A three-dimensional digital dissection method and system for vertebrate embryos

Abstract

The present invention proposes a three-dimensional digital dissection method and system for vertebrate embryos, which belongs to the field of CT imaging technology. The method comprises: fixing an embryo specimen; pre-processing the fixed embryo specimen; scanning the pre-processed embryo specimen using a micro-CT scanning system to obtain an embryo three-dimensional digital model; and performing virtual digital dissection on the region of interest based on the embryo three-dimensional digital model. The present invention can efficiently obtain high-precision three-dimensional structures of vertebrate embryos, and on this basis, realize non-destructive and accurate digital dissection.

Description

Technical Field

[0001] The invention belongs to the technical field of CT imaging, and in particular relates to a three-dimensional digital dissection method and system for vertebrate embryos. Background Art

[0002] The microscopic structure of organisms reflects their development process. Researchers focus on studying biological phenotypic characteristics and related phylogeny to explore the origin and evolution of life. CT has been widely used in non-destructive biological research. CT imaging uses X-rays to penetrate samples of different densities. Materials of different densities have different absorption capacities for X-rays, and reconstructs the three-dimensional digital structure of organisms. The segmentation, reconstruction and extraction of three-dimensional digital models are studied to achieve non-destructive digital anatomy.

[0003] In a series of scientific studies on organisms using CT to construct three-dimensional models, the three-dimensional structure of embryos during the development of different categories has received increasing attention. Embryo research provides important evidence for animal systematic development and evolution, and is also very important for animal taxonomy research. At present, morphological studies of embryos are usually obtained by comparative dissection. Dissection is a destructive study of specimens cut open with a specific knife. For the microscopic structural information of the embryo, wax embedding, staining, sectioning, microscopy and other methods are usually used to observe and study the similarities and differences in morphological changes during the individual development of embryos of various categories of animals. From a single slice to a series of slices, they are combined into a three-dimensional model to explore their phylogenetic relationships. At present, the following problems exist:

[0004] Vertebrate embryos are very small, and manual dissection by operators is very demanding. Observation of specific microscopic morphological structures usually requires a microscope, which is closely related to the quality of the slices. In the process of embedding, staining and slicing, each link requires precision, and only when specific details are cut can they be observed. If they cannot be observed, a large amount of materials and more time are needed to repeat the experiment.

[0005] Based on this, it is very necessary to develop a three-dimensional digital model that can efficiently and clearly display the internal structure of the embryo and then perform accurate digital dissection. Summary of the invention

[0006] The purpose of the present invention is to provide a three-dimensional digital dissection method and system for vertebrate embryos, which can efficiently obtain high-precision three-dimensional structures of vertebrate embryos and achieve non-destructive and accurate digital dissection on this basis.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is:

[0008] A three-dimensional digital dissection method for vertebrate embryos, the method comprising:

[0009] Fixed embryo specimens;

[0010] Pre-treatment of fixed embryo specimens;

[0011] The pre-processed embryo specimens were scanned using a micro-CT scanning system to obtain a three-dimensional digital model of the embryo;

[0012] Based on the three-dimensional digital model of the embryo, a virtual digital dissection is performed on the region of interest.

[0013] Furthermore, the fixed embryo specimen comprises:

[0014] Obtain fresh embryo specimens;

[0015] Rinse fresh embryo specimens with saline;

[0016] The washed embryo specimen is immersed in 8-12% formalin solution, sealed and stored away from light for 36 to 72 hours to obtain a fixed embryo specimen.

[0017] Furthermore, the pre-treatment of the fixed embryo specimen includes:

[0018] Move the fixed embryo specimen into anhydrous alcohol and soak for 5-15 minutes for initial dehydration;

[0019] Transfer the dehydrated embryo specimens to 0.5% Lugol's reagent and soak for 12-36 hours for staining;

[0020] After staining, the embryo specimens were taken out and placed in gradient alcohol and immersed for 8-12 minutes for secondary dehydration.

[0021] The embryo specimen after secondary dehydration is transferred to the sample chamber of the CO2 critical point dryer to achieve drying;

[0022] After the dried embryo specimen is transferred to a CT fixation container, the opening of the CT fixation container is sealed with a sealing film.

[0023] Furthermore, the gradient alcohol includes: 30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol.

[0024] Furthermore, the soaking time in the primary dehydration treatment is 5 minutes; the soaking time in the dyeing treatment is 24 hours; and the soaking time in the secondary dehydration treatment is 10 minutes.

[0025] Furthermore, the method of scanning the pre-treated embryo specimen using a micro-CT scanning system to obtain a three-dimensional digital model of the embryo includes:

[0026] Scanning the pre-processed embryo specimens using a micro-CT scanning system to obtain scanning data;

[0027] Reconstruct the scanned data to obtain three-dimensional data of the embryo specimen;

[0028] The three-dimensional data of the embryo specimen is segmented and extracted based on three-dimensional visualization software, and a digitally anatomical three-dimensional model of the embryo is obtained by rendering.

[0029] Furthermore, the parameter settings of the micro-CT scanning system include: voltage of 80-110 KV, current of 70-150 uA, and resolution of 0.8-20 μm.

[0030] A three-dimensional digital dissection system for vertebrate embryos, the system comprising:

[0031] A fixing module, used for fixing embryo specimens;

[0032] Pre-processing module, used for pre-processing fixed embryo specimens;

[0033] A CT scanning module is used to scan the pre-processed embryo specimen using a micro-CT scanning system to obtain a three-dimensional digital model of the embryo;

[0034] The dissection module is used to perform virtual digital dissection on the region of interest based on the three-dimensional digital model of the embryo.

[0035] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0036] 1. High-precision three-dimensional structures of various types of embryo samples can be obtained non-destructively. Non-destructive testing, without the need for dissection or slicing, can preserve the subtle structural information of the embryo sample in a state close to its natural state for subsequent research.

[0037] 2. The contrast of the internal structure of the different embryo specimens prepared was significantly improved during CT scanning, and the internal structure of the embryo was clearly visible.

[0038] 3. Dehydrate first and then stain with low concentration reagents to ensure uniform staining and avoid over-staining. Then use gradient dehydration critical point drying to thoroughly dehydrate and dry completely, so that the sample remains in the state after staining and the internal structure is stable and clear during CT scanning.

[0039] 4. The present invention not only maintains the original appearance of the embryo to the greatest extent, making the three-dimensional model reconstructed by CT scanning clear and accurate, but also can quickly obtain a large number of three-dimensional models of vertebrate embryos in a short period of time.

[0040] 5. Perform precise digital dissection on the 3D model of the embryo, extract different regions of interest, mark them with different colors, and clearly study their fine structures, morphological characteristics, and relationships, avoiding the extremely demanding manual dissection method, and non-destructively obtain a complete 3D digital model of the embryo, achieving non-destructive, multi-angle, all-round, and unlimited precise digital dissection.

[0041] 6. After accumulating a large amount of data, it can support the establishment of a three-dimensional vertebrate embryo morphology database, trace the evolutionary process of vertebrates, and provide important evidence for the systematic development and evolution of vertebrates. It has broad prospects in the field of microscopic vertebrates. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Flowchart of the method for three-dimensional digital dissection of vertebrate embryos.

[0043] Figure 2 Comparison of 3D reconstructions of alligator embryos.

[0044] Figure 3 Comparison of 3D reconstructions of mouse embryos. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0046] The three-dimensional digital dissection method of the vertebrate embryo of the present invention is as follows: Figure 1 As shown, the steps include steps 1 to 4.

[0047] Step 1: Fix the embryo specimen.

[0048] Take a fresh embryo specimen, rinse it with physiological saline, then put it into 8-12% (preferably 10%) formalin solution, let the specimen sink into the solution, seal and protect from light, and fix it for 36-72 hours (preferably 48 hours).

[0049] Step 2: Pre-process the fixed embryo specimens.

[0050] The fixed embryo specimens were immersed in anhydrous alcohol for 5-15 minutes for primary dehydration, and then immersed in 0.5% Lugol's reagent (5% Lugol's reagent and distilled water diluted in a ratio of 1:9) for 12-36 hours for staining. Then, gradient alcohol (30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, 100% ethanol) was used to immerse for 8-12 minutes for secondary dehydration, and then moved to the sample chamber of the critical point dryer, and the ethanol was replaced by liquid carbon dioxide, and the carbon dioxide was vaporized and exhausted, and the dried specimens were obtained. The dried specimens were transferred to a special fixed container for CT small samples and sealed with a sealing film for storage. Ensure that the specimens will not absorb water and deform during long-term scanning.

[0051] Step 3: Use a micro-CT scanning system to scan the pre-processed embryo specimen to obtain a three-dimensional digital model of the embryo.

[0052] The processed specimens were transferred to the stage of the micro-CT and scanned with a CT device (GE v|tome|x m300&180). The scanning voltage can be 80-110KV, the current is 70-150uA, and the resolution is 0.8-20μm. A series of tomograms were exported through the software provided by the CT scanner, and then the embryo structure was segmented, reconstructed and extracted using the Drishti software to render a three-dimensional digital model of the embryo.

[0053] Step 4: Based on the three-dimensional digital model of the embryo, a virtual digital dissection is performed on the region of interest.

[0054] The present invention performs precise digital dissection on a three-dimensional model of the embryo, extracts different regions of interest, marks them with different colors, and clearly studies their fine structures, morphological features, and mutual relationships.

[0055] Below, alligator embryo specimens and mouse embryo specimens are used as examples to illustrate the three-dimensional digital dissection method of vertebrate embryos provided by the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0056] Among them, the following embodiments involve supplies including:

[0057] Fixative formula: 10 ml of formalin, 0.4 g of sodium dihydrogen phosphate (NaH2PO4 2H2O), 0.65 g of disodium hydrogen phosphate (Na2HPO4), and 100 ml of distilled water. Mix by stirring to prepare a 10% neutral buffered formalin fixative.

[0058] Lugol's reagent: (5% Lugol's reagent and distilled water diluted in a ratio of 1:9), Lugol's reagent is non-toxic, easy to obtain, has good penetration effect, and has good staining effect on vertebrate soft tissue. It is easy to use, has high experimental sensitivity, and greatly saves experimental time.

[0059] Gradient dehydrated ethanol: 30%, 50%, 70%, 85%, 90%, 95%, and 100% ethanol aqueous solutions were prepared using water and 100% ethanol.

[0060] Carbon dioxide critical point drying treatment: A critical point dryer (Leica EM CPD300) was used for drying.

[0061] Micro-CT scanning system: GE v|tome|x m300&180 scanner is used.

[0062] 3D model processing software: Drishti, Vayu, VGStudio, Avizo, Mimics and other 3D visualization reconstruction software.

[0063] Example 1: Three-dimensional digital dissection method of alligator embryos.

[0064] 1. Specimen fixation.

[0065] Take a fresh alligator embryo specimen, rinse it with physiological saline, and then put it into 8-12% (preferably 10%) formalin solution, let the specimen sink into the solution, seal and protect from light, and fix it for 36-72 hours (preferably 48 hours).

[0066] 2. Specimen pretreatment.

[0067] Use forceps to remove the fixed embryo specimens and immerse them in anhydrous alcohol for 5-15 minutes for dehydration. Use forceps to transfer the dehydrated embryo specimens to 0.5% Lugol's reagent and immerse them for 24 hours for staining; after staining, remove the embryos and immerse them in gradient alcohol (30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, 100% ethanol) for 10 minutes for gradient dehydration. Use forceps to quickly transfer the dehydrated embryo samples to the sample chamber of the CO2 critical point dryer, and dry them by evaporating CO2. After drying, transfer them to a suitable CT fixation container and seal the opening with a sealing film.

[0068] 3. CT scan reconstruction.

[0069] The alligator embryo specimens before and after the treatment in step 2 were scanned with a micro-CT scanner (GE v|tome|x m300&180). The scanning voltage was 110KV, the current was 120uA, the continuous scanning method was adopted, the step length was 0.3°, the frame rate was 1, and 1200 360° images were scanned in 20 minutes. After the sample was scanned, it was reconstructed by the built-in software to obtain the three-dimensional data of the embryo specimen, and then segmented and extracted by the open source three-dimensional visualization software Drishti, and the digital anatomical embryo three-dimensional model 1c was rendered.

[0070] 4. Digital anatomy.

[0071] Based on the three-dimensional digital model of the embryo, the region of interest is dissected, rendered, and observed from multiple angles.

[0072] Figure 2 This is a comparison of the 3D reconstruction of the alligator embryo. Figure 2 The figure a in the middle shows a CT tomogram obtained by direct CT scanning without processing in step 2. The pixel size is 12.372 μm, and the internal bone morphology is unclear. Figure 2 The CT tomogram in b is processed in step 2. Its pixel size is 13.642 μm, and the internal bone organs are clearly shaped. Figure 2 Figure c is a 3D structure image rendered by Drishti software, which shows different structures segmented from the overall structure. By comparison, it can be seen that the micro-CT contrast of the alligator embryo after staining with Lugol's reagent and critical point drying with carbon dioxide has been significantly improved, and the morphology of the internal organs is clearly visible. The 3D software is used to quickly reconstruct a complete and accurate 3D model of the alligator embryo. Dissection is performed on the 3D model of the alligator embryo, and different regions of interest are extracted and marked with different colors to clearly study its fine structure, morphological characteristics, and relationships.

[0073] Example 2: Three-dimensional digital dissection method of mouse embryos.

[0074] 1. Specimen fixation.

[0075] Take a fresh mouse embryo specimen, rinse it with physiological saline, then put it into 8-12% formalin solution, let the specimen sink into the solution, seal and store in a dark place, and fix it for 36-72 hours (preferably 48 hours).

[0076] 2. Specimen processing.

[0077] Use forceps to remove the fixed embryo specimens and soak them in anhydrous alcohol for 5-15 minutes for dehydration. Use forceps to transfer the dehydrated embryo specimens to 0.5% Lugol's reagent and soak them for 24 hours for staining; after staining, remove the embryos and soak them in gradient alcohols of 30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol for 10 minutes respectively for gradient dehydration. Use forceps to quickly transfer the dehydrated embryo samples to the sample chamber of the CO2 critical point dryer, and dry them by evaporating CO2. After drying, transfer them to a suitable CT fixation container and seal the opening with a sealing film.

[0078] 3. CT scan reconstruction.

[0079] The mouse embryo specimens before and after the treatment in step 2 were scanned with a micro-CT scanner (GE v|tome|x m300&180). The scanning voltage can be 90KV, 110KV, the current is 70uA, 90uA, and the continuous scanning method is adopted. The step length is 0.3°, the frame rate is 1, and 1200 360° images are scanned in 20 minutes. After the sample is scanned, it is reconstructed by the built-in software to obtain the three-dimensional data of the embryo specimen, and then segmented and extracted by the open source three-dimensional visualization software Drishti, and the digital anatomical embryo three-dimensional model 2c is rendered.

[0080] 4. Digital anatomy.

[0081] Based on the three-dimensional digital model of the embryo, the area of ​​interest is dissected and observed from multiple angles.

[0082] Figure 3 Comparison of three-dimensional reconstructions of mouse embryos. Figure 3 The figure a in the middle shows a CT tomogram obtained by directly scanning a mouse embryo without the treatment in step 2. The pixel size is 6.188 μm, and the internal bone morphology is unclear. Figure 3 Middle b is a CT tomogram of a mouse embryo after processing in step 2, with a pixel size of 5.434 μm and clear morphology of internal skeletal organs. Figure 3 Figure c is a 3D structure image rendered by Drishti software, which shows different structures segmented from the overall structure. By comparison, it can be seen that the micro-CT contrast of the mouse embryo after Lugol's reagent staining and carbon dioxide critical point drying has been significantly improved, and the internal organ morphology is clearly visible. The 3D software is used to quickly reconstruct a complete and accurate 3D model of the mouse embryo. Dissection is performed on the 3D model of the mouse embryo, and different regions of interest are extracted and marked with different colors to clearly study its fine structure, morphological characteristics, and mutual relationships.

[0083] In summary, the present invention can be used to quickly obtain a large number of three-dimensional models of vertebrate embryos in a short period of time, and perform accurate digital dissection. It avoids the extremely demanding manual dissection method, obtains a complete three-dimensional digital model of the embryo without loss, and realizes non-destructive, multi-angle, all-round, and unlimited accurate digital dissection. After accumulating a large amount of data, it can support the establishment of a three-dimensional vertebrate embryo morphology database, trace the evolution of vertebrates, and provide important evidence for the systematic development and evolution of vertebrates.

[0084] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology in this field to understand the content of the present invention and implement it, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A three-dimensional digital dissection method for vertebrate embryos, It is characterized in that The method comprises: Fixed alligator embryo specimens; Pre-treating the fixed alligator embryo specimen; wherein the pre-treating of the fixed alligator embryo specimen comprises: The fixed alligator embryo specimens were moved into anhydrous alcohol and immersed for 5-15 minutes for primary dehydration; Transfer the dehydrated alligator embryo specimens to 0.5% Lugol's reagent and soak for 12-36 hours for staining; After staining, the alligator embryo specimens were taken out and placed in gradient alcohols for 8-12 minutes for secondary dehydration. The alligator embryo specimens after secondary dehydration were transferred to CO 2 Drying is achieved in the sample chamber of the critical point dryer; After transferring the dried alligator embryo specimen to a CT fixation container, the opening of the CT fixation container is sealed with a sealing film; The alligator embryo specimens after pretreatment were scanned using a micro-CT scanning system to obtain a three-dimensional digital model of the embryo; Based on the three-dimensional digital model of the embryo, a virtual digital dissection is performed on the region of interest.

2. The method according to claim 1, It is characterized in that The fixed alligator embryo specimen comprises: Obtain fresh alligator embryo specimens; Rinse fresh alligator embryo specimens with saline; The washed alligator embryo specimen is immersed in 8-12% formalin solution, sealed and darkened for 36 to 72 hours to obtain a fixed alligator embryo specimen.

3. The method according to claim 1, It is characterized in that The gradient alcohol includes: 30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol, 90% ethanol, 95% ethanol and 100% ethanol.

4. The method according to claim 1, It is characterized in that The soaking time in the primary dehydration treatment is 5 minutes; the soaking time in the dyeing treatment is 24 hours; and the soaking time in the secondary dehydration treatment is 10 minutes.

5. The method according to claim 1, It is characterized in that The method comprises scanning the pre-treated alligator embryo specimen using a micro-CT scanning system to obtain a three-dimensional digital model of the embryo, including: The alligator embryo specimens after pretreatment were scanned using a micro-CT scanning system to obtain scanning data; Reconstruct the scanned data to obtain the three-dimensional data of the alligator embryo specimen; The three-dimensional data of the alligator embryo specimen is segmented and extracted based on three-dimensional visualization software, and a digitally anatomical three-dimensional model of the embryo is rendered.

6. The method according to claim 5, It is characterized in that The parameter settings of the micro-CT scanning system include: voltage of 80-110 KV, current of 70-150 uA, and resolution of 0.8-20 μm.

7. A three-dimensional digital dissection system for vertebrate embryos, It is characterized in that The system comprises: A fixing module, used to fix alligator embryo specimens; The pre-processing module is used for pre-processing the fixed alligator embryo specimen; wherein the pre-processing of the fixed alligator embryo specimen includes: The fixed alligator embryo specimens were moved into anhydrous alcohol and immersed for 5-15 minutes for primary dehydration; Transfer the dehydrated alligator embryo specimens to 0.5% Lugol's reagent and soak for 12-36 hours for staining; After staining, the alligator embryo specimens were taken out and placed in gradient alcohols for 8-12 minutes for secondary dehydration. The alligator embryo specimens after secondary dehydration were transferred to CO 2 Drying is achieved in the sample chamber of the critical point dryer; After transferring the dried alligator embryo specimen to a CT fixation container, the opening of the CT fixation container is sealed with a sealing film; A CT scanning module is used to scan the pre-processed alligator embryo specimen using a micro-CT scanning system to obtain a three-dimensional digital model of the embryo; The dissection module is used to perform virtual digital dissection on the region of interest based on the three-dimensional digital model of the embryo.

Images