An improved method for sectioning of tree stem samples obtained by the microcoring method
The improved sliding sectioning method solves the problem of poor section quality in the micro-tree core method, and realizes rapid, low-cost, and efficient section preparation, which is suitable for tree growth dynamics research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 王文礼
- Filing Date
- 2025-01-13
- Publication Date
- 2026-07-14
AI Technical Summary
Existing techniques for slicing tree trunk samples obtained through the micro-tree core method suffer from problems such as poor slice quality, complicated operation, high cost, and unsuitability for rapid, large-scale slicing.
A modified sliding sectioning method was adopted, which includes sample softening, orientation determination, fixation, staining and mounting steps. The sample was fixed using a softening fixative and hot melt adhesive, and the section orientation was determined by electron microscopy. Staining was performed using safranin and alsin blue solutions, and the slides were mounted using neutral resin.
It enables the rapid and large-scale acquisition of high-quality tree stem sections, simplifies the operation process, reduces costs, and is suitable for the study of tree growth dynamics.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of hardwood slicing technology, and more particularly to a method for preparing slices of tree trunk samples obtained by the micro-core method. Background Technology
[0002] Micro-core sampling is a commonly used experimental method for monitoring the radial growth process of trees. Using a micro-growth cone, tissue samples (bark, phloem, and xylem) are periodically collected at the tree's diameter at breast height (DBH). By preparing biological sections and observing them under a microscope, the number, size, and morphological changes of cells in the cambium and xylem at each stage of differentiation can be recorded in detail. Its advantages include minimal damage to the tree, continuous sampling and monitoring of the development process of the cambium and xylem, and the ability to calculate the specific times of the onset and termination of xylem activity using models.
[0003] After obtaining tree samples using the micro-core method, the samples are then sectioned in the laboratory. In current research on tree growth dynamics, paraffin sectioning and slip sectioning are often chosen in combination with the micro-core method as a fundamental experimental approach. The paraffin sectioning method is a microscopic slide preparation method using paraffin as an embedding agent. The specific steps include: sample collection → fixation with FAA fixative for 24 hours → dehydration (50%, 60%, 70%, 80%, 90%, 100%, 100% ethanol, 1-2 hours per stage) → clearing (1 / 2 xylene + 1 / 2 ethanol, pure xylene, pure xylene, 2 hours per stage). h) → Embedding in paraffin (1 / 2 paraffin + 2 / 1 clearing agent for 24 hours → pure paraffin at 65°C for 4 hours, repeated twice → embedding, cooling at room temperature, then transferring to a 16°C refrigerator to cool and solidify → trimming the wax block) → sectioning and drying (rotary microtome sectioning → 1% gelatin mounting → drying on a 39°C drying table for 2-3 days) → dewaxing and rehydration (Ton bio-clearing agent treatment → sequential 100%, 95%, 85% ethanol, 5 min each) → staining (1% safranin staining → ethanol rinsing → 0.5% Fast Green staining) → dehydration (100% ethanol treatment) → mounting (neutral resin mounting → drying in a constant temperature oven to accelerate air drying). This method allows for clearer and more effective observation of the xylem structure; the sliding sectioning method can section hardwoods using only a sliding microtome, is simple and easy to implement, and is widely used in the preparation of plant material sections. However, tree growth dynamics studies are time-consuming, requiring weekly sample collection. The samples are numerous and the wood is hard. Paraffin sectioning uses many reagents, including numerous carcinogens and toxic substances, and is complex and costly. While slip sectioning can rapidly process large numbers of tree samples, it still has certain limitations. Therefore, for tree trunk samples obtained through the micro-core method, the slip sectioning technique needs methodological adjustments and improvements to facilitate subsequent research on tree growth dynamics. Summary of the Invention
[0004] The purpose of this invention is to provide an improved method for slide sectioning of tree trunks, in order to solve the technical problem of producing a large number of slide samples while ensuring the quality of the sections.
[0005] To achieve the above objectives, the technical solution adopted by this invention is as follows: A method for obtaining slip sections of tree stems using the micro-core method includes the following steps: (1) Sampling of stem tissue: A trephor was used to collect stem tissue samples from trees with a diameter at breast height of approximately 1.3 m. The samples included the phloem, cambium, and xylem regions. (2) Sample softening: After obtaining tree trunk samples using the micro-core method, they are immediately placed in 50 mL test tubes containing a softening fixative (ethanol: glycerol = 1:1). This method is particularly suitable for areas with high rainfall. Soaking in the dehydrating agent should be done for at least one week. This simplifies subsequent dehydration steps in the laboratory, facilitating subsequent slide sectioning.
[0006] (3) Determine the sample orientation: The distinction between cross-section, radial section, and tangential section in wood anatomy is difficult to make in the cylindrical shape of micro-cores. However, subsequent cell growth statistics are based on cross-sections. Therefore, it is necessary to identify the cross-section of the micro-core before section preparation. The following methods can be used to identify the cross-section of the micro-core: Place the microtree core under a 4x objective electron microscope, flip the microtree core to find the cross section, and only in the cross section can the tightly packed cell structure be observed.
[0007] (4) Sample fixation: After determining the orientation of the micro-tree core, use a hot melt glue gun to spread a layer of hot melt glue on a 3cm cube of wood. Place the micro-tree core on top and then completely enclose it with hot melt glue. When using hot melt glue to fix the sample, pay attention to whether there are air bubbles. If there are air bubbles, cover them with another layer of hot melt glue and wait for the hot melt glue to solidify for subsequent sectioning.
[0008] (5) Slide away slices: The solidified wood block containing the micro-tree core sample was fixed on a slide microtome, and then sectioning began.
[0009] (6) Staining and mounting: Staining was performed using safranin and alsin blue solutions. The staining time needed to be adjusted for different tree species. Generally, safranin staining for about 1 minute was suitable, followed by washing with 50% ethanol until no more red precipitate appeared on the sections. Alsin blue staining for about 2 minutes was suitable, followed by washing with water until no more blue precipitate appeared on the sections. The sections were mounted with neutral resin and photographed after the resin solidified. The alsin blue solution was prepared as follows: 0.5g alsin blue, 100ml 95% ethanol. The safranin solution was prepared as follows: 0.05g safranin, 100ml water.
[0010] The technical solution of this invention firstly softens the micro-tree core sample by using a softening fixative. Secondly, an electron microscope is used to observe the cross-sectional direction of the tree core, avoiding sections with radial and tangential sections that are not conducive to subsequent cell statistics. The sample is fixed with hot melt adhesive, and the sections obtained by the slip sectioning method can obtain a large number of sections with clear and complete cell tissue structures more quickly than the paraffin sectioning method. This is more conducive to subsequent statistics and research on tree growth dynamics and lays the foundation for exploring the growth process of tree trunks. Attached Figure Description
[0011] Figure 1 Cross section of micro-tree core under an electron microscope Figure 2 Micro-tree core section under an electron microscope Figure 3 String-section of a micro-tree core under an electron microscope Figure 4 micro-tree core sample Figure 5 Example of hot melt adhesive fixing of micro-tree core samples Figure 6 Micrograph of a cross-section of the stem tissue of *Juniperus alpinus* in Example 1. Figure 7 Micrograph of a cross-section of the stem tissue of Abies simonii in Example 2. Detailed Implementation
[0012] The following embodiments are only for further detailed description of the present invention, but do not constitute any limitation on the present invention.
[0013] Example 1: Method for Slicing Microstructure Cores of Cypress 'Alpine' (1) Sampling of stem tissue: A stem tissue sample was collected from the cypress at a diameter at breast height of approximately 1.3 m using a Trephor. The sample included the phloem, cambium, and xylem regions. (2) Sample softening: After obtaining alpine cypress stem samples using the micro-core method, they were immediately placed in 50 mL test tubes containing a dehydrating agent (ethanol: glycerol = 1:1). This method is particularly suitable for areas with high rainfall, and the samples should be soaked in the dehydrating agent for at least one week. This simplifies the subsequent dehydration steps in the laboratory, facilitating slide sectioning.
[0014] (3) Determine the sample orientation: Place the microtree core under a 4x objective electron microscope, flip the microtree core to find the cross section, and only in the cross section can the tightly packed cell structure be observed.
[0015] (4) Sample fixation: After determining the orientation of the micro-tree core, use a hot melt glue gun to spread a layer of hot melt glue on a 3cm cube. Place the micro-tree core on top and then completely enclose it with hot melt glue. When using hot melt glue to fix the sample, pay attention to whether there are air bubbles. If there are air bubbles, puncture them and cover them with another layer of hot melt glue to facilitate subsequent sectioning.
[0016] (5) Slide away slices: Secure the wooden block to the slicer, smooth the raised surface to a flat surface, and then begin slicing.
[0017] (6) Staining and mounting: Safranin staining time is generally about 1 minute, followed by washing with 50% ethanol until no more red precipitate appears on the slides; Alsin blue staining time is generally about 1 minute, followed by washing with water until no more blue precipitate appears on the slides. Mount the slides with neutral resin, and after the resin has solidified, take photographs and perform subsequent experiments.
[0018] Example 2: The core tissue of *Abies cuspidatum* microtrees is obtained by sliding sectioning. (1) Sampling of stem tissue: A stem tissue sample was collected from approximately 1.3 m in diameter at breast height of the *Abies hygrophora* var. *acrylonitrile* using a trephor. The sample included the phloem, cambium, and xylem regions. (2) Sample softening: After obtaining stem samples of Abies lanceolata using the micro-core method, they were immediately placed into 50 mL test tubes containing a dehydrating agent (ethanol: glycerol = 1:1). This method is particularly suitable for areas with high rainfall, and the samples should be soaked in the dehydrating agent for at least one week. This simplifies the subsequent dehydration steps in the laboratory, facilitating subsequent slide sectioning.
[0019] (3) Determine the sample orientation: Place the microtree core under a 4x objective electron microscope, flip the microtree core to find the cross section, and only in the cross section can the tightly packed cell structure be observed.
[0020] (4) Sample fixation: After determining the orientation of the micro-tree core, use a hot melt glue gun to spread a layer of hot melt glue on a 3cm cube. Place the micro-tree core on top and then completely enclose it with hot melt glue. When using hot melt glue to fix the sample, pay attention to whether there are air bubbles. If there are air bubbles, puncture them and cover them with another layer of hot melt glue to facilitate subsequent sectioning.
[0021] (5) Slide away slices: Secure the wooden block to the slicer, smooth the raised surface to a flat surface, and then begin slicing.
[0022] (6) Staining and mounting: Safranin staining time is generally about 1 minute, followed by washing with 50% ethanol until no more red precipitate appears on the slides; Alsin blue staining time is generally about 2 minutes, followed by washing with water until no more blue precipitate appears on the slides. Mount the slides with neutral resin, and after the resin has solidified, take photographs and perform subsequent experiments.
Claims
1. A method for preparing sections of tree trunk samples obtained by the micro-tree core method, characterized in that, Includes the following steps: 1) Sampling of stem tissue: A trephor was used to collect stem tissue samples from trees with a diameter at breast height of approximately 1.3 m. The samples included the phloem, cambium, and xylem regions. 2) Sample softening: After obtaining tree trunk samples using the micro-core method, immediately place them into 50mL test tubes containing a dehydrating agent (ethanol:glycerol = 1:1) and soak them in the dehydrating agent for at least one week. This method is particularly suitable for areas with high rainfall, simplifying subsequent dehydration steps in the laboratory for easier slide sectioning. 2.3) Determine the sample orientation: The obtained micro-tree cores are cylindrical and have both transverse and radial sections. Subsequent cell growth statistics are based on the transverse sections; therefore, it is necessary to identify the transverse sections of the micro-tree cores before section preparation. The transverse sections of the micro-tree cores are identified using the following method: Place the microtree core under a 4x objective electron microscope, flip the microtree core to find the cross section, and only in the cross section can the tightly packed cell structure be observed. 3.4) Sample fixation: After determining the orientation of the micro tree core, use a hot melt glue gun to spread a layer of hot melt glue on a cube with a side length of 5cm, place the micro tree core on it, and then use hot melt glue to completely wrap and seal the micro tree core. 4.5) Slide away slices: Fix the wooden block onto the slicer, smooth the raised, solidified hot melt adhesive surface into a flat surface, and then begin slicing. 5.6) Staining and mounting: Staining was performed using safranin and alsin blue solutions. The staining time needed to be adjusted for different tree species; generally, about 1 minute for safranin and about 2 minutes for alsin blue were appropriate. The slides were then mounted with neutral resin, and photographs and other subsequent experiments were conducted after the resin had solidified.
6. The method for preparing sections of tree trunk samples obtained by the micro-tree core method according to claim 1, characterized in that: In step 2), the volume ratio of ethanol to glycerol solution required for preparing the softening fixative is 1:1, and the soaking time in the softening fixative should be more than one week.
7. The method for preparing sections of tree trunk samples obtained by the micro-tree core method according to claim 2, characterized in that: In step 3), an electron microscope is used to identify the cross-section of the micro-tree core.
8. The method for preparing sections of tree trunk samples obtained by the micro-tree core method according to claim 3, characterized in that: In step 4), hot melt adhesive is used to fix and wrap the micro-tree core sample.
9. A method for preparing sections of tree trunk samples obtained by the micro-tree core method according to claim 4, characterized in that: In step 6), the staining time for safranin is generally about 1 minute, and for Alsin blue, it is about 2 minutes. The Alsin blue staining solution is prepared as follows: 0.5g Alsin blue, 100ml 95% ethanol. The safranin staining solution is prepared as follows: 0.05g safranin, 100ml water.