我们要感谢普朗克社会为这项研究提供资金, 以及最近在系考古、普朗克人类历史科学研究所建立了一个稳定的同位素实验室。

下面的议定书遵循了在普朗克人类历史科学研究所的光同位素质谱实验室的指导方针。应寻求国家和国际委员会的适当伦理许可, 以便进行分析, 涉及濒危的现代或历史动物区标本, 并利用当代利益攸关者感兴趣的考古和动物区材料.本文所用的样品为考古和化石标本。在这项研究中没有使用活着的人类, 而且对任何破坏性分析都获得了充分的道德、体制和政府许可。
1. 散装取样
注: 对于人和动物来说, 散装牙釉质取样的基本方法是在牙齿的颊缘上应用钻头。
<ol><li>使用铝合金射爆机或通过钻头安装, 清洁牙齿的外表面 (~ 0.1 毫米)。使用手持钻头, 用一个干净的菱形钻头 (见下文), 甚至形状, 通过一个恰克连接到钻机设置。确保温和和均匀的磨损作为一个凹槽平行于整个增长轴 (图 1和 图 2)。</li>
	<li>尝试, 在可能的情况下, 应用方法对同一牙部分为每个人或动物取样。 注: 在人类 paleodietary 研究中, 第三磨牙通常被认为是晚期少年/成人饮食的代表, 而第一磨牙是由于断奶影响而避免的23。每个分类的首选牙齿, 以及所代表的生命周期, 将因所研究的现有样本和问题而异。</li>
	<li>在通风良好的房间里钻, 同时戴上口罩, 避免吸入牙釉质粉。戴上护目镜来保护眼睛。请注意, 根据用于分析、预处理协议和牙齿大小的设备, 钻孔试样的用量将有所不同。为了防止加热试样, 在低速下钻。 注: 大约4-8 毫克的样品粉是利用在线气体制备和引种系统进行同位素比质谱和成岩试验的多项分析的适当目标。要知道, 所选的钻速将取决于样品的脆性, 并需要一定程度的试验和误差。一般情况下, 在手持式钻头上, 有两个五棒的强度是足够的。在一块干净的铝箔或称重纸上钻上牙粉。</li>
	<li>收集产生的搪瓷粉末, 并将其转移到一个1.5 毫升微离心管, 已 tared 在适当的准确平衡之前钻探。将每个管标为样本指定 (图 3)。在实验室笔记本和电子数据库中记录样本数和搪瓷重量。</li>
	<li>在对每颗新牙进行钻孔前, 先用0.5 米 HCl 在强健的组织上清洗使用过的钻头。然后用一个强健的组织用有机溶剂 (如丙酮、甲醇或乙醇) 清洗钻头。</li>
	<li>在钻孔后, 使用专用的真空吸尘器或簸箕和刷子彻底清洁工作区。用甲醇擦拭取样空间。真空或轻轻喷涂钻头与压缩空气, 以消除灰尘和样品之间的粉末样品。</li>
</ol>2. 顺序抽样
注意: 顺序取样可以用多种形式来处理, 并取决于所取样的分类、牙齿的大小以及所需的时间分辨率。
<ol><li>通过除去一层非常薄 (~ 0.1 毫米) 的外层牙釉质, 清洁被取样的牙齿表面。</li>
	<li>沿颊面垂直于齿的生长轴上钻样品, 从搪瓷根部连接到冠顶部。钻垂直于生长轴的水平带, 每个试样在搪瓷层上产生1-2 毫米宽凹槽 (图 1和 图 4)。注意避免将搪瓷钻入牙本质, 因为它会污染样品和由此产生的测量结果。 注: 取样的数量将因所需的分辨率而异, 而 c. 10 则是研究δ13c 和δ18O 值的季节变化的一个适当目标, hypsodont 的大小是家养的牛。</li>
	<li>为每个样品选择合适的钻头, 因为取样线的宽度由钻头的直径决定。</li>
	<li>使用钻机设置, 以更细腻的牙齿需要更多的增量样本 (图 2) (e., 人的牙齿), 以及更稳定的钻探。设置钻机支架, 使钻具稳固, 钻头向下指向。 注: 虽然四样品可以很容易地从一个永久性的人类磨牙使用手持方法24, 更精细的取样需要一个钻机或 CO2激光消融方法, 将不会在这里讨论14。另一种常见的方法是使用半自动化 microsampling25。</li>
	<li>握住牙齿或附着在夹子上。按牙釉质对钻头和施加压力。样品是沿颊面从顶部 (咬合面) 渐进地钻到牙冠根部 (ERJ) 的位置。多次重复相同的取样钻孔策略, 多次钻取平行于上一样品线的每条连续线。用一个小的圆柱形金刚石钻头, 用于细齿, 如绵羊臼齿, 直径为1毫米 (图 1C)。</li>
	<li>使用卡尺测量每个取样线的距离, 并记录此距离以进行比较. ERJ</li>
	<li>按照上面的步骤 1.3-1.6。 注: 如果对细小、细腻的牙齿进行分析, 不采用预处理和成岩检查方法, 那么小至1-4 毫克的样品将在在线气体制备和碳酸盐分析的引种设置中产生可靠结果。改进后的自动外围系统可以方便地使用更低的取样重量, 但不可避免地受到研究的搪瓷 (4 至 5 wt%)26、27中碳酸盐的比例的限制。研究人员应与实验室进行协商, 以测定样品的数量, 以确定所需的样品量。</li>
</ol>3. 傅里叶变换红外光谱分析/衰减总反射率法
<ol><li>对于衰减的总反射率法, 建立一个样品室背景, 无论是在真空或在正常的大气条件。</li>
	<li>用刮刀将大约1毫克的牙釉质放在样品室中的金刚石晶体上。降低和安全的样品后, 直到有牢固的连接之间的岗位, 样品, 和钻石板。根据设置或可用性, 关闭取样室, 不论是否建立真空。</li>
	<li>测量样品的 FTIR 光谱64倍的波数范围在400和 4000 cm-1之间。所需的复制数量将根据研究的目标而变化, 尽管对三种复制的分析, 理想情况下, 采用不同的整除数, 然后返回到离心管外壳的样本, 将确保可靠的结果。</li>
	<li>在每个整除和每个样品之间用甲醇清洁刮刀。</li>
	<li>使用可用软件执行基线更正。软件会自动从产生的 FTIR 剖面中减去样品室背景。为确保更好的重现性, 应考虑到只有最低吸光度为0.06 的最高磷酸盐波段为1035厘米-1的搪瓷光谱。</li>
	<li>通过检查711厘米-1的峰值, 监测所有样品中常见的次生污染碳酸盐方解石的存在。</li>
	<li>经过分析, 仔细地将样品返回到其离心管使用刮刀或吸入装置, 并进入下一阶段的预处理或分析。</li>
	<li>将原始波长和强度数据导出为. csv 文件 (或类似的), 并用于计算利息指数 (常用指数包括 a 位磷酸盐指数、B 区磷酸盐指数、磷酸盐结晶度指数、磷酸酯指数的水酰胺和共3/PO4指数17,18,19) (表 1)。</li>
	<li>将来自化石或考古样本的 FTIR 结果与现代动物牙釉质的生长数据集进行比较, 现在可在文献19、20中确定成岩蚀变的性质和程度。搪瓷取样。</li>
</ol>4. 简单醋酸 (0.1 米) 前处理
<ol><li>使用适当的平衡, 在离心管中为每个样品称量搪瓷粉。相应地给管子贴上标签。搪瓷粉必须与相同尺寸的颗粒磨成相同的度数。 注: 许多研究人员利用氧化剂, 如稀释漂白剂 (次氯酸钠) 或30% 过氧化氢 (H2O2) 从样品中去除有机物, 此时应添加。然而, 这些试剂不仅可以改变样品的稳定碳和氧同位素值, 而且在在线气体制备和引种系统中, 骨胶原样品的光谱表明100% 磷酸在碳酸盐中的应用测量不与有机样品反应 (图 5), 这表明这一步骤是不必要的。</li>
	<li>用吸管将0.1 毫升的0.1 米醋酸 (每1毫克的搪瓷) 添加到每个样品中。避免用吸管接触样品。如果样品和吸管接触, 使用新的吸管为下一个样品, 以避免交叉污染。</li>
	<li>鼓动, 要么通过摇晃或电子搅拌器 (图 3), 然后留下样品坐10分钟. 搪瓷粉不应留在醋酸长时间 (> 4 小时)。</li>
	<li>将样品放在离心中, 在 13700 x g 处放置2分钟。 注: 低成本替代方法是使用微型离心机4分钟, 在 3500 x g, 它持有较少的样品, 但在处理大型样本集时耗时较少。</li>
	<li>当离心程序停止时, 用干净的吸管用2毫升的超纯水代替乙酸, 然后离心 13700 x g 的样品2分钟。</li>
	<li>把超纯水再换两次, 就像以前一样, 测试是否中立。用吸管除去剩余的液体 (不干扰上清)。 注: 共三水洗与超纯水是进入中性的标准程序。样品应清洗, 直到达到中性。</li>
	<li>切割 Parafilm (1 厘米 x 1 厘米) 和放置在每个离心管。用锋利的物体在中心做一个小孔, 这样样品就会干得适当。</li>
	<li>将管子放在冷冻干燥器中, 除去剩余的液体。 注: 如果冷冻干燥机不可用, 温和烘箱干燥 (40 °c) 也是可能的, 不应对稳定的碳和氧同位素数据产生任何不利影响。</li>
	<li>干燥后, 取出 Parafilm 并关闭离心盖。确保管标签是正确的, 并在必要时重新编写。 注: 这是在储存前的最后阶段, 在进行计量时, 在有限资金的实验室可能是在其他地方进行分析的样品分发之前的最后阶段。</li>
</ol>5. 称量和测量样品和标准
<ol><li>使用刮刀, 重量约2毫克的搪瓷粉到锡盘上的平衡敏感到0.001 毫克 (图 6)。然后仔细将样品转移到磷酸硼硅酸盐玻璃瓶中。由于在牙釉质中碳酸盐的比例相对较小, 因此需要大量的搪瓷来产生可靠的结果。</li>
	<li>用甲醇清洁样品之间的刮刀, 并对每个样品使用新鲜的、干净的圆盘或称量容器。</li>
	<li>衡量国际标准的0.2 毫克, 例如原子能机构 NBS18、原子能机构603、原子能机构 CO8 或默克碳酸钙3。一个内部标准由一颗大牙的匀质搪瓷制成, 也可以使用20。对于完整的76个样品和标准, 使用61个样本和15标准, 这应该是均匀分布在整个运行。几项研究报告了20、28考古研究中适当数量的标准和细节。 注: 在室内标准中对搪瓷的反复预处理和分析, 横跨一系列的考古样本, 也可能为所研究的20样品提供适当的准确度测量。</li>
	<li>拧紧特殊设计的盖子, 可进入针, 直到隔膜是紧的, 但不吸入到瓶子里。不要过度收紧盖子, 因为隔膜上的压力会变得太强, 在分析过程中最终会导致一根折断的针。将瓶子放在加热块中列出的顺序中, 温度为70摄氏度。样品的顺序和重量必须可靠地记录入计算机软件。</li>
	<li>通过以下三步骤测量样品: 1) 冲洗填充样品与纯氦, 2) 添加磷酸, 3) 测量样品。<ol>
<li>用纯氦 (5.0 级) 冲洗样品, 流100毫升/分, 5 分钟即可冲洗出周围空气。</li>
		<li>将4-5 滴磷酸 (99%, 密度1.85 克/毫升) 添加到含碳酸盐的样品中。样品和 H3PO4之间的反应开始。在反应期间, CO2被释放运载同位素价值的碳酸离子 CO32-样品。</li>
		<li>等待1小时每样品直到 CO2的平衡被到达。1小时后, 应测量样品以获得可靠的结果。在收购过程中, 样品气体的混合物, 他传递到设备。干燥阶段从样品气体混合物去除水。</li>
		<li>用已知的同位素组成, 从 CO2参考气体的三峰开始测量样品。峰值强度应为 4000 mv, 以匹配采样峰值的强度 (介于4000到 8000 mv 之间)。三参考气体峰值应该是二十年代长和三十年代分开。请遵循定义示例的度量间隔。测量样品10次为 5 s 每和五十五年代分开。确保峰值高度随着时间的推移而减少, 表明样品/氦混合物的正确传输 (图 5)。 注: 在测量过程中, 软件通过比较已知同位素值、峰值高度和参考气体峰值面积与样品峰值高度和面积, 来计算样品的同位素组成。计算了样品的原始13c/12c 和18o/16o 组成。</li>
	</ol>
</li>
	<li>测量后对样品进行规范化。 注意: 这一点很重要, 因为在引入质谱仪时, 参考气体不经历与样品气体相同的化学和物理路径。因此, 必须将样品另外校准为国际标准 (步骤 5.3), 在运行中接受相同的物理和化学处理, 作为样本本身20。这些国际碳酸盐标准使样品的两点校准能够在国际三角洲测量尺度上, 并在整个给定的运行过程中对精度和重复性进行评估。</li>
</ol>

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<li>Roberts, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calling+all+archaeologists%3A+guidelines+for+terminology%2C+methodology%2C+data+handling%2C+and+reporting+when+undertaking+and+reviewing+stable+isotope+applications+in+archaeology%5BTitle%5D)+AND+%22Rapid+Communications+in+Mass+Spectrometry%22%5BJournal%5D)">Calling all archaeologists: guidelines for terminology, methodology, data handling, and reporting when undertaking and reviewing stable isotope applications in archaeology.</a> Rapid Communications in Mass Spectrometry. , (2018).</li>
<li>Snoeck, C., Pellegrini, M. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S0009254115300528">Comparing bioapatite carbonate pre-treatments for isotopic measurements: Part 1-Impact on structure and chemical composition.</a> Chemical Geology. 417, 394-403 (2015).</li>
<li>Pellegrini, M., Snoeck, C. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S0009254115300851">Comparing bioapatite carbonate pre-treatments for isotopic measurements: Part 2-Impact on carbon and oxygen isotope compositions.</a> Chemical Geology. 420, 88-96 (2016).</li>
<li>Wright, L. E., Schwarcz, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stable+carbon+and+oxygen+isotopes+in+human+tooth+enamel%3A+identifying+breastfeeding+and+weaning+in+prehistory%5BTitle%5D)+AND+%22American+Journal+of+physical+anthropology%22%5BJournal%5D)">Stable carbon and oxygen isotopes in human tooth enamel: identifying breastfeeding and weaning in prehistory.</a> American Journal of physical anthropology. 106 (1), 1-18 (1998).</li>
<li>Roberts, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fruits+of+the+forest%3A+Human+stable+isotope+ecology+and+rainforest+adaptations+in+Late+Pleistocene+and+Holocene+%28%E2%88%BC+36+to+3+ka%29+Sri+Lanka%5BTitle%5D)+AND+%22Journal+of+human+evolution%22%5BJournal%5D)">Fruits of the forest: Human stable isotope ecology and rainforest adaptations in Late Pleistocene and Holocene (∼ 36 to 3 ka) Sri Lanka.</a> Journal of human evolution. 106, 102-118 (2017).</li>
<li>Zazzo, A., Balasse, M., Patterson, W. P. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S0016703705001808">High-resolution δ13C intratooth profiles in bovine enamel: Implications for mineralization pattern and isotopic attenuation.</a> Geochimica et Cosmochimica Acta. 69 (14), 3631-3642 (2005).</li>
<li>Sydney-Zax, M., Mayer, I., Deutsch, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Carbonate+content+in+developing+human+and+bovine+enamel%5BTitle%5D)+AND+%22Journal+of+dental+research%22%5BJournal%5D)">Carbonate content in developing human and bovine enamel.</a> Journal of dental research. 70 (5), 913-916 (1991).</li>
<li>Rink, W. J., Schwarcz, H. P. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S030544038570026X">Tests for diagenesis in tooth enamel: ESR dating signals and carbonate contents.</a> Journal of Archaeological Science. 22 (2), 251-255 (1995).</li>
<li>Szpak, P., Metcalfe, J. Z., Macdonald, R. A. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S2352409X16306824">Best practices for calibrating and reporting stable isotope measurements in archaeology.</a> Journal of Archaeological Science: Reports. 13, 609-616 (2017).</li>
<li>Wright, L. E., Schwarcz, H. P. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S030544039890351X">Correspondence between stable carbon, oxygen and nitrogen isotopes in human tooth enamel and dentine: infant diets at Kaminaljuyu.</a> Journal of Archaeological Science. 26 (9), 1159-1170 (1999).</li>
<li>Schoeninger, M. J., Hallin, K., Reeser, H., Valley, J. W., Fournelle, J. <a target="_blank" href="https://onlinelibrary.wiley.com/doi/abs/10.1002/oa.653">Isotopic alteration of mammalian tooth enamel.</a> International Journal of Osteoarchaeology. 13 (1-2), 11-19 (2003).</li>
</ol>

作者声明没有利益冲突。

成功取样 (散装和递增) 牙列的挑战依赖于获得有关钻探技术和样品准备的知识, 同时投资于相对低廉的设备。当有关于取样和预处理方法的明确指示时, 这些挑战很容易逾越。在这篇文章中, 我们希望能以一种清晰、简明的方式向新的研究人员传播这些方法。在对有价值的考古和古生物样品进行分析和取样之前, 首次应用这些方法的学者应在可接近的现代动物区系进行实践。
?...

对牙釉质碳酸盐进行了稳定的碳氧同位素分析, 研究了过去人类膳食摄取量、断奶和流动性, 以及区系对植被、动物运动和牲畜 foddering 的依赖性。这些应用已被全面讨论和审查的各种环境条件表明, 当地干旱, 温度, 水源和植被组成的影响,1,2, 3,4,5,6。考古学和古生物学中潜在应用的多样性, 以及对牙釉质碳酸盐的良好保存, 使其成为稳定同位素工作3的吸引材料。在以前的一些出版物1、7中简要介绍了取样、预处理和成岩作用筛选方法。然而, 全面的口头和视觉演示仍然很大程度上无法使用, 特别是在考古科学实验室之外的人, 以及在有限的资金范围内的实验室小组中, 这种技术的应用兴趣正在增加。5。
牙釉质主要由羟基磷灰石 (bioapatite) 晶粒8 , 比骨中的更大, 使其更抗死后成岩离子置换和污染3。现代研究表明, 稳定的碳同位素 (δ13C) 测量的动物性牙釉质可靠记录的牲畜饮食和行为9,10。牙釉质的稳定氧同位素 (δ18O) 值由摄入水的氧同位素组成决定, 其中包括动植物食品中的水分、饮水、呼吸以及对水的各种环境影响。这可能导致进一步同位素分馏 (例如, 干旱, 温度, 海拔, 降雨量, 大陆位置)11。这使得它成为考古学、古生态和古生物研究中一种流行的饮食和环境重建方法。
牙釉质形成期相对较短 (年), 根据取样的牙齿不同而有所不同。为人, 第一磨牙牙釉质 mineralizes 在诞生和3岁之间, 前磨牙矿化在1.5 和7岁之间, 第二磨牙矿化在2.5 和8岁之间, 并且第三磨牙矿化在青春期期间, 在7和16年之间12.考虑到牙釉质在其形成期逐渐形成, 可以在整个生长轴上大量取样或依次取样, 以调查在形成期间发生的饮食和环境变化13。.按时间顺序排列的饮食变化在一个给定的牙齿之内是可观察的为人和其他动物1,14, 提供信息关于年际季节和膳食变化。
虽然搪瓷通常抗成岩作用, 但由于埋藏环境的同位素修改是可能的, 并已观察到15,16, 使实验检查和预处理选择有用。虽然它不是唯一可用的方法, 傅里叶变换红外光谱 (FTIR), 特别是在衰减传输模式, 已成为一种快速, 廉价, 相对容易接近的方法来评估埋藏初探改变牙釉质,特别在古生物上下文17,18,19,20。然而, 在地球化学或材料科学领域以外的许多人, 详细的议定书和记录标准仍然相对无法获得。
反应时间和研究人员在牙釉质预处理过程中所使用的化学物质在文献中也有很大的差异, 通常考虑到这种变异对样品21的稳定碳和氧同位素值的作用有限. ,22。在这里, 我们报告的方法, 使用稀醋酸 (0.1 米) 的预处理搪瓷粉末样品。然而, 由于预处理所产生的同位素测量的差异对牙釉质来说相对较小, 因此研究人员最好遵循它们希望将数据与11进行比较的数据集的协议。此外, 在采用小顺序取样的情况下, 特别是在全新世样品上, 不可以选择预处理 (经过试验成岩试验), 以避免样本损耗。
虽然我们在这里报告的方法并不是新的, 但对我们的知识来说, 这是第一次对牙釉质进行大量和连续抽样、预处理选择和成岩检查方法 (以 FTIR 的形式) 的完整的书面和可视文档。已被广泛地提供给不同的学术受众。虽然我们希望我们的努力将使这种方法更容易获得更多的个人和实验室, 研究人员谁想申请和出版这项技术必须知道最低报告标准, 成岩考虑, 并介绍要求综述了其他20, 以及潜在的解释复杂性, 将是独特的研究区域, 分类群分析, 和时间段5。

<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

人类和动物牙釉质碳酸盐的稳定碳氧同位素分析已在 paleodietary、古生态和环境研究中得到应用, 从最近的历史时期追溯到1000万年前。散装方法为搪瓷矿化时期提供了代表性的样品, 而在牙齿内的顺序取样可以跟踪这一时期的饮食和环境变化。虽然这些方法在考古学、生态学和古生物学中得到了广泛的应用和描述, 但没有明确的指导方针来帮助选择必要的实验室设备, 并详尽地描述实验室取样和协议。在本文中, 我们记录文本和视觉, 整个过程从抽样通过预处理和成岩筛选, 使该方法更广泛地提供给研究人员考虑其应用在各种实验室设置。

采用上述抽样方法, 制备了 bioapatite 的增釉样品。牙釉质中 bioapatite 的分析取决于取样的准确性, 无论是批量还是增量。在这种情况下, 我们选择了从不同的气候带展示考古标本 (两只绵羊) 的结果。从绵羊第二磨牙中分析了增量样本, 并从 ERJ 开始标记 (图 4)。增量示例位置已编号, 每个位置都以 mm 从 ERJ 的距离度量 (图 7)。
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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

用于稳定碳氧同位素分析的牙釉质碳酸盐的取样与预处理

对人和动物牙釉质进行了稳定的碳氧同位素分析, 作为个体饮食和环境重建的代表。在这里, 我们提供了详细的描述和视觉文件的散装和顺序牙釉质取样以及预处理的考古和古生物样品。

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

Research

JoVE Journal

Biochemistry

13.9K 次观看.  Max Planck Institute for the Science of Human History. 对人和动物牙釉质进行了稳定的碳氧同位素分析, 作为个体饮食和环境重建的代表。在这里, 我们提供了详细的描述和视觉文件的散装和顺序牙釉质取样以及预处理的考古和古生物样品。

视频: 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.

测量碳为基础的污染物矿化结合使用CO<sub&gt; 2</sub&gt;助焊剂和放射性碳分析

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

科研

环境科学

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.

农业经营条件下土壤侵蚀和沉积与土壤碳酸盐浓度关系的测量与制图

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.

表征，气相炭用苯聚羧酸的定量和特定化合物同位素分析（BPCA）

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

化学

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.

用同步快速反应层析成像动态孔隙规模油藏条件下成像碳酸盐

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

工程学

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.

高炉矿渣的双向稳定物价:沉淀碳酸钙和沸石重金属吸附剂的合成

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.

监测农业土壤中经修正的硅酸盐矿物风化造成的致病性无机碳积累。

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.

分离石英颗粒用于第四纪沉积物的光学刺激发光（OSL）测年，用于古环境研究

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).

优化的骨采样方案，用于从考古遗迹中提取古代DNA

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

遗传学

<meta charset="utf8"></head><body>使用外植体模型对骨形成进行多模态分析

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.

<meta charset="utf8"></head><body>作者聚焦:在骨再生研究中表征组织矿化的高级技术

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.