CN111999329A - Sample table for imaging of cryoelectron microscope - Google Patents
Sample table for imaging of cryoelectron microscope Download PDFInfo
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- CN111999329A CN111999329A CN202010883086.2A CN202010883086A CN111999329A CN 111999329 A CN111999329 A CN 111999329A CN 202010883086 A CN202010883086 A CN 202010883086A CN 111999329 A CN111999329 A CN 111999329A
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- base
- sample
- imaging
- electron microscope
- freezing
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- 238000003384 imaging method Methods 0.000 title claims abstract description 27
- 239000007769 metal material Substances 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 2
- 238000001493 electron microscopy Methods 0.000 claims 3
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical group C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 claims 1
- 238000000386 microscopy Methods 0.000 claims 1
- 230000008014 freezing Effects 0.000 abstract description 27
- 238000007710 freezing Methods 0.000 abstract description 27
- 230000005540 biological transmission Effects 0.000 abstract description 5
- 239000000523 sample Substances 0.000 description 47
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000010884 ion-beam technique Methods 0.000 description 4
- 239000012472 biological sample Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000011065 in-situ storage Methods 0.000 description 3
- 229910052594 sapphire Inorganic materials 0.000 description 3
- 239000010980 sapphire Substances 0.000 description 3
- 238000003325 tomography Methods 0.000 description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 1
- 239000004020 conductor Substances 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
Images
Classifications
-
- 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
- G01N23/22—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 measuring secondary emission from the material
- G01N23/225—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 measuring secondary emission from the material using electron or ion
- G01N23/2251—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 measuring secondary emission from the material using electron or ion using incident electron beams, e.g. scanning electron microscopy [SEM]
-
- 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
- G01N23/22—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 measuring secondary emission from the material
- G01N23/2202—Preparing specimens therefor
-
- 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
- G01N23/22—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 measuring secondary emission from the material
- G01N23/2204—Specimen supports therefor; Sample conveying means therefore
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention discloses a sample platform for imaging of a cryoelectron microscope, which comprises a base and a detachable filling part, wherein the base is of a disc-shaped structure, a convex edge is arranged on the base, a cavity for accommodating a sample is formed by the convex edge and the base in a surrounding mode, and the filling part can be used for filling a stepped structure formed by the outer side wall of the convex edge and the base during high-pressure freezing and is taken down during pre-thinning. The invention can accommodate and load a high-end frozen transmission electron microscope sample loading ring, can be stably fixed between the sample loading ring and the sample loading base, can be grabbed by a manipulator in an electron microscope Autoloader, and can be placed in a lens cone for imaging. Therefore, the sample stage provided by the invention can be applied to a freezing electron microscope of an Autoloader.
Description
Technical Field
The invention relates to the field of biological sample detection, in particular to a sample table for imaging of a cryoelectron microscope.
Background
In recent years, due to the development of cryo-electron tomography (cryo-ET), in-situ structure biological research has been rapidly developed, the state of the research object is closer to physiological conditions, and the resolution of the analysis structure is also increasing. The final objective is to analyze the structure of biological macromolecules and molecular machines in situ, thereby elucidating the nature of life activities at different levels of molecules, organelles, cells, and the like.
The thickness of a sample is generally required to be below 500nm by a frozen electron tomography technology, otherwise, excessive inelastic scattered electrons are generated during imaging, so that the signal-to-noise ratio of a picture is seriously reduced, and subsequent image processing is difficult to perform. Since the thickness of most cell and tissue samples is far over 500nm, the technical route taken by such samples is freeze fixation-Cryo-focused ion beam (Cryo-FIB) thinning-Cryo-tomography. Generally, some small cells are frozen and fixed on a special grid for an electron microscope by a rapid freezing mode, and then are thinned by a frozen focused ion beam to be subjected to frozen electron microscope imaging. However, the freezing depth of the rapid freezing is at most ten to several microns, and for tissues and some large-scale cells with the thickness of tens to hundreds of microns, the simple rapid freezing can not well fix the structure of the sample, so the high-pressure freezing technology is required. And (3) the sample block generated after high-pressure freezing is subjected to block repairing of a freezing microtome, is thinned to hundreds of nanometers by freezing focused ion beams, and is finally sent into a freezing electron microscope for imaging.
In order to improve the imaging resolution, people use a cryo-electron microscope with an Autoloader, such as FEI Titan Kros, for imaging, and such electron microscopes have high resolution and can obtain a more real and clear in-situ structure. However, the sample carrier used for the electron microscope is a carrier net, the sample carrier used for high-pressure freezing is a sample stage, and the Autoloader cannot carry the sample stage, so the existing sample stage cannot be applied to the cryomicroscope of the Autoloader.
Disclosure of Invention
The invention aims to provide a sample table for imaging of a freezing electron microscope, and the sample table is used for solving the problem that the conventional sample table cannot be applied to the freezing electron microscope of an Autoloader.
According to an embodiment of the invention, a sample stage for imaging of a cryoelectron microscope is provided, which comprises a base and a detachable filling part, wherein the base is of a disc-shaped structure, a convex edge is arranged on the base, the convex edge and the base enclose a cavity for accommodating a sample, and the filling part can fill a stepped structure formed by the outer side wall of the convex edge and the base during high-pressure freezing and is taken down during pre-thinning.
Specifically, the convex edge and the filling part are both of annular structures, and the filling part can be sleeved on the outer side wall of the convex edge.
Specifically, the center of the convex edge coincides with the center of the base.
In particular, the height of the filling part is the same as the height of the convex edge outer side wall.
Specifically, the base and the filling member are made of a heat-conductive metal material.
The embodiment of the invention provides a sample table for imaging of a freezing electron microscope, which can accommodate and load a high-end freezing transmission electron microscope sample ring, can be stably fixed between the sample ring and a sample base, can be grabbed by a manipulator in an electron microscope Autoloader, and can be placed in a lens barrel for imaging. Therefore, the sample stage provided by the invention can be applied to a freezing electron microscope of an Autoloader.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a structural diagram of a sample stage for imaging by a cryoelectron microscope according to an embodiment of the present invention;
FIG. 2 is a structural view of a base;
FIG. 3 is a block diagram of the filling member;
fig. 4 is a usage state diagram of a sample stage for cryoelectron microscope imaging according to an embodiment of the present invention.
Wherein, the sample loading device comprises a base 1, a filling part 2, a convex edge 3, a cavity 4, a sample loading base 5 and a sample loading ring 6.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to an embodiment of the invention, referring to fig. 1 to 3, a sample stage for imaging of a cryoelectron microscope is provided, which includes a base 1 and a detachable filling member 2, wherein the base 1 is a disc-shaped structure, a convex edge 3 is provided on the base 1, the convex edge 3 and the base 1 enclose a cavity 4 for accommodating a sample, and the filling member 2 can fill a stepped structure formed by an outer side wall of the convex edge 3 and the base 1 during high-pressure freezing and is taken down during a pre-thinning process.
Wherein, protruding edge 3 and filling member 2 are the loop configuration, and filling member 2 can overlap and establish on protruding lateral wall along 3, makes things convenient for the staff installation and takes off filling member 2. The center of the convex edge 3 coincides with the center of the base 1. The height of the filling part 2 is the same as the height of the outer side wall of the ledge 3. The base 1 and the filling member 2 are made of a heat conductive metal material, which may be gold-plated with pure copper/silver/copper, but is not limited to the above heat conductive material, and the embodiment is not limited thereto. The size of the sample stage can be set by actual requirements, and the embodiment is not limited.
The preparation process of the sample slice comprises the following steps: firstly, in a high-pressure freezing part, a filling part 2 is sleeved on the outer side wall of a convex edge 3, so that the filling part 2 fills the stepped structure formed by the outer side wall of the convex edge 3 and a base 1, a biological sample to be frozen is placed in a cavity 4, a cryoprotectant is added to fill the cavity 4, and then a sapphire sheet is covered on the cavity 4 to form a whole, and the whole is placed in a high-pressure freezer for freezing and fixing. The filling member 2 fills a space formed between the sapphire sheet and the base 1, and improves the freezing effect and the freezing speed.
Second, the frozen whole was transferred to a cryomicrotome in liquid nitrogen. The assembly of the base 1 and the lantern ring is pre-thinned after the sapphire sheet is taken down, about one third to one half of the assembly is trimmed, namely, the assembly is trimmed from a circle to a semicircle/large semicircle, the surface of the frozen biological sample is exposed, and the assembly is thinned from about hundred micrometers to dozens of micrometers. After the semi-circular shape is trimmed, the filling component 2 can be easily taken down, and the subsequent use of the sample table is not influenced. The purpose of this step is to expose the sample, take off the lantern ring, facilitate subsequent FIB cutting and electron microscope imaging.
Then, as shown in fig. 4, a sample loading ring 6(c-clip) and a sample loading base 5(AutoGrid) are loaded on the base 1 of the pre-thinned sample stage, the sample stage is stably fixed between the two to form a sample loading ring 6-sample stage-sample loading base 5 whole, and the whole is transferred to a scanning electron microscope freezing chamber in liquid nitrogen to be subjected to cryo-focused ion beam (cryo-FIB) thinning. The thinned sample has the thickness of about 200nm and can meet the requirements of transmission electron microscope imaging.
And finally, the whole of the sample loading ring 6, the sample stage and the sample loading base 5 is grabbed by a manipulator in the electron microscope Autoloader and is placed into a lens barrel for carrying out frozen transmission electron microscope imaging and data collection.
The embodiment of the invention provides a sample table for imaging of a freezing electron microscope, which can accommodate and load a high-end freezing transmission electron microscope sample ring 6, can be stably fixed between the sample ring 6 and a sample base 5, can be grabbed by a manipulator in an electron microscope Autoloader and can be placed in a lens cone for imaging. Therefore, the sample stage provided by the invention can be applied to a freezing electron microscope of an Autoloader.
Other embodiments of the invention will be apparent to those skilled in the art from 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 following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
It will be understood that the invention is not limited to the precise arrangements described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof. The scope of the invention is limited only by the appended claims.
Claims (5)
1. The utility model provides a be used for frozen electron microscope imaging to use sample platform, a serial communication port, including base (1) and detachable filling member (2), base (1) is discoid structure, be equipped with protruding edge (3) on base (1), protruding edge (3) and base (1) enclose into cavity (4) that are used for the holding sample, filling member (2) can be right when high pressure is frozen protruding lateral wall along (3) is filled with the stair structure that base (1) formed to take off at the attenuate in-process in advance.
2. The sample stage for cryoelectron microscopy imaging as defined in claim 1, wherein the protruding edge (3) and the filling member (2) are both ring structures, and the filling member (2) can be sleeved on the outer side wall of the protruding edge (3).
3. Sample stage for cryo-electron microscopy imaging as defined in claim 2, characterized in that the center of the ledge (3) coincides with the center of the base (1).
4. Sample stage for cryo-electron microscopy imaging as defined in claim 1, characterized in that the height of the filling part (2) is the same as the height of the outer side wall of the ledge (3).
5. Sample stage for cryo-electron microscopy imaging as defined in claim 1, characterized in that the base (1) and the filling part (2) are made of a heat conducting metal material.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2020102629579 | 2020-04-07 | ||
| CN202010262957.9A CN111562281A (en) | 2020-04-07 | 2020-04-07 | Sample table for imaging of cryoelectron microscope |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111999329A true CN111999329A (en) | 2020-11-27 |
Family
ID=72071422
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010262957.9A Withdrawn CN111562281A (en) | 2020-04-07 | 2020-04-07 | Sample table for imaging of cryoelectron microscope |
| CN202021846827.1U Active CN212646539U (en) | 2020-04-07 | 2020-08-28 | Sample table for imaging of cryoelectron microscope |
| CN202010883086.2A Pending CN111999329A (en) | 2020-04-07 | 2020-08-28 | Sample table for imaging of cryoelectron microscope |
Family Applications Before (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010262957.9A Withdrawn CN111562281A (en) | 2020-04-07 | 2020-04-07 | Sample table for imaging of cryoelectron microscope |
| CN202021846827.1U Active CN212646539U (en) | 2020-04-07 | 2020-08-28 | Sample table for imaging of cryoelectron microscope |
Country Status (1)
| Country | Link |
|---|---|
| CN (3) | CN111562281A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113252717A (en) * | 2021-06-15 | 2021-08-13 | 中国科学院地质与地球物理研究所 | Intelligent preparation system and method for frozen electron microscope sample and electronic equipment |
-
2020
- 2020-04-07 CN CN202010262957.9A patent/CN111562281A/en not_active Withdrawn
- 2020-08-28 CN CN202021846827.1U patent/CN212646539U/en active Active
- 2020-08-28 CN CN202010883086.2A patent/CN111999329A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113252717A (en) * | 2021-06-15 | 2021-08-13 | 中国科学院地质与地球物理研究所 | Intelligent preparation system and method for frozen electron microscope sample and electronic equipment |
| CN113252717B (en) * | 2021-06-15 | 2021-09-10 | 中国科学院地质与地球物理研究所 | Intelligent preparation system and method for frozen electron microscope sample and electronic equipment |
Also Published As
| Publication number | Publication date |
|---|---|
| CN212646539U (en) | 2021-03-02 |
| CN111562281A (en) | 2020-08-21 |
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