JP2004198347A - High-pressure freezer and sample support for freeze-fracturing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample support for freeze fracturing a high-pressure freezer capable of being commonly used for the high-pressure freezer and a freeze fracturing device. <P>SOLUTION: A disk-like recession part (sample mounting part) 11c is formed in a sample part 11b of a carrier 11. A biosample 8 is mounted onto the sample mounting part 11c. A through hole 11d for introducing pressure is opened at the center of the carrier 11. One end of the through hole 11d communicates with a through hole 5d, and the other end of the through hole 11d communicates with the sample mounting part 11c. A lid 12 of the support 10 is formed in a disk shape, and a recession 12a of the shape of the bottom of a pan is formed in one surface of the lid 12. The hole diameter of the recession 12a is approximately the sample as the hole diameter of the sample mounting part 11c, and its surface in which the recession 12a is formed is opposed to the biosample 8. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sample holder for freeze breaking of a high-pressure freezing apparatus used for preparing a sample such as a transmission electron microscope.
[0002]
2. Description of the Related Art A high-pressure freezing apparatus is used for preparing a sample of a transmission electron microscope, and is an apparatus for freezing a biological sample containing water under high pressure.
[0003]
For example, under a high pressure of 200 MPa (2000 bar), the freezing point of water drops and the nuclei that form ice crystals cannot be formed up to about −90 ° C., so that the freezing depth of ice-free crystals of a biological sample reaches several hundred μm. Since the size of cells in a biological sample is usually several μm to 30 μm, for example, if the freezing depth of ice-free crystals is 200 μm, a cell structure having a thickness of about 10 cells (assuming a cell size of 20 μm) is destroyed. Observation is possible without being performed.
[0004]
On the other hand, when a biological sample is frozen under normal pressure, the ice-free frozen depth of the sample is as shallow as 10 to 20 μm, and only an ice-free frozen layer having a thickness of one cell is obtained. For these reasons, the high-pressure freezing apparatus has attracted particular attention at present.
[0005]
FIG. 1 shows a conventional high-pressure freezing apparatus. In FIG. 1, reference numeral 1 denotes a chamber. In a processing chamber 2 in the chamber 1, a high-pressure device 3 and a coolant injection device 4 are arranged. The pot holding rod 6 holding the pot 5 is mounted on the rod mounting section 7 on the chamber 1 side.
[0006]
As shown in the perspective view of FIG. 1, the pot 5 has an open space portion 5a, a disk-shaped fixing portion 5b, and a connecting portion 5c connected to the fixing portion 5b. A through-hole 5d for introducing pressure is formed at the center of the fixing portion 5b and the connecting portion 5c, and the through-hole 5d is connected to the high-pressure device 3 at the end of the connecting portion 5c.
[0007]
Further, a holding screw 5e is screwed into the pot 5, and the disc-shaped carrier 9 holding the biological sample 8 is fixed to the fixing portion 5b by tightening the holding screw 5e. The biological sample 8 is placed on a disc-shaped concave portion 9a formed in the carrier 9, and the surface of the biological sample 8 faces the pressure introducing through-hole 5d. The carrier 9 and the fixing portion 5b are connected to each other so that the concave portion 9a is sealed with respect to the open space portion 5a.
[0008]
In the apparatus having such a configuration, when the biological sample 8 is frozen under high pressure, first, a fluid (for example, metal cyclohexane) is supplied from the high-pressure device 3 to the through-hole 5d, and the fluid is applied to the biological sample 8 through the fluid in an amount of 0.4 to 0.4. A pressure of 0.5 Mpa is applied. Next, a high pressure (for example, 200 MPa) from the high-pressure device 3 is applied to the sample 8 via the fluid in the through hole 5d.
[0009]
Then, under the high pressure, a coolant (for example, liquid nitrogen) is injected from the coolant injection device 4, and the liquid nitrogen is sprayed on the carrier 9 and the fixed portion 5b through the open space 5a. Thus, the biological sample 8 is frozen under high pressure.
[0010]
A high-pressure freezing apparatus as shown in FIG. 1 is known, for example, from Patent Document 1 below.
[0011]
[Patent Document 1]
JP 2001-318035 A
By the way, as a device used for preparing a sample of a transmission electron microscope, there is a freeze breaking device apart from the high-pressure freezing device described above. As shown in FIG. 2 (a), this freezing / cleaving device is a device for cutting a frozen sample by moving a cutting knife in the direction of an arrow. By using such a freeze-fracture apparatus, a frozen sample can be broken along the cell surface, and a sample for a transmission electron microscope having an uneven cell surface can be obtained.
[0013]
Therefore, the present inventor applied the carrier 9 to the sample holder of the freeze-splitting device in order to cut the sample 8 high-pressure frozen by the high-pressure freezing device of FIG. 1 by the freeze-severing device shown in FIG. I thought about setting. However, as shown in FIG. 2B, the surface of the frozen sample 8 is flush with the upper surface of the carrier 9, so that the sample cannot be cut by the cutting knife.
[0014]
In the first place, the carrier 9 in the high-pressure freezing device is not made for use in the freeze-cutting device. Usually, the sample 8 that has been subjected to high-pressure freezing in the high-pressure freezing apparatus is immersed together with the carrier 9 in a replacement treatment solution for two to three days for freezing replacement processing. Then, the sample 8 having undergone the substitution process is embedded in an embedding material, and thereafter, is thinly cut with a sharp microtome to obtain a sample for a transmission electron microscope. As described above, the carrier 9 in the high-pressure freezing apparatus shown in FIG. 1 is made for freezing replacement, and is not made for the freezing and breaking apparatus.
[0015]
The present invention has been made in view of such a point, and an object of the present invention is to provide a sample holder for freeze-cutting of a high-pressure freezing device that can be used commonly for a high-pressure freezing device and a freeze-cutting device.
[0016]
According to a first aspect of the present invention, there is provided a freeze-cleaving sample holder which has a sample mounting portion, and a carrier having a through-hole for introducing pressure through the sample mounting portion. And a lid placed on the carrier so as to cover the sample placed on the sample mounting portion.
[0017]
Embodiments of the present invention will be described below with reference to the drawings.
[0018]
FIG. 3 is a diagram showing an example of the sample holder for freeze-fracturing of the present invention. 3, the same components as those in FIG. 1 are denoted by the same reference numerals as those in FIG. 1, and a description thereof will be omitted.
[0019]
In FIG. 3, reference numeral 10 denotes a sample holder for freeze-fracture of the present invention, and the configuration other than the holder 10 is the same as that of FIG. The holder 10 includes a carrier 11 and a lid 12, and the holder 10 holding the biological sample 8 is fixed to the fixing portion 5b by tightening the holding screw 5e.
[0020]
The carrier 11 of the holder 10 has a disk-shaped flange 11a and a disk-shaped sample portion 11b, and the diameter of the flange 11a is substantially the same as the diameter of the fixed portion 5b. As shown in the perspective view of FIG. 3, a disc-shaped concave portion (sample mounting portion) 11c is formed in the sample portion 11b, and the sample mounting portion 11c has a hole diameter of about 1.4 mm. The depth of the mounting portion 11c is about 0.2 mm (that is, 200 μm). The biological sample 8 is mounted on the sample mounting portion 11c. Further, a through hole 11d for pressure introduction is formed at the center of the carrier 11, one end of the through hole 11d communicates with the through hole 5d, and the other end of the through hole 11d is connected to the sample mounting hole. It communicates with the part 11c.
[0021]
On the other hand, the lid 12 of the holder 10 is formed in a disk shape, and the diameter of the lid 12 is substantially the same as the diameter of the sample portion 11b. As shown in the perspective view of FIG. 3, a pot-shaped depression 12a is formed on one side of the lid 12, and the hole diameter of the depression 12a is substantially the same as the hole diameter of the sample mounting portion 11c. The lid 12 is placed on the sample portion 11b of the carrier 11 such that the surface on which the depression 12a is formed faces the biological sample 8.
[0022]
When the biological sample 8 is set on the sample mounting portion 11c, the biological sample 8 is set so that the surface of the biological sample 8 is higher than the upper surface of the sample portion 11b. The portion 12a is also filled with the biological sample 8. Then, the carrier 11 and the lid 12 are sealed so that the room filled with the biological sample 8, that is, the room formed by the depression 12 a and the sample mounting portion 11 c is sealed with respect to the open space 5 a. Are connected to each other.
[0023]
In the apparatus having such a configuration, when the biological sample 8 is frozen under high pressure, first, a fluid (for example, metal cyclohexane) is supplied from the high-pressure device 3 to the through holes 5d and 11d. A pressure of 4 to 0.5 Mpa is applied. Next, a high pressure (for example, 200 Mpa) from the high-pressure device 3 is applied to the sample 8 through the fluid in the through holes 5d and 11d. Then, under the high pressure, a coolant (for example, liquid nitrogen) is ejected from the coolant ejecting device 4, and the liquid nitrogen is sprayed onto the carrier 11 and the lid 12 through the open space 5a. As a result, a frozen sample 8 having an ice-free frozen layer of about 200 μm on the side of the through hole 11d is obtained.
[0024]
When the biological sample 8 is frozen under high pressure in this way, the pot 5 is taken out to a liquid nitrogen tank (not shown) outside the chamber 1. Then, the sample maker loosens the holding screw 5e of the pot 5 in liquid nitrogen using an auxiliary device, and takes out the carrier 11 containing the frozen sample 8 and the lid 12 from the pot 5. Further, the sample maker attaches the carrier 11 with the lid 12 to the sample holder 13 of the freeze-fracture apparatus in liquid nitrogen as shown in FIG.
[0025]
In FIG. 4, reference numeral 13 denotes a sample holder of the freeze cleaving apparatus, and a screw 13a is cut on a side surface of the sample holder 13. On the upper surface of the sample holder 13, a disc-shaped concave portion (carrier mounting portion) 13b for mounting the carrier 11 is formed. The sample maker grasps the carrier 11 in liquid nitrogen using an auxiliary device, and sets the carrier 11 with the lid 12 on the carrier mounting portion 13b.
[0026]
Then, the sample maker attaches the cap 14 to the sample holder 13 in liquid nitrogen using an auxiliary device. A screw is cut inside the cap 14, and a hole 15 slightly larger than the diameter of the lid 12 is formed on the upper surface of the cap 14.
[0027]
Thereafter, the sample creator takes out the sample holder 13 from the liquid nitrogen and attaches the sample holder 13 to the sample stage of the freeze cleaving device. FIG. 5 is a diagram (cross-sectional view) showing the sample holder 13 set on the sample stage 16 of the freeze cleaving apparatus. The sample stage 16 has a cooling mechanism, and the frozen sample 8 is cooled to a low temperature by the cooling mechanism. The sample chamber of the freeze cleaving device in which the sample holder 13 is disposed is exhausted by an exhaust device.
[0028]
As can be seen from FIG. 5, the upper surface of the carrier 11 and the upper surface of the cap 14 are substantially flush with each other, and the lid 12 projects higher than the upper surface of the cap 14. In addition, the surface of the frozen sample 8 rising higher than the upper surface of the carrier 11 projects higher than the upper surface of the cap 14. In such a state, the cutting knife of the freeze cutting device is moved in the direction of the arrow in the figure, and the cutting knife collides with the lid 12. Due to this collision, the lid 12 is removed from the carrier 11, and the frozen sample 8 raised above the upper surface of the cap 14 is cut by the cutting knife.
[0029]
Due to this cleavage, the frozen sample 8 is broken along the upper surface of the carrier 11, and at that time, the frozen sample 8 is broken along the cell surface. As a result, a frozen sample 8 having an uneven cell surface is obtained. Further, the thickness of the frozen sample 8 broken along the upper surface of the carrier 11 is about 200 μm, which is the same as the depth of the sample mounting portion 11c. The cell surface in the frozen layer of ice crystals. Therefore, a cell surface in which the cell structure is not destroyed is obtained. Since the sample chamber of the freeze cleaving apparatus is evacuated, the cut surface (cell surface) does not become frosted after the sample is cleaved, and the cell surface is maintained in a good state without being destroyed.
[0030]
After such sample cutting, gold vapor deposition processing is continuously performed on the cut surface of the sample 8 in the sample chamber of the freeze cutting device. Gold deposition in a so-called replica method is performed. After the gold deposition process, the sample holder 13 is removed from the sample stage 16, and the sample 8 is immersed in the sample solution. As a result, the sample 8 is melted, and only the gold coating film deposited on the surface of the sample 8 remains. Then, the gold coating film is set on a sample holder of a transmission electron microscope, and an image is observed by a transmission electron microscope.
[0031]
As described above, an example of the sample holder for freeze-breaking of the present invention has been described with reference to FIGS. 3 to 5. However, if the sample holder 10 for freeze-breaking is used, the sample 8 can be satisfactorily high-pressure frozen in a high-pressure freezing apparatus. can do. Further, the surface of the sample 8 rises higher than the upper surface of the carrier 11 by the depression 12a of the lid 12 of the sample holder 10 for freeze-fracturing, so that the frozen sample frozen under high pressure can be surely fractured by the freeze-fracturing apparatus. As described above, the sample holder 10 for freeze-fracturing of the present invention can be used commonly for the high-pressure freezing device and the freeze-fracturing device.
[0032]
In the above example, the depression 12a is formed in the lid 12, but the biological sample 8 may be cut even by using a flat lid having no such depression. That is, when the flat lid is removed from the carrier 11 by the collision of the cutting knife in the cleavage in FIG. 5, the lid is removed with the surface layer of the frozen sample 8 adhered to the surface of the lid. Thus, even when the surface layer of the frozen sample is peeled off, it is possible to obtain a frozen sample in which an uneven cell surface (fracture surface) appears. Also in this case, since the frozen sample is covered with the lid until the fracture, the frozen sample does not come into contact with the air even if the carrier or the lid comes into contact with the air. Therefore, no frost is formed on the surface of the frozen sample, and destruction of the cell surface due to the frost can be prevented.
[0033]
FIG. 6 is a diagram showing another example of the present invention. In FIG. 6, reference numeral 17 denotes a pot, and coolant passage holes 18 are formed above and below the pot 17. The fixing portion 19 of the pot 17 has a through hole 20 for introducing pressure. A disc-shaped carrier 22 is placed in the disc-shaped recess 21 formed in the fixing portion 19.
[0034]
On the upper surface of the carrier 22, a disc-shaped concave portion (sample mounting portion) 23 is formed, and a biological sample 24 is mounted thereon. The carrier 22 is provided with a pressure introduction through-hole 25. One end of the through-hole 25 communicates with the sample mounting portion 23, and the other end communicates with the through-hole 20. The carrier 22 is pressed by a holding screw 26, and a depression 27 is formed in the holding screw 26. The depression forms a heaped portion of the sample 24.
[0035]
Such a pot 17 is mounted on the high-pressure freezing apparatus shown in FIG. 3, and in a state where the sample 24 is applied with the high pressure from the high-pressure apparatus 3, the liquid nitrogen from the coolant injection apparatus 4 receives the press screw 26 and the like. , And the biological sample 24 is frozen at high pressure. In this case, the liquid nitrogen is sprayed from a direction perpendicular to the sample surface, and is evenly sprayed on the surface of the thinly formed press screw 26. Therefore, particularly in this example, the biological sample 24 can be efficiently cooled, and the sample can be rapidly frozen.
[Brief description of the drawings]
FIG. 1 is a view showing a conventional high-pressure freezing apparatus.
FIG. 2 is a view for explaining a conventional problem.
FIG. 3 is a diagram showing an example of the present invention.
4 is a view showing a sample holder of the freeze-splitting apparatus, and is a view showing a sample holder on which the sample holder for freeze-splitting of FIG. 3 is set.
FIG. 5 is a diagram (cross-sectional view) showing a sample holder set on a sample stage of the freeze-slicing apparatus.
FIG. 6 is a diagram showing another example of the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Chamber, 2 ... Processing chamber, 3 ... High pressure apparatus, 4 ... Coolant injection apparatus, 5 ... Pot, 5a ... Open space part, 5b ... Fixed part, 5c ... Connection part, 5d ... Through-hole, 5e ... Holding screw , 6: pot holding rod, 7: rod mounting portion, 8: sample, 9: carrier, 9a: concave portion, 10: freezing sample holder, 11: carrier, 11a: flange portion, 11b: sample portion, 11c ... Sample mounting portion, 11d through hole, 12 lid, 12a recess, 13 sample holder, 13a screw, 13b recess, 14 cap, 15 hole, 16 sample stage, 17 pot, 18 Coolant passage hole, 19: fixing portion, 20: through hole, 21: disk-shaped concave portion, 22: carrier, 23: sample mounting portion, 24: sample, 25: pressure introduction through hole, 26: holding screw, 27: Depression

Claims (5)

A carrier having a sample mounting portion and having a through hole for introducing pressure communicating with the sample mounting portion,
A sample holder for freeze-fracture of a high-pressure freezing device, comprising: a lid placed on the carrier so as to cover the sample placed on the sample mounting portion. 2. The sample holder for freeze breaking of a high-pressure freezing apparatus according to claim 1, wherein a depression is formed on a surface of the lid facing the sample. The freeze-fracturing sample holder can be mounted on a sample holder of a freeze-fracturing device, and the freeze-fracturing sample holder is configured such that the lid is detached from the carrier by collision of a cutting knife of the freeze-fracturing device. 3. The sample holder for freeze breaking of a high-pressure freezing apparatus according to claim 1, wherein the sample holder is held by a sample holder. A carrier having a sample mounting portion and having a through hole for introducing pressure communicating with the sample mounting portion,
A lid placed on the carrier so as to cover the sample placed on the sample mounting portion,
A pot having a pressure introduction through-hole communicating with a through-hole of the carrier, while holding the carrier and the lid so that the lid does not come off the carrier,
A pressure supply device connected to the pressure introduction through-hole of the pot,
Means for cooling the carrier. The high-pressure freezing apparatus according to claim 4, wherein a depression is formed on a surface of the lid facing the sample.

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