US3761709A - Method and apparatus for observing biological specimens using a scanning electron microscope - Google Patents

Method and apparatus for observing biological specimens using a scanning electron microscope Download PDF

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Publication number
US3761709A
US3761709A US00232405A US23240572A US3761709A US 3761709 A US3761709 A US 3761709A US 00232405 A US00232405 A US 00232405A US 23240572 A US23240572 A US 23240572A US 3761709 A US3761709 A US 3761709A
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United States
Prior art keywords
specimen
chamber
stage
frozen
holder
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.)
Expired - Lifetime
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US00232405A
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English (en)
Inventor
Y Hasegawa
Y Nagasawa
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Jeol Ltd
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Jeol Ltd
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Filing date
Publication date
Priority claimed from JP1757871U external-priority patent/JPS5120680Y2/ja
Priority claimed from JP10057771U external-priority patent/JPS5127716Y2/ja
Priority claimed from JP46087408A external-priority patent/JPS5116252B2/ja
Application filed by Jeol Ltd filed Critical Jeol Ltd
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Publication of US3761709A publication Critical patent/US3761709A/en
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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
    • H01J37/02—Details
    • H01J37/20—Means for supporting or positioning the object or the material; Means for adjusting diaphragms or lenses associated with the support
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
    • H01J37/02—Details
    • H01J37/18—Vacuum locks ; Means for obtaining or maintaining the desired pressure within the vessel

Definitions

  • a pre-evacuation chamber is provided adjacent the electron optical column of the scanning electron microscope.
  • the sample is quenched in liquid gas to be frozen. Thereafter, the sample is transferred to the pre-evacuation chamber and manipulated prior to placement in the specimen stage of the microscope to avoid the formation of frost or to remove the frost on the sample surface.
  • the above objects are made possible by placing certain devices and mechanisms, depending on the object in view, in the pre-evacuation chamber of a scanning electron microscope.
  • FIG. I shows the evacuation system of a typical scanning electron microscope
  • FIGS. 2 and 3 illustrate how the specimen is frozen according to this invention
  • FIG. 4 shows an embodiment for observing a frost free cross-section of the frozen specimen
  • FIGS. 5 and 6 show embodiments for removing the frost from the surface of the frozen specimen.
  • FIG. 7 shows an embodiment for preventing the formation of frost during specimen exchange.
  • an electron optical column I includes an electron gun chamber la and specimen chamber lb.
  • Column 1 is roughly evacuated by rotary pump 2 through valve 3 and completely evacuated by diffusion pump 4 and rotary pump 5 through valve 6.
  • a pre-evacuation chamber 7 is partitioned from the specimen chamber in column 1 by a door 8 and from the atmosphere by a cover 9.
  • the chamber 7 is evacuated by rotary pump 2 through valve 10 and is leaked through valve 11.
  • the following embodiment of the invention relates to the pre-evacuation chamber.
  • a biological specimen 12 is fixed onto a specimen holder 13, into which a holder exchanging rod 14 is screwed.
  • the holder is plunged into liquid nitrogen 15 or liquid Freon in the vessel 16.
  • the specimen is then enclosed by means of a cover 17 (shown in FIG. 3) while still submerged in the refrigerant, after which, the sealed specimen is transferred into the pre-evacuation chamber 7.
  • the cover 17 is removed under vacuum by means of exchanging rod 14, which is mounted with ball 18 and socket 19, so as to force the cover off the holder by pushing it up against projection arm 20 forming part of the pre-evacuation chamber 7. Removal of the cover is facilitated by the provision of a glass viewing window 21 forming part of the cover 9.
  • an interim stage 22 is maintained in the cold state by means of a liquid nitrogen tank 23 and a heat conducting rod 24.
  • An optical microscope 25 for observing the specimen mounted on the interim specimen stage 22 is mounted on the walls of the preevacuation chamber.
  • a manipulating rod 26, supported by a ball 27 and socket 28 for ease of maneuverability is mounted in the chamber wall.
  • a knife 29 or needle is attached to the tip of the rod in order to cut or slice the frozen specimen. Since the knife 29 or needle must be maintained in the cold state during specimen preparation, it is necessary to either bring the knife or needle into direct contact with the stage 22 prior to carrying out the cutting or slicing operation or to connect it to the heat conducting rod 24 by means of heat conducting wire belt 30. In either case, this embodiment enables the frozen specimen to be cut or sliced as desired without frost forming on the newly exposed surface, thereby making it possible to observe the inner structure of the specimen in an almost natural state.
  • FIG. 5 there is shown an embodiment comprising a heating coil 31 provided in the vicinity of the interim stage upon which the specimen holder complete with specimen is mounted. Heating current is supplied to the heating coil 30 from a supply source 32 via insulators 33. By utilizing this embodiment, it is possible to defrost a whole area of the specimen surface.
  • FIG. 6 there is shown an embodiment comprising an infrared ray irradiating device 34 mounted in place of the manipulating rod shown in FIG. 4.
  • the device 34 is mounted on a roller 35, again for ease of maneuverability. Consequently, the infrared rays emitted from the lamp 36 are focused by two builtin lenses 37 and 38 and can be optionally directed on any desired part of the frozen specimen surface. In this way, it is possible to defrost the specific area for observation hardly affecting the temperature of the specimen as a whole.
  • the embodiment as shown in FIG. 7 is designed to prevent frost forming in the specimen chamber during specimen exchange.
  • this invention employs the method whereby dry gas from a gas supply source 39 is supplied to the chamber via the leak valve 11 during the time the specimen is being exchanged.
  • An apparatus for treating a frozen biological specimen or the like in a scanning electron microscope having a specimen chamber and a specimen stage therein comprising a pre-evacuation chamber partitioned from the specimen chamber of the microscope, an interim stage disposed within said pre-evacuation chamber, a specimen holder for holding the specimen, said holder being mounted on said interim stage, means for maintaining the interim stage and the holder sufficiently cold to maintain the sample frozen, a cutting tool attached to the pre-evacuation chamber for cutting the frozen specimen, a heating means for defrosting a surface of the frozen specimen, and means for transferring the treated specimen from the interim stage to the specimen stage of the specimen chamber.
  • heating means comprises a heating coil arranged around the frozen specimen mounted on the interim stage.
  • heating means comprises an infrared ray irradiating device equipped with focusing means for selectively defrosting a given area of the surface of the frozen specimen mounted on the interim stage.
  • An apparatus including means for keeping said cutting tool sufficiently cold so as not to unfreeze the sample surface on contact.
  • An apparatus for treating a frozen biological specimen or the like in a scanning electron microscope having a specimen chamber and a specimen stage therein comprising a pre-evacuation chamber partitioned from the specimen chamber of the microscope, an interim stage housed in said pre-evacuation chamber, a specimen holder holding the specimen, said holder being mounted on said interim stage, means for maintaining the interim stage and the holder sufficiently cold to maintain the sample frozen, a cutting tool attached to the pre-evacuation chamber for cutting the frozen specimen, a heating means for defrosting a surface of the frozen specimen, an optical microscope mounted in the wall of the pre-evacuation chamber for observing the specimen from outside the chamber, and means for transferring the treated specimen from the interim stage to the specimen stage of the specimen chamber.
  • An apparatus for treating a frozen biological specimen or the like in a scanning electron microscope having a specimen chamber and a specimen stage therein comprising a pre-evacuation chamber partitioned from the specimen chamber of the microscope, an interim stage housed in said pre-evacuation chamber, a specimenholder for holding the specimen, said holder being mounted on said interim stage, means for maintaining the interim stage and the holder sufficiently cold to maintain the sample frozen, means for treating the frozen specimen, means for transferring the treated specimen from the interim stage to the specimen stage of the specimen chamber, and a means for supplying the preevacuation chamber of the microscope with dry gas during specimen exchange.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Sampling And Sample Adjustment (AREA)
US00232405A 1971-03-16 1972-03-07 Method and apparatus for observing biological specimens using a scanning electron microscope Expired - Lifetime US3761709A (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP1757871U JPS5120680Y2 (enrdf_load_stackoverflow) 1971-03-16 1971-03-16
JP10057771U JPS5127716Y2 (enrdf_load_stackoverflow) 1971-10-29 1971-10-29
JP46087408A JPS5116252B2 (enrdf_load_stackoverflow) 1971-11-02 1971-11-02

Publications (1)

Publication Number Publication Date
US3761709A true US3761709A (en) 1973-09-25

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US00232405A Expired - Lifetime US3761709A (en) 1971-03-16 1972-03-07 Method and apparatus for observing biological specimens using a scanning electron microscope

Country Status (4)

Country Link
US (1) US3761709A (enrdf_load_stackoverflow)
DE (1) DE2211423C3 (enrdf_load_stackoverflow)
FR (1) FR2130141B1 (enrdf_load_stackoverflow)
GB (1) GB1391336A (enrdf_load_stackoverflow)

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3858049A (en) * 1973-09-17 1974-12-31 Etec Corp Method and apparatus for sem specimen coating and transfer
US3885158A (en) * 1973-10-23 1975-05-20 Harris Corp Specimen block and specimen block holder
US3952203A (en) * 1972-07-21 1976-04-20 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Object adjustment device for a charged particle beam apparatus
US3980885A (en) * 1974-09-06 1976-09-14 Vincent William Steward Diagnosis by proton bombardment
US3984683A (en) * 1975-05-27 1976-10-05 Rca Corporation Apparatus and method for analyzing biological cells for malignancy
US4227080A (en) * 1977-12-05 1980-10-07 Hitachi, Ltd. Device for shifting frozen specimen, for use in scanning type electron microscope
US4284894A (en) * 1979-02-17 1981-08-18 C. Reichert Optische Werke, Ag Cold chamber for the working objects for microscopic and electron microscopic investigations
US4349242A (en) * 1980-02-15 1982-09-14 Nihon Denshi Kabushiki Kaisha Specimen observation apparatus including an optical microscope and a scanning electron microscope
US4749868A (en) * 1985-10-11 1988-06-07 Kureha Chemical Industry Company Limited Method of processing a sample containing water under scanning electron microscope and apparatus thereof
US4916314A (en) * 1988-09-23 1990-04-10 Amoco Corporation Method and apparatus for analyzing components of selected fluid inclusions
US4948979A (en) * 1987-12-21 1990-08-14 Kabushiki Kaisha Toshiba Vacuum device for handling workpieces
US5039864A (en) * 1989-08-11 1991-08-13 Jeol Ltd. Device for replacing electron microscope specimens
US5210424A (en) * 1990-12-20 1993-05-11 Siemens Nixdorf Informationssysteme Ag Cooling means for components in a vacuum chamber
US5362964A (en) * 1993-07-30 1994-11-08 Electroscan Corporation Environmental scanning electron microscope
US5412211A (en) * 1993-07-30 1995-05-02 Electroscan Corporation Environmental scanning electron microscope
US5986270A (en) * 1996-12-23 1999-11-16 U.S. Philips Corporation Particle-optical apparatus including a low-temperature specimen holder
US20040041094A1 (en) * 2002-09-02 2004-03-04 Johannes Petrus Geurts Remco Theodorus Method of obtaining a particle-optical image of a sample in a particle-optical device
US20100230590A1 (en) * 2006-06-07 2010-09-16 Fei Company Compact Scanning Electron Microscope

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2417213A (en) * 1944-12-28 1947-03-11 Rca Corp Device for indirect heating of materials
US3374349A (en) * 1966-11-14 1968-03-19 Victor G. Macres Electron probe having a specific shortfocal length magnetic lens and light microscope
US3483373A (en) * 1966-07-28 1969-12-09 Siemens Ag Airlock assembly for corpuscular ray devices

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2826701A (en) * 1954-09-01 1958-03-11 Gen Electric Low temperature chamber for electronoptics instruments
FR1520418A (fr) * 1965-11-25 1968-04-12 Balzers Patent Beteilig Ag Microscope à émission électronique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2417213A (en) * 1944-12-28 1947-03-11 Rca Corp Device for indirect heating of materials
US3483373A (en) * 1966-07-28 1969-12-09 Siemens Ag Airlock assembly for corpuscular ray devices
US3374349A (en) * 1966-11-14 1968-03-19 Victor G. Macres Electron probe having a specific shortfocal length magnetic lens and light microscope

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Cryofacture of Biological Material, Haggis, Scanning Electron Microscopy Conference, IIT Research Inst., Chicago, Ill. April, 1970 *
Scanning Electron Microscopy of Labile Biological Material..., Echlin et al. Scanning Electron Microscopy Conference, IIT Research Inst. 4/1970 *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3952203A (en) * 1972-07-21 1976-04-20 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Object adjustment device for a charged particle beam apparatus
US3858049A (en) * 1973-09-17 1974-12-31 Etec Corp Method and apparatus for sem specimen coating and transfer
US3885158A (en) * 1973-10-23 1975-05-20 Harris Corp Specimen block and specimen block holder
US3980885A (en) * 1974-09-06 1976-09-14 Vincent William Steward Diagnosis by proton bombardment
US3984683A (en) * 1975-05-27 1976-10-05 Rca Corporation Apparatus and method for analyzing biological cells for malignancy
US4227080A (en) * 1977-12-05 1980-10-07 Hitachi, Ltd. Device for shifting frozen specimen, for use in scanning type electron microscope
US4284894A (en) * 1979-02-17 1981-08-18 C. Reichert Optische Werke, Ag Cold chamber for the working objects for microscopic and electron microscopic investigations
US4349242A (en) * 1980-02-15 1982-09-14 Nihon Denshi Kabushiki Kaisha Specimen observation apparatus including an optical microscope and a scanning electron microscope
US4749868A (en) * 1985-10-11 1988-06-07 Kureha Chemical Industry Company Limited Method of processing a sample containing water under scanning electron microscope and apparatus thereof
US4948979A (en) * 1987-12-21 1990-08-14 Kabushiki Kaisha Toshiba Vacuum device for handling workpieces
US4916314A (en) * 1988-09-23 1990-04-10 Amoco Corporation Method and apparatus for analyzing components of selected fluid inclusions
US5039864A (en) * 1989-08-11 1991-08-13 Jeol Ltd. Device for replacing electron microscope specimens
US5210424A (en) * 1990-12-20 1993-05-11 Siemens Nixdorf Informationssysteme Ag Cooling means for components in a vacuum chamber
US5362964A (en) * 1993-07-30 1994-11-08 Electroscan Corporation Environmental scanning electron microscope
US5412211A (en) * 1993-07-30 1995-05-02 Electroscan Corporation Environmental scanning electron microscope
US5986270A (en) * 1996-12-23 1999-11-16 U.S. Philips Corporation Particle-optical apparatus including a low-temperature specimen holder
US20040041094A1 (en) * 2002-09-02 2004-03-04 Johannes Petrus Geurts Remco Theodorus Method of obtaining a particle-optical image of a sample in a particle-optical device
US6888136B2 (en) * 2002-09-02 2005-05-03 Fei Company Method of obtaining a particle-optical image of a sample in a particle-optical device
EP1394834A3 (en) * 2002-09-02 2005-11-16 FEI Company Method of obtaining an image of a sample in a particle-optical device
US20100230590A1 (en) * 2006-06-07 2010-09-16 Fei Company Compact Scanning Electron Microscope

Also Published As

Publication number Publication date
DE2211423B2 (de) 1979-03-15
DE2211423C3 (de) 1979-11-08
GB1391336A (en) 1975-04-23
FR2130141A1 (enrdf_load_stackoverflow) 1972-11-03
FR2130141B1 (enrdf_load_stackoverflow) 1976-06-11
DE2211423A1 (de) 1972-09-21

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