We would like to thank the Max Planck Society for funding this research as well as the recent setting up of a Stable Isotope laboratory at the Department Archaeology, Max Planck Institute for the Science of Human History.

The following protocol follows the guidelines of the Light Isotope Mass Spectrometry Laboratory at the Max Planck Institute for the Science of Human History. Appropriate ethics permissions from national and international committees should be sought for analyses involving endangered modern or historical faunal specimens, and for the use of archaeological and faunal material of interest to contemporary stakeholders. In this paper, the samples used were archaeological and fossil specimens. No living humans were used in this study and full ethical, institutional, and governmental permissions have been gained for any destructive analysis.
1. Bulk Sampling
Note: For humans and animals, the basic method of bulk tooth enamel sampling is the application of a drill to the buccal edge of the tooth.
<ol>
	<li>Clean the outer surface of the tooth (~0.1 mm) using an aluminum shot blaster or by gentle abrasion using the drill set-up. Use a hand-held drill with a clean diamond-tipped drill bit (see below), of even shape, attached via a chuck to the drill set-up. Ensure that gentle and even abrasion is performed as a groove parallel to the entire growth axis (Figure 1 and Figure 2).</li>
	<li>Attempt, where possible, to apply the method to the same tooth portion for each human or animal sampled. 
	Note: In human paleodietary research, third molars are generally favored as representative of late juvenile/adult diet while first molars are avoided due to weaning effects23. The preferred tooth for each taxon, and the period of life represented, will vary with the available samples and question under study.</li>
	<li>Drill in a well-ventilated room while wearing a mask to avoid the inhalation of tooth enamel powder. Wear goggles to protect eyes. Be aware that the amount of sample powder drilled will vary depending on the equipment used for analysis, pretreatment protocol, and size of the tooth. Drill at low speed in order to prevent heating the sample. 
	Note: Approximately, 4-8 mg of sample powder is an appropriate goal for multiple analyses using an online gas preparation and introduction system for isotope ratio mass spectrometry and diagenetic testing. Be aware that the chosen drill speed will depend on the fragility of the sample and will require some level of trial and error. In general, on a hand-held drill, two out of five bars of strength are sufficient. Drill the tooth powder over a piece of clean aluminum foil or weighing paper.</li>
	<li>Collect the resulting enamel powder and transfer it into a 1.5 mL micro-centrifuge tube that has been tared on an appropriately accurate balance prior to drilling. Label each tube with the sample designation (Figure 3). Record the sample numbers and enamel weight in a laboratory notebook as well as an electronic database.</li>
	<li>Before drilling each new tooth, clean the utilized drill bits first using 0.5 M HCl on a robust tissue. Then wash the drill bit with an organic solvent (such as acetone, methanol, or ethanol) using a robust tissue.</li>
	<li>Following a drilling sitting, clean the workspace thoroughly using a dedicated vacuum cleaner, or a dustpan and brush. Wipe the sampling space with methanol. Vacuum or lightly spray the drill with compressed air to remove dust and sample powder between samples.</li>
</ol>
2. Sequential Sampling
Note: Sequential sampling can be approached in a variety of forms and will depend on the taxon being sampled, the size of the tooth, as well as the desired temporal resolution.
<ol>
	<li>Clean the surface of the tooth being sampled by removing a very thin (~0.1 mm) layer of outer tooth enamel.</li>
	<li>Drill the samples along the buccal surface perpendicular to the growth axis of the tooth, running from the enamel root junction to the top of the crown. Drill horizontal bands perpendicular to the growth axis, with each sample resulting in a 1-2 mm wide groove across the enamel layer (Figure 1 and Figure 4). Take care to avoid drilling through the enamel into the dentine as it would contaminate the sample and the resulting measurement. 
	Note: The number of samples taken will vary depending on the resolution desired but c. 10 represents an appropriate goal to study seasonal changes in &#948;13C and &#948;18O values for a hypsodont the size of domesticated cattle.</li>
	<li>Choose the appropriate drill bit for each sample, as the width of the sampling line is determined by the diameter of the drill bit.</li>
	<li>Use a rig set-up for more delicate teeth requiring a greater number of incremental samples (Figure 2) (e.g., human teeth), as well as for more stable drilling. Set up the rig stand that holds the drill securely, with the drill bit pointing downwards. 
	Note: While four samples can easily be obtained from a permanent human molar using a hand-held approach24, more refined sampling requires a rig or CO2 laser ablation approach that will not be discussed here14. Another common approach is to use a semi-automated microsampling25.</li>
	<li>Hold the tooth or attach to a clamp. Press the tooth enamel against the drill bit and apply pressure. Samples are drilled incrementally along the buccal surface from the apex (occlusal surface) to the base of the crown at the location of the enamel-root junction (ERJ). Replicate the same sample drilling strategy multiple times drilling each consecutive line parallel to the previous sample line. Use a small cylindrical diamond drill bit for delicate teeth, such as sheep molars, with a diameter of 1 mm (Figure 1C).</li>
	<li>Measure the distance of each sampling line from the ERJ using calipers and record this distance for comparison.</li>
	<li>Follow steps 1.3-1.6 above. 
	Note: If small, delicate teeth are being analyzed and pretreatment and diagenetic check methods are not applied, samples as small as 1-4 mg will produce reliable results on an on-line gas preparation and introduction set-up for carbonate analysis. Modified automated-peripheral systems can facilitate the use of even lower sample weights, but are inevitably limited by the proportion of carbonate in the studied enamel (4 to 5 wt%)26,27. Researchers should consult with the laboratory where samples are going to be measured to determine the amount of sample required.</li>
</ol>
3. Fourier Transform Infrared Spectrometry/Attenuated Total Reflectance method
<ol>
	<li>For the Attenuated Total Reflectance method, establish a sample chamber background, either under vacuum or in normal atmospheric conditions.</li>
	<li>Place approximately 1 mg of tooth enamel over the diamond crystal in the sample chamber using a spatula. Lower and secure the sample post until there is a firm connection between the post, the sample, and the diamond plate. Close the sample chamber, with or without a vacuum being established, depending on the set-up or availability.</li>
	<li>Measure the FTIR spectra of the sample 64 times for the wavenumber ranging between 400 and 4,000 cm-1. The desired number of replicates will vary based on the goals of the study, though the analysis of three replicates, ideally with different aliquots being taken and then returned to the microcentrifuge tube housing the sample, will ensure robust results.</li>
	<li>Clean the spatula with methanol between each aliquot and between each sample.</li>
	<li>Perform a baseline correction using the available software. The software automatically subtracts the sample chamber background from the resulting FTIR profile. To ensure better reproducibility, only enamel spectra with a minimum absorbance of 0.06 for the highest phosphate band at ~1035 cm-1 should be taken into account.</li>
	<li>Monitor the presence of the common secondary contaminant carbonate calcite in all samples by checking for a peak at 711 cm-1.</li>
	<li>Following the analysis, carefully return the samples to their microcentrifuge tubes using a spatula or suction device and take onto the next stage of pretreatment or analysis.</li>
	<li>Export the raw wavelength and intensity data as a .csv file (or similar) and use to calculate the indices of interest (commonly used indices include A-site Phosphate Index, B-site Phosphate Index, Phosphate Crystallinity Index, Water-Amide on Phosphate Index, and CO3/PO4 index17,18,19) (Table 1).</li>
	<li>Compare the FTIR results from the fossil or archaeological samples to growing datasets of modern faunal tooth enamel now available in the literature19,20 to determine the nature and extent of diagenetic alteration to the enamel sampled.</li>
</ol>
4. Simple Acetic Acid (0.1 M) Pretreatment
<ol>
	<li>Weigh the enamel powder for each sample in a microcentrifuge tube using a balance as appropriate. Label the tube accordingly. Enamel powder must be ground to the same degree, with particles of a similar size. 
	Note: Many researchers utilize an oxidizing agent, such as dilute bleach (NaClO) or 30% hydrogen peroxide (H2O2) to remove organics from a sample and this should be added at this point. However, not only may these reagents alter the stable carbon and oxygen isotope values of a sample, but spectra of bone collagen samples in an on-line gas preparation and introduction system demonstrate that the 100% phosphoric acid utilized in carbonate measurement does not react with organic samples (Figure 5), suggesting that this step is unnecessary.</li>
	<li>Add 0.1 mL of 0.1 M acetic acid (per 1 mg of enamel) to each sample using a pipette. Avoid touching the samples with the pipette. If the sample and pipette come into contact, use a new pipette for the next sample to avoid cross contamination.</li>
	<li>Agitate, either through shaking or an electronic agitator (Figure 3) and then leave the samples to sit for 10 min. Enamel powder should not be left in acetic acid for an extended period of time (&#62;4 h).</li>
	<li>Place the samples in a microcentrifuge for 2 min at 13,700 x g. 
	Note: A low-cost alternative is the use of a mini-centrifuge for 4 min at 3,500 x g, which holds fewer samples but is less time consuming when dealing with large sample sets.</li>
	<li>When the microcentrifuge program stops, replace the acetic acid with 2 mL of ultrapure water using a clean pipette, then microcentrifuge the samples for 2 min at 13,700 x g.</li>
	<li>Change the ultrapure water two more times, as before, and test for neutrality. Remove the remaining fluid using a pipette (without disturbing the supernatant). 
	Note: A total of three washes with ultrapure water is the standard procedure to get to neutrality. The sample should be washed until neutrality is reached.</li>
	<li>Cut sheets of Parafilm (1 cm &#215; 1 cm) and place over each microcentrifuge tube. Make a small hole in the center using a sharp object so that the sample dries appropriately.</li>
	<li>Place the tubes in a freeze drier to remove any remaining fluid. 
	Note: If a freeze drier is not available, gentle oven drying (40 &#176;C) is also a possibility and should not have any adverse effects on the stable carbon and oxygen isotope data.</li>
	<li>Once dry, remove the Parafilm and close the microcentrifuge lids. Make sure that the tube labeling is correct and re-write if necessary. 
	Note: This is the final stage prior to storage for weighing out, which in labs with limited funds may be the final stage prior to the distribution of the samples for analysis elsewhere.</li>
</ol>
5. Weighing and Measuring Samples and Standards
<ol>
	<li>Using a spatula, weigh out approximately 2 mg of enamel powder onto a tin disc on a balance sensitive to 0.001 mg (Figure 6). Then carefully transfer the sample into a phosphoric acid-resistant borosilicate glass vial. This amount of enamel is required to yield reliable results due to the relatively small proportion of carbonate in tooth enamel.</li>
	<li>Clean the spatula between samples using methanol and use a fresh, clean disc or weighing vessel for each sample.</li>
	<li>Weigh out 0.2 mg of an international standard such as IAEA NBS18, IAEA 603, IAEA CO8, or Merck CaCO3. An in-house standard made from the homogenized enamel of a large tooth can be used as well20. For a full run of 76 samples and standards, use 61 samples and 15 standards, which should be evenly distributed over the whole run. Several studies have reported appropriate numbers of standards and details in archaeological research20,28. 
	Note: The repeated pretreatment and analysis of an enamel in house standard, across a number of runs of archaeological samples, is also likely to provide an appropriate measure of accuracy for the samples being studied20.</li>
	<li>Tighten the specially-designed lids, accessible to the needle until the septum is tight but not sucked into the vial. Do not over tighten the lid as the pressure on the septum will get too strong and can ultimately result in a broken needle during analysis. Place the vials in a listed order within a heating block at 70 &#176;C. The order and weights of samples must be reliably recorded for entry into the computer software.</li>
	<li>Measure the samples by following three steps: 1) flush fill the samples with pure Helium, 2) add phosphoric acid, 3) measure the sample.
	<ol>
		<li>Flush the samples with pure helium (grade 5.0) with a flow of 100 mL/min for 5 min to flush out ambient air.</li>
		<li>Add 4-5 drops of phosphoric acid (99%, density 1.85 g/mL) to the carbonate-containing sample. Reaction between the sample and H3PO4 begins. During the reaction, CO2 is released which carries the isotopic value of the carbonate ion CO32- of the sample.</li>
		<li>Wait 1 h per sample until the equilibration of CO2 is reached. After 1 h, the samples should be measured to obtain reliable results. During the acquisition, a mixture of sample gas and He passes to the device. A drying stage removes water from the sample gas mixture.</li>
		<li>Measure the sample by starting with three peaks of CO2 reference gas with a known isotopic composition. Peak intensity should be 4000 mV to match the intensity of the sample peaks (between 4000 to 8000 mV). The three reference gas peaks should be 20 s long and 30 s apart. Follow with defining the measurement interval of the sample. Measure the sample 10 times for 5 s each and 55 s apart. Ensure that peak height decreases over time indicating proper transport of the sample/helium mixture (Figure 5). 
		Note: During measurement, the software calculates the isotopic composition of the sample by comparing the known isotopic value, the peak height, and area of the peak of the reference gas with the peak height and area of the peaks of the samples. The raw 13C/12C and 18O/16O composition of the sample is calculated.</li>
	</ol>
	</li>
	<li>Normalize the samples after measurement. 
	Note: This is important because the reference gas does not undergo the same chemical and physical path as the sample gas when introduced into the mass spectrometer. Therefore, it is essential that the samples are additionally calibrated to international standards (step 5.3) that undergo the same physical and chemical treatment within a run as the sample itself20. These international carbonate standards enable the two-point calibration of samples onto the international delta measurement scale and the assessment of precision and repeatability throughout a given run.</li>
</ol>

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The authors declare no conflict of interest.

The challenges of successful sampling (bulk and incremental) of dentition relies on the access to knowledge regarding drilling techniques and sample preparation, alongside the investment in relatively inexpensive equipment. These challenges are easily surmountable when clear instructions are available concerning&#160;sampling and pretreatment approaches. In this article, we hope to have disseminated these in a clear, concise fashion for researchers new to these methods. Scholars applying these methods for the first time should practice on accessible modern fauna prior to the analysis and sampling of valuable archeological and paleontological samples.
The sampling of human and animal tooth enamel carbonate for stable isotope analysis is a simple procedure that has been undertaken at multiple laboratories. However, there is a tendency for techniques and technology associated with the drilling of dentition to vary by laboratory and be included in a broader set of insider technical knowledge that is not shared openly. Incremental sampling has major advantages, permitting the identification of detailed intra-individual variation in dietary intake and the ingestion of water. This is illustrated by compelling the differences found between individuals from different regions as dietary and environmental information is preserved in tooth enamel bioapatite. In our representative data, significant isotopic variation is evident between the sheep from tropical grasslands compared to the sheep from temperate dry-steppe grasslands (Figure 8).
Critical steps within the protocol are related to the accuracy in drilling, preservation of tooth enamel, and pretreatment techniques. Small inaccuracies in drilling, for example, through the enamel into the tooth dentine, could result in hugely variable isotope measurements29. The preservation of tooth enamel can be checked through a variety of methods, including the estimated carbonate proportion of a given sample measured, as well as the FTIR set-up discussed here. Researchers should also inform the laboratories of the burial environment, specifically whether water-logged or in acidic soils, which can affect the structural preservation of fossil tooth enamel. The hardness of tooth enamel should be considered as an initial indicator of preservation, which may only become evident during drilling. Enamel that is soft and easily drilled suggests that the bioapatite crystal lattice may have degraded and should be checked with FTIR or other means reported in the literature30. The variation in sample pretreatment seems to result in limited isotopic variation in tooth enamel21,22. Therefore, we suggest the use of simple protocols (e.g., 0.1 M acetic acid for less than 4 h followed by washing with distilled water).
There are several limitations to the technique, associated with the sampling design and interpretation. Drilling sequential samples is a skill that takes some time to master. A clear understanding of the taxa and tooth to be analyzed is essential in formulating a sampling design2,25. Further, drilling of samples can take a considerable amount of time to complete. However, the resulting carbon and oxygen stable isotope values for sequentially sampled dentition allow the researchers to track dietary and environmental changes. As these changes are related to natural seasonal variations, often in ancient periods, thoughtful interpretations that are contextualized within an understanding of variation in isotopic reference sets are integral to this research6.
In the article, we have demonstrated incremental sampling and bulk sampling of both human and sheep dentition. Further, we instruct the researchers on pretreatment methods for both sample sets. Incremental sampling method can be successfully applied to ancient and modern fauna with similar enamel growth and mineralization (e.g., cattle and horses). Pretreatment of enamel bioapatite as shown in the article can be used on samples from a cross-section of ancient remains. The most important lesson from our sampling procedure is bulk and incremental sampling of dentition, which is not easily explained in a document. Further demonstrations could democratize other archaeological isotope sampling and pre-treatment approaches (e.g., bone collagen extractions or the sampling of archaeological pottery for stable isotope measurements of fatty acids) enhancing the spread of knowledge and technology in this field. Such democratization should not, however, be seen as a complete replacement for consultation with the experts, or the available literature, to establish the standards of measurement and interpretation in a given context20,28.

Stable carbon and oxygen isotope analyses of tooth enamel carbonate has been used to study past human dietary intake, weaning, and mobility, as well as faunal reliance on vegetation, the movement of animals, and livestock foddering. These applications have been comprehensively discussed and reviewed for a variety of environmental conditions indicating the effects of local aridity, temperature, water sources, and vegetation compositions1,2,3,4,5,6. The diversity of potential applications in archaeology and paleontology, as well as the good preservation of tooth enamel carbonate, has made it an attractive material for stable isotope work3. Methods of sampling, pretreatment, and diagenesis screening are briefly described in a number of previous publications1,7. However, thorough verbal and visual demonstrations remain largely unavailable, particularly to people outside of archaeological science laboratories and among laboratory groups with limited funding where the interest in the use of this technique is increasing5.
Tooth enamel is primarily made up of hydroxyapatite (bioapatite) crystallites8 larger than those in bone, making it more resistant to post-mortem diagenetic ionic substitutions and contamination3. Modern studies have demonstrated that stable carbon isotope (&#948;13C) measurements of faunal tooth enamel reliably record animal diet and behavior9,10. The stable oxygen isotope (&#948;18O) value of tooth enamel is determined by the oxygen isotopic composition of ingested water, which includes water in plant and animal foods, drinking water, respiration, as well as various environmental impacts on the water which can lead to further isotopic fractionation (e.g., aridity, temperature, altitude, rainfall amount, continental location)11. This has made it a popular method for dietary and environmental reconstruction in archeological, paleoecological, and paleontological research.
The period of tooth enamel formation is relatively short (years) and differs depending on the tooth being sampled. For humans, first molar enamel mineralizes between birth and 3 years of age, premolars mineralize between 1.5 and 7 years of age, second molars mineralize between 2.5 and 8 years of age, and third molars mineralize during adolescence, between 7 and 16 years12. Given that tooth enamel forms incrementally over its period of formation, it can be sampled in bulk along the entire growth axis or sampled sequentially in order to investigate the changes in diet and environment that have occurred during the formation period13. Chronologically-ordered dietary change within a given tooth is observable for humans and other animals1,14, providing information regarding inter-annual seasonal and dietary variation.
While enamel is usually resistant to diagenesis, isotopic modifications resulting from the burial environment are possible and have been observed15,16, making experimental checks and pretreatment choices useful. While it is not the only available method, Fourier transform infrared spectroscopy (FTIR), particularly in Attenuated Transmission Mode, has emerged as a quick, inexpensive, and relatively accessible method for assessing taphonomic alteration in tooth enamel, particularly in paleontological contexts17,18,19,20. However, detailed protocols and recording standards remain relatively inaccessible to many people outside the fields of geochemistry or material science.
Reaction times and the chemicals employed by researchers in the pretreatment of tooth enamel also vary considerably in the literature, often with limited consideration as to what this variability may do to stable carbon and oxygen isotope values of the sample21,22. Here, we report an approach that uses dilute acetic acid (0.1 M) for the pretreatment of enamel powder samples. However, given that the differences in isotopic measurements resulting from pretreatment are relatively minor for tooth enamel, it is best for the researchers to follow the protocols for datasets with which they wish to compare their data to11. Furthermore, where small sequential samples are taken, particularly on Holocene samples, no pretreatment may be chosen (following pilot diagenetic tests) to avoid sample wastage.
Although the methods we report here are by no means new, to our knowledge, this is the first time that a thorough written and visual documentation of bulk and sequential sampling, pretreatment choices, and diagenetic check methods (in the form of FTIR) for tooth enamel have been made widely available to a varied academic audience. While we hope our efforts will make this approach more easily accessible to a wider number of individuals and laboratories, researchers who want to apply and publish this technique must be aware of minimum reporting standards, diagenetic considerations, and presentation requirements overviewed elsewhere20, as well as potential interpretive complexities that will be unique to their study region, taxa analyzed, and time period5.

<table><tbody><tr><td>Dremel Micro</td><td>Dremel</td><td></td><td>https://www.dremel.com/en_US/products/-/show-product/tools/8050-micro</td></tr><tr><td>Diamond-tipped drill bit</td><td>Dremel</td><td></td><td>https://www.dremel.com/en_US/products/-/show-product/accessories/7122-diamond-wheel-point</td></tr><tr><td>1.5 mL micro-centrifuge tube</td><td>Sigma Aldrich</td><td></td><td>https://www.sigmaaldrich.com/catalog/product/sigma/t2422?lang=de&amp;amp;region=DE&amp;amp;gclid=EAIaIQ&lt;br/&gt;obChMI7pHRpauW2QIV77ftCh1p1&lt;br/&gt;wjhEAAYASAAEgKzkvD_BwE</td></tr><tr><td>Methanol</td><td></td><td></td><td>Linear Formula:&amp;nbsp;CH&lt;sub&gt;3&lt;/sub&gt;OH</td></tr><tr><td>Acetic Acid</td><td></td><td></td><td>Linear Formula:&amp;nbsp;CH&lt;sub&gt;3&lt;/sub&gt;CO&lt;sub&gt;2&lt;/sub&gt;H</td></tr><tr><td>Dremel rig set-up (workstation)</td><td>Dremel</td><td></td><td>https://www.dremel.com/en_US/products/-/show-product/tools/220-01-workstation</td></tr><tr><td>Microcentrifuge</td><td>Thermo Scientific</td><td></td><td>http://www.thermofisher.com/order/catalog/product/75002401</td></tr><tr><td>Mini-centrifuge</td><td>Sprout</td><td></td><td>http://www.heathrowscientific.com/sprout-mini-centrifuge-4</td></tr><tr><td>Freeze drier</td><td>Zirbus Technology</td><td></td><td>http://www.zirbus.com</td></tr></tbody></table>

sampling and pretreatment of tooth enamel carbonate for stable carbon and oxygen isotope analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Using the sampling procedure presented above, incremental enamel bioapatite samples were prepared. The analysis of bioapatite in enamel depends on the accuracy of sampling, whether bulk or incremental. In this case, we have chosen to present the results of archaeological samples (two sheep) from different climatic zones. Incremental samples were analyzed from sheep second molars and labelled starting at the ERJ (Figure 4). Incremental sample locations were numbered, and each location was measured as its distance in mm from the ERJ (Figure 7).
Diverse carbon and oxygen stable isotope results from the two sheep confirm that they lived in different environments, in this case a tropical grassland (A) and a temperate dry-steppe grassland (B), respectively. Incremental &#948;18O values for sheep A show a narrow range between 3.3 to 5.1&#8240;, suggesting the ingestion of water sources with similar isotopic values and a lack of strong seasonal shifts in precipitation (Figure 8). In contrast, &#948;18O values for sheep B have a high amplitude of variation, ranging from &#9472;5.2 to &#9472;13.1&#8240;, indicating strong seasonal variation in precipitation. Stable carbon isotope values suggest strong differences in ingested vegetation between samples, with sheep A having a diet consisting mainly of C4 plants, while sheep B ingested primarily C3 vegetation. These sheep were specifically chosen to demonstrate environmental variation evident in incremental oxygen and carbon stable isotope results.
Human teeth are similarly sampled from the ERJ to the crown along the growth axis. Incremental &#948;18O and &#948;13C values for a human tooth from a rainforest environment are highly constrained, within a 2&#8240; range. This suggests a lack of variation in foraging strategies over the period of enamel mineralization (Figure 9).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58002/58002fig1.jpg" /> 
Figure 1: Drilling a Tooth. (A) Photo of a tooth being drilled on a rig set up. (B) Photo of enamel powder being collected in tin foil and placed into a 1.5 mL micro-centrifuge tube (with proper label). (C) Photo of different drill bits available for incremental sampling. <a href="https://www.jove.com/files/ftp_upload/58002/58002fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58002/58002fig2.jpg" /> 
Figure 2: Rig set up. Photo of rig set up with the drill in place. <a href="https://www.jove.com/files/ftp_upload/58002/58002fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58002/58002fig3.jpg" /> 
Figure 3: Sample preparation. Sample being placed into a micro-centrifuge tube and agitated on a vortex after chemicals were added. <a href="https://www.jove.com/files/ftp_upload/58002/58002fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58002/58002fig4.jpg" /> 
Figure 4: Incrementally sampled sheep teeth. Sheep teeth (A and B) that were incrementally sampled. <a href="https://www.jove.com/files/ftp_upload/58002/58002fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58002/58002fig5.jpg" /> 
Figure 5: Chromatogram of gasbench run. Photo of a chromatogram of one sample displaying intensity of reference gas peaks and sample peaks over time. Detected masses are 44, 45 and 46. The first three peaks are CO2 reference gas peaks with a known isotopic composition. Ten peaks that follow are sample peaks decreasing in intensity. Peaks should always be separated by several seconds to ensure a strict discrimination between the peaks and hence a clean peak integration. Numbers on top of each peak state the time (s) of peak detection. <a href="https://www.jove.com/files/ftp_upload/58002/58002fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58002/58002fig6.jpg" /> 
Figure 6: Weighing carbonate sample. Photo of sample being weighted into glass vials using a spatula. <a href="https://www.jove.com/files/ftp_upload/58002/58002fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58002/58002fig7.jpg" /> 
Figure 7: Incremental samples on a sheep tooth. Incremental samples along the growth axis of the tooth from the ERJ to the top of the crown plotted alongside carbon and oxygen stable isotope values. <a href="https://www.jove.com/files/ftp_upload/58002/58002fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58002/58002fig8.jpg" /> 
Figure 8: Sheep enamel carbonate isotope results. Stable oxygen and carbon isotope values for two incrementally sampled sheep teeth. <a href="https://www.jove.com/files/ftp_upload/58002/58002fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/58002/58002fig9.jpg" /> 
Figure 9: Human enamel carbonate isotope results. Stable oxygen and carbon isotope values for an incrementally sampled human tooth. <a href="https://www.jove.com/files/ftp_upload/58002/58002fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PCI (Phosphate Crystallinity Index)</td>
			<td rowspan="4"><img alt="Equation 1" src="/files/ftp_upload/58002/58002eq1.jpg" /></td>
			<td>Sponheimer and Lee-Thorp, 1999b</td>
		</tr>
		<tr>
			<td>other names:</td>
			<td></td>
		</tr>
		<tr>
			<td>CIIR (Crystallinity Index InfraRed)</td>
			<td>Shemesh, 1990</td>
		</tr>
		<tr>
			<td>IRSF (InfraRed Splitting Factor)</td>
			<td>Weiner and Bar-Yosef, 1990</td>
		</tr>
		<tr>
			<td>BPI (B-carbonate on Phosphate Index)</td>
			<td><img alt="Equation 2" src="/files/ftp_upload/58002/58002eq2.jpg" /></td>
			<td>LeGeros, 1991</td>
		</tr>
		<tr>
			<td>API (A-carbonate on Phosphate Index)</td>
			<td><img alt="Equation 3" src="/files/ftp_upload/58002/58002eq3.jpg" /></td>
			<td>Sponheimer and Lee-Thorp, 1999b</td>
		</tr>
		<tr>
			<td>BAI (relative amount of B- to A-site carbonate)</td>
			<td><img alt="Equation 4" src="/files/ftp_upload/58002/58002eq4.jpg" /></td>
			<td>Sponheimer, 1999; Sponheimer and Lee-Thorp, 1999b</td>
		</tr>
		<tr>
			<td>WAMPI (Water-Amide on Phosphate Index)&#173;</td>
			<td><img alt="Equation 5" src="/files/ftp_upload/58002/58002eq5.jpg" /></td>
			<td>Roche et al., 2010</td>
		</tr>
	</tbody>
</table>
Table 1: Empirical indices that characterize the crystal-chemical properties of enamel bioapatite. We recommend using the empirical indices from Sponheimer (1999), Sponheimer and Lee-Thorp (1999), and Roche et al. (2010) to characterize the crystal-chemical properties of enamel bioapatite.

Watch this Scientific Journal Video about Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis at JoVE.com

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

sampling-pretreatment-tooth-enamel-carbonate-for-stable-carbon-oxygen

Nanyang Technological University

Research

JoVE Journal

Biochemistry

13.9K Views.  Max Planck Institute for the Science of Human History. Stabiele koolstof en zuurstof isotoop analyse van mens en dier tandglazuur carbonaat is gebruikt als een proxy voor afzonderlijke voeding en milieu wederopbouw. Hier, wij bieden een gedetailleerde beschrijving en visuele documentatie van bulk en sequentiële tooth glazuur bemonstering alsmede voorbehandeling van archeologisch en paleontologisch monsters.

Video: Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate the environment. This radiocarbon signal &#8212; or rather the absence of signal &#8212; is evenly distributed throughout a contaminant source pool (unlike an added tracer) and is not impacted by biological, chemical or physical processes (e.g., the 14C radioactive decay rate is immutable). If the fossil-derived contaminant is fully degraded to CO2, a harmless end-product, that CO2 will contain no radiocarbon. CO2 derived from natural organic matter (NOM) degradation will reflect the NOM radiocarbon content (usually &#60;30,000 years old). Given a known radiocarbon content for NOM (a site background), a two end-member mixing model can be used to determine the CO2 derived from a fossil source in a given soil gas or groundwater sample. Coupling the percent CO2 derived from the contaminant with the CO2 respiration rate provides an estimate for the total amount of contaminant degraded per unit time. Finally, determining a zone of influence (ZOI) representing the volume from which site CO2 is collected allows determining the contaminant degradation per unit time and volume. Along with estimates for total contaminant mass, this can ultimately be used to calculate time-to-remediate or otherwise used by site managers for decision-making.

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A protocol is described wherein CO2 mineralized from organic contaminant (derived from petroleum feedstocks) biodegradation is trapped, quantified, and analyzed for 14C content. A model is developed to determine CO2 capture zone's spatial extent. Spatial and temporal measurements allow integrating contaminant mineralization rates for predicting remediation extent and time.

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate ...

Environment

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle.
Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyC&#39;s characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA

We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC.

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as ...

Chemistry

Dynamic Pore-scale Reservoir-condition Imaging of Reaction in Carbonates Using Synchrotron Fast Tomography

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve geologic seals and allow CO2 to escape. However, the dissolution processes at reservoir conditions are poorly understood. Thus, time-resolved experiments are needed to observe and predict the nature and rate of dissolution at the pore scale. Synchrotron fast tomography is a method of taking high-resolution time-resolved images of complex pore structures much more quickly than traditional &#181;-CT. The Diamond Lightsource Pink Beam was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 hours. The images were segmented and the porosity and permeability were measured using image analysis and network extraction. Porosity increased uniformly along the length of the sample; however, the rate of increase of both porosity and permeability slowed at later times.

Synchrotron fast tomography was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 h.

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve ...

Engineering

Two-way Valorization of Blast Furnace Slag: Synthesis of Precipitated Calcium Carbonate and Zeolitic Heavy Metal Adsorbent

The aim of this work is to present a zero-waste process for storing CO2 in a stable and benign mineral form while producing zeolitic minerals with sufficient heavy metal adsorption capacity. To this end, blast furnace slag, a residue from iron-making, is utilized as the starting material. Calcium is selectively extracted from the slag by leaching with acetic acid (2 M CH3COOH) as the extraction agent. The filtered leachate is subsequently physico-chemically purified and then carbonated to form precipitated calcium carbonate (PCC) of high purity (&lt;2 wt% non-calcium impurities, according to ICP-MS analysis). Sodium hydroxide is added to neutralize the regenerated acetate. The morphological properties of the resulting calcitic PCC are tuned for its potential application as a filler in papermaking. In parallel, the residual solids from the extraction stage are subjected to hydrothermal conversion in a caustic solution (2 M NaOH) that leads to the predominant formation of a particular zeolitic mineral phase (detected by XRD), namely analcime (NaAlSi2O6&#8729;H2O). Based on its ability to adsorb Ni2+, as reported from batch adsorption experiments and ICP-OES analysis, this product can potentially be used in wastewater treatment or for environmental remediation applications.

A protocol for the parallel production of precipitated calcium carbonate and zeolitic material from blast furnace slag via mineral carbonation and alkaline hydrothermal conversion, respectively, is presented. The performance of the zeolitic material towards nickel adsorption is tested.

The aim of this work is to present a zero-waste process for storing CO2 in a stable and benign mineral form while producing zeolitic minerals with ...

Monitoring Pedogenic Inorganic Carbon Accumulation Due to Weathering of Amended Silicate Minerals in Agricultural Soils.

The present study aims to demonstrate a systematic procedure for monitoring inorganic carbon induced by enhanced weathering of comminuted rocks in agricultural soils. To this end, the core soil samples taken at different depth (including 0-15 cm, 15-30 cm, and 30-60 cm profiles) are collected from an agriculture field, the topsoil of which has already been enriched with an alkaline earth metal silicate containing mineral (such as wollastonite). After transporting to the laboratory, the soil samples are air-dried and sieved. Then, the inorganic carbon content of the samples is determined by a volumetric method called calcimetry. The representative results presented herein showed five folded increments of inorganic carbon content in the soils amended with the Ca-silicate compared to control soils. This compositional change was accompanied by more than 1 unit of pH increase in the amended soils, implying high dissolution of the silicate. Mineralogical and morphological analyses, as well as elemental composition, further corroborate the increase in the inorganic carbon content of silicate-amended soils. The sampling and analysis methods presented in this study can be adopted by researchers and professionals looking to trace pedogenic inorganic carbon changes in soils and subsoils, including those amended with other suitable silicate rocks such as basalt and olivine. These methods can also be exploited as tools for verifying soil inorganic carbon sequestration by private and governmental entities to certify and award carbon credits.

The verification method described here is adaptable for monitoring pedogenic inorganic carbon sequestration in various agricultural soils amended with alkaline earth metal silicate-containing rocks, such as wollastonite, basalt, and olivine. This type of validation is essential for carbon credit programs, which can benefit farmers that sequester carbon in their fields.

The present study aims to demonstrate a systematic procedure for monitoring inorganic carbon induced by enhanced weathering of comminuted rocks in ...

Isolation of Quartz Grains for Optically Stimulated Luminescence (OSL) Dating of Quaternary Sediments for Paleoenvironmental Research

Optically stimulated luminescence (OSL) dating quantifies the time since mineral grains were deposited and shielded from additional light or heat exposure, which effectively resets the luminescence clock. The systematics of OSL dating is based on the dosimetric properties of common minerals, like quartz and feldspar. The acquired luminescence with exposure to natural ionizing radiation after burial provides a depositional age for many Quaternary sedimentary systems, spanning the past 0.5 Ma. This contribution details the procedures for separating pure quartz grains of a known range of particle sizes to facilitate luminescence analysis with small or single grain aliquots. Specifically, protocols are given for the needed data and interpretations for effective OSL dating of terrestrial sediment cores or sample tubes from exposures. These cores, 5-20 m long in 1.2 m sections, are split lengthwise and crown-cut leaving 80% of core volume undisturbed, which facilitates sampling of light-protected sediment for OSL dating deep within the core. Sediment samples are then subjected to a series of physical separations to obtain a certain grain-size interval (e.g., 150-250 &#181;m). Magnetic minerals are removed in wet and dry states using magnets. A series of chemical digestions starts with soaking in H2O2 to remove organic matter, followed by HCl exposure to remove carbonate minerals, followed by density separation. Subsequently, grains are soaked in HF for 80 min and after in HCl to render solely quartz grains. The mineralogic purity (&#62;99%) of the quartz extract is quantified with grain petrographic assessment and Raman spectroscopy. Repeating this quartz isolation procedure may be necessary with sediment that contains &#60;15% quartz grains. Excitation of the purified quartz grains by LED-derived blue and IR light allows calculations of the fast and IR depletion ratios, which are metrics to assess the dominance of luminescence emissions from quartz.

Isolation of Quartz Grains for Optically Stimulated Luminescence OSL Dating of Quaternary Sediments for Paleoenvironmental Research

This protocol is for the isolation of quartz grains by size for luminescence dating of sediments. Outlined are physical cleansing and chemical digestions by soaking sequentially in H2O2, HCl, HF, and HCl again to isolate quartz grains. The quartz purity is quantified with microscopic assessment, Raman spectroscopy, and IR depletion ratio.

Optically stimulated luminescence (OSL) dating quantifies the time since mineral grains were deposited and shielded from additional light or heat ...

Optimized Bone Sampling Protocols for the Retrieval of Ancient DNA from Archaeological Remains

The methods presented here seek to maximize the chances for the recovery of human DNA from ancient archaeological remains while limiting input sample material. This was done by targeting anatomical sampling locations previously determined to yield the highest amounts of ancient DNA (aDNA) in a comparative analysis of DNA recovery across the skeleton. Prior research has suggested that these protocols maximize the chances for the successful recovery of ancient human and pathogen DNA from archaeological remains. DNA yields were previously assessed by Parker et al. 2020 in a broad survey of aDNA preservation across multiple skeletal elements from 11 individuals recovered from the medieval (radiocarbon dated to a period of circa (ca.) 1040-1400 CE, calibrated 2-sigma range) graveyard at Krakauer Berg, an abandoned medieval settlement near Pei&#223;en Germany. These eight sampling spots, which span five skeletal elements (pars petrosa, permanent molars, thoracic vertebra, distal phalanx, and talus) successfully yielded high-quality ancient human DNA, where yields were significantly greater than the overall average across all elements and individuals. Yields were adequate for use in most common downstream population genetic analyses. Our results support the preferential use of these anatomical sampling locations for most studies involving the analyses of ancient human DNA from archaeological remains. Implementation of these methods will help to minimize the destruction of precious archaeological specimens.

The protocol presents a series of best practice protocols for the collection of bone powder from eight recommended anatomical sampling locations (specific locations on a given skeletal element) across five different skeletal elements from medieval individuals (radiocarbon dated to a period of ca. 1040-1400 CE, calibrated 2-sigma range).

The methods presented here seek to maximize the chances for the recovery of human DNA from ancient archaeological remains while limiting input sample ...

Genetics

Multimodal Analysis of Bone Formation Using Explant Models

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

Author Spotlight: Advanced Techniques for Characterizing Tissue Mineralization in Bone Regeneration Research

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous ...

Bioengineering

Microhardness Protocol for Evaluating Rodent Oral Disease

Microhardness Measurements on Tooth and Alveolar Bone in Rodent Oral Disease Models

The mechanical property, microhardness, is evaluated in dental enamel, dentin, and bone in oral disease models, including dental fluorosis and periodontitis. Micro-CT (&#181;CT) provides 3D imaging information (volume and mineral density) and scanning electron microscopy (SEM) produces microstructure images (enamel prism and bone lacuna-canalicular). Complementarily to structural analysis by &#181;CT and SEM, microhardness is one of the informative parameters to evaluate how structural changes alter mechanical properties. Despite being a useful parameter, studies on microhardness of alveolar bone in oral diseases are limited. To date, divergent microhardness measurement methods have been reported. Since microhardness values vary depending on the sample preparation (polishing and flat surface) and indentation sites, diverse protocols can cause discrepancies among studies. Standardization of the microhardness protocol is essential for consistent and accurate evaluation in oral disease models. In the present study, we demonstrate a standardized protocol for microhardness analysis in tooth and alveolar bone. Specimens used are as follows: for the dental fluorosis model, incisors were collected from mice treated with/without fluoride-containing water for 6 weeks; for ligature-induced periodontal bone resorption (L-PBR) model, alveolar bones with periodontal bone resorption were collected from mice ligated on the maxillary 2nd molar. At 2 weeks after the ligation, the maxilla was collected. Vickers hardness was analyzed in these specimens according to the standardized protocol. The protocol provides detailed materials and methods for resin embedding, serial polishing, and indentation sites for incisors and alveolar. To the best of our knowledge, this is the first standardized microhardness protocol to evaluate the mechanical properties of tooth and alveolar bone in rodent oral disease models.

Author Spotlight: Establishing an Accurate Microhardness Testing Protocol for Craniofacial Tissues

Microhardness is a mechanical property and an informative parameter for evaluating hard tissue pathophysiology. Here, we demonstrate a standardized protocol (sample preparation, polishing, flat surface, and indentation sites) for microhardness analysis in tooth and alveolar bone in rodent oral disease models, namely, dental fluorosis, and ligature-induced periodontal bone resorption.

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Samenvatting

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