Preparation method of scanning electron microscope sample for observing microscopic characteristics of lower surface of Artemisia leaf plant

Abstract

The invention relates to a preparation method of a scanning electron microscope sample for observing microscopic characteristics of the lower surface of a folium artemisiae argyi plant, and can effectively solve the problems that the epidermis is difficult to tear and dissociate due to the fact that the folium artemisiae argyi plant is a paper leaf and is thin, even if non-glandular hair on the lower surface can be successfully removed and the epidermis can be separated out, the epidermis cannot be separated out, and the microscopic characteristics of the lower surface of the folium artemisiae argyi plant cannot be observed. In order to solve the problem that the microscopic characteristics of the stem and the glandular hair of the T-shaped non-glandular hair are difficult to observe due to the removal of the glandular hair and the whole T-shaped non-glandular hair, the technical scheme for solving the problem is as follows: the method comprises the following steps of: 1) shearing leaves; 2) methanol fixation; the preparation method comprises the following steps of 1, sample preparation, 2, sample preparation, 3, chloral hydrate treatment, 4, phosphate buffer solution rinsing, 5, pectinase solution treatment, 6, ultrasonic treatment, 7, rinsing and 8, sheet sealing. The preparation method is simple in operation, low in cost, small in workload, high in success rate and clear in observation, observation of microscopic characteristics of the lower surfaces of various folium artemisiae argyi plants can be successfully achieved, and the preparation method is an innovation of the preparation method of the artemisia folium artemisiae argyi plant structure observation sample.

Description

Technical Field

[0001] The invention relates to the field of plant microscopic identification and observation, in particular to a scanning electron microscope sample preparation method for observing microscopic features of the lower surface of leaves of Artemisia wormwood plants. Background Art

[0002] Mugwort is a commonly used Chinese medicine, produced in most parts of the country. Mugwort is dried and crushed to make "mugwort wool", which is used to make moxa sticks for moxibustion. There are more than ten kinds of plants in the Artemisia genus, including wild mugwort, Mongolian mugwort, May mugwort, Artemisia selengensis, southern mugwort, Que mugwort, northern mugwort, and broad-leaved mountain mugwort. The lower surface of their leaves is densely covered with non-glandular hairs like mugwort, so they are often used as counterfeits or substitutes of mugwort. These similar plants to mugwort also have medicinal or edible value. So far, the research on the leaf epidermis characteristics of mugwort plants is not in-depth enough. In the relevant fields at home and abroad, the microscopic identification of the leaves of mugwort plants is still very difficult. Some characteristics of the leaf epidermis, such as the type and density of hairs, stomatal type, stomatal index, and the pattern of the periclival wall of epidermal cells, are of great reference value for the identification of mugwort plants. The common feature of these Artemisia wormwood plants represented by Artemisia argyi is that the lower surface is densely covered with extremely long T-shaped non-glandular hairs on the apical cells. These non-glandular hairs cover a large number of glandular hairs, while the upper surface often has only a small number of T-shaped non-glandular hairs. The glandular hairs on the lower surface of the leaves of Artemisia wormwood plants and the microscopic characteristics of the apical cells and stalks of the T-shaped non-glandular hairs have important reference value in identifying Artemisia wormwood plants.

[0003] Observation of leaf epidermal characteristics using an optical microscope During the preparation of leaf epidermal sections, it is difficult to tear off and dissociate the epidermis because the leaves of mugwort plants are papery and thin. Even if the non-glandular hairs on the lower surface can be successfully removed and the epidermis can be separated, the stomata and epidermal cells can be observed. However, due to the removal of the glandular hairs and the entire T-shaped non-glandular hairs (including the apical cells and stalks), it is difficult to observe the microscopic characteristics of the stalks and glandular hairs of the T-shaped non-glandular hairs. Compared with the optical microscope, the scanning electron microscope can observe the stereoscopic characteristics and ultra-fine details of the glandular hairs, non-glandular hairs, stomata and epidermal cells on the leaf surface. These microscopic characteristics are very valuable for the microscopic identification of mugwort leaves. However, it is easier to observe the upper epidermis of mugwort leaves using a scanning electron microscope; while the lower epidermis is densely covered with non-glandular hairs and covers the glandular hairs, stomata and epidermal cells distributed below it. Therefore, when the lower surface is directly observed using a scanning electron microscope, only the apical cells of the T-shaped non-glandular hairs can be seen. Therefore, it is very necessary to design a sample preparation method specifically for observing the microscopic features of the lower surface of the leaves of mugwort plants, which can be used to observe the glandular hairs, T-shaped non-glandular hair stalks, stomata, epidermal cells, etc. on the lower surface using a scanning electron microscope, but there has been no public report so far. Summary of the invention

[0004] In view of the above situation, in order to solve the defects of the prior art, the purpose of the present invention is to provide a scanning electron microscope sample preparation method for observing the microscopic features of the lower surface of the leaves of Artemisia wormwood plants, which can effectively solve the problem that since Artemisia wormwood plants have papery leaves and thin leaves, it is difficult to tear off and dissociate the epidermis. Even if the non-glandular hairs on the lower surface can be successfully removed and the epidermis can be separated, it will be difficult to observe the microscopic features of the stalk and glandular hairs of the T-shaped non-glandular hairs due to the removal of the glandular hairs and the entire T-shaped non-glandular hairs (including the apical cells and the stalk).

[0005] The technical solution provided by the present invention is a method for preparing a scanning electron microscope sample for observing the microscopic features of the lower surface of leaves of Artemisia sphaerocephala plants, comprising the following steps: 1) Cut leaves: Cut mature leaves from Artemisia spp. and cut 5-10mm thick leaves without thick veins on both sides of the midrib of the leaf. 2 blade; 2) Methanol fixation: Place the cut leaves in methanol for 24-48 hours; 3) Chloral hydrate treatment: transfer the fixed leaves into 50%-60% chloral hydrate solution and soak for 1-3 hours; 4) Phosphate buffer rinse: Transfer the leaves to pH 4.0 phosphate buffer and rinse for 5-15 minutes; 5) Pectinase solution treatment: Take out the rinsed leaves and soak them in a 0.1%-0.5% pectinase solution prepared with a phosphate buffer solution with a pH of 4.0 at 45-50°C for 2-3 hours; 6) Ultrasonic treatment: The leaves were removed from the pectinase solution and transferred to a container containing a phosphate buffer solution with a pH of 4.0. A layer of emery cloth was placed at the bottom of the container. The lower surface of the leaves was in close contact with the emery cloth. The container was ultrasonically treated in an ultrasonic cleaning machine for 30-60 minutes. 7) Rinse: Remove the leaves from the phosphate buffer and rinse them with phosphate buffer at pH 4.0 for 1-2 minutes; 8) Sealing: Add the rinsed leaves to 30%, 50%, 70%, 85%, 95%, and 100% ethanol in turn, treat for 10-30 minutes at each concentration, then treat with isoamyl acetate for 10-30 minutes, dry with a carbon dioxide critical point dryer, spray with gold, and observe with a scanning electron microscope.

[0006] The invention has the advantages of simple operation, low cost, small workload, high success rate, clear observation and good effect. It can satisfactorily solve the observation of microscopic features of the lower surfaces of leaves of various mugwort plants, and can observe the glandular hairs, stalks of T-shaped non-glandular hairs, stomata, epidermal cells and the like on the lower surface under a scanning electron microscope. It does not require high operation technology and can achieve ideal effects. It is an innovation in the sample preparation method for structural observation of Artemisia mugwort plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 This is a scanning electron microscope observation example of the lower surface of Artemisia argyi of the present invention. DETAILED DESCRIPTION

[0008] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. Example 1

[0009] The present invention may further include the following steps when implemented: 1) Cut leaves: Cut mature leaves from Artemisia argyi plants, and cut 5mm thick leaves without thick veins on both sides of the midrib of the Artemisia argyi leaves. 2 blade; 2) Methanol fixation: The cut Artemisia argyi leaves were placed in methanol for fixation for 24 hours; 3) Chloral hydrate treatment: transfer the fixed Artemisia argyi leaves into 50% chloral hydrate solution and soak for 1 hour; 4) Phosphate buffer rinse: Transfer the leaves to pH 4.0 phosphate buffer and rinse for 5 minutes; 5) Pectinase solution treatment: Take out the rinsed Artemisia argyi leaves and soak them in a 0.1% pectinase solution prepared with phosphate buffer at pH 4.0 at 45°C for 2 hours; 6) Ultrasonic treatment: Take the Artemisia argyi leaves out of the pectinase solution and transfer them to a container containing a phosphate buffer solution with a pH of 4.0. A layer of coarse sandpaper is placed at the bottom of the container. The lower surface of the Artemisia argyi leaves is in close contact with the coarse sandpaper. The container is ultrasonically treated in an ultrasonic cleaning machine for 30 minutes. The ultrasonic frequency is 33-40kHz, the power is 100-400W, and the temperature is controlled below 50°C. 7) Rinse: Remove the Artemisia argyi leaves from the phosphate buffer and rinse them with phosphate buffer at pH 4.0 for 1 min; 8) Sealing: Add the rinsed Artemisia argyi leaves into 30%, 50%, 70%, 85%, 95% and 100% ethanol in turn, treat for 10 minutes at each concentration, then treat with isoamyl acetate for 10 minutes, dry with a carbon dioxide critical point dryer, spray with gold, and observe with a scanning electron microscope. Example 2

[0010] The present invention may further include the following steps when implemented: 1) Cut leaves: Cut mature leaves from the Artemisia mongolica plant, and cut 8mm thick leaves without thick veins on both sides of the midrib of the Artemisia mongolica leaf. 2 blade; 2) Methanol fixation: The cut Artemisia mongolica leaves were placed in methanol for fixation for 36 hours; 3) Chloral hydrate treatment: transfer the fixed Artemisia mongolica leaves into 55% chloral hydrate solution and soak for 2 hours; 4) Phosphate buffer rinse: Transfer the leaves of Artemisia mongolica to phosphate buffer with a pH of 4.0 and rinse for 10 minutes; 5) Pectinase solution treatment: Take out the rinsed Artemisia mongolica leaves and soak them in a 0.3% pectinase solution prepared with a phosphate buffer solution with a pH of 4.0 at 48°C for 2.5 hours; 6) Ultrasonic treatment: The leaves of Artemisia mongolica were taken out from the pectinase solution and transferred to a container containing a phosphate buffer solution with a pH of 4.0. A layer of coarse sandpaper was placed at the bottom of the container. The lower surface of the leaves of Artemisia mongolica was in close contact with the coarse sandpaper. The container was ultrasonically treated in an ultrasonic cleaning machine for 45 minutes. The ultrasonic frequency was 33-40kHz, the power was 100-400W, and the temperature was controlled below 50°C. 7) Rinse: Remove the leaves of Artemisia mongolica from the phosphate buffer and rinse them with phosphate buffer at pH 4.0 for 1.5 min; 8) Sealing: Add the rinsed Artemisia mongolica leaves into 30%, 50%, 70%, 85%, 95% and 100% ethanol in turn, treat for 20 minutes at each concentration, then treat with isoamyl acetate for 20 minutes, dry with a carbon dioxide critical point dryer, spray with gold, and observe with a scanning electron microscope. Example 3

[0011] The present invention may further include the following steps when implemented: 1) Cut leaves: Cut mature leaves from the broad-leaved Artemisia plant, and cut 10mm thick leaves without thick veins on both sides of the midrib of the broad-leaved Artemisia leaf. 2 blade; 2) Methanol fixation: The cut Artemisia latifolia leaves were placed in methanol for fixation for 48 hours; 3) Chloral hydrate treatment: Transfer the fixed Artemisia latifolia leaves into 60% chloral hydrate solution and soak for 3 hours; 4) Phosphate buffer rinse: Transfer the leaves of Artemisia latifolia to phosphate buffer with a pH of 4.0 and rinse for 15 minutes; 5) Pectinase solution treatment: Take out the rinsed Artemisia latifolia leaves and soak them in a 0.5% pectinase solution prepared with phosphate buffer at pH 4.0 at 50°C for 3 hours; 6) Ultrasonic treatment: Take the Artemisia latifolia leaves out of the pectinase solution and transfer them to a container containing a phosphate buffer solution with a pH of 4.0. Place a layer of coarse sandpaper at the bottom of the container. The lower surface of the Artemisia latifolia leaves is facing downward and in close contact with the coarse sandpaper. The container is ultrasonically treated in an ultrasonic cleaning machine for 60 minutes. The ultrasonic frequency is 33-40kHz, the power is 100-400W, and the temperature is controlled below 50°C. 7) Rinse: Take out the leaves of Artemisia latifolia from the phosphate buffer and rinse them with phosphate buffer with pH 4.0 for 2 minutes; 8) Sealing: Add the rinsed Artemisia latifolia leaves into 30%, 50%, 70%, 85%, 95% and 100% ethanol in turn, treat for 30 minutes at each concentration, then treat with isoamyl acetate for 30 minutes, dry with a carbon dioxide critical point dryer, spray with gold, and observe with a scanning electron microscope.

[0012] The scanning electron microscope sample preparation method for observing the microscopic features of the lower surface of the leaves of Artemisia sphaerocephala plants of the present invention can be used to remove the top cells of the T-shaped non-glandular hairs on the lower surface of the leaves of Artemisia sphaerocephala plants, and then after dehydration, drying and gold spraying, the glandular hairs, the stalks of the T-shaped non-glandular hairs, the stomata, and the epidermal cells on the lower surface can be directly observed by a scanning electron microscope. The scanning electron microscope sample processing method for the leaves of Artemisia sphaerocephala plants densely covered with T-shaped non-glandular hairs has four key technologies: first, using methanol as a fixative can maintain the morphological features of the leaf epidermis; second, transferring the methanol fixation to a chloral hydrate solution liquid, which is beneficial to maintain the characteristic morphology of the leaf surface; thirdly, after the chloral hydrate treatment, it is transferred to a phosphate buffer solution with a pH of 4.0 to remove the chloral hydrate, while maintaining the leaf surface morphology as a transition to the subsequent aqueous solution; fourthly, under the conditions of pH 4.0 and 45-50°C, after the leaves are fully treated with pectinase, the ultrasonic treatment vibration generates a force on the top cells of the T-shaped non-glandular hairs on the lower surface, and at the same time, under the friction force generated by the coarse sandpaper particles, the top cells can be removed, exposing the glandular hairs on the lower surface, the stalks of the T-shaped non-glandular hairs, stomata, epidermal cells, etc.

[0013] In the experiment, the present invention adopts a variety of plant leaves with dense hairs, such as Artemisia argyi, Artemisia argyi, Artemisia latifolia, etc., and takes Example 1 as an example. After repeated trials and experiments, the results show that the sample processing method can remove the top cells of T-shaped non-glandular hairs, expose the glandular hairs on the lower surface, the stalks of T-shaped non-glandular hairs, stomata, epidermal cells, etc., and at the same time can maintain the morphological characteristics of these structures on the leaf surface (such as Figure 1As shown), the existing technical problems are solved satisfactorily. At the same time, other embodiments are also used to observe mugwort and Artemisia mongolica, and the same or similar results are obtained, indicating that the method is stable and reliable, and has a strong use value. Moreover, after experiments and field applications, less materials are required, reagents are saved, and the cost is reduced by more than 90%. It is very easy to operate and the success rate is as high as more than 99%, achieving very satisfactory technical effects. It can effectively solve the sample processing problems encountered in clearly observing the lower surface of mugwort plants with a scanning electron microscope. It is widely used in the morphological observation and research of the leaf epidermis of various mugwort plants. It is of great value for the classification, identification, plant ecology, plant physiology and other morphological and anatomical research related to the research institutes of plants and medicinal plants. It is a major innovation in the sample preparation method for the structural observation of Artemisia wormwood plants, and has significant economic and social benefits.

[0014] It should be pointed out that the above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession can make changes or modify the technical contents disclosed above into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A method for preparing a scanning electron microscope sample for observing the microscopic features of the lower surface of leaves of Artemisia sphaerocephala plants, characterized in that: The following steps are involved: 1) Cut leaves: Cut mature leaves from Artemisia spp. and cut 5-10mm thick leaves without thick veins on both sides of the midrib of the leaf. 2 blade; 2) Methanol fixation: Place the cut leaves in methanol for 24-48 hours; 3) Chloral hydrate treatment: transfer the fixed leaves into 50%-60% chloral hydrate solution and soak for 1-3 hours; 4) Phosphate buffer rinse: Transfer the leaves to pH 4.0 phosphate buffer and rinse for 5-15 minutes; 5) Pectinase solution treatment: Take out the rinsed leaves and soak them in a 0.1%-0.5% pectinase solution prepared with a phosphate buffer solution with a pH of 4.0 at 45-50°C for 2-3 hours; 6) Ultrasonic treatment: The leaves were removed from the pectinase solution and transferred to a container containing a phosphate buffer solution with a pH of 4.

0. A layer of emery cloth was placed at the bottom of the container. The lower surface of the leaves was in close contact with the emery cloth. The container was ultrasonically treated in an ultrasonic cleaning machine for 30-60 minutes. 7) Rinse: Remove the leaves from the phosphate buffer and rinse them with phosphate buffer at pH 4.0 for 1-2 minutes; 8) Sealing: Add the rinsed leaves to 30%, 50%, 70%, 85%, 95%, and 100% ethanol in turn, treat for 10-30 minutes at each concentration, then treat with isoamyl acetate for 10-30 minutes, dry with a carbon dioxide critical point dryer, spray with gold, and observe with a scanning electron microscope.

2. The method for preparing a scanning electron microscope sample for observing the microscopic features of the lower surface of leaves of Artemisia sphagnum mugwort plants according to claim 1, characterized in that: The following steps are involved: 1) Cut leaves: Cut mature leaves from Artemisia argyi plants, and cut 5mm thick leaves without thick veins on both sides of the midrib of the Artemisia argyi leaves. 2 blade; 2) Methanol fixation: The cut Artemisia argyi leaves were placed in methanol for fixation for 24 hours; 3) Chloral hydrate treatment: transfer the fixed Artemisia argyi leaves into 50% chloral hydrate solution and soak for 1 hour; 4) Phosphate buffer rinse: Transfer the leaves to pH 4.0 phosphate buffer and rinse for 5 minutes; 5) Pectinase solution treatment: Take out the rinsed Artemisia argyi leaves and soak them in a 0.1% pectinase solution prepared with phosphate buffer at pH 4.0 at 45°C for 2 hours; 6) Ultrasonic treatment: Take the Artemisia argyi leaves out of the pectinase solution and transfer them to a container containing a phosphate buffer solution with a pH of 4.

0. A layer of coarse sandpaper is placed at the bottom of the container. The lower surface of the Artemisia argyi leaves is in close contact with the coarse sandpaper. The container is ultrasonically treated in an ultrasonic cleaning machine for 30 minutes. The ultrasonic frequency is 33-40kHz, the power is 100-400W, and the temperature is controlled below 50°C. 7) Rinse: Remove the Artemisia argyi leaves from the phosphate buffer and rinse them with phosphate buffer at pH 4.0 for 1 min; 8) Sealing: Add the rinsed Artemisia argyi leaves into 30%, 50%, 70%, 85%, 95% and 100% ethanol in turn, treat for 10 minutes at each concentration, then treat with isoamyl acetate for 10 minutes, dry with a carbon dioxide critical point dryer, spray with gold, and observe with a scanning electron microscope.

3. The method for preparing a scanning electron microscope sample for observing the microscopic features of the lower surface of leaves of Artemisia sphaerocephala plants according to claim 1, characterized in that: The following steps are involved: 1) Cut leaves: Cut mature leaves from the Artemisia mongolica plant, and cut 8mm thick leaves without thick veins on both sides of the midrib of the Artemisia mongolica leaf. 2 blade; 2) Methanol fixation: The cut Artemisia mongolica leaves were placed in methanol for fixation for 36 hours; 3) Chloral hydrate treatment: transfer the fixed Artemisia mongolica leaves into 55% chloral hydrate solution and soak for 2 hours; 4) Phosphate buffer rinse: Transfer the leaves of Artemisia mongolica to phosphate buffer with a pH of 4.0 and rinse for 10 minutes; 5) Pectinase solution treatment: Take out the rinsed Artemisia mongolica leaves and soak them in a 0.3% pectinase solution prepared with a phosphate buffer solution with a pH of 4.0 at 48°C for 2.5 hours; 6) Ultrasonic treatment: Take the Artemisia mongolica leaves out of the pectinase solution and transfer them to a container containing a phosphate buffer solution with a pH of 4.

0. Place a layer of emery cloth at the bottom of the container. The lower surface of the Artemisia mongolica leaves is in close contact with the emery cloth. The container is ultrasonically treated in an ultrasonic cleaning machine for 45 min. The ultrasonic frequency is 33-40 kHz, the power is 100-400 W, and the temperature is controlled below 50°C. 7) Rinse: Remove the leaves of Artemisia mongolica from the phosphate buffer and rinse them with phosphate buffer at pH 4.0 for 1.5 min; 8) Sealing: Add the rinsed Artemisia mongolica leaves into 30%, 50%, 70%, 85%, 95% and 100% ethanol in turn, treat for 20 minutes at each concentration, then treat with isoamyl acetate for 20 minutes, dry with a carbon dioxide critical point dryer, spray with gold, and observe with a scanning electron microscope.

4. The method for preparing a scanning electron microscope sample for observing the microscopic features of the lower surface of leaves of Artemisia sphagnum mugwort plants according to claim 1, characterized in that: The following steps are involved: 1) Cut leaves: Cut mature leaves from the broad-leaved Artemisia plant, and cut 10mm thick leaves without thick veins on both sides of the midrib of the broad-leaved Artemisia leaf. 2 blade; 2) Methanol fixation: The cut Artemisia latifolia leaves were placed in methanol for fixation for 48 hours; 3) Chloral hydrate treatment: Transfer the fixed Artemisia latifolia leaves into 60% chloral hydrate solution and soak for 3 hours; 4) Phosphate buffer rinse: Transfer the leaves of Artemisia latifolia to phosphate buffer with a pH of 4.0 and rinse for 15 minutes; 5) Pectinase solution treatment: Take out the rinsed Artemisia latifolia leaves and soak them in a 0.5% pectinase solution prepared with phosphate buffer at pH 4.0 at 50°C for 3 hours; 6) Ultrasonic treatment: Take the Artemisia latifolia leaves out of the pectinase solution and transfer them to a container containing a phosphate buffer solution with a pH of 4.

0. Place a layer of coarse sandpaper at the bottom of the container. The lower surface of the Artemisia latifolia leaves is facing downward and in close contact with the coarse sandpaper. The container is ultrasonically treated in an ultrasonic cleaning machine for 60 minutes. The ultrasonic frequency is 33-40kHz, the power is 100-400W, and the temperature is controlled below 50°C. 7) Rinse: Take out the leaves of Artemisia latifolia from the phosphate buffer and rinse them with phosphate buffer with pH 4.0 for 2 minutes; 8) Sealing: Add the rinsed Artemisia latifolia leaves into 30%, 50%, 70%, 85%, 95% and 100% ethanol in turn, treat for 30 minutes at each concentration, then treat with isoamyl acetate for 30 minutes, dry with a carbon dioxide critical point dryer, spray with gold, and observe with a scanning electron microscope.

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