JP3781723B2 - Sample holder for freezing breakage of high-pressure freezing apparatus and high-pressure freezing apparatus - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a sample holder for freezing breakage of a high-pressure freezing apparatus used in sample preparation such as a transmission electron microscope.
[0002]
[Prior art]
The high-pressure freezing apparatus is used in the preparation of a transmission electron microscope sample, and is an apparatus for freezing water-containing biological samples and the like under high pressure.
[0003]
For example, at a high pressure of 200 Mpa (2000 bar), the freezing point of water decreases, and nuclei that form ice crystals cannot be formed up to about −90 ° C. Therefore, the depth of ice-freezing of biological samples reaches several hundred μm. Since the cell size of a biological sample is usually several μm to 30 μm, for example, if the ice-free crystal freezing depth is 200 μm, the cell structure with a thickness of about 10 cells (with a cell size of 20 μm) is destroyed. Observation is possible without being done.
[0004]
On the other hand, when a biological sample is frozen under normal pressure, the ice-free crystal freezing depth of the sample is as shallow as 10 to 20 μm, and only an ice-free crystal frozen layer having a thickness of one cell is obtained. For these reasons, high-pressure freezing devices are currently attracting particular attention.
[0005]
FIG. 1 is a view showing a conventional high-pressure freezing apparatus. In FIG. 1, 1 is a chamber. In the processing chamber 2 in the chamber 1, a high-pressure device 3 and a coolant injection device 4 are arranged. A pot holding rod 6 holding the pot 5 is mounted on the rod mounting portion 7 on the chamber 1 side.
[0006]
As shown in the perspective view of FIG. 1, the pot 5 has an open space 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 opened at the center of the fixed 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 presser screw 5e is screwed into the pot 5, and the discoid carrier 9 holding the biological sample 8 is fixed to the fixing part 5b by tightening the presser screw 5e. The biological sample 8 is placed on a disk-shaped recess 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 fixed 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 subjected to high-pressure freezing, first, a fluid (for example, metal cyclohexane) is supplied from the high-pressure apparatus 3 to the through hole 5d, and 0.4 to 0.4 is supplied to the biological sample 8 through the fluid. 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 through the fluid in the through hole 5d.
[0009]
Under the high pressure, a coolant (for example, liquid nitrogen) is sprayed from the coolant spray device 4, and the liquid nitrogen is sprayed onto the carrier 9 and the fixed portion 5b through the open space 5a. Thus, the biological sample 8 is frozen at high pressure.
[0010]
A high-pressure freezing apparatus as shown in FIG. 1 is known, for example, in Patent Document 1 below.
[0011]
[Patent Document 1]
Japanese Patent Laid-Open No. 2001-318035
[Problems to be solved by the invention]
By the way, as an apparatus used for sample preparation of a transmission electron microscope, there is a freezing cleaving apparatus in addition to the high-pressure freezing apparatus described above. This frozen cleaving apparatus is an apparatus that cleaves a frozen sample by moving a cleaving knife in the direction of an arrow as shown in FIG. If such a freezing cleaving apparatus is used, the frozen sample can be cracked along the cell surface, and a sample for a transmission electron microscope in which an uneven cell surface appears can be obtained.
[0013]
Therefore, the inventor of the present invention uses the carrier 9 as a sample holder of the freezing cleaving apparatus in order to cleave the sample 8 that has been high-pressure frozen by the high-pressure freezing apparatus of FIG. 1 using the freezing cleaving apparatus 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 cleaved with a cleaving knife.
[0014]
In the first place, the carrier 9 in the high-pressure freezing device is not made for the purpose of being used in the freezing and breaking device. Usually, the sample 8 that has been subjected to high-pressure freezing in the high-pressure freezing apparatus is immersed in the replacement processing solution for 2 to 3 days together with the carrier 9 for the freeze replacement processing. Then, the sample 8 that has been subjected to the replacement process is embedded with an embedding material, and then the cutting edge 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 of FIG. 1 is made for freeze replacement, and is not made for the freezing cleaving apparatus.
[0015]
The present invention has been made in view of the above points, and an object of the present invention is to provide a sample holder for freezing breakage of a high-pressure freezing apparatus that can be used in common for a high-pressure freezing apparatus and a freezing breaking apparatus.
[0016]
[Means for Solving the Problems]
The sample holder for freezing and fracturing of the present invention that achieves this object has a concave sample mounting portion, a carrier having a pressure introducing through hole that communicates with the sample mounting portion, and the sample mounting portion. It is a lid placed on the carrier so as to cover the placed sample, and includes a lid having a recess formed on a surface facing the sample .
[0017]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0018]
FIG. 3 is a diagram showing an example of the sample holder for freezing and breaking according to the present invention. In FIG. 3, the same components as those in FIG. 1 are assigned the same numbers as in FIG.
[0019]
In FIG. 3, 10 is a sample holder for freezing and cutting of the present invention, and the configuration other than the holder 10 is the same as FIG. The holding body 10 includes a carrier 11 and a lid 12, and the holding body 10 holding the biological sample 8 is fixed to the fixing portion 5b by tightening the presser screw 5e.
[0020]
The carrier 11 of the holding body 10 has a disk-shaped flange portion 11a and a disk-shaped sample portion 11b, and the diameter of the flange portion 11a is substantially the same as the diameter of the fixed portion 5b. Further, as shown in the perspective view of FIG. 3, the sample portion 11b is formed with a disk-shaped recess (sample placement portion) 11c, and the hole diameter of the sample placement portion 11c is 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 placed on the sample mounting portion 11c. Further, a through hole 11d for introducing pressure 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 the sample mounting. It leads to part 11c.
[0021]
On the other hand, the lid 12 of the holder 10 is formed in a disc 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 in FIG. 3, a pan-bottom depression 12a is formed on one surface 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 11 b of the carrier 11 so that the surface on which such a depression 12 a 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. Therefore, as shown in FIG. The portion 12 a is also filled with the biological sample 8. The room filled with the biological sample 8, that is, the room formed by the depression 12a and the sample placement part 11c is sealed with respect to the open space part 5a so that the carrier 11 and the lid 12 are sealed. Are connected to each other.
[0023]
In the apparatus having such a configuration, when the biological sample 8 is subjected to high-pressure freezing, first, a fluid (for example, metal cyclohexane) is supplied from the high-pressure apparatus 3 to the through holes 5d and 11d, and the biological sample 8 is supplied to the biological sample 8 through the fluid. 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. Under the high pressure, a coolant (for example, liquid nitrogen) is sprayed from the coolant spray 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 crystal frozen layer of about 200 μm on the through hole 11d side is obtained.
[0024]
Thus, when the biological sample 8 is frozen at high pressure, the pot 5 is taken out into a liquid nitrogen tank (not shown) outside the chamber 1. Then, the sample creator loosens the holding screw 5e of the pot 5 in liquid nitrogen using an auxiliary device, and takes out the carrier 11 and the lid 12 containing the frozen sample 8 from the pot 5. Further, the sample creator attaches the carrier 11 with the lid 12 to the sample holder 13 of the freeze cleaving 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 13 a is cut on the side surface of the sample holder 13. In addition, a disk-shaped concave portion (carrier placement portion) 13 b for placing the carrier 11 is formed on the upper surface of the sample holder 13. The sample creator grips the carrier 11 in liquid nitrogen using an auxiliary device, and sets the carrier 11 with the lid 12 on the carrier placement portion 13b.
[0026]
Then, the sample creator attaches the cap 14 to the sample holder 13 using an auxiliary device in liquid nitrogen. A screw is cut inside the cap 14, and a hole 15 slightly larger than the diameter of the lid 12 is formed in the upper surface of the cap 14.
[0027]
Thereafter, the sample creator removes the sample holder 13 from the liquid nitrogen and attaches the sample holder 13 to the sample stage of the freeze cleaving apparatus. FIG. 5 is a diagram (sectional view) showing the sample holder 13 set on the sample stage 16 of the freezing cleaving apparatus. The sample stage 16 includes a cooling mechanism, and the frozen sample 8 is cooled to a low temperature by the cooling mechanism. In addition, the sample chamber of the freeze cleaving apparatus in which the sample holder 13 is disposed is exhausted by an exhaust apparatus.
[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 on the same plane, and the lid 12 protrudes higher than the upper surface of the cap 14. Further, the surface of the frozen sample 8 raised above the upper surface of the carrier 11 protrudes higher than the upper surface of the cap 14. In such a state, the cleaving knife of the freezing cleaving apparatus is moved in the direction of the arrow in the figure, and the cleaving knife collides with the lid 12. The lid 12 is removed from the carrier 11 by this collision, and the frozen sample 8 raised above the upper surface of the cap 14 is cleaved by the cleaving knife.
[0029]
By this cleaving, the frozen sample 8 is cracked along the upper surface of the carrier 11, and the frozen sample 8 is cracked along the cell surface. As a result, the frozen sample 8 on which the uneven cell surface appears is obtained. In addition, the thickness of the frozen sample 8 cracked 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. It is a cell surface in an ice crystal frozen layer. For this reason, a cell surface in which the cell structure is not destroyed is obtained. In addition, since the sample chamber of the freezing cleaving apparatus is evacuated, frost does not form on the fractured surface (cell surface) after the sample cleaving, and the cell surface is maintained in a good state without being destroyed.
[0030]
After such a sample cleaving is performed, a gold vapor deposition process is subsequently performed on the cleaved surface of the sample 8 in the sample chamber of the freezing cleaving apparatus. Gold deposition in a so-called replica method is performed. After the gold vapor 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, this gold coating film is set on a sample holder of a transmission electron microscope, and image observation with a transmission electron microscope is performed.
[0031]
As mentioned above, although an example of the sample holder for freezing cutting of this invention was demonstrated using FIGS. 3-5, if the sample holding body 10 for freezing cutting mentioned above is used, the sample 8 will be favorably high-pressure freezing in a high-pressure freezing apparatus. can do. Furthermore, since the surface of the sample 8 rises higher than the upper surface of the carrier 11 by the recess 12a of the lid 12 of the sample holder 10 for freezing cleaving, the frozen sample frozen at high pressure can be reliably cleaved by the freezing cleaving apparatus. Thus, the sample holder 10 for freezing and cleaving of the present invention can be used in common for the high-pressure freezing apparatus and the freezing cleaving apparatus.
[0032]
In the above example, the depression 12a is formed in the lid 12, but the biological sample 8 may be cleaved even if a flat lid without such a depression is used. In the cleaving in FIG. 5, when the flat lid is removed from the carrier 11 by the collision of the cleaving knife, the lid is removed with the surface layer of the frozen sample 8 stuck 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 having an uneven cell surface (split section). Also in this case, since the frozen sample is covered with the lid until it is cleaved, the frozen sample is not exposed to the atmosphere even if the carrier or the lid is exposed to the atmosphere. For this reason, frost does not form on the surface of the frozen sample, and destruction of the cell surface due to frost can be prevented.
[0033]
FIG. 6 shows another example of the present invention. In FIG. 6, reference numeral 17 denotes a pot, and coolant passing holes 18 are formed above and below the pot 17. A through hole 20 for introducing pressure is formed in the fixing portion 19 of the pot 17. A disc-shaped carrier 22 is placed in a disc-shaped recess 21 formed in the fixed portion 19.
[0034]
On the upper surface of the carrier 22, a disk-shaped concave portion (sample placement portion) 23 is formed, and a biological sample 24 is placed thereon. The carrier 22 has a pressure introducing 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 presser screw 26, and a recess 27 is formed in the presser screw 26. A pile of the sample 24 is created by this depression.
[0035]
Such a pot 17 is attached to the high-pressure freezing device shown in FIG. 3, and liquid nitrogen from the coolant injection device 4 is pressed into the holding screw 26 and the like in a state where a high pressure from the high-pressure device 3 is applied to the sample 24. The biological sample 24 is frozen at high pressure. In this case, liquid nitrogen is sprayed from a direction perpendicular to the sample surface, and is sprayed uniformly on the surface of the presser screw 26 made thin. For this reason, especially 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 diagram for explaining a conventional problem.
FIG. 3 is a diagram showing an example of the present invention.
4 is a diagram showing a sample holder of the freeze cleaving apparatus, and is a diagram showing a sample holder on which the frozen cleaving sample holder shown in FIG. 3 is set. FIG.
FIG. 5 is a diagram (cross-sectional view) showing a sample holder set on a sample stage of a freeze cleaving 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 ... Fixing part, 5c ... Connection part, 5d ... Through-hole, 5e ... Holding screw , 6 ... Pot holding rod, 7 ... Rod mounting part, 8 ... Sample, 9 ... Carrier, 9a ... Recessed part, 10 ... Sample holder for freezing, 11 ... Carrier, 11a ... Gutter, 11b ... Sample part, 11c ... Sample mounting portion, 11d ... through hole, 12 ... lid, 12a ... dent, 13 ... sample holder, 13a ... screw, 13b ... concave, 14 ... cap, 15 ... hole, 16 ... sample stage, 17 ... pot, 18 ... Coolant passage hole, 19 ... fixed portion, 20 ... through hole, 21 ... disc-shaped recess, 22 ... carrier, 23 ... sample mounting portion, 24 ... sample, 25 ... pressure introducing through hole, 26 ... presser screw, 27 ... Hollow

Claims (3)

A carrier having a concave sample mounting portion and having a through hole for introducing pressure leading to the sample mounting portion;
A lid placed on the carrier so as to cover a sample placed on the sample mounting section, and for freezing cleaving of a high-pressure freezing apparatus provided with a lid formed with a recess on a surface facing the sample Sample holder.   The frozen cleaving sample holder can be attached to a sample holder of a freezing cleaving apparatus, and the freezing cleaving sample holder can be removed from the carrier by the collision of a cleaving knife of the freezing cleaving apparatus. The sample holder for freezing cleaving of a high-pressure freezing apparatus according to claim 1, wherein the sample holder is held by a sample holder. A carrier having a concave sample mounting portion and having a through hole for introducing pressure leading to the sample mounting portion;
A lid placed on the carrier so as to cover the sample placed on the sample placement unit, and a lid having a depression formed on the surface facing the sample;
Holding the carrier and the lid so that the lid does not come off from the carrier, and a pot having a pressure introducing through hole communicating with the through hole of the carrier;
A pressure supply device connected to the pressure introducing through hole of the pot;
A high-pressure freezing apparatus provided with means for cooling the carrier.

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