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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopic+evidence+for+dietary+variability+in+the+early+hominin+Paranthropus+robustus.">Isotopic evidence for dietary variability in the early hominin Paranthropus robustus.</a> Science. 314 (5801), 980-982 (2006).</li><li>Lee-Thorp, J., Sponheimer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+case+studies+used+to+reassess+the+reliability+of+fossil+bone+and+enamel+isotope+signals+for+paleodietary+studies.">Three case studies used to reassess the reliability of fossil bone and enamel isotope signals for paleodietary studies.</a> Journal of Anthropological Archaeology. 22 (3), 208-216 (2003).</li><li>Zazzo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+and+enamel+carbonate+diagenesis:+a+radiocarbon+prospective.">Bone and enamel carbonate diagenesis: a radiocarbon prospective.</a> Palaeogeography, Palaeoclimatology, Palaeoecology. 416, 168-178 (2014).</li><li>Sponheimer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Isotopic paleoecology of the Makapansgat Limeworks fauna (Australopithecus africanus, South Africa). , (1999).</li><li>Sponheimer, M., Lee-Thorp, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+enamel+carbonate+environments+during+fossilization.">Alteration of enamel carbonate environments during fossilization.</a> Journal of Archaeological Science. 26 (2), 143-150 (1999).</li><li>Roche, D., S&#233;galen, L., Balan, E., Delattre, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+assessment+of+Miocene-Pliocene+tooth+enamel+from+Tugen+Hills+(Kenyan+Rift+Valley)+through+FTIR,+chemical+and+stable-isotope+analyses.">Preservation assessment of Miocene-Pliocene tooth enamel from Tugen Hills (Kenyan Rift Valley) through FTIR, chemical and stable-isotope analyses.</a> Journal of Archaeological Science. 37 (7), 1690-1699 (2010).</li><li>Roberts, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calling+all+archaeologists:+guidelines+for+terminology,+methodology,+data+handling,+and+reporting+when+undertaking+and+reviewing+stable+isotope+applications+in+archaeology.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+bioapatite+carbonate+pre-treatments+for+isotopic+measurements:+Part+1-Impact+on+structure+and+chemical+composition.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+bioapatite+carbonate+pre-treatments+for+isotopic+measurements:+Part+2-Impact+on+carbon+and+oxygen+isotope+compositions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+carbon+and+oxygen+isotopes+in+human+tooth+enamel:+identifying+breastfeeding+and+weaning+in+prehistory.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruits+of+the+forest:+Human+stable+isotope+ecology+and+rainforest+adaptations+in+Late+Pleistocene+and+Holocene+(&#8764;+36+to+3+ka)+Sri+Lanka.">Fruits of the forest: Human stable isotope ecology and rainforest adaptations in Late Pleistocene and Holocene (&#8764; 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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+&#948;13C+intratooth+profiles+in+bovine+enamel:+Implications+for+mineralization+pattern+and+isotopic+attenuation.">High-resolution &#948;13C 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbonate+content+in+developing+human+and+bovine+enamel.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tests+for+diagenesis+in+tooth+enamel:+ESR+dating+signals+and+carbonate+contents.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Best+practices+for+calibrating+and+reporting+stable+isotope+measurements+in+archaeology.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correspondence+between+stable+carbon,+oxygen+and+nitrogen+isotopes+in+human+tooth+enamel+and+dentine:+infant+diets+at+Kaminaljuyu.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopic+alteration+of+mammalian+tooth+enamel.">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&#38;region=DE&#38;gclid=EAIaIQobChMI7pHRpauW2QIV77ftCh1p1wjhEAAYASAAEgKzkvD_BwE</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td></td>
			<td></td>
			<td>Linear Formula:&#160;CH3OH</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td></td>
			<td></td>
			<td>Linear Formula:&#160;CH3CO2H</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)。
<p ...

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

13.8K 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

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

科研

生物化学

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

核小体的重建与差异同位素标记的姐妹组蛋白

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

人视网膜解剖与 RPE-脉络膜的蛋白质组分析

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). SM is a common sphingolipid composed of a phosphorylcholine and a ceramide as the hydrophilic and hydrophobic component, respectively. A number of SM species are present in mammalian cells due to a variety in the sphingoid long chain base (LCB) and an N-acyl moiety in the ceramide. In this report, we show a method of estimating the number of carbon and double bonds in a LCB and an N-acyl moiety based on their corresponding product ions in MS/MS/MS (MS3) experiments. In addition, we present a quantitative analysis method for SM using two stable isotopically labeled SM species, which facilitates determining the range used in SM quantitation. The present method will be useful in characterizing a variety of SM species in biological samples and industrial products such as cosmetics.

Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

用两个稳定 Isotopically 标记鞘磷脂种鞘磷脂的定量和定性方法

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

电化学检测氘动力学同位素对希瓦氏菌 oneidensis MR-1 细胞外电子传输的影响

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

操纵活细胞构建稳定的3D 蜂窝组件, 无需人工支架

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

低氧环境下人巨噬细胞极化的蛋白质组学分析

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...