Section preparation device and section preparation method

Abstract

To enable stable preparation of high-quality thick sections.SOLUTION: A section preparation device 1 is provided, comprising a blade 3 for slicing a sample to prepare sections, a liquid storage unit 2 for storing a liquid E for floating the sections therein, and steam generation means 5 for generating steam D. The steam generation means 5 includes a heater 5a provided near the blade 3, and the heater 5a heats the liquid E near the blade 3.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a section preparation device and a section preparation method. [Background technology]

[0002] Conventionally, a technique for producing sections thicker than ultrathin sections for transmission electron microscopes (TEM) has been proposed (see, for example, Non-Patent Documents 1 and 2). Thick sections are useful for identifying the three-dimensional position of a region of interest, for example, a region to be observed by TEM, within a sample.

[0003] Sections are made by cutting a resin block in which a sample is embedded with a diamond knife. The thicker the section, the greater the force applied to the diamond knife during cutting, so it is difficult to make thick sections in the same way as ultrathin sections. In Non-Patent Documents 1 and 2, in order to make it possible to make sections with a thickness of 2 μm to 10 μm, a heater and infrared light are combined with a diamond knife, the blade of the diamond knife is heated by the heater, and the resin block itself is heated by the infrared light. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Georg Kislinger, 5 others, “Multiscale ATUM-FIB Microscopy Enables Targeted UltrastructuralAnalysis at Isotropic Resolution”, iScience, July 24, 2020, Volume 23, Issue 7 [Non-Patent Document 2] Georg Kislinger, 5 others, “ATUM-FIB microscopy fortargeting and multiscale imaging of rare events in mouse cortex”, STAR Protocols, December 16, 2020, Volume 1, Issue 3 Summary of the Invention [Problem to be solved by the invention]

[0005] In order to create flat, high-quality thick slices, the resin block needs to have a suitable hardness. In Non-Patent Documents 1 and 2, the entire resin block is heated by infrared light. Furthermore, the entire water in the boat of the diamond knife is heated by a heater arranged at the rear of the diamond knife, which heats the blade arranged at the front end of the diamond knife, so that a large amount of water vapor is generated and the entire resin block or a wide area becomes hydrated. Therefore, the entire resin block becomes soft, and wrinkles called chattering are easily generated in the slices, making it difficult to stably create flat, high-quality thick slices.

[0006] The present invention has been made in consideration of the above-mentioned circumstances, and has an object to provide a section preparation device and a section preparation method that are capable of stably preparing high-quality thick sections. [Means for solving the problem]

[0007] One aspect of the present invention is a section creation device comprising a blade for cutting a sample to create a section, a liquid storage section for storing a liquid in which the section floats, and steam generating means for generating steam, the steam generating means comprising a heater positioned near the blade, and the heater heating the liquid in the vicinity of the blade. Another aspect of the invention is a method of sectioning that includes the steps of generating steam adjacent to a blade and cutting a sample with the blade in the steam to create a section. Effect of the Invention

[0008] The present invention has the effect of enabling high-quality thick sections to be stably produced. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a perspective view of a sectioning device according to an embodiment. [Diagram 2] 2A is a plan view, FIG. 2B is a front view, and FIG. 2C is a side view of the section producing device of FIG. [Diagram 3] FIG. 2 is a diagram showing a state in which liquid is stored in a boat of the sectioning device of FIG. 1. [Figure 4] FIG. 3 is a vertical cross-sectional view of the section producing device taken along line II in FIG. 2, illustrating the state in which the section producing device is used. [Diagram 5] 1 is a flowchart of a sectioning method according to an embodiment. [Figure 6] FIG. 2 is a side view of a modified example of the sectioning device of FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A slice preparation device and a slice preparation method according to an embodiment of the present invention will be described below with reference to the drawings. The section preparation device 1 according to this embodiment is mounted on a known microtome or ultramicrotome for use, and cuts a block B of resin containing a sample A to prepare a section C (see FIG. 4). The section preparation device 1 is also called a knife, and when it has a diamond blade 3 as described later, it is also called a diamond knife.

[0011] The thickness of a slice that can be made with a general diamond knife is at most 0.5 μm to 2.0 μm. The slice making device 1 is suitably used for making a slice C that is thicker than a general diamond knife. Sample A is embedded in a block of hardened resin B. Sample A is, for example, aquatic microorganisms, cells or tissues of animals and plants, bacteria, viruses, materials such as polymeric materials, metals, composite materials, ruins, clay minerals, etc. The resin is a known resin that is generally used for ultrathin sections.

[0012] As shown in Figures 1 and 2, the section preparation device 1 comprises a boat (liquid storage section) 2 with an open top, a blade 3 fixed to the boat 2, a fixing section 4 provided on the boat 2, and a steam generating means 5 for generating steam D in the vicinity of the blade 3.

[0013] The boat 2 has a front-rear direction, a left-right direction, and a top-down direction which are perpendicular to each other, with the front-rear direction and the left-right direction corresponding to the horizontal direction, and the top-down direction corresponding to the vertical direction. In Figures 1 and 2, the X direction is the front-rear direction, the Y direction is the left-right direction, and the Z direction is the top-down direction. In the following description, the side in the X direction where the blade 3 is located is the front side of the boat 2, and the other end side in the X direction is the rear side of the boat 2. Figure 2(a) is a plan view seen from above, Figure 2(b) is a side view seen from the left side, and Figure 2(c) is a front view seen from the front side.

[0014] The boat 2 has a generally rectangular box shape in a plan view from above, and has a tank 6 surrounded by a front wall 6a, a rear wall 6b, and a pair of side walls 6c, 6d. As shown in Figs. 3 and 4, water (liquid) E for floating the slices C can be stored in the tank 6. The boat 2 may have other shapes and structures. The boat 2 is formed from a material having high thermal conductivity, for example, a metal such as aluminum or stainless steel.

[0015] The tank 6 has a shallow portion 61 provided in an area near the blade 3, and a deep portion 62 provided in an area away from the blade 3 and deeper than the shallow portion 61. Specifically, the bottom surface of the tank 6 has different heights at the front end and other portions rearward of the front end, and the bottom surface of the front end is higher than the bottom surfaces of other portions. The shallow portion 61 is provided at the front end of the tank 6, and the deep portion 62 is provided rearward of the shallow portion 61.

[0016] The blade 3 is disposed at the front end of the boat 2. The upper end of the blade 3 is made of diamond. The cutting edge 3a at the upper end of the blade 3 is disposed at approximately the same height as the upper ends of the side walls 6a, 6b, 6c, and 6d, and is disposed on approximately the same plane as the front end surface of the boat 2. When water E is stored in the tank 6 up to the upper ends of the side walls 6a, 6b, 6c, and 6d, the water E also comes into contact with the cutting edge 3a, and the piece C cut by the blade 3 floats on the surface of the water E (see FIG. 4).

[0017] The fixing part 4 is a part for fixing the sectioning device 1 to the stage of a microtome or ultramicrotome, and is provided below the boat 2. The fixing part 4 is integral with the boat 2 and is formed from the same material as the boat 2.

[0018] The steam generating means 5 includes a heater 5a disposed near the blade 3. The heater 5a is connected to a power source (not shown) by a lead wire 5b. The steam generating means 5 may further include a controller (not shown) that controls the temperature of the heater 5a. The heater 5a is, for example, rod-shaped, inserted into a hole 2a penetrating the boat 2 in the left-right direction Y, and fixed to the boat 2 with a screw 7. The hole 2a is provided directly below the bottom surface of the shallow portion 61, and the heater 5a is disposed parallel to the blade tip 3a near the rear side of the blade 3. In order to effectively generate steam near the blade 3, the heater 5a preferably extends on both sides of the blade 3 in the left-right direction Y.

[0019] The heater 5a generates water vapor D by heating the water E in the tank 6. As shown in Fig. 4, when cutting the block B, the tip surface B1 of the block B, which is in close proximity to and in contact with the blade 3, comes into contact with the water vapor D, and moisture permeates into the block B from the tip surface B1. Here, by disposing the heater 5a only at the front end of the boat 2, the water E is heated to a high temperature at the front end of the tank 6 to generate steam, while heating of the water E and generation of steam are suppressed at the rear side of the tank 6. As a result, an appropriate amount of steam is generated in a limited area near the blade 3, and the penetration of the moisture is limited only to the surface layer of the tip face B1, for example to a depth of several μm from the tip face B1, preventing moisture absorption in other parts of the block B. Furthermore, the absorption of moisture in the surface layer by the steam D does not cause an increase in the temperature of the block B. Therefore, the block B softens only in the hydrated surface layer, while the other parts of the block B maintain a high hardness.

[0020] The depth of the water E in the tank 6 is shallow at a shallow portion 61 at the front end and deep at a deep portion 62 at the rear. Therefore, the water E is quickly heated to a high temperature by the heater 5a at the front end of the tank 6, and the temperature of the water E increases more slowly in the rear portion of the tank 6. This creates a temperature gradient in the water E in the tank 6, where the temperature decreases from the front end to the rear side, i.e., as the water moves away from the blade 3 in the front-rear direction X.

[0021] Furthermore, the boat 2, which is made of a material with high thermal conductivity such as metal, has a heat dissipation property that dissipates heat of the water E to the outside from the outer surface of the boat 2. Therefore, in the rear part of the tank 6, the temperature rise of the water E is suppressed by heat dissipation, and the formation of the temperature gradient is further promoted. That is, the shallow portion 61, the deep portion 62, and the boat 2 constitute a temperature gradient forming means. These temperature gradient forming means suppress the generation of steam in the portions of the tank 6 other than the front end portion away from the blade 3, thereby suppressing the excessive generation of steam D and the drop in the level of the water E in the tank 6.

[0022] The amount of water vapor D generated depends on the heating temperature of the water E by the heater 5a. In one example, the heating temperature of the water E in the vicinity of the blade 3 is preferably higher than room temperature. In one example, the heating temperature is 30°C or higher. In addition, the heating temperature of the water E is preferably a temperature at which no air bubbles are generated, for example, approximately 90°C or lower. To control the temperature of the water E, the section preparation device 1 may further include a temperature sensor (not shown) such as a thermocouple that measures the temperature in the vicinity of the blade 3. The temperature sensor is preferably disposed in the vicinity of the blade 3, and may be disposed, for example, in a groove (not shown) formed in the front end surface of the boat 2.

[0023] Next, a section preparation method according to one embodiment of the present invention will be described. As shown in Figure 5, the slice preparation method of this embodiment includes step S1 of generating water vapor D near the blade 3, step S2 of cutting a block B containing a sample A in the water vapor D to prepare a slice C, and step S3 of recovering the slice C. The sectioning method is performed using the sectioning device 1 and a conventional ultramicrotome. A microtome may be used instead of the ultramicrotome. The sectioning device 1 is fixed to the stage of the ultramicrotome at the fixing part 4, and the block B is fixed to the arm of the ultramicrotome. Water E is supplied into the boat 2 up to the top ends of the side walls 6a, 6b, 6c, and 6d.

[0024] In step S1, the heater 5a is turned on to increase the temperature of the heater 5a. This heats the water E at the front end of the boat 2, creating a temperature gradient in the water E in the boat 2 and generating water vapor D in the vicinity of the blade 3. Next, in step S2, the ultramicrotome is operated while generating steam D to cut the block B and create a section C. Specifically, the arm is lowered and the tip surface B1 of the block B is cut by the blade 3, and then the arm is raised. The thickness of the section C is, for example, 2 μm to 10 μm. The section C cut from the block B floats on the water surface. When making serial sections, step S2 is repeated multiple times, and multiple sections C arranged in a row float on the water surface.

[0025] Next, in step S3, the section C on the water surface is collected on a substrate such as a cover glass, a slide glass, or a silicon wafer. Since the thick section C allows observation of the sample A without staining, it is, for example, subjected to optical microscope observation immediately after collection. Staining the sections can cause contamination of the sections. In addition, staining is generally performed by dropping a stain onto the sections attached to a substrate and heating them, but during this process the sections can peel off from the substrate. By eliminating the need for staining, these problems can be prevented.

[0026] In this case, the thicker the slice, the greater the force applied to the blade when cutting the block, so it is difficult to produce a thick slice using a typical diamond knife. According to this embodiment, the surface layer of the tip surface B1 of the block B is moistened by the steam D generated by the steam generating means 5 near the blade 3, and the surface layer is softened and the sliding of the blade 3 relative to the block B is improved. This makes it possible to easily cut even thick slices C.

[0027] In addition, the heater 5a disposed at the front end of the boat 2 generates an appropriate amount of water vapor D near the blade 3. Furthermore, the temperature gradient forming means 2, 61, 62 suppress the generation of water vapor at a position away from the blade 3, thereby more appropriately limiting the range and amount of water vapor D. Such water vapor D hydrates only the surface layer of the tip surface B1 of the block B without increasing the temperature of the block B, and the rigidity of the entire block B is maintained. This prevents chattering caused by the deflection of the block B during cutting from occurring in the slice C. In addition, the water absorbed in the block B improves the sliding of the blade 3 during cutting, thereby improving the cutting ability even for thick slices C. In addition, even if wrinkles or curls occur in the slice C during cutting, the slice C naturally stretches and becomes flat during cutting because the slice C is softened by hydration. Therefore, flat and high-quality slices C with no or few wrinkles or curls can be stably produced.

[0028] Conventionally, in order to smooth out wrinkles and curls in the slices, the slices were placed on a water drop and heated with a heater after collection. According to the present embodiment, such a process is not necessary, and the collected slices C can be used immediately in the next process. Furthermore, if block B were to be softened by heating itself, the bending of block B during cutting would make it easier for wrinkles called chattering to occur in slice C, making it difficult to produce high-quality slices C. Furthermore, if the entire water E in the tank 6 were heated to generate steam D, the water E in the tank 6 would decrease rapidly, causing the blade tip 3a to dry out and the slice C to no longer float on the water surface. This is particularly disadvantageous when making serial slices.

[0029] Furthermore, according to this embodiment, the water E in the boat 2 is heated by the heater 5a, so that the slices C floating on the water surface are heated. As a result, even if the slices C are wrinkled or curled, the slices C will naturally stretch while floating on the water surface, and good quality slices C can be obtained more stably. Furthermore, according to this embodiment, in the preparation of serial sections, the dry tip surface B1 newly exposed by cutting comes into contact with water vapor D when the next section C is cut, which causes the surface layer to become hydrated. In this manner, the cutting of the tip surface B1 and the hydration of the surface layer are repeated, so that high-quality serial sections can be prepared.

[0030] Furthermore, according to this embodiment, since the diamond blade 3 has very high thermal conductivity, the temperature of the blade 3 is maintained lower than the temperatures of the water E and the boat 2, even though the heater 5a is disposed near the blade 3. Therefore, the sample A is prevented from being affected by heat during cutting. This is advantageous in the case of a sample A that is easily affected by heat, such as a biological sample.

[0031] Furthermore, according to this embodiment, the temperature gradient of the water E by the temperature gradient forming means 2, 61, 62 suppresses the generation of unnecessary water vapor that does not contribute to the production of good quality sections C, thereby suppressing a drop in the water level. Therefore, it is possible to produce serial sections without the need to supply water to the tank 6 midway. This makes it possible to produce serial sections with constant thickness and other conditions under conditions where the temperature of the water E and the amount of water vapor D are constant. If the water level drops, the blade tip 3a will dry out and the slice C will no longer float on the water surface, so it is necessary to supply water to the tank 6. Since supplying water causes turbulence on the water surface, it is preferable not to supply water, especially while preparing serial sections.

[0032] Furthermore, according to this embodiment, when it becomes necessary to supply water to the tub 6 due to a drop in the water level or the like, the water can be supplied to the tub 6 from the rear side while maintaining a constant temperature of the water E near the blade 3. That is, the water E supplied to the rear end of the tub 6 is heated while moving forward, and is sufficiently heated by the time it reaches the vicinity of the blade 3. Therefore, water can be supplied without affecting the generation of steam D.

[0033] The resin of block B may be either hydrophobic or hydrophilic. Hydrophilic resins are more likely to absorb water and soften than hydrophobic resins. Therefore, depending on the thickness of the slice C to be produced, it may be possible to select whether to use a hydrophobic resin or a hydrophilic resin. For example, when a thicker slice is to be produced, a hydrophilic resin may be used.

[0034] In the above embodiment, the slice preparation device 1 is equipped with a heat dissipative boat 2 and a shallow portion 61 and a deep portion 62 as a temperature gradient forming means, but instead of or in addition to this, it may be equipped with a cooling means for cooling the water E at a position away from the blade 3. 6 shows an example of the cooling means 8. The cooling means 8 has a tube 8a for cooling water, and the tube 8a is inserted into a hole 2b that penetrates the rear end of the boat 2 in the left-right direction Y. The water E at the rear end of the boat 2 is cooled by the cooling water flowing inside the tube 8a, forming a temperature gradient.

[0035] In the above embodiment, the slice producing device 1 may include a cooling means 8 for cooling the water E in the tank 6 for the purpose of controlling the temperature of the water E and the amount of water vapor D generated with higher precision. Since the heater 5a only has a function of increasing the temperature, it may be difficult to accurately control the temperature of the water E and the amount of water vapor D generated by the heater 5a alone. By adding a cooling means, it is possible to prevent excessive heating of the water E and excessive generation of water vapor D. The cooling means 8 in this case may be provided at the rear end of the boat 2 as shown in FIG. 6, or may be provided at the front end of the boat 2 together with the heater 5a.

[0036] When the cooling means 8 is provided at the rear end of the boat 2, the positions of the heater 5a and the cooling means 8 may be interchangeable. That is, the cooling means 8 may be disposed at the front end of the boat 2, and the heater 5a may be disposed at the rear end of the boat 2. For example, both the heater 5a and the tube 8a may be removably fixed in the hole 2a by the screw 7, and similarly, both the heater 5a and the tube 8a may be removably fixed in the hole 2b by the screw (not shown). When preparing ultrathin sections, it may be preferable to cool the blade 3 and the block B. By making the positions of the heater 5a and the cooling means 8 interchangeable, the section preparation device 1 can be used for purposes other than preparation of thick sections C.

[0037] In the above embodiment, the slice producing device 1 may further include a heat insulating means for suppressing a decrease in the temperature of the water E in the vicinity of the blade 3. As described above, when the boat 2 and the fixing part 4 are made of a metal with high heat dissipation properties, heat dissipation also occurs at the front end of the boat 2. The heat shielding means suppresses heat dissipation at the front end of the boat 2. This allows the heater 5a to efficiently heat the water E near the blade 3 to a higher temperature, making it possible to form a larger temperature gradient.

[0038] An example of the heat shielding means is a heat shielding member provided on at least the surface of the fixed part 4. The heat shielding member is made of a material with low thermal conductivity, such as a resin. The fixed part 4 is a part that comes into contact with a metallic stage of an ultramicrotome or a microtome, and heat is likely to escape from the front end of the boat 2 to the stage via the fixed part 4. By providing a heat shielding member on the surface of the fixed part 4 that comes into contact with the stage, heat dissipation from the front end of the boat 2 can be effectively suppressed.

[0039] In the above embodiment, the thick section C is prepared, but instead, an ultrathin section may be prepared. In this case, the conditions are adjusted so that the block B is hydrated from the tip surface B1 to a depth suitable for cutting the ultrathin section. For example, the amount of evaporation per unit time may be reduced by lowering the temperature of the heater 5a, or the block B may be dried by a drying means.

[0040] The drying means applies dry gas to the surface of the block B at a timing different from the timing when the blade 3 cuts the tip surface B1. For example, the drying means includes a tube having a nozzle at its tip, a pump that supplies air to the tube, and a dehumidifying member such as silica gel that is placed inside the tube, and the dry air dehumidified by the dehumidifying member is discharged from the nozzle. The dry gas may be a gas other than air, for example, a non-volatile gas such as nitrogen. In a typical ultramicrotome, block B is moved back and forth in the vertical direction, and when sample A descends, it is cut by blade 3. Therefore, it is preferable that the drying gas hits block B only when sample A ascends. Also, to prevent the drying gas from stirring the water surface, it is preferable that the drying gas is discharged from the bottom of block B upward.

[0041] Although the embodiments of the present invention and their modified examples have been described in detail above with reference to the drawings, the specific configuration of the present invention is not limited to the above-mentioned embodiments and modified examples, and various design changes are possible within the scope that does not deviate from the gist of the present invention. For example, the heater 5a may have a shape other than a rod shape and may be disposed at a position other than the hole 2a. For example, the heater 5a may be provided on the bottom surface or the front wall 6a of the tank 6, or may be disposed in the water E without being fixed to the boat 2. At least a part of the boat 2 may be made of a material other than metal. For example, at least a part of the front end of the boat 2 may be made of a material with low thermal conductivity, such as resin.

[0042] In addition, the temperature gradient forming means may form a temperature gradient in a direction other than the front-to-back direction depending on the shape of the boat 2. For example, if the boat 2 has a large dimension in the left-to-right direction Y, a temperature gradient may be formed in which the temperature decreases as the boat 2 moves away from the blade 3 in the left-to-right direction Y. Furthermore, although the steam generating means 5 generates steam D by heating the water E in the tank 6, steam may be generated in the vicinity of the blade 3 by other methods. For example, the boat 2 may have a steam port that opens in the vicinity of the blade 3, and the steam generating means 5 may supply steam to the steam port from a steam source separate from the boat 2 via a steam path. Furthermore, the slice preparation device 1 can be used to prepare slices for any purpose, for example, to prepare serial slices for array tomography or slices for pathological diagnosis. [Explanation of symbols]

[0043] 1 Sectioning device 2 Boat (liquid storage, temperature gradient forming means) 3 Blades 5. Steam generating means 5a Heater 6 tanks 61 Shallow part (temperature gradient forming means) 62 Deep (Means for forming temperature gradient) 8 Cooling means (temperature gradient forming means) Sample A C-intercept D. Water vapor (steam) E. Water (liquid)

Claims

1. A blade for cutting the sample to create a section; A liquid reservoir for storing a liquid in which the slice is floated; A steam generating means for generating steam, The vapor generating means comprises a heater disposed adjacent the blade, the heater heating the liquid adjacent the blade.

2. 2. The sectioning device according to claim 1, further comprising a temperature gradient forming means for forming a temperature gradient in the liquid such that the temperature decreases with increasing distance from the blade.

3. the liquid storage portion has a shallow portion provided in a region near the blade and a deep portion provided in a region away from the blade and deeper than the shallow portion, 3. The sectioning device according to claim 2, wherein said temperature gradient forming means comprises said shallow portion and said deep portion.

4. the liquid storage portion is made of a material having high thermal conductivity and has a heat dissipation property for dissipating heat of the liquid to the outside; 3. The sectioning device according to claim 2, wherein the temperature gradient generating means comprises the liquid reservoir.

5. 3. The sectioning apparatus of claim 2, wherein said temperature gradient creating means comprises cooling means for cooling said liquid at a location remote from said blade.

6. generating steam adjacent to the blade; and cutting the sample with the blade in the steam to create a section.

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