WO2024152399A1 - 一种增强高淀粉含量种子显微ct对比度的方法 - Google Patents

一种增强高淀粉含量种子显微ct对比度的方法 Download PDF

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WO2024152399A1
WO2024152399A1 PCT/CN2023/076745 CN2023076745W WO2024152399A1 WO 2024152399 A1 WO2024152399 A1 WO 2024152399A1 CN 2023076745 W CN2023076745 W CN 2023076745W WO 2024152399 A1 WO2024152399 A1 WO 2024152399A1
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seeds
micro
starch content
phosphotungstic acid
treatment
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English (en)
French (fr)
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徐秀苹
冯旻
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中国科学院植物研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/0098—Plants or trees
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/02—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material
    • G01N23/04—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material and forming images of the material
    • G01N23/046—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material and forming images of the material using tomography, e.g. computed tomography [CT]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/302—Stain compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/305—Fixative compositions

Definitions

  • the present invention belongs to the field of biotechnology, and particularly relates to a method for enhancing the micro-CT contrast of seeds with high starch content.
  • a common method for studying the morphological structure of seed starch or endosperm cells is to slice seeds in the filling stage and observe the exposed starch grains and endosperm cell structure under an optical microscope.
  • a common method for studying the morphological structure of seed starch or endosperm cells is to slice seeds in the filling stage and observe the exposed starch grains and endosperm cell structure under an optical microscope.
  • Due to the high hardness of mature seeds it is extremely difficult to make slices. Therefore, how to directly observe the arrangement of endosperm cells in seeds with high starch content without destroying the structure of mature seeds is one of the technical difficulties encountered in the current research technology of mature seed morphological structure.
  • CN111398000A discloses a method for enhancing the contrast of high starch content seeds by micro-CT.
  • the method can directly observe the morphological structure of seeds without destroying the structure of mature seeds.
  • the method is only applicable to seeds with low starch content, such as mung bean seeds (starch content not exceeding 50%).
  • seeds with high starch content since the density inside the seeds is uniform, there is no difference in the ray transmittance.
  • the starch part of the reconstructed image has a uniform texture and the structure cannot be distinguished at all. Therefore, a method for effectively enhancing the contrast of high starch content seeds by micro-CT is needed.
  • the purpose of the present invention is to provide a method for enhancing the microscopic CT contrast of seeds with high starch content, which combines shaking table pretreatment with soaking high starch content seeds in phosphotungstic acid solution, optimizes the treatment conditions, and then performs carbon dioxide critical point drying, thereby greatly improving the microscopic CT contrast of seeds with high starch content.
  • the present invention provides the following technical solutions:
  • the method for enhancing the micro-CT contrast of high starch content seeds comprises the following steps step:
  • step 2 sequentially dehydrating and carbon dioxide critical point drying the high starch content seeds after the fixation and dyeing treatment in step 2);
  • step 3 performing micro-CT scanning on the high starch content seeds treated in step 3);
  • the starch content of the high-starch content seeds is more than 60%;
  • step 2) the conditions for the fixation and staining treatment are as follows:
  • the temperature is 4°C;
  • the time is 30-60 days;
  • the phosphotungstic acid solution is replaced and shaken, and then the fixation and staining process is continued; the phosphotungstic acid solution is replaced once every two weeks.
  • the high-starch content seeds are selected from rice seeds (starch content of about 70%), purple rice seeds (starch content of 70%), millet seeds (starch content of 70%), oat seeds (starch content of 60%), glutinous rice seeds (starch content of 75%), sorghum seeds (starch content of 70%) or red rice seeds (starch content of 60%).
  • step 1) the mass concentration of phosphotungstic acid in the phosphotungstic acid solution is 1-10%, such as 1%, 5% or 10%.
  • step 1) the composition of each 100 mL of the FAA fixative solution is as follows: 90 mL of 50% ethanol aqueous solution; 5 mL of glacial acetic acid; and 5 mL of formaldehyde.
  • step 2) the shaking table treatment conditions are as follows: temperature of 4° C.; 7.5-8.5 h, preferably 8 h; rotation speed of 40-80 rpm, preferably 60 rpm.
  • step 2) different high starch content seeds are treated with different fixation and dyeing times to achieve better contrast; specifically, the fixation and dyeing times are as follows:
  • the high starch content seeds are rice seeds, purple rice seeds, oat seeds, glutinous rice seeds, and red rice seeds
  • the time for the fixation and dyeing treatment is 39-41 days
  • the time of the fixation and dyeing treatment is 30-32 days;
  • the time for the fixation and dyeing treatment is 58-60 days.
  • step 3 the dehydration treatment is carried out with an ethanol aqueous solution; further, the dehydration treatment is carried out with ethanol aqueous solutions with volume fractions of 70%, 80%, 90% and 100% in sequence for gradient dehydration, each treatment lasting 10-20 minutes.
  • step 4 the conditions of the micro-CT scanning are as follows: resolution of 0.56-1.15 ⁇ m; scanning voltage of 40-100 kV, preferably 100 kV; scanning step length of 0.2°; and 2 images are taken at each angle.
  • step 4 different resolutions and scanning voltages are set for different high starch content seeds to achieve better contrast; the resolution and scanning voltage of the micro-CT scanning are performed as follows:
  • the resolution of the micro-CT scan was 0.88 ⁇ m and the scanning voltage was 100 kV;
  • the resolution of the micro-CT scan was 0.95 ⁇ m and the scanning voltage was 100 kV;
  • the resolution of the micro-CT scan was 0.68 ⁇ m and the scanning voltage was 41 kV;
  • the resolution of the micro-CT scan was 0.88 ⁇ m and the scanning voltage was 100 kV;
  • the resolution of the micro-CT scan was 0.88 ⁇ m and the scanning voltage was 100 kV;
  • the resolution of the micro-CT scan was 1.15 ⁇ m and the scanning voltage was 100 kV;
  • the resolution of the micro-CT scan was 0.74 ⁇ m and the scanning voltage was 100 kV.
  • the seed coat of the high starch content seeds is cut.
  • FIG. 1 is a cross section of a partially enlarged rice seed treated with FAA fixative, 10% cesium iodide, 10% phosphotungstic acid, and 10% phosphotungstic acid, and all of which were critical point dried (from left to right).
  • FIG. 2 is a cross section of a partially enlarged portion of purple rice seeds treated with FAA fixative, 10% cesium iodide, Lugos solution, 10% phosphotungstic acid, and 10% phosphotungstic acid, and all of which were critical point dried (from left to right).
  • FIG. 3 shows cross sections (from left to right) of millet seeds treated with FAA fixative, 10% cesium iodide, and 10% phosphotungstic acid solution, respectively, and all of which were critical point dried.
  • FIG. 4 shows cross sections (from left to right) of oat seeds treated with FAA fixative and 10% phosphotungstic acid solution, respectively, and then critical point dried.
  • FIG. 5 shows cross sections (from left to right) of glutinous rice seeds treated with FAA fixative and 10% phosphotungstic acid solution, respectively, and after critical point drying.
  • FIG. 6 shows cross-sections of sorghum seeds treated with FAA fixative and 10% phosphotungstic acid solution, respectively, and after critical point drying (from left to right).
  • FIG. 7 shows cross sections (from left to right) of red rice seeds treated with FAA fixative and 10% phosphotungstic acid solution, respectively, and then critical point dried.
  • FIG. 8 shows cross-sections (from left to right) of mung bean seeds treated with FAA fixative and 10% phosphotungstic acid solution, respectively, and then critical point dried.
  • composition of the FAA fixative (100 mL) is: 90 mL of 50% ethanol aqueous solution, 5 mL of glacial acetic acid, and 5 mL of formaldehyde.
  • Preparation of phosphotungstic acid solution dissolve phosphotungstic acid in FAA fixative to obtain a phosphotungstic acid solution with a mass concentration of 10%.
  • Carbon dioxide critical point drying treatment A critical point drying apparatus (CPD300, Leica) was used for the treatment.
  • Micro-CT scanning Skyscan 1172, Bruker Corporation.
  • the rice seeds were placed in FAA fixative, 10% cesium iodide solution, and 10% phosphotungstic acid solution, respectively, and inverted several times to remove bubbles until the samples were sunk into the solution.
  • the samples were placed on a shaker and shaken at 4°C for 8 hours at 60 rpm, and then fixed at 4°C for 40 days. During this period, the solution was changed every two weeks. After each change of the solution, the samples were placed on a shaker and shaken at 4°C for 8 hours at 60 rpm, and then continued to be fixed at 4°C.
  • Gradient ethanol dehydration was then carried out using 70%, 80%, 90%, and 100% ethanol aqueous solutions, with each gradient treatment lasting 20 minutes.
  • the dehydrated samples were subjected to carbon dioxide critical point drying treatment.
  • the dried samples were subjected to micro-CT scanning.
  • the resolution of samples treated with FAA fixative, 10% cesium iodide solution, and 10% phosphotungstic acid solution was 0.88 ⁇ m, and the scanning voltages were 40 kV, 100 kV, and 100 kV, respectively.
  • the local magnification resolution of the samples treated with 10% phosphotungstic acid solution was 0.56 ⁇ m, and the scanning voltage was 100 kV.
  • the scanning step size was 0.2°, and two images were taken at each angle.
  • FIG. 1 After the sample was scanned and reconstructed using NRecon software, a cross-sectional image was obtained, as shown in Figure 1, wherein the upper left image is a cross-sectional image of rice seeds treated with FAA fixative, the upper right image is a cross-sectional image of rice seeds treated with 10% cesium iodide solution, the lower left image is a cross-sectional image of rice seeds treated with 10% phosphotungstic acid, and the lower right image is a partially enlarged cross-sectional image of rice seeds treated with 10% phosphotungstic acid.
  • the micro-CT contrast of rice seeds treated with phosphotungstic acid and dried at the critical point of carbon dioxide has been significantly improved, and the endosperm cell morphology is clearly visible.
  • Purple rice seeds were placed in FAA fixative, 10% cesium iodide solution, Lugos solution, and 10% phosphotungstic acid solution, respectively, and inverted several times to remove bubbles until the sample sank into the solution.
  • the sample was placed on a shaker at 4°C and shaken for 8 hours at 60 rpm, and then fixed at 4°C for 40 days; during this period, the solution was replaced every two weeks, and after each solution replacement, the sample was placed on a shaker at 4°C and shaken for 8 hours at 60 rpm, and then continued to be fixed at 4°C; then, 70%, 80%, 90% and 100% ethanol aqueous solutions were used for gradient ethanol dehydration, each gradient treatment lasted 20 minutes, and the dehydrated samples were subjected to carbon dioxide critical point drying treatment.
  • the dried samples were subjected to micro-CT scanning.
  • the resolution of samples treated with FAA fixative, 10% cesium iodide solution, Lugos solution, and 10% phosphotungstic acid solution was 0.95 ⁇ m, and the scanning voltages were 43 kV, 100 kV, 100 kV, and 100 kV, respectively.
  • the local magnification resolution of the sample treated with 10% phosphotungstic acid was 0.56 ⁇ m, and the scanning voltage was 100 kV.
  • the scanning step size was 0.2°, and two images were taken at each angle.
  • FIG2 After the sample was scanned and reconstructed using NRecon software, a cross-sectional image was obtained, as shown in FIG2 , wherein the upper row, from left to right, shows the cross-sections of purple rice seeds treated with FAA fixative, 10% cesium iodide, and Lugos solution, and the lower row, from left to right, shows the cross-sections of purple rice seeds that were locally enlarged after being treated with 10% phosphotungstic acid solution and 10% phosphotungstic acid solution.
  • the micro-CT contrast of rice seeds treated with phosphotungstic acid and dried at the critical point of carbon dioxide has been significantly improved, and the endosperm cell morphology is clearly visible.
  • Millet seeds were placed in FAA fixative, 10% cesium iodide solution, and 10% phosphotungstic acid solution, respectively, and inverted several times to remove bubbles until the sample sank into the solution, and then placed on a shaker at 4°C for 8 hours, 60 rpm, and then fixed at 4°C for 30 days; during this period, the solution was replaced every two weeks, and after each solution replacement, the sample was placed on a shaker at 4°C for 8 hours, 60 rpm, and then continued to be fixed at 4°C; then, 70%, 80%, 90% and 100% ethanol aqueous solutions were used for gradient ethanol dehydration, each gradient treatment lasted 20 minutes, and the dehydrated samples were subjected to carbon dioxide critical point drying treatment.
  • the dried samples were subjected to micro-CT scanning.
  • the scanning voltage of each processed sample was 41 kV, the resolution was 0.68 ⁇ m, the scanning step was 0.2°, and two images were taken at each angle.
  • the oat seeds were placed in FAA fixative and 10% phosphotungstic acid solution, respectively, and inverted several times to remove bubbles until the sample sank into the solution.
  • the sample was placed on a shaker and shaken at 4°C for 8 hours at 60 rpm, and then fixed at 4°C for 40 days. During this period, the solution was changed every two weeks. After each change of the solution, the sample was placed on a shaker and shaken at 4°C for 8 hours at 60 rpm, and then continued to be fixed at 4°C. Then, 70%, 80%, 90% and 100% ethanol aqueous solutions were used for gradient ethanol dehydration, and each gradient treatment lasted 20 minutes. The dehydrated samples were subjected to carbon dioxide critical point drying treatment.
  • the dried samples were subjected to micro-CT scanning.
  • the scanning voltage for the samples treated with FAA fixative was 40 kV, and the scanning voltage for the samples treated with 10% phosphotungstic acid was 100 kV.
  • the resolution was 0.88 ⁇ m, the scanning step was 0.2°, and two images were taken at each angle.
  • FIG4 After the samples were scanned and reconstructed using NRecon software, a cross-sectional image was obtained, as shown in FIG4 , where, from left to right, they are cross-sectional images of oat seeds treated with FAA fixative and 10% phosphotungstic acid solution.
  • FIG4 By comparing the images, it can be seen that the micro-CT contrast of the oat seeds treated with phosphotungstic acid and dried at the critical point of carbon dioxide has been significantly improved, and the endosperm cell morphology is clearly visible.
  • the dried samples were subjected to micro-CT scanning.
  • the scanning voltage for the samples treated with FAA fixative was 43 kV
  • the scanning voltage for the samples treated with 10% phosphotungstic acid was 100 kV.
  • the resolution was 0.88 ⁇ m
  • the scanning step was 0.2°
  • two images were taken at each angle.
  • FIG5 After the samples were scanned and reconstructed using NRecon software, a cross-sectional image was obtained, as shown in FIG5 , where, from left to right, they are cross-sectional images of glutinous rice seeds treated with FAA fixative and 10% phosphotungstic acid solution.
  • FIG5 By comparing the images, it can be seen that the micro-CT contrast of glutinous rice seeds treated with phosphotungstic acid and dried at the critical point of carbon dioxide has been significantly improved, and the endosperm cell morphology is clearly visible.
  • Sorghum seeds seed coat cut
  • Sorghum seeds were placed in FAA fixative and 10% phosphotungstic acid solution respectively, and inverted several times to remove bubbles until the sample sank into the solution, and then placed on a shaker at 4°C for 8 hours, 60 rpm, and then fixed at 4°C for 60 days; during this period, the solution was replaced every two weeks, and after each solution replacement, the sample was placed on a shaker at 4°C for 8 hours, 60 rpm, and then continued to be fixed at 4°C; then, 70%, 80%, 90% and 100% ethanol aqueous solutions were used for gradient ethanol dehydration, each gradient treatment lasting 20 minutes, and the dehydrated samples were subjected to carbon dioxide critical point drying treatment.
  • the dried samples were subjected to micro-CT scanning.
  • the scanning voltage for the samples treated with FAA fixative was 40 kV, and the scanning voltage for the samples treated with 10% phosphotungstic acid was 100 kV.
  • the resolution was 1.15 ⁇ m, the scanning step was 0.2°, and two images were taken at each angle.
  • FIG6 a cross-sectional image was obtained, as shown in FIG6 , where, from left to right, are the cross-sections of sorghum seeds treated with FAA fixative and 10% phosphotungstic acid solution.
  • the red rice seeds were placed in FAA fixative and 10% phosphotungstic acid solution, respectively, and inverted several times to remove bubbles until the sample was sunk into the solution, and then placed on a shaker at 4°C for 8 hours, 60 rpm, and then fixed at 4°C for 40 days; during this period, the solution was changed every two weeks, and after each change of the solution, the seeds were placed on a shaker at 4°C for 8 hours, 60 rpm, and then continued to be fixed at 4°C; then, 70%, 80%, 90% and 100% ethanol aqueous solutions were used for gradient ethanol dehydration, each gradient treatment lasted 20 minutes, and the dehydrated samples were subjected to carbon dioxide critical point drying treatment.
  • the dried samples were subjected to micro-CT scanning.
  • the scanning voltage for the samples treated with FAA fixative was 40 kV, and the scanning voltage for the samples treated with 10% phosphotungstic acid was 100 kV.
  • the resolution was 0.74 ⁇ m, the scanning step was 0.2°, and two images were taken at each angle.
  • FIG7 After the sample was scanned and reconstructed using NRecon software, a cross-sectional image was obtained, as shown in FIG7 , where, from left to right, are the cross-sectional images of glutinous rice seeds treated with FAA fixative and 10% phosphotungstic acid solution.
  • the mung bean seeds were placed in FAA fixative and 10% phosphotungstic acid solution, respectively, and inverted several times to remove bubbles until the sample sank into the solution.
  • the sample was placed on a shaker and shaken at 4°C for 8 hours at 60 rpm, and then fixed at 4°C for 60 days. During this period, the solution was replaced every two weeks. After each solution replacement, the sample was placed on a shaker and shaken at 4°C for 8 hours at 60 rpm, and then continued to be fixed at 4°C. Then, 70%, 80%, 90% and 100% ethanol aqueous solutions were used for gradient ethanol dehydration, and each gradient treatment lasted for 20 minutes. The dehydrated samples were subjected to carbon dioxide critical point drying treatment.
  • the dried samples were subjected to micro-CT scanning, with the scanning voltage of 40 kV for the samples treated with FAA fixative and 100 kV for the samples treated with 10% phosphotungstic acid, with a resolution of 1.76 ⁇ m and a 0.5 mm aluminum sheet.
  • the scanning step length was 0.2°, and two images were taken at each angle.
  • the present invention also investigated the influence of different operating conditions on the micro-CT contrast enhancement effect of seeds with high starch content.
  • the research results show that adopting the operating conditions of the above steps can obtain higher micro-CT contrast.
  • the present invention combines the shaking table treatment method with the phosphotungstic acid solution soaking treatment method to improve the fixation and dyeing effect of seeds with high starch content, and then uses carbon dioxide critical point drying to greatly improve the microscopic CT contrast of seeds with high starch content.
  • the present invention further determines the operating conditions of shaking table treatment and fixation and staining treatment to improve the micro-CT contrast of seeds with high starch content.

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Abstract

一种增强高淀粉含量种子显微CT对比度的方法，包括如下步骤：1)将磷钨酸溶于FAA固定液中得到磷钨酸溶液；2)将待测的高淀粉含量种子浸泡磷钨酸溶液中，先进行摇床处理，再进行固定和染色处理；3)经步骤2)固定和染色处理后的高淀粉含量种子依次进行脱水和二氧化碳临界点干燥；4)将步骤3)处理后的高淀粉含量种子进行显微CT扫描。将摇床预处理与采用磷钨酸溶液浸泡高淀粉含量种子处理手段相结合，并优化处理条件，然后再进行二氧化碳临界点干燥，提高了高淀粉含量种子的显微CT对比度。

Description

一种增强高淀粉含量种子显微CT对比度的方法
相关申请交叉引用
本申请要求由申请人中国科学院植物研究所于2023年1月17日提交 的、题目为一种增强高淀粉含量种子显微CT对比度的方法的中国专利申 请申请号为202310077757.X的优先权，该申请的全部内容通过引用并入 本文。
技术领域
本申请属于生物技术领域，特别涉及一种增强高淀粉含量种子显微CT对比度的方法。
背景技术
研究种子淀粉或胚乳细胞形态结构的常用方法是将灌浆期的种子制成切片，露出的淀粉粒和胚乳细胞结构，在光学显微镜下进行观察。但成熟种子因硬度大，制作切片极其困难，因此如何不破坏成熟种子的结构而直接观察高淀粉含量种子胚乳细胞的排布状态，是目前成熟种子形态结构研究技术中遇到的技术难题之一。
CN111398000A公开一种增强高淀粉含量种子显微CT对比度的方法，该方法能够不破坏成熟种子的结构而直接观察种子的形态结构，但该方法仅适用于淀粉含量较低的种子，如绿豆种子(淀粉含量不超过50％)；而对于高淀粉含量的种子，由于其种子内部密度均一，导致射线透过率没有差别，重构后的图片淀粉部分为均一质地，根本无法区分结构。因此需要一种有效增强高淀粉含量种子显微CT对比度的方法。
发明内容
本发明的目的是提供一种增强高淀粉含量种子显微CT对比度的方法，将摇床预处理与采用磷钨酸溶液浸泡高淀粉含量种子处理手段相结合，并优化处理条件，再进行二氧化碳临界点干燥，从而极大地提高了高淀粉含量种子的显微CT对比度。
为实现上述目的，本发明提供如下技术方案：
本发明提供的增强高淀粉含量种子显微CT对比度的方法，包括如下 步骤：
1)将磷钨酸溶于FAA固定液中得到磷钨酸溶液；
2)将待测的高淀粉含量种子浸泡所述磷钨酸溶液中，先进行摇床处理，再进行固定和染色处理；
3)经步骤2)固定和染色处理后的高淀粉含量种子依次进行脱水和二氧化碳临界点干燥；
4)将步骤3)处理后的高淀粉含量种子进行显微CT扫描；
其中：
所述高淀粉含量种子的淀粉含量60％以上；
步骤2)中，所述固定和染色处理的条件如下：
温度为4℃；
时间为30-60天；
所述固定和染色处理期间，更换磷钨酸溶液并进行摇床处理，然后继续固定和染色处理；所述磷钨酸溶液的更换频率为每两周更换一次。
所述高淀粉含量种子选自水稻种子(淀粉含量70％左右)、紫米种子(淀粉含量为70％)、小米种子(淀粉含量为70％)、燕麦种子(淀粉含量60％)、糯米种子(淀粉75％)、高梁种子(淀粉含量为70％)或红米种子(淀粉含量为60％)。
步骤1)中，所述磷钨酸溶液中磷钨酸的质量浓度为1-10％，如1％、5％或10％。
步骤1)中，每100mL所述FAA固定液的组成如下：50％乙醇水溶液90mL；冰乙酸5mL；甲醛5mL。
步骤2)中，所述摇床处理的条件如下：温度为4℃；7.5-8.5h，优选为8h；转速为40-80转/min，优选为60转/min。
步骤2)中，不同的高淀粉含量种子选择不同的固定和染色处理时间以达到较好的对比度；具体地，所述固定和染色处理的时间按照如下进行：
当所述高淀粉含量种子为水稻种子、紫米种子、燕麦种子、糯米种子、红米种子，所述固定和染色处理的时间为39-41天；
当所述高淀粉含量种子为小米种子，所述固定和染色处理的时间为30-32天；
当所述高淀粉含量种子为高梁种子，所述固定和染色处理的时间为58-60天。
步骤3)中，所述脱水处理采用乙醇水溶液进行；进一步地，所述脱水处理依次采用体积分数为70％、80％、90％和100％的乙醇水溶液进行梯度脱水，各处理10-20分钟。
步骤4)中，所述显微CT扫描的条件如下：分辨率为0.56-1.15μm；扫描电压为40-100kV，优选为100kV；扫描步长为0.2°；每个角度均成像2幅。
步骤4)中，不同的高淀粉含量种子设置不同的分辨率和扫描电压以达到较好的对比度；所述显微CT扫描的分辨率和扫描电压按照如下进行：
对于水稻种子，所述显微CT扫描的分辨率为0.88μm，扫描电压为100kV；
对于紫米种子，所述显微CT扫描的分辨率为0.95μm，扫描电压为100kV；
对于小米种子，所述显微CT扫描的分辨率为0.68μm，扫描电压为41kV；
对于燕麦种子，所述显微CT扫描的分辨率为0.88μm，扫描电压为100kV；
对于糯米种子，所述显微CT扫描的分辨率为0.88μm，扫描电压为100kV；
对于高梁种子，所述显微CT扫描的分辨率为1.15μm，扫描电压为100kV；
对于红米种子，所述显微CT扫描的分辨率为0.74μm，扫描电压为100kV。
本发明中，在进行所述步骤1)之前，将所述高淀粉含量种子的种皮切破。
附图说明
图1为分别经FAA固定液、10％碘化铯、10％磷钨酸、10％磷钨酸处理局部放大的水稻种子，并都经临界点干燥后的样品横切面(从左至右)。
图2为分别经FAA固定液、10％碘化铯、Lugos溶液、10％磷钨酸、10％磷钨酸处理局部放大的紫米种子，并都经临界点干燥后的样品横切面(从左至右)。
图3为分别经FAA固定液、10％碘化铯、10％磷钨酸溶液处理的小米种子，并都经临界点干燥后的样品横切面(从左至右)。
图4为分别经FAA固定液、10％磷钨酸溶液处理的燕麦种子，并都经临界点干燥后的样品横切面(从左至右)。
图5为分别经FAA固定液、10％磷钨酸溶液处理的糯米种子，并都经临界点干燥后的样品横切面(从左至右)。
图6为分别经FAA固定液、10％磷钨酸溶液处理的高梁种子，并都经临界点干燥后的样品横切面(从左至右)。
图7为分别经FAA固定液、10％磷钨酸溶液处理的红米种子，并都经临界点干燥后的样品横切面(从左至右)。
图8为分别经FAA固定液、10％磷钨酸溶液处理的绿豆种子，并都经临界点干燥后的样品横切面(从左至右)。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下实施例中：
所述FAA固定液(100mL)的组成为：50％乙醇水溶液90mL，冰乙酸5mL，甲醛5mL。
磷钨酸溶液的配制：将磷钨酸溶于FAA固定液中，得到质量浓度为10％的磷钨酸溶液。
碘化铯溶液的配制：将碘化铯溶于FAA固定液中，得到质量浓度为10％的碘化铯溶液。
Lugos溶液的配制：将4g碘化钾、2g I2加入到50ml FAA固定液中，得到Lugos溶液。
二氧化碳临界点干燥处理：采用临界点干燥仪(CPD300，徕卡)进行处理。
显微CT扫描：采用Skyscan1172，布鲁克公司。
试验例1、水稻种子的显微CT对比度的增强处理
将水稻种子分别投入至FAA固定液、10％碘化铯溶液、10％磷钨酸溶液中，上下颠倒几次去除气泡，至样品沉入溶液中，放在摇床上4℃摇8小时，60转/min，之后放于4℃固定40天；期间每两周更换一次溶液，每更换一次溶液后均须放在摇床上4℃摇8小时，60转/min，之后继续于4℃固定；然后采用70％、80％、90％和100％乙醇水溶液进行梯度乙醇脱水，各梯度处理20分钟，脱水后的样品进行二氧化碳临界点干燥处理。
干燥后的样品进行显微CT扫描，FAA固定液、10％碘化铯溶液、10％磷钨酸溶液处理的分辨率为0.88μm，扫描电压分别为40kV、100kV、100kV，10％磷钨酸溶液处理样品的局部放大分辨率为0.56μm，扫描电压为100kV；扫描步长均为0.2°，每个角度均成像2幅。
样品扫描后经NRecon软件重构，得到横切面图片，如图1所示，其中，左上图为FAA固定液处理的水稻种子横切面，右上图为经10％碘化铯溶液处理后的水稻种子的横切面，左下图为经10％磷钨酸处理后的水稻种子的横切面，右下图为经10％磷钨酸处理后的水稻种子局部放大的横切面；对比四幅图可以看出，经磷钨酸处理和二氧化碳临界点干燥后的水稻种子的显微CT对比度得到了显著的提高，胚乳细胞形态清晰可见。
试验例2、紫米种子的显微CT对比度的增强处理
将紫米种子分别投入至FAA固定液、10％碘化铯溶液、Lugos溶液、10％磷钨酸溶液中，上下颠倒几次去除气泡，至样品沉入溶液中，放在摇床上4℃摇8小时，60转/min，之后放于4℃固定40天；期间每两周更换一次溶液，每更换一次溶液后均须放在摇床上4℃摇8小时，60转/min，之后继续于4℃固定；然后采用70％、80％、90％和100％乙醇水溶液进行梯度乙醇脱水，各梯度处理20分钟，脱水后的样品进行二氧化碳临界点干燥处理。
干燥后的样品进行显微CT扫描，FAA固定液、10％碘化铯溶液、Lugos溶液、10％磷钨酸溶液处理的分辨率为0.95μm，扫描电压分别为43kV、100kV、100kV、100kV，10％磷钨酸处理样品的局部放大分辨率为0.56μm，扫描电压为100kV；扫描步长均为0.2°，每个角度均成像2幅。
样品扫描后经NRecon软件重构，得到横切面图片，如图2所示，其中，上排从左至右依次为FAA固定液、10％碘化铯、Lugos溶液处理的紫米种子横切面，下排从左至右依次为10％磷钨酸溶液和10％磷钨酸溶液处理后局部放大的紫米种子横切面，对比各图可以看出，经磷钨酸处理和二氧化碳临界点干燥后的水稻种子的显微CT对比度得到了显著的提高，胚乳细胞形态清晰可见。
试验例3、小米种子的显微CT对比度的增强处理
将小米种子分别投入至FAA固定液、10％碘化铯溶液、10％磷钨酸溶液中，上下颠倒几次去除气泡，至样品沉入溶液中，放在摇床上4℃摇8小时，60转/min，之后放于4℃固定30天；期间每两周更换一次溶液，每更换一次溶液后均须放在摇床上4℃摇8小时，60转/min，之后继续于4℃固定；然后采用70％、80％、90％和100％乙醇水溶液进行梯度乙醇脱水，各梯度处理20分钟，脱水后的样品进行二氧化碳临界点干燥处理。
干燥后的样品进行显微CT扫描，各处理样品的扫描电压均为41kV，分辨率均为0.68μm，扫描步长均为0.2°，每个角度均成像2幅。
样品扫描后经NRecon软件重构，得到横切面图片，如图3所示，其中，从左至右依次为FAA固定液、10％碘化铯溶液、10％磷钨酸溶液处理后的小米种子横切面，对比各图可以看出，经磷钨酸处理和二氧化碳临界点干燥后的小米种子的显微CT对比度得到了显著的提高，胚乳细胞形态清晰可见。由于小米种子较小，染色处理的时间也较短。
试验例4、燕麦种子的显微CT对比度的增强处理
将燕麦种子分别投入至FAA固定液、10％磷钨酸溶液中，上下颠倒几次去除气泡，至样品沉入溶液中，放在摇床上4℃摇8小时，60转/min，之后放于4℃固定40天；期间每两周更换一次溶液，每更换一次溶液后均须放在摇床上4℃摇8小时，60转/min，之后继续于4℃固定；然后采用70％、80％、90％和100％乙醇水溶液进行梯度乙醇脱水，各梯度处理20分钟，脱水后的样品进行二氧化碳临界点干燥处理。
干燥后的样品进行显微CT扫描，FAA固定液处理样品的扫描电压为40kV，10％磷钨酸处理样品的扫描电压为100kV，分辨率均为0.88μm，扫描步长均为0.2°，每个角度均成像2幅。
样品扫描后经NRecon软件重构，得到横切面图片，如图4所示，其中，从左至右依次为FAA固定液、10％磷钨酸溶液处理后的燕麦种子横切面，对比各图可以看出，经磷钨酸处理和二氧化碳临界点干燥后的燕麦种子的显微CT对比度得到了显著的提高，胚乳细胞形态清晰可见。
试验例5、糯米种子的显微CT对比度的增强处理
将糯米种子分别投入至FAA固定液、10％磷钨酸溶液中，上下颠倒几次去除气泡，至样品沉入溶液中，放在摇床上4℃摇8小时，60转/min，之后放于4℃固定40天；期间每两周更换一次溶液，每更换一次溶液后均须放在摇床上4℃摇8小时，60转/min，之后继续于4℃固定；然后采用70％、 80％、90％和100％乙醇水溶液进行梯度乙醇脱水，各梯度处理20分钟，脱水后的样品进行二氧化碳临界点干燥处理。
干燥后的样品进行显微CT扫描，FAA固定液处理样品的扫描电压为43kV，10％磷钨酸处理样品的扫描电压为100kV，分辨率均为0.88μm，扫描步长均为0.2°，每个角度均成像2幅。
样品扫描后经NRecon软件重构，得到横切面图片，如图5所示，其中，从左至右依次为FAA固定液、10％磷钨酸溶液处理后的糯米种子横切面，对比各图可以看出，经磷钨酸处理和二氧化碳临界点干燥后的糯米种子的显微CT对比度得到了显著的提高，胚乳细胞形态清晰可见。
试验例6、高梁种子的显微CT对比度的增强处理
将高梁种子(切破种皮)分别投入至FAA固定液、10％磷钨酸溶液中，上下颠倒几次去除气泡，至样品沉入溶液中，放在摇床上4℃摇8小时，60转/min，之后放于4℃固定60天；期间每两周更换一次溶液，每更换一次溶液后均须放在摇床上4℃摇8小时，60转/min，之后继续于4℃固定；然后采用70％、80％、90％和100％乙醇水溶液进行梯度乙醇脱水，各梯度处理20分钟，脱水后的样品进行二氧化碳临界点干燥处理。
干燥后的样品进行显微CT扫描，FAA固定液处理样品的扫描电压为40kV，10％磷钨酸处理样品的扫描电压为100kV，分辨率均为1.15μm，扫描步长均为0.2°，每个角度均成像2幅。
样品扫描后经NRecon软件重构，得到横切面图片，如图6所示，其中，从左至右依次为FAA固定液、10％磷钨酸溶液处理后的高梁种子的横切面，对比两图可以看出，经磷钨酸处理和二氧化碳临界点干燥后的高梁种子的显微CT对比度得到了显著的提高，淀粉胚乳细胞形态清晰可见。
试验例7、红米种子的显微CT对比度的增强处理
将红米种子分别投入至FAA固定液、10％磷钨酸溶液中，上下颠倒几次去除气泡，至样品沉入溶液中，放在摇床上4℃摇8小时，60转/min，之后放于4℃固定40天；期间每两周更换一次溶液，每更换一次溶液后均须放在摇床上4℃摇8小时，60转/min，之后继续于4℃固定；然后采用70％、80％、90％和100％乙醇水溶液进行梯度乙醇脱水，各梯度处理20分钟，脱水后的样品进行二氧化碳临界点干燥处理。
干燥后的样品进行显微CT扫描，FAA固定液处理样品扫描电压为40kV，10％磷钨酸处理扫描电压为100kV，分辨率均为0.74μm，扫描步长均为0.2°，每个角度均成像2幅。
样品扫描后经NRecon软件重构，得到横切面图片，如图7所示，其中，从左至右依次为FAA固定液、10％磷钨酸溶液处理后的糯米种子的横切面，对比两图可以看出，经磷钨酸处理和二氧化碳临界点干燥后的糯米种子的显微CT对比度得到了显著的提高，胚乳细胞形态清晰可见。
试验例8、绿豆种子的显微CT对比度的增强处理
将绿豆种子分别投入至FAA固定液、10％磷钨酸溶液中，上下颠倒几次去除气泡，至样品沉入溶液中，放在摇床上4℃摇8小时，60转/min，之后放于4℃固定60天；期间每两周更换一次溶液，每更换一次溶液后均须放在摇床上4℃摇8小时，60转/min，之后继续于4℃固定；然后采用70％、80％、90％和100％乙醇水溶液进行梯度乙醇脱水，各梯度处理20分钟，脱水后的样品进行二氧化碳临界点干燥处理。
干燥后的样品进行显微CT扫描，FAA固定液处理样品的扫描电压为40kV，10％磷钨酸处理样品的扫描电压为100kV，分辨率均为1.76μm，加0.5mm铝片。扫描步长均为0.2°，每个角度均成像2幅。
样品扫描后经NRecon软件重构，得到横切面图片，如图8所示，其中，从左至右依次为FAA固定液、10％磷钨酸溶液处理后的绿豆种子的横切面，对比两图可以看出，经磷钨酸处理和二氧化碳临界点干燥后的绿豆种子的显微CT对比度得到了改善，细节增多，但改善效果差于高淀粉含量种子。由此说明，本发明所述的处理方法能够显著提高高淀粉含量种子的显微CT对比度。
本发明在研究过程中还考察了不同操作条件对高淀粉含量种子的显微CT对比度增强效果的影响，研究结果表明，采用上述各步骤的操作条件能够获得更高的显微CT对比度。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业应用
本申请具有如下有益技术效果：
1、本发明将摇床处理手段与采用磷钨酸溶液浸泡处理手段相结合，提高高淀粉含量种子的固定和染色效果，再经二氧化碳临界点干燥，极大地提高了高淀粉含量种子的显微CT对比度。
2、本发明进一步确定了摇床处理及固定和染色处理的操作条件，以提高了高淀粉含量种子的显微CT对比度。

Claims (9)

  1. 一种增强高淀粉含量种子显微CT对比度的方法，包括如下步骤：
    1)将磷钨酸溶于FAA固定液中得到磷钨酸溶液；
    2)将待测的高淀粉含量种子浸泡所述磷钨酸溶液中，先进行摇床处理，再进行固定和染色处理；
    3)经步骤2)固定和染色处理后的高淀粉含量种子依次进行脱水和二氧化碳临界点干燥；
    4)将步骤3)处理后的高淀粉含量种子进行显微CT扫描；
    其中：
    所述高淀粉含量种子的淀粉含量60％以上；
    步骤2)中，所述固定和染色处理的条件如下：
    温度为4℃；时间为30-60天；
    所述固定和染色处理期间，更换磷钨酸溶液并进行摇床处理，然后继续固定和染色处理；所述磷钨酸溶液的更换频率为每两周更换一次。
  2. 根据权利要求书1所述的方法，其特征在于：所述高淀粉含量种子选自水稻种子、紫米种子、小米种子、燕麦种子、糯米种子、高梁种子或红米种子。
  3. 根据权利要求书1所述的方法，其特征在于：步骤1)中，所述磷钨酸溶液中磷钨酸的质量浓度为1-10％；
    每100mL所述FAA固定液的组成如下：
    50％乙醇水溶液90mL；
    冰乙酸5mL；
    甲醛5mL。
  4. 根据权利要求书1-3任一项所述的方法，其特征在于：步骤2)中，所述摇床处理的条件如下：
    温度为4℃；
    时间为7.5-8.5h；
    转速为40-80转/min。
  5. 根据权利要求书1-4中任一项所述的方法，其特征在于：步骤2)中，所述固定和染色处理的时间按照如下进行：
    当所述高淀粉含量种子为水稻种子、紫米种子、燕麦种子、糯米种子 或红米种子，所述固定和染色处理的时间为39-41天；
    当所述高淀粉含量种子为小米种子，所述固定和染色处理的时间为30-32天；
    当所述高淀粉含量种子为高梁种子，所述固定和染色处理的时间为58-60天。
  6. 根据权利要求书1-5中任一项所述的方法，其特征在于：步骤3)中，所述脱水处理采用乙醇水溶液进行；
    所述脱水处理依次采用体积分数为70％、80％、90％和100％的乙醇水溶液进行梯度脱水，各处理10-20分钟。
  7. 根据权利要求书1-6中任一项所述的方法，其特征在于：步骤4)中，所述显微CT扫描的条件如下：
    分辨率为0.56-1.15μm；
    扫描电压为40-100kV；
    扫描步长为0.2°；
    每个角度均成像2幅。
  8. 根据权利要求书7所述的方法，其特征在于：步骤4)中，所述显微CT扫描的分辨率和扫描电压按照如下进行：
    对于水稻种子，所述显微CT扫描的分辨率为0.88μm，扫描电压为100kV；
    对于紫米种子，所述显微CT扫描的分辨率为0.95μm，扫描电压为100kV；
    对于小米种子，所述显微CT扫描的分辨率为0.68μm，扫描电压为41kV；
    对于燕麦种子，所述显微CT扫描的分辨率为0.88μm，扫描电压为100kV；
    对于糯米种子，所述显微CT扫描的分辨率为0.88μm，扫描电压为100kV；
    对于高梁种子，所述显微CT扫描的分辨率为1.15μm，扫描电压为100kV；
    对于红米种子，所述显微CT扫描的分辨率为0.74μm，扫描电压为 100kV。
  9. 根据权利要求书1-8中任一项所述的方法，其特征在于：在进行所述步骤1)之前，将所述高淀粉含量种子的种皮切破。
PCT/CN2023/076745 2023-01-17 2023-02-17 一种增强高淀粉含量种子显微ct对比度的方法 WO2024152399A1 (zh)

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