U.S. patent number 3,761,709 [Application Number 05/232,405] was granted by the patent office on 1973-09-25 for method and apparatus for observing biological specimens using a scanning electron microscope.
This patent grant is currently assigned to Nihon Denshi Kabushiki Kaisha. Invention is credited to Yoichi Hasegawa, Yuji Nagasawa.
United States Patent |
3,761,709 |
Hasegawa , et al. |
September 25, 1973 |
METHOD AND APPARATUS FOR OBSERVING BIOLOGICAL SPECIMENS USING A
SCANNING ELECTRON MICROSCOPE
Abstract
A biological specimen is prepared for electron scanning by
plunging it into liquid nitrogen or liquid Freon. The frozen
specimen together with its holder is transferred into the
pre-evacuation chamber of a scanning electron microscope and
treated according to the purpose of observation. When this is
completed, the specimen is transferred to the specimen stage in the
specimen chamber, the stage being maintained cold. The scanning
electron beam is applied and the specimen image is observed.
Inventors: |
Hasegawa; Yoichi (Tokyo,
JA), Nagasawa; Yuji (Tokyo, JA) |
Assignee: |
Nihon Denshi Kabushiki Kaisha
(Tokyo, JA)
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Family
ID: |
27281891 |
Appl.
No.: |
05/232,405 |
Filed: |
March 7, 1972 |
Foreign Application Priority Data
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Mar 16, 1971 [JA] |
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46/17578 |
Oct 29, 1971 [JA] |
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46/100577 |
Nov 2, 1971 [JA] |
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46/87408 |
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Current U.S.
Class: |
250/442.11;
850/14; 850/29; 250/443.1; 359/395 |
Current CPC
Class: |
H01J
37/18 (20130101); H01J 37/20 (20130101) |
Current International
Class: |
H01J
37/18 (20060101); H01J 37/20 (20060101); H01J
37/02 (20060101); H01j 037/18 () |
Field of
Search: |
;250/49.5A,49.5B |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
"Scanning Electron Microscopy of Labile Biological Material...,"
Echlin et al. Scanning Electron Microscopy Conference, IIT Research
Inst. 4/1970 .
"Cryofacture of Biological Material," Haggis, Scanning Electron
Microscopy Conference, IIT Research Inst., Chicago, Ill. April,
1970.
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Primary Examiner: Lawrence; James W.
Assistant Examiner: Church; C. E.
Claims
We claim:
1. 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.
2. An apparatus according to claim 1 in which said heating means
comprises a heating coil arranged around the frozen specimen
mounted on the interim stage.
3. An apparatus according to claim 1 in which said 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.
4. An apparatus according to claim 1 including means for keeping
said cutting tool sufficiently cold so as not to unfreeze the
sample surface on contact.
5. 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.
6. 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 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 pre-evacuation chamber of
the microscope with dry gas during specimen exchange.
Description
BACKGROUND
As compared with a conventional transmission type electron
microscope, a scanning electron microscope has the advantage of
being able to produce a stereo image of the specimen configuration.
In this case, however, since bulk specimens are used, the presence
of water removed by sublimation in the microscope column results in
damage to the specimen which adversely affects the natural
three-dimensional configuration. This particularly applies to
biological and chemical samples where the water content is high. In
an effort to solve this problem, quench freezing of the specimen
has recently been introduced. Quench freezing has a twofold
advantage in that in addition to eliminating the possibility of
damage due to sublimation, the specimen surface requires no coating
in order to carry out observation. It is, however, necessary to
prevent frost from forming on the surface of the frozen specimen or
alternatively to effectively remove the frost from said
surface.
Accordingly, it is an advantage of this invention to prevent the
formation of frost on the specimen surface or to facilitate the
removal of frost forming on the specimen surface. It is another
advantage of this invention to prevent the formation of frost on
the cross-section surface of the specimen where it is desired to
carry out observation of the section. It is yet another advantage
of this invention to prevent frost from forming on the interim
specimen stage housed in the pre-evacuation chamber or on the inner
wall of the pre-evacuation chamber during specimen exchange.
BRIEF DESCRIPTION
Briefly, according to this invention, 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.
In accordance with a preferred embodiment of this invention, 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.
THE DRAWINGS
The invention will now be further described with reference to the
accompanying drawings, in which:
FIG. 1 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; and,
FIG. 7 shows an embodiment for preventing the formation of frost
during specimen exchange.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring to FIG. 1, an electron optical column 1 includes an
electron gun chamber 1a and specimen chamber 1b. 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.
Referring to FIG. 2, a biological specimen 12 is fixed onto a
specimen holder 13, into which a holder exchanging rod 14 is
screwed. Thus assembled, 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. Next, as shown in FIG. 3, 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. When this is completed, the specimen
is transferred to the specimen stage in the specimen chamber in
column 1, the stage being maintained in the cold state, the
scanning electron beam is applied, and the specimen image is
observed.
Referring to FIG. 4, 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
pre-evacuation 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.
Referring to 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.
Referring to 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 built-in
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. In the
conventional apparatus, it is necessary to expose the
pre-evacuation chamber to the atmsophere in order to retrieve the
examined specimen and replace it with a new one. Consequently, the
interim specimen stage becomes coated with frost; moreover, the
sublimation of this frost, as a result of subsequent evacuation,
causes frost to form on the new specimen. To prevent this from
occurring, 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.
Having thus described the invention with the detail and
particularity as required by the Patent Laws, what is desired
protected by Letters Patent is set forth in the following
claims.
* * * * *