A method for quickly cleaning fresh pollen secretions
By using fixation with a fixative solution and ultrasonic cleaning with phosphate buffer and ethanol aqueous solution, the problem of cleaning fresh pollen secretions was solved, ensuring the integrity of pollen structure and clarity of patterns, and providing high-quality scanning electron microscope images.
Patent Information
- Application Number
- CN202210432093.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-04-22
- Publication Date
- 2025-08-01
- Estimated Expiration
- 2042-04-22
AI Technical Summary
Existing technologies cannot quickly and effectively clean fresh pollen secretions without damaging the morphology and structure of the pollen, resulting in the inability to clearly observe pollen patterns under a scanning electron microscope.
After fixing fresh anthers with fixative, they were rinsed with phosphate buffer and ultrasonically cleaned with ethanol aqueous solution. After multiple dehydration and heat treatments, the integrity of the pollen structure was ensured, and secretions were thoroughly removed.
It achieves complete preservation of pollen morphology and structure and thorough removal of secretions, providing clear images for observing pollen ornamentation and meeting the needs of taxonomists.
Smart Images
Figure CN114894591B_ABST
Abstract
Description
Technical Field:
[0001] The present invention belongs to the field of electron microscopes, and particularly relates to a method for quickly cleaning fresh pollen secretions. Background Art:
[0002] Plant pollen is a unique structure of seed plants, and pollen ornamentation is of great significance in taxonomy. However, the pollen walls of some plants secrete mucus that completely covers the pollen ornamentation, and the true pollen morphological structure cannot be seen at all under a scanning electron microscope, which brings a lot of troubles to taxonomists. Since the fresh pollen excretes mucus that will wrap the pollen completely, dealing with these secretions for fresh plant pollen is a very difficult experiment. First, it is necessary to ensure that the pollen morphological structure does not deform and the outer wall ornamentation is not damaged, and at the same time, the secretions need to be removed completely. At present, there is no effective method for quickly cleaning fresh pollen secretions. Summary of the Invention:
[0003] The purpose of the present invention is to provide a method for quickly cleaning fresh pollen secretions.
[0004] The method for quickly cleaning fresh pollen secretions of the present invention is to fix fresh flowers with a fixing solution, then open the anthers to take pollen, wash it with a phosphate buffer solution, and then dehydrate to obtain a scanning electron microscope sample. The characteristic is that the pollen washed with the phosphate buffer solution is ultrasonically cleaned several times with an ethanol aqueous solution after partial dehydration, then heated at a constant temperature, then replaced with an ethanol aqueous solution, ultrasonically cleaned several times, and then subjected to subsequent dehydration to obtain a pre-treated scanning electron microscope sample.
[0005] Preferably, the pollen washed with the phosphate buffer solution is dehydrated with ethanol aqueous solutions with a volume fraction of 30% and 50%, and then 70% ethanol aqueous solution is added for ultrasonic cleaning three times. The first time is 10 min, the second time is 5 min, and the third time is 3 min. Each time, the supernatant is centrifuged and discarded, and new 70% ethanol aqueous solution is added. Then it is heated at 50 °C for 2 h, and then ultrasonically cleaned 2 times, each time for 3 min, and the supernatant is centrifuged and discarded each time, and new 70% ethanol aqueous solution is added. Then it is dehydrated with 80% ethanol aqueous solution, 90% ethanol aqueous solution, and 100% ethanol to obtain a pre-treated scanning electron microscope sample.
[0006] Preferably, the fixing of the fresh flowers with the fixing solution is to put the fresh anthers into the FAA fixing solution, evacuate and then fix for more than 12 h.
[0007] Preferably, the addition of 70% ethanol aqueous solution for ultrasonic cleaning is carried out three times with different times.
[0008] Preferably, the addition of new 70% ethanol aqueous solution needs to be heated.
[0009] Preferably, the heating at 50°C for 2 h is heating in a constant-temperature metal bath at 50°C for 2 h.
[0010] Preferably, after the heating, add a new 70% ethanol aqueous solution by volume again and ultrasonically clean it twice.
[0011] The rinsing with phosphate buffer solution is to rinse three times with 0.1 M phosphate buffer solution, 20 min each time, and centrifuge for 5 min at 5000 rpm each time.
[0012] The dehydration of the pollen rinsed with phosphate buffer solution with 30% and 50% ethanol aqueous solutions by volume is dehydration with 30% and 50% ethanol aqueous solutions by volume, 10 min each time.
[0013] The subsequent dehydration with 80% ethanol aqueous solution by volume, 90% ethanol aqueous solution by volume, and 100% ethanol is dehydration with 80% ethanol aqueous solution by volume for 10 min each time, 90% ethanol aqueous solution by volume for 10 min each time, and 100% ethanol for 5 times, 10 min each time.
[0014] The present invention can not only ensure that the pollen morphology and structure remain unchanged and the outer wall ornamentation is not damaged, but also clean the secretions completely. It provides high-quality pictures for taxonomists and is of great significance in taxonomy. Description of the drawings:
[0015] Figure 1 It is a scanning electron microscope image of the pollen morphological structure of Lactuca indica L. var. ludens (Maxim.) C. Shih, directly coated on the stage without treatment;
[0016] Figure 2 It is a scanning electron microscope image of the pollen morphological structure of Lactuca indica L. var. ludens (Maxim.) C. Shih, by conventional methods;
[0017] Figure 3 It is a scanning electron microscope image of the pollen morphological structure of Lactuca indica L. var. ludens (Maxim.) C. Shih, by the method of the present invention;
[0018] Figure 4 It is a scanning electron microscope image of the pollen morphological structure of Costus argenteus Wall., directly coated on the stage without treatment;
[0019] Figure 5 It is a scanning electron microscope image of the pollen morphological structure of Costus argenteus Wall., by conventional methods;
[0020] Figure 6 It is a scanning electron microscope image of the pollen morphological structure of Costus argenteus Wall., by the method of the present invention;
[0021] Figure 7 It is a scanning electron microscope image of the pollen morphological structure of Lactuca sativa L., directly coated on the stage without treatment;
[0022] Figure 8 It is a scanning electron microscope image of the morphological structure of lettuce flower pollen, using conventional methods;
[0023] Figure 9 It is a scanning electron microscope image of the morphological structure of lettuce flower pollen, using the method of the present invention;
[0024] Figure 10 It is a scanning electron microscope image of the morphological structure of Yunnan lettuce flower pollen, directly coated on the stage without treatment;
[0025] Figure 11 It is a scanning electron microscope image of the morphological structure of Yunnan lettuce flower pollen, using conventional methods;
[0026] Figure 12 It is a scanning electron microscope image of the morphological structure of Yunnan lettuce flower pollen, using the method of the present invention;. Specific implementation manners:
[0027] The following examples are some illustrations of the present invention and should not be regarded as limitations of the present invention.
[0028] Example 1:
[0029] Select 4 Zingiberaceae plant pollens with the most mucilage secreted on the exine and unclear ornamentation as test materials, as shown in Table 1.
[0030] Table 1 Names and collection locations of experimental materials
[0031]
[0032]
[0033] I. Collect fresh anthers in the field and put them into FAA fixative (since the samples are collected in the field for a long time and cannot be fixed with the conventional scanning electron microscope fixative glutaraldehyde, the main reason is that glutaraldehyde requires storage in a 4°C refrigerator and the samples collected in the field do not meet the requirements, so FAA fixative is selected).
[0034] II. The following steps are completed in the laboratory after collection:
[0035] 1. Take fresh anthers and put them into fresh FAA fixative, evacuate and fix for another 12 h.
[0036] 2. Open the anthers and place the pollen in a 1.5 ml centrifuge tube. Rinse three times with 0.1 M phosphate buffer (20 min each time), centrifuge for 5 min at 5000 rpm each time.
[0037] 3. Dehydrate with ethanol at 30% and 50% for 10 min each time, centrifuge for 5 min at 5000 rpm, pour off the supernatant and add 70% ethanol.
[0038] 4. Conduct ultrasonic cleaning three times with 70% ethanol. The first time is for 10 minutes, the second time is for 5 minutes, and the third time is for 3 minutes. Centrifuge each time (5000 rpm), pour off the supernatant, and add fresh 70% ethanol.
[0039] 5. Heat at 50 °C in a thermostatic metal bath for 2 hours.
[0040] 6. Conduct ultrasonic cleaning twice, each time for 3 minutes. Centrifuge each time (5000 rpm), pour off the supernatant, and add fresh 70% ethanol.
[0041] 7. Continue with serial dehydration: 80% ethanol for 10 minutes, 90% ethanol for 10 minutes, and 100% ethanol 5 times, each time for 10 minutes. Transfer to a special sample basket.
[0042] 8. Perform critical point drying and coat the stage.
[0043] 9. Deposit 10 nm of platinum using an ion sputtering instrument.
[0044] 10. Observe and take pictures using a scanning electron microscope.
[0045] III. Instrument Numbers Used:
[0046] 1. Ultrasonic: WIGGENS 59HZ 250W
[0047] 2. Centrifuge: eppendorf Centrifuge 5425
[0048] 3. Thermostatic metal bath: JS-400A
[0049] 3. Critical point dryer: LEICA EM CPD300
[0050] 4. Ion sputtering instrument: LEICA EM ACE600
[0051] 5. Scanning electron microscope: JEOL JSM-6360LV
[0052] IV. The main steps of this method are steps 4, 5, and 6. The other steps are conventional methods.
[0053] 1. In step 4, ultrasonic cleaning is performed three times with different durations each time. The 10-minute duration for the first time is to ultrasonically remove some secreted mucus. The gradually shortened durations for the second and third times are to prevent excessive ultrasonic damage to the pollen outer wall structure. Repeatedly replacing 70% ethanol is to pour out the cleaning waste liquid and allow the fresh 70% ethanol to clean better. Ultrasonic cleaning in 70% ethanol has the best effect because 70% ethanol has a certain fixing effect on cell tissues, and 70% ethanol is also used in the preparation of FAA fixative.
[0054] 2. Step 5 is heating. The most crucial part is to heat at 50°C for 2 hours. If the time is too long, the pollen morphological structure will be damaged, and if it is too short, the secretion cannot be completely removed. It is preferred to control the temperature at 50°C and the time at 2 hours just to avoid damaging the pollen morphological structure. So far, there has been no literature reporting a method of using fresh plant pollen heating to remove secretions for sample preparation.
[0055] 3. Step 6 is cleaning. Ultrasonic cleaning is carried out twice again to better clean the pollen secretions more thoroughly.
[0056] To verify that the efficiency of this invented method is very good, the following experimental comparisons are carried out:
[0057] 1. The untreated pollen is directly adhered to the stage;
[0058] 2. Treated by the conventional method, that is, without heating treatment, directly dehydrated with 70% ethanol without cleaning, which means steps 4, 5, and 6 of the present invention are omitted compared.
[0059] The results of the comparative scanning electron microscope images are as follows.
[0060] 1. The pollen of Costus tonkinensis. As Figure 1 shown, the morphological structure of the untreated Costus tonkinensis pollen directly coated on the stage: belonging to the genus Costus of the family Costaceae, the pollen is adhered to each other, and the secretion contaminates the ornamentation. As Figure 2 shown, the morphological structure of the Costus tonkinensis pollen treated by the conventional method: the pollen secretion is not completely removed, and the pollen morphological structure is deformed. As Figure 3 shown, the morphological structure of the Costus tonkinensis pollen treated by the treatment method of the present invention: the effect is very obvious and clean, the pollen has long spines, spiral grooves, and the smooth surface at the bottom has clear ornamentation. It provides high-quality pictures for taxonomists and has important significance in taxonomy.
[0061] 2. The pollen of Costus argenteus. As Figure 4 shown, the morphological structure of the untreated Costus argenteus pollen directly coated on the stage: belonging to the genus Costus of the family Costaceae, the pollen is adhered to each other, and the secretion contaminates the ornamentation. As Figure 5 shown, the morphological structure of the Costus argenteus pollen treated by the conventional method: the pollen secretion is not completely removed, and the pollen morphological structure is deformed. As Figure 6 shown, the morphological structure of the Costus argenteus pollen treated by the treatment method of the present invention: the effect is very obvious and clean, the pollen has tubercles, long spiral narrow grooves, and the smooth surface at the bottom has clear ornamentation. It provides high-quality pictures for taxonomists and has important significance in taxonomy.
[0062] 3. The pollen of Costus speciosus. As Figure 7 shown, the morphological structure of the untreated Costus speciosus pollen directly coated on the stage: belonging to the genus Costus of the family Costaceae, the pollen is adhered to each other, and the secretion contaminates the ornamentation. As Figure 8As shown, the morphological structure of the pollen of Lactuca sativa var. angustana treated by the conventional method: the pollen secretion was not completely removed, and the morphological structure of the pollen was deformed. As Figure 9 As shown, the morphological structure of the pollen of Lactuca sativa var. angustana treated by the method of the present invention: the effect is very obvious and clean, the pollen is smooth, with long grooves, short grooves, and the surface ornamentation of the thin-walled area is clear. It provides high-quality pictures for taxonomists and is of great significance in taxonomy.
[0063] 4. Pollen of Lactuca sativa var. angustana in Yunnan. As Figure 10 As shown, the morphological structure of the pollen of Lactuca sativa var. angustana in Yunnan directly smeared on the stage without treatment: belonging to the genus Lactuca in the family Costaceae, the pollen grains are adhered to each other, and the secretion contaminates the ornamentation. As Figure 11 As shown, the morphological structure of the pollen of Lactuca sativa var. angustana in Yunnan treated by the conventional method: the pollen secretion was not completely removed, and the morphological structure of the pollen was deformed. As Figure 12 As shown, the morphological structure of the pollen of Lactuca sativa var. angustana in Yunnan treated by the method of the present invention: the effect is very obvious and clean, the pollen is tuberculate, with long and wide grooves, and the surface ornamentation at the bottom is clear. It provides high-quality pictures for taxonomists and is of great significance in taxonomy.
Claims
1. A method for quickly cleaning fresh pollen secretions, characterized in that, After fixing fresh flower anthers with a fixing solution, open the anthers to obtain pollen, wash it with phosphate buffer solution. The pollen washed with phosphate buffer solution is dehydrated with 30% and 50% ethanol aqueous solutions by volume fraction, and then 70% ethanol aqueous solution by volume fraction is added for ultrasonic cleaning three times, 10 minutes for the first time, 5 minutes for the second time, and 3 minutes for the third time. Each time, centrifuge and pour off the supernatant, add a new 70% ethanol aqueous solution by volume fraction, then heat at 50 °C for 2 hours, and then perform ultrasonic cleaning 2 times, 3 minutes each time. Each time, centrifuge and pour off the supernatant and add a new 70% ethanol aqueous solution by volume fraction. Then, dehydrate it with 80% ethanol aqueous solution, 90% ethanol aqueous solution, and 100% ethanol by volume fraction according to the conventional method, and perform critical point drying to obtain a pre-treated scanning electron microscope sample.
2. The method for rapidly cleaning fresh pollen secretions according to claim 1, wherein, The fixation of fresh flower anthers with a fixing solution mentioned above means putting fresh anthers into FAA fixing solution, evacuating and fixing for more than 12 hours.
3. The method for quickly cleaning fresh pollen secretions according to claim 1, characterized in that, The rinsing with phosphate buffer solution mentioned above means rinsing three times with 0.1M phosphate buffer solution, 20 minutes each time, and centrifuging for 5 minutes at 5000 rpm each time.
4. The method for quickly cleaning fresh pollen secretions according to claim 1, characterized in that, The dehydration of the pollen rinsed with phosphate buffer solution with 30% and 50% ethanol aqueous solutions by volume fraction mentioned above means dehydrating with 30% and 50% ethanol aqueous solutions by volume fraction, 10 minutes each time, centrifuging for 5 minutes at 5000 rpm, pouring off the supernatant and adding 70% ethanol.
5. The method for quickly cleaning fresh pollen secretions according to claim 1, characterized in that, The heating at 50 °C for 2 hours mentioned above means heating at 50 °C for 2 hours in a constant temperature metal bath.
6. The method for quickly cleaning fresh pollen secretions according to claim 1, characterized in that, The dehydration with 80% ethanol aqueous solution, 90% ethanol aqueous solution, and 100% ethanol by volume fraction mentioned above means dehydrating with 80% ethanol aqueous solution for 10 minutes each time, 90% ethanol aqueous solution for 10 minutes each time, and 100% ethanol for 5 times, 10 minutes each time.