Sequence grinding three-dimensional imaging method and device for insect structure

Abstract

The invention relates to a sequence grinding three-dimensional imaging method and device for an insect structure. The method comprises the following steps: S1, dehydrating a sample; s2, performing embedding medium penetration treatment on the sample; s3, performing embedding and curing treatment to obtain a firm insect sample block; s4, fixing the insect sample block, performing sequence grinding, and photographing the cross section at the same time to obtain a sequence cross section image; and S5, importing the sequence section image into three-dimensional imaging software, and reconstructing to generate a three-dimensional image of the sample. According to the method, the sequence grinding method is innovatively applied to three-dimensional imaging of insect structures, high-resolution section images can be obtained by using a micron-sized grinding device and a high-resolution digital camera, boundaries among complex structures can be clearly defined, so that the obtained data is more objective, and according to the high-resolution sequence section images, the three-dimensional imaging of the insect structures can be realized. A more accurate three-dimensional image can be reconstructed and generated by utilizing mainstream three-dimensional imaging software in the prior art.

Description

Technical Field

[0001] The present application relates to the technical field of three-dimensional construction of insect structures, and in particular to a method and device for serial grinding three-dimensional imaging of insect structures. Background Art

[0002] When studying arthropods, three-dimensional digital modeling of biological samples is a commonly used technical means. There are many modeling technologies in the existing technology, such as confocal laser scanning microscope (CLSM), micro-computed tomography (Micro-CT), laser ablation tomography (LAT scanning) and tissue sectioning technology.

[0003] The principle of CLSM is to use a laser beam to focus on a point on the sample and scan the area around this point. Only the light at the focus is collected and converted into an image, thereby improving the clarity and resolution of the image. It is particularly suitable for observing the fine structure of living cells or fixed cells, molecular and ion dynamics, etc., but the imaging effect on thicker specimens is limited because of the large fluorescence interference from outside the focus, photobleaching of the labeled dye, and high-intensity lasers that cause the dye to fade rapidly during continuous scanning, affecting the observation effect; for thick specimens, it is difficult to clearly define the boundaries of fine structures.

[0004] The principle of Micro-CT is to irradiate samples from different angles with X-rays and measure the intensity changes after the X-rays penetrate the sample. The computer uses these data to reconstruct a three-dimensional image of the sample. It has the advantage of high resolution and is suitable for tiny samples. However, the low attenuation of X-rays by soft tissue can easily lead to poor imaging effects, and contrast agents are needed to enhance the contrast.

[0005] LAT scanning technology combines the principles of laser ablation and tomography. It obtains three-dimensional information of the sample by laser ablating the sample surface and scanning layer by layer. However, thermal damage or chemical changes may occur during the laser ablation process, affecting the original structure of the sample. The ablation rate and accuracy may be affected by the laser parameters and sample properties.

[0006] The principle of tissue sectioning technology is to cut biological tissue samples into very thin slices for observation under a microscope. It usually includes steps such as fixation, dehydration, embedding, sectioning and staining. The sectioned tissue samples can clearly show their internal structure and cellular components. It is mainly used to prepare sample slices suitable for observation. However, the operation steps are complicated and time-consuming, and skilled technicians are required. When handling harder samples, the slices are prone to curling and breaking, resulting in structural deformation and artifacts.

[0007] Insects are known for their excellent movement ability, which is due to their exoskeleton structure. The hard outer skin provides support, while the inner skin gives elasticity. In the past, many three-dimensional structural studies, biomechanical studies, and bionics studies have explored the inner and outer skins of arthropods. Because the inner and outer skins fit closely and have similar materials, the above-mentioned mainstream scanning methods are difficult to clearly define the boundary between the inner and outer skins. They often rely on artificial distinctions. Such data lack objectivity. When performing three-dimensional digital modeling based on these cross-sectional images, it is also difficult to obtain an accurate three-dimensional model. Summary of the invention

[0008] In order to solve the technical problem that the mainstream three-dimensional digital modeling method in the prior art is difficult to obtain a three-dimensional model of the fine structure of an insect, the present invention provides a serial grinding three-dimensional imaging method and device for an insect structure.

[0009] On the one hand, the technical solution provided by the present application is as follows: a method for serial grinding three-dimensional imaging of insect structures, comprising the following steps: S1, dehydration of samples; S2, the samples are treated with embedding agent infiltration; S3, embedding and solidification treatment to obtain a solid insect sample block; S4, fix the insect sample block and perform serial grinding, illuminate the cross section with ultraviolet light or visible light, and then take pictures to obtain serial cross-section images; S5, importing the serial cross-sectional images into three-dimensional imaging software to reconstruct and generate a three-dimensional image of the sample.

[0010] By adopting the above technical solution, the serial grinding method is a three-dimensional reconstruction technology used in paleontology. The fossils are polished layer by layer through delicate techniques, and only a very thin layer is polished off each time. Then the polished surface is photographed to obtain a high-resolution cross-sectional image. This method is very helpful in discovering details, but it is devastating to the fossils and is not suitable for rare fossil samples. The present application innovatively applies the serial grinding method to the three-dimensional imaging of insect structures. The premise of using the serial grinding method is to make the insect into an insect sample block as solid as a fossil. The insect sample of the present application can obtain a solid insect sample block after dehydration, embedding agent penetration, embedding and curing treatment, thereby making the serial grinding method feasible. High-resolution cross-sectional images can be obtained using a micron-level grinding device and a high-resolution digital camera. The axial and radial resolutions can reach the micron level, and the boundaries between complex structures can be clearly defined. That is, this accuracy is sufficient to accurately distinguish the inner cuticle and the outer cuticle in the exoskeleton of insect arthropods. Especially under ultraviolet illumination, the difference between the inner cuticle and the outer cuticle is very obvious and no longer relies on artificial distinction. The data obtained in this way is more objective. Based on these high-resolution serial cross-sectional images, using the mainstream three-dimensional imaging software in the prior art, more accurate three-dimensional images can be reconstructed and generated, which is particularly suitable for rapid or large-scale three-dimensional reconstruction of insect structures. The present application can also capture true color high-resolution cross-sectional images, which provide more structural information than pseudo-color or monochrome images. The serial grinding method does not have the risk of deformation, fracture, and artifacts caused by slice folding. It has high grinding efficiency and imaging efficiency. It can complete the grinding and imaging of hundreds of sections per hour with low cost. It is particularly beneficial for the study of insect functional morphology. It also has important application value for biological three-dimensional structure finite element analysis (FEA analysis), 3D geometric morphology research, and elastic distribution analysis within biological skeletal structure. At the same time, this method can also be used for imaging other biological samples.

[0011] Preferably, in step S1, the sample is preserved with 75% ethanol, and then dehydrated in 85%, 95% and 100% ethanol in sequence, with each concentration treatment time being 30 to 60 minutes, and finally further dehydrated in 100% ethanol for 4 to 24 hours, with the dehydration temperature being 50°C±10°C.

[0012] Preferably, in step S2, the dehydrated sample is sequentially placed in embedding agents of different concentrations for gradient infiltration, first in a solution of 20% embedding agent and 80% ethanol for 1 to 3 hours; then in a solution of 50% embedding agent and 50% ethanol for 1 to 3 hours; then in a solution of 80% embedding agent and 20% ethanol for 1 to 3 hours; then in a solution of 100% embedding agent for 1 to 3 hours; finally in a 100% embedding agent for 6 to 48 hours, the infiltration temperature is 50°C±5°C, and the embedding agent is a UV-cured acrylic resin.

[0013] Preferably, in step S3, the embedding agent comprises 50 parts of polyurethane acrylate, 47-48 parts of 4-acryloylmorpholine, 1 part of (2,4,6-trimethylbenzoyl)diphenylphosphine oxide and 1-2 parts of white nanoscale color paste, all of which are parts by mass.

[0014] Preferably, in step S3, the embedding agent is filled in the silicone mold, and then the infiltrated sample is immersed in the embedding agent; then a vacuum treatment is performed to eliminate bubbles inside the sample, the vacuum degree is required to be -50 to -75 kPa, and the time is 30 minutes ± 10 minutes; then a two-step curing process is adopted to avoid overheating, sample deformation and bubble formation. The first step is to first use low-intensity 365nm ultraviolet light for preliminary curing, the curing time is 2 to 6 hours, and the ultraviolet light intensity ranges from 100 to 1000μW / cm². The second time, high-intensity ultraviolet light is used for secondary curing, the time is 2 to 48 hours, and the ultraviolet light intensity ranges from 1000μW / cm² and above.

[0015] Preferably, during the curing process of step S3, an ice bag or a cooling plate is placed under the silicone mold to reduce the sample temperature, thereby ensuring the sample quality.

[0016] On the other hand, the present application also provides another technical solution: a device for implementing the above-mentioned serial grinding three-dimensional imaging method of the insect structure, comprising a mounting platform, a sample driving module, a serial grinding device and an imaging module, wherein the sample driving module, the serial grinding device and the imaging module are all arranged on the mounting platform; the sample driving module comprises an X-axis feeding module, a Y-axis moving module and a clamp, wherein the clamp is arranged on the X-axis feeding module for clamping the insect sample block, the X-axis feeding module is arranged on the Y-axis moving module for gradually advancing the insect sample block toward the serial grinding device, the serial grinding device and the imaging module are arranged horizontally in parallel, the Y-axis moving module is used to repeatedly move the insect sample block to the serial grinding device and the imaging module, the serial grinding device is used to perform serial grinding on the insect sample block, and the imaging module is used to take pictures of the cross-section of the ground insect sample block to obtain a serial cross-section image.

[0017] By adopting the above technical solution, the present application uses the X-direction feeding module to gradually advance the insect sample block to make it close to the grinding disk, so that the insect sample block can be gradually ground, and each time a layer is ground, the cross-section is imaged immediately until all cross-sections of the insect sample in the insect sample block are imaged. The device described in the present application is low in cost and cost-effective, forming another key advantage, which is convenient for widespread popularization and extensive testing.

[0018] Preferably, the sequential grinding device includes a driving motor, a grinding disc, a power controller and a grinding liquid dripping mechanism, the grinding disc is arranged on the main shaft of the driving motor, the power controller is used to control the movement of the driving motor, and the grinding liquid dripping mechanism is used to drip the grinding liquid onto the grinding disc and / or the insect sample block.

[0019] Preferably, the Y-direction moving module further includes a front stopper and a rear stopper for limiting the precise position of the forward and backward movement of the X-direction feeding module.

[0020] Preferably, the imaging module includes an optical objective lens, a digital camera, an ultraviolet and visible light generator and an adjustable base, wherein the optical objective lens, the digital camera and the ultraviolet and visible light generator are all arranged on the adjustable base, and the adjustable base is arranged on a mounting platform, the optical objective lens is used to magnify the cross-section of the insect sample block, and the digital camera is located at the rear end of the optical objective lens, and is used to take pictures of the magnified cross-section of the insect sample block.

[0021] In summary, the present application includes at least one of the following beneficial technical effects: 1. This application innovatively applies the serial grinding method to the three-dimensional imaging of insect structures. The serial grinding method can obtain high-resolution cross-sectional images and clearly define the boundaries between complex structures. That is, this accuracy is sufficient to accurately distinguish the inner and outer cuticles in the exoskeletons of insects and arthropods. Especially under ultraviolet illumination, the difference between the inner and outer cuticles is very obvious and no longer depends on artificial distinction. The data obtained in this way is more objective. Based on these high-resolution serial cross-sectional images, more accurate three-dimensional images can be reconstructed; 2. The present application can also capture true color high-resolution cross-sectional images, which provide more structural information than pseudo color or monochrome images.

[0022] 3. The serial grinding method does not have the risk of deformation and fracture and artifacts caused by slice folding. It has high grinding efficiency and imaging efficiency. It can complete the grinding and imaging of hundreds of sections per hour. It is low-cost and is particularly beneficial for the study of insect functional morphology. 4. The device described in this application is low-cost and cost-effective, which is another key advantage and facilitates widespread popularization and testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a stereoscopic diagram of the three-dimensional imaging device described in an embodiment of the present application (grinding position); Figure 2 is a front view (grinding position) of the three-dimensional imaging device according to an embodiment of the present application; Figure 3 is a stereogram of the three-dimensional imaging device described in the embodiment of the present application (photographing position); Figure 4is a front view (photographing position) of the three-dimensional imaging device according to an embodiment of the present application; Figure 5 is a three-dimensional diagram of a sample driving module according to an embodiment of the present application; Figure 6 is a three-dimensional diagram of the sequential grinding device described in the embodiment of the present application; Figure 7 It is a stereoscopic diagram of the imaging module described in the embodiment of the present application.

[0024] Explanation of the reference numerals: 1. mounting platform; 2. sample driving module; 21. X-axis feeding module; 211. X-axis feeding module controller; 22. Y-axis moving module; 221. front stopper; 222. rear stopper; 23. fixture; 24. XYR-axis adjusting device; 3. sequential grinding device; 31. driving motor; 32. grinding disc; 33. grinding liquid dripping mechanism; 34. Z-axis adjusting device; 4. imaging module; 41. optical objective lens; 42. digital camera; 43. ultraviolet and visible light generator; 44. adjustable base; 441. X-axis moving module; 442. Z-axis moving module; 45. camera controller; 46. tube mirror; 5. monitor; 100. insect sample block. DETAILED DESCRIPTION

[0025] The following is combined with Figures 1 to 7 This application is described in further detail.

[0026] The present application embodiment discloses a method for sequential grinding three-dimensional imaging of insect structures, comprising the following steps: S1, dehydration of samples; S2, the samples are treated with embedding agent infiltration; S3, performing embedding and solidification treatment to obtain a solid insect sample block, wherein a cubic sample block is preferably used for convenience of clamping and fixing; S4, fix the insect sample block and perform serial grinding, illuminate the cross section with ultraviolet light or visible light, and then take pictures to obtain serial cross-section images; S5, importing the serial cross-sectional images into three-dimensional imaging software to reconstruct and generate a three-dimensional image of the sample.

[0027] In this embodiment, in step S1, the sample is preserved with 75% ethanol, and then dehydrated with 85%, 95% and 100% ethanol in sequence, with each concentration treatment time being 30 minutes to 60 minutes, and finally further dehydrated in 100% ethanol for 4 to 24 hours, with the dehydration temperature being 50°C ± 10°C. This application adopts gradient ethanol dehydration, and uses ethanol solutions of different concentrations to dehydrate the sample step by step. By gradually transitioning from low concentration to high concentration of ethanol, the shrinkage, deformation and even rupture of the sample tissue caused by rapid dehydration can be effectively avoided, thereby ensuring the morphological integrity of the sample.

[0028] In this embodiment, in step S2, the dehydrated samples are sequentially placed in embedding agents of different concentrations (ultraviolet-curable acrylic resin can be used) for gradient infiltration, first in a solution of 20% embedding agent and 80% ethanol for 1 to 3 hours; then in a solution of 50% embedding agent and 50% ethanol for 1 to 3 hours; then in a solution of 80% embedding agent and 20% ethanol for 1 to 3 hours; then in a 100% embedding agent for 1 to 3 hours; finally, in a 100% embedding agent for 6 to 48 hours, and the infiltration temperature is 50°C ± 5°C. For insect samples with larger bodies and higher degrees of hardening, magnetic stirring can also be used to accelerate the infiltration. The reason for using gradient infiltration in this application is mainly to avoid deformation or even rupture of sample tissue due to rapid expansion, and to ensure the morphological integrity of the sample. ‌ In this embodiment, in step S3, the embedding agent includes 50 parts of polyurethane acrylate (as an oligomer), 47-48 parts of 4-acryloylmorpholine (as a monomer), 1 part of (2,4,6-trimethylbenzoyl) diphenylphosphine oxide (as a photoinitiator), and 1-2 parts of white nanoscale color paste, all of which are parts by mass. For small insects with a body width of less than 5 mm, 0.5-1 part of fluorescent color paste should also be added to enhance the background contrast of the serial cross-sectional image.

[0029] After curing, polyurethane acrylate has the high wear resistance, adhesion, flexibility, high peel strength and excellent low temperature resistance of polyurethane and the excellent optical properties and weather resistance of polyacrylate. It is a radiation curing material with excellent comprehensive performance. 4-Acryloylmorpholine, as a functional monomer, participates in copolymerization or coupling reaction with other compounds to form cross-linked polymers, improve the mechanical properties and chemical resistance of the material, and can also be used as a photosensitive monomer to participate in photopolymerization. Under the irradiation of ultraviolet light, it can undergo polymerization reaction. The high mass ratio of 4-acryloylmorpholine is the key to keep the material relatively strong after curing. Photoinitiator is one of the important components, which plays a decisive role in the curing rate. After being irradiated by ultraviolet light, the photoinitiator absorbs the energy of light, initiates chain polymerization, and cross-links and cures oligomers and monomers, which has the advantages of fast, environmental protection and energy saving.

[0030] In this embodiment, in step S3, the embedding agent is filled in the silicone mold, and then the infiltrated sample is immersed in the embedding agent; then a vacuum treatment is performed to eliminate bubbles inside the sample, and the vacuum degree is required to be -50 to -75 kPa, and the time is 30 minutes ± 10 minutes; then a two-step curing process is adopted to avoid overheating, sample deformation and bubble formation. The first step is to first use low-intensity 365nm ultraviolet light for preliminary curing, and the curing time is 2 to 6 hours, and the ultraviolet light intensity ranges from 100 to 1000μW / cm². The second time, high-intensity ultraviolet light is used for secondary curing, and the time is 2 to 48 hours, and the ultraviolet light intensity ranges from 1000μW / cm² and above.

[0031] In this embodiment, during the curing process of step S3, an ice bag or a cooling plate is placed under the silicone mold to reduce the sample temperature, thereby ensuring the sample quality.

[0032] In this embodiment, in step S5, since the resolution of the cross-sectional image obtained in the present application is very high, after being imported into the mainstream three-dimensional imaging software, the serial cross-sectional images can be automatically aligned and volume rendered, or the volume rendering can be generated using the automatic threshold function to form a three-dimensional image. Further manual segmentation of different structures can also be performed to construct a three-dimensional model and complete the three-dimensional reconstruction of different insect body structures.

[0033] Serial grinding is a three-dimensional reconstruction technology used in paleontology. The fossils are polished layer by layer with delicate techniques, and only a very thin layer is removed each time. The polished surface is then photographed to obtain a high-resolution cross-sectional image. This method is very helpful in discovering details, but it is devastating to the fossils and is not suitable for rare fossil samples. The present application innovatively applies the serial grinding method to the three-dimensional imaging of insect structures. The premise of using the serial grinding method is to make the insect into an insect sample block as solid as a fossil. The insect sample of the present application can obtain a solid insect sample block after dehydration, embedding agent penetration, embedding and curing treatment, thereby making the serial grinding method feasible. High-resolution cross-sectional images can be obtained using a micron-level grinding device and a high-resolution digital camera. The axial and radial resolutions can reach the micron level, and the boundaries between complex structures can be clearly defined. That is, this accuracy is sufficient to accurately distinguish the inner and outer cuticles in the exoskeletons of insects and arthropods. Especially under ultraviolet illumination, the difference between the inner and outer cuticles is very obvious and no longer relies on artificial distinction. The data obtained in this way is more objective. Based on these high-resolution serial cross-sectional images, using the mainstream three-dimensional imaging software in the prior art, more accurate three-dimensional images can be reconstructed and generated, which is particularly suitable for rapid or large-scale three-dimensional reconstruction of insect structures. The present application can also capture true color high-resolution cross-sectional images, which provide more structural information than pseudo-color or monochrome images. The serial grinding method does not have the risk of deformation, fracture, and artifacts caused by slice folding. It has high grinding efficiency and imaging efficiency. It can complete the grinding and imaging of hundreds of sections per hour with low cost. It is particularly beneficial for the study of insect functional morphology. It also has important application value for biological three-dimensional structure finite element analysis (FEA analysis), 3D geometric morphology research, and elastic distribution analysis within biological skeletal structure. At the same time, this method can also be used for imaging other biological samples.

[0034] Reference Figures 1 to 4 The present application also discloses a device for realizing the above-mentioned method of sequential grinding three-dimensional imaging of insect structures, comprising a mounting platform 1, a sample driving module 2, a sequential grinding device 3 and an imaging module 4, wherein the sample driving module 2, the sequential grinding device 3 and the imaging module 4 are all arranged on the mounting platform 1. The present application also provides a monitor 5 for displaying the parameters and status of the acquired cross-sectional images.

[0035] Reference Figure 1 , Figure 2 and Figure 5As shown, the sample driving module 2 includes an X-direction feeding module 21, a Y-direction moving module 22 and a clamp 23. The clamp 23 is arranged on the X-direction feeding module 21 to clamp the insect sample block 100. The X-direction feeding module 21 is arranged on the Y-direction moving module 22 to gradually advance the insect sample block 100 to the sequence grinding device 3. The sequence grinding device 3 and the imaging module 4 are arranged horizontally in parallel. The Y-direction moving module 22 is used to repeatedly move the insect sample block 100 to the sequence grinding device 3 and the imaging module 4. The sequence grinding device 3 is used to perform sequence grinding on the insect sample block 100, and the imaging module 4 is used to take pictures of the cross section of the ground insect sample block 100 to obtain a sequence cross-sectional image. The present application controls the movement of the X-direction feeding module 21 through an X-direction feeding module controller 211; an XYR direction adjustment device 24 is also provided below the clamp 23 to adjust the position of the insect sample block to ensure that the sample cross section can match the grinding disc 32.

[0036] Reference Figure 5 The clamp 23 is used to clamp the insect sample block 100 and adjust the angle of the insect sample block 100. The X-direction feeding module 21 preferably adopts a micron-level feeding module. The Y-direction moving module 22 can move the insect sample block 100 to the sequence grinding device 3 for grinding, and can also move the insect sample block 100 to the imaging module 4 for photographing. The SBR linear guide is preferably used to ensure the accuracy of linear motion. The Y-direction moving module 22 also includes a front stopper 221 and a rear stopper 222, which are used to limit the precise position of the forward and backward movement of the X-direction feeding module 21 to ensure the accuracy of each grinding position and photographing position.

[0037] Reference Figure 6 The sequential grinding device 3 includes a driving motor 31, a grinding disc 32, a power controller (not shown), a grinding liquid dripping mechanism 33 and a Z-direction adjusting device 34. The grinding disc 32 is arranged on the main shaft of the driving motor 31. The power controller is used to control the movement of the driving motor 31. The grinding liquid dripping mechanism 33 is used to drip the grinding liquid on the grinding disc 32 and / or the insect sample block 100. The Z-direction adjusting device 34 is used to adjust the position of the grinding disc 32 to ensure that the sample section can match the grinding disc 32. The grinding disc 32 adopts a replaceable grinding disc 32 with a variable diameter. The surfaces of different grinding discs 32 are inlaid with diamond particles of 1000, 2000, and 3000 meshes. Among them, the grinding disc 32 of 1000 mesh is suitable for larger insect samples, while the grinding discs 32 of 2000 and 3000 meshes are more suitable for small samples. The grinding liquid required in the grinding process is stored in a test tube in the grinding liquid dripping mechanism 33, and drips at a speed of 1-2 drops per second through adjustment.

[0038] Reference Figure 7The imaging module 4 includes an optical objective lens 41, a digital camera 42, an ultraviolet and visible light generator 43 and an adjustable base 44. The optical objective lens 41, the digital camera 42 and the ultraviolet and visible light generator 43 are all arranged on the adjustable base 44. The adjustable base 44 is arranged on the mounting platform 1 and is used to adjust the position of the digital camera 42 to ensure that the imaging surface is consistent with the sample section; the optical objective lens 41 is used to magnify the section of the insect sample block 100, and the digital camera 42 is located at the rear end of the optical objective lens 41 and is used to take pictures of the magnified section of the insect sample block 100. Under ultraviolet light illumination, the difference between the inner epidermis and the outer epidermis is very obvious. According to the size of the insect sample, the optical objective lens 41 can be selected from 1-5 times, 5 times, 10 times and 20 times optical objective lenses 41. The shooting position of the digital camera 42 can be adjusted through the adjustable base 44 to ensure the imaging quality. The adjustable base 44 should include an X-direction moving module 441 and a Z-direction moving module 442 to achieve the X-direction movement adjustment function and the lifting adjustment function. In this embodiment, a tube lens 46 is also provided between the optical objective lens 41 and the digital camera 42 to cooperate with the optical objective lens 41 to form an image, and the digital camera 42 is controlled by the camera controller 45 to take pictures.

[0039] The present application utilizes the X-direction feeding module 21 to gradually advance the insect sample block 100 to make it close to the grinding disc 32, so that the insect sample block 100 can be gradually ground, and each time a layer is ground, the cross-section is imaged immediately until all cross-sections of the insect sample in the insect sample block 100 are imaged. The device described in the present application is low in cost and cost-effective, which forms another key advantage and facilitates widespread popularization and testing.

[0040] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for serial grinding three-dimensional imaging of insect structures, characterized in that: The following steps are involved: S1, dehydration of samples; S2, the samples are treated with embedding agent infiltration; S3, embedding and solidification treatment to obtain a solid insect sample block; S4, fix the insect sample block and perform serial grinding, illuminate the cross section with ultraviolet light or visible light, and then take pictures to obtain serial cross-section images; S5, importing the serial cross-sectional images into three-dimensional imaging software to reconstruct and generate a three-dimensional image of the sample.

2. The method for sequential grinding three-dimensional imaging of insect structures according to claim 1, characterized in that: In step S1, the sample is preserved with 75% ethanol, and then dehydrated in 85%, 95% and 100% ethanol in sequence, with each concentration treatment time being 30 minutes to 60 minutes, and finally further dehydrated in 100% ethanol for 4 to 24 hours, with the dehydration temperature being 50°C±10°C.

3. The method for sequential grinding three-dimensional imaging of insect structures according to claim 1, characterized in that: In step S2, the dehydrated sample is sequentially placed in embedding agents of different concentrations for gradient infiltration, first in a solution of 20% embedding agent and 80% ethanol for 1 to 3 hours; then in a solution of 50% embedding agent and 50% ethanol for 1 to 3 hours; then in a solution of 80% embedding agent and 20% ethanol for 1 to 3 hours; then in a solution of 100% embedding agent for 1 to 3 hours; finally in a 100% embedding agent for 6 to 48 hours, the infiltration temperature is 50°C±5°C, and the embedding agent is a UV-cured acrylic resin.

4. The method for sequential grinding three-dimensional imaging of insect structures according to claim 3, characterized in that: In step S3, the embedding agent includes 50 parts of polyurethane acrylate, 47-48 parts of 4-acryloylmorpholine, 1 part of (2,4,6-trimethylbenzoyl)diphenylphosphine oxide, and 1-2 parts of white nanoscale color paste, all of which are parts by mass.

5. The method for sequential grinding three-dimensional imaging of insect structures according to claim 1, characterized in that: In step S3, the embedding agent is filled in the silicone mold, and then the infiltrated sample is immersed in the embedding agent; then a vacuum treatment is performed to eliminate bubbles inside the sample, and the vacuum degree is required to be -50 to -75 kPa, and the time is 30 minutes ± 10 minutes; then a two-step curing process is adopted to avoid overheating, sample deformation and bubble formation. The first step is to first use low-intensity 365nm ultraviolet light for preliminary curing, and the curing time is 2 to 6 hours, and the ultraviolet light intensity ranges from 100 to 1000μW / cm². The second time, high-intensity ultraviolet light is used for secondary curing, and the time is 2 to 48 hours, and the ultraviolet light intensity ranges from 1000μW / cm² and above.

6. The method for sequential grinding three-dimensional imaging of insect structures according to claim 5, characterized in that: During the curing process of step S3, an ice bag or a cooling plate is placed under the silicone mold to reduce the sample temperature, thereby ensuring the sample quality.

7. A device for implementing the method for serial grinding three-dimensional imaging of insect structures as claimed in any one of claims 1 to 6, characterized in that: The invention comprises an installation platform, a sample driving module, a sequential grinding device and an imaging module, wherein the sample driving module, the sequential grinding device and the imaging module are all arranged on the installation platform; the sample driving module comprises an X-direction feeding module, a Y-direction moving module and a clamp, wherein the clamp is arranged on the X-direction feeding module and is used to clamp the insect sample block; the X-direction feeding module is arranged on the Y-direction moving module and is used to gradually push the insect sample block toward the sequential grinding device; the sequential grinding device and the imaging module are arranged horizontally in parallel; the Y-direction moving module is used to repeatedly move the insect sample block to the sequential grinding device and the imaging module; the sequential grinding device is used to perform sequential grinding on the insect sample block; and the imaging module is used to take pictures of the cross-section of the ground insect sample block to obtain a sequential cross-section image.

8. The device according to claim 7, characterized in that The sequential grinding device includes a driving motor, a grinding disc, a power controller and a grinding liquid dripping mechanism, wherein the grinding disc is arranged on the main shaft of the driving motor, the power controller is used to control the movement of the driving motor, and the grinding liquid dripping mechanism is used to drip the grinding liquid onto the grinding disc and / or the insect sample block.

9. The device according to claim 7, characterized in that The Y-direction moving module also includes a front stopper and a rear stopper, which are used to limit the precise position of the forward and backward movement of the X-direction feeding module.

10. The device according to claim 7, characterized in that The imaging module comprises an optical objective lens, a digital camera, an ultraviolet and visible light generator and an adjustable base, wherein the optical objective lens, the digital camera and the ultraviolet and visible light generator are all arranged on the adjustable base, and the adjustable base is arranged on a mounting platform. The optical objective lens is used to magnify the cross section of the insect sample block, and the digital camera is located at the rear end of the optical objective lens and is used to take pictures of the magnified cross section of the insect sample block.

Images