Pretreatment method of Jiangfish tooth slice for micro-chemical analysis

Abstract

The invention discloses a pretreatment method of a gusset-fish tooth slice for micro-chemical analysis. The pretreatment method comprises the following steps: collecting mandibular teeth of a dead gusset-fish individual; cleaning the teeth, removing soft tissues on the surfaces and in tooth roots, and airing the cleaned teeth; the teeth are fixed on a base made of epoxy resin blocks, and the base is fixed in a mold; preparing an epoxy resin reagent, and embedding the teeth in epoxy resin; a low-speed precise cutting machine is used for cutting the buccal side and the lingual side of the tooth along the coronal plane, so that the central axis of the coronal plane of the tooth is contained in a slice with the thickness of 3 mm; further grinding and polishing one side of the slice, and fixing the slice on a glass slide; and grinding and polishing the other side of the slice, cleaning, and drying. The dolphin tooth slice obtained through the pretreatment method has the advantages of being smooth in surface, precise in polishing, not prone to breaking and cracking, capable of achieving growth axial surface maximization and the like, and can be used for microchemical analysis and growth layer group analysis of dolphin teeth.

Description

Technical Field

[0001] The invention belongs to the technical field of animal models, and in particular relates to a pre-treatment method of finless porpoise tooth slices for microchemical analysis. Background Art

[0002] The Yangtze finless porpoise (Neophocaena) is a small toothed whale found in tropical waters from the Persian Gulf in the west to temperate coastal waters and some rivers in the east to Japan. Its main species include the Indo-Pacific finless porpoise (Neophocaena phocaenoides) and the narrow-ridged finless porpoise (N.asiaeorientalis). Recent studies have suggested that the narrow-ridged finless porpoise be further divided into two independent 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 World Conservation Union (IUCN). In 2021, the Yangtze finless porpoise was upgraded to a national first-class key protected wild animal, highlighting the urgency of its protection. However, research on the biological characteristics and ecological habits of finless porpoises, such as growth and development, changes in diet, and river and lake migration, is still insufficient, which restricts the in-depth advancement of protection and rescue work. Therefore, it is urgent to develop methods to trace key characteristics in the life history of individual finless porpoises.

[0003] Teeth play an important role in the study of age determination, migration trajectory, and dietary changes of many terrestrial and marine mammals. Tooth tissue is highly mineralized and metabolically inert. It is mainly composed of dentin, with the surface covered by enamel and the internal pulp cavity surrounded by dentin. About 72% of the composition of dentin is inorganic minerals, mainly hydroxyapatite, 10% is water, and the remaining about 20% is organic matter. Dentin begins to form in the fetal period of animals and continues to grow into the pulp cavity. During this process, it will form a clearly identifiable growth layer group according to the change of growth rate. Since teeth can record the elemental and isotopic information of the environment they have experienced during their growth, and these trace elements and isotopes will not change once deposited in the teeth, teeth have become "recorders" for studying environmental changes throughout their life course. By making thin slices containing the growth axis of dentin and analyzing its microchemical composition along the growth axis (from the tip of the tooth to the pulp cavity), the physiological state or environmental changes of the individual finless porpoise can be tracked in its life course.

[0004] Tooth microchemical analysis requires advanced analytical equipment, such as laser ablation inductively coupled plasma mass spectrometer (LA-ICPMS) and laser ablation multi-collector inductively coupled plasma mass spectrometer (LA-MC-ICPMS). These instruments are often used in the study of metals and minerals. However, the sample processing methods of minerals or metals often only consider the exposed surface and the flatness and polishing of the surface, but rarely consider the specific research axis, which is not completely applicable to biological tissues such as animal teeth. In animal tooth research, direct cutting or grinding is often used to obtain the maximum growth axis plane, which is conducive to the observation of growth layer groups and improve the resolution of microchemical analysis. However, for smaller samples such as finless porpoise teeth, this operation is difficult and easily causes tooth fracture and damage. In addition, these methods often do not meet the analysis requirements of precision instruments, such as insufficient surface flatness and polishing, and the sample slices after grinding are easy to break and crack. Therefore, overcoming the technical difficulties of sample pretreatment in the microchemical analysis of finless porpoise teeth is of great significance for promoting the microchemical research of finless porpoise teeth and further promoting the ecological research of finless porpoises. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a pretreatment method for finless porpoise tooth slices for microchemical analysis. In combination with the needs of tooth growth layer analysis and microchemical analysis, resin embedding, precision cutting, fixed grinding and polishing and other processing methods are combined to optimize the tooth pretreatment method. The tooth slices have the advantages of smooth surface, precise polishing, not easy to break or crack, and the ability to maximize the growth axis surface, and can be used for microchemical analysis and growth layer group analysis of finless porpoise teeth.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for pre-processing finless porpoise tooth slices for microchemical analysis comprises the following steps:

[0008] Step 1) Tooth extraction: Collect the straighter teeth in the middle of the left and right rows of the lower jaw of the dead finless porpoise. If the individual has teeth missing due to severe decay, take the adjacent straighter teeth. If there are too many missing teeth, select the teeth that are not severely worn.

[0009] Step 2) Teeth cleaning: Pull out the teeth, use hydrogen peroxide and instruments to remove the soft tissue attached to the tooth surface, and use instruments to reach into the pulp cavity to remove the soft tissue inside the pulp cavity; then use deionized water to clean the outer surface of the teeth and the inside of the pulp cavity;

[0010] Step 3) Tooth embedding: fix the tooth on the resin block, and fix the resin block with the tooth fixed in the mold, pour in the cold embedding resin reagent, embed the tooth in the middle of the resin, and guide out the bubbles in the pulp cavity; finally, put the mold embedding the finless porpoise tooth into the oven and place it until the resin is completely solidified and hardened;

[0011] Step 4) Tooth slicing: remove the resin block from the mold, grind the surface flat, and use a low-speed precision cutting machine to cut off the buccal and lingual sides of the tooth along the coronal surface of the tooth. During the cutting process, the saw blade is parallel to the coronal surface of the tooth, and the longest axis from the cusp to the root is retained 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 the cutting marks, and then stick this side on a glass slide; after fixing, finely grind the upper surface of the tooth slice, and after the surface is gradually smooth, polish it with a polishing cloth and a silica suspension; then wash and dry it.

[0013] Preferably, in step 2), the deionized water is used for cleaning for 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-mounted resin block into small blocks of 0.5 cm square; apply the mixed AB glue on the upper surface of the resin block, lay 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 an internal length, width and height of 1 cm. After placement, let it stand for 30 to 40 minutes until the AB glue is fully cured; prepare the cold-mounted resin and the coagulant in a mass ratio of 7:1 and stir evenly; pour the prepared resin reagent into the mold to embed the tooth in the middle of the resin; use a single clean hard-bristle brush to extend into the pulp cavity to remove the bubbles therein; finally, put the mold with the embedded finless porpoise tooth into a 37°C oven 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 embedded with the teeth is taken out from the mold, and the surface is polished and smoothed using 1200 mesh, 2000 mesh, and 4000 mesh sandpaper in sequence, and the buccal and lingual sides of the teeth are cut off along the coronal plane of the teeth using a low-speed precision cutting machine with a micrometer. During the cutting process, the saw blade is parallel to the coronal plane of the teeth, and the longest axis from the cusp to the root is retained in the 3 mm thick slice to maximize the area of ​​the coronal plane after cutting.

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

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

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

[0019] The pretreatment method of the finless porpoise tooth slice for microchemical analysis of the present invention fills the gap in the pretreatment method for tooth samples in the previous microchemical analysis of solid samples. The method combines the growth characteristics and material properties of the finless porpoise tooth itself, introduces the cold embedding processing technology used in the pretreatment of solid sample microchemical analysis, and the prepared embedding material has the characteristics of strong hardness, good support, high transparency, good thermal stability, etc., which ensures that it is not easy to disintegrate during the pretreatment process of the tooth. The surface flatness of the tooth slice is high, which is convenient for grinding and polishing, and convenient for observing the growth layer group structure, and can be used for analysis instruments with high instantaneous temperature such as laser ablation. In addition, the introduction of precision cutting technology can retain the largest growth axis plane of the tooth in a thinner slice, thereby meeting the sample conditions required for microchemical analysis and retaining the structural characteristics of the growth layer group of the finless porpoise tooth. The finless porpoise tooth sample treated by this method can meet the analysis requirements of various instruments such as 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 group analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a photo of the teeth of the left lower jaw of a deceased finless porpoise;

[0021] Figure 2 This is a photo of a finless porpoise tooth embedded in an epoxy resin block;

[0022] Figure 3 This is a slice of a finless porpoise tooth pasted on a glass slide;

[0023] Figure 4 for Figure 3 A partial enlarged view of a slice of a middle finless porpoise's tooth. DETAILED DESCRIPTION

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

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

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] Example 1

[0028] A method for pre-processing finless porpoise tooth slices for microchemical analysis, comprising the following specific steps:

[0029] (1) Tooth extraction

[0030] The straighter teeth in the middle of the left and right rows of the lower jaw of dead finless porpoises were collected; if the individual was severely decayed and had missing teeth in that position, the adjacent straighter teeth were collected as much as possible; if too many teeth were missing, the teeth that were not severely worn were selected as much as possible.

[0031] The finless porpoise tooth specimen in this embodiment comes 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 part of the left row of the finless porpoise's lower jaw. Figure 1 As shown, store in 1.5 mL centrifuge tubes and keep in a -20°C refrigerator until use.

[0032] (2) Teeth cleaning

[0033] Pull out the tooth, use hydrogen peroxide and a toothbrush to remove the soft tissue attached to the tooth surface, use the bristles of a plastic brush to reach into the pulp cavity, and remove the nerves and other tissues in the pulp cavity; then use deionized water to clean the outer surface of the tooth and the inside of the pulp cavity. The details are as follows:

[0034] After removing the teeth, use tweezers to clamp the teeth and use a toothbrush in hydrogen peroxide to remove organic matter on the tooth surface. Then use deionized water to wash for 5 to 6 minutes to further remove the remaining organic matter. Then place the teeth in an open 1.5 mL centrifuge tube and dry them in an oven at 37°C for 20 to 24 hours.

[0035] (3) Tooth embedding

[0036] Fix the tooth on the resin block, and fix the resin block with the tooth fixed in the mold, pour in the cold mounting resin reagent, embed the tooth in the middle of the resin, and remove the bubbles in the pulp cavity; finally, put the mold embedded with the finless porpoise tooth into the oven and place it until the resin is completely solidified and hardened. The details are as follows:

[0037] Use a cutting machine to cut the solidified cold-mounted resin block into small pieces of 0.5 cm square; apply the mixed AB glue (Swiss ergo.7450 epoxy resin AB glue) on the upper surface of the resin block, place the tooth flat on the AB glue, and after confirming that the tooth is flat, let it stand for 5 minutes to wait for the AB glue to solidify, so as to fix the tooth on the resin block; then use AB glue to fix the resin block on the bottom of the silicone mold with an internal length, width and height of 1 cm. After placement, let it stand for 5 minutes to wait for the AB glue to fully solidify; mix the cold-mounted resin (EpoFix Resin, Struers) and the coagulant (EpoFix Hardener, Struers Company) was prepared in a disposable plastic cup at a mass ratio of 7:1, and stirred continuously in the same direction to avoid bubbles. The prepared resin reagent was poured into the mold to embed the tooth in the middle of the resin. A single clean hard-bristle brush was inserted into the pulp cavity to remove the bubbles. Finally, the mold with the embedded finless porpoise tooth was placed in an oven at 37°C for more than 12 hours until the resin was completely solidified and hardened. Figure 2 shown.

[0038] (4) Tooth slices

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

[0040] The square resin block with embedded teeth was taken out of the mold. At this time, the contact surface between the resin block and the mold was smooth and transparent, and the contact surface with the air might have pits. Use 1200 mesh, 2000 mesh, and 4000 mesh sandpaper to grind the surface flat. Use a low-speed precision cutting machine with a micrometer to cut along the coronal surface of the tooth. Before cutting, use the clamp of the cutting machine to tighten the resin block with embedded teeth, 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, and retain the longest axis from the cusp to the root in the 3 mm thick slice to maximize the area of ​​the coronal surface after cutting.

[0041] (5) Grinding and polishing

[0042] Use sandpaper to finely grind one side of the slice to remove the cutting marks, then use AB glue to stick this side to a glass slide with a sample label written with a marker; after fixation, finely grind the upper surface of the tooth slice, and after the surface is gradually smooth, use a polishing cloth and silica suspension to polish; after polishing, use a cutting machine to cut off the resin and glass except for the label and the tooth, wash with deionized water and dry it. The details are as follows:

[0043] Use 2400-mesh and 4000-mesh sandpaper to finely grind one side of the slice to remove the cutting marks, then use AB glue to stick this side to a glass slide with a sample label written with a marker, taking care to cover the label with resin and avoid the tooth covering the label; finely grind the upper surface of the tooth slice, and after the surface is gradually smooth, use polishing cloth and silica suspension to polish; after polishing, use a cutter to cut off excess glass and resin, rinse the tooth slice with deionized water, place it in a clean beaker and use Milli-Q water (resistivity 18.2 MΩ·cm) ultrasonic cleaning for 5 minutes, then take out the tooth slice and rinse it with Milli-Q water for 2 minutes, place the cleaned tooth slice in a culture dish, and put absorbent paper under the slice; place the tooth slice in the culture dish in an oven at 37°C and dry it for 12 hours to obtain the finless porpoise tooth slice.

[0044] The final finless porpoise tooth slices are as follows Figure 3 , 4 As shown in the figure, it can be seen that the largest growth axis plane of the finless porpoise tooth is retained in the thinner slice, which maximizes the integrity of the tooth analysis surface and retains the growth characteristics of the tooth, which can meet the sample condition requirements for microchemical analysis. The finless porpoise tooth slices processed by the method of the present invention can meet the requirements of laser ablation inductively coupled plasma mass spectrometry analysis (multi-element content), laser ablation multi-receiver inductively coupled plasma mass spectrometry analysis ( 87 Sr / 86 Sr ratio) and other analysis needs, and ensure that the slices after microchemical analysis can be used for age identification after simple reprocessing, and the microchemical analysis results can be correlated with the growth and development sequence.

[0045] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. A method for pre-processing finless porpoise tooth slices for microchemical analysis, characterized in that: The following steps are involved: Step 1) Tooth extraction: Collect the straighter teeth in the middle of the left and right rows of the lower jaw of the dead finless porpoise; if the individual has severe decay and the teeth in this position are missing, take the adjacent straighter teeth; If too many teeth are missing, choose teeth that are not severely worn; Step 2) Teeth cleaning: Pull out the teeth, use hydrogen peroxide and instruments to remove the soft tissue attached to the tooth surface, and use instruments to reach into the pulp cavity to remove the soft tissue inside the pulp cavity; then use deionized water to clean the outer surface of the teeth and the inside of the pulp cavity; Step 3) Tooth embedding: fix the tooth on the resin block, and fix the resin block with the tooth fixed in the mold, pour in the cold embedding resin reagent, embed the tooth in the middle of the resin, and guide out the bubbles in the pulp cavity; finally, put the mold embedding the finless porpoise tooth into the oven and place it until the resin is completely solidified and hardened; Step 4) Tooth slicing: remove the resin block from the mold, grind the surface flat, and use a low-speed precision cutting machine to cut off the buccal and lingual sides of the tooth along the coronal surface of the tooth. During the cutting process, the saw blade is parallel to the coronal surface of the tooth, and the longest axis from the cusp to the root is retained in the slice to maximize the area of ​​the coronal surface after cutting; Step 5) Grinding and polishing: Use sandpaper to finely grind one side of the slice to remove the cutting marks, and then stick this side on a glass slide; after fixing, finely grind the upper surface of the tooth slice, and after the surface is gradually smooth, polish it with a polishing cloth and a silica suspension; then wash and dry it.

2. The method for pre-treating finless porpoise tooth slices for microchemical analysis according to claim 1, characterized in that: In the step 2), the teeth are rinsed with deionized water for 5 to 6 minutes, and then placed in an oven at 37° C. to dry for 20 to 24 hours.

3. The method for pre-treating finless porpoise tooth slices for microchemical analysis according to claim 1, characterized in that: The specific operation of step 3) is as follows: using a cutting machine to cut the solidified cold-mounting resin block into small blocks of 0.5 cm square; applying the mixed AB glue on the upper surface of the resin block, placing the tooth flat on the resin block, and then using AB glue to fix the resin block to the bottom of a silicone mold with an internal length, width and height of 1 cm. After placement, let it stand for 30 to 40 minutes to allow the AB glue to fully solidify; preparing the cold-mounting resin and the coagulant in a mass ratio of 7:1 and stirring evenly; pouring 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 bubbles inside; finally, place the mold containing the finless porpoise tooth in an oven at 37°C for more than 12 hours until the resin is completely solidified and hardened.

4. The method for pre-treating finless porpoise tooth slices for microchemical analysis according to claim 1, characterized in that: The specific operation of step 4) is as follows: the square resin block embedded with the tooth is taken out from the mold, and the surface is polished and smoothed using 1200 mesh, 2000 mesh, and 4000 mesh sandpaper in sequence, and the buccal and lingual sides of the tooth are cut off along the coronal surface of the tooth using a low-speed precision cutting machine with a micrometer. During the cutting process, the saw blade is parallel to the coronal surface of the tooth, and the longest axis from the cusp to the root is retained in the 3 mm thick slice to maximize the area of ​​the coronal surface after cutting.

5. The method for pre-treating finless porpoise tooth slices for microchemical analysis according to claim 1, characterized in that: The specific operation of step 5) is as follows: use 2400 mesh and 4000 mesh sandpaper to finely grind one side of the slice to remove the cutting marks, and then stick this side to a slide with a sample label with AB glue; finely grind the upper surface of the tooth slice, and after the surface is gradually smooth, polish it with a polishing cloth and a silica suspension; after polishing, use a cutting machine to cut off excess glass and resin, rinse the tooth slice with deionized water, place it in a clean beaker and use Milli-Q water ultrasonic cleaning for 5 minutes, then take out the tooth slice and rinse it with Milli-Q water for 2 minutes, place the cleaned tooth slice in a culture dish, and put absorbent paper under the slice; place the tooth slice in the culture dish in an oven at 37°C and dry it for 12 hours.

6. The method for pre-treating finless porpoise tooth slices for microchemical analysis according to claim 5, characterized in that: The resistivity of the Milli-Q water is 18.2 MΩ·cm.

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