CN114486432A - Novel high-flux half-moon-shaped carrier net for freezing double-beam extraction of transmission electron microscope sample - Google Patents
Novel high-flux half-moon-shaped carrier net for freezing double-beam extraction of transmission electron microscope sample Download PDFInfo
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- CN114486432A CN114486432A CN202210074439.3A CN202210074439A CN114486432A CN 114486432 A CN114486432 A CN 114486432A CN 202210074439 A CN202210074439 A CN 202210074439A CN 114486432 A CN114486432 A CN 114486432A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
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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
Technical Field
The invention provides a novel high-flux half-moon-shaped carrier net for freezing double-beam extraction of a transmission electron microscope sample, and belongs to the technical field of frozen electron tomography.
Background
The frozen electron tomography technology is a high-resolution and trans-scale in-situ frozen electron microscope technology, and can obtain in-situ three-dimensional high-resolution ultrastructure of cell and tissue samples, in-situ structure information of biomacromolecules and in-situ interaction information of protein machines. Compared with the traditional cryoelectron microscope method, the method has the advantages that the accurate Z-axis information is added to the original data of the frozen electron tomography, so that the requirements on the purity and the assembly strictness of the sample are lower, the sample does not need to be separated from the original environment in the sample preparation process, and the obtained structure has more physiological significance. With its increasing sophistication, the gap in structural biology from viruses to bacteria, cells and even tissue dimensions will be filled. However, this technique requires that the thickness of the sample is 300nm or less, and a thinner sample of 150nm or less is required for obtaining high-resolution information, but in order to investigate the structure of the in-situ state of the biological tissue sample, it is generally necessary to freeze and fix the biological tissue sample by a technique such as high-pressure freezing. The thickness of the fixed sample is generally about 100 μm, and how to prepare a high-quality biological tissue sample slice suitable for the research of the frozen electron tomography technology is an important technical problem in the field of in-situ structure biology. The latest nano mechanical extraction technology in the freezing focusing ion beam can realize the slicing extraction of biological tissue samples under the condition of whole-course freezing, and solves the difficulty of preparing thicker biological tissue samples.
The method for preparing the frozen transmission electron microscope sample by adopting the frozen focused ion beam nano mechanical extraction technology comprises the following general steps: 1. depositing a platinum (Pt) protective layer on the surface of the biological sample; 2. etching the region of interest by using an ion beam to form a cut block; 3. etching the bottom and two sides of the cut block by using ion beams, and only leaving a few microns on one side to be connected with the cut block as a support; 4. the tip of a mechanical arm (usually a tungsten needle tip) is close to the cutting block, and a thin slice is adhered to the tungsten needle tip by adopting a Pt deposition or reverse deposition method; 5. etching one side left in the step 3 by using ion beams, and separating the cut block from the sample; 6. cutting the block to approach one tooth of the half-moon-shaped carrier net through a mechanical control system; 7. sticking the cut blocks on a focusing ion beam carrier by adopting a reverse deposition method; 8. cutting off and separating the tungsten needle and the thin sheet by adopting focused ion beams to finish sample extraction; 9. and further thinning the thin sheet to be below 200nm by adopting focused ion beams to finish the preparation of the frozen transmission electron microscope sample.
It can be seen from the sample preparation step that, unlike conventional freezing focused ion beam etching, the extraction of the frozen transmission electron microscope sample involves not only the thinning of the sample by etching, but also the transfer and adhesion of frozen blocks, so that the design of the novel grid is particularly important, and the quality and efficiency of the extraction of the frozen sample are closely related to directly determining whether the experiment can be carried out or not. The carrier web of a common commercial focused ion beam is generally half-moon shaped with four teeth in the middle of the half-moon so that the extracted cut pieces can only be secured to one side of the teeth. Only one end of the sample is fixed, so that the thin sheet is very easy to bend and distort under stress in the thinning process of the sample, the quality of the thin sheet is influenced, and the quality of data collection of the transmission electron microscope is influenced finally. On the other hand, due to the limitation of the needle inserting angle, only 4 samples can be adhered at one time, the efficiency is low, and the preparation requirements of a large number of samples cannot be met. Therefore, new grid is urgently needed to meet the requirement of preparing high-quality frozen slices and to improve the flux as much as possible under the condition that the conditions allow.
As shown in the schematic diagram of FIG. 1, the commercial half-moon shaped mesh carrier has a four-tooth structure, a tooth width of about 0.16mm, a length of about 0.8mm, and a tooth pitch of about 0.12 mm. After the frozen sample is extracted, it is attached to the sides of the teeth, as shown in fig. 2, to effect transfer of the sample.
Because the spacing between the existing commercial mesh-carrying teeth is too large, usually larger than 0.16mm (the area of the cut block is usually 3-40 μm), two-side carrying cannot be realized, the frozen sample can only be adhered to one side of the teeth, and only one end can support the sample, so that the sample can be bent and curled in the process of thinning, and the thinner the sample, the more serious the bending and curling, as shown in fig. 3. The sample has the following defects of bending and curling in the thinning process: (1) the sample can not be thinned continuously, if the sample is thinned continuously, the stress of the sheet is uneven and the sheet is damaged, and the usable area of the sheet is greatly reduced. (2) The quality of the transmission electron microscope data collection is affected after the bending or the curling occurs. In addition, because the commercialized half-moon-shaped carrier net can only be bonded on one side, the existing commercialized carrier net can load 4 samples at most at each time, the flux is low, the carrier net needs to be replaced repeatedly when preparing samples, and the experimental efficiency is influenced.
Disclosure of Invention
In order to overcome the defects of the prior art of the traditional commercial focused ion beam grid, the invention aims to provide a novel high-flux half-moon-shaped grid for freezing a double-beam extraction transmission electron microscope sample, wherein the grid can realize the simultaneous fixation of two ends of a cut block and is not easy to bend and curl after being thinned. Meanwhile, the bonding of a plurality of samples of one grid can be realized, and the extraction flux of the frozen samples is improved. The novel freezing focusing ion beam grid has the characteristics of high quality and high flux of prepared samples, simple structure, easy processing, convenient use and high matching degree with the original sample platform.
Aiming at the problems that the two ends of the conventional commercialized carrying net cannot be bonded and the flux is low, the novel high-flux semilunar carrying net for the frozen double-beam extraction transmission electron microscope sample comprises a semilunar gold, copper or molybdenum carrying net, and the radius of the carrying net is 1.5 mm.
There are two structures:
four teeth are arranged at the chord position of the grid; gaps are reserved between adjacent teeth; the teeth are also provided with gaps vertical to the strings; each tooth is 0.16mm in width and 0.8mm in length; the width of the gap is 0.008-0.03mm, and the length is 0.3-0.8 mm. The width of the gap is adjustable.
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 width of the gap is 0.08-0.03mm, and the length is 0.3-0.8 mm. The width of the long teeth is adjustable.
The invention may also be implemented in other ways, such as:
1. the sample is not attached to the surface of the teeth by means of a gap, for example, by cutting a groove in the middle of the teeth, and then the block is clamped in the groove to play a role in fixing.
2. The gap is not rectangular, and for example, the gap is triangular or circular for sample mounting.
3. On the basis of the invention, the designed size is modified properly to realize similar functions.
4. The invention is designed by adopting the carrying net made of other materials.
The invention provides a novel grid with strong expandability, high flux, simple structure, convenient processing and convenient use, and the grid can be combined with frozen focused ion beams to realize the preparation of high-quality and high-flux frozen transmission electron microscope samples.
Drawings
FIG. 1 is a schematic view of a commercial half-moon shaped carrier web of the prior art;
FIG. 2 is a scanning electron microscope image of a commercial half-moon shaped mesh bonded sample of the prior art;
FIG. 3 is a scanning electron microscope image of a thinned sample carried by a commercial half-moon-shaped grid in the prior art;
FIG. 4 is a schematic view of a first grid structure according to the present invention;
FIG. 5 is a scanning electron micrograph of a first grid according to the present invention;
FIG. 6 is a scanning electron micrograph of a first grid according to the present invention after loading a sample thereon;
FIG. 7 is a schematic view of a second grid structure according to the present invention;
FIG. 8 is a scanning electron micrograph of a second grid according to the present invention;
FIG. 9 is a scanning electron micrograph of a sample mounted on a second mounting net according to the present invention.
Detailed Description
The specific technical scheme of the invention is described by combining the embodiment.
A novel high-flux half-moon-shaped carrier net for freezing double-beam extraction of a transmission electron microscope sample comprises a half-moon-shaped carrier net 1 made of gold, copper or molybdenum, wherein the radius of the carrier net 1 is 1.5 mm.
There are two structures:
the first is to process four teeth 2 with the width of 0.16mm and the length of 0.8mm on a carrier net 1 with the radius of 1.5mm, and the gap 3 between the teeth 2 is 0.12 mm. On the basis, a gap 4 with the width of 0.008-0.03mm and the length of 0.3-0.8mm is processed on each tooth 2.
The extracted sample can be fixed at two ends simultaneously, the problem that the sample is bent and curled during thinning is solved, the schematic structural diagram is shown in fig. 4, the scanning electron microscope photo of the sample is shown in fig. 5, and the scanning electron microscope photo after carrying the sample is shown in fig. 6. The example in fig. 4-6 is only one of the implementation examples in this type of carrier network, and the present invention is not limited to this implementation example scope.
The minimum value of 0.008mm of the width of the gap 4 is designed according to that the width of the sheet is not less than 0.008mm when data of a transmission electron microscope are collected. The maximum width of 0.03mm is selected according to the efficiency of cutting the sample by the Ga ion beam and the firmness of the bonding when the sample is extracted, the cutting with the too large width value is time-consuming, and the sample is dropped when the sample is extracted due to the too heavy cutting block. The width of the tooth gap 4 can also be selected according to the size of the sample. If other ion source double-beam electron microscope with high cutting efficiency is adopted, such as xenon ion source, the width range can be increased to 0.008-0.1 mm. The length of the gap 4 is selected based on the length of the sample that is not obscured by large angle tilts during cryo-electron microscopy data collection.
The second is that a plurality of gaps 6 with the width of 0.008-0.03mm and the length of 0.3-0.8mm are directly processed on the carrier net 1 with the radius of 1.5mm, and the number of the strip teeth 5 and the gaps 6 between every two gaps 6 can be adjusted. The structure schematic diagram is shown in fig. 7, and the scanning electron micrograph of the object is shown in fig. 8. Fig. 9 shows a scanning electron micrograph of the mounted sample. The examples in fig. 7 to 9 are only one of the embodiments of this type of carrier web, and the number of the gaps 6 and the elongated teeth 5 are not limited to this embodiment, and the scope of the present invention is not limited to this embodiment. The minimum value of the width of the gap 6 is 0.008mm, the width of the sheet is not less than 0.008mm when data of a transmission electron microscope are collected, the maximum value of the width of the gap 6 is 0.03mm, the width is determined according to the efficiency of cutting a sample by the Ga ion beam and the firmness of bonding when the sample is extracted, the time is better when the width value is too large, and the sample falls off when the sample is extracted due to too heavy cutting. The width of the gap in the tooth may also be selected based on the size of the sample. The length of the gap is selected based on the length of the sample that is tilted at a large angle without obscuring the sample during cryo-electron microscopy data collection. If other ion source double-beam electron microscope with high cutting efficiency is adopted, such as xenon ion source, the width range of the gap 6 can be increased to 0.008-0.1 mm.
The first type of net carries can realize both sides bonding, and the second type of net carries the space also can realize both sides bonding and at least loading 8 samples, improves system appearance efficiency greatly.
The two types of grid screens are not limited to the preparation of a frozen double-beam electron microscope sample, but are also suitable for the preparation of a normal-temperature double-beam electron microscope sample.
The two types of carrier webs are not limited to the dimensions of the embodiment, the width and length of the gaps, the width and length of the teeth, the size of the tooth spaces, the number of gaps, etc. are adjustable, and fine adjustment of the dimensions is within the scope of the present patent.
Claims (7)
1. The novel high-flux half-moon-shaped grid for freezing double-beam extraction of the transmission electron microscope sample is characterized by comprising a grid (1), wherein four teeth (2) are arranged at the chord positions of the grid (1); gaps (3) are reserved between adjacent teeth (2); the teeth (2) are also provided with gaps (4) vertical to the strings;
or a plurality of gaps (6) are formed in the chord position of the carrier net (1), the gaps (6) are perpendicular to the chord, and long teeth (5) are formed between the adjacent gaps (6).
2. The novel high-flux half-moon-shaped grid for the frozen dual-beam extraction of transmission electron microscope samples as claimed in claim 1, wherein the grid (1) is made of gold, copper or molybdenum.
3. The new high-flux half-moon-shaped grid for the frozen dual-beam extraction of TEM samples as claimed in claim 1 or 2, wherein the radius of the grid (1) is 1.5 mm.
4. The new high-throughput half-moon-shaped mesh for the frozen dual-beam extraction of TEM samples as claimed in claim 3, wherein each of said teeth (2) has a width of 0.16mm and a length of 0.8 mm; the width of the gap (4) is 0.008-0.03mm, and the length is 0.3-0.8 mm.
5. The new high-throughput half-moon grid for frozen dual-beam extraction of TEM samples as claimed in claim 4, wherein the width of said gap (3) is adjustable.
6. The novel high-flux half-moon-shaped mesh for the frozen dual-beam extraction of TEM samples as claimed in claim 3, wherein said gap (6) has a width of 0.08-0.03mm and a length of 0.3-0.8 mm.
7. The new high flux half-moon grid for frozen dual-beam extraction of TEM samples as claimed in claim 6, wherein the width of said elongated teeth (5) is adjustable.
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