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WO2009145377A1 - Station de travail pour un microscope électronique à cryotransmission - Google Patents

Station de travail pour un microscope électronique à cryotransmission Download PDF

Info

Publication number
WO2009145377A1
WO2009145377A1 PCT/KR2008/003061 KR2008003061W WO2009145377A1 WO 2009145377 A1 WO2009145377 A1 WO 2009145377A1 KR 2008003061 W KR2008003061 W KR 2008003061W WO 2009145377 A1 WO2009145377 A1 WO 2009145377A1
Authority
WO
WIPO (PCT)
Prior art keywords
holder
stand
workstation
liquid nitrogen
vessel
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.)
Ceased
Application number
PCT/KR2008/003061
Other languages
English (en)
Inventor
Sun-Gyu Lee
Jong-Man Jeung
Youn-Joong Kim
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Korea Basic Science Institute KBSI
Original Assignee
Korea Basic Science Institute KBSI
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Korea Basic Science Institute KBSI filed Critical Korea Basic Science Institute KBSI
Priority to PCT/KR2008/003061 priority Critical patent/WO2009145377A1/fr
Publication of WO2009145377A1 publication Critical patent/WO2009145377A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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
    • G01N1/42—Low-temperature sample treatment, e.g. cryofixation
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
    • H01J2237/20—Positioning, supporting, modifying or maintaining the physical state of objects being observed or treated
    • H01J2237/2001—Maintaining constant desired temperature
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
    • H01J2237/20—Positioning, supporting, modifying or maintaining the physical state of objects being observed or treated
    • H01J2237/2005—Seal mechanisms

Definitions

  • the present invention relates, in general, to a workstation for cryogenic transmission electron microscopy, in which a biological sample is observed at low temperature using a transmission electron microscope or a high-voltage electron microscope (HVEM) and, more particularly, to a workstation for cryogenic transmission electron microscopy, in which a biological sample processed at low temperature is more rapidly and efficiently loaded onto a holder of the workstation, thereby making it possible to improve efficiency and reliability of the cryogenic transmission electron microscopy during which it is important to maintain the low temperature.
  • HVEM high-voltage electron microscope
  • Recent science and engineering technologies take an interest in the submicrometer microstructure of a material departing from a process.
  • an instrument such as a transmission electron microscope or an atomic force microscope (AFM)
  • FAM atomic force microscope
  • technologies that directly observe organic materials such as polymers or biological samples using the transmission electron microscope also arouse much interest.
  • the direct observation of the organic materials using the transmission electron microscope, etc. is more difficult compared with the observation of inorganic materials. This is because the organic materials have a melting point lower than the inorganic materials.
  • physical chemical processing such as staining is required.
  • the present invention is mainly interested in technology that processes a biological sample such as a cell into a sample capable of direct observation using the transmission electron microscope or a high-voltage electron microscope (HVEM) .
  • a biological sample such as a cell into a sample capable of direct observation using the transmission electron microscope or a high-voltage electron microscope (HVEM) .
  • HVEM high-voltage electron microscope
  • There are several methods of processing the biological sample for example, a staining method of drying the biological sample and staining the dried biological sample, a chemical fixation method of injecting and fixing a fixing solution into a fine cell structure, and so on.
  • the staining method the staining itself is impossible for certain kinds of biological samples, or a staining agent deforms a structure of the biological sample making it difficult to observe a prototype of the biological sample.
  • the chemical fixation method the speed at which a fixing solution is penetrated into a cell is slow, and thus it is difficult to observe a cell structure the way the prototype should be,
  • a technique developed in order to overcome these drawbacks of the related arts is to rapidly cool a biological sample using liquid nitrogen or liquid helium to maintain a prototype of the biological sample without deformation, and then observe the biological sample using, for instance, a transmission electron microscope.
  • This technique is commonly called “cryogenic transmission electron microscopy (cryo-TEM)," and its availability is known through several monographs.
  • FIG. 1 illustrates a conventional workstation 1 used when a rapidly frozen sample is loaded onto a holder under low temperature.
  • the workstation 1 generally includes a stand 11, an insulated vessel 12 disposed on one side of an upper surface of the stand 11, and a Dewar vessel 13 disposed on the other side of the stand 11.
  • a holder insertion socket 16 is formed on a sidewall of the insulated vessel 12 which faces the Dewar vessel 13. Similarly, the Dewar vessel 13 is provided with a holder coupling hole 20 at a position opposite the holder insertion socket 16.
  • the Dewar vessel 13 includes a charging port (not shown) capable of charging liquid nitrogen and a cap 14 capable of opening and closing the charging port at an upper end thereof.
  • a holder 15 mounted on a transmission electron microscope is fitted into the holder insertion socket 16 and the holder coupling hole 20, and is kept cold by liquid nitrogen charged into the insulated vessel 12 and the Dewar vessel 13. After the workstation 1 is set, the biological sample rapidly cooled by the vitrification device is loaded onto an end of the holder 15 through the sample loading port 18 using tweezers.
  • the conventional workstation having the aforementioned configuration has the following problems.
  • an 0-ring made of rubber material is interposed between the holder and the holder insertion socket.
  • this rubber 0-ring comes into contact with the low-temperature liquid nitrogen, the 0-ring becomes rigid making it difficult to maintain the airtightness, and it takes a long time to separate the holder from the holder insertion socket.
  • the moisture and liquid nitrogen in the insulated vessel mix to generate blocks of ice during the process of charging the liquid nitrogen into the insulated vessel. When these ice blocks become attached to the sample, it is difficult to obtain and observe a desired image from the sample through the transmission electron microscope.
  • a means capable of properly adjusting the behavior of the liquid nitrogen charged into the insulated vessel is also required.
  • an embodiment of the present invention is to provide a workstation for cryogenic transmission electron microscopy, which is configured to eliminate a freezing phenomenon occurring on the top of a transparent cover when water vapor in the atmosphere is cooled due to a low temperature of liquid nitrogen when a rapidly frozen sample is loaded onto a holder under low temperature.
  • Another embodiment of the present invention is to provide a workstation for cryogenic transmission electron microscopy, which is configured to not only efficiently protect an 0-ring made of rubber material interposed between a holder and a holder insertion socket from low- temperature liquid nitrogen but also to prevent a sample from being contaminated by blocks of ice generated on the top of a transparent cover generated by the mixing of moisture with liquid nitrogen in an insulated vessel in the process of charging the liquid nitrogen into the insulated vessel .
  • a workstation for cryogenic transmission electron microscopy which includes: a stand, upper and lower surfaces of which are flat; an insulated vessel disposed on one side of the upper surface of the stand, and including a holder insertion socket on a sidewall thereof, wherein a transparent cover having a liquid nitrogen injection port and a sample loading port is detachably mounted on an upper end of the insulated vessel, and includes a step on an outer circumference of an upper surface thereof; a Dewar vessel disposed on another side of the upper surface of the stand so as to face the holder insertion socket, including a charging port for charging liquid nitrogen and a cap for opening and closing the charging port at an upper end thereof, and having a holder coupling hole in a sidewall thereof which faces the insulated vessel; and a holder, opposite ends of which are coupled to the holder insertion socket and the holder coupling hole.
  • a step is formed along the outer circumference of the upper portion of the transparent cover, so that the space, in which the liquid nitrogen discharged from the interior of the insulated vessel can be confined, is formed.
  • the liquid nitrogen which is confined in a space of the upper portion of the transparent cover, serves as the boundary layer for preventing moisture in the atmosphere from being frozen on the upper surface of the transparent cover.
  • the transparent cover is made of acryl. Besides the acryl material, any plastic material will do as long as it can be used at low temperature.
  • the workstation further includes a height adjuster, which is disposed on the lower surface of the stand on which the Dewar vessel is disposed.
  • the height adjuster functions to incline the stand such that the height at which the Dewar vessel is installed is higher than that at which the insulated vessel is installed.
  • the liquid nitrogen can be prevented from coming into direct contact with the CD- ring interposed between the holder and the holder insertion socket, and the blocks of ice generated by mixing of the moisture with the liquid nitrogen in the insulated vessel can be collected on the side opposite the sample because of the inclination of the stand, and thus the sample can be prevented from being contaminated. Consequently, the second object of the present invention is accomplished by this configuration.
  • the height adjuster includes two short rods, each of which has a regular hexagonal cross section, and a long rod connecting the two short rods at a position deviating from the center of the regular hexagonal cross section of each short rod in a direction perpendicular to one of six sides of the regular hexagonal cross section of each short rod, and having a circular cross section.
  • the long rod is in contact with the lower surface of the stand. Since the long rod deviates from the center of the regular hexagonal cross section of each short rod, height of the long rod can be adjusted in four steps.
  • the step is formed along the outer circumference of the upper portion of the transparent cover having the sample loading port, so that there is formed a space inside which the liquid nitrogen discharged from the interior of the insulated vessel can be confined.
  • the liquid nitrogen which is confined in a space of the upper portion of the transparent cover, serves as the boundary layer for preventing moisture in the atmosphere from being frozen on the upper surface of the transparent cover.
  • the height adjuster is disposed on the lower surface of the stand on which the Dewar vessel is disposed, and functions to incline the stand such that the height at which the Dewar vessel is installed is higher than that at which the insulated vessel is installed.
  • the liquid nitrogen can be prevented from being directly contacted with the 0-ring interposed between the holder and the holder insertion socket, and the blocks of ice generated by the mixing of the moisture with the liquid nitrogen in the insulated vessel can be collected on the side opposite the sample because of the inclination of the stand, and thus the sample can be prevented from being contaminated.
  • FIG. 1 is a cross-sectional view illustrating a conventional workstation for cryogenic transmission electron microscopy
  • FIG. 2 is a cross-sectional view illustrating a workstation for cryogenic transmission electron microscopy according to an exemplary embodiment of the present invention.
  • FIG. 3 is a detailed view illustrating a height adjuster according to an exemplary embodiment of the present invention. [Best Mode]
  • FIG. 2 is a cross-sectional view illustrating a workstation 100 for cryogenic transmission electron microscopy according to an exemplary embodiment of the present invention.
  • the workstation 100 for cryogenic transmission electron microscopy of the present invention generally includes a stand 101, an insulated vessel 102, a Dewar vessel 103, and a holder 5.
  • the stand 101 has the shape of a plate, upper and lower surfaces of which are flat, and functions as a base on which the insulated vessel 102 and the Dewar vessel 103 are disposed.
  • the insulated vessel 102 is disposed on one side of the upper surface of the stand 101, and is provided with a holder insertion socket 106 on a sidewall thereof.
  • a transparent cover 107 has a liquid nitrogen injection port 109 and a sample loading port 108, and is detachably mounted on an upper end of the insulated vessel 102.
  • the transparent cover 107 is provided with a step 107' on the outer circumference of an upper surface thereof.
  • a space is formed at the upper portion of the transparent cover 107 by the step 107' . Nitrogen gas, formed by evaporation and discharge of the liquid nitrogen in the insulated vessel 102, is confined in a space of the upper portion of the transparent cover 107.
  • the nitrogen gas thus confined serves as a boundary layer for preventing moisture in the air from becoming frozen on the upper surface of the transparent cover 107.
  • the transparent cover is preferably made of acryl. Besides acryl material, any plastic material will do as long as it can be used at low temperature.
  • the Dewar vessel 103 is disposed on the other side of the stand 101 so as to face the holder insertion socket 106, and is provided with a charging port (not shown) capable of charging liquid nitrogen and a cap 104 capable of opening and closing the charging port at an upper end thereof.
  • the Dewar vessel 103 is provided with a holder coupling hole 111 in a sidewall thereof which faces the insulated vessel 102.
  • the liquid nitrogen charged in the Dewar vessel 103 cools the holder 105 fitted into the holder coupling hole 111.
  • the holder 105 onto which the biological sample is loaded is coupled to the holder insertion socket 106 and the holder coupling hole 111 at opposite ends thereof, wherein the end of the holder 105 onto which the biological sample is loaded is inserted into the holder insertion socket 106.
  • the first end of the holder 105 inserted into the holder insertion socket 106 is provided with an O-ring 112 made of rubber material in order to maintain airtightness on an outer circumference thereof.
  • the workstation for cryogenic transmission electron microscopy further includes a height adjuster 110 on a lower surface of the stand 101 on which the Dewar vessel 103 is disposed.
  • the height adjuster 110 functions to incline the stand 101 such that the height at which the Dewar vessel 103 is installed is higher than that at which the insulated vessel 102 is installed.
  • the liquid nitrogen can be prevented from coming into direct contact with the O-ring 112 interposed between the first end of the holder 105 and the holder insertion socket 106, and blocks of ice generated by mixture of moisture and liquid nitrogen in the insulated vessel 102 in the process of charging the liquid nitrogen into the insulated vessel 102 can be collected on the side opposite the sample because of the inclination of the stand, and thus the sample can be prevented from contamination .
  • the height adjuster 110 includes two short rods 110' , each of which has a regular hexagonal cross section, and a long rod 110", which connects the two short rods 110' at a position deviating from the center of the regular hexagonal cross section of each short rod 110' in a direction perpendicular to one of six sides of the regular hexagonal cross section of each short rod 110' and has a circular cross section.
  • the lower surface of the stand 101 is supported in contact with the long rod 110" alone. Since the long rod 110" deviates from the center of the regular hexagonal cross section of each short rod 110' , the height of the long rod 110" can be adjusted in four steps.
  • FIG. 3 illustrates the case in which the long rod 110" is highest from the ground.
  • the height adjuster can be made of aluminum.
  • the opposite ends of the holder 105 are coupled to the holder insertion socket 106 and the holder coupling hole 111 of the work station 100, respectively.
  • the first end of the holder 105, onto which the biological sample is loaded, is inserted into the holder insertion socket 106.
  • the height adjuster 110 is installed on the lower surface of the stand 101 on which the Dewar vessel 103 is disposed.
  • the inclination of the stand 101 is properly adjusted using the height adjuster 110 which is capable of performing a four-step height adjustment process .
  • liquid nitrogen is charged through the liquid nitrogen injection port 109 of the insulated vessel 102. Further, liquid nitrogen is charged through the charging inlet (not shown) opened by taking off the cap 104 of the Dewar vessel 103.
  • the sample is loaded onto the first end of the holder 105 through the sample loading port 108 of the insulated vessel 102 using tweezers. At this time, the liquid nitrogen continues to be injected into the insulated vessel 102 so as to keep it under low temperature conditions.
  • the holder 105 is separated from the workstation, and then is transferred to a transmission electron microscope.
  • the workstation for cryogenic transmission electron microscopy which observes the biological sample at low temperature using the transmission electron microscope or HVEM, can more rapidly and efficiently load the biological sample, processed at low temperature, on the holder of the workstation, and thus is useful for the cryogenic testing in which it is important to maintain a low temperature.

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  • 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

L'invention porte sur une station de travail pour une microscopie électronique à transmission cryogénique. De l'azote liquide, qui est confiné dans un espace défini par l'étage d'un couvercle transparent monté de façon détachable sur un récipient isolé, sert de couche limite pour empêcher l'humidité dans l'atmosphère de se congeler sur le dessus du couvercle transparent. En outre, un organe de réglage de hauteur est disposé sur la surface inférieure d'un socle sur lequel un vase de Dewar est disposé, et incline le socle de telle sorte que la hauteur d'installation du vase de Dewar est supérieure à celle du récipient isolé. Ainsi, l'azote liquide ne peut pas venir en contact direct avec un joint torique interposé entre un support et une douille d'introduction de support, et des blocs de glace générés par le mélange d'humidité avec l'azote liquide peuvent être collectés sur un côté, pour ainsi empêcher une contamination de l'échantillon.
PCT/KR2008/003061 2008-05-30 2008-05-30 Station de travail pour un microscope électronique à cryotransmission Ceased WO2009145377A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/KR2008/003061 WO2009145377A1 (fr) 2008-05-30 2008-05-30 Station de travail pour un microscope électronique à cryotransmission

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2008/003061 WO2009145377A1 (fr) 2008-05-30 2008-05-30 Station de travail pour un microscope électronique à cryotransmission

Publications (1)

Publication Number Publication Date
WO2009145377A1 true WO2009145377A1 (fr) 2009-12-03

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PCT/KR2008/003061 Ceased WO2009145377A1 (fr) 2008-05-30 2008-05-30 Station de travail pour un microscope électronique à cryotransmission

Country Status (1)

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WO (1) WO2009145377A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102014110722A1 (de) * 2014-07-29 2016-02-04 Leica Mikrosysteme Gmbh Ladestation zum Umladen von gefrorenen Proben bei tiefen Temperaturen
CN106814095A (zh) * 2017-02-12 2017-06-09 马新军 用于扫描电镜的冷冻制样装置
EP3477680A1 (fr) * 2017-10-30 2019-05-01 Gatan, Inc. Support de cryotransfert et poste de travail
EP3247987B1 (fr) 2015-01-20 2020-09-02 Leica Mikrosysteme GmbH Dispositif de transfert d'échantillons

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6410925B1 (en) * 2000-07-31 2002-06-25 Gatan, Inc. Single tilt rotation cryotransfer holder for electron microscopes
WO2002077612A1 (fr) * 2001-03-22 2002-10-03 Universiteit Maastricht Dispositif permettant de preparer des echantillons destines a un microscope cryoelectronique
JP2005158288A (ja) * 2003-11-20 2005-06-16 Canon Inc 電子顕微鏡用試料冷却ホルダ
EP1852889A2 (fr) * 2006-05-01 2007-11-07 FEI Company Appareil d'optique corpusculaire doté d'un thermocontact

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6410925B1 (en) * 2000-07-31 2002-06-25 Gatan, Inc. Single tilt rotation cryotransfer holder for electron microscopes
WO2002077612A1 (fr) * 2001-03-22 2002-10-03 Universiteit Maastricht Dispositif permettant de preparer des echantillons destines a un microscope cryoelectronique
JP2005158288A (ja) * 2003-11-20 2005-06-16 Canon Inc 電子顕微鏡用試料冷却ホルダ
EP1852889A2 (fr) * 2006-05-01 2007-11-07 FEI Company Appareil d'optique corpusculaire doté d'un thermocontact

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102014110722A1 (de) * 2014-07-29 2016-02-04 Leica Mikrosysteme Gmbh Ladestation zum Umladen von gefrorenen Proben bei tiefen Temperaturen
DE102014110722B4 (de) * 2014-07-29 2016-06-09 Leica Mikrosysteme Gmbh Ladestation zum Umladen von gefrorenen Proben bei tiefen Temperaturen
US10217603B2 (en) 2014-07-29 2019-02-26 Leica Mikrosysteme Gmbh Loading station for transferring frozen samples at low temperatures
EP3247987B1 (fr) 2015-01-20 2020-09-02 Leica Mikrosysteme GmbH Dispositif de transfert d'échantillons
CN106814095B (zh) * 2017-02-12 2019-05-24 马新军 用于扫描电镜的冷冻制样装置
CN106814095A (zh) * 2017-02-12 2017-06-09 马新军 用于扫描电镜的冷冻制样装置
CN109720737A (zh) * 2017-10-30 2019-05-07 加坦公司 低温转移保持器和工作台
EP3477680A1 (fr) * 2017-10-30 2019-05-01 Gatan, Inc. Support de cryotransfert et poste de travail
JP2019083191A (ja) * 2017-10-30 2019-05-30 ガタン インコーポレイテッドＧａｔａｎ，Ｉｎｃ． クライオトランスファーホルダ及びワークステーション
CN109720737B (zh) * 2017-10-30 2020-10-16 加坦公司 低温转移保持器和工作台
US10832887B2 (en) 2017-10-30 2020-11-10 Gatan, Inc. Cryotransfer holder and workstation
EP4174903A1 (fr) * 2017-10-30 2023-05-03 Gatan, Inc. Support de cryotransfert et poste de travail
US11908655B2 (en) 2017-10-30 2024-02-20 Gatan, Inc. Cryotransfer holder and workstation

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