A method of observing and / or counting plant leaf trichomes

Abstract

The present application belongs to the field of microscopic observation, and relates to a method for observing and / or counting plant leaf glandular hairs. The method comprises the following steps: flattening a plant leaf, drying, and observing and / or counting using an optical stereomicroscope. The method can well solve the problem of uneven leaf, and also well solve the problem of non-glandular hair blocking the field of view, and realizes efficient observation of plant leaf morphology and density.

Description

Technical Field

[0001] This invention belongs to the field of microscopic observation technology, specifically relating to a method for observing and / or counting glandular hairs on plant leaves. Background Technology

[0002] The leaves are the primary source of aroma in aromatic medicinal plants, and their surfaces are covered with numerous non-glandular and glandular hairs. Glandular hairs are important secretory structures in plants, closely related to aroma release, biological resistance, and the accumulation of aromatic substances. Therefore, observing the morphology and distribution density of glandular hairs in plant leaves is of significant practical and research value. However, some aromatic medicinal plants have long and dense non-glandular hairs covering their leaf surfaces, and their leaves are wavy. These two factors have long hindered the accurate and comprehensive observation of the morphology and density of glandular hairs.

[0003] Regarding the observation of epidermal hairs in *Erigeron asiatica*, to eliminate interference from non-glandular hairs, existing techniques involve ventilating in a cool, shaded place for a period of time to allow the non-glandular hairs to dry and shrink. However, although the non-glandular hairs shrink, they still exist. Whether using a conventional optical stereomicroscope, fluorescence microscope, or scanning electron microscope, the obstruction caused by the non-glandular hairs severely interferes with the observation and counting of glandular hairs. When the density of non-glandular hairs is high and they completely cover the area, even after shrinkage, they still obstruct the field of view, making it impossible to see the glandular hairs. Furthermore, the wavy texture of *Erigeron asiatica* leaves, with the depth of field of a conventional lens being less than the range of leaf undulation, results in unclear views in parts of the field of view, making it impossible to fully count the density of glandular hairs within the field of view. After drying the leaves in the shade, existing techniques also use a pressing ring to flatten the leaves. This not only exacerbates the shrinkage during the drying process, increasing the difficulty of flattening, but also causes the leaves to become brittle, leading to breakage during forced flattening and preventing a completely clear field of view. Another technique is to use field emission scanning electron microscopy to obtain a clear field of view. However, scanning electron microscopes are expensive, sample pretreatment steps are complicated, there is a certain technical threshold, and various reagents and gold sputtering are required. Although a clear field of view can be obtained, the problem of non-glandular hair occlusion cannot be solved, and there are also fundamental defects.

[0004] Therefore, there is still a lack of an efficient method for observing the morphology and distribution density of glandular hairs in plant leaves. Summary of the Invention

[0005] The purpose of this invention is to provide a method for observing and / or counting glandular hairs on plant leaves. The method described in this invention effectively solves the problem of uneven leaves and also effectively addresses the issue of non-glandular hairs obstructing the field of vision, enabling efficient observation of plant leaf morphology and density.

[0006] This invention provides a method for observing and / or counting glandular hairs on plant leaves, comprising the following steps:

[0007] The plant leaves were flattened, dried, and observed and / or statistically analyzed using an optical stereomicroscope.

[0008] Preferably, the leaves of the plant have glandular hairs and non-glandular hairs, and the plant includes sage, sage, tobacco, or nudiflora.

[0009] Preferably, the leaf is selected from the area near the vein in the middle of the leaf.

[0010] Preferably, the flattening process includes using double-sided tape to attach the plant leaves to the flat surface.

[0011] Preferably, the plane includes the plane of the glass slide.

[0012] Preferably, the drying process includes air drying.

[0013] Preferably, the air-drying time is 2 to 48 hours.

[0014] Preferably, the drying process also includes sticking on non-glandular hairs.

[0015] Preferably, the process of removing non-glandular hairs includes using adhesive tape to remove them.

[0016] Preferably, observation is performed using an optical stereo microscope, including:

[0017] After taking pictures using an optical stereo microscope, combine them with the depth-of-field extension function for observation; or take pictures directly using a super-depth-of-field optical stereo microscope for observation.

[0018] This invention provides a method for observing and / or counting glandular hairs on plant leaves. The method involves first flattening the leaf, then drying it, and finally observing it under an optical stereomicroscope, enabling efficient observation and counting of glandular hairs. When the density of glandular hairs is moderate or low, it is not necessary to use tape to remove non-glandular hairs; they can be counted directly. If the density of epidermal hairs is high and non-glandular hairs severely obstruct the field of view, tape can be used to remove non-glandular hairs, exposing the glandular hairs for counting. If, even after flattening and air-drying, the leaf still has unevenness, causing partial blurring, depth-of-field extension or direct use of a super-depth-of-field stereomicroscope can be employed to obtain a completely clear photograph. This method effectively solves the problem of uneven leaves and also effectively addresses the problem of non-glandular hairs obstructing the field of view. The materials used in the experimental operation are simple and readily available, and the method has low equipment requirements, requiring only an optical stereomicroscope. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The images shown are the results obtained by the three methods provided in Embodiment 1 of the present invention. A is the result obtained by processing leaves with low non-glandular hair density using the method described in Group 1-1; B is the result obtained by processing leaves with low non-glandular hair density using the method described in Group 1-2; C is the result obtained by processing leaves with low non-glandular hair density using the method described in Group 1-3 of the present invention; and D is the result obtained by processing leaves with high non-glandular hair density using the method described in Group 1-1.

[0021] Figure 2 This is a schematic diagram of the process of first flattening and then air-drying according to Embodiment 1 of the present invention;

[0022] Figure 3 The images shown are the results obtained by the three methods provided in Embodiment 2 of the present invention. A is the result obtained by processing leaves with low non-glandular hair density using the method described in Group 2-1; B is the result obtained by processing leaves using the method described in Group 2-2; C is the result obtained by processing leaves with low glandular hair density using Group 2-3 of the present invention; and D is the result obtained by processing samples with high non-glandular hair density using the method described in Group 2-1.

[0023] Figure 4 The image shows the effect of using adhesive tape for hair removal provided in Embodiment 3 of the present invention. In this image, A is the result of group 3-1 after processing the sample with high non-glandular hair density, and B is the result of group 3-2 after processing the sample with high non-glandular hair density.

[0024] Figure 5 This is an image showing the effect of using depth-of-field compositing technology on the results provided in Embodiment 4 of the present invention; wherein, A is an image of the result after processing a sample with low non-glandular hair density using the method described in Group 4-1, B is an image of depth-of-field compositing after taking multiple photos of a sample with low non-glandular hair density using the method described in Group 4-2, and C is an image of depth-of-field compositing after taking multiple photos of a sample with high non-glandular hair density using the method described in Group 4-2.

[0025] Figure 6The images show the effects of different methods used to treat the glandular hairs on leaves of *Artemisia argyi* in Example 5 of this invention. Image A shows the result of treating a sample with high non-glandular hair density using the method described in Example 1, Group 1-1 (air-drying followed by flattening, no hair removal, and no depth-of-field extension). Image B shows the result of treating a sample with high non-glandular hair density using the method described in Example 4, Group 4-2 (flattening followed by air-drying, hair removal with tape, and depth-of-field extension). Image C shows the result of treating a sample with medium non-glandular hair density using the method described in Example 4, Group 4-1 (flattening followed by air-drying, no hair removal, and no depth-of-field extension). Detailed Implementation

[0026] This invention provides a method for observing and / or counting glandular hairs on plant leaves, comprising the following steps:

[0027] The plant leaves were flattened, dried, and observed and / or statistically analyzed using an optical stereomicroscope.

[0028] This invention involves flattening plant leaves. The plant leaves have glandular hairs and non-glandular hairs, and the plant preferably includes *Artemisia argyi*, *Sage*, tobacco, or *Callicarpa nudiflora*. The leaves are preferably selected from the area near the central vein of the leaf. More preferably, the leaves are cut into 3cm × 2cm squares. Fresh leaves are preferably collected for this process, and the flattening operation is performed immediately while the leaves are still fresh and flexible. Flattening preferably involves using double-sided tape to attach the plant leaves to a flat surface. When it is necessary to observe the upper surface of the leaf, the lower surface of the leaf is attached to the flat surface using double-sided tape; when it is necessary to observe the lower surface of the leaf, the upper surface of the leaf is attached to the flat surface using double-sided tape. The flat surface preferably includes the plane of a glass slide. The flat surface can also be a sample stage. After flattening, the leaf is preferably gently pressed with the fingertip to ensure it is fully flat, maximizing flatness without breaking or damaging the glandular hairs.

[0029] After flattening, the leaf is dried according to the present invention. In the present invention, the drying preferably includes air drying. In the present invention, the air drying time is preferably 2-48 hours, more preferably 3-24 hours. In the present invention, the humidity during the air drying process is preferably 40-75%. In the present invention, the temperature during the air drying process is preferably 15-28°C. In the present invention, the air drying preferably avoids direct sunlight or other strong light. The drying method of the present invention can make non-glandular hairs brittle. When the drying method of the present invention is combined with the subsequent removal of non-glandular hairs, the non-glandular hairs can be completely removed, achieving a thorough and unobstructed effect. In the present invention, the drying also causes slight dehydration of the mesophyll cells, softening the tissue, which will be stretched and flattened by the adhesion and adsorption of the double-sided tape.

[0030] In this invention, the drying process preferably includes adhering to non-glandular hairs. This adhering preferably involves using adhesive tape. In this invention, after drying (i.e., air-drying and dehydration), the non-glandular hairs are brittle, long, and easily broken. This invention utilizes adhesive tape to adhere to the non-glandular hairs, leaving residual non-glandular hairs and exposing short glandular hairs. In this invention, if the epidermal hair density is high and non-glandular hairs severely obstruct the field of view, adhesive tape is used to adhere the non-glandular hairs, exposing the glandular hairs; if the glandular hair density is moderate or low during observation, it is not necessary to use adhesive tape to adhere the non-glandular hairs, and observation and / or counting can be performed directly. When using adhesive tape to adhere non-glandular hairs, this invention preferably uses repeated application and tearing of the tape on the leaf surface to adhere the non-glandular hairs and expose the glandular hairs, allowing for observation and counting of the number of glandular hairs. This invention, combined with a uniform field of view area, allows for the calculation of glandular hair density.

[0031] In this invention, observation is preferably performed using an optical stereomicroscope, including:

[0032] After taking pictures using an optical stereo microscope, combine them with the depth-of-field extension function for observation; or take pictures directly using a super-depth-of-field optical stereo microscope for observation.

[0033] In this invention, if observation reveals that even after flattening and air-drying, the leaves still exhibit undulations, causing partial blurring of the field of view, a depth-of-field extension can be used, or a super-depth-of-field stereomicroscope can be directly employed to obtain a completely clear photograph, which can then be observed and / or statistically analyzed. Combining the flattening, drying, and hair removal processes of this invention with depth-of-field extension overcomes the problem of shallow depth of field in optical microscopes without depth-of-field extension, which prevents clear observation of leaf curvature and causes blurred vision. It also solves the problem of non-glandular hairs obstructing the field of view. In this invention, the depth-of-field extension preferably includes operation using the depth-of-field extension function in computer software such as Photoshop.

[0034] In this invention, the statistics preferably include statistical analysis of glandular trichome density, and the formula for calculating the glandular trichome density is preferably as shown in Formula I:

[0035] Glandular hair density D = Number of glandular hairs N / Visual field area S = N / (L × W) × 1,000,000 (hairs / mm²) -2 ), formula I.

[0036] The visual field area S = visual field length L (μm) × visual field width W (μm).

[0037] The number of glandular hairs, N (number of hairs), was statistically analyzed using the method of this invention.

[0038] To further illustrate the present invention, a method for observing and / or counting glandular hairs on plant leaves provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] Comparison of eye-level and cross-sectional views

[0041] Fresh leaves of Artemisia argyi were collected and divided into three groups as shown in Table 1. The groups were then treated separately, and photographs were taken after treatment. The results are shown in Table 1.

[0042] Table 1 Comparison of Level View and Cross-Sectional Effects

[0043]

[0044] Figure 1 The images show the results obtained using three different methods. A shows the results obtained after processing leaves with low non-glandular hair density using the method described in Group 1-1. The results indicate that after air-drying and then flattening, the leaves are not flat enough, and some areas of the field of view are blurred, making it impossible to accurately count the glandular hairs. B shows the results obtained after processing using the method described in Group 1-2. The results indicate that after cross-section observation, only epidermal hairs can be observed from the cross-section; due to the lack of leaf area data, the glandular hair density cannot be calculated and recorded. C shows the results obtained after processing using the method of this invention (Group 1-3). The results indicate that after flattening and then air-drying, the entire field of view is clear, and the glandular hairs can be accurately counted for some samples with low non-glandular hair density. D shows the results obtained after processing samples with high non-glandular hair density using the method described in Group 1-1. The results indicate that when the non-glandular hair density is high, simply air-drying without hair removal, although the non-glandular hairs shrink, still obstructs the field of view, making it impossible to count the glandular hairs.

[0045] The above results indicate that the method described in Group 1-1, which first involves "ventilating the leaves in a cool, shaded place for a period of time to allow the non-glandular hairs on the leaf surface to dry and shrink," still results in the non-glandular hairs shrinking and becoming smaller, but they still exist and severely interfere with the observation and counting of glandular hairs. When the density of non-glandular hairs is very high, and they completely cover the leaf, even after drying and shrinking, they still cover the leaf, making it impossible to see or count the glandular hairs. Flattening the leaves using a pressing ring after air-drying not only exacerbates the shrinkage during the drying process, increasing the difficulty of flattening, but also easily causes brittleness, leading to leaf breakage during forced flattening and preventing a completely clear field of view. Furthermore, combining the method described in Group 1-1 with depth-of-field synthesis technology results in image alignment difficulties due to the interference of non-glandular hairs, making it impossible to synthesize a clear super-depth-of-field image.

[0046] The method of the present invention has a greater advantage, as it can obtain a completely clear field of view and accurately count the density of glandular hairs.

[0047] A schematic diagram illustrating the method of applying the adhesive flat and then air-drying according to the present invention is shown below. Figure 2 As shown, A is a frontal view and B is a rear view. According to... Figure 2 It can be seen that the slide area is only related to the area of ​​the mounting medium. Making one slide can meet the needs of multi-point observation and statistics. When the slide area is large enough, the entire leaf can be attached to the slide and the observation and statistics of the glandular hairs of the whole leaf can be completed.

[0048] Example 2

[0049] The Influence of the Order of Drying and Leveling Processes and Leveling Methods

[0050] Fresh leaves of Artemisia argyi were collected and divided into three groups as shown in Table 2. The groups were then treated separately, and photos were taken after treatment. The results are shown in Table 2.

[0051] Table 2. Results of the Influence of Drying and Leveling Sequence and Leveling Methods on the Results of Drying and Leveling Processes

[0052]

[0053]

[0054] Note: Example 1 group 1-1 and Example 2 group 2-1 are actually one set of experiments, but they are shown in different tables for easy comparison; similarly, Example 1 group 1-3 and Example 2 group 2-3 are also one set of experiments.

[0055] Figure 3 The images show the results obtained by three methods. A shows the result after processing leaves with low non-glandular hair density using the method described in group 2-1. The results indicate that after air-drying and then flattening, the leaves are not flat enough, and some areas of the field of view are blurred, making it impossible to accurately count the glandular hairs. B shows the result after processing leaves using the method described in group 2-2. The results indicate that after air-drying, the leaves are curled and brittle, and the tissue breaks into small pieces during the flattening process, making slide preparation impossible. C shows the result after processing leaves with low glandular hair density using group 2-3 of this invention. The results indicate that after flattening and then air-drying, the entire field of view is clear, and the glandular hairs can be accurately counted. D shows the result after processing samples with high non-glandular hair density using the method described in group 2-1. The results indicate that when the non-glandular hair density is high, simply air-drying and flattening with a pressing ring without hair removal, although the non-glandular hairs shrink, still obstruct the field of view, making it impossible to count the glandular hairs.

[0056] according to Figure 3 It is evident that the "flatten first, then dry" operation sequence of this invention is crucial, as it is key to obtaining a completely clear field of vision and accurately calculating the density of glandular hairs.

[0057] Example 3

[0058] The effects of using tape for hair removal

[0059] Fresh leaves of Artemisia argyi were collected and divided into two groups as shown in Table 3. The groups were then treated separately, and photographs were taken after treatment. The results are shown in Table 3.

[0060] Table 3. Results of the effect of using adhesive tape for hair removal

[0061]

[0062]

[0063] Figure 4 The images show the effects of using adhesive tape on hair removal. In Group A, the results of processing samples with high non-glandular hair density in Group 3-1 are shown. The results indicate that air drying alone, without hair removal, causes non-glandular hair to shrink but still obstructs the field of view, making it impossible to count the density of glandular hair. In Group B, the results of processing samples with high non-glandular hair density using the method described in Group 3-2 are shown. The results indicate that after air drying, hair removal removes non-glandular hair and fully exposes glandular hair in the field of view, allowing for the counting of glandular hair density.

[0064] Example 4

[0065] The effect of using depth-of-field compositing technology

[0066] The depth-of-field compositing technology described in this invention can be accomplished using a regular optical stereo microscope in conjunction with Photoshop, or it can be accomplished using the built-in function of a super depth-of-field optical stereo microscope.

[0067] Fresh leaves of Artemisia argyi were collected and divided into two groups as shown in Table 4. The groups were then treated separately, and photos were taken after treatment. The results are shown in Table 4.

[0068] Table 4 shows the impact of using depth-of-field composition.

[0069]

[0070] Figure 5The images show the effect of depth-of-field composite technology on the results. A represents the image taken after processing a sample with low non-glandular hair density using the method described in Group 4-1. The results show that when the sample is flattened and air-dried before being taken under a stereomicroscope, without depth-of-field composite, some areas of the field of view are blurred. B represents a composite image taken after processing a sample with low non-glandular hair density using the method described in Group 4-2. The results show that when the sample is flattened and air-dried before being taken under a stereomicroscope, combined with depth-of-field composite, the entire field of view is clear. C represents a composite image taken after processing a sample with high non-glandular hair density using the method described in Group 4-2. The results show that when the sample is flattened and air-dried before being taken under a stereomicroscope, combined with depth-of-field composite, the entire field of view is clear. However, due to the obstruction of non-glandular hair, it is impossible to observe and count glandular hair. Therefore, hair removal is necessary for samples with high non-glandular hair density.

[0071] Example 5

[0072] The leaves of *Artemisia argyi* were treated using the method of Example 1 Group 1-1 (i.e., Example 2 Group 2-1) and the method of this invention, respectively, and the results are as follows: Figure 6 As shown. Figure 6 Images show the effects of different methods on the glandular hairs on *Artemisia argyi* leaves. Image A shows the result of processing samples with high non-glandular hair density using the method described in Example 1, Group 1-1 (air-drying followed by flattening, no hair removal, and no depth-of-field extension). Image B shows the result of processing samples with high non-glandular hair density using the method described in Example 4, Group 4-2 (flattening followed by air-drying, hair removal with tape, and depth-of-field extension). Image C shows the result of processing samples with medium non-glandular hair density using the method described in Example 4, Group 4-1 (flattening followed by air-drying, no hair removal, and no depth-of-field extension). The results indicate that the method described in Example 4, Group 4-1 significantly improves the problems of non-glandular hair occlusion and partial blurring caused by leaf undulation compared to existing technologies. Group 4-2 further improves the partial blurring of the field of view for leaves with severe undulation, achieving the goal of accurately calculating glandular hair density.

[0073] In summary, the abundance of non-glandular hairs on the epidermis of plant leaves can obstruct the observation of glandular hairs; leaf curling and undulation can also lead to a blurred field of view. Both of these situations make it impossible to statistically determine glandular hair density. This invention provides a method to solve the above problems:

[0074] First, lay all the leaves flat and let them air dry. Then, during observation, if the density of epidermal hairs is high and non-glandular hairs severely obstruct the field of view, use tape to stick the non-glandular hairs to expose the glandular hairs, and then count the glandular hair density. If the density of glandular hairs is moderate or low during observation, it is not necessary to stick the non-glandular hairs with tape, and the density can be counted directly. If, during observation, even after flattening and air drying, the leaves are still undulating, causing partial blurring of the field of view, you can use depth-of-field extension or directly use a super depth-of-field stereomicroscope to take a completely clear picture, and then count the glandular hair density.

[0075] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for observing and / or counting glandular trichomes on plant leaves, characterized in that, Includes the following steps: The plant leaves were laid flat, air-dried, and observed and / or statistically analyzed using an optical stereomicroscope. The observation using an optical stereo microscope includes: taking pictures using an optical stereo microscope, combining them with a depth-of-field expansion function, and then observing them. Alternatively, images can be taken directly using a super depth-of-field optical stereo microscope for observation. The statistics include the statistical analysis of glandular hair density; The leaves of the plant have glandular hairs and non-glandular hairs, and the plant is Artemisia argyi, Sage, Tobacco, or Callicarpa nudiflora; the leaves are selected from the area near the veins in the middle of the leaf. The process of air-drying also includes sticking on non-glandular hairs.

2. The method according to claim 1, characterized in that, The flattening process involves using double-sided tape to attach the plant leaves to a flat surface.

3. The method according to claim 2, characterized in that, The plane includes the plane of the glass slide.

4. The method according to claim 1, characterized in that, The air-drying time is 2 to 48 hours.

5. The method according to claim 1, characterized in that, The removal of non-glandular hairs includes using adhesive tape to remove non-glandular hairs.

Images