during the thinning process, and the thinner the sample, the more serious the bending and curling, as shown in Figure 3. The bending and curling of the sample during the thinning process will have the following shortcomings: (1) The sample cannot continue to be thinned. If the thinning continues, the sheet will be unevenly stressed and damaged, and the usable area of the sheet will be greatly reduced. (2) Bending or curling affects the quality of TEM data collection. In addition, since the commercial half-moon-shaped carrier grid can only be bonded on one side, the existing commercial carrier grid can load up to 4 samples at a time, and the throughput is low, which leads to the need to replace the carrier grid repeatedly when preparing samples, which affects experimental efficiency.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned deficiencies of the conventional commercialized focused ion beam carrier grids, the object of the present invention is to provide a novel high-throughput meniscus carrier grid for freezing double-beam extraction TEM samples, and the carrier grid can realize dicing Both ends are fixed at the same time, and it is not easy to bend and curl after thinning. At the same time, the bonding of multiple samples on one carrier network can be realized, which improves the throughput of frozen sample extraction. The novel frozen focused ion beam carrier grid has the characteristics of high quality of prepared samples, high throughput, simple structure, easy processing, convenient use, and high matching degree with the original sample stage.

Aiming at the problems that the existing commercial carrier nets cannot be bonded at both ends and the flux is low, the present invention provides a novel high-throughput half-moon-shaped carrier network for freezing double-beam extraction transmission electron microscopy samples, including half-moon-shaped gold, copper or molybdenum The carrier net, the radius of the carrier net is 1.5mm.

There are two structures:

There are four teeth at the chord of the carrier net; a gap is left between adjacent teeth; a gap perpendicular to the chord is also opened on the teeth; each of the teeth is 0.16mm wide and 0.8mm long; Said gap is 0.008-0.03mm wide and 0.3-0.8mm long. The width of the gap is adjustable.

Alternatively, a plurality of gaps are opened at the chords of the carrier net, the gaps are perpendicular to the chords, and long teeth are formed between adjacent gaps. The width of the gap is 0.08-0.03mm, and the length is 0.3-0.8mm. The width of the long tooth is adjustable.

The present invention can also be implemented in other ways, such as:

1. Lay the sample on the surface of the tooth in the form of no gap, for example, cut a groove in the middle of the tooth, and then clamp the cut block in the groove to fix it.

2. The voids are not in the form of rectangles, such as triangles, circles and other shapes for sample loading.

3. On the basis of the present invention, the size of the design is appropriately modified to achieve similar functions.

4. On the basis of the present invention, the carrier net of other materials is used for design.

The invention provides a novel carrier network with strong scalability, high flux, simple structure, convenient processing and convenient use. The carrier network is combined with a frozen focused ion beam, which can realize high-quality and high-flux frozen transmission electron microscope samples. preparation.

Description of drawings

1 is a schematic diagram of a commercialized half-moon carrier network in the prior art;

Fig. 2 is the scanning electron microscope image after the commercialized half-moon-shaped carrier grid has bonded samples in the prior art;

Fig. 3 is the scanning electron microscope image after the thinning of the sample carried on the commercialized half-moon-shaped carrier net in the prior art;

4 is a schematic diagram of a first carrier network structure of the present invention;

Fig. 5 is the scanning electron microscope photograph of the first kind of carrier network of the present invention;

Fig. 6 is the scanning electron microscope photograph after the first kind of carrier net of the present invention is loaded with sample;

7 is a schematic diagram of a second carrier network structure of the present invention;

Fig. 8 is the scanning electron microscope photograph of the second kind of carrier network of the present invention;

FIG. 9 is a scanning electron microscope photograph of the second type of carrier grid of the present invention after the sample is mounted.

Detailed ways

The specific technical solutions of the present invention are described with reference to the embodiments.

The novel high-throughput half-moon-shaped carrier grid for freezing double-beam extraction TEM samples includes a half-moon-shaped carrier grid 1 of gold, copper or molybdenum, and the radius of the carrier grid 1 is 1.5 mm.

There are two structures:

The first is to process four teeth 2 with a width of 0.16 mm and a length of 0.8 mm on a carrier net 1 with a radius of 1.5 mm, and the gap 3 between the teeth 2 is 0.12 mm. On this basis, a gap 4 with a width of 0.008-0.03mm and a length of 0.3-0.8mm is machined on each tooth 2 .

The extracted sample can be fixed at both ends at the same time, which solves the problem of bending and curling of the sample during thinning. The schematic diagram of its structure is shown in Figure 4, and its actual SEM photo is shown in Figure 5. The photo is shown in Figure 6. The example in FIG. 4 to FIG. 6 is only one of the implementation cases in this type of carrier network, and the present invention is not limited to the scope of this implementation example.

The minimum value of the width of the slit 4 is 0.008mm, and the design is based on the fact that the width of the sheet is not less than 0.008mm when the transmission electron microscope data is collected. The selection of the maximum width of 0.03mm is based on the efficiency of Ga ion beam cutting the sample and the firmness of the adhesion when extracting the sample. If the width value is too large, it will take more time to cut. In addition, if the cutting block is too heavy, it will also cause the sample to be extracted when the sample is extracted. fall off. The width of the tooth gap 4 can also be selected according to the size of the sample. If other ion source dual beam electron microscopes with high cutting efficiency are used, such as xenon ion source, the width range can be increased to 0.008-0.1mm. The length of the slit 4 is selected according to the length of tilting at a large angle without blocking the sample during the collection of cryo-EM data.

The second is to directly process a plurality of gaps 6 with a width of 0.008-0.03mm and a length of 0.3-0.8mm on the carrier net 1 with a radius of 1.5mm, and long teeth 5 and gaps 6 between each two gaps 6 The number can be adjusted. The schematic diagram of its structure is shown in FIG. 7 , and its actual scanning electron microscope photo is shown in FIG. 8 . The SEM photo of the loaded sample is shown in Figure 9. The example in FIG. 7 to FIG. 9 is only one of the implementation cases in this type of carrier network. The number of the gaps 6 and the long teeth 5 in the present invention are not limited to this implementation example, and the present invention is not limited to this implementation example. scope. The minimum value of the width of the gap 6 is 0.008mm, and the width of the sheet is not less than 0.008mm when the TEM data is collected, and the maximum value of the width of the gap 6 is 0.03mm. It is determined by the firmness of the connection. If the width value is too large, it will take more time to cut. In addition, if the cutting block is too heavy, the sample will fall off when the sample is extracted. The width of the voids in the teeth can also be selected according to the size of the sample. The choice of the gap length is based on the length of the large tilt angle without obscuring the sample during cryo-EM data collection. If other ion source dual beam electron microscopes with high cutting efficiency are used, such as xenon ion source, the width of the gap 6 can be increased to 0.008-0.1mm.

The first type of carrier net can be bonded on both sides, and the gaps of the second carrier net can also be bonded on both sides and loaded with at least 8 samples, which greatly improves the sample preparation efficiency.

The two types of carrier grids described are not limited to the preparation of frozen double-beam electron microscope samples, but are also applicable to the preparation of double-beam electron microscope samples at room temperature.

The two types of carrier nets described are not limited to the dimensions in the implementation case. The width and length of the gap, the width and length of the teeth, the size of the tooth spacing, the number of gaps, etc. are all adjustable, and the fine adjustment of the size is thrown in this patent. within the scope of protection.

Claims (7)

1. The novel high-throughput half-moon-shaped carrier net of the frozen double-beam extraction TEM sample is characterized in that, it comprises a carrier net (1), and is provided with four teeth (2) at the chord of the carrier net (1); adjacent A gap (3) is left between the teeth (2) of the tooth (2); a gap (4) perpendicular to the chord is also opened on the teeth (2); Alternatively, a plurality of gaps (6) are opened at the chord of the carrier net (1), the gaps (6) are perpendicular to the chord, and long teeth (5) are formed between adjacent gaps (6). 2 . The novel high-throughput half-moon-shaped carrier grid for freezing double-beam extraction TEM samples according to claim 1 , wherein the carrier grid (1) is made of gold, copper or molybdenum. 3 . 3. The novel high-throughput half-moon-shaped carrier net for freezing double-beam extraction TEM samples according to claim 1 or 2, characterized in that, the radius of the carrier net (1) is 1.5 mm. 4. The novel high-throughput half-moon-shaped carrier screen for freezing double-beam extraction TEM samples according to claim 3, wherein each of the teeth (2) is 0.16mm wide and 0.8mm long; Said gap (4) is 0.008-0.03mm wide and 0.3-0.8mm long. 5. The novel high-flux half-moon-shaped carrier net for freezing double-beam extraction TEM samples according to claim 4, characterized in that the width of the gap (3) is adjustable. 6. The novel high-throughput half-moon-shaped carrier screen for freezing double-beam extraction TEM samples according to claim 3, wherein the width of the gap (6) is 0.08-0.03mm, and the length is 0.3-0.8mm . 7 . The novel high-throughput half-moon-shaped carrier net for freezing double-beam extraction TEM samples according to claim 6 , wherein the width of the elongated teeth ( 5 ) is adjustable. 8 .

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