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。

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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 ...

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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

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

生物发光成像评估的免疫反应，植入心脏组织工程（东隧）

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

科研

生物工程

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5&deg;) and wide angles (typically &gt; 5&deg;). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

小型和广角X-射线散射研究生物大分子的解决方案

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

人体软组织的生物力学特性采用压痕和拉伸试验

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

使用微X射线计算机断层扫描与磷酸制备可视化人类纤维肌组织

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...

Four-Dimensional CT Analysis Using Sequential 3D-3D Registration

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data remains difficult because segmentation in all volumetric frames is time-consuming. We aimed to analyze joint kinematics using a sequential 3D-3D registration technique to provide the kinematics of the moving bone with respect to the fixed bone semiautomatically using 4DCT DICOM data and existing software. Surface data of the source bones are reconstructed from 3DCT. The trimmed surface data are respectively matched with surface data from the first frame in 4DCT. These trimmed surfaces are sequentially matched until the last frame. These processes provide positional information for target bones in all frames of the 4DCT. Once the coordinate systems of the target bones are decided, translation and rotation angles between any two bones can be calculated. This 4DCT analysis offers advantages in kinematic analyses of complex structures such as carpal or tarsal bones. However, fast or large-scale motions cannot be traced because of motion artifacts.

We analyzed joint kinematics from four-dimensional computed tomography data. The sequential 3D-3D registration method semiautomatically provides the kinematics of the moving bone with respect to the subject bone from four-dimensional computed tomography data.

使用顺序 3D-3D 注册进行四维 CT 分析

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data ...

3D Imaging of Soft-Tissue Samples using an X-ray Specific Staining Method and Nanoscopic Computed Tomography

We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described protocol is easy to apply, fast and suitable for larger soft-tissue samples. The presented methodology enables the characterization of crucial tissue structures in three dimensions and is demonstrated on a whole mouse kidney. The multiscale approach allows to image the entire mouse kidney and supports the selection of further volumes of interest, which are acquired with higher resolutions ranging into the nanometer range. Thereby, soft-tissue morphology with a similar detail level as the corresponding histological light microscopy images is reproduced. Deeper insights into the 3D configuration of tissue structures are achieved without impeding further investigations through histological methods.

A protocol for 3D visualization of microscopic tissue structures by using an X-ray specific staining method designed for X-ray computed tomography is presented.

使用 X 射线特定染色方法和纳米计算机断层扫描对软组织样本进行 3D 成像

We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described ...

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create anatomically accurate head phantoms with realistic brain imaging properties because standard fabrication methods are not optimized to replicate any patient-specific anatomical detail and 3D printing materials are not optimized for imaging properties. In order to test and validate a novel navigation system for use during brain tumor surgery, an anatomically accurate phantom with realistic imaging and mechanical properties was required. Therefore, a phantom was developed using real patient data as input and 3D printing of molds to fabricate a patient-specific head phantom comprising the skull, brain and tumor with both ultrasound and X-ray contrast. The phantom also had mechanical properties that allowed the phantom tissue to be manipulated in a similar manner to how human brain tissue is handled during surgery. The phantom was successfully tested during a surgical simulation in a virtual operating room.
The phantom fabrication method uses commercially available materials and is easy to reproduce. The 3D printing files can be readily shared, and the technique can be adapted to encompass many different types of tumor.

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

患者特异性聚乙烯酒精幻影制造与超声波和X射线对比脑肿瘤手术规划

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create ...

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 ...

Viscoelastic Characterization of Soft Tissue-Mimicking Gelatin Phantoms using Indentation and Magnetic Resonance Elastography

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the biomechanics-related mechanisms of disease, injury, and development. The mechanical testing method is the most straightforward way for tissue characterization and is considered as verification for in vivo measurement. Among the many ex vivo mechanical testing techniques, the indentation test provides a reliable way, especially for samples that are small, hard to fix, and viscoelastic such as brain tissue. Magnetic resonance elastography (MRE) is a clinically used method to measure the biomechanical properties of soft tissues. Based on shear wave propagation in soft tissues recorded using MRE, viscoelastic properties of soft tissues can be estimated in vivo based on wave equation. Here, the viscoelastic properties of gelatin phantoms with two different concentrations were measured by MRE and indentation. The protocols of phantom fabrication, testing, and modulus estimation have been presented.

This article presents a demonstration and summary of protocols of making gelatin phantoms that mimic soft tissues, and the corresponding viscoelastic characterization using indentation and magnetic resonance elastography.