Method for observing three-dimensional structure of mating system of brown planthopper by combining microscopic CT with FIB-SEM

Abstract

The invention discloses a method for observing a three-dimensional structure of a brown planthopper mating system by combining micro-CT with FIB-SEM, the method comprises sample treatment and microscope observation, the sample treatment: performing tissue fixation on the brown planthopper in mating at 2-6 DEG C by using a stationary liquid; microscopic CT (computed tomography) observation: observing the treated sample by utilizing microscopic CT; and FIB-SEM observation: observing the treated sample by using the FIB-SEM. The method is free of tissue damage, rapid and efficient, the three-dimensional characteristics of the reproductive system can be observed, the structure of any plane can be reconstructed and observed, and repeatability is high; according to the method, the basic structure of the insect can be comprehensively understood from outside to inside and from plane to three-dimensional, the target sample can be observed on the whole by combining the microscopic CT with the FIB-SEM, the key structure can be observed more carefully, and an important basis is provided for comprehensively observing the brown planthopper mating system structure.

Description

Technical Field

[0001] The invention belongs to the field of biological application technology, and in particular relates to a method for observing the three-dimensional structure of a mating system of a brown planthopper by combining micro-CT with FIB-SEM. Background Art

[0002] Brown planthopper is a migratory rice monophagous pest. The study of brown planthopper is crucial both from the perspective of biological evolution and insect control. At present, there are two methods commonly used to study the mating system of brown planthopper, anatomical method and transmission electron microscopy.

[0003] Anatomy is the most commonly used method to observe the phenotype of insect mating system. The anatomical method can clearly identify the external morphological structure of the mating system. This method is quick and simple, and has low requirements for experimental conditions. Transmission electron microscopy is to observe the insect mating system using a transmission electron microscope. It can directly observe the internal ultrastructure of the insect mating system and can be magnified 100 to 10,000 times. However, the use of anatomical methods for observation will destroy the most original internal structure of brown planthoppers, and the mating system cannot be observed at the microscopic level with the naked eye. The preparation process of the transmission electron microscopy method is relatively cumbersome, and only a single plane image can be observed. It is acceptable for structures at known locations that need to be observed, but for unknown structures, the content of a single slice is too one-sided and cannot be fully observed and analyzed. Because the structure of the mating system of brown planthoppers is very complex, it is impossible to observe it in a three-dimensional and comprehensive way from the structure using any of the above methods.

[0004] Micro-CT is a relatively new technology that can clearly understand the internal microscopic structure of a sample without destroying it, with an accuracy of micrometer level. Its characteristics are that it can take stereoscopic photos, observe the information of each cross section inside the entire insect, and perform 2D and 3D analysis on the target structure that needs to be observed. Micro-CT was first used in the field of biomedicine to study the structure of bones and teeth. Since 2005, Greco and other talents have applied micro-CT technology to the study of bee ovaries. So far, there has been no report on the use of micro-CT technology to study the three-dimensional structure of the reproductive system of brown planthoppers during mating.

[0005] Focused ion beam scanning electron microscopy (FIB-SEM) is a high-resolution imaging tool that combines focused ion beam (FIB) and scanning electron microscopy (SEM) technologies. It combines the ability of FIB to precisely cut samples with the ability of SEM to perform high-resolution imaging, and is widely used in materials science, nanotechnology, biology, and other fields. The core advantage of FIB-SEM technology is that it can simultaneously cut, image, and analyze samples, making it an important tool for high-resolution structural analysis. So far, there have been no reports on the use of FIB-SEM technology to study the three-dimensional structure of the reproductive system of brown planthoppers during mating. Summary of the invention

[0006] In order to make up for the shortcomings of the prior art, the present invention aims to provide a method for observing the three-dimensional structure of the mating system of brown planthoppers by using micro-CT combined with FIB-SEM (focused ion beam scanning electron microscope), wherein the brown planthopper samples are fixed with a fixative, then treated with reagents such as sodium dimethyl arsenate, and finally the reproductive system of the mating brown planthoppers is scanned by micro-CT, so as to achieve a comprehensive understanding of the basic structure of the insects from the outside to the inside and from the plane to the three-dimensional, so as to solve the problems of cumbersome operation and incomplete observation results of the existing observation methods.

[0007] To achieve the above object, the present invention can be implemented through the following technical solutions:

[0008] The method of using micro-CT combined with FIB-SEM to observe the three-dimensional structure of the mating system of brown planthopper comprises the following steps:

[0009] Sample processing: Use fixative to fix the mating brown planthoppers at a temperature of 2℃ to 6℃;

[0010] Micro-CT observation: Observe the processed samples using micro-CT;

[0011] FIB-SEM observation: Use FIB-SEM to observe the processed samples.

[0012] Furthermore, the specific process of the sample processing is as follows:

[0013] S1. Fix the mating brown planthoppers with liquid nitrogen, and then fix the samples in glutaraldehyde solution for 24 hours;

[0014] S2. The brown planthopper samples were washed in sodium dimethylarsenate buffer and treated with a solution containing equal volumes of OsO4 and potassium ferrocyanide on ice;

[0015] S3. After rinsing with double distilled water (ddH2O), the sample was incubated in thiocarbazide solution at room temperature;

[0016] S4. Rinse the sample with double distilled water (ddH2O) and treat with OsO4 aqueous solution at room temperature;

[0017] S5. Wash the tissue blocks with double distilled water (ddH2O) and soak the samples in 0.5% to 1.5% uranyl acetate solution overnight;

[0018] S6. After washing the samples with double distilled water (ddH2O), the samples were incubated in a solution of lead nitrate-aspartic acid, then dehydrated in increasing levels of ethanol, and finally embedded in EPON 812 resin for 48 hours.

[0019] Furthermore, in step S1, the glutaraldehyde solution uses 0.1M sodium dimethylarsenate buffer as solvent, and the glutaraldehyde concentration is 2% to 3%.

[0020] Furthermore, in step S2, the OsO4 and potassium ferrocyanide solution uses 0.3M sodium dimethyl arsenate as solvent, OsO4 is 1% to 3% and potassium ferrocyanide solution is 1% to 5%; the treatment is carried out on ice for 0.5 to 1.5 hours.

[0021] Furthermore, in step S3, the concentration of the thiocarbazide solution is 0.5% to 1.5%, and the incubation time is 15 to 25 minutes.

[0022] Furthermore, in step S4, the concentration of the OsO4 aqueous solution is 1% to 3%, and the treatment time is 25 to 35 minutes.

[0023] Furthermore, in step S6, lead nitrate is dissolved in 0.03 ml of aspartic acid at pH 5.5, with a concentration of 0.6% to 0.7%.

[0024] Furthermore, in step S6, the incubated sample is dehydrated in 30%, 50%, 70%, 90%, and 100% ethanol in increasing levels, each dehydration treatment lasts for 30 minutes, and finally embedded in EPON 812 resin at 55°C to 65°C for 48 hours.

[0025] Furthermore, the target reproductive system includes the reproductive system of a mating brown planthopper; before the sample is processed, the brown planthopper sample is first frozen in liquid nitrogen, and then the sample is placed in a fixative at room temperature.

[0026] Furthermore, for both micro-CT observation and FIB-SEM observation, the processed samples were directly glued to the sample stage for observation and scanning, and the taken images were used to reconstruct the brown planthopper samples in three dimensions using the amira software.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] (1) The method of the present invention is non-invasive, rapid and efficient, can observe the three-dimensional characteristics of the mating system, and can reconstruct and observe the structure of any plane with strong repeatability. Through this method, a comprehensive understanding of the basic structure of insects from the outside to the inside and from the plane to the three-dimensional can be obtained, which provides an important basis and technical means for the comprehensive observation of the mating system structure of brown planthoppers.

[0029] (2) The method of the present invention uses micro-CT combined with FIB-SEM to conduct morphological observation of the reproductive system during mating, and establishes a 3D model of the reproductive organs of male and female brown planthoppers during mating. The external morphology and internal detail characteristics of the sample can be obtained simultaneously, the image resolution is high, and the spatial vision of the 3D model obtained by modeling and reconstruction is good; the sample preparation process is simple, the operation is convenient, the space occupied by the instrument is small, and it has a very good promotion prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is an electron microscope image of the brown planthopper of the present invention during mating (from the receptors in the ovipositor and the penis of the male brown planthopper);

[0031] Figure 2 The electron microscope images of the brown planthopper of the present invention during mating (sections from different positions);

[0032] Figure 3 It is a three-dimensional reconstruction image of the brown planthopper during mating of the present invention (including a reproductive system image of the brown planthopper during mating). DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] In this example, micro-CT combined with FIB-SEM was used to observe the three-dimensional structure of the reproductive organs of mating brown planthoppers, and the specific steps were as follows:

[0035] Preparation of fixative solution: dissolve glutaraldehyde in sodium dimethylarsenate buffer (0.1M) to a concentration of 2.5%; dissolve 2% OsO4 and 3% potassium ferrocyanide in 0.3M sodium dimethylarsenate to a concentration of 2% OsO4 and 3% potassium ferrocyanide; dissolve uranyl acetate in water to a concentration of 1%; dissolve thiocarbazide in water to a concentration of 1%; dissolve lead nitrate in 0.03M L-aspartic acid (pH5.5) to a concentration of 0.66%.

[0036] Pretreatment: Mating brown planthoppers were frozen in liquid nitrogen.

[0037] Fixative treatment: Brown planthoppers frozen in liquid nitrogen were immersed in the prepared glutaraldehyde solution at room temperature for 24 hours, and then the brown planthopper samples were washed in sodium dimethylarsenate buffer (0.1M) and treated with an equal volume of 2% OsO4 and 3% potassium ferrocyanide solution on ice for 1 hour; after rinsing with double distilled water (ddH2O), the samples were incubated in 1% thiocarbazide solution at room temperature for 20 minutes; then, the samples were rinsed with ddH2O and treated with 2% OsO4 aqueous solution at room temperature for 30 minutes; tissue blocks were washed with ddH2O and immersed in 1% uranyl acetate solution at 4°C overnight; after washing the samples with ddH2O, the samples were incubated in 0.66% lead nitrate at 60°C for 30 minutes, and then dehydrated in a stepwise increasing ethanol series of 30%, 50%, 70%, 90%, and 100% for 30 minutes at each step, and finally embedded in EPON 812 resin at 60°C for 48 hours.

[0038] Micro-CT observation and FIB-SEM observation: After the mating brown planthoppers were processed, they were directly glued to the sample stage for observation and scanning, and the images were reconstructed into three-dimensional volumes using the amira software.

[0039] Figure 1 and Figure 2 Shown is an electron microscope view of the brown planthopper during mating. Figure 1 Showing the receptors in the ovipositor and the penis of the male brown planthopper, Figure 2 Shown are sections from different locations, including the penis and sperm of a male brown planthopper, and the reproductive tract of a female brown planthopper. Figure 3 Shown is a three-dimensional reconstruction of the brown planthopper during mating of the present invention (including a diagram of the reproductive system of the brown planthopper during mating).

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for observing the three-dimensional structure of the mating system of brown planthopper using micro-CT combined with FIB-SEM, characterized in that: The following steps are involved: Sample processing: Use fixative to fix the mating brown planthoppers at a temperature of 2℃ to 6℃; Micro-CT observation: Observe the processed samples using micro-CT; FIB-SEM observation: Use FIB-SEM to observe the processed samples.

2. The method for observing the three-dimensional structure of the mating system of brown planthopper by using micro-CT combined with FIB-SEM as claimed in claim 1, characterized in that: The specific process of sample processing is as follows: S1. Fix the mating brown planthoppers with liquid nitrogen, and then fix the samples in glutaraldehyde solution for 24 hours; S2. The brown planthopper samples were washed in sodium dimethylarsenate buffer and treated with a solution containing equal volumes of OsO4 and potassium ferrocyanide on ice; S3. After rinsing with double distilled water (ddH2O), the sample was incubated in thiocarbazide solution at room temperature; S4. Rinse the sample with double distilled water (ddH2O) and treat with an aqueous solution of OsO4 at room temperature; S5. Wash the tissue blocks with double distilled water (ddH2O) and soak the samples in 0.5% to 1.5% uranyl acetate solution overnight. S6. After washing the samples with double distilled water (ddH2O), the samples were incubated in a solution of aspartic acid containing lead nitrate, then dehydrated in increasing levels of ethanol, and finally flat-embedded with EPON 812 resin for 48 hours.

3. The method for observing the three-dimensional structure of the mating system of brown planthopper by using micro-CT combined with FIB-SEM as claimed in claim 2, characterized in that: In step S1, the glutaraldehyde solution uses 0.1 M sodium dimethylarsenate buffer as solvent, and the glutaraldehyde concentration is 2% to 3%.

4. The method for observing the three-dimensional structure of the mating system of brown planthopper by using micro-CT combined with FIB-SEM as claimed in claim 2, characterized in that: In step S2, the OsO4 and potassium ferrocyanide solution is prepared by using 0.3M sodium dimethylarsenate as solvent, with 1% to 3% OsO4 and 1% to 5% potassium ferrocyanide solution; the treatment is carried out on ice for 0.5 to 1.5 hours.

5. The method for observing the three-dimensional structure of the mating system of brown planthopper by using micro-CT combined with FIB-SEM as claimed in claim 2, characterized in that: The concentration of the thiocarbohydrazide solution in step S3 is 0.5% to 1.5%, and the incubation time is 15 to 25 minutes.

6. The method for observing the three-dimensional structure of the mating system of brown planthopper by using micro-CT combined with FIB-SEM as claimed in claim 2, characterized in that: In step S4, the concentration of the OsO4 aqueous solution is 1% to 3%, and the treatment time is 25 to 35 minutes.

7. The method of observing the three-dimensional structure of the mating system of brown planthopper by using micro-CT combined with FIB-SEM as claimed in claim 2, characterized in that: In step S6, lead nitrate is dissolved in 0.03 ml of aspartic acid at pH 5.5, with a concentration of 0.6% to 0.7%.

8. The method of observing the three-dimensional structure of the mating system of brown planthopper by using micro-CT combined with FIB-SEM as claimed in claim 2, characterized in that: In step S6, the incubated samples are dehydrated in ethanol at increasing concentrations of 30%, 50%, 70%, 90%, and 100%, with each dehydration treatment lasting 30 minutes, and finally embedded in EPON 812 resin at 55° C. to 65° C. for 48 hours.

9. A method for observing the three-dimensional structure of the mating system of brown planthopper using micro-CT combined with FIB-SEM according to any one of claims 1 to 8, characterized in that: The target reproductive system included the mating system of mating brown planthoppers. Before sample processing, the brown planthopper samples were frozen in liquid nitrogen and then placed in fixative at room temperature.

10. The method for observing the three-dimensional structure of the mating system of brown planthopper by using micro-CT combined with FIB-SEM as claimed in claim 9, characterized in that: Both micro-CT observation and FIB-SEM observation were performed by sticking the processed samples directly onto the sample stage for observation and scanning. The images were taken and three-dimensionally reconstructed using the amira software.

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