CN116067741A - Method for enhancing high-starch-content seed micro CT contrast - Google Patents
Method for enhancing high-starch-content seed micro CT contrast Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 26
- 230000002708 enhancing effect Effects 0.000 title claims abstract description 9
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 claims abstract description 69
- 229920002472 Starch Polymers 0.000 claims abstract description 54
- 235000019698 starch Nutrition 0.000 claims abstract description 54
- 239000008107 starch Substances 0.000 claims abstract description 54
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 46
- 238000002591 computed tomography Methods 0.000 claims abstract description 30
- 239000001569 carbon dioxide Substances 0.000 claims abstract description 23
- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 23
- 238000004043 dyeing Methods 0.000 claims abstract description 22
- 238000001035 drying Methods 0.000 claims abstract description 4
- 238000002791 soaking Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 117
- 240000007594 Oryza sativa Species 0.000 claims description 47
- 235000007164 Oryza sativa Nutrition 0.000 claims description 47
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 38
- 235000009566 rice Nutrition 0.000 claims description 38
- 208000005156 Dehydration Diseases 0.000 claims description 14
- 230000018044 dehydration Effects 0.000 claims description 14
- 238000006297 dehydration reaction Methods 0.000 claims description 14
- 239000000834 fixative Substances 0.000 claims description 13
- 244000062793 Sorghum vulgare Species 0.000 claims description 12
- 235000019713 millet Nutrition 0.000 claims description 12
- 240000006394 Sorghum bicolor Species 0.000 claims description 11
- 235000011684 Sorghum saccharatum Nutrition 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 11
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 9
- 235000007189 Oryza longistaminata Nutrition 0.000 claims description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 4
- 229960000583 acetic acid Drugs 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 238000010186 staining Methods 0.000 claims description 2
- 238000000352 supercritical drying Methods 0.000 abstract description 18
- XQPRBTXUXXVTKB-UHFFFAOYSA-M caesium iodide Chemical compound [I-].[Cs+] XQPRBTXUXXVTKB-UHFFFAOYSA-M 0.000 description 14
- 238000012360 testing method Methods 0.000 description 8
- 240000004922 Vigna radiata Species 0.000 description 6
- 235000010721 Vigna radiata var radiata Nutrition 0.000 description 6
- 235000011469 Vigna radiata var sublobata Nutrition 0.000 description 6
- 238000010306 acid treatment Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
- 230000003321 amplification Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
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- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000010603 microCT Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
Images
Classifications
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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
- 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
Abstract
The invention discloses a method for enhancing the micro CT contrast of seeds with high starch content. The method for enhancing the micro CT contrast of the seeds with high starch content provided by the invention comprises the following steps: 1) Dissolving phosphotungstic acid in FAA fixing solution to obtain a phosphotungstic acid solution; 2) Soaking the seeds with high starch content to be detected in the phosphotungstic acid solution, carrying out shaking table treatment, and then carrying out fixing and dyeing treatment; 3) Sequentially dehydrating and drying the seeds with high starch content subjected to the fixing and dyeing treatment in the step 2) at a critical point of carbon dioxide; 4) And (3) carrying out micro CT scanning on the seeds with high starch content treated in the step (3). According to the invention, the pretreatment of the shaking table is combined with the treatment means of immersing the seeds with high starch content by adopting the phosphotungstic acid solution, the treatment conditions are optimized, and then the carbon dioxide critical point drying is carried out, so that the microscopic CT contrast ratio of the seeds with high starch content is greatly improved.
Description
Technical Field
The invention relates to a method for enhancing the micro CT contrast of seeds with high starch content, belonging to the field of biotechnology.
Background
The usual method for studying the morphological structure of seed starch or endosperm cells is to slice the seed in the filling stage, and observe the exposed starch grains and endosperm cell structure under an optical microscope. However, the mature seeds are extremely difficult to manufacture into slices due to high hardness, so that how to directly observe the arrangement state of endosperm cells of seeds with high starch content without damaging the structure of the mature seeds is one of the technical problems encountered in the prior art of researching the morphological structure of the mature seeds.
CN111398000a discloses a method for enhancing the micro CT contrast of high starch content seeds, which can directly observe the morphological structure of seeds without damaging the structure of mature seeds, but is only applicable to seeds with lower starch content, such as mung bean seeds (starch content is not more than 50%); for seeds with high starch content, the internal density of the seeds is uniform, so that the transmittance of rays is not different, the starch part of the reconstructed picture is uniform, and the structure cannot be distinguished at all. There is therefore a need for a method that effectively enhances the micro-CT contrast of high starch seed.
Disclosure of Invention
The invention aims to provide a method for enhancing the microscopic CT contrast of high-starch-content seeds, which combines the pretreatment of a shaking table with the treatment of immersing the high-starch-content seeds by using a phosphotungstic acid solution, optimizes the treatment conditions, and then carries out carbon dioxide critical point drying, thereby greatly improving the microscopic CT contrast of the high-starch-content seeds.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the method for enhancing the micro CT contrast of the seeds with high starch content provided by the invention comprises the following steps:
1) Dissolving phosphotungstic acid in FAA fixing solution to obtain a phosphotungstic acid solution;
2) Soaking the seeds with high starch content to be detected in the phosphotungstic acid solution, carrying out shaking table treatment, and then carrying out fixing and dyeing treatment;
3) Sequentially dehydrating and drying the seeds with high starch content subjected to the fixing and dyeing treatment in the step 2) at a critical point of carbon dioxide;
4) Carrying out micro CT scanning on the seeds with high starch content treated in the step 3);
wherein:
the starch content of the high starch content seed is more than 60 percent;
in step 2), the conditions of the fixing and dyeing treatment are as follows:
the temperature is 4 ℃;
the time is 30-60 days;
during the fixing and dyeing treatment, replacing the phosphotungstic acid solution and carrying out shaking table treatment, and then continuing the fixing and dyeing treatment; the replacement frequency of the phosphotungstic acid solution is that the phosphotungstic acid solution is replaced every two weeks.
The high starch content seed is selected from rice seed (about 70% starch content), purple rice seed (70% starch content), millet seed (70% starch content), oat seed (60% starch content), glutinous rice seed (75% starch), sorghum seed (70% starch content) or red rice seed (60% starch content).
In step 1), the mass concentration of the phosphotungstic acid in the phosphotungstic acid solution is 1-10%, such as 1%, 5% or 10%.
In step 1), the composition of the FAA fixative per 100mL is as follows: 90mL of 50% ethanol aqueous solution; glacial acetic acid 5mL; formaldehyde 5mL.
In the step 2), the conditions of the shaking table treatment are as follows: the temperature is 4 ℃;7.5 to 8.5 hours, preferably 8 hours; the rotation speed is 40-80 rpm, preferably 60 rpm.
In step 2), different fixing and dyeing treatment times are selected for different seeds with high starch content to achieve better contrast; specifically, the time of the fixing and dyeing treatment is performed as follows:
when the high starch content seeds are rice seeds, purple rice seeds, oat seeds, glutinous rice seeds and red rice seeds, the fixing and dyeing treatment time is 39-41 days;
when the high starch content seeds are millet seeds, the fixing and dyeing treatment time is 30-32 days;
when the high starch seed is sorghum seed, the fixing and staining process takes 58-60 days.
In the step 3), the dehydration treatment is carried out by adopting an ethanol water solution; further, the dehydration treatment sequentially adopts ethanol water solutions with volume fractions of 70%, 80%, 90% and 100% to carry out gradient dehydration, and each treatment lasts for 10-20 minutes.
In step 4), the conditions of the micro CT scan are as follows: resolution is 0.56-1.15 μm; the scanning voltage is 40-100kV, preferably 100kV; the scanning step length is 0.2 degrees; each angle imaged 2 frames.
In the step 4), different high starch content seeds are provided with different resolutions and scanning voltages so as to achieve better contrast; the resolution and scan voltage of the micro CT scan are as follows:
for rice seeds, the resolution of the micro CT scanning is 0.88 mu m, and the scanning voltage is 100kV;
for purple rice seeds, the resolution of the micro CT scanning is 0.95 mu m, and the scanning voltage is 100kV;
for millet seeds, the resolution of the micro CT scanning is 0.68 mu m, and the scanning voltage is 41kV;
for oat seeds, the resolution of the micro CT scanning is 0.88 mu m, and the scanning voltage is 100kV;
for glutinous rice seeds, the resolution of the micro CT scanning is 0.88 mu m, and the scanning voltage is 100kV;
for sorghum seeds, the resolution of the micro CT scanning is 1.15 mu m, and the scanning voltage is 100kV;
for red rice seeds, the resolution of the micro CT scanning is 0.74 μm, and the scanning voltage is 100kV.
In the present invention, the seed coat of the high starch content seed is cut open prior to performing the step 1).
The beneficial effects obtained by the invention are as follows:
1. according to the invention, the shaking table treatment means is combined with the phosphotungstic acid solution soaking treatment means, so that the fixation and dyeing effects of the seeds with high starch content are improved, and the microscopic CT contrast ratio of the seeds with high starch content is greatly improved through carbon dioxide critical point drying.
2. The invention further determines the operation conditions of shaking table treatment, fixation and dyeing treatment so as to improve the microscopic CT contrast of the seeds with high starch content.
Drawings
FIG. 1 shows cross sections (from left to right) of samples obtained by treating locally enlarged rice seeds with FAA fixative, 10% cesium iodide, 10% phosphotungstic acid, and 10% phosphotungstic acid, respectively, and drying at critical points.
FIG. 2 shows cross sections (from left to right) of samples obtained by treating locally enlarged purple rice seeds with FAA fixative, 10% cesium iodide, lugos solution, 10% phosphotungstic acid, respectively, and all being critical point dried.
FIG. 3 shows cross sections (from left to right) of samples of millet seeds treated with FAA fixative, 10% cesium iodide, 10% phosphotungstic acid solution, respectively, and all dried at the critical point.
FIG. 4 shows cross sections (from left to right) of samples of oat seed treated with FAA fixative, 10% phosphotungstic acid solution, respectively, and all dried at the critical point.
FIG. 5 shows cross sections (from left to right) of samples of glutinous rice seeds treated with FAA fixative solution and 10% phosphotungstic acid solution, respectively, and all dried at critical points.
FIG. 6 shows cross sections (from left to right) of samples of sorghum seeds treated with FAA fixative, 10% phosphotungstic acid solution, respectively, and all dried at the critical point.
FIG. 7 shows cross sections (from left to right) of samples of red rice seeds treated with FAA fixative and 10% phosphotungstic acid solution, respectively, and all dried at the critical point.
FIG. 8 shows cross sections (from left to right) of samples of mung bean seeds treated with FAA fixative and 10% phosphotungstic acid solution, respectively, and all dried at the critical point.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In the following examples:
the FAA fixative (100 mL) comprises the following components: 90mL of 50% ethanol aqueous solution, 5mL of glacial acetic acid and 5mL of formaldehyde.
Preparing a phosphotungstic acid solution: and dissolving phosphotungstic acid in the FAA fixing solution to obtain a phosphotungstic acid solution with the mass concentration of 10%.
Preparation of cesium iodide solution: cesium iodide was dissolved in the FAA fixative to give a cesium iodide solution with a mass concentration of 10%.
Preparation of Lugos solution: 4g of potassium iodide, 2g I 2 Added to 50ml of FAA fixative to give Lugos solution.
Carbon dioxide critical point drying treatment: the treatment was performed using a critical point dryer (CPD 300, leka).
Micro CT scanning: skyscan1172, bruce, inc. was used.
Test example 1 enhancement treatment of micro CT contrast of Rice seed
Respectively adding rice seeds into FAA fixing solution, 10% cesium iodide solution and 10% phosphotungstic acid solution, reversing the above steps for several times to remove bubbles until the samples are immersed into the solution, shaking the samples on a shaking table at 4 ℃ for 8 hours, rotating the samples for 60 revolutions per minute, and fixing the samples at 4 ℃ for 40 days; the solution is replaced every two weeks, the solution is placed on a shaking table for 8 hours at 4 ℃ after being replaced every two weeks, the rotation speed is 60 r/min, and then the fixation is continued at 4 ℃; then, carrying out gradient ethanol dehydration by adopting 70%, 80%, 90% and 100% ethanol aqueous solutions, carrying out gradient treatment for 20 minutes, and carrying out carbon dioxide critical point drying treatment on the dehydrated sample.
Carrying out microscopic CT scanning on the dried sample, wherein the resolution of the FAA fixing solution, the 10% cesium iodide solution and the 10% phosphotungstic acid solution is 0.88 mu m, the scanning voltages are 40kV, 100kV and 100kV respectively, the local amplification resolution of the 10% phosphotungstic acid solution is 0.56 mu m, and the scanning voltage is 100kV; the scanning step sizes are all 0.2 degrees, and each angle is imaged by 2 frames.
Reconstructing the sample after scanning by using an NRecon software to obtain a cross section picture, as shown in fig. 1, wherein the upper left image is a cross section of the rice seed treated by the FAA fixing solution, the upper right image is a cross section of the rice seed treated by the 10% cesium iodide solution, the lower left image is a cross section of the rice seed treated by the 10% phosphotungstic acid, and the lower right image is a partially amplified cross section of the rice seed treated by the 10% phosphotungstic acid; comparing the four figures, it can be seen that the microscopic CT contrast of the rice seeds after phosphotungstic acid treatment and carbon dioxide critical point drying is obviously improved, and the endosperm cell morphology is clearly visible.
Test example 2 enhancement treatment of micro CT contrast of purple Rice seed
Respectively adding purple rice seeds into FAA fixing solution, 10% cesium iodide solution, lugos solution and 10% phosphotungstic acid solution, reversing the steps up and down for several times to remove bubbles until the samples are immersed into the solution, shaking the samples on a shaking table at 4 ℃ for 8 hours, rotating the samples at 60 revolutions per minute, and fixing the samples at 4 ℃ for 40 days; the solution is replaced every two weeks, the solution is placed on a shaking table for 8 hours at 4 ℃ after being replaced every two weeks, the rotation speed is 60 r/min, and then the fixation is continued at 4 ℃; then, carrying out gradient ethanol dehydration by adopting 70%, 80%, 90% and 100% ethanol aqueous solutions, carrying out gradient treatment for 20 minutes, and carrying out carbon dioxide critical point drying treatment on the dehydrated sample.
Carrying out microscopic CT scanning on the dried sample, wherein the resolution of the FAA fixing solution, the 10% cesium iodide solution, the Lugos solution and the 10% phosphotungstic acid solution is 0.95 mu m, the scanning voltages are respectively 43kV, 100kV and 100kV, the local amplification resolution of the 10% phosphotungstic acid treated sample is 0.56 mu m, and the scanning voltage is 100kV; the scanning step sizes are all 0.2 degrees, and each angle is imaged by 2 frames.
After sample scanning, reconstructing by using an NRecon software to obtain a cross section picture, as shown in fig. 2, wherein the cross section of the purple rice seed treated by the FAA fixing solution, the 10% cesium iodide and the Lugos solution is sequentially arranged from left to right, the cross section of the purple rice seed treated by the 10% phosphotungstic acid solution and the 10% phosphotungstic acid solution is sequentially arranged from left to right and is locally amplified, and compared with the pictures, the microscopic CT contrast of the rice seed treated by the phosphotungstic acid and dried by the carbon dioxide critical point is obviously improved, and the endosperm cell morphology is clearly visible.
Test example 3 enhancement treatment of micro CT contrast of millet seeds
Respectively adding millet seeds into FAA fixing solution, 10% cesium iodide solution and 10% phosphotungstic acid solution, reversing the steps for several times to remove bubbles, immersing the sample in the solution, shaking the sample on a shaking table at 4 ℃ for 8 hours, rotating the sample at 60 revolutions per minute, and fixing the sample at 4 ℃ for 30 days; the solution is replaced every two weeks, the solution is placed on a shaking table for 8 hours at 4 ℃ after being replaced every two weeks, the rotation speed is 60 r/min, and then the fixation is continued at 4 ℃; then, carrying out gradient ethanol dehydration by adopting 70%, 80%, 90% and 100% ethanol aqueous solutions, carrying out gradient treatment for 20 minutes, and carrying out carbon dioxide critical point drying treatment on the dehydrated sample.
The dried samples were subjected to micro CT scanning at a scanning voltage of 41kV, a resolution of 0.68 μm and a scanning step of 0.2℃for each sample, and 2 images were obtained for each angle.
After sample scanning, reconstructing by using an NRecon software to obtain a cross section picture, as shown in fig. 3, wherein the cross section of the millet seeds treated by FAA fixing solution, 10% cesium iodide solution and 10% phosphotungstic acid solution is sequentially from left to right, and comparing the pictures, the microscopic CT contrast of the millet seeds treated by phosphotungstic acid and dried by using carbon dioxide critical points is obviously improved, and the endosperm cell morphology is clearly visible. The time for the dyeing process is also shorter, since the millet seeds are smaller.
Test example 4 enhancement of micro CT contrast of oat seed
Respectively adding oat seeds into FAA fixing solution and 10% phosphotungstic acid solution, reversing the steps for several times to remove bubbles until the samples are immersed into the solution, shaking the solution for 8 hours at 4 ℃ on a shaking table for 60 revolutions per minute, and then fixing the solution at 4 ℃ for 40 days; the solution is replaced every two weeks, the solution is placed on a shaking table for 8 hours at 4 ℃ after being replaced every two weeks, the rotation speed is 60 r/min, and then the fixation is continued at 4 ℃; then, carrying out gradient ethanol dehydration by adopting 70%, 80%, 90% and 100% ethanol aqueous solutions, carrying out gradient treatment for 20 minutes, and carrying out carbon dioxide critical point drying treatment on the dehydrated sample.
And carrying out microscopic CT scanning on the dried sample, wherein the scanning voltage of the FAA fixing solution treated sample is 40kV, the scanning voltage of the 10% phosphotungstic acid treated sample is 100kV, the resolution is 0.88 mu m, the scanning step length is 0.2 degrees, and each angle is imaged for 2 frames.
After sample scanning, reconstructing by using an NRecon software to obtain a cross section picture, as shown in fig. 4, wherein the cross section of the oat seed is sequentially processed by FAA fixing solution and 10% phosphotungstic acid solution from left to right, and comparing the pictures, the microscopic CT contrast of the oat seed after phosphotungstic acid treatment and carbon dioxide critical point drying is obviously improved, and the endosperm cell morphology is clearly visible.
Test example 5 enhancement treatment of micro CT contrast of Oryza Glutinosa seed
Respectively adding glutinous rice seeds into FAA fixing solution and 10% phosphotungstic acid solution, reversing the steps for several times to remove bubbles until the samples are immersed into the solution, shaking the samples on a shaking table at 4 ℃ for 8 hours, rotating the samples at 60 revolutions per minute, and fixing the samples at 4 ℃ for 40 days; the solution is replaced every two weeks, the solution is placed on a shaking table for 8 hours at 4 ℃ after being replaced every two weeks, the rotation speed is 60 r/min, and then the fixation is continued at 4 ℃; then, carrying out gradient ethanol dehydration by adopting 70%, 80%, 90% and 100% ethanol aqueous solutions, carrying out gradient treatment for 20 minutes, and carrying out carbon dioxide critical point drying treatment on the dehydrated sample.
And carrying out microscopic CT scanning on the dried sample, wherein the scanning voltage of the FAA fixing solution treated sample is 43kV, the scanning voltage of the 10% phosphotungstic acid treated sample is 100kV, the resolution is 0.88 mu m, the scanning step length is 0.2 degrees, and each angle is imaged for 2 frames.
After the sample is scanned, the cross section picture is obtained through reconstructing an NRecon software, as shown in fig. 5, wherein the cross section of the glutinous rice seed is sequentially processed by FAA fixing solution and 10% phosphotungstic acid solution from left to right, and compared with the pictures, the microscopic CT contrast of the glutinous rice seed after phosphotungstic acid treatment and carbon dioxide critical point drying is obviously improved, and the endosperm cell morphology is clearly visible.
Test example 6 enhancement treatment of micro CT contrast of sorghum seeds
Respectively putting sorghum seeds (seed coat is cut) into FAA fixing solution and 10% phosphotungstic acid solution, reversing the steps up and down for several times to remove bubbles until the samples are immersed into the solution, shaking the samples on a shaking table at 4 ℃ for 8 hours, rotating the samples for 60 revolutions/min, and fixing the samples at 4 ℃ for 60 days; the solution is replaced every two weeks, the solution is placed on a shaking table for 8 hours at 4 ℃ after being replaced every two weeks, the rotation speed is 60 r/min, and then the fixation is continued at 4 ℃; then, carrying out gradient ethanol dehydration by adopting 70%, 80%, 90% and 100% ethanol aqueous solutions, carrying out gradient treatment for 20 minutes, and carrying out carbon dioxide critical point drying treatment on the dehydrated sample.
And carrying out microscopic CT scanning on the dried sample, wherein the scanning voltage of the sample treated by the FAA fixing solution is 40kV, the scanning voltage of the sample treated by 10% phosphotungstic acid is 100kV, the resolution is 1.15 mu m, the scanning step length is 0.2 DEG, and each angle is imaged for 2 pieces.
After sample scanning, reconstructing by using an NRecon software to obtain a cross section picture, as shown in fig. 6, wherein the cross section of the sorghum seed is sequentially processed by FAA fixing solution and 10% phosphotungstic acid solution from left to right, and comparing the two pictures, it can be seen that the microscopic CT contrast of the sorghum seed after phosphotungstic acid treatment and carbon dioxide critical point drying is obviously improved, and the starchy endosperm cell morphology is clearly visible.
Test example 7 enhancement treatment of micro CT contrast of Red Rice seed
Respectively adding red rice seeds into FAA fixing solution and 10% phosphotungstic acid solution, reversing the steps for several times to remove bubbles until the sample is immersed into the solution, shaking the solution for 8 hours at 4 ℃ on a shaking table, rotating the solution for 60 revolutions per minute, and fixing the solution at 4 ℃ for 40 days; the solution is replaced every two weeks, the solution is placed on a shaking table for 8 hours at 4 ℃ after being replaced every two weeks, the rotation speed is 60 r/min, and then the fixation is continued at 4 ℃; then, carrying out gradient ethanol dehydration by adopting 70%, 80%, 90% and 100% ethanol aqueous solutions, carrying out gradient treatment for 20 minutes, and carrying out carbon dioxide critical point drying treatment on the dehydrated sample.
And carrying out microscopic CT scanning on the dried sample, wherein the scanning voltage of the FAA fixing solution treated sample is 40kV, the scanning voltage of the 10% phosphotungstic acid treated sample is 100kV, the resolution is 0.74 mu m, the scanning step length is 0.2 degrees, and each angle is imaged for 2 pieces.
After sample scanning, reconstructing by using an NRecon software to obtain a cross section picture, as shown in fig. 7, wherein the cross section of the glutinous rice seed is sequentially processed by FAA fixing solution and 10% phosphotungstic acid solution from left to right, and comparing the two pictures, it can be seen that the microscopic CT contrast of the glutinous rice seed after phosphotungstic acid treatment and carbon dioxide critical point drying is obviously improved, and the endosperm cell morphology is clearly visible.
Test example 8 enhancement treatment of micro CT contrast of mung bean seeds
Respectively adding semen Phaseoli Radiati seed into FAA fixing solution and 10% phosphotungstic acid solution, reversing upside down for several times to remove air bubbles until the sample is immersed into the solution, shaking at 4deg.C for 8 hr, 60 rpm, and fixing at 4deg.C for 60 days; the solution is replaced every two weeks, the solution is placed on a shaking table for 8 hours at 4 ℃ after being replaced every two weeks, the rotation speed is 60 r/min, and then the fixation is continued at 4 ℃; then, carrying out gradient ethanol dehydration by adopting 70%, 80%, 90% and 100% ethanol aqueous solutions, carrying out gradient treatment for 20 minutes, and carrying out carbon dioxide critical point drying treatment on the dehydrated sample.
And carrying out microscopic CT scanning on the dried sample, wherein the scanning voltage of the FAA fixing solution treated sample is 40kV, the scanning voltage of the 10% phosphotungstic acid treated sample is 100kV, the resolution is 1.76 mu m, and 0.5mm aluminum sheet is added. The scanning step sizes are all 0.2 degrees, and each angle is imaged by 2 frames.
After sample scanning, reconstructing by using an NRecon software to obtain a cross section picture, as shown in fig. 8, wherein the cross section of the mung bean seeds treated by the FAA fixing solution and the 10% phosphotungstic acid solution is sequentially from left to right, and comparing the two pictures, it can be seen that the microscopic CT contrast of the mung bean seeds treated by the phosphotungstic acid treatment and the carbon dioxide critical point drying is improved, the detail is increased, but the improvement effect is worse than that of the mung bean seeds with high starch content. Therefore, the treatment method disclosed by the invention can obviously improve the microscopic CT contrast of the seeds with high starch content.
The invention also examines the influence of different operation conditions on the enhancement effect of the micro CT contrast of the seeds with high starch content in the research process, and the research result shows that the operation conditions of the steps can obtain higher micro CT contrast.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (9)
1. A method for enhancing the micro CT contrast of high starch seed comprising the steps of:
1) Dissolving phosphotungstic acid in FAA fixing solution to obtain a phosphotungstic acid solution;
2) Soaking the seeds with high starch content to be detected in the phosphotungstic acid solution, carrying out shaking table treatment, and then carrying out fixing and dyeing treatment;
3) Sequentially dehydrating and drying the seeds with high starch content subjected to the fixing and dyeing treatment in the step 2) at a critical point of carbon dioxide;
4) Carrying out micro CT scanning on the seeds with high starch content treated in the step 3);
wherein:
the starch content of the high starch content seed is more than 60 percent;
in step 2), the conditions of the fixing and dyeing treatment are as follows:
the temperature is 4 ℃; the time is 30-60 days;
during the fixing and dyeing treatment, replacing the phosphotungstic acid solution and carrying out shaking table treatment, and then continuing the fixing and dyeing treatment; the replacement frequency of the phosphotungstic acid solution is that the phosphotungstic acid solution is replaced every two weeks.
2. The method according to claim 1, characterized in that: the high starch content seed is selected from rice seed, purple rice seed, millet seed, oat seed, glutinous rice seed, sorghum seed or red rice seed.
3. The method according to claim 1, characterized in that: in the step 1), the mass concentration of the phosphotungstic acid in the phosphotungstic acid solution is 1-10%;
the composition of the FAA fixative per 100mL is as follows:
90mL of 50% ethanol aqueous solution;
glacial acetic acid 5mL;
formaldehyde 5mL.
4. A method according to any one of claims 1-3, characterized in that: in the step 2), the conditions of the shaking table treatment are as follows:
the temperature is 4 ℃;
the time is 7.5-8.5h;
the rotating speed is 40-80 rpm.
5. The method according to any one of claims 1-4, wherein: in step 2), the fixing and dyeing process is performed as follows:
when the high starch content seeds are rice seeds, purple rice seeds, oat seeds, glutinous rice seeds or red rice seeds, the fixing and dyeing treatment time is 39-41 days;
when the high starch content seeds are millet seeds, the fixing and dyeing treatment time is 30-32 days;
when the high starch seed is sorghum seed, the fixing and staining process takes 58-60 days.
6. The method according to any one of claims 1-5, wherein: in the step 3), the dehydration treatment is carried out by adopting an ethanol water solution;
the dehydration treatment sequentially adopts ethanol water solutions with volume fractions of 70%, 80%, 90% and 100% to carry out gradient dehydration, and each treatment lasts for 10-20 minutes.
7. The method according to any one of claims 1-6, wherein: in step 4), the conditions of the micro CT scan are as follows:
resolution is 0.56-1.15 μm;
the scanning voltage is 40-100kV;
the scanning step length is 0.2 degrees;
each angle imaged 2 frames.
8. The method of claim 7, wherein: in step 4), the resolution and scanning voltage of the micro CT scan are performed as follows:
for rice seeds, the resolution of the micro CT scanning is 0.88 mu m, and the scanning voltage is 100kV;
for purple rice seeds, the resolution of the micro CT scanning is 0.95 mu m, and the scanning voltage is 100kV;
for millet seeds, the resolution of the micro CT scanning is 0.68 mu m, and the scanning voltage is 41kV;
for oat seeds, the resolution of the micro CT scanning is 0.88 mu m, and the scanning voltage is 100kV;
for glutinous rice seeds, the resolution of the micro CT scanning is 0.88 mu m, and the scanning voltage is 100kV;
for sorghum seeds, the resolution of the micro CT scanning is 1.15 mu m, and the scanning voltage is 100kV;
for red rice seeds, the resolution of the micro CT scanning is 0.74 μm, and the scanning voltage is 100kV.
9. The method according to any one of claims 1-8, wherein: cutting the seed coat of the high starch content seed prior to performing step 1).
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