Embedding container and embedding method

Abstract

The present invention provides an embedding container capable of providing an additional approach to methods for embedding cells and tissues. This embedding container 1 comprises: a filter 11 which has a surface on which an object 100 to be embedded by an embedding agent can be placed, and through which a chemical solution in which the object 100 is immersed can permeate; and a base material 10 that supports the filter 11 and accommodates the object 100. A space 12 that can be filled with the embedding agent is formed on the rear-surface side of the filter 11.

Description

Embedding container and embedding method

[0001] The present disclosure relates to an embedding container and an embedding method.

[0002] In cell biology and medical research, specimens are prepared by fixing cells, tissues, etc. with formalin and embedding them in an embedding medium such as paraffin. For example, Patent Document 1 discloses a "tissue cassette assembly for embedding cell blocks." Further techniques for embedding cells and tissues are desired.

[0003] Patent No. 5204810

[0004] The embedding container according to the embodiment has a surface on which an object to be embedded in an embedding agent can be placed, and is equipped with a filter that is permeable to the chemical solution in which the object is immersed, and a base material that supports the filter and contains the object, and a space is formed on the back side of the filter that can be filled with the embedding agent.

[0005] FIG. 1 is a conceptual diagram showing the configuration of an embedding container according to an embodiment. FIG. 2 is a block diagram showing a specimen preparation device that executes a specimen preparation process for preparing a specimen from an object accommodated in the embedding container shown in FIG. 1. FIG. 3 is a flowchart showing the specimen preparation process for preparing a specimen from an object accommodated in the embedding container shown in FIG. 1. FIG. 4 is a diagram showing the process of S102 shown in FIG. 3. FIG. 5 is a diagram showing the process of S103 shown in FIG. 3. FIG. 6 is a diagram showing the process of S104 shown in FIG. 3. FIG. 7 is a diagram showing the process of S109 shown in FIG. 3. (a) is a conceptual diagram showing the configuration of a conventional embedding container, (b) is a diagram showing the embedding container shown in (a) filled with paraffin, (c) is a diagram showing the state (part 1) in which the paraffin filled in the embedding container shown in (a) has solidified, (d) is a diagram showing the state (part 2) in which the paraffin filled in the embedding container shown in (a) has solidified, and (e) is a diagram showing the state (part 3) in which the paraffin filled in the embedding container shown in (a) has solidified. 1A is a diagram showing the embedding container shown in FIG. 1 filled with paraffin, and FIG. 1B is a diagram showing the state in which the paraffin filled in the embedding container shown in FIG. 1 has solidified. FIG. 10 is a diagram showing a thin-sectioning process using a microtome. FIG. 11 is a conceptual diagram showing the configuration of an embedding container according to a first modified example. FIG. 12 is a conceptual diagram showing the configuration of an embedding container according to a second modified example. FIG. 13 is a conceptual diagram showing the configuration of an embedding container according to a third modified example. FIG. 14 is a conceptual diagram showing the configuration of an embedding container according to a fourth modified example. FIG. 15 is a diagram (part 1) showing a specimen preparation process according to a modified example, in which FIG. 1A shows a process corresponding to step S101 shown in FIG. 3 , FIG. 1B shows a process corresponding to steps S102 and S103 shown in FIG. 3 , FIG. 1C shows a process of attaching the embedding container to tape, FIG. 1D shows a process corresponding to step S104 shown in FIG. 3 , and FIG. 1E shows a process of placing the embedding container on a slide glass. 16A and 16B are diagrams (part 2) showing a specimen preparation process according to a modified example, in which (a) shows the process corresponding to step S105 shown in Fig. 3, (b) shows the process of peeling the tape from the slide glass, (c) shows the process corresponding to steps S106 to S108 shown in Fig. 3, and (d) shows the process corresponding to step S109 shown in Fig. 3.(a) is a plan view of an embedding container related to the fifth modified example, (b) is a side view of the embedding container shown in Figure 17(a), (c) is a perspective view of the embedding container shown in (a), (d) is a cross-sectional perspective view along line A-A shown in (c), and (e) is a cross-sectional view of the filling hole shown in (a). (a) is a plan view of an embedding container according to the sixth modified example, (b) is a plan view of an embedding container according to the seventh modified example, (c) is a plan view of an embedding container according to the eighth modified example, (d) is a plan view of an embedding container according to the ninth modified example, (e) is a plan view of an embedding container according to the tenth modified example, (f) is a plan view of an embedding container according to the eleventh modified example, (g) is a plan view of an embedding container according to the twelfth modified example, (h) is a plan view of an embedding container according to the thirteenth modified example, (i) is a plan view of an embedding container according to the fourteenth modified example, (j) is a plan view of an embedding container according to the fifteenth modified example, and (k) is a plan view of an embedding container according to the sixteenth modified example.

[0006] The embedding container according to the present disclosure will be described below with reference to the drawings. However, it should be noted that the technical scope of the present disclosure is not limited to the embodiments, but extends to the inventions described in the claims and their equivalents.

[0007] FIG. 1 is a conceptual diagram showing the configuration of an embedding container including a determination device according to an embodiment. The embedding container 1 has a base material 10 and a filter 11, and is used when preparing a specimen by embedding an object 100 placed on the filter 11 after pre-processing the object 100. The base material 10 supports the filter 11 and accommodates the object 100. The filter 11 is capable of receiving the object 100 on its surface and is permeable to a chemical solution in which the object 100 is immersed. The object 100 placed on the surface of the filter 11 is embedded in an embedding medium (not shown). The embedding container 1 is cut together with the object 100 placed on the surface of the filter 11 while the object 100 is embedded in the embedding medium, thereby preparing a specimen of the object 100 that can be observed using a microscope or the like.

[0008] The substrate 10 is made of at least one of plastic, paper, wood, and fiber, and has an inner wall formed with a locking portion for locking the filter 11. The substrate 10 may have a shape of, but is not limited to, a hollow cylinder, a hollow polygonal prism, a hollow truncated cone, or a hollow truncated polygonal pyramid.

[0009] In the substrate 10, a space 12 is formed on the side of the filter 11 opposite the surface on which the object 100 is mounted, i.e., on the back side of the filter 11, which space 12 can be filled with an embedding agent to embed the object 100. The volume of the space 12 formed on the back side of the filter 11 is determined according to the rate of decrease in volume of the embedding agent, which is filled into the space 12 as a high-temperature liquid, as the temperature of the embedding agent decreases and the embedding agent solidifies. The volume of the space 12 is determined so that the entire back side of the filter 11 is covered with the embedding agent when the embedding agent filled into the substrate 10 to embed the object 100 solidifies.

[0010] The substrate 10 is made of at least one of plastic, paper, wood, and fiber, which facilitates cutting of the embedding container 1 when preparing a specimen containing the object 100. The plastic may be a thermoplastic resin or a thermosetting resin. For example, the plastic forming the substrate 10 may include, but is not limited to, one or more of silicone rubber, polyacetal (POM), polycarbonate (PC), polypropylene (PP), polyethylene terephthalate (PET), polyurethane (PU), polymethyl methacrylate (PMMA), polystyrene (PS), and polyvinyl chloride (PVC). The substrate 10 is preferably made of an optically transparent material.

[0011] When the substrate 10 is made of plastic, the substrate 10 is easy to process and may be manufactured by any method such as compression molding, injection molding, extrusion molding, etc. When the substrate 10 is made of wood, the substrate 10 may be manufactured by any method such as thermoforming, etc. When the substrate 10 is made of paper or fiber, the substrate 10 may be manufactured by any method such as lamination.

[0012] The filter 11 is a porous member having a flat plate shape that allows the chemical solution in which the object 100 is immersed to pass through, and is disposed so that the back surface of the filter 11 is spaced apart from the lower end of the substrate 10. The filter 11 may be a mesh filter. The filter 11 may be made of any material, as long as it is not corroded by the chemical solution in which the object 100 is immersed and can withstand the pressure associated with the introduction and discharge of the chemical solution in which the object 100 is immersed. The filter 11 is formed from at least one of plastic, paper, wood, and fiber. As an example, the filter 11 is formed from a plastic such as polytetrafluoroethylene (PFTE), polyimide (PI), or polyethersulfone (PES). The filter 11 may also be formed from fiber such as a nonwoven fabric.

[0013] The filter 11 may be coated on the surface facing the object 100 so as to have affinity for the object 100. The coating material for coating the filter 11 may be, but is not limited to, poly-L-lysine, poly-D-lysine, an extracellular matrix (laminin, fibronectin, collagen, or the like), or proteoglycan.

[0014] The thickness of the filter 11 may be 20 μm or more, preferably 200 μm or less, and more preferably 100 μm or less. When the thickness of the filter 11 is 100 μm or more and 200 μm or less, the filter 11 is easy to manufacture and handle. When the thickness of the filter 11 is 20 μm or more and less than 100 μm, less pressure is required to communicate with the chemical solution in which the object 100 is immersed, making it easier to handle solutions such as embedding agents.

[0015] The object 100 is a living organism from which a specimen is to be prepared. The living organism may be an organic living organism. For example, the living organism is a cell. For example, the cell is an animal cell or a plant cell. For example, the cell is a living cell or a dead cell.

[0016] The organism may be an organism other than a cell. As an example, the organism includes at least one of a single cell, a spheroid (aggregate of cells), a biological tissue, an organoid (aggregate of cells), an organism (such as a microorganism), a fungus, and an algae. The organism may include an organelle within a cell. The size of the organism may be, but is not limited to, a major axis of 0.5 mm or less, 1 mm or less, 1.5 mm or less, 2 mm or less, 2.5 mm or less, or 3 mm or less. The size of the organism may be a major axis of 100 μm or less, which is difficult to visually confirm, or 500 μm or less, which is the size of a microstructure.

[0017] 2 is a block diagram showing a specimen preparation device 200 that executes a specimen preparation process for preparing a specimen from the object 100 accommodated in the embedding container 1. The specimen preparation device 200 embeds the object 100 accommodated in the embedding container 1 and prepares a specimen from the embedded object 100. The specimen preparation device 200 includes a communication unit 201, a storage unit 202, an input unit 203, an output unit 204, a control unit 205, a movement unit 206, an organism manipulation unit 207, an introduction unit 208, a cutting unit 209, and a specimen preparation unit 210.

[0018] The communication unit 201 is a component that enables the specimen preparation device 200 to communicate with an external device such as a host control device of the specimen preparation device 200, and includes a communication interface circuit. The communication interface circuit is, for example, a communication interface circuit for a wired LAN, a wireless LAN, LTE (Long Term Evolution), or the like. The communication unit 201 supplies data received from the external device to the control unit 205, and transmits data supplied from the control unit 205 to the external device.

[0019] The storage unit 202 includes, for example, at least one of a semiconductor storage device, a magnetic tape device, a magnetic disk device, and an optical disk device. The storage unit 202 stores an operating system program, a driver program, an application program, data, and the like used in processing by the control unit 205. For example, the storage unit 202 stores a specimen preparation program that causes the control unit 205 to execute a specimen preparation process that embeds the object 100 contained in the embedding container 1 and prepares a specimen from the embedded object 100. The storage unit 202 also stores various information used in the specimen preparation process. The specimen preparation program may be installed into the storage unit 202 from a computer-readable portable storage medium such as a CD-ROM or a DVD-ROM using a known setup program.

[0020] The input unit 203 may be any device capable of inputting data, such as a touch panel or a keyboard. An operator using the specimen preparation device 200 can input letters, numbers, symbols, and the like using the input unit 203. When operated by the operator, the input unit 203 generates a signal corresponding to the operation. The generated signal is then supplied to the control unit 205 as an instruction from the operator.

[0021] The output unit 204 may be any device capable of displaying video, images, etc., such as a liquid crystal display or an organic EL display. The output unit 204 displays video corresponding to video data supplied from the control unit 205, images corresponding to image data, etc. The output unit 204 may also be an output device that prints video, images, text, etc. on a display medium such as paper.

[0022] The control unit 205 includes one or more processors and their peripheral circuits. The control unit 205 is, for example, a CPU, and comprehensively controls the overall operation of the specimen preparation device 200. The control unit 205 executes processing based on programs (such as driver programs, operating system programs, and application programs) stored in the storage unit 202. The control unit 205 can also execute multiple programs (such as application programs) in parallel.

[0023] The moving unit 206 is controlled by the control unit 205 in accordance with an operator's instruction input to the input unit 203, and includes an actuator that moves the embedding container 1. The moving unit 206 places the embedding container 1 inside the specimen preparation device 200 from outside the specimen preparation device 200, and also removes the embedding container 1 placed inside the specimen preparation device 200 and moves it to an embedding dish disposed in the specimen preparation unit 210. The operations of the moving unit 206 are not limited to these, and the moving unit 206 can move the embedding container 1 from any location to any location depending on the purpose.

[0024] The organism manipulation unit 207 is controlled by the control unit 205 in accordance with instructions from an operator input to the input unit 203, and is equipped with a manipulator having a nozzle that manipulates the object 100 contained in the embedding container 1. The organism manipulation unit 207 further includes a pump that supplies and sucks gas into the nozzle flow path, a support unit that moves the nozzle, and the like.

[0025] The introduction unit 208 is controlled by the control unit 205 in response to instructions from an operator input to the input unit 203, and includes a syringe for introducing a liquid into the embedding container 1 and a manifold for accommodating the embedding container 1. The introduction unit 208 introduces a liquid 160 into the embedding container 1 and discharges the liquid 160 from the embedding container 1. For example, the introduction unit 208 introduces a treatment liquid for treating the object 100 into the embedding container 1 and introduces an embedding agent for embedding the object 100 into the embedding container 1.

[0026] The cutting unit 209 is equipped with a cutting tool, and is controlled by the control unit 205 in accordance with instructions from the operator input to the input unit 203, to cut the embedded object 100 with the cutting tool to create a specimen.

[0027] The specimen preparation section 210 performs extension, deparaffinization, staining, dehydration and clearing, and sealing processes on the specimen prepared in the cutting section 209, thereby making the specimen prepared in the cutting section 209 in a state that can be observed by microscope observation, etc.

[0028] Fig. 3 is a flowchart showing a specimen preparation process for preparing a specimen from the object 100 accommodated in the embedding container 1. Fig. 4 is a diagram showing the process of S102 shown in Fig. 3, Fig. 5 is a diagram showing the process of S103 shown in Fig. 3, Fig. 6 is a diagram showing the process of S104 shown in Fig. 3, and Fig. 7 is a diagram showing the process of S109 shown in Fig. 3. The specimen preparation process shown in Fig. 3 is executed mainly by the control unit 205 in cooperation with each element of the specimen preparation device 200, based on a control program stored in advance in the storage unit 202. Note that the specimen preparation process shown in Fig. 3 is one aspect of the embedding method according to the embodiment.

[0029] First, the control unit 205 places the object 100, which has settled in a solvent liquid that dissolves the object 100, on the surface of the filter 11 via the moving unit 206 and the organism manipulation unit 207 (S101). The control unit 205 forms a bubble at the tip of the nozzle of the organism manipulation unit 207, and brings the object 100 into contact with the gas-liquid interface between the formed bubble and the solvent liquid, thereby recovering the object 100 from the solvent liquid. The control unit 205 may also recover the object 100 by supplying or exhausting a fluid containing a liquid or gas to or from the flow path of the nozzle of the organism manipulation unit 207. The organism manipulation unit 207 may also be a dispenser such as a pipette. Next, the control unit 205 places the recovered object 100 on the surface of the filter 11. The control unit 205 places the object 100 on the surface of the filter 11. Next, the control unit 205 moves the embedding container 1 into the manifold 110 of the introduction unit 208, with the object 100 placed on the surface of the filter 11. The control unit 205 moves the embedding container 1 via the movement unit 206.

[0030] Next, the control unit 205 performs a chemical pretreatment on the object 100 using various treatment liquids (S102). The control unit 205 introduces various treatment liquids 111 into the embedding container 1 housed in the manifold 110 via syringes to perform pretreatment on the object 100 placed in the embedding container 1. The treatment liquids 111 used in the pretreatment of the object 100 include a fixative, a dehydrating liquid, and a degreasing liquid. The fixative used in the pretreatment may be any of formalin, glutaraldehyde, Bouin's solution, Carnoy's solution, and methanol. The dehydrating liquid used in the pretreatment may be an alcohol such as ethanol, and may be ethanol of any concentration, such as absolute ethanol, 90% ethanol, 80% ethanol, or 70% ethanol. The degreasing liquid used in the pretreatment may be xylene.

[0031] Next, the control unit 205 embeds the object 100 together with the embedding container 1 (S103). The control unit 205 moves the embedding container 1, with the object 100 preprocessed in S102 placed thereon, to the embedding dish 120 arranged in the sampling unit 210. The control unit 205 fills the embedding container 1 contained in the embedding dish 120 with an embedding medium 121 to embed the object 100 placed in the embedding container 1. The embedding medium 121 is, for example, paraffin. The control unit 205 fills the embedding container 1 with liquefied paraffin heated to approximately 60°C until the object 100 is fully immersed, and leaves the embedding container 1 filled with paraffin until the temperature of the heated paraffin drops and the paraffin solidifies. Note that the embedding medium 121 may be celloidin instead of paraffin.

[0032] Next, the control unit 205 thinly slices the object 100 embedded together with the embedding container 1 in the process of S103 to prepare a specimen (S104). The control unit 205 moves the object 100 embedded together with the embedding container 1 from the embedding dish 120 via the moving unit 206. The control unit 205 inverts and positions the embedding container 1 so that the filter 11 is positioned above the object 100. The control unit 205 cuts the inverted embedded object from above using the cutting tool 125 to prepare a thin film including a cross section of the object 100 as a specimen.

[0033] Next, the control unit 205 stretches the specimen prepared in the process of S104 (S105). The control unit 205 moves the specimen prepared in the process of S104 to a water bath filled with water using the moving unit 206, and floats the specimen in the water filled in the water bath, thereby stretching the specimen. The stretched specimen is then moved onto a slide glass by the moving unit 206 and left to dry.

[0034] Next, the control unit 205 deparaffinizes the specimen stretched in the process of S105 (S106). The control unit 205 moves the specimen together with the slide glass using the moving unit 206, and deparaffinizes the specimen by sequentially immersing the specimen in doses filled with xylene, 100% ethanol, and 70% ethanol, respectively.

[0035] Next, the control unit 205 stains the specimen deparaffinized in the process of S106 with hematoxylin-eosin staining (HE staining) (S107). The control unit 205 moves the specimen together with the slide glass using the moving unit 206 and sequentially immerses the specimen in doses filled with hematoxylin solution and eosin solution, respectively, to stain the specimen. Note that the control unit 205 may stain the specimen with a known HE staining method such as Klüver-Barrera staining (KB staining), Fontana-Masson staining, Giemsa staining, Gram staining, or toluidine blue staining.

[0036] Next, the specimen stained in the process of S107 is dehydrated and cleared (S108). The control unit 205 moves the specimen together with the slide glass using the moving unit 206, and dehydrates and clears the specimen by sequentially immersing the specimen in doses filled with 70% ethanol, 100% ethanol, and xylene, respectively.

[0037] The control unit 205 then mounts the specimen 131 dehydrated and cleared in the process of S108 on the glass slide 130 (S109). The specimen 131 is mounted by placing a mounting medium on the glass slide 130 on which the dehydrated and cleared specimen is placed, and then placing a cover glass 132 on the mounting medium placed on the glass slide 130. The mounting medium may contain glycerin, or a commercially available mounting medium may be used. The periphery of the cover glass 132 may be sealed with commercially available nail polish or the like.

[0038] The embedding container 1 has a space on the back side of the filter 11 that can be filled with an embedding agent, thereby preventing the formation of voids that could trap air bubbles on the front side of the filter 11 and preventing deformation of the filter 11. The embedding container 1 prevents the formation of voids on the front side of the filter 11 and prevents deformation of the filter 11, thereby preventing the object 100 from peeling off from the filter 11 and the position of the object 100 from becoming unclear.

[0039] Fig. 8(a) is a conceptual diagram showing the configuration of a conventional embedding container, Fig. 8(b) is a diagram showing the state in which the embedding container shown in Fig. 8(a) is filled with paraffin, Fig. 8(c) is a diagram showing the state in which the paraffin filled in the embedding container shown in Fig. 8(a) has solidified (part 1), Fig. 8(d) is a diagram showing the state in which the paraffin filled in the embedding container shown in Fig. 8(a) has solidified (part 2), and Fig. 8(e) is a diagram showing the state in which the paraffin filled in the embedding container shown in Fig. 8(a) has solidified (part 3).

[0040] The embedding container 101 has a base material 102 and a filter 103, and like the embedding container 1, is used when preparing a specimen by embedding an object 100 placed on the filter 103 after pre-processing the object 100. The base material 102 and the filter 103 are made of the same materials as the base material 10 and the filter 11, and have the same configurations and functions as the base material 10 and the filter 11.

[0041] The embedding container 101 differs from the embedding container 1 in the arrangement of the filter 103. In the embedding container 1, the filter 11 has a space 12 on the back side thereof that can be filled with an embedding medium, whereas in the embedding container 101, the filter 103 does not have a space on the back side thereof that can be filled with an embedding medium.

[0042] As shown in Fig. 8(a), the object 100 is placed on the surface of the filter 11 of the embedding container 1. The pre-processing shown in S102 in Fig. 3 is performed on the object 100 while it is placed on the surface of the filter 103 of the embedding container 101. After the pre-processing, the object 100 is embedded in paraffin 104, which is a high-temperature liquid, as shown in Fig. 8(b). The embedding container 101 is left standing until the temperature of the paraffin 104 in which the object 100 is embedded drops and the paraffin 104 solidifies.

[0043] When the paraffin 104 in which the object 100 is embedded solidifies, the volume of the paraffin 104 shrinks, which may cause various changes in the object 100 and the area around the object 100. In the example shown in Fig. 8(c), the volume of the paraffin 104 shrinks, which causes a gap 105 to form between the filter 103 and the object 100 and the paraffin 104. In the example shown in Fig. 8(c), the gap 105 is formed between the filter 103 and the object 100 and the paraffin 104, which may cause the object 100 to peel off from the filter 103 and the paraffin 104 when the embedded object 100 is sliced.

[0044] In the example shown in Fig. 8(d), the volume of the paraffin 104 contracts, forming a void 105 between the filter 103 and the paraffin 104, and the object 100 separates from the filter 103. In the example shown in Fig. 8(d), because the void 105 is formed between the filter 103 and the paraffin 104, there is a risk that the object 100 will peel off from the filter 103 and the paraffin 104 when the embedded object 100 is sliced. In addition, in the example shown in Fig. 8(d), the object 100 separates from the filter 103 and moves from its initial position on the surface of the filter 103, making the position of the object 100 unclear.

[0045] In the example shown in Fig. 8(e), the filter 103 is deformed due to the contraction of the volume of the paraffin 104. In the example shown in Fig. 8(e), the filter 103 is deformed, and the position of the object 100 moves from the initial position before being embedded in the paraffin 104, making the position of the object 100 unclear.

[0046] FIG. 9(a) is a diagram showing the embedding container 1 filled with paraffin, and FIG. 9(b) is a diagram showing the embedding container 1 after the paraffin has solidified.

[0047] 9(a), the pretreated object 100 is placed on the filter 11 of the embedding container 1 and is then embedded in high-temperature liquid paraffin 13. The paraffin 13 that embeds the object 100 embeds the object 100 and fills the space 12. The embedding container 1 is left standing until the temperature of the paraffin 13 that embeds the object 100 drops and solidifies.

[0048] 9(b), in the embedding container 1, the paraffin 13 remains in the space 12 even after solidification, so that when the paraffin 13 in which the object 100 is embedded solidifies, the paraffin 13 remains in the space 12 despite its volume shrinking. In the embedding container 1, the paraffin 13 remains in the space 12 when solidified, so that there is a low risk of voids being formed between the filter 11 and the object 100 / paraffin 13, and there is also a low risk of the object 100 becoming separated from the filter 103.

[0049] Furthermore, the embedding container 1 forms the space 12 with the base material 10 and the filter 11, so that the space 12 that can be filled with an embedding medium can be easily formed. Specifically, by positioning the filter 11 so that the back surface of the filter 11 is spaced apart from the lower end of the base material 10, the space 12 that can be filled with an embedding medium can be easily formed.

[0050] Furthermore, in the embedding container 1, the filter 11 has a flat plate-like shape, which makes it easy to handle and place the filter 11, and also reduces the manufacturing cost of the filter 11 compared to when a filter with a complex shape is used.

[0051] The specimen preparation device 200 prepared the specimen by moving the embedding container 1 to the embedding dish 120 arranged in the specimen preparation section 210. However, the specimen preparation device according to the embodiment may be configured to perform pre-processing and embedding of the object 100 without moving the embedding container 1 to the embedding dish 120, and to perform thin-sectioning with the embedding container 1 directly attached to a microtome.

[0052] FIG. 10 is a diagram showing a thin-sectioning process using a microtome.

[0053] In the specimen preparation device according to the modified example, after pre-processing and embedding of the object 100, the object is attached directly to a microtome and then sliced. In the specimen preparation device according to the modified example, the embedding process is performed, and the interior of the housing 10 is filled with an embedding medium 123. Then, the housing 10 is cut with a cutting tool 125 while the end face of the housing 10 where the space 12 is formed is protruding from the microtome, thereby performing the slice process. Note that in the specimen preparation device according to the modified example, the embedding container 1 is attached directly to the microtome without being moved to the embedding dish 120 to perform the slice process, but instead, the pre-processing, embedding process, and slice process of the object 100 may be performed using a single microtome.

[0054] 11 is a conceptual diagram showing the configuration of an embedding container including a determination device according to the first modified example. The embedding container 2 differs from the embedding container 1 in that it includes an absorber 20. The configurations and functions of the components of the embedding container 2 other than the absorber 20 are the same as those of the components of the embedding container 1 that are assigned the same reference numerals, and therefore detailed explanations thereof will be omitted here.

[0055] The absorbent material 20 is a porous member made of a synthetic resin such as polyurethane, and is arranged to be contained in the space 12. The absorbent material 20 has a plurality of holes formed therein that allow the liquid that has passed through the filter 11 to pass therethrough. The absorbent material absorbs the embedding agent. The absorbent material 20 is arranged throughout the entire space 12, but may also be arranged in only a part of the space 12 as long as it is arranged so as to contact the entire back surface of the filter 11.

[0056] The specimen preparation process for preparing a specimen from the object 100 accommodated in the embedding container 2 is carried out in the same manner as the specimen preparation process shown in FIG.

[0057] The embedding container 2 is disposed in a space and includes an absorbent material 20 that absorbs the embedding medium, thereby preventing the filter 11 from deforming when the embedding medium solidifies. By preventing the filter 11 from deforming when the embedding medium solidifies, the embedding container 2 can prevent the position of the object 100 from becoming unclear.

[0058] 12 is a conceptual diagram showing the configuration of an embedding container including a determination device according to the second modification. The embedding container 3 differs from the embedding container 1 in that it includes a deflection preventive material 30. The configurations and functions of the components of the embedding container 3 other than the deflection preventive material 30 are the same as those of the components of the embedding container 1 that are assigned the same reference numerals, and therefore detailed explanations thereof will be omitted here.

[0059] The sagging prevention material 30 is made of at least one of plastic, paper, wood, and fiber, and has higher rigidity than the filter 11. The sagging prevention material 30 has a lattice shape and is formed with a plurality of holes 31 through which a chemical solution such as a treatment solution in which the object 100 is immersed can pass. The sagging prevention material 30 is disposed so that its front surface contacts the back surface of the filter 11 and its back surface is spaced apart from the lower end of the base material 10. The sagging prevention material 30 may be fastened by a fastening portion formed on the base material 10, or may be bonded to the back surface of the filter 11 by an adhesive member (not shown).

[0060] The specimen preparation process for preparing a specimen from the object 100 accommodated in the embedding container 3 is carried out in the same manner as the specimen preparation process shown in FIG.

[0061] The embedding container 3 is disposed in the space 12 and is provided with a deflection prevention material 30 that absorbs the embedding agent, thereby preventing the filter 11 from deforming when the embedding agent solidifies. The embedding container 2 prevents the filter 11 from deforming when the embedding agent solidifies, thereby preventing the position of the object 100 from becoming unclear.

[0062] Furthermore, the embedding container 3 has a plurality of holes 31 formed in the deflection-preventing material 30 through which the chemical solution in which the object 100 is immersed can pass, allowing the chemical solution, such as the treatment solution used in pretreatment, to pass through without clogging the deflection-preventing material 30. Because the embedding container 3 allows the chemical solution to pass through without clogging the deflection-preventing material 30, it is possible to prevent a decrease in the processing efficiency of pretreatment due to the placement of the deflection-preventing material 30.

[0063] Furthermore, since the filter 11 is positioned in the embedding container 3 so that its back surface is spaced apart from the lower end of the substrate 10, a sufficient amount of embedding material can be filled into the space 12 even though the anti-deflection material 30 is placed.

[0064] The deflection prevention material 30 only needs to have its front surface in contact with the back surface of the filter 11, and may be arranged so that its back surface coincides with the lower end of the substrate 10. For example, if the holes 31 formed in the deflection prevention material 30 allow a sufficient amount of embedding medium to be filled into the space 12, the deflection prevention material 30 may be arranged so that its back surface coincides with the lower end of the substrate 10. By arranging the deflection prevention material 30 so that its back surface coincides with the lower end of the substrate 10, the height of the embedding container 3 can be reduced, allowing it to be made more compact.

[0065] 13 is a conceptual diagram showing the configuration of an embedding container including a determination device according to a third modification. The embedding container 4 differs from the embedding container 1 in that it includes a space-forming member 40. The configurations and functions of the components of the embedding container 4 other than the space-forming member 40 are the same as those of the embedding container 1 with the same reference numerals, and therefore detailed explanations will be omitted here. In the embedding container 4, the filter 11 is positioned so that the back surface of the filter 11 coincides with the lower end of the substrate 10.

[0066] The space forming member 40 is a sealing material made of plastic such as an O-ring, and is placed on the base 42 so that its upper end is in contact with the lower surface of the substrate 10, forming a space 43 together with the substrate 10 and the base 42. The space 43 is sealed by being isolated from the outside by the substrate 10, the space forming member 40, and the base 42. The space forming member 40 is positioned by engaging with a recess formed in at least one of the lower surface of the substrate 10 and the upper surface of the base 42.

[0067] The specimen preparation process for preparing a specimen from the object 100 accommodated in the embedding container 4 is carried out in the same manner as the specimen preparation process shown in FIG.

[0068] The embedding container 4 forms a sealed space 43 on the back surface of the filter 11 by the substrate 10, the space-forming member 40, and the base 42, thereby preventing air from entering from the back surface of the filter 11 when the embedding medium solidifies. By preventing air from entering from the back surface of the filter 11 when the embedding medium solidifies, the embedding container 4 can prevent voids from being formed between the filter 11 and the object 100 or the solidified embedding medium. The embedding container 4 can prevent voids from being formed between the filter 11 and the object 100 or the solidified embedding medium, which would make it difficult to determine the position of the object 100.

[0069] Furthermore, since the filter 11 is positioned in the embedding container 4 so that its back surface coincides with the lower end of the substrate 10, the filter 11 can be easily positioned, and manufacturing efficiency can be improved compared to when the back surface does not coincide with the lower end of the substrate 10.

[0070] 14 is a conceptual diagram showing the configuration of an embedding container according to a fourth modification. The embedding container 5 includes a housing 50 and a filter 51. The filter 51 has a recess 52 formed in it, and a mounting area 53 on the bottom surface of the recess 52 allows the object 100 to be mounted thereon. The filter 51 functions as a guide that guides the object 100 to the mounting area 53. The object 100 mounted in the mounting area 53 is embedded in an embedding agent (not shown). The embedding container 5 is cut together with the object 100 mounted in the mounting area 53 while it is embedded in the embedding agent, thereby producing a specimen of the object 100 that can be observed under a microscope or the like.

[0071] Similar to the housing 10, the housing 50 is made of at least one of plastic, paper, wood, and fiber and has a cylindrical shape. The housing 50 has side surfaces arranged to surround the mounting area 53. A locking portion for locking the filter 51 is formed on the inner wall of the housing 50.

[0072] The filter 51 is a porous member, similar to the filter 11, and is fastened to the housing 50 by fastening portions formed on the inner wall of the housing 20 so as to form a space 12 on the rear surface. The filter 51 has a cylindrical central portion 54 and a cylindrical peripheral portion 55 arranged to surround the central portion 54. The height of the central portion 54 is lower than the height of the peripheral portion 55, and a recess 52 is formed above the central portion 54. The surface of the central portion 54 is a mounting area 53 on which the object 100 can be placed. The thickness of the central portion 54 may be 10 μm or more and 20 μm or less.

[0073] By placing a single filter 51 inside the housing 50, the embedding container 5 can form a guide section that guides the object 100 to the mounting area 53, thereby achieving the function of guiding the object 100 to the mounting area 12 located at the center of the housing 10 using a simple manufacturing method.

[0074] The embedding container 5 prevents the object 100 from being placed at the edge of the housing 10 by making the filter 51 function as a guide that guides the object 100 to the mounting area 53 located in the center of the housing 10. Preventing the object 100 from being placed at the edge of the housing 10 prevents the object 100 from interfering with the housing 10 and being damaged when the embedded object 100 is sliced. Furthermore, the embedding container 5 allows the object 100 to be placed in the mounting area 53 surrounded by the peripheral part 55 of the filter, thereby restricting movement of the object 100 when a chemical solution is passed through it.

[0075] In the specimen preparation process shown in Figure 3, the specimen is prepared by sequentially executing the processes of S101 to S109. However, in the specimen preparation process according to the embodiment, the processes of S104 to S109 may be performed by attaching the embedded embedding container to tape.

[0076] FIG. 15 is a diagram (part 1) showing a specimen preparation process according to a modified example, and FIG. 16 is a diagram (part 2) showing a specimen preparation process according to a modified example. FIG. 15(a) shows a process corresponding to step S101 shown in FIG. 3 , FIG. 15(b) shows a process corresponding to steps S102 and S103 shown in FIG. 3 , and FIG. 15(c) shows a process for attaching an embedding container to tape. FIG. 15(d) shows a process corresponding to step S104 shown in FIG. 3 , and FIG. 15(e) shows a process for placing an embedding container on a glass slide. FIG. 16(a) shows a process corresponding to step S105 shown in FIG. 3 , and FIG. 16(b) shows a process for peeling the tape from the glass slide. FIG. 16(c) shows a process corresponding to steps S106 to S108 shown in FIG. 3 , and FIG. 16(d) shows a process corresponding to step S109 shown in FIG. 3 .

[0077] First, the object is placed in an embedding container 6 shown in Fig. 15(a). Next, as shown in Fig. 15(b), the embedding container 6 with the object placed therein is placed in a microtome 60 capable of performing pre-processing and embedding. Next, the object placed in the embedding container 5 placed in the microtome 60 is subjected to pre-processing and then embedded. Next, after the object placed in the embedding container 6 has been embedded, tape 61 is attached to the embedding container 6 as shown in Fig. 15(c).

[0078] Next, as shown in Figure 15(d), the embedding material 62 adhering to the tape 61 peeled off from the embedding container 6 is cut to prepare a specimen. Next, as shown in Figure 15(e), the tape 61 is attached to a slide glass 63. By attaching the tape 61 to the slide glass 63, the specimen 64 attached to the tape 61 is placed on the slide glass 63. When the tape 61 is attached to the slide glass 63, a liquid is applied to the area where the specimen 64 will be placed. By applying the liquid to the area where the specimen 64 will be placed, the adhesive strength between the slide glass 63 and the specimen 64 is improved.

[0079] 16(a), the slide glass 63 with the tape 61 attached thereto is stored for a predetermined time, such as 12 hours, at a temperature higher than room temperature, such as 47° C. By storing the slide glass 63 at a temperature higher than room temperature for the predetermined time, the specimen 64 attached to the tape 61 stretches.

[0080] 16(b), the tape 61 attached to the slide glass 63 is peeled off from the slide glass 63. When the tape 61 is peeled off from the slide glass 63, the adhesive force between the slide glass 63 and the specimen 64 is stronger than the adhesive force between the tape 61 and the specimen 64, so the specimen 64 remains in a state of being disposed on the slide glass 63.

[0081] Next, as shown in Fig. 16(c), the specimen 64 placed on the slide glass 63 is stained, dehydrated, and cleared. Then, as shown in Fig. 16(d), the stained, dehydrated, and cleared specimen 64 is sealed with a cover glass 65, and the specimen preparation process is completed.

[0082] 15 and 16, the embedding object 62 adheres to the tape 61 peeled from the embedding container 6, and is cut on the attached tape 61 to prepare the specimen. However, the embedding container according to the embodiment may be configured so that the embedding container is cut to perform the thin-sectioning process.

[0083] Fig. 17(a) is a plan view of an embedding container according to a fifth modified example, Fig. 17(b) is a side view of the embedding container shown in Fig. 17(a), Fig. 17(c) is a perspective view of the embedding container shown in Fig. 17(a), Fig. 17(d) is a cross-sectional perspective view taken along line A-A shown in Fig. 17(c), and Fig. 17(e) is a cross-sectional view of the filling hole shown in Fig. 17(a).

[0084] The embedding container 7 includes a substrate 70, a filter 71, and a support plate 72. The substrate 70 has a single filling hole 73, a pair of first through holes 74 and 75, a pair of second through holes 76 and 77, and multiple recesses 78. The substrate 70 is formed from at least one of highly rigid plastic, paper, wood, and fiber. The plastic forming the substrate 70 includes, but is not limited to, one or more of silicone rubber, polyacetal (POM), polycarbonate (PC), polypropylene (PP), polyethylene terephthalate (PET), polyurethane (PU), polymethyl methacrylate (PMMA), polystyrene (PS), and polyvinyl chloride (PVC). The substrate 70 has a generally rectangular planar shape with arc-shaped ends in the longitudinal direction (DL) and a linear transverse direction (DS).

[0085] The filling hole 73 is arranged to penetrate from one surface 70a to the other surface 70b of the substrate 70, with the diameter of one opening 73a being shorter than the diameter of the other opening 73b, and at least a portion of the opening 73b being tapered. A pair of first through holes 74 and 75 are formed to penetrate from one surface 70a to the other surface 70b of the substrate 70, and are arranged along the longitudinal direction DL, with the filling hole 73 as the midpoint. The pair of first through holes 74 and 75 are formed so that the diameter of one opening 74a and 75a is longer than the diameter of the other opening 74b and 75b. A pair of second through holes 76 and 77 are formed to penetrate from one surface 70a to the other surface 70b of the substrate 70, and are arranged outside the pair of first through holes 74 and 75 along the longitudinal direction DL, with the filling hole 73 as the midpoint. The pair of first through holes 76 and 77 are formed so that the diameter of one opening 76a and 77a is longer than the diameter of the other opening 76b and 77b.

[0086] Each of the plurality of recesses 78 opens on one surface 70a of the substrate 70, and is arranged so as to surround the filling hole 73 and the pair of first through holes 74 and 75. The size and arrangement of the plurality of recesses 78 are determined so that the total areas per unit length in the longitudinal direction DL of the opening 73a of the filling hole 73, the openings 74a and 75a of the pair of first through holes 74 and 75, and the opening of the recess 78 are approximately the same.

[0087] The filter 71 is a porous film having a circular planar shape, and is disposed so as to cover one opening 73 a of the filling hole 73 .

[0088] The support plate 72 is formed from at least one of highly rigid plastic, paper, wood, and fiber, has the same planar shape as the substrate 70, and is positioned to face one surface 70a of the substrate 70. Plastics forming the support plate 72 include, but are not limited to, one or more of silicone rubber, polyacetal (POM), polycarbonate (PC), polypropylene (PP), polyethylene terephthalate (PET), polyurethane (PU), polymethyl methacrylate (PMMA), polystyrene (PS), and polyvinyl chloride (PVC). The support plate 72 supports the filter 71 by sandwiching the filter 71 together with the substrate 70. The support plate 72 has a plurality of through holes 72a formed at positions corresponding to the filling hole 73, the pair of first through holes 74 and 75, the pair of first through holes 76 and 77, and the plurality of recesses 78.

[0089] The embedding container 7 has a plurality of recesses 78 formed therein so that the total areas per unit length in the longitudinal direction DL of the opening 73 a of the filling hole 73, the openings 74 a and 75 a of the pair of first through holes 74 and 75, and the opening of the recess 78 are approximately the same. The embedding container 7 has a plurality of recesses 78 formed therein so that the total areas per unit length in the longitudinal direction DL of the openings 73 a, 74 a, and 75 a and the opening of the recess 78 are approximately the same. This results in a substantially uniform area per unit length in the longitudinal direction DL where the blade of a cutting tool cutting one surface 70 a of the base material 70 a abuts against the synthetic resin that forms the one surface 70 a. Because the embedding container 7 has a substantially uniform area per unit length in the longitudinal direction DL where the blade of the cutting tool abuts against the synthetic resin that forms the one surface 70 a, the cutting amounts per unit length of the support plate 72 and the base material 70 when cut along the longitudinal direction DL per unit length in the longitudinal direction DL are substantially the same. When the embedding container 7 is cut along the longitudinal direction DL, the cutting amounts per unit length of the support plate 72 and the base material 70 are approximately the same, and therefore the cutting forces applied to the cutting tools cutting the support plate 72 and the base material 70 are approximately the same. Because the embedding container 7 is cut with approximately the same cutting forces applied to the cutting tools cutting the support plate 72 and the base material 70, it is possible to prevent problems in cutting the embedded object caused by uneven cutting forces applied to the cutting tools.

[0090] Although the embedding container 7 has a single filling hole 73 formed therein, the embedding container according to the embodiment may have two or more filling holes formed therein.

[0091] Figure 18(a) is a plan view of an embedding container according to a sixth modified example, Figure 18(b) is a plan view of an embedding container according to a seventh modified example, Figure 18(c) is a plan view of an embedding container according to an eighth modified example, Figure 18(d) is a plan view of an embedding container according to a ninth modified example, Figure 18(e) is a plan view of an embedding container according to a tenth modified example, Figure 18(f) is a plan view of an embedding container according to an eleventh modified example, and Figure 18(g) is a plan view of an embedding container according to a twelfth modified example.

[0092] The embedding container 7a has three filling holes 73a and a plurality of recesses 78a whose total area per unit length in the longitudinal direction of the filling holes 73a and the recesses 78a is approximately the same and whose openings surround the periphery of the filling hole 73a. The embedding container 7b has six filling holes 73b and a plurality of recesses 78b whose total area per unit length in the longitudinal direction of the filling holes 73b and the recesses 78b is approximately the same and whose openings surround the periphery of the filling hole 73b. The embedding container 7c has eight filling holes 73c and a plurality of recesses 78c whose total area per unit length in the longitudinal direction of the filling holes 73c and the recesses 78c is approximately the same and whose openings surround the periphery of the filling hole 73c. The embedding container 7d has 12 filling holes 73d and multiple recesses 78d that have approximately the same total area per unit length in the longitudinal direction and are formed to surround the filling holes 73d. The embedding container 7e has 24 filling holes 73e and multiple recesses 78e that have approximately the same total area per unit length in the longitudinal direction and are formed to surround the filling holes 73e. The embedding container 7f has 48 filling holes 73f and multiple recesses 78f that have approximately the same total area per unit length in the longitudinal direction and are formed to surround the filling holes 73f. The embedding container 7g has 96 filling holes 73g formed therein, and multiple recesses 78g are formed so that the total area of ​​the openings of the filling holes 73g and recesses 78g per unit length in the longitudinal direction is approximately the same and so that they surround the filling holes 73g.

[0093] The embedding container according to the embodiment may have filling holes formed therein that have diameters different from the diameters of the filling holes 73, 73a, 73b, and 73c.

[0094] Figure 18(h) is a plan view of an embedding container according to the 13th modified example, Figure 18(i) is a plan view of an embedding container according to the 14th modified example, Figure 18(j) is a plan view of an embedding container according to the 15th modified example, and Figure 18(k) is a plan view of an embedding container according to the 16th modified example.

[0095] The embedding container 7h has a single filling hole 73h with a diameter longer than the filling hole 73 formed in the embedding container 7, and multiple recesses 78h are formed so that the total area of ​​the openings of the filling hole 73h and the recess 78h per unit length in the longitudinal direction is approximately the same and surrounds the periphery of the filling hole 73h. The embedding container 7i has three filling holes 73i with a diameter longer than the filling hole 73a formed in the embedding container 7a, and multiple recesses 78i are formed so that the total area of ​​the openings of the filling hole 73i and the recess 78i per unit length in the longitudinal direction is approximately the same and surrounds the periphery of the filling hole 73i. The embedding container 7j has six filling holes 73j with a diameter longer than the filling hole 73b formed in the embedding container 7b, and multiple recesses 78j are formed so that the total area of ​​the openings of the filling hole 73j and the recess 78j per unit length in the longitudinal direction is approximately the same and surrounds the periphery of the filling hole 73j. The embedding container 7k has three filling holes 73k with a diameter longer than the filling hole 73c formed in the embedding container 7c, and multiple recesses 78k are formed so that the total area of ​​the openings of the filling holes 73k and recesses 78k per unit length in the longitudinal direction is approximately the same and so that they surround the filling hole 73k.

[0096] The embedding containers 7a to 7k are formed so that the total area of ​​the multiple recesses and the filling holes per unit length in the longitudinal direction is approximately the same, so that, as with the embedding container 7, uneven cutting of the embedded object can be prevented.

[0097] 1 to 7, 7a to 7k embedding container 10, 70 base material 11, 51, 71 filter 12 space 100 object

Claims

1. An embedding container comprising: a filter on whose surface an object to be embedded in an embedding agent can be placed and through which a chemical solution in which the object will be immersed can pass; and a base material that supports the filter and contains the object, wherein a space that can be filled with embedding agent is formed on the back side of the filter.

2. The embedding container according to claim 1, further comprising an absorbent material disposed in the space and configured to absorb the embedding medium.

3. The embedding container according to claim 1, further comprising a sagging prevention material disposed in the space to prevent the filter from sagging.

4. The embedding container according to claim 3, wherein the anti-flexure material has a plurality of holes formed therein through which the chemical solution in which the object is immersed can pass.

5. The embedding container according to claim 3, wherein the anti-sagging material is positioned so that the back surface of the anti-sagging material is spaced apart from the lower end of the base material.

6. The embedding container according to claim 3, wherein the anti-sagging material is positioned so that the back surface of the anti-sagging material coincides with the lower end of the base material.

7. The embedding vessel according to claim 1, wherein the substrate forms the space together with the filter.

8. The embedding vessel according to claim 1, wherein the filter is positioned so that the rear surface of the filter is spaced apart from the lower end of the substrate.

9. The embedding container according to claim 1, further comprising a space forming member that forms the space together with the filter.

10. The embedding vessel of claim 9, wherein the filter is positioned so that the back surface of the filter coincides with the lower edge of the substrate.

11. A method for embedding an object in an embedding container comprising: a filter on whose surface an object to be embedded in an embedding agent can be placed and which is permeable to a chemical solution in which the object will be immersed; and a substrate for supporting the filter and containing the object, wherein a space capable of being filled with an embedding agent is formed on the back side of the filter, the method comprising the steps of placing the object on the surface of the filter, performing a pre-processing step of immersing the object placed on the surface of the filter in a liquid, and embedding the pre-processed object in the embedding agent.

12. An embedding device comprising: a storage section for storing an embedding container according to any one of claims 1 to 10; and an introduction section for introducing a processing liquid and / or an embedding medium into the embedding container stored in the storage section.

Images