CN114486432B - Novel high-flux semilunar shaped carrier net for freezing double-beam extraction transmission electron microscope sample - Google Patents

Abstract

The invention provides a new type of high-throughput half-moon-shaped carrier grid for frozen double-beam extraction of transmission electron microscope samples, which includes a half-moon-shaped gold, copper or molybdenum carrier grid. Four teeth are provided at the chord of the carrier grid, and the four teeth are vertical. on the string; there is a gap between adjacent teeth; the teeth are also provided with gaps perpendicular to the string; or, there are multiple gaps at the string of the load net, the gaps are perpendicular to the string, and adjacent gaps are formed between Long teeth. The invention provides a new carrier grid that is reliable, has a simple structure, is easy to process, and is easy to use. The carrier grid is combined with a cryo-focused ion beam to realize the preparation of high-quality, high-throughput cryo-transmission electron microscope samples.

Description

A new high-throughput half-moon carrier grid for cryogenic double-beam extraction of transmission electron microscopy samples

Technical field

The invention provides a new high-throughput half-moon carrier network for freezing double-beam extraction of transmission electron microscope samples, and belongs to the technical field of cryo-electron tomography.

Background technique

Cryoelectron tomography is a high-resolution, cross-scale in-situ cryo-electron microscopy technology that can obtain in-situ three-dimensional high-resolution ultrastructure of cells and tissue samples, in-situ structural information of biological macromolecules, and in-situ interactions of protein machines. function information. Compared with the traditional cryo-electron microscopy method, the raw data of cryo-electron tomography adds precise Z-axis information, so the requirements for sample purity and assembly stringency are lower, and there is no need to remove the sample from its native environment during the preparation process. The structure is more physiological. As it becomes more complete, gaps in structural biology from viruses to bacteria, cells and even tissue dimensions will be filled. However, this technology requires that the thickness of the sample must be below 300nm, and obtaining high-resolution information requires a thinner sample below 150nm. However, in order to study the structure of biological tissue samples in situ, it is usually necessary to use high-pressure freezing and other technologies to conduct biological tissue samples. Freeze fixed. The thickness of fixed samples is generally around 100 μm. How to prepare high-quality biological tissue sample sections suitable for cryo-electron tomography research is an important technical issue facing the field of in situ structural biology. The latest nanomechanical extraction technology in cryo-focused ion beam can realize slice extraction of biological tissue samples under full freezing conditions, solving the difficulty of preparing thick biological tissue samples.

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

It can be seen from the sample preparation steps that, unlike conventional cryo-focused ion beam etching, extracting cryo-TEM samples not only involves sample etching and thinning, but also involves the transfer and bonding of cryo-sections, so the design of the new carrier grid appears to be It is particularly important and closely related to the quality and efficiency of frozen sample extraction, which directly determines whether the experiment can be carried out. The common commercial focused ion beam carrier grid is generally half-moon-shaped, with four teeth in the middle of the half-moon, so the extracted slices can only be fixed to one side of the teeth. Only one end of the sample is fixed, which makes the sheet easily bent and twisted due to stress during the thinning process, affecting the quality of the sheet and ultimately the quality of transmission electron microscopy data collection. On the other hand, due to the limitation of the needle insertion angle, only four samples can be adhered at one time, which is inefficient and cannot meet the preparation requirements of a large number of samples. Therefore, there is an urgent need for new carrier grids to meet the requirements for the preparation of high-quality frozen sections and, if conditions permit, to increase throughput as much as possible.

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

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

Contents of the invention

In order to overcome the above-mentioned shortcomings of the existing technology of traditional commercial focused ion beam grids, the purpose of the present invention is to provide a new high-throughput half-moon shaped grid for frozen double-beam extraction of transmission electron microscopy samples, which grid can achieve dicing Both ends are fixed at the same time, making it less likely to bend and curl after thinning. At the same time, it can realize the bonding of multiple samples on one carrier network, improving the throughput of frozen sample extraction. This new type of cryo-focused ion beam carrier grid has the characteristics of high sample quality, high throughput, simple structure, easy processing, convenient use, and high matching with the original sample stage.

In order to solve the problems that the existing commercial carrier grid cannot be bonded at both ends and has low flux, the present invention provides a new high-throughput half-moon-shaped carrier grid for frozen double-beam extraction of transmission electron microscopy samples, including half-moon-shaped gold, copper or molybdenum. Carrier network, the radius of the carrier network is 1.5mm.

There are two structures:

Four teeth are provided at the string of the loaded net; there are gaps between adjacent teeth; there are also gaps perpendicular to the string on the teeth; each tooth is 0.16mm wide and 0.8mm long; so The width of the gap is 0.008-0.03mm, and the length is 0.3-0.8mm. The width of the gap is adjustable.

Alternatively, a plurality of gaps are provided at the chords of the loaded 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 teeth is adjustable.

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

1. Put the sample on the surface of the tooth without any gaps, for example, cut a groove in the middle of the tooth, and then clamp the cut piece in the groove to fix it.

2. The gap is not in the form of a rectangle, for example, the sample is loaded in a triangle, circle, etc. shape.

3. Based on the present invention, make appropriate modifications to the designed dimensions to achieve similar functions.

4. Design using carrier nets of other materials based on the present invention.

The invention provides a new carrier grid with strong scalability, high throughput, simple structure, easy processing, and convenient use. The carrier grid is combined with a cryo-focused ion beam to realize high-quality, high-throughput frozen transmission electron microscopy samples. preparation.

Description of the drawings

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

Figure 2 is a scanning electron microscope image of a sample bonded to a commercial half-moon carrier grid in the prior art;

Figure 3 is a scanning electron microscope image of a thinned sample mounted on a commercial half-moon carrier network in the prior art;

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

Figure 5 is a scanning electron microscope photo of the first carrier network of the present invention;

Figure 6 is a scanning electron microscope photo of the first carrier network of the present invention after carrying a sample;

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

Figure 8 is a scanning electron microscope photo of the second type of carrier network of the present invention;

Figure 9 is a scanning electron microscope photograph of the second type of carrier network of the present invention after loading a sample.

Detailed ways

The specific technical solutions of the present invention will be described with reference to examples.

A new type of high-throughput half-moon-shaped carrier grid for frozen double-beam extraction of transmission electron microscopy samples includes a half-moon-shaped gold, copper or molybdenum carrier grid 1, 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 0.16mm and a length of 0.8mm on a carrier network 1 with a radius of 1.5mm, and the gap 3 between the teeth 2 is 0.12mm. On this basis, a gap 4 with a width of 0.008-0.03mm and a length of 0.3-0.8mm is processed 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. Its structural diagram is shown in Figure 4, and its physical scanning electron microscope photo is shown in Figure 5. The scanning electron microscope after loading the sample The photo is shown in Figure 6. The examples in Figures 4 to 6 are 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 gap 4 is 0.008mm. The design basis is that when collecting transmission electron microscopy data, the width of the slice should not be less than 0.008mm. The selection of the maximum width of 0.03mm is based on the efficiency of the Ga ion beam in cutting the sample and the firmness of the bonding when extracting the sample. Cutting with a width that is too large will be more time-consuming. In addition, cutting into pieces that are too heavy will also cause problems when extracting the sample. fall off. The width of the tooth gap 4 can also be selected according to the size of the sample. If other dual-beam electron microscopes with ion sources with high cutting efficiency are used, such as xenon ion sources, the width range can be increased to 0.008-0.1mm. The length of gap 4 is selected based on the length of the sample that can be tilted at a large angle during cryo-electron microscopy data collection without blocking the sample.

The second method is to directly process multiple gaps 6 with a width of 0.008-0.03mm and a length of 0.3-0.8mm on the carrier network 1 with a radius of 1.5mm, the long teeth 5 and the gaps 6 between each two gaps 6 The quantity is adjustable. Its structural schematic is shown in Figure 7, and its physical scanning electron microscope photo is shown in Figure 8. The SEM photo after loading the sample is shown in Figure 9. The examples in Figures 7 to 9 are only one of the implementation cases in this type of carrier network. The number of gaps 6 and the long teeth 5 in the present invention are not limited to this implementation example. The present invention is not limited to this implementation example. scope. The minimum value of the gap 6 width of 0.008mm is designed based on the fact that the width of the slice is not less than 0.008mm when collecting transmission electron microscopy data. The maximum value of the gap 6 width of 0.03mm is selected based on the efficiency of the Ga ion beam cutting the sample and the viscosity when extracting the sample. Determined by the firmness of the connection, it is more time-consuming to cut when the width is too large. In addition, cutting into pieces that are too heavy will cause the sample to fall off when extracting the sample. The width of the gaps in the teeth can also be selected based on the size of the sample. The selection of the gap length is based on the length that can be tilted at a large angle without blocking the sample when collecting cryo-EM data. If other ion source dual-beam electron microscopes with high cutting efficiency are used, such as xenon ion sources, the width range of gap 6 can be increased to 0.008-0.1mm.

The first type of carrier net can realize bonding on both sides, and similarly, the gap of the second type of carrier net can also realize bonding on both sides and load at least 8 samples, which greatly improves the sample preparation efficiency.

The two types of carrier grids are not limited to the preparation of frozen double-beam electron microscope samples, but are also suitable for the preparation of normal temperature double-beam electron microscope samples.

The two types of carrier nets are not limited to the sizes in the implementation cases. The width and length of the gaps, the tooth width and length, the size of the tooth spacing, the number of gaps, etc. are all adjustable. Fine adjustments in size are left in this patent. within the scope of protection.

Claims (5)

1. A new type of high-throughput half-moon shaped carrier grid for freezing double-beam extraction of transmission electron microscopy samples, which is characterized by including a carrier grid (1) with four teeth (2) at the chord of the carrier grid (1); adjacent There is a gap (3) between the teeth (2); the teeth (2) are also provided with a gap (4) perpendicular to the string. 2. The novel high-throughput half-moon-shaped carrier grid for frozen double-beam extraction of transmission electron microscopy samples according to claim 1, characterized in that the material of the carrier grid (1) is gold, copper or molybdenum. 3. The novel high-throughput half-moon-shaped carrier grid for freezing double-beam extraction of transmission electron microscopy samples according to claim 1 or 2, characterized in that the radius of the carrier grid (1) is 1.5 mm. 4. The novel high-throughput half-moon carrier grid for frozen double-beam extraction of transmission electron microscopy samples according to claim 3, characterized in that each of the teeth (2) is 0.16 mm wide and 0.8 mm long; The width of the gap (4) is 0.008-0.03 mm, and the length is 0.3-0.8 mm. 5. The novel high-throughput half-moon carrier grid for freezing double-beam extraction of transmission electron microscopy samples according to claim 4, characterized in that the width of the gap (3) is adjustable.