CN114486432B - Novel high-flux semilunar shaped carrier net for freezing double-beam extraction transmission electron microscope sample - Google Patents
Novel high-flux semilunar shaped carrier net for freezing double-beam extraction transmission electron microscope sample Download PDFInfo
- Publication number
- CN114486432B CN114486432B CN202210074439.3A CN202210074439A CN114486432B CN 114486432 B CN114486432 B CN 114486432B CN 202210074439 A CN202210074439 A CN 202210074439A CN 114486432 B CN114486432 B CN 114486432B
- Authority
- CN
- China
- Prior art keywords
- electron microscope
- transmission electron
- sample
- teeth
- net
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N23/20008—Constructional details of analysers, e.g. characterised by X-ray source, detector or optical system; Accessories therefor; Preparing specimens therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention provides a novel high-flux semilunar shaped carrier net for freezing double-beam extraction transmission electron microscope samples, which comprises a semilunar shaped gold, copper or molybdenum carrier net, wherein four teeth are arranged at the chord of the carrier net and are perpendicular to the chord; gaps are reserved between adjacent teeth; the teeth are also provided with slits perpendicular to the strings; or a plurality of gaps are formed at the positions of the strings of the carrier net, the gaps are perpendicular to the strings, and strip teeth are formed between the adjacent gaps. The invention provides a novel carrying net which is simple in structure, convenient to process and convenient to use, and can be combined with a frozen focused ion beam to prepare a high-quality and high-flux frozen transmission electron microscope sample.
Description
Technical Field
The invention provides a novel high-flux semilunar shaped carrier net for freezing double-beam extraction transmission electron microscope samples, and belongs to the technical field of freezing 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 biological macromolecules and in-situ interaction information of protein machines. Compared with the traditional freeze electron microscope method, the original data of the freeze electron tomography increases accurate Z-axis information, 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 process of preparing the sample, and the obtained structure has more physiological significance. As it becomes increasingly perfect, the gap in structural biology from viral to bacterial, cellular and even tissue dimensions will be filled. However, this technique requires that the thickness of the sample must be 300nm or less and that the sample be thinner than 150nm for obtaining high resolution information, but in order to study the structure of the biological tissue sample in situ, it is generally necessary to freeze-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 high-quality biological tissue sample slices suitable for the research of the frozen electron tomography technology is an important technical problem facing the field of in-situ structure biology. The latest nano mechanical extraction technology in the frozen focused ion beam can realize the slice extraction of the biological tissue sample under the whole-process frozen condition, and solves the difficulty of thicker biological tissue sample preparation.
The general steps for preparing frozen transmission electron microscope samples by adopting frozen focused ion beam nano mechanical extraction technology are as follows: 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; 3. etching the bottom and two sides of the cut block by using an ion beam, wherein only one side is connected with the cut block by a few micrometers to serve as a support; 4. the tip of a manipulator (usually a tungsten needle point) is close to the cutting block, and a Pt deposition or reverse deposition method is adopted to adhere the thin sheet to the tungsten needle point; 5. etching one side left in the step 3 by using an ion beam, and separating the cut piece from the sample; 6. cutting into pieces close to one tooth of the half-moon-shaped carrier net through a mechanical control system; 7. sticking the cut blocks on a focused ion beam carrier net by adopting a reverse deposition method; 8. cutting off and separating the tungsten needle from the thin sheet by adopting a focused ion beam to finish sample extraction; 9. and adopting a focused ion beam to further thin the sheet to below 200nm to finish the preparation of the frozen transmission electron microscope sample.
It can be seen from the sample preparation step that, unlike conventional frozen focused ion beam etching, the extraction of frozen transmission electron microscope samples involves not only sample etching thinning but also transfer and bonding of frozen sections, so the design of the novel carrier net is particularly important, and is closely related to the quality and efficiency of frozen sample extraction, and whether directly determined experiments can be performed 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-month so the extracted cut pieces can only be affixed to one side of the teeth. The sample is fixed at one end only, so that the thin sheet is extremely easy to bend and twist due to stress in the thinning process of the sample, the quality of the thin sheet is affected, and finally the quality of transmission electron microscope data collection is affected. On the other hand, due to the limitation of the needle insertion angle, only 4 samples can be adhered at one time, the efficiency is low, and the preparation requirement of a large number of samples cannot be realized. There is therefore an urgent need for new carrier webs to meet the high quality frozen section preparation and to maximize throughput where conditions permit.
As shown in the schematic diagram of FIG. 1, the conventional commercial half-moon shaped carrier has a four-tooth structure with a tooth width of about 0.16mm, a length of about 0.8mm, and a tooth space of about 0.12mm. After the frozen sample is extracted, it is glued to the sides of the teeth, as shown in fig. 2, and the transfer of the sample is achieved.
Since the existing commercial carrier mesh has too large a tooth space, usually more than 0.16mm (the dicing area is usually 3-40 μm), two-side carrying cannot be realized, the frozen sample can only be adhered to one side of the teeth, so that only one end can support the sample, bending and curling of the sample can occur during thinning, and the thinner the sample is, the more serious the bending and curling is, as shown in fig. 3. The sample is bent and curled during the thinning process, and the following defects are caused: (1) If the sample is continuously thinned, the tablet is stressed unevenly and damaged, and the usable area of the tablet is greatly reduced. (2) After bending or curling, the quality of the transmission electron microscope data collection is affected. In addition, because the commercial half-moon-shaped carrier net can only be bonded on one side, the current commercial carrier net can be loaded with 4 samples at most each time, the flux is low, the carrier net needs to be replaced repeatedly when the samples are prepared, and the experimental efficiency is affected.
Disclosure of Invention
In order to overcome the defects of the prior art of the traditional commercial focused ion beam carrier net, the invention aims to provide a novel high-flux half-moon-shaped carrier net for freezing double-beam extraction transmission electron microscope samples, which can realize the simultaneous fixation of two ends of a cut block and is not easy to bend and curl after thinning. Meanwhile, the bonding of a plurality of samples of one carrier net can be realized, and the flux of frozen sample extraction is improved. The novel frozen focused ion beam carrier network has the characteristics of high quality of prepared samples, high flux, simple structure, easy processing, convenient use and high matching degree with the original sample stage.
Aiming at the problems that the two ends of the existing commercial carrier net cannot be adhered and the flux is low, the novel high-flux half-moon-shaped carrier net for frozen double-beam extraction transmission electron microscope samples provided by the invention comprises half-moon-shaped gold, copper or molybdenum carrier nets, and the radius of the carrier net is 1.5mm.
There are two structures:
four teeth are arranged at the chord of the carrying net; gaps are reserved between adjacent teeth; the teeth are also provided with slits perpendicular to the strings; each tooth width is 0.16mm, and the length is 0.8mm; the gap width is 0.008-0.03mm, and the length is 0.3-0.8mm. The width of the gap is adjustable.
Or a plurality of gaps are formed at the positions of the strings of the carrier net, the gaps are perpendicular to the strings, and strip teeth are formed between the adjacent gaps. The width of the gap is 0.08-0.03mm, and the length is 0.3-0.8mm. The width of the strip teeth is adjustable.
The invention may also be embodied in other forms, such as:
1. the sample is put on the surface of the tooth without a gap, for example, a groove is formed in the middle of the tooth, and then the cut piece is clamped in the groove to play a role of fixing.
2. The voids are not rectangular, and are formed in a triangular shape, a circular shape, or the like, for example, for sample mounting.
3. The invention is based on the design size is modified to achieve similar functions.
4. The invention is designed by adopting a carrier net of other materials.
The invention provides a novel carrying net which has strong expandability, high flux, simple structure, convenient processing and convenient use, and can realize the preparation of high-quality and high-flux frozen transmission electron microscope samples by combining frozen focused ion beams.
Drawings
FIG. 1 is a schematic diagram of a prior art commercial half moon carrier network;
FIG. 2 is a scanning electron microscope image of a prior art commercial half-moon shaped carrier web bonded sample;
FIG. 3 is a scanning electron microscope image of a sample carried by a commercial half-moon shaped carrying net after thinning in the prior art;
FIG. 4 is a schematic view of a first carrier network according to the present invention;
FIG. 5 is a scanning electron microscope photograph of a first carrier web of the present invention;
FIG. 6 is a scanning electron microscope photograph of a first type of the present invention after a sample is carried on a carrier web;
FIG. 7 is a schematic diagram of a second carrier network according to the present invention;
FIG. 8 is a scanning electron microscope photograph of a second carrier web of the present invention;
fig. 9 is a scanning electron microscope photograph of a second type of the present invention after a sample is carried on a carrier web.
Detailed Description
The specific technical scheme of the invention is described by combining the embodiments.
The novel high-flux semilunar shaped carrier net for frozen double-beam extraction transmission electron microscope samples comprises a semilunar shaped gold, copper or molybdenum carrier net 1, wherein the radius of the carrier net 1 is 1.5mm.
There are two structures:
the first is to machine four teeth 2 with a width of 0.16mm and a length of 0.8mm on a carrier web 1 with a radius of 1.5mm, the gaps 3 between the teeth 2 being 0.12mm. 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 realize the simultaneous fixation of two ends, solves the problems of bending and curling of the sample during thinning, and has a structure shown in a schematic diagram in fig. 4, an actual scanning electron microscope photo shown in fig. 5, and a scanning electron microscope photo after carrying the sample shown in fig. 6. The examples of fig. 4-6 are only one of the embodiments in such a carrier network, and the invention is not limited in scope to this embodiment.
Wherein the minimum value of the width of the gap 4 is 0.008mm, and the design basis is that the width of the sheet is not less than 0.008mm when the transmission electron microscope data is collected. The maximum width of 0.03mm is determined according to the efficiency of cutting the sample by the Ga ion beam and the firmness of bonding when extracting the sample, the cutting is relatively time-consuming when the width is too large, and the sample is also dropped when extracting the sample due to too heavy cutting. The width of the tooth gap 4 may 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 0.008-0.1mm. The length of the gap 4 is selected according to the length of the sample which is not blocked by the large-angle tilting during the data collection of the frozen electron microscope.
The second is to directly process a plurality of gaps 6 with the width of 0.008-0.03mm and the length of 0.3-0.8mm on a 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 are adjustable. The schematic structure is shown in fig. 7, and the actual scanning electron microscope photograph is shown in fig. 8. Fig. 9 shows a scanning electron microscope photograph after mounting the sample. The example of fig. 7-9 is only one of the embodiments of the carrier network, and the number of voids 6 and the elongated teeth 5 of the present invention are not limited to this embodiment, but are 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 the transmission electron microscope data is used for collecting data, the maximum value of the width of the gap 6 is 0.03mm, the selection is determined according to the efficiency of cutting samples by the Ga ion beam and the firmness of bonding when the samples are extracted, the time is relatively good when the width value is too large for cutting, and in addition, the samples fall off when the samples are extracted due to too heavy cutting. The width of the gap in the teeth may also be selected according to the size of the sample. The gap length is selected according to the length of the sample which is inclined at a large angle without shielding during the data collection of the cryo-electron microscope. If other ion source double beam electron microscope with high cutting efficiency is adopted, such as xenon ion source, the width of the gap 6 can be increased to 0.008-0.1mm.
The first type of carrying net can realize two-sided bonding, and the second type of carrying net gap can also realize two-sided bonding and at least load 8 samples, so that the sample preparation efficiency is greatly improved.
The two kinds of carrier nets are not only limited to frozen double-beam electron microscope sample preparation, but also are applicable to normal-temperature double-beam electron microscope sample preparation.
The two kinds of carrier nets are not limited to the sizes in the embodiment, the widths and lengths of the gaps, the widths and lengths of the teeth, the sizes of the intervals between the teeth, the number of the gaps and the like are adjustable, and the fine adjustment of the sizes is thrown into the protection scope of the patent.
Claims (5)
1. The novel high-flux half-moon shaped carrying net for freezing double-beam extraction transmission electron microscope samples is characterized by comprising a carrying net (1), wherein four teeth (2) are arranged at the chord position of the carrying net (1); gaps (3) are reserved between adjacent teeth (2); the teeth (2) are also provided with slits (4) perpendicular to the strings.
2. The novel high-throughput semilunar shaped carrier net for frozen double-beam extraction transmission electron microscope samples according to claim 1, wherein the carrier net (1) is made of gold, copper or molybdenum.
3. The novel high throughput half moon shaped mesh for frozen dual beam extraction transmission electron microscope samples according to claim 1 or 2, wherein the mesh (1) has a radius of 1.5mm.
4. A novel high throughput half moon shaped carrier web for frozen dual beam extraction transmission electron microscope samples according to claim 3 wherein each of said teeth (2) is 0.16mm wide and 0.8mm long; the gap (4) is 0.008-0.03-mm in width and 0.3-0.8-mm in length.
5. The novel high throughput meniscus of frozen dual-beam extraction transmission electron microscope samples of claim 4, wherein the gap (3) is adjustable in width.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210074439.3A CN114486432B (en) | 2022-01-21 | 2022-01-21 | Novel high-flux semilunar shaped carrier net for freezing double-beam extraction transmission electron microscope sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210074439.3A CN114486432B (en) | 2022-01-21 | 2022-01-21 | Novel high-flux semilunar shaped carrier net for freezing double-beam extraction transmission electron microscope sample |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114486432A CN114486432A (en) | 2022-05-13 |
| CN114486432B true CN114486432B (en) | 2023-09-29 |
Family
ID=81473240
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210074439.3A Active CN114486432B (en) | 2022-01-21 | 2022-01-21 | Novel high-flux semilunar shaped carrier net for freezing double-beam extraction transmission electron microscope sample |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114486432B (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4815842A (en) * | 1986-05-29 | 1989-03-28 | Acton Research Corporation | Spectrograph for evaluating contamination of optical components in space |
| CA2463176A1 (en) * | 1998-04-17 | 1999-10-17 | Applera Corporation | Electrophoresis method and apparatus for separating bio-organic molecules |
| JP2004111097A (en) * | 2002-09-13 | 2004-04-08 | Sharp Corp | Sample stage for focused ion beam device and method for forming lamina sample |
| KR20070065502A (en) * | 2005-12-20 | 2007-06-25 | 삼성전자주식회사 | Manufacturing apparatus for tem-sample and manufacturing method for tem-sample using the same |
| CN201166633Y (en) * | 2008-01-18 | 2008-12-17 | 北京工业大学 | Loading network for measuring electrology of transmission electric mirror base on phase-change material |
| CN102779712A (en) * | 2012-08-07 | 2012-11-14 | 中国石油大学(北京) | Thermally driven transmission electron microscope grid made of intelligent composite material |
| JP2013164521A (en) * | 2012-02-10 | 2013-08-22 | Sharp Corp | Capsule toner manufacturing method and capsule toner |
| CN212722273U (en) * | 2020-07-07 | 2021-03-16 | 西安交通大学 | Device for extracting transmission electron microscope sample by using focused ion beam |
| CN113109370A (en) * | 2021-03-29 | 2021-07-13 | 北京大学 | Porous transmission electron microscope supporting film, ultra-flat graphene electron microscope carrier net and preparation method thereof |
| CN113218980A (en) * | 2021-04-27 | 2021-08-06 | 苏州鲲腾智能科技有限公司 | High-resolution method for synchronously acquiring and analyzing structure and component information of integrated circuit |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11370360B2 (en) * | 2019-12-25 | 2022-06-28 | Panasonic Intellectual Property Management Co., Ltd. | Display system |
-
2022
- 2022-01-21 CN CN202210074439.3A patent/CN114486432B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4815842A (en) * | 1986-05-29 | 1989-03-28 | Acton Research Corporation | Spectrograph for evaluating contamination of optical components in space |
| CA2463176A1 (en) * | 1998-04-17 | 1999-10-17 | Applera Corporation | Electrophoresis method and apparatus for separating bio-organic molecules |
| JP2004111097A (en) * | 2002-09-13 | 2004-04-08 | Sharp Corp | Sample stage for focused ion beam device and method for forming lamina sample |
| KR20070065502A (en) * | 2005-12-20 | 2007-06-25 | 삼성전자주식회사 | Manufacturing apparatus for tem-sample and manufacturing method for tem-sample using the same |
| CN201166633Y (en) * | 2008-01-18 | 2008-12-17 | 北京工业大学 | Loading network for measuring electrology of transmission electric mirror base on phase-change material |
| JP2013164521A (en) * | 2012-02-10 | 2013-08-22 | Sharp Corp | Capsule toner manufacturing method and capsule toner |
| CN102779712A (en) * | 2012-08-07 | 2012-11-14 | 中国石油大学(北京) | Thermally driven transmission electron microscope grid made of intelligent composite material |
| CN212722273U (en) * | 2020-07-07 | 2021-03-16 | 西安交通大学 | Device for extracting transmission electron microscope sample by using focused ion beam |
| CN113109370A (en) * | 2021-03-29 | 2021-07-13 | 北京大学 | Porous transmission electron microscope supporting film, ultra-flat graphene electron microscope carrier net and preparation method thereof |
| CN113218980A (en) * | 2021-04-27 | 2021-08-06 | 苏州鲲腾智能科技有限公司 | High-resolution method for synchronously acquiring and analyzing structure and component information of integrated circuit |
Non-Patent Citations (3)
| Title |
|---|
| High-Throughput Correlative Electrochemistry-Microscopy at a Transmission Electron Microscopy Grid Electrode;Isabel M. Ornelas等;《Analytical Chemistry》;第91卷;全文 * |
| 光刻胶灰化用于全息离子束刻蚀光栅制作;徐向东,洪义麟,傅绍军;《真空科学与技术》(第05期);全文 * |
| 利用冷冻聚焦离子束制备冷冻含水切片技术问题探讨;贾星;;《分析测试技术与仪器》(第03期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114486432A (en) | 2022-05-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hsieh et al. | Towards high-resolution three-dimensional imaging of native mammalian tissue: electron tomography of frozen-hydrated rat liver sections | |
| JP4625909B2 (en) | Tissue array block manufacturing method, the tissue array sheet manufacturing method, the tissue array block, tissue array chip, a tissue array block making unit, and tissue array sheet manufacturing apparatus | |
| US8293497B2 (en) | Microdissection method and microdissection system | |
| Seagull et al. | The organization of cortical microtubule arrays in the radish root hair | |
| EP3076150A1 (en) | Methods and apparatuses for sectioning and imaging samples | |
| US20140135236A1 (en) | Matrix for receiving a tissue sample and use thereof | |
| CN114486432B (en) | Novel high-flux semilunar shaped carrier net for freezing double-beam extraction transmission electron microscope sample | |
| Burghardt et al. | Advanced electron microscopic techniques provide a deeper insight into the peculiar features of podocytes | |
| CN109342288A (en) | Characterization method of shale reservoir pore space | |
| CN102967497B (en) | Position observation in plant leaf blade micromechanism and the method that blade is processed | |
| JP2003194681A (en) | Tem sample preparation method | |
| Karreman et al. | VIS2FIX: a high‐speed fixation method for immuno‐electron microscopy | |
| Esposito | Complementary techniques: laser capture microdissection—increasing specificity of gene expression profiling of cancer specimens | |
| Schalek et al. | Imaging a 1 mm3Volume of Rat Cortex Using a MultiBeam SEM | |
| Tkadletz et al. | Efficient preparation of microtip arrays for atom probe tomography using fs-laser processing | |
| Palisaitis | Use of cleaved wedge geometry for plan‐view transmission electron microscopy sample preparation | |
| CN110849921B (en) | Method for preparing interface transmission electron microscope sample of composite material | |
| Fernández et al. | Tissue macroarrays (" microchops") for gene expression analysis | |
| JP2001289747A (en) | Apparatus and method for lamina sample preparation | |
| McKenzie et al. | Focused ion beam sample preparation for atom probe tomography | |
| Kolovou et al. | A new method for cryo-sectioning cell monolayers using a correlative workflow | |
| EP2784472A1 (en) | Cell sorting device and cell sorting method | |
| CN113433346A (en) | Preparation method of transmission electron microscope thin-film material section sample based on ultrathin slice | |
| Mourik et al. | Towards the imaging of Weibel–Palade body biogenesis by serial block face‐scanning electron microscopy | |
| CN114964969A (en) | Preparation method of hundred-micron-sized transmission electron microscope sample |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |