CN104568565A - Sample clamping tool and transfer box for frozen water-containing slices prepared by focused ion beam method - Google Patents
Sample clamping tool and transfer box for frozen water-containing slices prepared by focused ion beam method Download PDFInfo
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- CN104568565A CN104568565A CN201510010319.7A CN201510010319A CN104568565A CN 104568565 A CN104568565 A CN 104568565A CN 201510010319 A CN201510010319 A CN 201510010319A CN 104568565 A CN104568565 A CN 104568565A
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Abstract
The invention discloses a set of sample clamp and transfer box for biological frozen water-containing slices prepared by a focused ion beam method. The sample clamp and transfer box comprises a sample clamping device and a transfer device. The sample clamp and transfer box disclosed by the invention has the innovations that ice pollution of the biological frozen water-containing samples in the slice preparation and transport process can be effectively reduced, and the cutting thickness of the prepared sample slices is less than 300nm. The sample prepared by the device can be applied to directly installing a common electron microscope of a support grid and performing special transmission electron microscope imaging on the support grid.
Description
Technical field
The present invention relates to the imaging technique sample preparation of Ice mapping electron tomography and in situ TEM field of sample preparation.
Background technology
Cryotronics fault imaging three-dimensionalreconstruction (cryo-ET) technology is the important technology of development structure biology and RESEARCH ON CELL-BIOLOGY.This technology can obtain truer and locate and interactional information close to natural cell interior high-resolution three-dimension structure and protein macromolecule, is the important supplementary means of proteomics research.Normally used sample preparation methods---, there are problems in freezing microtome section technology, and as the compression of sample in section and collection process, chatter, shrinkage, fracture and to carry net adhesive ability poor etc., these defects have had a strong impact on the development of this research field.A kind of preparation method-focused ion beam method (focused ion beam-FIB) of freezing moisture section sample newly prepares freezing moisture section in recent years, it is the atomic collision generation sputtering effect adopting Ga ion and material, cut thin effect to reach to sample, the main equipment used is Double electron beam scanning Electronic Speculum (electron gun and ion gun are housed and in the angle of about 52 °) and freezing preparation system.Proved this method feasibility by the seminar of the M.Marko of the U.S. the earliest in 2006, in the W.Baumeister seminar of Germany in 2010 and the P.Zhang seminar of the U.S. in 2012, these technology are further developed and applied.In freezing moisture section sample preparation process, sample clamping tool and delivery means be ensure this technology carry out smoothly, and obtain smooth, thickness is suitable and the necessary experimental facilities of low stain freezing microtome section sample.The clamping used in the research of above seminar and transmitting device are all that the transmission electron microscope special according to this seminar is developed voluntarily, and year network capacity is fragile, does not have widespread use character.Now commercially not relevant product.
Summary of the invention
The present invention designs sample clamping tool and the divert cassette of a set of easy to use, highly versatile, directly can clamp common transmission electron microscope carrier net (copper mesh or molybdenum net) or special transmission electron microscope carrier net (c-ring+c-clip is netted in special the carrying of the Titan Krois transmission electron microscope as FEI Co.), and the section sample prepared is shifted easily and stores.
One aspect of the present invention provides a kind of equipment, and described equipment comprises sample clamping tool and sample divert cassette.
Sample clamping tool is made up of high heat conduction fine copper (red copper), and it comprises top cover, tool body, cylindrical seat, carries net brace groove and spring pressuring plate.
Top cover can free switch, and can cover sample clamping tool top (comprise and carry net brace groove and a spring pressuring plate) completely.
Cylindrical seat is positioned at bottom clamping device, and its diameter can regulate, and can be connected with the instrument that shuttles back and forth (shuttle) of the freezing transmission equipment of different model.
The size of carrying net brace groove also can change according to the loaded size and shape difference of carrying net.
The angle that groove pre-dumping is 38 °, object parallels with focused ion beam (FIB) in Double electron beam scanning Electronic Speculum, facilitates micro Process to operate.
Sample divert cassette, outside is made up of insulating material polystyrene, and inside is made up of stainless steel, comprises Electronic Speculum and carries net memory storage standing groove, hollow out platform, sample clamping tool standing groove, sample divert cassette support and divert cassette shell.
Sample divert cassette has one or more Electronic Speculum and carries net memory storage standing groove, and it carries net memory storage for placing Electronic Speculum.In one embodiment, divert cassette has three Electronic Speculum and carries net memory storage standing groove, three Electronic Speculum can be placed and carry net memory storage, described Electronic Speculum carries net memory storage can place 4 years nets, therefore in divert cassette, device 3 × 4=12 can carry net sample altogether, under liquid nitrogen environment, in convenient experiment, carry the switching manipulation of net memory storage.
Sample clamping tool standing groove is used for fixing described sample clamping tool, and it makes whole device can more stably loading and unloading net.
Hollow out platform and support can make part liquid nitrogen flow into bottom sample box, and whole device is kept at low ambient temperatures for a long time, and make package unit firm, the carrying out of convenient experiment.
The present invention has following innovative point:
1. keep low temperature
The present invention adopts red copper derby as the material of sample clamping tool, the heat produced can be conducted in time by conduction of heat in process, makes the net that carries of freezing sample continue to remain on liquid nitrogen temperature.
2. reduce the pollution of sample
With switch anti-pollution lid closely on sample clamping tool of the present invention, only open in cutting process, can ensure that in transmission with transfer process, reduce sample contacts with extraneous, prevents ice crystal from polluting.
3. divert cassette easily
The present invention researches and develops the clamping of a set of cryogenic sample, can will carry network folder easily and be loaded in sample clamping tool, and the freezing moisture section sample prepared is taken out and stored from clamping device in delivery means in liquid nitrogen environment.
Use sample clamp and the delivery means of the present invention's design, effectively can simplify practical operation, also significantly can reduce the ice of freezing moisture section in preparation and transmitting procedure simultaneously and pollute, thus acquire more large-area section observable region, experimental implementation is simple, is widely used.
More specifically, the invention provides the following:
1. carry the equipment of net for shifting and store Electronic Speculum, described equipment comprises sample clamping tool and sample divert cassette, wherein
Described sample clamping tool comprises: top cover (1), and described top cover can access with convenient-loading net by free switch, and makes when closing to carry net from pollution and destruction; Tool body (2); Cylindrical seat (3), described cylindrical seat is positioned at the bottom of described clamping device, the below of described tool body, and the diameter of described cylindrical seat conforms to the instrument of shuttling back and forth of the freezing transmission equipment by coupled different model; Carry net brace groove (4), described year net brace groove is used for supporting carries net, and its size conforms to the size of netting of carrying that will load; With spring pressuring plate (5), when carrying net and being loaded in described carrying in net brace groove, described spring pressuring plate clamps the described lateral edges carrying net, makes it in transmitting procedure, keep stable, and described top cover can cover described year net brace groove and described spring pressuring plate when closed, and
Described sample divert cassette comprises: one or more Electronic Speculum carries net memory storage standing groove (6), and described one or more Electronic Speculum is carried net memory storage standing groove and carried net memory storage for placing Electronic Speculum; Hollow out platform (7), described hollow out platform carries net memory storage standing groove and the sample clamping tool standing groove (8) for fixing described sample clamping tool for carrying described one or more Electronic Speculum, and liquid nitrogen by described hollow out platform hollow part thus and be not carried in described one or more Electronic Speculum Electronic Speculum of carrying on net memory storage standing groove and carry net memory storage and be carried in the sample clamping tool on described sample clamping tool standing groove; Sample divert cassette support (9), the below that described sample divert cassette support is positioned at described hollow out platform is stablized to keep described sample divert cassette in order to support described hollow out platform; With divert cassette shell (10), described divert cassette shell surrounds described hollow out platform and described sample divert cassette support, and net memory storage is carried and the sample clamping tool be carried on described sample clamping tool standing groove remains on low temperature to make the liquid nitrogen through described hollow out platform by being carried in described one or more Electronic Speculum Electronic Speculum of carrying on net memory storage standing groove in the space that described hollow out platform, described sample divert cassette support are provided for holding liquid nitrogen together with described divert cassette shell.
2. the equipment according to 1, wherein said sample clamping tool is processed by highly heat-conductive material, so that the heat produced in experimentation can conducting fast, what keep sample is in low-temperature condition.
3. the equipment according to 2, wherein said highly heat-conductive material is red copper.
4. the equipment according to 1, wherein in described sample divert cassette, described divert cassette shell (10) is made up of polystyrene, and the remainder of described sample divert cassette is made up of stainless steel.
5. the equipment according to 1, the angle of inclination of wherein said year net brace groove keeps 38 ° with parallel with focused ion beam (FIB) in Double electron beam scanning Electronic Speculum.
6. the equipment according to 1, wherein said sample divert cassette has 3 Electronic Speculum and carries net memory storage standing groove.
7. the equipment according to 1, wherein said sample clamping tool standing groove is fixed described sample clamping tool and is verted to enable the described sample clamping tool be fixed carry out 360 °, to facilitate described sample clamping tool and to carry the handling of netting.
8. the equipment according to 1, wherein said one or more Electronic Speculum carries plane residing for net memory storage standing groove higher than the plane residing for described sample clamping tool standing groove.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of sample clamping tool, and illustrated sample clamping tool comprises: top cover (1), tool body (2), cylindrical seat (3), year net brace groove (4) and spring pressuring plate (5); Figure (a): sample clamping tool open mode, wherein carries net and has been loaded on clamping device; Figure (b): the store status that clamping device top cover is closed.
Fig. 2 is sample divert cassette schematic diagram, illustrated sample divert cassette comprises: Electronic Speculum carries net memory storage standing groove (6), hollow out platform (7), sample clamping tool standing groove (8), sample divert cassette support (9) and divert cassette shell (10), wherein in Fig. 2 (a), sample clamping tool has been loaded on sample clamping tool standing groove (8).
Fig. 3 is the result figure of the experiment according to one embodiment of the invention, wherein, and A: the thickness of the freezing moisture section after right-angle view FIB is thinning; B: side observe carry net grid in thinning after sample; C and D: electron diffraction pattern; And, E and F: the structure of cell junctions and inner organelle.
Embodiment
Below in conjunction with the drawings and specific embodiments, illustrate the present invention further: experimental facilities is the Helios Nanolab 600i Double electron beam scanning Electronic Speculum that FEI Co. produces, and is equipped with the freezing preparation system of PP3000T that Quorum company produces.Sample clamping tool is installed in divert cassette, and by its overall immersion liquid nitrogen, keeps liquid nitrogen liquid level about 0.5cm place more than clamping device top.First use tweezers will carry net to carry net memory storage from Electronic Speculum and take out and be installed on sample clamping tool, then close the top cover of clamping device in case stagnant ice pollution.Again clamping device is connected by the transmitting device of cylindrical seat with freezing preparation system, the clamping device being fixed with sample is sent into Double electron beam scanning Electronic Speculum and prepares chamber, open the lid of clamping device, the thinning processing of focused ion beam is carried out to sample.After freezing moisture section preparation completes freezing moisture section, close the lid of clamping device, and fast sample and clamping device are together transferred in the clamping device standing groove in the divert cassette that is immersed in liquid nitrogen.After placing, under liquid nitrogen environment, open the top cover of the clamping device in divert cassette, take out freezing moisture section and carry net, be returned to the Electronic Speculum be in liquid nitrogen and carry in net memory storage, in order to later stage transmission electron microscope imaging.
Embodiment 1
One, sample is prepared
Use and throw Refrigeration Technique and carry on the net by sample snap frozen to 200 object molybdenums, as related to cell sample, then suggestion adopts and drips the method that sample blots from the back side and prepare carrying online front, thinner to ensure sample ice sheet.
Two, cutting process
1. under liquid nitrogen environment, net will be carried and be installed on clamping device, then transfer to being mounted with the sample clamping tool carrying net in sample divert cassette, and close protective cover.
2. sample clamping tool to be transferred in the large sample preparation cup of PP3000T and to be connected with sample transferring stick, and vacuumizing.
3., after sample enters PP3000T sample preparation chamber, after spraying plating Pt metal, be transferred to scanning electron microscope sem cryogenic sample platform.
4. adopt focused ion beam reduction to carry out thinning cutting to freezing aqueous specimen, cutting parameter: under FIB voltage 30kV, to cut with electric current 0.79nA-80pA respectively.
5. thinningly to complete, take out the freezing moisture section sample completed and carry out transmission electron microscope imaging or Cord blood.
Claims (8)
1. carry the equipment of net for shifting and store Electronic Speculum, described equipment comprises sample clamping tool and sample divert cassette, wherein
Described sample clamping tool comprises: top cover (1), and described top cover can access with convenient-loading net by free switch, and makes when closing to carry net from pollution and destruction; Tool body (2); Cylindrical seat (3), described cylindrical seat is positioned at the bottom of described clamping device, the below of described tool body, and the diameter of described cylindrical seat conforms to the instrument of shuttling back and forth of the freezing transmission equipment by coupled different model; Carry net brace groove (4), described year net brace groove is used for supporting carries net, and its size conforms to the size of netting of carrying that will load; With spring pressuring plate (5), when carrying net and being loaded in described carrying in net brace groove, described spring pressuring plate clamps the described lateral edges carrying net, makes it in transmitting procedure, keep stable, and described top cover can cover described year net brace groove and described spring pressuring plate when closed, and
Described sample divert cassette comprises: one or more Electronic Speculum carries net memory storage standing groove (6), and described one or more Electronic Speculum is carried net memory storage standing groove and carried net memory storage for placing Electronic Speculum; Hollow out platform (7), described hollow out platform carries net memory storage standing groove and the sample clamping tool standing groove (8) for fixing described sample clamping tool for carrying described one or more Electronic Speculum, and liquid nitrogen by described hollow out platform hollow part thus and be not carried in described one or more Electronic Speculum Electronic Speculum of carrying on net memory storage standing groove and carry net memory storage and be carried in the sample clamping tool on described sample clamping tool standing groove; Sample divert cassette support (9), the below that described sample divert cassette support is positioned at described hollow out platform is stablized to keep described sample divert cassette in order to support described hollow out platform; With divert cassette shell (10), described divert cassette shell surrounds described hollow out platform and described sample divert cassette support, and net memory storage is carried and the sample clamping tool be carried on described sample clamping tool standing groove remains on low temperature to make the liquid nitrogen through described hollow out platform by being carried in described one or more Electronic Speculum Electronic Speculum of carrying on net memory storage standing groove in the space that described hollow out platform, described sample divert cassette support are provided for holding liquid nitrogen together with described divert cassette shell.
2. equipment according to claim 1, wherein said sample clamping tool is processed by highly heat-conductive material.
3. equipment according to claim 2, wherein said highly heat-conductive material is red copper.
4. equipment according to claim 1, wherein in described sample divert cassette, described divert cassette shell (10) is made up of polystyrene, and the remainder of described sample divert cassette is made up of stainless steel.
5. equipment according to claim 1, the angle of inclination of wherein said year net brace groove keeps 38 ° with parallel with focused ion beam (FIB) in Double electron beam scanning Electronic Speculum.
6. equipment according to claim 1, wherein said sample divert cassette has 3 Electronic Speculum and carries net memory storage standing groove.
7. equipment according to claim 1, wherein said sample clamping tool standing groove is fixed described sample clamping tool and is verted to enable the described sample clamping tool be fixed carry out 360 °.
8. equipment according to claim 1, wherein said one or more Electronic Speculum carries plane residing for net memory storage standing groove higher than the plane residing for described sample clamping tool standing groove.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510010319.7A CN104568565B (en) | 2014-08-28 | 2015-01-09 | Focused ion beam method prepares the sample clamping tool and transfer box of the aqueous section of freezing |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2014104291769 | 2014-08-28 | ||
| CN201410429176.9A CN104198242A (en) | 2014-08-28 | 2014-08-28 | Sample clamping tool and transferring box for preparing frozen moisture contained chips by focused ion beam method |
| CN201510010319.7A CN104568565B (en) | 2014-08-28 | 2015-01-09 | Focused ion beam method prepares the sample clamping tool and transfer box of the aqueous section of freezing |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104568565A true CN104568565A (en) | 2015-04-29 |
| CN104568565B CN104568565B (en) | 2017-06-06 |
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Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410429176.9A Pending CN104198242A (en) | 2014-08-28 | 2014-08-28 | Sample clamping tool and transferring box for preparing frozen moisture contained chips by focused ion beam method |
| CN201510010319.7A Active CN104568565B (en) | 2014-08-28 | 2015-01-09 | Focused ion beam method prepares the sample clamping tool and transfer box of the aqueous section of freezing |
| CN201520014069.XU Withdrawn - After Issue CN204556364U (en) | 2014-08-28 | 2015-01-09 | The equipment of net is carried for shifting and store Electronic Speculum |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410429176.9A Pending CN104198242A (en) | 2014-08-28 | 2014-08-28 | Sample clamping tool and transferring box for preparing frozen moisture contained chips by focused ion beam method |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201520014069.XU Withdrawn - After Issue CN204556364U (en) | 2014-08-28 | 2015-01-09 | The equipment of net is carried for shifting and store Electronic Speculum |
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| CN (3) | CN104198242A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106404512A (en) * | 2016-10-19 | 2017-02-15 | 中国科学院生物物理研究所 | Carrying network for transmission electron microscope |
| CN110006934A (en) * | 2017-12-28 | 2019-07-12 | Fei 公司 | Pass through the methods, devices and systems of plasma focus Ion Beam Treatment biology cryogenic sample |
| CN110823931A (en) * | 2019-10-16 | 2020-02-21 | 中国科学院生物物理研究所 | Preparation method of frozen electron microscope sample |
| CN118010408A (en) * | 2024-04-09 | 2024-05-10 | 河南工学院 | Concrete strength detection device |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104198242A (en) * | 2014-08-28 | 2014-12-10 | 中国科学院生物物理研究所 | Sample clamping tool and transferring box for preparing frozen moisture contained chips by focused ion beam method |
| WO2018073242A1 (en) * | 2016-10-17 | 2018-04-26 | Universität Basel | Lossless cryo-grid preparation by controlled sample evaporation |
| CN110514685A (en) * | 2019-09-29 | 2019-11-29 | 广西师范大学 | A kind of loading instrument for the test of in-situ scanning Electronic Speculum |
| CN115575364B (en) * | 2022-09-30 | 2023-08-04 | 中国科学院生物物理研究所 | Ion beam processing method based on optical microscopic imaging |
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-
2014
- 2014-08-28 CN CN201410429176.9A patent/CN104198242A/en active Pending
-
2015
- 2015-01-09 CN CN201510010319.7A patent/CN104568565B/en active Active
- 2015-01-09 CN CN201520014069.XU patent/CN204556364U/en not_active Withdrawn - After Issue
Patent Citations (6)
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| EP2009421A1 (en) * | 2007-06-29 | 2008-12-31 | FEI Company | Method for separating a lamella for TEM inspection from a core sample |
| CN101344463A (en) * | 2007-07-12 | 2009-01-14 | 陶革新 | Operation dynamic display timing control four-point refrigeration automatic tool setting system of freezing microtome |
| CN201199207Y (en) * | 2008-04-14 | 2009-02-25 | 李静 | Desktop totally-enclosed semiautomatic constant-temperature rapid environment protection energy-saving frozen slicer |
| CN201623016U (en) * | 2009-11-10 | 2010-11-03 | 中芯国际集成电路制造(上海)有限公司 | Clamp and sample box with the same |
| CN103196719A (en) * | 2013-03-17 | 2013-07-10 | 中国科学院苏州纳米技术与纳米仿生研究所 | Micro/nano-particle sample carrying mesh storage device for transmission electron microscope, and sample preparation method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106404512A (en) * | 2016-10-19 | 2017-02-15 | 中国科学院生物物理研究所 | Carrying network for transmission electron microscope |
| CN110006934A (en) * | 2017-12-28 | 2019-07-12 | Fei 公司 | Pass through the methods, devices and systems of plasma focus Ion Beam Treatment biology cryogenic sample |
| US11735404B2 (en) | 2017-12-28 | 2023-08-22 | Fei Company | Method, device and system for the treatment of biological cryogenic samples by plasma focused ion beams |
| CN110823931A (en) * | 2019-10-16 | 2020-02-21 | 中国科学院生物物理研究所 | Preparation method of frozen electron microscope sample |
| CN118010408A (en) * | 2024-04-09 | 2024-05-10 | 河南工学院 | Concrete strength detection device |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104198242A (en) | 2014-12-10 |
| CN204556364U (en) | 2015-08-12 |
| CN104568565B (en) | 2017-06-06 |
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