作者要感谢由美国陆军ERDC军事工程6.1基础研究发展计划和ERDC中心指导研究计划提供的这项工作的财政支持。作者还要感谢ERDC岩土与结构实验室的混凝土和材料分会的工作人员和设施配套的实验工作。允许出版被授予由主任，岩土与结构实验室。

1，鱼鳞样品制备在这项研究中，量表由美国陆军工程师研究与发展中心（ERDC）环境实验室在中间的长度（29 个尾柱）从大约600毫米长的雀鳝（A.抹刀 ）获得。鱼鳞是按照卫生研究院（NIH）动物保健指导的ERDC和研究所获得的。 <ol><li>物料 记录在该研究中获得的鳞的鱼的空间位置。请务必遵守相应的组织或获得的生物标本，如美国国立卫生研究院动物保健指导政府的指导方针。存储该秤在适当的介质，如磷酸盐缓冲盐水溶液以保持水合，一旦它们从鱼除去维持矿物质含量。避免长时间存储，可能导致矿物质损失，这可能会影响纳米压入数据。使用中等毛刷和tweezers从硬鳞除去任何软组织。 </li><li>试样安装和切片 检查鱼鳞的短轴的利用FTIR的横截面图（ 图1）和纳米压痕要求第一安装规模的硬介质，如双组分环氧树脂组成的树脂和固化剂。使用通用型RT固化环氧低峰值温度如商业通用型环氧本研究中有小于55℃的峰值温度下使用</li></ol><img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51535/51535fig1highres.jpg" src="/files/ftp_upload/51535/51535fig1.jpg" /> A的图1中X-射线CT图像抹刀刻度描绘检查在本研究中A的短轴横截面用锅铲纳米压痕和红外光谱[A（前），P（后），D（背），V（腹）]。&lt;/ P&gt; <ol start="2"><li><ol><li>按住鱼鳞中使用市售的塑料样品架一个直径32毫米的样品的模具。这将保持正确的方向，同时安装在环氧树脂样品。 </li><li>一旦样品在模具中保持，倒入样品上的未固化的环氧树脂，然后让环氧根据制造商的指示治愈。 </li><li>后的环氧树脂固化后，部分使用金刚石刀片的高精度切断已安装的样品看到，在试样的中线。 </li><li>超声处理在蒸馏水中15分钟，从样品除去任何碎屑。 </li></ol></li><li>抛光的纳米压痕和红外 获得光滑平整的表面进行纳米压痕， 如图2所示，下面的抛光步骤和参数如下提示基于与所述抛光机制造和测试样本的讨论。然而这些参数可能需要调整基于响应不同的生物材料如材料去除率。在抛光步骤之间蒸馏水洗澡样品超声至关重要的是确保从粗抛光步骤颗粒不会引入到随后的精细抛光步骤。 </li></ol><img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51535/51535fig2highres.jpg" src="/files/ftp_upload/51535/51535fig2.jpg" /> 抛光短轴横截面A的图2。图片锅铲规模安装在环氧树脂。 <ol start="4"><li><ol><li>粗抛光用15微米的SiC垫和水作为润滑剂，直到样品是平面使用的7磅的每分钟转速（rpm）200转和速度的自动抛光头的力。 </li><li>超声处理的样品在蒸馏水浴中15分钟。 </li><li>中间抛光用使用水的润滑剂以130rpm的压板速度为6微米的SiC垫和7磅的力5分钟。 </li><li>在蒸馏水中15分钟，浴超声处理的样品。 </li><li>抛光用使用水的润滑剂以130rpm的压板速度和7磅5分钟的力的1微米的SiC板。 </li><li>超声处理的样品在蒸馏水浴中15分钟。 </li><li>最后的抛光具有使用适当的抛光垫，例如一个高密度，非织造的，低小睡多孔，制造商建议为50纳米混悬剂的聚氨酯50纳米的胶态二氧化硅悬浮液。抛光以130rpm为7磅5分钟的力的速度。 </li><li>超声处理的样品在蒸馏水浴中15分钟。 </li></ol></li></ol> 2，纳米压痕测试<ol><li>每批每次测试前校准纳米压痕系统制造商的指引。校准应该包括确定该系统的面积函数的Berkovich压头和框架的刚度。此外，在该步骤执行一个显微镜对压头校准为确保缩进关联到所选择的显微镜位置。 </li><li>样品加载到压痕，并使用光学显微镜的控件上的压痕，使样品成为关注的焦点。 </li><li>使用软件控制来移动样品的位置为第一缩进。理想情况下，这是在从沿标尺的横截面的中心线的ganoine层的边缘环氧约10微米。 </li><li>执行4个并行的行缩进的间隔为15μm分开，以获得统计学显著数据设置开始在这个位置。在纳米压痕设置为5亿元，装卸率0.1分钟/秒，30秒的保持时间，而5微米的每一行的最小间距缩进的最大负荷。缩进的行应该设置来执行正交于ganoine表面，并应指定缩进足够数量的跨尺度的横截面行进在遍历约10微米到对EPOXŸ过去的骨层。 </li><li>当批处理结束时，有压痕创建基准缩进为100 MN在第一个和最后一个缩进最大负载，这应该是在环氧树脂的ganoine层之前和骨层后，分别。这些相关联的开始和结束点为缩进的每一行。 </li><li>纳米压痕后，将样品放回PBS溶液，以避免进一步的脱水。 </li><li>使用纳米压痕软件确定的基础上，奥利弗-法尔法13的弹性模量和硬度，如果与时间无关的材料响应是观察。否则，保持时间可能需要被扩展，以克服从卸载太快观察到蠕变。 </li></ol> 3，空间分辨的ATR-FTIR光谱利用滑盖上连接到一个红外显微镜ATR附件的一个建议的方法来收集空间分辨傅立叶变换的LA的红外（FTIR）光谱Y一代的鱼鳞样。在ATR附件允许采集高质量的光谱具有非常小（〜10微米2）的空间分辨率，这是无法实现的任何其他的FTIR技术。同一抛光的样品（ 图2），用于纳米压痕实验制备用于这些实验中。 <ol><li>选择具有表面和尺寸适合于红外显微镜被用于分析的样品，以确保高质量的光谱是由ATR-FTIR spectromicroscopy获得的。 </li><li>准备红外显微镜来收集数据。显微红外要求的红外信号的校准相同的采样条件，将用于测量的样品下。通常，这包括制冷探测器，并允许时间来稳定以及收集在相同的环境条件下，所有的背景光谱和样品光谱。这可以是特别重要的，因为CO 2和水蒸汽中的空气可以ðramatically影响红外光谱。同样重要的是，确保该仪器的光学对准。 </li><li>收集合适的背景光谱减去样品对。对于这些实验，抛光，镀金显微镜载玻片用作背景的红外spectromicroscopy。 </li><li>使用合适的目标，着眼于样品和选择感兴趣的分析的面积。 </li><li>一旦一个感兴趣的区域被发现，附加ATR附件的红外显微镜物镜，提高样品，直到它与ATR内反射元素亲密接触，并收集样品光谱。 </li><li>收集红外光谱后，执行所需的必要标准的数据处理。 </li></ol> 4，X射线计算机断层扫描（CT） <ol><li>获取并准备规模在1.1节中讨论</li><li>扫描仪设置<ol><li>根据制造商的规格热身的X射线源。 </li><li>设置X射线电压和50千伏和160μA，分别电流。 </li><li>设置曝光时间为1,450毫秒。 </li><li>选择1.0mm的铝过滤器。 </li><li>在装载前的样品，进行平场校正，当X射线源处于关闭状态（暗场）和（明场）。 </li></ol></li><li>山和负载标本 秤必须安装在一个方式，以便它们不会移动或移动整个扫描的长度。这些样品还必须使用的材料几乎是透明的，以X射线装。泡沫塑料和封口膜的组合可以用于固定规模的CT阶段。 <ol><li>刚性地装入样本，使得最长的尺寸是平行于所述检测器。 </li><li>安装的试样固定在扫描阶段。 </li><li>样本位置，使得它会在转动整个扫描的中心。 </li><li>选择最高的分辨率，使整个刻度是在视场（FOV）的字段，在这种情况7.5微米。 </li></ol></li><li>采集设置 执行扫描，在此研究中具有0.25°的旋转步骤和第15帧的平均值，如果较低的分辨率是可以接受的，增加步长大小和/或减少平均以减少总的扫描时间的帧。 </li><li>重构参数 一旦得到一个数据集，重建的透视投影图像来创建包含横截面图像的数据组。选择除以下SKYSCAN的NRecon软件的默认设置。 <ol><li>更改振铃伪影校正到20。 </li><li>改变射束硬化校正到25％。 </li><li>调整CS静态旋转，使横截面图像的水平。 </li></ol></li><li>影像处理 使用SKYSCAN的CTAN软件来获得最终的3D灰度图像。调整灰度范围至适当水平，从聚苯乙烯泡沫塑料和封口膜取下工件。 </li></ol>准备通过抛光的纳米压痕和micro-/nano-structure鉴定标本，利用扫描电子显微镜（SEM）下检视。低真空模式被利用，以减少试样脱水和需要应用的导电涂料。在抛光的试样中使用的能量色散型X射线光谱（EDX）扫描电镜成像结合进行局部化学分析。 EDX分析在这是由纳米压痕，以提供化学和机械性能之间的相关性分析，在同一行/网格进行。新鲜断裂面还审议通过SEM提供有关存在于鱼鳞的生物矿化结构的形态和方向更好的信息。为了提高观察断裂面的纳米尺度结构的分辨率，标本溅涂金（Au）和成像在高真空模式。以下提供所使用的程序的其他详细信息。 <ol><li>抛光表面的SEM成像<ol><li>放置抛光试样进SEM室和泵腔室进入低真空模式下为0.1〜0.5毫巴的压力腔室。 </li><li>调整工作距离约5.0毫米。 </li><li>启动高压（HV）并导航到的标本感兴趣区域，包括ganoine和骨亚层或其它感兴趣的区域之间的过渡地带。 </li><li>在15 kV高压及约3.9 nA的束流获取图像。 </li><li>聚焦图像，并执行所有必要的路线和stigmation调整。 </li><li>从感兴趣的相关放大倍数（通常250X〜10,000倍）使用低真空背散射电子（BSE）的检测器中的识别（ 即 ，致密骨的生物矿物含量和密度的变化与多孔骨，以帮助至少三个区域捕捉图像）。 </li></ol></li><li>断裂面的SEM成像<ol><li>使用双面胶带碳与断裂表面朝上加盖新鲜断裂试样，以90°的扫描电镜存根。 </li><li>溅射涂层用金，以提供对断裂面的子纳米厚的导电层。 </li><li>将试件放入扫描电镜室和泵室成高真空模式。 </li><li>调节工作距离，以在3.0和5.0毫米。 </li><li>启动高压并导航到对试样感兴趣的区域。在这种情况下，利率的主要领域是存在于ganoine和硬骨层结构。 </li><li>在5千伏和15千伏高压和0.24 nA的提高分辨率较低的电子束电流之间获取图像。 </li><li>首先着眼标本和进行初步比对。 </li><li>增加放大倍数大于5,000 X和开关正常场发射镜头插入浸入/超高分辨率（UHR）镜头。 </li><li>执行UHR路线和stigmation调整。 </li><li>从至少三个区域Ø捕获图像f权益有关的放大倍率（通常是5,000×250,000倍）使用通过二次电子（SE）模式下操作镜头检测器（TLD）。 </li></ol></li><li>抛光表面的EDX分析（与扫描电镜成像相结合进行）。这些参数是材料依赖性的，需要进行调整，所以EDX相互作用空间的大小，以纳米压痕相互作用体积类似由莫塞尔14所讨论的。 <ol><li>导航到对试件磨平感兴趣区域，其中包括在缩进的每行的末尾由基准标记表明纳米压痕网格。 </li><li>确保HV是至少15千伏，离子束电流为至少3.9 Na和工作距离大于5.0毫米。 </li><li>捕捉区域使用EDX分析的背散射电子像。 </li><li>用EDX分析软件，捕捉相同的图像在定位区域沿压痕线进行化学分析，以帮助。 </li><li>使用"线分析"技术，POsition一行沿着缩进感兴趣的行开始在第一个缩进，并在最后结束的缩进进行化学分析。 </li><li>指定要放置沿线分析的点的数量。它最好是用分析点和缩进都存在，以提供化学成分和机械性能之间的直接空间相关性的相同的号码。 </li><li>当行被定位和指定点正确，发起用EDX软件的线分析。 </li><li>当行分析完成后，确定将要由沿着对试样的抛光表面中的指定行所获得的点的光谱量化感兴趣的元素。 </li><li>一旦利息部分都已经确定，进行背景校正占轫致辐射等功效。 </li><li>选择该软件的反卷积分析选项，以获得定量分析上沿指定路线到泉每个点TIFY的化学组成在每个点。 </li><li>保存定量化学分析结果连同该分析中使用纳米压痕测量机械性能的空间相关性，以帮助指定的行的图象。 </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+technique+for+determining+hardness+and+elastic+modulus+using+load+and+displacement+sensing+indentation+experiments.">An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments.</a> J Mater Res. 7, 1564-1583 (1992).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Agilent Technologies Nano Indenter G200 User's Guide. , (2012).</li><li>Ulm, F. J., 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=Statistical+indentation+techniques+for+hydrated+nanocomposites:+concrete,+bone,+and+shale.">Statistical indentation techniques for hydrated nanocomposites: concrete, bone, and shale.</a> J. Amer. Cer. Soc. 90, 2677-2692 (2007).</li><li>Randall, N. X., Vandamme, M., Ulm, F. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoindentation+analysis+as+a+two-dimensional+tool+for+mapping+the+mechanical+properties+of+complex+surfaces.">Nanoindentation analysis as a two-dimensional tool for mapping the mechanical properties of complex surfaces.</a> J. Mater. Res. 24, 679-690 (2009).</li><li>Oyen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoindentation+of+Biological+and+Biomimetic+Materials.">Nanoindentation of Biological and Biomimetic Materials.</a> Exper. Tech. 37, 73-87 (2013).</li></ol>

作者什么都没有透露。

从实验的角度来看，研究人员需要记住，与天然存在的生物材料，如矿化鳞鱼，在鱼的报道规模的空间位置工作时是至关重要的，因为以前的研究已经显示出矿化鱼鳞的机械性能主要取决于到那里的量表位于鱼4。 矿化生物材料的机械性能也已证明是依赖于样品4的水合状态。试图比较已​​适当的水分，以公布的结果在公开文献中，使用了干燥的化石样品新鲜样品时，这限制了该技术的有用性。因此，长时间的测试时间需要避免以减少脱水的上一个样本的机械性能纳米压痕过程的影响。材料具体试点研究建议，以确保实验换货运行时间是足够的最小不改变材料的力学行为。湿细胞纳米压痕将是一个较好的方法，以保持材料的恒定水合状态，如果该测试设备允许的话。 在本研究中使用的纳米压痕法，其计算出的弹性模量从卸载曲线假定该材料表现为线性弹性各向同性材料。该技术可用于与各种压头尖端。然而，三面Berkovich压头为65.35°的半视角被用于本研究。替代的提示，如立方角（半角= 35.36°）是适用于本手稿提出的程序，但由于立方角尖比Berkovich压头的裂纹更加尖锐可以在样品中产生低得多的负荷比在尖端的Berkovich。 抛光是必不可少的步骤，以获得光滑的，平坦的表面以最小的表面活性Ë粗糙度不会影响纳米压痕效果。在这个手稿提交的抛光步骤，可能需要根据所用抛光机的类型进行修改，一个建议的程序。然而，关键的步骤，以确保准确的纳米压痕数据是，表面粗糙度最小化，并且对于这个特定的材料制成的50nm的最终抛光须获得光滑平整的表面的压痕深度被探测。 缩进的间距也确保不被从先前的凹痕发生的材料形变的影响纳米压痕准确数据。用于本研究中的设备的压痕用户手册建议缩进间距应至少为20-30X的最大穿透深度为Berkovich压压头15。替代材料，所要求的压痕间距将需要根据所施加的负荷和最大的压痕深度被确定为在打开前面所讨论的文学16,17。另外，被选择的保持时间为这种材料克服观察探测允许要使用的纳米压痕软件的奥利弗 - 法尔分析方法的不同阶段的材料的任何蠕变。然而，通过Oyen的18所讨论的替代分析方法可用于当时间依赖材料的响应可以不与合适的保持时间克服生物材料。 为了实现从X射线CT的高分辨率结果，一些设置必须进行优化。本文概述了一个非常具体的关于鱼鳞使用了一个独特的尺寸和分层厚度参数。具有不同的样品尺寸，这些设置将需要进行调整，以获得最高质量的数据集。选择每个参数的过程中，应随附正在使用的计算机的用户手册中明确规定。扫描设定值（电压，电流，曝光，过滤器的选择）和重建的设置（环状伪影，束硬化）可能需要进行修改，以适应各种其它样本大小和几何形状。 X射线CT提供的整体尺度形态学的识别覆盖材料仅发生在秤没有相互重叠的骨层ganoine层的图像。的X射线CT图像也识别该ganoine层由横跨尺度的非均匀的厚度，甚至表现出凹坑缺乏ganoine层完全的。 有趣的是，空间相关的SEM / EDX化学分析纳米压痕的数据确定的，而不是一个更渐进的过渡观察到的P的矿化鱼鳞急剧离散的2层之间的过渡senagalus（在Bruet 等 2）。 纳米压痕的组合，FTIR，EDX和SEM提供的机械性能，化学分析和结构信息进行确认外层为ganoine与搪瓷般的形态和化学。此外，这些技术证实内层为材料的骨层。 总之，本研究中所概述的方法中确定的程序和相应的结果来检查A的矿化鱼鳞刮刀从大块结构到纳米结构和化学组成。

研究人员正在研究生物材料结构，并试图阐明的设计原则，其提供结构的生物材料具有改进的机械性能，例如更高的韧性和时相比，其各成分的强度。对装甲鱼鳞为设计原则进行的调查真鲷7，Polypterus senagalus 2,6， 海象鱼身上3，鲤鱼 4，和Atractosteus锅铲 1表明有必要扩大现有的实验方法的应用研究结构响应和微观结构特征，由于详细的标准方法不能用于这些类型的材料和实验。 在讨论的不同的装甲鱼的鳞片，A。锅铲是一个历史顶点美国8中央的捕食者，是种高LY矿化尺度。该物种交流的肌肉块皮肤质量相比，同等规模的前面提到的9鱼以获得改进的捕食防御系统。根据页面和伯尔10，A。锅铲是第三大的淡水鱼在北美的白鲟（ 鲟transmontanus）和大西洋鲟（ 鲟oxyrhynchus）是较大的品种。 A的高矿化鳞鱼锅铲只是最近正在研究中。 Thompson和骨纤维11建议GAR秤的形态都具有由ganoine外层，漫骨层，板层骨层的三层结构组成。在A.当前的研究锅铲尺度并没有区分骨层为弥漫性或板层骨地区，但刚刚研究了骨区域作为一个单一的内层1,12。 在本研究中，本程序为在vestigating的微观结构，纳米结构，化学组成，及A的秤的机械特性的空间分布锅铲基于红外光谱的结果，扫描电镜，X射线CT和纳米压痕技术的介绍。

<table><tbody><tr><td>Epoxy resin</td><td>Buehler</td><td>701-501512</td><td></td></tr><tr><td>Epoxy hardener</td><td>Buehler</td><td>703-501528</td><td></td></tr><tr><td>Samplkups</td><td>Buheler</td><td>20-8180</td><td></td></tr><tr><td>SamplKlips I</td><td>Buehler</td><td>20-4100-100S</td><td></td></tr><tr><td>High precision cut-off saw</td><td>Buehler</td><td>Isomet</td><td></td></tr><tr><td>UltraMet 2002 sonic cleaner</td><td>Buehler</td><td>B2510R-MT</td><td></td></tr><tr><td>Polisher</td><td>Buehler</td><td>49-1750-160</td><td></td></tr><tr><td>1,200 grit (15 &amp;mu;m) SiC paper</td><td>Struers</td><td>40400012</td><td></td></tr><tr><td>4,000 grit (6 &amp;mu;m) SiC paper</td><td>Struers</td><td>40400014</td><td></td></tr><tr><td>50 nm colloidal silica</td><td>Buehler</td><td>40-10075</td><td></td></tr><tr><td>Chemomet polishing pad for 50 nm suspension</td><td>Buehler</td><td>40-7918</td><td></td></tr><tr><td>Nanoindenter</td><td>MTS</td><td>G200</td><td></td></tr><tr><td>FTIR continuum microscope</td><td>Thermo Nicollet</td><td>6700</td><td></td></tr><tr><td>X-ray computed tomography</td><td>Skyscan</td><td>Skyscan 1173</td><td></td></tr><tr><td>SEM</td><td>FEI</td><td>NovaNanoSEM 630</td><td></td></tr><tr><td>EDX</td><td>Bruker</td><td>AXS Xflash detector 4010</td><td></td></tr><tr><td>Sputter coater</td><td>Denton</td><td>Desk II</td><td></td></tr></tbody></table>

characterization of multi-layered fish scales (atractosteus spatula) using nanoindentation, x-ray ct, ftir, and sem

保护生物材料，如矿化鳞鱼，腹弹，羊角，鹿角和龟板的分层体系结构提供了潜力引导防护材料和系统的设计在未来的独特设计原则。了解这些材料体系的结构与性能的关系在微型和纳米其中故障启动是至关重要的。目前，实验技术，如纳米压痕，X射线CT，扫描电镜和研究人员提供了一种方法来关联这些材料系统1-6的层次微观力学行为。然而，一个良好定义的标准程序制备试样矿化生物材料是当前不可用。在本研究中，该方法用于探测空间相关的化学，结构和A的多层规模的机械性能利用纳米压痕锅铲 ，FTIR，SEM，有连接能量色散X射线（EDX）元素分析和X-射线CT呈现。

图3描绘了在空间上相关nanoinidentation / SEM / EDX分析跨越约800微米长的短轴线的横截面进行的平均结果。在大约60微米厚ganoine层，计算出压痕的69.0 GPa和3.3 GPA硬度的平均弹性模量。在纳米压痕确定的14.3 GPA在0.5 GPa和硬度为约740微米厚的骨层的平均弹性模量。 EDX测定碳，氧，钙和磷，这是通常出现在矿化鳞片。然而，ganoine和骨层中所含的化学成分可量化的差别。在骨层中观察到碳尖峰可能是由于该区域不被作为高度矿化，从而导致轻微的增加，碳也引起了BSE图像的整体亮度观测降低。具体来说，ganoine层&#39;，S是指钙原子浓度比:P为1.71出现了类似的羟基磷灰石具有1.67的理论比例。骨层的平均钙:P比降低至1.51表示从ganoine层中的矿化量的减少。 红外光谱在图4中的骨层和ganoine层确定的主要官能团如酰胺基，羧基，磷酸根，和羰基。具体地说，FTIR证实了目测羟基磷灰石签名中的内（骨）层的外侧（ganoine）层和胶原签名。峰在3,500-3,000厘米-1由于NH伸缩和NH弯曲1,550至1,500 -1之间代表骨层酰胺基团。在波数1,470-1,365厘米的区域峰-1代表酰胺取代的烷基。此外，一个独特的C = O伸缩在1641厘米-1观察到的骨层上。豌豆从3,000-2,500厘米-1 KS表示羧基。无论是骨骼和ganoine层"光谱产生的近1,079.33厘米-1表示拉伸磷酸盐的明显峰。 在图5中的X射线CT图像捕获的ganoine层不覆盖骨层，其中鳞片彼此重叠。较亮的灰色ganoine层表明致密，坚硬，并且更严厉的阶段，而较暗的灰色骨层表明密度较小和较少僵硬阶段。此外，该X射线CT图像计算机辅助识别的非均匀性ganoine层厚度。实际上，清楚的凹坑被观察到接近ganoine层，它不覆盖骨层在所有的中心。 在断裂面进行蚀刻以H 3 PO 4的图6A中的SEM图像显示组织为ganoine层的分层图案的纳米结构。这种纳米棒组织结构关联到从FTIR对ganoine面积得到的羟基磷灰石的签名。 图6A示出了断裂面的典型较低的放大倍率SEM显微照片清楚地识别与虚线的ganoine和骨层之间的过渡。 图6B描绘了断裂面的较高放大倍数的SEM图像与H 3 PO 4蚀刻之后。蚀刻之后，在所述外ganoine层取向的纳米棒是清晰可辨的，而纤维状纳米结构中的骨层观察。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51535/51535fig3highres.jpg" src="/files/ftp_upload/51535/51535fig3.jpg" /> 图3。模量和硬度值数据空间相关，SEM / EDX化学成分。 图4从外（ganoine）和内部（骨）层收集红外光谱。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51535/51535fig5highres.jpg" src="/files/ftp_upload/51535/51535fig5.jpg" /> 图5透视图上的外（ganoine）点蚀的CT图象层，覆盖所述内（骨）层。 <img alt="图6" fo:content-width="5in" fo:src="/files/ftp_upload/51535/51535fig6highres.jpg" src="/files/ftp_upload/51535/51535fig6.jpg" /> 图6（A）低倍率的SEM图像典型的断裂面，（B）在外层（ganoine）纳米棒的高放大倍数的图像，并在内侧（B纤维ONY）层.. <a href="https://www.jove.com/files/ftp_upload/51535/51535fig6highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM at JoVE.com

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

本文介绍了用于探测空间相关的化学，结构和Atractosteus抹刀的多层规模（A.刮刀 ），使用纳米压痕机械性能的方法中，傅里叶变换红外光谱（FTIR），扫描电子显微镜（SEM）和X-射线计算机断层摄影（X射线CT）。实验结果已被用于研究生物防护材料的设计原则。

表征多层鱼鳞（中<em&gt; Atractosteus锅铲</em&gt;）基于纳米压痕技术，X射线CT，红外光谱和SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

characterization-multi-layered-fish-scales-atractosteus-spatula-using

Research

JoVE Journal

Bioengineering

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

15.1K 次观看.  U.S. Army Engineer Research and Development Center. 本文介绍了用于探测空间相关的化学，结构和Atractosteus抹刀的多层规模（A.刮刀 ），使用纳米压痕机械性能的方法中，傅里叶变换红外光谱（FTIR），扫描电子显微镜（SEM）和X-射线计算机断层摄影（X射线CT）。实验结果已被用于研究生物防护材料的设计原则。 

视频: Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

Microfocus X-ray CT (microCT) Imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha)

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. Non-destructive microfocus X-ray computed tomography (microCT) imaging has become a powerful and emerging protocol in biology, due to technological advancements in sample staining methods and innovations in microCT hardware, processing computers, and data analysis software. However, this protocol is not commonly used, as it is in the medical and industrial fields. One of the reasons for this limited use is the lack of a simple and comprehensible manual that covers all of the necessary steps: sample collection, fixation, staining, mounting, scanning, and data analyses. Another reason is the vast diversity of metazoans, particularly marine invertebrates. Because of marine invertebrates&#8217; diverse sizes, morphologies, and physiologies, it is crucial to adjust experimental conditions and hardware configurations at each step, depending on the sample. Here, microCT imaging methods are explained in detail using three phylogenetically diverse marine invertebrates: Actinia equina (Anthozoa, Cnidaria), Harmothoe sp. (Polychaeta, Annelida), and Xenoturbella japonica (Xenoturbellida, Xenacoelomorpha). Suggestions on performing microCT imaging on various animals are also provided.

Microfocus X-ray CT microCT Imaging of Actinia equina Cnidaria, Harmothoe sp. Annelida, and Xenoturbella japonica Xenacoelomorpha

Here, protocols for performing microfocus X-ray computed tomography (microCT) imaging of three marine invertebrate animals are explained in detail. This study describes steps such as sample fixation, staining, mounting, scanning, image reconstruction, and data analyses. Suggestions on how the protocol can be adjusted for different samples are also provided.

微聚焦 X 射线 CT (微CT) 阿克蒂尼亚 equina (Cnidaria),哈莫特霍sp. (安妮利达) 和Xenoturbella japonica (西纳科洛莫卡) 成像

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. ...

科研

环境科学

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

基于海胆大白鲨A接地采样:仿生为设计一个协议

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

生物工程

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

原子力显微镜悬臂式纳米压痕:空气和流体中纳米级机械性能测量

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

工程学

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

测量海洋海绵针力学性能的毫米尺度弯曲试验系统

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

表征使用原子力显微镜，冲击压痕和流变测定脑组织的多尺度力学性能

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

神经科学

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design&#160;and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (&#956;m). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young&#39;s Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with &#181;m spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells.
In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer&#39;s guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

The protocol presents a complete workflow for soft material nanoindentation experiments, including hydrogels and cells. First, the experimental steps to acquire force spectroscopy data are detailed; then, the analysis of such data is detailed through a newly developed open-source Python software, which is free to download from GitHub.

软物质纳米压痕的实验和数据分析工作流程

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft ...

<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.

在离体模型中全面表征组织矿化

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

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

钙化事件的表征在海洋Tubeworm使用实时光学和电子显微镜技术

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

表征多层鱼鳞（中

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原子力显微镜悬臂式纳米压痕:空气和流体中纳米级机械性能测量

测量海洋海绵针力学性能的毫米尺度弯曲试验系统

表征使用原子力显微镜，冲击压痕和流变测定脑组织的多尺度力学性能

软物质纳米压痕的实验和数据分析工作流程

在离体模型中全面表征组织矿化

在离体模型中全面表征组织矿化

在离体模型中全面表征组织矿化

钙化事件的表征在海洋Tubeworm使用实时光学和电子显微镜技术