Method for observing glandular hairs of aina by scanning electron microscope

Abstract

The application provides a method for observing glandular hairs of Ainsliaea sinensis by using a scanning electron microscope. The method comprises the following steps: (1) selecting a region to be measured in a leaf blade, cutting a sample, and immersing the sample in an aldehyde fixing solution for fixation and immersion; (2) performing gradient dehydration by using an ethanol solution, performing replacement by using isopentyl acetate, and then performing drying by using a critical point drying instrument; (3) removing non-glandular hairs by using adhesive tape; and (4) then spraying gold and observing under a scanning electron microscope. By using the method, the observation and distribution density statistics of the glandular hairs of Ainsliaea sinensis can be simply, efficiently and accurately completed. By using the method, the problem of uneven leaf blade is well solved, and the problem of non-glandular hairs shielding and interfering with the visual field is completely solved.

Description

Technical Field

[0001] This invention relates to the field of plant observation, and in particular to a method for observing glandular hairs of *Erigeron davidii* using a scanning electron microscope. Background Technology

[0002] *Artemisia annua* is an aromatic medicinal plant, with its aroma primarily originating from its leaves. The leaf surface is covered with numerous non-glandular and glandular hairs. Glandular hairs are an important secretory structure in *Artemisia annua*, closely related to aroma release, biological resistance, and the accumulation of aromatic substances. Therefore, observing the morphology and distribution density of glandular hairs in *Artemisia annua* leaves is of significant practical and research value. However, the non-glandular hairs covering the leaf surface of *Artemisia annua* are long and dense, and the leaves are wavy. These two factors have long hindered the accurate and comprehensive observation of the morphology and density of glandular hairs. Existing techniques for observing epidermal hairs in *Artemisia annua* have fundamental flaws. First, regarding the problem of non-glandular hairs obscuring and interfering with glandular hairs, the old technique involved ventilating the plant in a cool, shaded place for a period of time to allow the non-glandular hairs to dry and shrink. Although the non-glandular hairs shrink in this method, they still exist and severely interfere with the observation and statistical analysis of glandular hairs. When the density of non-glandular hairs is very high and they completely cover the area, even if the non-glandular hairs shrink, they still obstruct the field of vision, making it impossible to see the glandular hairs. Secondly, given the wavy and undulating nature of the leaves of *Artemisia argyi*, the depth of field of ordinary lenses is less than the range of leaf undulation, resulting in some unclear fields of vision and making it impossible to fully count the density of glandular hairs within the field of vision. The old technique of first air-drying the leaves and then using a pressing ring to flatten them not only exacerbates the wrinkling during the air-drying process and increases the difficulty of flattening, but also, due to the highly brittle nature of *Artemisia argyi* leaves after drying, the leaves break during the forced flattening process, making it impossible to obtain a completely clear field of vision.

[0003] Specifically, CN105466931A discloses a portable device and a method for rapidly assessing the quality of *Artemisia* var. *marginata*. The method involves "first ventilating the leaves in a cool, shaded place for a period of time until the non-glandular hairs on the leaf surface dry and shrink." While ventilation in the shade causes the non-glandular hairs to dry and shrink, they still exist and significantly interfere with the observation and counting of glandular hairs. When the density of non-glandular hairs is high and they completely cover the plant, even after drying and shrinking, they still cover the glandular hairs, making them invisible and impossible to count. CN205608304U discloses a portable device for assessing epidermal hairs in *Artemisia* var. *marginata* plants. However, because the obtained images are cross-sectional, only the epidermal hairs can be observed from the cross-section, lacking leaf area data, making it impossible to calculate and record glandular hair density. The paper "Observation and Study of Glandular Hair Morphology of Four Lamiaceae Plants Based on Field Emission Scanning Electron Microscopy" published by Liu Ruohan et al. uses conventional scanning electron microscopy to observe the glandular hairs on the surface of leaves of peppermint, patchouli, spearmint, and dandelion. The method observed that the glandular hairs of the plants were not covered by a large number of non-glandular hairs, and there were no problems with leaf undulation or fragility after drying. Therefore, no special operation is required. In addition, it can be seen from the results photos in the paper that the epidermal cells in the samples were all wrinkled and deformed, and the original morphology of the cells could not be well displayed. This method is not suitable for leaves of dandelion, which are covered by non-glandular hairs, have undulating leaves, and are brittle and fragile after drying. Summary of the Invention

[0004] In view of this, the present invention proposes a method for observing glandular hairs of *Ipomoea aquatica* using a scanning electron microscope, which allows for clearer observation of leaf glandular hairs and effective calculation and recording of glandular hair density.

[0005] The technical solution of this invention is implemented as follows:

[0006] A method for observing glandular hairs of *Echinochloa crus-galli* using scanning electron microscopy includes the following steps:

[0007] (1) Select the area near the vein in the middle of the leaf of *Artemisia argyi*, cut out the sample, and immerse it in aldehyde fixative for fixation.

[0008] (2) After being dehydrated by gradient ethanol solution, isoamyl acetate was replaced and then dried using a critical point dryer. Liquid carbon dioxide was used to replace isoamyl acetate in the sample, and then the carbon dioxide was changed from liquid to critical state to gas and released from the sample to achieve the drying effect.

[0009] (3) Use tape to stick off non-glandular hair;

[0010] (4) Then spray gold and observe it under a scanning electron microscope.

[0011] Furthermore, the sample size is (0.4~1.2)×(0.4~1.2)cm.

[0012] Furthermore, the fixed soaking time is 20-30 hours, preferably 24 hours.

[0013] Furthermore, the aldehyde fixative is an FAA fixative or a paraformaldehyde fixative.

[0014] Furthermore, the FAA fixative is a 50% to 70% FAA fixative.

[0015] Furthermore, the paraformaldehyde fixative is a 1% to 8% paraformaldehyde fixative.

[0016] Furthermore, the solubility of the ethanol solution is 70 v / v% to 100 v / v.

[0017] Furthermore, the gradient dehydration time is 7 to 14 hours.

[0018] Furthermore, the concentrations of the gradient dehydrated ethanol solution are 70 v / v%, 80 v / v%, 85 v / v%, 90 v / v%, 95 v / v%, 100 v / v% (first time), and 100 v / v% (second time), with each concentration lasting 1 to 2 hours.

[0019] Furthermore, the outgassing rate of the critical point dryer is controlled to be ≤2 ml / s, and the drying time of the critical point dryer is 2 to 4 hours, preferably 3 to 4 hours, and more preferably 3 hours. Controlling the outgassing rate allows the sample to dry gently and slowly, avoiding excessively rapid changes in liquid carbon dioxide from liquid to gaseous in delicate structures such as epidermal cells, non-glandular trichomes, and glandular trichomes, which could damage the surface structure due to surface tension.

[0020] Furthermore, the specific procedure for using adhesive tape to adhere non-glandular hairs is as follows: Select transparent adhesive tape, pinch one end of the adhesive side of the tape with your thumb and forefinger, spreading the adhesive side of the tape outward in an Ω shape, and then use the protruding part to approach and adhere the non-glandular hairs. The advantage of this method is that when the adhesive side of the tape is in an Ω shape near the sample, the elastic arc formed by the tape provides a buffer during the transmission of force from the hand to the sample, thus allowing for more precise and gentle control of the adhesion force. The conventional method is to directly press the smooth side of the tape with your fingers, pressing the adhesive side against the sample; this method can easily damage the sample.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] (1) The method of this invention, which employs the procedure of "fixation first, slow dehydration, then hair adhesion, followed by gold spraying and scanning electron microscopy," greatly optimizes the observation effect of glandular hairs in *Erigeron davidii*, enabling simple, efficient, and accurate statistical analysis of glandular hair distribution density. This method not only effectively solves the problem of uneven leaves in *Erigeron davidii*, but also completely eliminates the problem of non-glandular hairs obstructing the field of view. Furthermore, it overcomes the issues of shallow depth of field in optical microscopes and tissue deformation caused by conventional scanning electron microscopy procedures.

[0023] (2) This invention uses a gradient alcohol soaking followed by critical point drying to fully dehydrate and dry the material, making the non-glandular hairs fully and completely dry and brittle. This changes the old method of simply "drying the leaves in a cool, ventilated place for a period of time until the non-glandular hairs on the leaf surface dry and shrink." The purpose of the old method is to shrink the non-glandular hairs and reduce their occlusion. The purpose of this invention is to make the non-glandular hairs brittle, so that they can be completely removed. At the same time, the remaining structure does not deform or wrinkle, which can well display the morphology of the glandular hairs and other epidermal cells.

[0024] (3) This invention utilizes the characteristics of non-glandular hairs being brittle, long, and easily broken after dehydration. After sufficient dehydration, non-glandular hairs are gently adhered to the dry environment immediately using adhesive tape, leaving the remnants of non-glandular hairs and exposing short glandular hairs, which greatly improves the observation effect of the glandular hairs of Artemisia argyi. Attached Figure Description

[0025] Figure 1 Scanning electron microscope images before and after treatment with adhesive tape to remove non-glandular hairs in this embodiment of the invention;

[0026] Figure 2 Scanning electron microscope images of glandular hairs of *Echinochloa chinensis* observed by the method of Example 1 and Comparative Example 1 of this invention.

[0027] Figure 3 The effect of different fixation methods on observation results (scanning electron microscope images);

[0028] Figure 4 The effect of different dehydration methods on the observation results (scanning electron microscope images);

[0029] Figure 5 The effect of using and not using tape on the observation results (scanning electron microscope image). Detailed Implementation

[0030] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0031] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0033] The 70% FAA fixative and 4% paraformaldehyde fixative used in the embodiments of this invention are both commercially available products.

[0034] The critical point dryer used in this embodiment of the invention is a Quorum K850.

[0035] Example 1

[0036] A method for observing glandular hairs of Echinocactus glaucus using scanning electron microscopy

[0037] First fix the fibers, then slowly dehydrate them, then attach the fibers, and finally spray with gold and use a scanning electron microscope.

[0038] 1. Specific operations:

[0039] (1) Collect fresh leaves of Artemisia argyi, select the area near the vein in the middle of the leaf, cut out a 1×1cm square, and immerse it in 70v / v% FAA fixative for 24 hours;

[0040] (2) The sample was dehydrated for 1 hour at each of the following concentrations using a gradient of ethanol solution (v / v = 70%, 80%, 85%, 90%, 95%, 100%, 100%). For the second 100% concentration, a fresh anhydrous ethanol solution was used. After 1 hour of replacement with isoamyl acetate, the sample was dried for 3 hours using a critical point dryer. The outgassing rate was controlled to be ≤2 ml / s to ensure that the organic solvent was fully replaced during the immersion in the solution and to achieve dynamic balance between the intake and exhaust during the purification process. This also prevented the liquid carbon dioxide in the fine structures such as epidermal cells, non-glandular hairs, and glandular hairs from changing from liquid to gas too quickly, which could damage the surface structure due to surface tension.

[0041] (3) To use adhesive tape to stick non-glandular hairs, the specific procedure is as follows: Select transparent adhesive tape, pinch one end of the adhesive side of the tape with your thumb and forefinger, so that the adhesive side of the tape spreads outward in an Ω shape, and use the protruding part to approach and stick the non-glandular hairs. The advantage of this method is that when the adhesive side of the tape is in an Ω shape and approaches the sample, the force of the hand is buffered by the arc formed by the tape during the process of being transmitted to the sample, so the sticking force can be controlled more precisely. The conventional method is to directly press the smooth side of the tape with your fingers, so that the adhesive side is pressed against the sample. This operation method is prone to damaging the sample.

[0042] (4) Then spray gold and observe under a scanning electron microscope to count the number of glandular hairs.

[0043] The leaves of *Ipomoea aquatica* have a high density of non-glandular hairs, which can easily obscure the glandular hairs. This invention adds a hair removal step before gold spraying, which involves gently and repeatedly sticking and tearing tape onto the leaf surface to remove the non-glandular hairs and expose the glandular hairs. This allows for observation and counting of the number of glandular hairs. By combining the area of ​​the same field of view, the glandular hair density can be calculated as: glandular hair density = number of glandular hairs / area.

[0044] like Figure 1 As shown, Figure 1 A: Non-glandular hair obscuring the view under scanning electron microscopy; Figure 1 B: Diagram of hair removal procedure; Figure 1 C: Scanning electron microscopy reveals all glandular hairs clearly visible across the entire field of view after hair removal; Figure 1 D: Non-glandular hair obscuring the view under scanning electron microscopy; Figure 1 E, F: Clear and well-formed glandular hairs exposed under a scanning electron microscope after hair removal.

[0045] Comparative Example 1 - Observation Method of Optical Stereomicroscope

[0046] Fresh leaves of Artemisia argyi were collected, dried in the shade, and then pressed flat (see CN 105466931 A). They were then photographed directly using an optical stereomicroscope.

[0047] Specific operation: Dry the leaves of Artemisia argyi in a cool and ventilated place for 10 hours. Place the dried leaves on the cutting ring and put the lens fixing tube on top so that the leaves are positioned between the lens and the pressure ring.

[0048] II. Observation Results

[0049] The observation results of the method of the present invention are compared with those of the optical stereomicroscope observation method as shown in Table 1 below:

[0050] Table 1. Observation effects of the method of the present invention and the observation method of optical stereomicroscope.

[0051]

[0052] This invention not only effectively solves the problem of uneven leaf surfaces but also completely eliminates the issue of non-glandular hairs obstructing the field of view. This technique only requires slowing down the drying process and adding a step of using adhesive tape to adhere the non-glandular hairs, based on the conventional scanning electron microscope sample processing procedure, to achieve excellent observation results. The required tape is simple and readily available. Therefore, this technique enables the observation and distribution density statistics of glandular hairs in *Aegilops henryi* to be completed simply, efficiently, and accurately.

[0053] In addition, the leaf cutting size is (0.4~1.2)×(0.4~1.2)cm, the fixation time with fixative is 20~30h, the replacement with isoamyl acetate is 1~48h, the dehydration time of each concentration of gradient ethanol solution is 1~2h, and the drying time is 2~4h. Based on Example 1, adjustments within the above ranges can achieve the purpose of the present invention.

[0054] Comparative study examples

[0055] Fresh leaves of Artemisia argyi from the same batch were collected, and the effects of fixation method, drying method, and hair removal on the observation results were studied.

[0056] 1. The effect of fixation (using scanning electron microscopy)

[0057] Comparative Study Example 1-1: After air drying, rapid dehydration was performed. No tape was used to stick the non-glandular hairs. After gold spraying, the images were taken directly using a scanning electron microscope. Other treatments were the same as in Example 1.

[0058] Comparative Study Examples 1-2: After fixation with 70% FAA fixative, the hairs were rapidly dehydrated. No tape was used to adhere the non-glandular hairs. After gold spraying, the hairs were directly photographed using a scanning electron microscope. Other treatments were the same as in Example 1.

[0059] Comparative Studies Examples 1-3: After fixation with 4% paraformaldehyde fixative, the hairs were rapidly dehydrated. No tape was used to adhere the non-glandular hairs. After gold spraying, the hairs were directly photographed using a scanning electron microscope. Other treatments were the same as in Example 1.

[0060] The specific operation for the above rapid dehydration is as follows: Dehydration is performed for 15 minutes at each concentration of ethanol solution gradient (v / v = 70%, 80%, 85%, 90%, 95%, 100%).

[0061] Table 2 shows the results of the study on the effects of different fixation methods combined with rapid dehydration on the observed effects.

[0062]

[0063] like Figure 3 As shown, where, Figure 3 A shows that after air drying, routine rapid dehydration causes severe deformation of non-glandular hairs, and when the density of non-glandular hairs is high, they obstruct the field of vision, making it impossible to see glandular hairs.

[0064] Figure 3 B shows that although FAA fixation was performed and glandular trichomes could be identified, routine rapid dehydration still caused severe deformation of non-glandular trichomes, glandular trichomes, and the epidermis, failing to properly display the original morphology of the tissues and cells. Figure 3As can be seen from D, although it was fixed by FAA, routine rapid dehydration caused severe tissue deformation, and when the density of non-glandular trichomes was high, it obstructed the field of vision, making it impossible to see the glandular trichomes.

[0065] Figure 3 As can be seen from C, although it was fixed with 4% paraformaldehyde, the conventional rapid dehydration still caused severe deformation of non-glandular trichomes, glandular trichomes and epidermis, and could not well show the original morphology of tissues and cells.

[0066] 2. Effect of dehydration rate (using scanning electron microscopy)

[0067] Comparative Study Example 2-1: After fixation with 70% FAA fixative, rapid dehydration was performed, i.e., dehydration with ethanol solution gradient (v / v = 70%, 80%, 85%, 90%, 95%, 100%) for 15 minutes at each concentration, followed by rapid drying with a critical point dryer for 30 minutes, with an evaporation rate ≥10 ml / s. No tape was used to stick the non-glandular hairs. After gold sputtering, the images were taken directly using a scanning electron microscope. Other treatments were the same as in Example 1.

[0068] Comparative Study Example 2-2: After fixation with 4% paraformaldehyde fixative, the hair was slowly dehydrated using a gradient dehydration method and slowly dried using a critical point dryer as in Example 1. No tape was used to stick the non-glandular hairs. After gold spraying, the hairs were directly photographed using a scanning electron microscope. Other treatments were the same as in Example 1.

[0069] Comparative Study Examples 2-3: After fixation with 70% FAA fixative, the hairs were slowly dehydrated using a gradient dehydration method and then slowly dried using a critical point dryer, as in Example 1. No tape was used to adhere the non-glandular hairs. After gold sputtering, the hairs were directly photographed using a scanning electron microscope. Other treatments were the same as in Example 1.

[0070] Table 3 shows the results of the study on the effect of dehydration rate on the observed effects.

[0071]

[0072] like Figure 4 As shown, where,

[0073] Figure 4 As can be seen from A, even with FAA fixation, rapid dehydration still leads to tissue shrinkage;

[0074] Figure 4 B shows that the tissue morphology remained intact after fixation with 4% paraformaldehyde and slow dehydration.

[0075] Figure 4 C shows that the tissue morphology remained intact after FAA fixation and slow dehydration;

[0076] Figure 4As can be seen from D, after FAA fixation and slow dehydration, the tissue morphology remains intact. However, when the density of non-glandular trichomes is high, the glandular trichomes cannot be seen due to obstruction, so it is impossible to observe and count the glandular trichomes.

[0077] 3. The effects of using tape for hair removal

[0078] Comparative Study Example 3-1: No tape was used to stick the non-glandular hairs, and other treatments were the same as in Example 1.

[0079] Comparative Study Example 3-2: Same as Example 1.

[0080] Table 4. Results of the study on the effect of using tape on the observation results.

[0081]

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for observing glandular hairs of *Echinochloa crus-galli* using a scanning electron microscope, characterized in that, Includes the following steps: (1) Select the area to be tested from the leaves of Artemisia argyi, cut out the sample, and immerse it in aldehyde fixative for fixation; (2) After dehydration with gradient ethanol solution and replacement with isoamyl acetate, the product is dried using a critical point dryer; (3) Use tape to stick off non-glandular hairs; (4) Then sputter gold and observe under a scanning electron microscope; The concentrations of the gradient ethanol solution are 70v / v%, 80v / v%, 85v / v%, 90v / v%, 95v / v%, 100v / v%, and 100v / v, that is, 100v / v% dehydration twice, each concentration for 1 to 2 hours; The fixation soaking time is 20-30 hours; the aldehyde fixative is FAA fixative or paraformaldehyde fixative. The venting rate of the critical point dryer should be ≤2ml / s; The critical point dryer has a drying time of 2-4 hours. The specific operation of using tape to stick non-glandular hair is as follows: Select transparent tape, pinch one end of the adhesive side of the tape with your thumb and forefinger, so that the adhesive side of the tape is spread outward in an Ω shape, and use the protruding part to approach and stick the non-glandular hair.

2. The method for observing glandular hairs of *Echinochloa crus-galli* using a scanning electron microscope according to claim 1, characterized in that... The sample size is (0.4~1.2)×(0.4~1.2)cm.

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