JP2023022846A - Sample preparation method, freeze pressurization device, and observation method - Google Patents

Abstract

To provide a sample preparation method for suppressing formation of an ice crystal, a freeze pressurization device and an observation method.SOLUTION: A sample preparation method includes: a step for filling a space inside of a sample preparation device 10 comprising a first housing member 11a and a second housing member 11b having a cavity unit and an opening unit with one surface of the cavity unit opened with an object to be observed and water; a step of fixing the opening of the first housing member 11a and the opening of the second housing member 11b together; a step of cooling the sample preparation device 10; and a step of dividing the object to be observed frozen by the step of cooling, by separating the first housing member 11a and the second housing member 11b.SELECTED DRAWING: Figure 4

Description

Applied for the application of Article 30, Paragraph 2 of the Patent Law Presented at the 60th High Pressure Symposium hosted by the High Pressure Society of Japan on October 23, 2019. Presented at the 10th Ionic Liquid Symposium hosted by the Ionic Liquid Research Group on November 21, 2019.

TECHNICAL FIELD The present invention relates to a sample preparation method, a freezing pressurization device, and an observation method.

When observing a substance with a high water content such as food, plants, micro-organisms, cells, etc. as an object to be observed with a microscope, the object to be observed is frozen and cut to prepare a sample for observation. A freeze-fracture sample preparation technique is known (for example, Patent Document 1, etc.).

However, when the water contained in the object to be observed freezes, ice crystals are generated and the object to be observed is destroyed, so there is a problem that proper observation cannot be performed. In order to suppress such destruction of the observed object, a method of suppressing the formation of ice crystals by using an organic solvent or the like has been proposed, but the organic solvent may affect the observed object. I didn't go to observe.

In this regard, Non-Patent Document 1, Patent Document 2, and the like disclose freezing techniques for suppressing the formation of ice crystals. Here, a method for producing a sample for observation with a scanning electron microscope (SEM) disclosed in Non-Patent Document 1 will be described with reference to FIG. FIG. 10 is a diagram showing an outline of preparation of an observation sample in the prior art.

In the conventional technique described in Non-Patent Document 1, a freeze-fractured sample is prepared using a plate-type sample preparing apparatus 50 as shown in FIG. 10(a). As shown in FIG. 10(b), the conventional plate-type specimen preparation apparatus 50 is configured to be divisible into two plate-type housing members 51a and 51b. The flat-plate storage members 51a and 51b have recesses with open tops, and by connecting the two, a sample preparation area as shown in FIG. 10(a) is formed.

FIGS. 10(c)-(e) show each step of the freeze-fracture sample preparation method. First, as shown in FIG. 10(c), an object to be observed and water are placed in the hollow portion of the plate-type sample preparation apparatus 50 to fill the hollow portion with water. At this time, the object to be observed is arranged so that the site to be observed is arranged at the boundary between the flat plate-shaped housing members 51a and 51b.

After that, as shown in FIG. 10(d), the plate-type specimen preparation apparatus 50 is cooled to freeze the object to be observed and water. As a method for cooling the plate-shaped sample preparation device 50, there is a method of bringing a cooled metal block into contact with the plate-shaped sample preparation device 50. FIG. At this time, the water in the plate-type specimen preparation apparatus 50 freezes from the outer portion, and cooling proceeds further while unfrozen water and the object to be observed are included. Therefore, when the water or the moisture content of the object to be observed freezes, the volume expansion is suppressed, and the formation of ice crystals is suppressed.

After freezing the contents of the plate-shaped sample preparation device 50, the plate-shaped storage members 51a and 51b are divided as shown in FIG. 10(e). In the flat plate-shaped storage members 51a and 51b, since the part of the recessed portion that is connected to the other flat-plate storage member 51 is narrow, the object to be observed is easily broken when the flat-plate storage member 51 is divided. It has a structure. The object to be observed cut in this way is vacuum-dried and then coated with osmium to form a sample for microscopic observation.

As in the prior art described with reference to FIG. 10, it is possible to prepare a sample in which the formation of ice crystals is suppressed by applying pressure when frozen. However, in the prior art such as Non-Patent Document 1, rapid cooling at a cooling rate of 10 K / s is required, and only small objects of 1 mm or less (about 0.06 ml) can be used to prepare samples. couldn't.

Therefore, there has been a demand for further improvements in the convenience of freeze-fracture specimen preparation techniques that suppress the formation of ice crystals.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a sample preparation method, a freezing pressurization apparatus, and an observation method that suppress the formation of ice crystals.

That is, according to the present invention,
An object to be observed and water are put into a space inside a sample preparation device comprising a first member and a second member having a hollow portion and an opening with one side of the hollow portion open, and the space is filled with water. filling into
aligning and fixing the opening of the first member and the opening of the second member;
a step of cooling the sample preparation device;
and a step of separating the first member and the second member and cutting the frozen observation object by the cooling step.

According to the present invention, it is possible to provide a sample preparation method, a freezing pressurization apparatus, and an observation method that suppress the formation of ice crystals.

1 is a perspective view showing the structure of a sample preparation apparatus according to this embodiment; FIG. FIG. 2 is a diagram showing the structure of a housing member that constitutes the sample preparation apparatus of the present embodiment; FIG. 2 is a diagram showing the structure of a fixing member that constitutes the sample preparation apparatus of the present embodiment; 4A and 4B are diagrams for explaining preparation of an observation sample according to the present embodiment; FIG. SEM images of samples produced in this embodiment and comparative examples thereof. FIG. 4 is a diagram showing a first modification of the housing member that constitutes the sample preparation apparatus of the present embodiment; The figure which shows the 2nd modification of the accommodating member which comprises the sample preparation apparatus of this embodiment. FIG. 10 is a diagram showing a third modification of the housing member and a modification of the fixing member, which constitute the sample preparation apparatus of the present embodiment; FIG. 10 is a diagram showing a third modification of the housing member and a modification of the fixing member, which constitute the sample preparation apparatus of the present embodiment; FIG. 11 is a diagram showing an outline of preparation of an observation sample in the conventional technology;

The present invention will be described below with reference to embodiments, but the present invention is not limited to the embodiments described later. In addition, in each figure referred to below, the same reference numerals are used for common elements, and description thereof will be omitted as appropriate.

In addition, in the embodiments described below, a case is exemplified in which a sample for observation with a scanning electron microscope (SEM) is prepared using micro-organisms as an object to be observed, but the embodiments are not particularly limited. do not have. Therefore, in addition to micro-organisms, foods, plants, cells, gel-like substances, etc. may be used as objects to be observed. Further, as an application thereof, the present invention may be applied in fields such as chemistry and pharmaceuticals. Also, the observation device may be a device other than the SEM.

FIG. 1 is a perspective view showing the structure of a sample preparation apparatus 10 of this embodiment. A sample preparation apparatus 10 of the present embodiment is composed of a storage section and a fixing section, and as shown in FIG. A fixing member 12 and a screw 13 are provided for fixing the first housing member 11a and the second housing member 11b. A closed space can be formed by combining the first housing member 11a and the second housing member 11b. In addition, in FIG. 1, the screw 13 for fixing is shown as an example of a hexagon socket head bolt, but the embodiment is not particularly limited. In the embodiments described below, the object to be observed and the water contained in the sample preparation device 10 may be collectively referred to as "contents."

By cooling the sample preparation apparatus 10 shown in FIG. 1 and freezing and breaking the contained contents, it is possible to prepare a sample for microscopic observation in which formation of ice crystals is suppressed. Each part constituting the sample preparation apparatus 10 of the present embodiment shown in FIG. 1 will be described below with reference to FIGS. 2 and 3. FIG. FIG. 2 is a view showing the structure of a housing member 11 that constitutes the sample preparation apparatus 10 of this embodiment, and FIG. 3 is a view that shows the structure of a fixing member 12 that constitutes the sample preparation apparatus 10 of this embodiment. be.

First, the housing member 11 shown in FIG. 2 will be described. FIG. 2(a) shows a projection view of the housing member 11. FIG. The upper part of FIG. 2(a) shows a top view of the housing member 11, and the lower part of FIG. 2(a) shows a cross-sectional view of the housing member 11 taken along line AA' in the upper part of FIG. 2(a). . 2B shows a perspective view of the housing member 11, and FIG.

As shown in FIGS. 2A and 2B, the housing member 11 has a structure including a hollow portion for housing an object to be observed and the like, and an opening in which one side of the hollow portion is open. Further, as shown in FIG. 2, the hollow portion of the housing member 11 may be provided with a recess for facilitating fixation of the position of the housed object to be observed.

In the sample preparation apparatus 10 of the present embodiment, the openings of the two housing members 11a and 11b are aligned and fixed so that the cavities of the housing members 11a and 11b are connected to accommodate the object to be observed and water. A closed space (hereinafter simply referred to as “space”) is formed for Since this space is filled with the content and sealed, the expansion of the content when frozen is suppressed by the inner wall of the housing member 11, and the content can be pressurized. As a result, it is possible to suppress the formation of ice crystals in the observed object.

The shapes of the first housing member 11a and the second housing member 11b, which constitute the sample preparation apparatus 10 of the present embodiment, do not necessarily have to be the same, and they may have different shapes.

In addition, the size of the hollow portion of the containing member 11 is not particularly limited as long as the contents can be sufficiently frozen. Compared to the conventional techniques such as Document 2 and Non-Patent Document 1, a sample can be produced even for a much larger object to be observed.

Next, the fixing member 12 shown in FIG. 3 will be described. FIG. 3A shows a projection view of the fixing member 12. FIG. 3B shows a perspective view of the fixing member 12. FIG.

As shown in FIGS. 3A and 3B, the fixing member 12 of the present embodiment is a frame-shaped member, and is provided with a through screw hole in the upper portion thereof. In the sample preparation apparatus 10 of the present embodiment, two housing members 11a and 11b are housed in the frame of the fixing member 12 with their openings aligned, and the housing members 11a and 11b are tightly closed by tightening them with screws 13. It can be fixed (see Figure 1).

In addition, the shape of the fixing member 12 is not limited to a frame shape as shown in FIG. That is, the fixing member 12 may have a structure in which the two holding members 11a and 11b with the openings aligned are pressed from above and below by occupying the screws 13. It may be member 12 .

In addition, since each member constituting the sample preparation apparatus 10 according to the embodiment to be described has a simple structure, it is easy to process during manufacturing, and is excellent in terms of manufacturing cost.

As described above, the sample preparation device 10 having the structure shown in FIGS. 1 to 3 can prepare a sample in which the formation of ice crystals is suppressed. Next, preparation of a freeze-fractured sample by the sample preparation apparatus 10 of this embodiment will be described with reference to FIG. FIG. 4 is a diagram for explaining preparation of an observation sample in this embodiment. The sample preparation in this embodiment is performed in the order of FIGS. 4(a) to 4(c).

First, as shown in FIG. 4A, a loading step and a fixing step are performed. That is, in the loading step, the object to be observed and water are put into the first containing member 11a and the second containing member 11b, and the hollow portions of the first containing member 11a and the second containing member 11b are filled with the contents. do. In order to prevent contamination of the sample with impurities, the water to be filled is preferably pure water or ultrapure water (the term "water" in the described embodiment means pure water and ultrapure water). shall also include).

After the loading step, in the fixing step, the opening of the first housing member 11a and the opening of the second housing member 11b are aligned, housed inside the fixing member 12, and tightened with the screws 13 to secure the second housing member 11b. The first housing member 11a and the second housing member 11b are fixed. Thus, the sample preparation apparatus 10 having the form shown in FIG. 1 is constructed.

In a preferred embodiment, the loading step and fixing step shown in FIG. 4(a) may be performed in water. As a result, air does not enter the sealed space of the sample preparation apparatus 10, and the space is filled with water, so that pressurization by expansion can be effectively performed.

Further, in order to improve the airtightness of the sealed space, a gasket may be inserted between each housing member 11 when fixing the first housing member 11a and the second housing member 11b. . The gasket material is not particularly limited, but pressure-sensitive paper, copper, or the like can be used, for example. By using pressure-sensitive paper that is colored according to the applied pressure (for example, "Prescale (registered trademark)" manufactured by Fuji Film Co., Ltd.) as a gasket, the first housing member 11a and the second housing member 11a are separated from each other in the fixing step. It can be visually recognized that the member 11b is fixed with an appropriate pressure. Also, by inserting a copper gasket, it is possible to improve heat conduction in the cooling process, which will be described later, and to perform efficient cooling. In particular, it was confirmed that when a copper gasket was inserted, the formation of ice crystals was suppressed more effectively than when pressure-sensitive paper was used.

After the loading process and the fixing process, the cooling process is performed as shown in FIG. FIG. 4(b) is an enlarged cross-sectional view of the first housing member 11a and the second housing member 11b of the sample preparation apparatus 10. FIG. As shown in FIG. 4(b), the closed space of the sample preparation device 10 is filled with contents to fill the inside. Therefore, when the sample preparation device 10 is cooled and the contents are frozen, the inside is pressurized due to the expansion of the contents due to freezing. This can suppress the formation of ice crystals.

A method for cooling the sample preparation apparatus 10 in the cooling step is not particularly limited, and various methods can be adopted. An example of the cooling method is a method of immersing the sample preparation device 10 in liquid nitrogen, but a cooling material other than liquid nitrogen may be used.

After the contents are frozen by the cooling process, the cutting process shown in FIG. 4(c) is performed. In the cutting step, the sample preparation device 10 is released and the first housing member 11a and the second housing member 11b are separated. In addition, since the ice that has expanded due to freezing is held down by the inner wall of the storage portion, in the cutting step, the first storage member 11a and the second storage member 11b are grasped and separated by folding the connecting portions. As a result, the frozen object to be observed is cleaved, and the cleaved surface can be exposed. In addition, in order to facilitate the separation of the storage members 11, a blade may be inserted into the connecting portion to cut the ice before cutting. The cut surface exposed by the cutting step serves as an observation surface in microscopic observation. Thereafter, the object to be measured is vacuum-dried and coated with osmium to complete a sample for SEM observation, which can be observed by SEM.

Through the steps described with reference to FIGS. 4A and 4B, it is possible to prepare an observation sample while suppressing damage due to formation of ice crystals even for an object to be observed containing moisture. In the prior art shown in FIG. 10, only a small sample of 1 mm or less (approximately 0.06 ml) can be produced. Observation specimens can be prepared even for large objects to be observed. Furthermore, in the cooling step for sample preparation in the present embodiment, the formation of ice crystals can be suppressed even if the cooling rate is lower than that of the prior art shown in FIG.

FIG. 5 is an SEM image of a sample produced in this embodiment and its comparative example. The sample in FIG. 5 is obtained by freezing and breaking a 2% agar gel as an object to be observed. FIGS. 5(A) and (a-1) to (a-3) are SEM images of a sample prepared by freezing and breaking a 2% agar gel by the method of the present embodiment. FIGS. 5B and 5B and 5B-1 to 5B-3 are comparative examples with this embodiment, and are images of 2% agar gel immersed in liquid nitrogen and observed with SEM.

First, FIGS. 5(A) and (a-1) to (a-3) will be described. FIG. 5A is an SEM image of the entire sample produced by the method of this embodiment. 5(a-1) to (a-3) are SEM images in which positions 1 to 3 in FIG. 5(A) are enlarged, respectively. FIG. 5(a-1) is an SEM image near the outside of the sample, and it was observed that the network structure of the agar was slightly damaged. FIG. 5(a-2) is an SEM image of a portion about 1 mm inside from the surface of the sample, and a good network structure peculiar to agar was observed. Further, FIG. 5(a-3) is an SEM image of the vicinity of the center of the sample, in which a good network structure peculiar to agar was observed. That is, in any part of the sample prepared by the method of the present embodiment, a network structure peculiar to agar was observed, and in particular, in FIGS. was observed, it was confirmed that the method of the present embodiment suppresses the formation of ice crystals during freezing.

Next, FIGS. 5B and 5B-1 to 5B-3 will be described. FIG. 5(B) is an SEM image of a sample prepared by immersing a 2% agar gel in liquid nitrogen. 5(b-1) to (b-3) are SEM images in which positions 1 to 3 in FIG. 5(B) are enlarged, respectively, and FIG. 5(b-1) is the outside of the sample. FIG. 5(b-2) shows the part about 1 mm inside from the sample surface, and FIG. 5(a-3) shows the vicinity of the center of the sample. As shown in FIGS. 5(b-1) and 5(b-2), the portion near the outer periphery of the sample prepared by immersing 2% agar gel in liquid nitrogen is damaged by the generation of ice crystals. was confirmed. In addition, as shown in FIG. 5(b-3), a network structure of agar can be seen near the center of the sample. Damage confirmed.

As shown in FIG. 5, the sample prepared by the method of the present embodiment was confirmed to have a network structure unique to agar, and it was confirmed that the formation of ice crystals was suppressed. Therefore, it was confirmed that, by the method of the present embodiment, a sample could be produced by suppressing the formation of ice crystals even with an object to be observed having a high water content.

In the embodiments described so far, the housing member 11 having the shape illustrated in FIG. 2 has been described. However, the shape of the housing member 11 is not limited to that shown in FIG. 2, and may be, for example, a shape such as each modification shown below.

First, a first modified example will be described. FIG. 6 is a diagram showing a first modification of the housing member 11 that constitutes the sample preparation apparatus 10 of this embodiment. FIG. 6(a) is a top projection view and a side cross-sectional projection view of a first modified example of the housing member 11, and FIG. 6(b) is a perspective view and a cross-sectional perspective view. As shown in FIG. 6, the housing member 11 in the first modified example has a shape in which the cavity narrows toward the opening. With such a shape, the area where the opening of the first housing member 11a and the opening of the second housing member 11b are connected becomes small, so that the housing members 11 can be easily separated. The cutting process can be easily performed.

Next, a second modified example will be described. FIG. 7 is a diagram showing a second modification of the housing member 11 that constitutes the sample preparation apparatus 10 of this embodiment. FIG. 7(a) is a top projection view and a side cross-sectional projection view of a second modification of the housing member 11, and FIG. 7(b) is configured by the housing member 11 according to the second modification. 1 is a perspective view of a sample preparation device 10; FIG. A housing member 11 according to a second modification shown in FIG. 7 has a shape in which the housing portion and the fixing portion of the present embodiment are integrated.

As shown in FIG. 7(a), the housing member 11 of the second modification includes the cavity and the opening described in FIG. 2 and the like, and further includes a flange on the opening surface of the opening. The flange portion is provided with holes evenly distributed on the same circumference. The flange portion has a function corresponding to the fixing member 12, and can fix the first housing member 11a and the second housing member 11b by being screwed in the fixing process. The positions and number of holes in the flange portion are not limited to those shown in FIG. 7, and may be arbitrary. Also, the holes in the flange portion may be tapped screw holes.

The storage member 11 according to the second modification constitutes the sample preparation device 10 by combining them as shown in the left diagram of FIG. 7(b). That is, the housing member 11 with flanges is used as the first housing member 11a and the second housing member 11b, and the holes of the opening and the flange are aligned and fixed with screws 13 and nuts 13a. As a result, the sample preparation apparatus 10 can be configured as shown in the right diagram of FIG. 7(b). By using the housing member 11 according to the second modification, the sample preparation apparatus 10 can be made without the fixing member 12 .

Next, a third modified example will be described. 8 and 9 are diagrams showing a third modified example of the housing member 11 and a modified example of the fixing member 12 that constitute the sample preparation apparatus 10 of this embodiment.

In a third modification of the housing member 11, as shown in FIGS. 8A and 8B, the housing member 11a and the second housing member 11b of the table 1 have different shapes. That is, the first housing member 11a has the same shape as the housing member 11 shown in FIG. In addition, as shown in FIG. 8A, it is preferable to provide a hole for inserting a pin 14 as described later in the first housing member 11a. Further, as shown in FIG. 8B, the second housing member 11b is provided with a tapered hole in the housing member 11 shown in FIG. . As shown in FIG. 8C, the fixing member 12 has the same shape as that shown in FIG. may be provided.

A sample preparation apparatus 10 using the above-described housing member 11 and fixing member 12 is configured as shown in FIG. 9(a). FIG. 9(a) shows a loading step and a fixing step in the sample preparation apparatus 10 using each member shown in FIG. Here, as shown in FIG. 9A, the screw for fixing the housing member 11 and the fixing member 12 is a screw 13' having a tapered tip (hereinafter referred to as a "tapered screw 13'"). ) is used. The tapered screw 13' has a tapered shape at the tip corresponding to the tapered portion of the second housing member 11b. The tapered portion is in close contact.

FIG. 9B is a cross-sectional view of the sample preparation device 10. FIG. Note that the object to be observed, water, and the like are omitted in FIG. 9B. As shown in FIG. 9(b), by using the tapered screw 13', it is possible to fix the containing members 11a and 11b, to seal the space, and to pressurize in the cooling process. Further, by inserting the pin 14 into the first housing member 11a and the fixing member 12, it is possible to prevent the respective members from being displaced.

By using the sample preparation apparatus 10 having the configuration shown in FIGS. 8 and 9, the workability of each step can be improved.

According to the embodiments of the present invention described above, it is possible to provide a sample preparation method, a freezing pressurization device, and an observation method that suppress the formation of ice crystals. In addition, since the present embodiment can suppress the formation of ice crystals with a simple structure, it can contribute to cost reduction. Furthermore, significantly larger samples can be made than in the prior art.

As described above, the present invention has been described with embodiments, but the present invention is not limited to the above-described embodiments, and within the scope of embodiments that can be conceived by those skilled in the art, as long as the actions and effects of the present invention are exhibited. , are within the scope of the present invention. Further, the sample preparation device according to the described embodiments is not limited to one used for preparing observation samples, and may be a device for pressurizing contents by freezing water.

10 ... sample preparation device,
11 ... accommodation member,
12... Fixing member,
13... screw,
13'...tapered screw,
13a ... nut,
14... pin,
50... Plate type sample preparation device,
51... Flat plate type housing member

JP 2013-253957 A JP 2014-25732 A

Hirohiko Ohta et al. (2011): New freeze-drying method for SEM sample preparation using cooling water. Journal of the Society of Medical and Biological Electron Microscopy, 25(1):9-13.

Claims (11)

An object to be observed and water are put into a space inside a sample preparation device comprising a first member and a second member having a hollow portion and an opening with one side of the hollow portion open, and the space is filled with water. filling into
aligning and fixing the opening of the first member and the opening of the second member;
a step of cooling the sample preparation device;
and a step of separating the first member and the second member, and cutting the frozen observation object by the cooling step. 2. The sample preparation method according to claim 1, wherein said filling step is performed in water. 2. The step of breaking the first member and the second member by folding a portion where the first member and the second member are connected to separate the first member and the second member. 3. or the sample preparation method according to 2. a first accommodating portion and a second accommodating portion each having a hollow portion and an opening in which one surface of the hollow portion is open;
a fixing part that fixes the first accommodation part and the second accommodation part,
Having a space formed by combining the opening of the first accommodating portion and the opening of the second accommodating portion and fixing it by the fixing portion,
Freezing pressure device. 5. The freeze pressurization device of claim 4, wherein said cavity narrows towards said opening. 6. The freezing and pressurizing device according to claim 4, wherein the first accommodating portion and the second accommodating portion are each configured integrally with the fixing portion. The second accommodating portion has a tapered portion that opens on the opposite side of the opening, and a screw having a shape corresponding to the tapered portion connects the first accommodating portion and the second accommodating portion. 6. A freezing pressurization device according to claim 4 or 5, characterized in that it is fixed. Any one of claims 4 to 7, characterized in that a gasket is inserted between the opening of the first accommodating portion and the opening of the second accommodating portion and fixed by the fixing portion. 2. The freeze pressurization device according to item 1. 9. The freezing pressurization device according to claim 8, wherein the material of said gasket is copper. 9. The freeze pressurization device of claim 8, wherein the gasket is colored according to the pressure applied. An object to be observed and water are put into a space inside a sample preparation device comprising a first member and a second member having a hollow portion and an opening with one side of the hollow portion open, and the space is filled with water. filling into
aligning and fixing the opening of the first member and the opening of the second member;
a step of cooling the sample preparation device;
a step of separating the first member and the second member and cutting the frozen observation object by the cooling step;
and a step of observing a fractured surface of the object to be observed that has been fractured by the fractured step.

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