A pretreatment method for beluga teeth section for microchemical analysis

Abstract

The application discloses a pretreatment method for a Neophocaena asiaeorientalis tooth section for micro-chemical analysis, and comprises the following steps: collecting mandibular teeth of a dead Neophocaena asiaeorientalis; cleaning the teeth to remove soft tissues on the surface and inside the tooth root, and drying the cleaned teeth; fixing the teeth on a base made of an epoxy resin block, and fixing the base in a mold; configuring an epoxy resin reagent, embedding the teeth in the epoxy resin; cutting the buccal side and the lingual side of the teeth along the coronal surface by using a low-speed precision cutting machine, so that the central axis of the coronal surface of the teeth is contained in a 3mm-thick section; further grinding and polishing one side of the section, fixing the section on a glass slide; grinding and polishing the other side of the section, and obtaining the section after cleaning and drying. The Neophocaena asiaeorientalis tooth section obtained by the pretreatment method has the advantages of smooth surface, precise polishing, difficulty in breaking and cracking, and maximization of growth axis surface, and can be used for micro-chemical analysis and growth layer group analysis of Neophocaena asiaeorientalis teeth.

Description

Technical Field

[0001] This invention belongs to the field of animal model technology, specifically relating to a pretreatment method for finless porpoise tooth sections used for microchemical analysis. Background Technology

[0002] The finless porpoise (Neophocaena) is a small toothed whale distributed in tropical waters from the Persian Gulf in the west to temperate coastal waters and some rivers from Japan in the east. Its main species include the Indo-Pacific finless porpoise (Neophocaena phocaenoides) and the narrow-ridged finless porpoise (N. asiaeorientalis). Recent studies have suggested further classifying the narrow-ridged finless porpoise into two separate species: the East Asian finless porpoise (Nasunameri) and the Yangtze finless porpoise (Naasiaeorientalis). Currently, the Yangtze finless porpoise and the East Asian finless porpoise are listed as critically endangered and endangered species respectively by the International Union for Conservation of Nature (IUCN). In 2021, the Yangtze finless porpoise was upgraded to a Class I protected wild animal in China, highlighting the urgency of its conservation. However, research on the biological characteristics and ecological habits of finless porpoises, such as growth and development, dietary changes, and migration between rivers and lakes, remains insufficient, hindering in-depth conservation and rescue efforts. Therefore, it is urgent to develop methods to trace key characteristics in the life history of individual finless porpoises.

[0003] Teeth play a crucial role in studies of age determination, migration patterns, and dietary changes in many terrestrial and marine mammals. Highly mineralized and metabolically inert, tooth tissue is primarily composed of dentin, covered by enamel, and surrounded by the pulp cavity. Dentin is composed of approximately 72% inorganic minerals, mainly hydroxyapatite, 10% water, and the remaining 20% ​​organic matter. Dentin begins to form during the fetal stage and continues to grow into the pulp cavity, forming clearly identifiable growth layers according to changes in its growth rate. Because teeth record elemental and isotopic information from their environment during growth, and these trace elements and isotopes remain unchanged once deposited, teeth serve as "recorders" for studying environmental changes throughout their lifespan. By preparing thin slices containing the dentin growth axis and analyzing their microchemical composition along the growth axis (from the cusp to the pulp cavity), it is possible to track the physiological state or environmental changes of an individual Yangtze finless porpoise throughout its lifespan.

[0004] Dental microchemical analysis relies on advanced analytical equipment, such as laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) and laser ablation multiple receiver inductively coupled plasma mass spectrometry (LA-MC-ICPMS). These instruments are commonly used in the study of metals and minerals; however, sample preparation methods for minerals or metals often only consider the exposed surface and its smoothness and polishing, with less consideration for the specific research axis, and are not entirely suitable for biological tissues like animal teeth. In animal tooth research, direct cutting or grinding is often used to maximize the growth axis plane, facilitating the observation of growth layers and improving the resolution of microchemical analysis. However, for smaller samples such as those from finless porpoises, this operation is difficult, easily causing tooth breakage and damage. Furthermore, these methods often do not meet the analytical requirements of precision instruments, such as insufficient surface smoothness and polishing, and the tendency for ground sample slices to break and crumble. Therefore, overcoming the technical challenges of sample pretreatment in the microchemical analysis of finless porpoise teeth is of great significance for advancing microchemical research on finless porpoise teeth and further promoting finless porpoise ecology. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a pretreatment method for finless porpoise tooth sections used for microchemical analysis. Combining the needs of tooth growth layer analysis and microchemical analysis, this method integrates resin embedding, precision cutting, fixation, grinding, and polishing to optimize the tooth pretreatment process. The resulting tooth sections have advantages such as smooth surface, precise polishing, resistance to breakage and fracture, and maximization of the growth axis. They can be used for microchemical analysis and growth layer analysis of finless porpoise teeth.

[0006] This invention is achieved through the following technical solution:

[0007] A pretreatment method for finless porpoise tooth sections used for microchemical analysis includes the following steps:

[0008] Step 1) Tooth Removal: Collect the straighter teeth from the middle of the left and right rows of the lower jaw of the deceased finless porpoise; if the individual is severely decayed and the tooth is missing in that position, take the adjacent straighter tooth; if too many teeth are missing, select the tooth with less wear.

[0009] Step 2) Tooth cleaning: Remove the tooth, use hydrogen peroxide and instruments to remove the soft tissue attached to the tooth surface, and use instruments to insert into the pulp cavity to remove the soft tissue inside the pulp cavity; then use deionized water to clean the outer surface of the tooth and the inside of the pulp cavity.

[0010] Step 3) Tooth embedding: Fix the tooth onto the resin block, and fix the resin block with the tooth in the mold. Pour in cold embedding resin reagent to embed the tooth in the middle of the resin and remove air bubbles from the pulp cavity. Finally, put the mold with the embedded finless porpoise tooth into the oven and let it sit until the resin is completely solidified and hardened.

[0011] Step 4) Tooth slice: Remove the resin block from the mold, smooth the surface, and use a low-speed precision cutter to cut off the buccal and lingual sides of the tooth along the coronal surface. During the cutting process, keep the saw blade parallel to the coronal surface of the tooth and retain the longest axis from the cusp to the root in the slice to maximize the area of ​​the coronal surface after cutting.

[0012] Step 5) Grinding and polishing: Use sandpaper to finely grind one side of the slice to remove cutting marks, then attach this side to the glass slide; after fixing, finely grind the upper surface of the dental slice, and after the surface gradually becomes smooth, polish it with a polishing cloth and silica suspension; after cleaning and drying, it is ready.

[0013] Preferably, in step 2), the time for cleaning with deionized water is 5 to 6 minutes, and then the teeth are placed in an oven at 37°C to dry for 20 to 24 hours.

[0014] Preferably, the specific operation of step 3) is as follows: Use a cutting machine to cut the solidified cold-mounting resin block into small pieces of 0.5cm square; apply the mixed AB glue to the upper surface of the resin block, place the tooth flat and fix it on the resin block, and then use AB glue to fix the resin block to the bottom of a silicone mold with an internal length, width and height of 1cm. After placement, let it stand for 30-40 minutes to allow the AB glue to fully cure; mix the cold-mounting resin and the accelerator at a mass ratio of 7:1 and stir evenly; pour the prepared resin reagent into the mold so that the tooth is embedded in the middle of the resin; use a single clean hard bristle brush to insert into the pulp cavity and remove the air bubbles; finally, place the mold with the embedded finless porpoise tooth in an oven at 37°C for more than 12 hours until the resin is completely solidified and hardened.

[0015] Preferably, the specific operation of step 4) is as follows: the square resin block containing the tooth is removed from the mold, and the surface is polished smooth by using 1200 grit, 2000 grit and 4000 grit sandpaper in sequence. The buccal and lingual sides of the tooth are cut off along the coronal surface of the tooth using a low-speed precision cutter with a micrometer. During the cutting process, the saw blade is kept parallel to the coronal surface of the tooth, and the longest axis from the cusp to the root is retained in the 3mm thick slice to maximize the area of ​​the coronal surface after cutting.

[0016] Preferably, the specific operation of step 5) is as follows: use 2400-grit and 4000-grit sandpaper to finely grind one side of the slide to remove cutting marks, and then use AB glue to stick this side to a glass slide with a sample label; finely grind the upper surface of the dental slide, and after the surface gradually becomes smooth, polish it with a polishing cloth and silica suspension; after polishing, use a cutter to cut off excess glass and resin, rinse the dental slide with deionized water, and then place it in a clean beaker for ultrasonic cleaning with Milli-Q water for 5 minutes. After that, take out the dental slide and rinse it with Milli-Q water for 2 minutes. Place the cleaned dental slide in a petri dish and place absorbent paper under the slide; place the dental slide in the petri dish in an oven at 37°C and dry it for 12 hours to obtain the final product.

[0017] Preferably, the resistivity of the Milli-Q water is 18.2 MΩ·cm.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention provides a pretreatment method for finless porpoise tooth sections used for microchemical analysis, filling a gap in previous methods for tooth sample pretreatment in solid sample microchemical analysis. This method combines the growth characteristics and material properties of finless porpoise teeth with a cold-mounting process used in solid sample microchemical analysis pretreatment. The resulting embedding material possesses high hardness, good support, high transparency, and good thermal stability, ensuring it does not easily disintegrate during tooth pretreatment. The tooth section has a high surface smoothness, facilitating grinding and polishing, and allowing for observation of the growth layer structure. It can also be used with analytical instruments that handle high instantaneous temperatures, such as laser ablation. Furthermore, the introduction of a precision cutting process preserves the largest growth axis plane of the tooth in a thinner section, thus meeting the sample conditions required for microchemical analysis while preserving the structural characteristics of the finless porpoise tooth growth layer. Finless porpoise tooth samples processed using this method can meet the analytical requirements of various instruments, including laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) and laser ablation multi-collector inductively coupled plasma mass spectrometry (LA-MC-ICPMS), and can be used for tooth growth layer analysis. Attached Figure Description

[0020] Figure 1 Photograph of the lower left jaw teeth of a deceased Yangtze finless porpoise;

[0021] Figure 2 Photographs of finless porpoise teeth embedded in epoxy resin blocks;

[0022] Figure 3 A section of a finless porpoise tooth pasted on a glass slide;

[0023] Figure 4 for Figure 3 A magnified view of a section of a tooth slice from a Yangtze finless porpoise. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0025] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Example 1

[0028] A pretreatment method for finless porpoise tooth sections used for microchemical analysis, the specific steps of which are as follows:

[0029] (1) Tooth removal

[0030] Collect the straighter teeth from the middle of the left and right rows of the lower jaw of dead finless porpoises; if the individual is severely decomposed and the teeth in that position are missing, then take the straighter teeth from the nearest adjacent teeth if possible; if too many teeth are missing, then choose the teeth that are not severely worn.

[0031] The finless porpoise teeth specimen in this embodiment were obtained from a dead finless porpoise found in the main stream of the Yangtze River in April 2023. After dissection, the teeth were extracted from the middle of the left row of the porpoise's lower jaw. Figure 1 As shown, store in 1.5mL centrifuge tubes and refrigerate at -20°C until use.

[0032] (2) Teeth cleaning

[0033] The tooth is extracted, and hydrogen peroxide and a toothbrush are used to remove the soft tissue attached to the tooth surface. A plastic-bristled brush is then used to penetrate the pulp chamber to remove nerves and other tissues. Afterwards, deionized water is used to clean the outer surface of the tooth and the inside of the pulp chamber. Details are as follows:

[0034] After the teeth are removed, they are held with tweezers and the organic matter on the surface of the teeth is removed with a toothbrush in hydrogen peroxide. The teeth are then rinsed with deionized water for 5-6 minutes to further remove any remaining organic matter. The teeth are then placed in an open 1.5 mL centrifuge tube and dried in an oven at 37°C for 20-24 hours.

[0035] (3) Tooth embedding

[0036] The tooth was fixed to a resin block, and the resin block with the tooth fixed was then fixed in a mold. Cold-mounting resin was poured in, embedding the tooth in the resin, and air bubbles in the pulp cavity were removed. Finally, the mold containing the finless porpoise tooth was placed in an oven until the resin completely solidified and hardened. Details are as follows:

[0037] Use a cutting machine to cut the solidified cold-mounted resin block into 0.5cm square pieces; apply the mixed AB glue (Swiss Ergo 7450 epoxy resin AB glue) to the surface of the resin block, place the tooth flat on the AB glue, and let it sit for 5 minutes to allow the AB glue to solidify, thus fixing the tooth to the resin block; then use AB glue to fix the resin block to the bottom of a silicone mold with internal dimensions of 1cm, and let it sit for 5 minutes after placement to allow the AB glue to fully cure; mix the cold-mounted resin (EpoFix Resin, Struers) with the accelerator (EpoFix). Hardener (Struers) was mixed in a 7:1 mass ratio into a disposable plastic cup, and stirred continuously in one direction to avoid generating air bubbles; the prepared resin was poured into the mold, embedding the tooth in the resin; a single clean, stiff-bristled brush was inserted into the pulp cavity to remove any air bubbles; finally, the mold containing the finless porpoise tooth was placed in a 37°C oven for at least 12 hours, until the resin had completely solidified and hardened. Figure 2 As shown.

[0038] (4) Dental slices

[0039] Remove the resin block from the mold, smooth the surface, and use a low-speed precision cutter with a micrometer to cut away the buccal and lingual sides of the tooth along the coronal plane. During the cutting process, keep the saw blade parallel to the coronal plane of the tooth, preserving the longest axis from the cusp to the root in the slice to maximize the area of ​​the coronal plane after cutting. Details are as follows:

[0040] Remove the square resin block containing the tooth from the mold. At this point, the surface of the resin block in contact with the mold is smooth and transparent, while the surface in contact with the air may have pits. Use 1200 grit, 2000 grit, and 4000 grit sandpaper to polish the surface smooth in sequence. Use a low-speed precision cutter with a micrometer along the coronal surface of the tooth. Before cutting, use the clamp of the cutter to fix the resin block containing the tooth. Adjust the angle of the resin block on the clamp so that the saw blade is parallel to the coronal surface of the tooth. Cut off the buccal and lingual sides of the tooth, keeping the longest axis from the cusp to the root in a 3mm thick slice to maximize the area of ​​the coronal surface after cutting.

[0041] (5) Grinding and polishing

[0042] Finely grind one side of the dental slide with sandpaper to remove cutting marks. Then, glue this side to a glass slide with a sample label written on it using AB glue. After fixing, finely grind the upper surface of the dental slide. Once the surface is gradually smooth, polish it with a polishing cloth and silica suspension. After polishing, use a cutting machine to cut off the resin and glass except for the label and the teeth. Rinse with deionized water and let it air dry. The final product is as follows:

[0043] Finely grind one side of the tooth section using 2400-grit and 4000-grit sandpaper to remove cutting marks. Then, glue this side to a glass slide with a sample label written on it using AB glue, making sure the resin covers the label and that the teeth do not obscure it. Finely grind the upper surface of the tooth section until it becomes smooth, then polish it with a polishing cloth and silica suspension. After polishing, use a cutter to remove excess glass and resin. Rinse the tooth section with deionized water and then place it in a clean beaker for ultrasonic cleaning with Milli-Q water (resistivity 18.2 MΩ·cm) for 5 minutes. After that, remove the tooth section and rinse it with Milli-Q water for 2 minutes. Place the cleaned tooth section in a petri dish and place absorbent paper under the section. Place the tooth section in the petri dish in an oven at 37°C for 12 hours to dry, obtaining the finless porpoise tooth section.

[0044] The final slice of the finless porpoise tooth is as follows Figure 3 , 4 As shown, the largest growth axis plane of the finless porpoise tooth is preserved in a relatively thin section, maximizing the integrity of the tooth analysis surface and retaining the tooth's growth characteristics, thus meeting the sample condition requirements for microchemical analysis. Finless porpoise tooth sections processed using the method of this invention can meet the requirements for laser ablation inductively coupled plasma mass spectrometry (ICP-MS) analysis (multi-element content) and laser ablation multi-receiver ICP-MS analysis (multi-element content). 87 Sr / 86 It meets the needs of various analyses such as Sr ratio, and ensures that the slices after microchemical analysis can be used for age identification after simple reprocessing, and correlates the microchemical analysis results with the growth and development sequence.

[0045] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A pretreatment method for finless porpoise tooth sections used for microchemical analysis, characterized in that, Includes the following steps: Step 1) Tooth Removal: Collect the straighter teeth from the middle of the left and right rows of the lower jaw of the deceased finless porpoise; if the individual is severely decomposed and the tooth is missing in that position, then take the adjacent straighter tooth. If too many teeth are missing, choose the teeth that are not severely worn. Step 2) Tooth cleaning: Remove the tooth, use hydrogen peroxide and instruments to remove the soft tissue attached to the tooth surface, and use instruments to insert into the pulp cavity to remove the soft tissue inside the pulp cavity; then use deionized water to clean the outer surface of the tooth and the inside of the pulp cavity. Step 3) Tooth embedding: Use a cutting machine to cut the solidified cold-mounting resin block into 0.5 cm square pieces; apply the mixed AB glue to the upper surface of the resin block, place the tooth flat and fix it on the resin block, and then use AB glue to fix the resin block to the bottom of the silicone mold with internal dimensions of 1 cm. After placement, let it stand for 30-40 minutes to allow the AB glue to fully cure; mix the cold-mounting resin and the accelerator at a mass ratio of 7:1 and stir evenly; pour the prepared resin reagent into the mold, so that the tooth is embedded in the middle of the resin; Use a single clean, stiff bristle to insert into the pulp cavity and remove any air bubbles; finally, place the mold containing the finless porpoise tooth in a 37°C oven for more than 12 hours until the resin is completely solidified and hardened. Step 4) Tooth sectioning: Remove the square resin block containing the tooth from the mold, and polish the surface smooth with 1200 grit, 2000 grit, and 4000 grit sandpaper in sequence. Use a low-speed precision cutter with a micrometer to cut off the buccal and lingual sides of the tooth along the coronal surface. During the cutting process, keep the saw blade parallel to the coronal surface of the tooth and retain the longest axis from the cusp to the root in the 3mm thick section to maximize the area of ​​the coronal surface after cutting. Step 5) Grinding and polishing: Use 2400-grit and 4000-grit sandpaper to finely grind one side of the section to remove cutting marks. Then, use AB glue to attach this side to a glass slide with a sample label. Finely grind the upper surface of the dental section. After the surface gradually becomes smooth, polish it with a polishing cloth and silica suspension. After polishing, use a cutter to cut off excess glass and resin. Rinse the dental section with deionized water and place it in a clean beaker for ultrasonic cleaning with Milli-Q water for 5 minutes. Then, take out the dental section and rinse it with Milli-Q water for 2 minutes. Place the cleaned dental section in a petri dish and place absorbent paper under the section. Place the dental section in the petri dish in an oven at 37°C and dry it for 12 hours.

2. The pretreatment method for finless porpoise tooth sections for microchemical analysis according to claim 1, characterized in that, In step 2), the teeth are cleaned with deionized water for 5-6 minutes, and then dried in an oven at 37°C for 20-24 hours.

3. The pretreatment method for finless porpoise tooth sections for microchemical analysis according to claim 1, characterized in that, Step 5) The resistivity of the Milli-Q water is 18.2 MΩ•cm.

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