Method for measuring internal mechanical properties of plant tissue based on atomic force microscope

Abstract

The invention discloses a method for measuring internal mechanical properties of plant tissues based on an atomic force microscope, and belongs to the field of plant tissue mechanical property detection. According to the method, a sample slice capable of ensuring the initial mechanical property is prepared through a freezing slicing technology, and the slice is fixed on a glass slide and does not fall off. And an atomic force microscope and an inverted fluorescence section microscope are combined together, so that precise positioning of a section measurement area is realized, and tissue cell morphology-mechanical property two-dimensional expression information is obtained. Therefore, the problems that in the prior art, the initial mechanical property test in the plant tissue cannot be achieved, in the test process, a sample moves and falls off, a probe measurement area cannot be accurately positioned, and only a force-distance curve can be output during measurement are solved.

Description

Technical Field

[0001] The present invention relates to the field of plant tissue mechanical property detection, and in particular to a method for measuring the internal mechanical properties of plant tissue based on an atomic force microscope. Background Art

[0002] The mechanical properties of biological tissues are core parameters for how organisms respond to external stimuli and regulate their functions. In plants, the mechanical properties of cell walls directly support their morphological structure and environmental adaptation. Cacti use lignin to strengthen their stems and retain water to resist drought, while apples soften their cell walls as they ripen, promoting fruit enlargement. In animal tissues, the elastic cushioning capacity of cartilage declines with age due to increased stiffness, while tumor tissues enhance malignant characteristics through hardening of the extracellular matrix. Abnormal hardening of breast cancer tissue has been confirmed by atomic force microscopy (AFM). These cross-species examples reveal that biomechanical properties are not only the physical foundation for maintaining structural stability but also a dynamic signaling system that regulates growth and differentiation.

[0003] As a key tool in biomechanical research, atomic force microscopy (AFM) can obtain parameters such as stiffness and viscosity of tissue surfaces through micro- and nano-scale probes, providing important data support for revealing the drought resistance of plant tissues or the pathological changes of animal tumor tissues. However, existing biomechanical research using atomic force microscopy has the following major problems: First, atomic force microscopy mainly performs mechanical measurements by direct contact between the probe and the sample surface. Current methods for measuring plant mechanical properties using atomic force microscopy are limited to the plant surface, and the mechanical properties of the internal plant tissues are currently unknown. Second, because plant samples are fresh and tender, it is impossible to maintain the integrity of the tissue samples during slicing. Existing technologies for plant slicing mainly focus on paraffin sections and semi-thin sections. However, these methods involve dehydration, chemical fixation, and high-temperature baking, which change and interfere with the mechanical properties of the tissues. Third, after existing plant sections are sliced, the samples cannot be fixed to the slide. When the atomic force microscope probe lightly touches the sample for measurement, there is a problem of sample detachment and movement. Fourth, the current atomic force microscope can only locate the target area of ​​the plant by "blind scanning" the probe on the sample surface to find the measurement area, which is time-consuming and easily damages the probe tip, and cannot achieve accurate positioning of the measurement area. At the same time, atomic force microscopy measurements of plants can only output force-distance curves, failing to link cell morphology with mechanical properties to explain the mechanism of force action during plant morphogenesis.

[0004] The above reasons make it impossible to study the internal mechanical properties of plant sample tissues. Therefore, it is necessary to provide a method for measuring the internal mechanical properties of plant tissues based on atomic force microscopy to solve the above problems. Summary of the Invention

[0005] Technical issues to be solved: In order to avoid the shortcomings of the existing technology, the present invention provides a method for measuring the internal mechanical properties of plant tissues based on atomic force microscopy, designs a frozen sectioning method, prepares sample slices that can ensure the initial mechanical properties, and realizes the fixation of the slices on the slide without falling off. By combining the atomic force microscope with an inverted fluorescent sectioning microscope, precise positioning of the slice measurement area is achieved, and two-dimensional expression information of tissue cell morphology and mechanical properties is obtained to solve the existing problems.

[0006] The technical solution of the present invention is: a method for measuring the internal mechanical properties of plant tissues based on atomic force microscopy, comprising the following steps: The atomic force microscope is integrated with an inverted fluorescence section microscope, and both the atomic force microscope and the inverted fluorescence section microscope are connected to a computer; Prepare a slice of the plant sample to be tested into an observation sample and place it on a stage. Use an inverted fluorescence slice microscope to take an optical cell morphology image of the slice, i.e., an optical image, and transmit it to a computer. Projecting the optical image calibration into the atomic force microscope and performing optical image matching calibration in the atomic force microscope; In the atomic force microscope, the target area of ​​the slice is selected according to the optical image, and the probe of the atomic force microscope is calibrated. After the probe is calibrated, the probe parameters are set, and the force-distance curve data is collected at each data collection point in the target area. The probe-collected data is transmitted to the computer for processing into a mechanical image. The computer matches the collected optical image and mechanical image to obtain the morphological and mechanical property information of the internal slices of the plant tissue; The slices of the plant sample to be tested are embedded by a gradient embedding method and then processed by freezing to obtain the slices.

[0007] A further technical solution of the present invention is that the slices of the plant sample to be tested are prepared by the following method: Pre-cool and wash samples; Immerse the sample in an OCT embedding medium of a set concentration for 10 to 30 minutes, and place the OCT embedding medium and the sample in a vacuum chamber for 10 to 30 minutes for infiltration. Repeat the embedding and infiltration twice, with increasing OCT embedding medium concentrations in each step. After permeation, the samples were transferred to 100% OCT embedding solution for directional fixation of the samples; The directionally fixed sample is freeze-solidified stepwise to obtain a freeze-solidified sample; The frozen solidified samples were serially sectioned at -20°C using a microtome with a thickness of 10-25 μm.

[0008] A further technical solution of the present invention is that the concentrations of the OCT embedding agent for the two times are 75% and 85% respectively.

[0009] A further technical solution of the present invention is: the step-by-step freezing and solidification method includes: first placing the sample at a constant temperature of -20°C for 10 minutes for pre-cooling and solidification, and then deep-freezing the pre-cooled and solidified sample with liquid nitrogen, the liquid nitrogen deep-freezing temperature is -196°C, and the liquid nitrogen deep-freezing time is 30 seconds.

[0010] A further technical solution of the present invention is: the method for pre-cooling and cleaning the sample includes: rinsing three times with 4°C distilled water, and then gently wiping the sample tissue surface along the axis with sterile absorbent paper to remove free water.

[0011] A further technical solution of the present invention is: the method of preparing a slice of a plant sample to be tested into an observation sample includes: selecting a target slice, attaching the slice to a glass slide, wherein the glass slide is a lysine-coated glass slide, and then rinsing off the OCT embedding agent on the slice after settling at room temperature for a certain period of time to obtain a slice fixed on the glass slide.

[0012] A further technical solution of the present invention is that the room temperature precipitation time is 10 to 30 minutes, and after the room temperature precipitation, the residual OCT embedding agent in the slice is rinsed with deionized water.

[0013] A further technical solution of the present invention is that the probe is a conical probe or a spherical probe, and the parameters of the probe are set as follows: acquisition force is 40-60 nN, acquisition speed is 100-150 μm / s, and lifting height is 2-4 μm.

[0014] A further technical solution of the present invention is: the method for a computer to process the data collected by the probe into a mechanical image is: the computer uses the Hertz model or the Sneddon model to fit the force-distance curve data of each data collection point collected by the probe, combined with the probe shape, to calculate the corresponding Young's modulus value, and then generate a slice Young's modulus diagram, that is, a mechanical image.

[0015] The beneficial effects of the present invention are: This invention provides a method for measuring the internal mechanical properties of plant tissues using atomic force microscopy. By utilizing plant tissue cryosectioning techniques, gradient embedding, and room-temperature precipitation, along with a two-dimensional measurement system formed by combining an atomic force microscope with an inverted fluorescence sectioning microscope, this method provides the most original, stable, and accurate method for measuring the internal mechanical properties of plant tissues, capable of interpreting biological processes from a dual perspective of cell morphology and mechanical properties. This method addresses the following issues with existing atomic force microscopes (AFMs) and measurement methods: the inability to measure the internal mechanical properties of plant tissues, the impact of traditional sectioning methods on plant tissue mechanical properties, the inability to accurately locate the probe measurement area, and the limited output of force-distance curves.

[0016] Compared with the prior art, the present invention has the following advantages: 1. The cryosectioning method of the present invention preserves the initial mechanical properties of plant tissues, facilitating precise measurement of the internal mechanical properties of plant tissues. The cryosectioning method proposed in the present invention utilizes a gradient embedding technique to completely embed the embedding solution into the tissue voids, maintaining not only the integrity of the tissue structure but also the plant's initial mechanical properties to the greatest extent possible. Cryosectioning then exposes the interior of the plant tissue, overcoming the interference with tissue mechanical properties caused by dehydration, chemical fixation, and high-temperature baking processes in existing sectioning methods.

[0017] 2. This invention improves the ability to secure sections to glass slides, solving the problem of sample section detachment and movement during mechanical measurements. By combining a lysine-coated glass slide with a room-temperature precipitation step to prepare the sectioned observation specimen, the present invention achieves the goal of securing the section to the glass slide without movement.

[0018] 3. The present invention can quickly locate the target area and realize two-dimensional data interpretation. The present invention integrates an atomic force microscope with an inverted fluorescence section microscope to form a two-dimensional measurement system. The inverted fluorescence section microscope can find plant section samples in the optical field of view, realize accurate positioning of the target area, improve the efficiency of probe measurement, and avoid unnecessary sample damage. At the same time, it can also obtain cell morphology information, which is combined with the mechanical properties of the atomic force microscope to comprehensively analyze biological processes, thereby obtaining morphological and mechanical property information of plant internal tissue sections, so as to deeply understand the mechanism and performance of plants under different environmental stresses. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 This is a flow chart of a method for measuring the internal mechanical properties of plant tissues based on atomic force microscopy according to the present invention; Figure 2 is the optical image of the slice; Figure 3 Mechanical images of slices. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] The present invention is a method embodiment of measuring the internal mechanical properties of plant tissues based on atomic force microscopy. This embodiment studies the internal mechanical properties of anther tissues of Arabidopsis thaliana. Early anthers were selected as experimental samples for internal mechanical measurements of tissues. Figure 1 As shown in the flowchart, the following steps are included: Step 1: Prepare frozen sections of the sample, select the target sections, and fix the sections on slides to prepare observation specimens.

[0023] This step uses a gradient embedding method to ensure that the internal tissue structure of the sample is intact and the initial mechanical properties are not affected; the embedded sample is then frozen and sectioned to expose the internal tissue of the sample; and the sample slices are then fixed on a lysine-coated slide using a room temperature precipitation method to prepare observation specimens for microscopic observation. The specific method is: Step 1.1 Pre-cool and wash the sample and remove residual water. Specifically, rinse the anther sample three times with 4°C distilled water, then gently wipe the tissue surface axially with sterile absorbent paper to remove free water.

[0024] Step 1.2: Completely immerse the specimen in 75% OCT embedding medium and allow it to stand for 10 minutes. Place the OCT embedding medium and the specimen in a vacuum chamber and vacuum it for 15 minutes. Remove the specimen and completely immerse it in 85% OCT embedding medium again and allow it to stand for 10 minutes. Place the OCT embedding medium and the specimen in a vacuum chamber and vacuum it for 15 minutes. This step allows the OCT embedding medium to penetrate the interstitial spaces of the specimen.

[0025] Step 1.3: Transfer the infiltrated sample from step 1.2 to 100% OCT embedding medium and fix the sample in a directional manner. This involves adjusting the angle of the sample so that its axis is perpendicular to the cross-section of the slice.

[0026] Step 1.4: Freeze-solidify the fixed specimen in a stepwise manner to obtain a frozen specimen. Specifically, pre-freeze the specimen at -20°C for 10 minutes. The pre-freeze-solidified specimen is then deep-frozen in liquid nitrogen at a temperature of -196°C for 30 seconds to maintain the integrity of the tissue structure.

[0027] Step 1.5 Place the embedded and frozen sample prepared in step 1.4 into a Leica CM1950 cryostat. The sample is serially sectioned at -20°C using the cryostat. The thickness of the prepared sections is 20 μm.

[0028] After sectioning in step 1.6, select a target sample slice and prepare the observation specimen. This is done by attaching the slice to a lysine-coated slide. After the slide settles at room temperature for 30 minutes, gently rinse the remaining embedding agent with deionized water to obtain a morphologically intact, firmly attached sample slice.

[0029] To enhance adhesion between frozen plant sections and glass slides, this example uses lysine-treated slides. This allows the positive surface charge of the slides to electrostatically attract the negatively charged cellular components in the tissue sections. Immediately after section preparation, the sections are spread flat on the lysine-treated slides and allowed to settle at room temperature for 30 minutes. This promotes thinning of the water film on the surface of the plant sections, ensuring full contact and adhesion between the tissue and the slide. This enhances electrostatic and physical adhesion, ensuring stability during subsequent atomic force measurements.

[0030] Step 2: Measure the internal mechanical properties of plant tissues: Integrate the atomic force microscope with the inverted fluorescence section microscope, collect optical images and mechanical images of the observation sample slices prepared in step 1, and match and fuse the collected optical images and mechanical images through a computer to obtain the sample slice tissue slice morphology-mechanical property information.

[0031] This step constructs a two-dimensional measurement system by integrating an atomic force microscope with an inverted fluorescence section microscope. The atomic force microscope provides a mechanical module, and the inverted fluorescence section microscope provides an optical module and an image overlap calibration module. That is, this step provides a plant slice mechanical measurement method based on an atomic force microscope and an inverted fluorescence section microscope, which can accurately locate the measurement target area and realize a two-dimensional interpretation of plant biological processes from the perspective of cell morphology and mechanical properties.

[0032] The specific method is: Step 2.1 Build a measurement system: Integrate an inverted fluorescence section microscope on the atomic force microscope. The atomic force microscope is used to collect force-distance curve data for each data collection point in the measurement target area of ​​the slice, and the inverted fluorescence section microscope is used to collect optical images of the measurement target area of ​​the slice. Both the atomic force microscope and the inverted fluorescence section microscope are connected to a computer.

[0033] Step 2.2 Optical image acquisition: Place the slice fixed on the slide on the stage, find the target anther under the inverted fluorescence microscope, locate the target area of ​​the slice, and take the optical cell morphology of the slice, i.e., the optical image, as shown in the figure. Figure 2and passed to the computer.

[0034] from Figure 2 As can be seen in the figure, the plant tissue is in a fresh sample state after sectioning, with intact cell structure and no detachment.

[0035] Step 2.3 Image calibration and projection: The optical image collected by the inverted fluorescence section microscope is calibrated and projected into the atomic force microscope to enable the measurement target area to be accurately found in the atomic force microscope.

[0036] Step 2.4 Mechanical image acquisition: Step 2.4.1 Collect force-distance curve data for each data collection point in the target area of ​​the slice measurement: In the atomic force microscope, the target measurement area of ​​the slice is selected according to the optical image, and then the probe of the atomic force microscope is calibrated. After the probe is calibrated, the probe measurement parameters are set. The probe collects mechanical information of each data collection point in the target measurement area of ​​the slice according to the set parameters, that is, force-distance curve data collection. The probe collected data is transmitted to the computer for processing.

[0037] Specifically, calibration involves importing the optical image from the inverted fluorescence microscopy microscope into the atomic force microscope image calibration module. This allows the optical image to be viewed within the atomic force microscope, and the mechanical measurement area to be selected within the optical image. Probe setting parameters include acquisition force, acquisition speed, and lift height. In this embodiment, a conical probe was used, with acquisition force set to 60 nN, acquisition speed set to 150 μm / s, and lift height set to 3.5 μm. Mechanical data was collected from each data acquisition point on the slice using the probe's set parameters and transmitted to the computer.

[0038] Step 2.4.2 Data processing: The computer processes the probe data. For each data collection point on the slice, the force-distance curve data (reflecting the deformation state of the material surface during the measurement process) is combined with the probe shape and the Sneddon model is used to fit and calculate the corresponding Young's modulus value. This generates a Young's modulus diagram of the slice, i.e., a mechanical image.

[0039] The Hertz model and Sneddon model are classic models used in atomic force microscopy to analyze the mechanical behavior of the contact between the probe and the sample. The model analysis software is built into the computer. For small deformations using a spherical probe, the Hertz model is used for fitting, and for large deformations using a conical probe, the Sneddon model is used for fitting.

[0040] Step 2.5 Image analysis and fusion: The computer performs dual-modal image superposition, matching and fusion on the optical image and mechanical image of the anther slice to present the morphological and mechanical property information of the internal slice of the plant tissue, i.e. Figure 2、 Figure 3 As shown, the distribution of mechanical hardness can be matched with the cell morphology diagram, thereby deeply analyzing the mechanical regulation process of anther growth morphology.

[0041] Figure 3 It can be seen that the anther mechanical information is collected completely, and Figure 2 Combined analysis revealed significant differences in the hardness of the inner reproductive cells and the outer anther epidermis.

[0042] It should be noted that in order to ensure biological repeatability, three anthers were cut separately, and a slice was selected from each of the three anthers. Each of the three slices was measured once to ensure the accuracy of the measurement.

[0043] This invention breaks through the barriers to measuring plant surface mechanics, enabling the exploration of the internal mechanical properties of plant tissues. Using cryosectioning, the internal tissues are exposed while preserving their original mechanical properties. By using lysine-coated slides combined with a room-temperature precipitation process, the sample remains intact, resulting in the production of an optimal sample standard for atomic force measurement of the internal mechanical properties of plant tissues. By constructing a two-dimensional measurement system based on an atomic force microscope and an inverted fluorescence sectioning microscope, the target region is precisely located, allowing for a dual-dimensional interpretation of plant biological processes from the perspectives of cell morphology and mechanical properties.

[0044] 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 in the scope of protection of the present invention.

Claims

1. A method for measuring the internal mechanical properties of plant tissues based on atomic force microscopy, characterized in that: The following steps are involved: The atomic force microscope is integrated with an inverted fluorescence section microscope, and both the atomic force microscope and the inverted fluorescence section microscope are connected to a computer; Prepare a slice of the plant sample to be tested into an observation sample and place it on a stage. Use an inverted fluorescence slice microscope to take an optical cell morphology image of the slice, i.e., an optical image, and transmit it to a computer. Projecting the optical image calibration into the atomic force microscope and performing optical image matching calibration in the atomic force microscope; In the atomic force microscope, the target area of ​​the slice is selected according to the optical image, and the probe of the atomic force microscope is calibrated. After the probe is calibrated, the probe parameters are set, and the force-distance curve data is collected at each data collection point in the target area. The probe-collected data is transmitted to the computer for processing into a mechanical image. The computer matches the collected optical image and mechanical image to obtain the morphological and mechanical property information of the internal slices of the plant tissue; The slices of the plant sample to be tested are embedded by a gradient embedding method and then processed by freezing to obtain the slices.

2. The method for measuring the internal mechanical properties of plant tissues based on atomic force microscopy according to claim 1, characterized in that: The slices of the plant sample to be tested are prepared by the following method: Pre-cool and wash samples; Immerse the sample in an OCT embedding medium of a set concentration for 10 to 30 minutes, and place the OCT embedding medium and the sample in a vacuum chamber for 10 to 30 minutes for infiltration. Repeat the embedding and infiltration twice, with increasing OCT embedding medium concentrations in each step. After permeation, the samples were transferred to 100% OCT embedding solution for directional fixation of the samples; The directionally fixed sample is freeze-solidified stepwise to obtain a freeze-solidified sample; The frozen solidified samples were serially sectioned at -20°C using a microtome with a thickness of 10-25 μm.

3. The method for measuring the internal mechanical properties of plant tissues based on atomic force microscopy according to claim 2, characterized in that: The concentrations of OCT embedding medium were 75% and 85% respectively.

4. The method for measuring the internal mechanical properties of plant tissues based on atomic force microscopy according to claim 2, characterized in that: The stepwise freezing and solidification method includes: firstly placing the sample at a constant temperature of -20°C for 10 minutes for pre-cooling and solidification, and then deep-freezing the pre-cooled and solidified sample with liquid nitrogen, wherein the temperature of the liquid nitrogen deep freezing is -196°C and the time of the liquid nitrogen deep freezing is 30 seconds.

5. The method for measuring the internal mechanical properties of plant tissues based on atomic force microscopy according to claim 2, characterized in that: The method for pre-cooling and cleaning the sample includes: rinsing three times with 4° C. distilled water, and then gently wiping the sample tissue surface along the axis with sterile absorbent paper to remove free water.

6. The method for measuring the internal mechanical properties of plant tissues based on atomic force microscopy according to claim 1, characterized in that: The method for preparing a slice of a plant sample to be tested into an observation sample includes: selecting a target slice, attaching the slice to a glass slide, wherein the glass slide is a lysine-coated glass slide, settling the slice at room temperature for a certain period of time, and then washing away the OCT embedding agent on the slice to obtain a slice fixed on the glass slide.

7. The method for measuring the internal mechanical properties of plant tissues based on atomic force microscopy according to claim 6, characterized in that: The room temperature precipitation time is 10 to 30 minutes. After the room temperature precipitation, the residual OCT embedding agent in the slices is rinsed with deionized water.

8. The method for measuring the internal mechanical properties of plant tissues based on atomic force microscopy according to claim 1, wherein: The probe is a conical probe or a spherical probe, and the parameters of the probe are set as follows: acquisition force of 40-60 nN, acquisition speed of 100-150 μm / s, and lifting height of 2-4 μm.

9. The method for measuring the internal mechanical properties of plant tissues based on atomic force microscopy according to claim 1, characterized in that: The method by which a computer processes the data collected by the probe into a mechanical image is as follows: the computer uses the Hertz model or the Sneddon model to fit the force-distance curve data of each data collection point collected by the probe, combined with the probe shape, to calculate the corresponding Young's modulus value, and then generates a slice Young's modulus diagram, that is, a mechanical image.

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