A specimen embedding method and device

Abstract

The present invention relates to a specimen embedding method and device, wherein a specimen embedding method comprises the following steps: adding embedding liquid into an embedding box one by one, starting an oscillator after each addition of embedding liquid, blowing low-speed air from the top of the embedding box to freeze the added embedding liquid; placing a specimen after the embedding liquid is frozen and formed for the first time, placing a positioning plate at each end of the embedding box, and the two positioning plates are respectively located at the top and bottom of the specimen; after the embedding liquid is added for the last time, covering the upper surface of the embedding liquid with a heat preservation plate, and blowing low-speed air through the air inlet and the exhaust port set at the two ends of the heat preservation plate to realize the specimen freezing and forming. The embedding liquid is added in multiple times, and the filling amount is planned according to the specimen size, so as to reduce the expansion of the specimen during the freezing process, and effectively prevent the specimen from moving in position due to the freezing expansion of the embedding liquid.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a specimen embedding method and device. Background Art

[0002] The statements in this section merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Cryosectioning is a method of rapidly cooling tissue to a certain hardness under low-temperature conditions and then slicing it. The entire specimen is frozen and then sliced. The image data of each slice is obtained and reconstructed in 3D to form a virtual digital image for use in medical teaching, research and other fields. It can save specimen resources and has been gradually promoted in the fields of medical research and teaching.

[0004] The specimen is an irregular object, and it is difficult to position it directly on the slicing equipment. It needs to be embedded to freeze and solidify the specimen into a regular object. The embedding process of the existing technology will pre-freeze a base, and then place the specimen together with the liquid embedding solution on the upper layer of the base and freeze it together. This method is simple and fast, but during the freezing and forming process of the specimen with the upper layer of embedding solution, it is affected by the inconsistent freezing speed of the surrounding ice cubes, causing squeezing and position movement, which will change the position of the specimen relative to the base, causing inaccurate positioning of subsequent frozen sectioning operations; secondly, the embedding solution will be mixed with gas during the filling process, and the freezing process will form a bubble cavity. After the specimen is sliced, the cavity will affect the quality of the acquired image. Summary of the invention

[0005] In order to solve the technical problems existing in the above-mentioned background technology, the present invention provides a specimen embedding method and device, wherein an embedding box and an oscillator are placed inside a freezing device, and embedding liquid is added and frozen gradually. Each time the embedding liquid is added, the air is discharged by the oscillator, and the specimen is positioned in cooperation with positioning plates placed at both ends of the embedding box.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a specimen embedding method, comprising the following steps:

[0008] Add embedding solution to the embedding box gradually, shake after each addition, and blow low-speed air from the top of the embedding box to freeze the added embedding solution;

[0009] After the embedding solution is frozen for the first time, the specimen is placed in the embedding box. A positioning plate is placed at each end of the embedding box, and the two positioning plates are located at the top and bottom of the specimen respectively.

[0010] After the embedding solution is added for the last time, a heat preservation plate is covered on the upper surface of the embedding solution, and low-speed air is blown in through the air inlet and exhaust ports set at both ends of the heat preservation plate to achieve specimen freezing molding.

[0011] Add embedding solution to the embedding box gradually, specifically:

[0012] When adding embedding solution for the first time, add it to a height of 6~10cm;

[0013] Starting from the second time, the height of each addition of embedding solution should not exceed 3 cm;

[0014] When adding embedding solution for the last time, the embedding solution height should exceed the upper surface of the specimen by at least 1 cm.

[0015] The air flow temperature of low-speed air does not exceed 4°C.

[0016] The embedding solution added for the first time is frozen to form a base, and the specimen is placed on the base. The remaining embedding solution freezes the specimen, the base, the positioning plate and the embedding box into a whole.

[0017] The second aspect of the present invention provides a device for implementing the above-mentioned embedding method, including a rectangular and horizontally arranged embedding box, the bottom of the embedding box is located in the groove of the oscillator, the embedding box and the oscillator are located inside the freezing chamber of the freezing device, the top of the embedding box is detachably connected to the insulation plate, and the two ends of the insulation plate are provided with an air inlet and an exhaust port connected to the interior of the embedding box.

[0018] Both ends of the embedding box in the length direction are provided with detachably connected positioning plates, and the two positioning plates are provided with positioning holes corresponding to the positions.

[0019] The positioning holes are located on the positioning plate.

[0020] A positioning line is connected between the positioning holes at corresponding positions of the two positioning plates.

[0021] The distance between two adjacent positioning holes on a set of positioning plates is the width or height of the specimen section.

[0022] The positioning wire is detachably connected to the positioning plate.

[0023] Compared with the prior art, one or more of the above technical solutions have the following beneficial effects:

[0024] 1. Add embedding solution in multiple times, plan the filling amount according to the specimen size, reduce the expansion of the specimen during the freezing process, and effectively avoid the specimen from moving due to the freezing expansion of the embedding solution.

[0025] 2. The oscillator located at the bottom of the embedding box can effectively avoid the generation of bubble cavities.

[0026] 3. After the first embedding solution is frozen, it forms a base at the bottom of the embedding box, ensuring that a low-temperature environment is formed at the bottom of the specimen first. Combined with the low-temperature and low-speed air blown in from the top of the embedding box, the bottom and surrounding areas of the embedding box are cooled down first, avoiding the situation where the top freezes before the bottom, thereby avoiding the internal stress caused by the upper layer of the specimen freezing first and further reducing the expansion caused by the freezing of the specimen.

[0027] 4. The use of positioning plates and positioning lines can determine the position and range of the target object through cross-section when it is impossible to observe the inside of the colored ice, providing convenience for subsequent precise processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0029] Figure 1 is a schematic diagram of the relative positions of a specimen and an embedding device provided by one or more embodiments of the present invention;

[0030] Figure 2 is a schematic diagram of the structure of a positioning plate in an embedding device provided by one or more embodiments of the present invention;

[0031] In the figure: 1. specimen, 2. base, 3. oscillator, 4. insulation board, 41. air inlet, 42. exhaust port, 5. freezing device, 6. embedding box, 7. positioning plate, 71. positioning hole, 72. groove. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0033] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0035] The specimen (which can be an animal or human body) is embedded to form a regular object, and then placed in a slicing device (such as a milling machine) for cryosectioning. By obtaining the image data of each slice (which can be a cross section, longitudinal section or sagittal section), a virtual digital image (such as a virtual digital human) is formed after 3D reconstruction for use in medical teaching, research and other fields, which can save specimen resources. The embedding process must ensure that the regular object does not contain bubbles that affect image recognition. At the same time, the specimen itself will expand during the too-fast freezing process, and some tiny tissues may be destroyed due to expansion. At the same time, the expansion process is accompanied by displacement, which will cause the relative position of the specimen to change, making it impossible for the subsequent image processing process to identify the width, height and cross-section center point of the specimen, which will lead to inaccurate virtual image data.

[0036] Therefore, the following embodiment proposes a specimen embedding method and device, wherein an embedding box and an oscillator are placed inside a freezing device, embedding liquid is added and frozen gradually, and the freezing speed is reduced; each time the embedding liquid is added, air is discharged through the oscillator to reduce the influence of bubbles; and the width, height and cross-section center point of the specimen are positioned in cooperation with positioning plates placed at both ends of the embedding box.

[0037] Embodiment 1:

[0038] like Figure 1 As shown, a specimen embedding method comprises the following steps:

[0039] The embedding solution is added gradually into the embedding box 6 placed horizontally. After each addition, the oscillator 3 is started, and low-speed air (flow rate 3-5 m / s, temperature 0-3°C) is blown into the embedding solution from the top of the embedding box 6 to freeze the added embedding solution;

[0040] After the embedding liquid is frozen and formed for the first time, the specimen 1 is placed, and a positioning plate 7 is placed at each of the front and rear ends of the embedding box 6;

[0041] After the embedding solution is added for the last time, a heat preservation plate is covered on the upper surface of the embedding solution, and low-speed air is blown in through the air inlet 41 and the air outlet 42 provided at both ends of the heat preservation plate to realize specimen freezing molding.

[0042] Take a human specimen with a length of 1.7m as an example:

[0043] The embedding liquid is added gradually into the horizontally placed embedding box 6, specifically:

[0044] When adding embedding solution for the first time, add a height of 6 to 10 cm to ensure that the base 2 formed after freezing has sufficient strength to bear the weight of the specimen and the rest of the embedding solution on top;

[0045] The height of the embedding solution added for the second time should be calculated based on the shape, thickness, tissue structure, pitch position, etc. of the target object. In this embodiment, it should not exceed 3 cm. If the specimen is thicker, more embedding solution needs to be added. However, it is necessary to ensure that after the second time, each time the embedding solution is added, it does not exceed 3 cm.

[0046] The embedding solution added for the last time should cover the upper surface of specimen 1 by at least 1 cm.

[0047] After each addition, the oscillator 3 is started, and low-speed air is blown into the top of the embedding box 6 to freeze the added embedding solution. Specifically:

[0048] The embedding box 6 is placed in the groove of the oscillator 3, and the embedding box 6 and the oscillator 3 are placed in the freezing chamber of a freezing device (which can be a large freezer). After each addition of the embedding solution, the oscillator 3 is started to exhaust the air to prevent the formation of bubble cavities; after the air in the added embedding solution is exhausted, the freezing device is started to blow low-speed air from the top of the embedding box 6 into the added embedding solution, and the air flow temperature does not exceed 4°C (the embedding solution used for specimen frozen sectioning has a specific composition and a freezing point of about 8°C) until the embedding solution is frozen to form ice cubes.

[0049] The embedding solution is added for the second time in multiple times according to the height of the specimen. In this embodiment, no more than 3 cm of embedding solution is added at one time, and it can also be added in multiple times.

[0050] like Figure 2 As shown, the edges of the positioning plates 7 arranged at the front and rear ends of the embedding box 6 are provided with positioning holes 71 corresponding to the positions, and positioning lines (which can be steel wires or any low-temperature resistant material) are connected between the positioning holes 71. After the specimen 1 is placed in the embedding box 6, the positioning line between the two positioning plates 7 forms a width dividing line of the specimen 1, so that the positioning line is the same width as the specimen 1. After the specimen 1 is frozen and formed, the positioning plates 7 are also frozen and fixed, and the width direction of the positioning plates 7 forms the positioning of the specimen 1 in the subsequent sectioning process; grooves 72 can also be set at the bottom and both sides of the positioning plates 7, and the positioning lines are connected between the grooves 72 of the two positioning plates 7. The width and height of the positioning plates 7 are used as the width and height scales of the specimen, and the intersection of the center lines in the two directions defines the center point to form the center point of the end face of the specimen 1, which is also used for positioning in the subsequent sectioning process.

[0051] The embedding liquid added for the first time is frozen to form a base 2, and the specimen 1 is placed on the base 2. The embedding liquid added for the second time fixes the specimen 1, the positioning plate 7, and the positioning line between the positioning plates 7 on the base 2. The embedding liquid added for the third time freezes the specimen 1, the embedding liquid, and the embedding box 6 into a whole as a reserve for the subsequent frozen sectioning process.

[0052] In the above embedding process, the embedding solution is added in multiple times, and the filling amount is planned according to the specimen size to reduce the expansion of the specimen during the freezing process, which can effectively prevent the specimen from moving due to the freezing expansion of the embedding solution.

[0053] The use of an oscillator can effectively avoid the generation of bubble cavities.

[0054] The embedding box first forms a base at the bottom to ensure that a low-temperature environment is formed at the bottom of the specimen first. Combined with the low-temperature and low-speed air blown in from the top of the embedding box, the bottom and surrounding areas of the embedding box are cooled down first to avoid the top freezing before the bottom, thereby avoiding the internal stress caused by the upper layer of the specimen freezing first and further reducing the expansion caused by the freezing of the specimen.

[0055] The use of positioning plates and positioning lines can determine the position and range of the target object through cross-section when the interior of the colored ice cannot be observed, providing convenience for subsequent precise processing.

[0056] After the specimen is embedded, its subsequent slicing process will form specimen cross-sectional image data. In order to make the boundary between the specimen image and the ice cube image clear and easy to distinguish, a pigment of a certain color will be added to the embedding solution. The introduction of the pigment will make it impossible to observe the specimen inside the ice cube during the embedding process. The two positioning plates are respectively located at the two ends of the embedding box (the top of the head and the sole of the foot of the human specimen in this embodiment). The positioning line between the two positioning plates will form four points in the cross-sectional image after slicing (after slicing, the steel wire cross section shows the original color of the metal, which is very obvious compared with the specimen cross section and the ice cube cross section. The cross section obtained by some milling cutting equipment can even obtain the four points of the steel wire cross section through reflection), which can show the position, width, height and center point of the specimen, thereby facilitating subsequent precise processing.

[0057] Embodiment 2:

[0058] The present embodiment provides an apparatus for implementing the above-mentioned embedding method, comprising a rectangular and horizontally arranged embedding box 6, the bottom of the embedding box 6 being located in the groove of the oscillator 3, the embedding box 6 and the oscillator 3 being located inside the freezing chamber of the freezing device 5, the top of the embedding box 6 being detachably connected to the insulation plate 4, the insulation plate 4 having an air inlet 41 and an exhaust port 42 connected to the interior of the embedding box 6 at both ends, detachably connected positioning plates being provided at both ends in the length direction of the embedding box 6, the two positioning plates being provided with positioning holes corresponding to the positions, and detachably connected positioning lines being provided in the positioning holes of the two positioning plates.

[0059] The above device is used to implement the embedding method of the first embodiment, the embedding box and the oscillator are placed inside the freezing device, the embedding solution is added and frozen gradually, and the freezing speed is reduced; each time the embedding solution is added, the air is discharged through the oscillator to reduce the influence of bubbles; the positioning plates placed at both ends of the embedding box are used to locate the width, height and cross-section center of the specimen.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A specimen embedding method, characterized in that: The following steps are involved: The embedding solution is added to the embedding box gradually, and the embedding solution is shaken after each addition. Low-speed air is blown into the top of the embedding box to freeze the added embedding solution; wherein the bottom of the embedding box is located in the groove of the oscillator, and the embedding box and the oscillator are located inside the freezing chamber of the freezing device; the low-speed air has a flow rate of 3 to 5 m / s and an air flow temperature not exceeding 4°C; After the embedding solution is frozen for the first time, the specimen is placed in the embedding box. A positioning plate is placed at each end of the embedding box, and the two positioning plates are located at the top and bottom of the specimen respectively. The embedding solution added for the first time forms a base at the bottom of the embedding box after freezing, ensuring that the bottom of the specimen forms a low-temperature environment first, and the bottom and surrounding of the embedding box are cooled down first with the low-temperature and low-speed air blown in from the top of the embedding box. Positioning holes corresponding to the positions are provided on the positioning plates, and positioning lines are connected between the positioning holes at corresponding positions of the two positioning plates; After the embedding solution is added for the last time, a heat preservation plate is covered on the upper surface of the embedding solution, and low-speed air is blown in through the air inlet and exhaust ports provided at both ends of the heat preservation plate to realize specimen freezing molding; The specimen embedding device comprises a rectangular and horizontally arranged embedding box, the bottom of which is located in the groove of the oscillator, the embedding box and the oscillator are located inside the freezing chamber of the freezing device, the top of the embedding box is detachably connected to the insulation plate, and at least two groups of detachably connected positioning plates are provided at both ends in the length direction of the embedding box; positioning holes corresponding to the positions are provided on the positioning plates; positioning lines are connected between the positioning holes at the corresponding positions of the two positioning plates; the distance between two adjacent positioning holes on a group of positioning plates is the width or height of the specimen section; the positioning line is detachably connected to the positioning plate; and air inlets and exhaust ports communicating with the interior of the embedding box are provided at both ends of the insulation plate.

2. A specimen embedding method according to claim 1, characterized in that: The step of adding embedding solution into the embedding box one by one comprises: When adding embedding solution for the first time, add it to a height of 6~10cm; Starting from the second time, the height of each addition of embedding solution should not exceed 3 cm; When adding embedding solution for the last time, the embedding solution height should exceed the upper surface of the specimen by at least 1 cm.

3. A specimen embedding method according to claim 1, characterized in that: The embedding solution added for the first time is frozen to form a base, and the specimen is placed on the base. The remaining embedding solution freezes the specimen, the base, the positioning plate and the embedding box into a whole.

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