A method for measuring plant cell-scale hygroscopic deformation based on cryo-scanning electron microscopy

Abstract

The present invention relates to a method for measuring plant cell-scale moisture absorption deformation based on a cryo-scanning electron microscope, comprising the steps of sample pretreatment, moisture absorption treatment, cell-scale image acquisition in a water-absorbed state, cell-scale image acquisition in a water-depleted state, and moisture absorption deformation measurement. The present invention introduces a cryo-scanning electron microscope to successively obtain images in a rehydrated state and a water-depleted state, thereby obtaining the cell-scale moisture absorption deformation result of Cleistogenes serratus, which can solve the problem that conventional instruments are difficult to accurately capture microscopic structural changes in water-depleted and rehydrated states due to rapid changes in tissues of water-driven plants, especially Cleistogenes serratus.

Description

Technical Field

[0001] The invention relates to the field of plant microscopic deformation research, and in particular to a method for measuring plant cell-scale moisture absorption deformation based on a cryo-scanning electron microscope. Background Art

[0002] Wind rollers are a type of herbaceous plant widely distributed in grasslands. During the dry and windy non-growing season, their above-ground residues spread with the wind, forming a unique land surface process in arid areas. During the growth process or at the end of the growing season, the above-ground parts of the plants usually form spherical or nearly spherical wind rollers by frequent branching of the stems, which move under the action of wind, such as Salsola and Tribulus truncatula, which are common in typical grasslands. However, unlike wind rollers that form spherical structures, Cleistogenes striata, a plant of the genus Cleistogenes in the Poaceae family widely distributed in arid and semi-arid areas, is the only wind roller plant reported to form wind rollers through geometric remodeling after the end of the growing season. The process of its stem node hygroscopic deformation is based on changes in morphology and structure to ensure that the wind rollers respond to environmental changes in a timely manner. Observation of this change feature helps to understand its adaptive mechanism and ecological function, and this rapid and extensive deformation ability may also provide inspiration for the creation of innovative and efficient artificial bionic materials.

[0003] Scanning electron microscopy (SEM) is an advanced microscopic imaging technology that can observe the microscopic morphology of the sample with high resolution while maintaining its original structure. Without damaging the sample, the morphology and structure of the sample surface can be restored in detail, with a large depth of field and three-dimensional sense. The depth of field of images at different magnifications is also different: the depth of field of an image magnified thousands of times can reach tens of microns, while that of an image magnified tens of thousands of times is only a few microns, so that the fine structures on the surface of various samples can be directly observed. However, for plant samples such as Cleistogenes striata that require water control for rapid dynamic observation, conventional electron microscope observation methods can no longer meet the needs. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for measuring plant cell-scale moisture absorption deformation based on cryo-scanning electron microscopy.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] The method for measuring plant cell-scale moisture absorption deformation based on cryo-scanning electron microscopy includes the following steps:

[0007] S1. Sample pretreatment

[0008] Obtaining a dry and curled single stem node of Cleistogenes scabra; longitudinally cutting the single stem node along the curling surface of the single stem node to obtain a sample with a longitudinal section;

[0009] S2. Moisture absorption treatment

[0010] The longitudinal section of the sample is facing upwards, and one end is fixed on the sample stage of a cryo-scanning electron microscope; water is dripped onto the sample until the sample is in a straight state;

[0011] S3. Acquisition of cell-scale images under water absorption

[0012] The sample is placed in liquid nitrogen mud for freezing and solidification, and then sublimated under low temperature and high vacuum conditions, so that the moisture on the surface of the sample is directly converted from solid to gas to reach a dry state; then the sample is placed in a cryo-electron microscope chamber, and the longitudinal section of the sample is imaged;

[0013] S4. Cell-scale imaging acquisition under dehydration

[0014] Open the cryo-electron microscope chamber, wait for the temperature in the chamber to return to room temperature, so that the sample can return to a curled state due to evaporation of water, and image the longitudinal section of the sample;

[0015] S5. Hygroscopic deformation determination

[0016] By comparing the cell-scale images in the water-absorbing state and the cell-scale images in the water-depleting state, the length change values ​​of the cells at the corresponding positions were obtained, and the shrinkage rate was calculated.

[0017] Furthermore, in step S1, the single stem node is frozen before longitudinal cutting.

[0018] Furthermore, in step S1, the sample is torn longitudinally using a blade and tweezers to obtain a longitudinal section of the sample. During the operation, the blade and tweezers are only in contact with the two ends of the sample to ensure that the middle part is not damaged or squeezed, thereby preserving the integrity of the observation area.

[0019] Furthermore, in step S2, after the sample is completely straightened from the dry and curled state, filter paper is used to absorb residual moisture around the sample.

[0020] Furthermore, in step S3, the freezing and solidification time is 5 to 10 seconds.

[0021] Furthermore, in step S3, the low temperature and high vacuum are specifically a temperature of -90 to -110°C and a vacuum degree of 9.59×10 -5 ~9.77×10 -5 mbar.

[0022] Furthermore, in step S3, the dry state is reached when the surface is clearly visible in the field of vision.

[0023] Specifically, the sublimation process in step S3 can avoid the problem of surface tension between the gas phase and the liquid phase, and thus causes less damage to the sample.

[0024] Furthermore, in step S3 and step S4, the imaging requirement is to observe neatly arranged microfibrils on the cell wall.

[0025] Furthermore, in step S5, cells with clear cell wall microfibrils are selected as observation cells, the length between two cell wall microfibrils of each cell in the axial direction of the sample is the cell length value of the cell, the difference between the cell length values ​​of the same cell in the water absorption state and the water dehydration state is the length change value of the cell, and the ratio of the length change value to the cell length value in the water absorption state is the shrinkage rate of the cell.

[0026] Furthermore, in step S5, the longitudinal section is divided into an inner side of the curl and an outer side of the curl, and a plurality of cells are selected from the inner side of the curl and the outer side of the curl respectively to calculate the average shrinkage rate of the plurality of cells respectively, so as to obtain the deformation result of the inner side of the curl and the deformation result of the outer side of the curl. Beneficial Effects

[0027] 1. In view of the problem that water drives the rapid changes of plant (especially Cleistogenes serrata) tissues, it is difficult for conventional instruments to accurately capture the changes in microstructures under dehydration and rehydration states. The present invention introduces a cryo-scanning electron microscope to obtain images in the rehydrated state and the dehydrated state, thereby obtaining the cellular scale hygroscopic deformation results of Cleistogenes serrata; further, the present invention divides the longitudinal section of Cleistogenes serrata into the inner curling side and the outer curling side, and calculates the deformation results respectively, which can more accurately describe the hygroscopic change principle of Cleistogenes serrata, and provide new ideas for the later research of artificial bionic materials.

[0028] 2. The present invention can avoid the use of the tert-butanol drying method in the prior art, and especially avoids the irreversible, cumbersome process and easy damage to the sample surface caused by using tert-butanol as a replacement agent to replace the water in the biological tissue, so that the present invention can reversibly observe the deformation of Cleistogenes striata before and after moisture absorption.

[0029] 3. The present invention also avoids the use of the critical point drying method in the prior art, especially avoiding the disadvantages of the critical point drying method that requires special critical point drying equipment, the operation is relatively complicated, and the equipment and maintenance costs are high. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A flow chart of sample preparation and observation according to an embodiment of the present invention;

[0031] Figure 2 It is a comparative analysis diagram of cell scale images in the water absorption state and the water loss state of an embodiment of the present invention;

[0032] Figure 3 It is the moisture absorption deformation result of the embodiment of the present invention. DETAILED DESCRIPTION

[0033] The present invention is described in detail below through specific embodiments, but the scope of the present invention is not limited to the

[0034] In order to make the above-mentioned objects, features and advantages of the present invention more clearly understood, the following embodiments are described in conjunction with the attached

[0035] The present invention is further described in detail with reference to the drawings and specific embodiments.

[0036] The method for measuring plant cell-scale moisture absorption deformation based on cryo-scanning electron microscopy includes the following steps:

[0037] (1) Take a single stem node of Cleistogenes scabra, which is completely dry and curled, and freeze it in liquid nitrogen for 1 minute. Immediately tear the sample longitudinally with the help of dissecting forceps to obtain a natural longitudinal section of the stem node.

[0038] (2) Use conductive glue to fix one end of the longitudinal section on the metal sample stage, and drop a drop of distilled water on the sample in mid-air. After 2 minutes, it can be observed that the sample is completely straightened after absorbing water. Connect the sample stage to the sample injection device of the cryo-scanning electron microscope, and quickly transfer it into the prepared liquid nitrogen mud (-196℃) and keep it for 10 seconds to achieve freezing and solidification of the sample. Subsequently, the frozen sample is transferred to the low-temperature preparation chamber and sublimated at -90℃ under high vacuum. The time is set to 10 minutes, so that the moisture on the surface of the sample is directly converted from solid to gas to achieve a drying effect (if the low-temperature freezing and sublimation effects are not obvious, the sublimation time can be appropriately extended until the surface can be clearly displayed in the field of vision).

[0039] (3) In order to improve the electron scattering ability of the sample and reduce the charging effect caused by the electron beam during observation, the frozen plant samples were coated, i.e., gold-sprayed. The voltage was set to 1.2 kV, the current was 5-10 mA, and the time was 30-60 s.

[0040] (4) The sample was transferred into a cryo-EM chamber that had been set to low temperature, the voltage was set to 1 kV, the TLD (secondary electron) detector was used, the SE mode was selected, the current was 86 pA, and the magnification was 4000-5000 times. At this time, the neatly arranged microfibrils on the cell wall can be observed, and the longitudinal section can be scanned and imaged, especially the inner and outer sides of the longitudinal section.

[0041] (5) Open the electron microscope chamber and wait for the temperature in the chamber to return to room temperature (25°C). At this time, the sample will return to its curled state due to evaporation of water. Reset the voltage to 1 kV, use the TLD (secondary electron) detector, select the SE mode, the current is 86 pA, the magnification is 4000-5000 times, find the inner and outer sides of the longitudinal section that has been low-temperature scanned, and scan the area again (see Figure 2 ).

[0042] (6) Measure the length between microfibrils on the cell wall in the same area before and after moisture absorption and calculate the shrinkage rate. Specifically, select three cells on the outside of the longitudinal section and two cells on the inside of the longitudinal section (see Figure 2 ), calculate the average shrinkage rate respectively, where, Figure 2 (c) is a scanning image of the longitudinal section under water absorption, (d) is a scanning image of the longitudinal section under water loss, (a) is an enlarged image of the outside of the longitudinal section in (c), (b) is an enlarged image of the outside of the longitudinal section in (d); (e) is an enlarged image of the inside of the longitudinal section in (c), and (f) is an enlarged image of the inside of the longitudinal section in (d). The results show that the shrinkage rate of the inner side of the longitudinal section is 18.64%, which is significantly higher than the 1.34% of the outer side of the longitudinal section (see Figure 3 ).

[0043] The embodiments of the present invention are described in detail above. The description of the above embodiments is only used to help understand the method of the present invention and its core concept, and does not limit the scope of implementation of the present invention. Therefore, all equivalent changes or modifications made according to the structure, characteristics and principles described in the patent scope of the present invention should be included in the scope of the patent application of the present invention. In summary, the content of this specification should not be understood as a limitation of the present invention.

Claims

1. A method for measuring plant cell scale moisture absorption deformation based on cryo-scanning electron microscopy, characterized in that: The steps include: S1. Sample pretreatment Obtaining a dry and curled single stem node of Cleistogenes scabra; longitudinally cutting the single stem node along the curling surface of the single stem node to obtain a sample with a longitudinal section; S2. Moisture absorption treatment The longitudinal section of the sample is facing upwards, and one end is fixed on the sample stage of a cryo-scanning electron microscope; water is dripped onto the sample until the sample is in a straight state; S3. Acquisition of cell-scale images under water absorption The sample is placed in liquid nitrogen mud for freezing and solidification, and then sublimated under low temperature and high vacuum conditions, so that the moisture on the surface of the sample is directly converted from solid to gas to reach a dry state; then the sample is placed in a cryo-electron microscope chamber, and the longitudinal section of the sample is imaged; S4. Cell-scale imaging acquisition under dehydration Open the cryo-electron microscope chamber, wait for the temperature in the chamber to return to room temperature, so that the sample can return to a curled state due to evaporation of water, and image the longitudinal section of the sample; S5. Hygroscopic deformation determination The cell scale image in the water absorption state and the cell scale image in the water loss state are compared to obtain the length change value of the cells at the corresponding position, and the shrinkage rate is calculated; the longitudinal section is divided into the inner side of the curl and the outer side of the curl, and several cells are selected from the inner side of the curl and the outer side of the curl respectively to calculate the average shrinkage rate of several cells respectively, and obtain the deformation result of the inner side of the curl and the deformation result of the outer side of the curl.

2. The method according to claim 1, characterized in that In step S1, the single stem node is frozen before longitudinal cutting.

3. The method according to claim 2, characterized in that In step S1, the sample is torn longitudinally using a blade and tweezers to obtain a longitudinal section of the sample. During the operation, the blade and tweezers are only in contact with the two ends of the sample to ensure that the middle part is not damaged or squeezed, thereby retaining the integrity of the observation area.

4. The method according to claim 1, characterized in that: In step S2, after the sample is completely straightened from the dry and curled state, filter paper is used to absorb the residual moisture around the sample.

5. The method according to claim 1, characterized in that: In step S3, the freezing and solidification time is 5 to 10 seconds.

6. The method according to claim 1, characterized in that In step S3, the low temperature and high vacuum are specifically -90 to -110°C and 9.59×10 -5 ~9.77×10 -5 mbar.

7. The method according to claim 1, characterized in that In step S3, the dry state is reached when the surface is clearly visible in the field of vision.

8. The method according to claim 1, characterized in that In step S3 and step S4, the imaging requirement is to observe neatly arranged microfibrils on the cell wall.

9. The method according to claim 1, characterized in that: In step S5, cells with clear cell wall microfibrils are selected as observation cells, the length between two cell wall microfibrils of each cell in the axial direction of the sample is the cell length value of the cell, the difference between the cell length values ​​of the same cell in the water absorption state and the water desorption state is the length change value of the cell, and the ratio of the length change value to the cell length value in the water absorption state is the shrinkage rate of the cell.

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