本论文中描述的研究在加拿大光源BMIT设施进行，该设施得到加拿大创新、自然科学和工程研究理事会、萨斯喀彻温大学、萨斯喀彻温省政府、加拿大西部经济多样化、加拿大国家研究理事会和加拿大卫生研究所的支持。作者要感谢加拿大光源公司的光束线科学家，特别是亚当·韦伯、丹尼斯·米勒、谢尔盖·加西洛夫和宁祖在SkyScan SRμCT和白光束显微镜系统的设置和故障排除方面给予的帮助。我们还要感谢托莱多大学医学和生命科学学院的贝丝·达尔泽尔和东北俄亥俄医科大学的杰弗里·温斯特鲁普博士为这项研究获取尸体样本。JM Andronowski通过阿克伦大学和刑事司法法医学国家司法研究与发展研究所提供的启动研究基金（2018-DU-BX-0188）得到支持。RA戴维斯由阿克伦大学提供的研究生助理支持。用于制芯和锯的设备和用品由阿克伦大学提供的启动资金购买，NSF向CW Holyoke提供EAR-1624242。

所有标本均来自托莱多大学、医学和生命科学学院以及东北俄亥俄医科大学（NEOMED）的防腐尸体捐赠者，并征得捐赠者本人或捐赠者近亲的知情同意。阿克伦大学保护人类主体机构审查委员会（IRB）认为，这些标本不受IRB的全面审查，因为它们不是从活体个体中获取的。所有捐助者均可获得人口信息，包括年龄、性别和死因。选定的个人没有记录影响骨骼的情况，也没有接触可能影响死亡时骨重塑的治疗方案。皮质骨骼样本取自19至101岁（平均=73.9岁）的现代男性和女性尸体的粪便。股骨中轴已被广泛研究，包括检查皮质孔隙20、21、22、23、24和骨组织25、26、27的物质密度的变化，因此成为微结构分析的常用场所。
1. 组织采购和捣碎
<ol><li>使用装有暴跌切割硬质合金刀片（用于复合材料）的振荡锯，从左股骨的中度诊断中获取约 7.5 厘米的骨块。</li>
	<li>在45°C的孵化器中，将股骨块浸泡在装有粉状蛋白酶的烤箱安全玻璃盘中，然后用自来水溶液浸泡1小时。</li>
	<li>孵化后，使用钝解剖或牙科工具小心切除剩余的软组织和围产期。 注意:避免使用锋利的工具（如手术刀）去除软组织。此类仪器可能会对 μCT 扫描中检测到的骨骼造成损坏，从而影响标本保存和扫描数据质量。</li>
	<li>将骨块放入超声波清洁器中5-10分钟，用20:1部分自来水清洗溶液（见 材料表），或使用手持式水牙线（例如水皮），以清除甲状腺中的碎屑或遮挡物。</li>
	<li>将骨块浸入标本杯中，并填充 70% 的乙醇。让骨头浸泡至少24小时，以去除脂质。 注:海琳也可以用来去除脂质。然而，长时间浸泡在xylenes中可能会使骨骼变得脆或粉笔，因为它是乳化剂。</li>
	<li>24小时后，从乙醇中去除骨块，在环境温度下进行24-48小时的空气干燥。 注:协议可在此处暂停。</li>
</ol>2. 组织剖腹产
<ol><li>将 75 x 25 mm 玻璃显微镜滑动放在设置为 140 °C 的热板上。 在滑梯中央熔化大量热环氧树脂（见 材料表）。<ol>
<li>如果为显微镜（+lt;50 μm）准备额外的薄块，骨块可能需要嵌入两部分环氧树脂中，以保存腹腔。此外，在实施此协议时，脆弱标本（如糖尿病骨或高度创伤标本）将标本嵌入环氧树脂中是必要的。 注:本协议中使用的骨骼样本是从防腐尸体标本中检索的。如果在验尸时或手术箱中收集新鲜标本，通过 SRμCT 检查软组织结构（例如血管），环氧树脂浸渍可能会诱发此类组织的损伤。在这些情况下，建议使用替代粘合剂或安装介质（例如双面胶带、建模粘土）。</li>
	</ol></li>
	<li>将骨块的劣质部分按入显微镜滑梯上的热环氧树脂中，骨块的长度垂直于滑动。来回移动样品，以便涂覆骨底，并确保与滑梯的安全粘附。</li>
	<li>让安装的标本在热板上休息约5分钟，让热环氧树脂进入毛孔和/或裂缝。 注意:滑梯上的环氧树脂应无气泡，以获得最佳粘附。要去除气泡，请在幻灯片上来回移动样品。气泡通常由于水和/或乙醇被困在骨头内逸出和蒸发而形成。</li>
	<li>使用钝力从热板中取出装有标本的滑梯，并在室温下冷却约 10 分钟。使用剃须刀刀片从滑梯边缘取出任何环氧树脂，以确保夹头充分抓住滑梯。</li>
	<li>将滑梯与粘附的样品连接到玻璃滑块上，并将夹头安装到慢速剖面锯的旋转臂上（见材料表，图 2）。 注:虽然本协议中使用了布勒 IsoMet 锯，但可使用其他精密分节锯来代替 IsoMet（例如，莱科、Exakt、智能切割、CT3、布勒彼得罗辛、井钻石线）。</li>
	<li>使用定位表盘调整旋转臂，以确保刀片触点并转动样本。放置标本，使骨骼的横截面被切成垂直于其长度。</li>
	<li>将重量添加到切割臂的远侧，以对抗手臂的重量。 注意:如果使用配重不足，样品可能会压在刀片上，导致刀片断裂。</li>
	<li>将切割液（20:1 部分水加入切割液）添加到锯的液体容器中。</li>
	<li>紧紧固定金刚石晶圆刀片，确保流体水平淹没刀片的切割部分。将速度设置为 200 RPM，然后慢慢将样品降低到刀片上（图 3）。</li>
	<li>确保刀片和夹头不会摆动和/或弹跳。如果注意到过度运动，请立即停止锯子并拧紧刀片和/或夹头手臂组件，然后再恢复切割。添加额外的配重，如果夹头是积极向上和向下移动。过度运动，包括可见的侧向运动，可能导致刀片断裂。</li>
	<li>第一个厚部分是一个"废物切割"，以提供一个定义明确的表面平行于每个额外的切口。初始废物切割后，抬起旋转臂，使用定位表盘将夹头向刀片移动 5 毫米。进一步厚的部分（约1毫米）显微镜可以进一步收集与此方法。 注:为了保存有价值的组织，可以省略废物削减。然而，当将样品边缘不均匀地分割时，至关重要的是，标本的峰值必须切线排列到芯钻位的边缘。<ol>
<li>分割时一定要考虑到刀片的路缘。例如，要从具有 0.5 mm 的刀片中获取 5 mm 部分，移动样品并将 5.5 mm 朝向刀片。</li>
	</ol></li>
	<li>剖面完成后，将玻璃滑梯与安装的标本放在热板上，以熔化热环氧树脂。这允许从幻灯片中快速移除骨块。 注:协议可在此处暂停。</li>
</ol>3. 样品芯
<ol><li>使用步骤 2.2-2.4 中描述的热环氧结合技术，将 5 毫米骨部分安装到浅铝锡（直径约 8 厘米）底部。</li>
	<li>将锡放在磨钻机的 XY 机器桌上（见材料表）和手紧固定夹（图 4）。</li>
	<li>将 2 毫米内径空心轴珠宝商的钻石尖芯钻头（见 材料表）插入磨钻夹头。调整深度限制器，防止通过锡（图5）进行腐蚀。</li>
	<li>将钻头下方骨样的中央前部对齐，同时避免与围庭、内皮或高度创伤区域密切接触。 注:自动选择前额股骨皮质是不可行的，因为皮质厚度因个体而异，尤其是随着年龄的增长。</li>
	<li>用蒸馏水填充锡，以完全覆盖样品。这可防止热积聚、样品燃烧和/或在腐蚀期间损坏钻头。 注:为了评估腐蚀引起的热损伤的可能性，当腐蚀位首先穿透骨骼表面时，红外温度计用于实现蒸馏水的温度读数。温度变化1°C，从22.9-23.9°C的10个样本核心为这个测试。因此，我们认为热引起的损害可以忽略不计。</li>
	<li>对于核心位和骨骼之间接触的最初几个实例，请施加温和的压力，以便在骨骼的上部表面佩戴戒指。这可防止钻头在制芯过程开始时偏转，并确保钻头正确放置。</li>
	<li>在钻探过程中，将钻头抬入和抬出样品，同时将钻头的尖端保持在水面之下。每隔几秒钟继续此技术，以清除被困的骨尘，并确保碎屑不会遮住钻头。 注意:如果核心正在形成圆锥形，很可能是由于 1） 允许足够的时间从腐蚀位冲洗骨尘，2） 腐蚀发生得太快。提高速度可能会从样品中分离出大块，并粉碎优越的方面。</li>
	<li>结核完成后，产生的骨芯可能卡在空心钻头（图6）中。使用一对细尖的钳子或一个小艾伦扳手将核心从位（图2）中移出。</li>
	<li>将核心样品存放在贴有标签的微中心管中，存放在凉爽干燥的位置，直到成像。</li>
</ol>4. 评估皮质骨芯骨微结构参数的图像处理程序
<ol><li>重建 μCT 图像<ol>
<li>在 https://www.bruker.com/products/microtomography.html 下载并安装最新的 NRecon 版本，用于重建 SRμCT 投影图像。</li>
		<li>选择桌面上的 NRecon 快捷方式，并会显示相关的 GPURecon 服务器。</li>
		<li>在弹出窗口中打开所需的数据集。如果窗口未显示，请选择数据查看器窗口左上角的文件夹图标。</li>
		<li>从 SRμCT 收购中选择第一个投影。在 输出下，删除 使用投资回报率 和 规模打开的选择。</li>
		<li>选择重建文件目的地。选择 浏览 并创建一个名为 Recon的新文件夹。选定的文件格式应为 BMP（8）。</li>
		<li>检查 错位补偿。 注意:此估计通常接近正确。粗糙的 3D 渲染可以通过上下移动箭头来手动调整重叠的图像，以便左右边缘尽可能紧密地对齐。</li>
		<li>在 "设置"下，选择所需的选择以应用 平滑、 光束硬化、CS 旋转、大于 FOV 的对象和环形工事算法。</li>
		<li>通过选择自动调整输出下的直方图。 注意:由此产生的图像可能变暗。</li>
		<li>选择 开始 开始处理重建。</li>
		<li>使用标准命名为运河/骨细胞拉库纳指数28。这些可能包括:总VOI组织体积（电视）、运河体积（Ca.V）、运河总数（Ca.N）、平均运河直径（Ca.Dm）、皮质孔隙度（Ca.V/TV），按百分比给出的、空白总数（N.Lc）和平均拉库纳体积（Lc.V）等。要确定每毫米3 （N.Lc/BV）的拉库纳尔密度，骨量 （BV） 计算为总体积减去运河体积 （TV-Ca.V）。 注:协议可在此处暂停。</li>
	</ol></li>
	<li>从重建的图像收集微结构数据<ol><li>在 https://www.bruker.com/products/microtomography/micro-ct-software/3dsuite.html 下载并安装最新版本的CT分析仪，以分析微结构参数。 注:CT分析器的免费版本功能有限。因此，建议购买完整的许可证，以进行更详细的分析。</li>
		<li>在图像|下属性|更改像素大小，确保像素大小与应用的 μCT 成像协议相匹配。 注:如果在 ImageJ 或类似程序中编辑图像，请注意，保存后，将修改 TIFF 文件中嵌入的标题，分析软件在导入数据集时将更改像素大小。</li>
		<li>选择 自定义处理 以创建任务列表（参见 补充材料）从扫描数据集分析骨骼微结构。使用CT分析仪专有的插件的骨细胞拉库纳网络参数的一般协议如下: 注:插件任务列表适用于样本是 FOV 中唯一可见的主题的数据集。如果标本周围有空空间，则需要应用投资回报率。否则，3D 分析和单个对象分析中收集的值将被人为地降低。<ol><li>在分析软件中打开自定义处理菜单之前，重新加载图像以重置和/或调整任何修改（例如，从 ImageJ 或类似内容中编辑）。</li>
			<li>为了减少图像中的噪音，请在 3D 空间中应用高斯低通滤镜，该滤波器具有圆形内核，半径为 2-3。 注:这些设置通过试验和错误测试应用于所报告的 SRμCT 实验的数据集。目标是为数据获得最高质量的重建。调整重建设置，以适应每个独特的实验设置。</li>
			<li>通过选择低值和高值来突出血管管，将全球灰度阈值应用于图像。 图 8B 和 8D 中看到的重建切片描绘了 0-155 的示例阈值。 注:类似于步骤 4.2.3.2，此处应用的阈值设置是通过广泛的试错选择的。阈值应针对使用的每个实验设置和 μCT 成像系统进行调整。</li>
			<li>去斑点 （除臭）去除3D空间中的白色斑点，这些斑点位于骨细胞漆的体积像素（voxel）大小范围内，以便仅隔离运河。 注:对于以0.9 μm像素大小拍摄的人类皮质骨的SRμCT扫描，骨细胞缺陷的下限为13个voxel。</li>
			<li>去除 2D空间中的任何黑色斑点，以清除运河中的文物。这些在 2D 中可能相当大，从而删除了 <15，000 像素的功能。</li>
			<li>使用 形态学操作 功能，根据图像质量，利用 2 或 3 半径的圆形内核在 3D 空间中展开毛孔，以分离被困在运河中的任何软组织。</li>
			<li>使用与步骤 4.2.3.5 相同的设置执行额外的 脱位 功能。以去除运河内孤立的软组织。</li>
			<li>从步骤 4.2.3.6 中侵蚀除缩。使用具有 2 或 3 半径的圆形内核的 形态学操作 功能。此步骤的半径必须与程序 4.2.3.6 中使用的半径匹配。</li>
			<li>运行 3D 分析 并选择用于计算血管体积的参数。一般来说，基本值将提供足够的信息。</li>
			<li>使用 "保存位图" 将已处理的图像保存到目录中的自定义子折叠器中。 注意:如果使用 Amira/Avizo、Dragonfly、Drishti 等程序从已处理的图像中创建 3D 重建图像，则建议将图像保存为单色（1 位）。</li>
			<li>使用 单个对象分析 功能计算血管管的数量并描述其大小、形状和方向。</li>
			<li>重复步骤4.2.3.1–4.2.3.3。重置骨细胞拉库纳分析的图像。</li>
			<li>使用 Despeckle 功能去除 3D 空间中的白色斑点，确保此类工件小于拉库纳尺寸的下限。此步骤可消除扫描中看似皮质毛孔的噪音，同时保留真正的骨细胞缺陷。对于 0.9 μm 像素大小的人类 SRμCT 扫描，此下限为 13 个 voxel。</li>
			<li>再次去除大于拉库纳大小上限的白色斑点。对于具有步骤 4.2.3.13 中列出的设置的人类 SRμCT 数据集，此限制为 2743 voxels。</li>
			<li>执行 3D 分析 ，以提取与骨细胞缺陷相关的微观结构信息。</li>
			<li>选择 "保存位图 "以保存已处理的图像，以便隔离骨细胞缺陷。</li>
			<li>执行 单个对象分析 ，以在选定的兴趣量 （VOI） 中计算 3D 中的骨细胞数量。 注:任务列表建立并测试后，CT分析器具有批量管理器（BatMan）功能，可用于加速数据提取并确保图像处理均匀。具有程序 4.2.3 示例设置的任务列表。可以在 补充材料中找到。</li>
		</ol></li>
	</ol></li>
</ol>

<ol>
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作者没有什么可透露的。

对于采购具有有限 FOV 设置的高分辨率 SRμCT 成像的均匀和圆柱形皮质骨芯样品，尚未制定全面、标准化的协议。此处详细的协议通过提供有关如何为 SRμCT 成像采购一致大小的皮质骨芯样本以及随后精确可视化和提取微结构数据的全面教程来填补这一空白。我们已经表明，我们的协议提供了一个更标准化和可靠的方法来采购皮质骨芯比以前描述的截断直线骨块的任意尺寸。因此，依靠手持式旋转?...

随着成像技术的最新发展，现在以极高的分辨率获取X射线成像数据是可行的。桌面微CT（μCT）系统是目前成像取消骨骼的标准，由于其无损性质1.然而，当皮质骨的显微结构特征成像时，μCT的使用更加有限。由于分辨率限制，桌面系统无法达到比皮质孔隙小的显微结构特征（如骨细胞孔）所需的分辨率。对于此应用，SRμCT 是理想的，因为这些系统的分辨率更高1。例如，加拿大光源 （CLS） 生物医学成像和治疗 （BMIT） 光束线2上的实验产生了体积小至 0.9 μm 的图像。先前的研究1，3，4，5已经利用这个分辨率从人类长骨皮质骨标本（图1）获得投影和随后的三维（3D）渲染，以量化骨细胞拉库纳尔密度4，6，7，8，9和变化的拉库纳尔形状和大小3在整个人类寿命和性别之间。进一步的研究已经证明，在人类10中存在奥斯丁带，这种现象以前在法医人类学文献中只与非人类哺乳动物有关。
为了实现超常的分辨率，X 射线束必须在视野 （FOV） 内进行精细聚焦，这通常将最大标本大小限制在直径几毫米以内。目前，文献中没有描述符合这些限制的骨样采购的全面、标准化的程序。FOV 中的中心标本对于确保 1） 样本在成像过程中旋转 180° 时保持中心位置至关重要，2） 扫描伪影因有限，因为没有图像截断。换句话说，FOV 以外的样品没有部分干扰光束进入 FOV 内部的焦点。如果发生这种情况，重建算法将被剥夺完全正确的重建所需的一些衰减数据。还值得注意的是，360°（全旋转）扫描将光束硬化的影响降至最低，但增加了成像过程中因错位和样品移动而引起的工件。因此，虽然 360° 扫描通常会生成更清洁的数据，但成像时间会翻倍，因此必须解决实验成本和数据质量之间的妥协问题。
骨成像实验的一个重要和经常被忽视的方面是在扫描前进行的准确和可复制的标本准备技术。将SRμCT方法纳入实验的研究简要地提到了他们的采样方案，但作者对收集标本的特定方法几乎没有提供细节。许多此类研究提到任意尺寸的直线骨块切割，但一般不提供有关使用3、4、10、11、12、13、14的工具或嵌入材料的进一步信息。一些研究人员通常使用手持式旋转工具（如Dremel），从感兴趣的区域（ROI）3、4、10、11、12、13、14中去除直线骨块。此方法可产生可能大于 FOV 的非统一大小的样本，增加了扫描工件和图像截断的可能性。此类标本通常需要使用精密的金刚石晶圆锯（例如布勒伊索梅特）进行进一步精炼。采购尺寸一致的样品（达到百分之二百/毫米）对于确保所获取的数据集质量最高且后续结果可复制至关重要。
样本采购方法的有限报告在尝试使用和/或验证先前研究中执行的方法时增加了一层额外的困难。目前，研究人员必须直接与作者联系，了解其取样程序的更多细节。此处详述的协议为生物医学研究人员提供了一种经过全面记录、可复制且经济高效的采样技术。本文的主要目标是提供一个全面的教程，说明如何使用磨钻压机和金刚石芯位采购大小一致的皮质骨芯样品，从而准确可视化和提取微结构数据。这种方法是从高压岩石力学15、16、17、18、19的硬材料块中常规收集均匀小直径（1-5毫米）圆柱体的程序中修改的。

<table><tbody><tr><td>1-1/8&quot; plunge cutting carbide for composites</td><td>Warrior</td><td>61812</td><td>28.6mm plunge</td></tr><tr><td>70% Ethanol</td><td>Fisher Scientific</td><td>BP8201500</td><td>3.8 Liters</td></tr><tr><td>Blunt-tipped forceps</td><td>Fisher Scientific</td><td>10-300</td><td></td></tr><tr><td>Centrifuge tubes</td><td>ThermoFisher</td><td>55398</td><td></td></tr><tr><td>Crystalbond 509-3 Epoxy</td><td>Ted Pella</td><td>821-3</td><td></td></tr><tr><td>CTAnalyser</td><td>Bruker microCT</td><td>v.1.15.4.0</td><td>Download and install at https://www.bruker.com/products/microtomography/micro-ct-software/3dsuite.html</td></tr><tr><td>Dental Tool Kit</td><td>Amazon</td><td>787269885110</td><td></td></tr><tr><td>Diamond wafering saw blade for composite material</td><td>Buehler</td><td>#11-4247</td><td></td></tr><tr><td>Drill Press</td><td>Jet Mill/Drill</td><td>350017</td><td>Model: JMD-15, benchtop drill presses are suitable substites, but typically lack a translatable machine table for positioning samples beneath the drill stem</td></tr><tr><td>Fine-tipped forceps</td><td>Fisher Scientific</td><td>22-327379</td><td></td></tr><tr><td>Fixturing clamps for XY machine table for mill/drill</td><td>MSC Industrial Supply</td><td>#04804571</td><td></td></tr><tr><td>Glass microscope slides</td><td>Ted Pella</td><td>26005</td><td>75x50mm slides, 1mm thick</td></tr><tr><td>Glass slide chuck</td><td>Buehler</td><td>#112488</td><td>Large enough to hold 75x50mm glass slides</td></tr><tr><td>Hot plate capable of reaching 140 &amp;deg;C</td><td>ThermoScientific</td><td>HP88850105</td><td></td></tr><tr><td>Incubator</td><td>NAPCO</td><td>Model 4200</td><td></td></tr><tr><td>Isocut Fluid</td><td>Buehler</td><td>111193032</td><td>Lubricant; 30mL</td></tr><tr><td>Jeweler&#039;s diamond coring drill bit</td><td>Otto Frei</td><td>#119.050</td><td>2mm inner diameter hollow stem coring bit</td></tr><tr><td>NRecon</td><td>Bruker microCT</td><td>v.1.6.10.2</td><td>Download and install at https://www.bruker.com/products/microtomography.html</td></tr><tr><td>Oscillating saw</td><td>Harbor Freight</td><td>62866</td><td></td></tr><tr><td>Oven-safe glass dishes</td><td>Pyrex</td><td>1117715</td><td>Glass food storage container</td></tr><tr><td>Precision slow-speed saw (Isomet 1000)</td><td>Buehler</td><td>111280160</td><td></td></tr><tr><td>Razor blades</td><td>Amazon</td><td>25181</td><td></td></tr><tr><td>Shallow aluminum tins</td><td>Amazon</td><td>B01MRWLD0R</td><td>~8cm diameter</td></tr><tr><td>Specimen cups</td><td>Amazon</td><td>616784425436 885334344729</td><td></td></tr><tr><td>Tergazyme detergent</td><td>Alconox</td><td>1304-1</td><td>1.8kg box</td></tr><tr><td>Ultrasonic cleaner</td><td>MTI Corporation</td><td>KJ201508006</td><td></td></tr></tbody></table>

a sectioning, coring, and image processing guide for high-throughput cortical bone sample procurement and analysis for synchrotron micro-ct

骨骼是一种动态和机械活性组织，在人的寿命中改变结构。骨骼重塑工艺的产品已经使用传统的二维技术进行了大量研究。通过桌面微计算断层扫描 （μCT） 和同步加速器辐射微计算断层扫描 （SRμCT） 的 X 射线成像技术的最新进展，使得获得比其他 3D 成像技术（例如 SEM） 更大的视野 （FOV） 高分辨率三维 （FOV） 扫描能够更完整地了解人类皮质骨骼内的微观结构。但是，标本应准确以 FOV 为中心，以限制已知影响数据分析的条纹伪影的外观。先前的研究已经报告采购了不规则形状的直线骨块，这些骨块由于边缘不均匀或图像截断而导致成像。我们应用了地质采样协议（科林），从人类粪便的前部获取一致大小的皮质骨芯标本，用于SRμCT实验。这种腐蚀方法效率高，对组织的破坏力极小。它创建均匀的圆柱形样品，可减少在旋转过程中的等轴测量性质的成像伪影物，并在整个扫描过程中为 X 射线束提供均匀的路径长度。对核心和不规则形状样品的X射线断层图像数据的图像处理证实了该技术改进皮质骨微构造的可视化和分析的潜力。该协议的目标是提供一种可靠且可重复的方法，用于提取皮质骨芯，该方法可适应各种类型的高分辨率骨成像实验。这项工作的首要目标是为 SRμCT 创建一个经济实惠、一致且简单明了的标准化皮质骨骼采购。相关领域的研究人员可以进一步调整这一程序，他们通常评估硬复合材料，如生物人类学、地球科学或材料科学。

所述的核心取样方法被证明是高效和高效的。使用此协议的科林标本允许在 CLS BMIT-BM 光束线2上采购 +300 一致大小的样品，FOV 为 +2 mm，尺寸为 1.49μm voxel。为了验证核心直径的一致性，沿人类前股骨核心子集（n=69）的长度（顶部、中间、底部）进行了三次测量。核心的平均直径为1.96±0.11毫米， 核心长度的平均变薄为0.06±0.06毫米/毫米为了强调其他硬复合材料的适用性，我...

Watch this Scientific Journal Video about A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT at JoVE.com

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

我们采用了地质（科灵）采样方案，从人类粪便的前部获取均匀大小的皮质骨标本，用于SRμCT实验。这种方法具有极小的破坏性、有效性，可产生圆柱形标本，最大限度地减少不规则样本形状的成像伪影物，并改进微结构可视化和分析。

同步加速器微CT高通量皮质骨样品采购和分析的剖面、科林和图像处理指南

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

a-sectioning-coring-image-processing-guide-for-high-throughput

Research

JoVE Journal

Biology

5.0K 次观看.  The University of Akron. 我们采用了地质（科灵）采样方案，从人类粪便的前部获取均匀大小的皮质骨标本，用于SRμCT实验。这种方法具有极小的破坏性、有效性，可产生圆柱形标本，最大限度地减少不规则样本形状的成像伪影物，并改进微结构可视化和分析。

视频: A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

科研

生物工程

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

造血干细胞的自动定量 - 在脱钙骨的组织学图像间质细胞的相互作用

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

发育生物学

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.

Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron tomography.

使用双光束扫描电子显微镜从玻璃体生物样品制备薄片进行冷冻电子断层扫描

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

生物化学

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different bone loss conditions. However, the standard approach for trabecular analysis of micro-CT images is slice-by-slice semi-automatic hand-contouring, which is labor intensive and error prone. Described here is an efficient method for automatic segmentation of trabecular bones according to the bone's outer boundaries, where trabecular bones can be identified and segmented automatically with accuracy with less operator bias when appropriate segmentation parameters are set. To profile satisfactory segmentation parameters, an image stack of segmentation results is displayed, where all possible combinations of the segmentation parameters are changed one by one in sequence, and segmentation results with associated parameters can easily be visually checked. As a quality-control feature of the plugin, simulated standard objects are quantified where the measured quantities can be compared with theoretical values. Layer-by-layer quantification of trabecular properties and trabecular thicknesses are reported by such a plugin, and the distributions of such properties within the selected regions can be profiled easily. Although layer-by-layer quantification retains more information about trabecular bones and facilitates further statistical analysis of structural changes, such measures are unavailable from the output of current commercial software, where only a single quantified value for each parameter is reported for each sample. Therefore, the described workflows are better approaches for analyzing trabecular bones with accuracy and efficiency.

We present a workflow for segmenting and quantifying trabecular bones for 2D and 3D images based on the bone&#39;s outer boundary using an ImageJ plugin. This approach is more efficient and accurate than the current manual hand-contouring approach, and provides layer-by-layer quantifications, which are not available in current commercial software.

用 Imagej 插件对小梁骨的外边界辅助分割和量化

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...

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 ...

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

一种基于微 ct 的小动物脑损伤和定位电极表征方法

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

神经科学

An Unbiased Approach of Sampling TEM Sections in Neuroscience

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative studies of the changes in densities and distributions of such features, an unbiased sampling protocol is vital for reliable results. Here, we present a workflow for the image acquisition of brain samples. The workflow allows systematic uniform random sampling within a defined brain region, and the images can be analyzed using a disector. This technique is much faster than extensive examination of serial sections but still presents a feasible approach to estimate the densities and distributions of ultrastructure features. Before embedding, stained vibratome sections were used as a reference to identify the brain region under investigation, which helped speed up the overall specimen preparation process. This approach was used for comparative studies investigating the effect of an enriched-housing environment on several ultrastructural parameters in the mouse brain. Based on the successful use of the workflow, we adapted it for the purpose of elemental analysis of brain samples. We optimized the protocol in terms of the time of user-interaction. Automating all the time-consuming steps by compiling a script for the open source software SerialEM helps the user to focus on the main work of acquiring the elemental maps. As in the original workflow, we paid attention to the unbiased sampling approach to guarantee reliable results.

We introduce a novel workflow for electron microscopy investigations of brain tissue. The method allows the user to examine neuronal features in an unbiased fashion. For elemental analysis, we also present a script that automatizes most of the workflow for randomized sampling.

神经科学中透射电镜切片采样的一种无偏方法

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative ...

骨折易感性机械测试的原则和方案

Practical Considerations for the Design, Execution, and Interpretation of Studies Involving Whole-Bone Bending Tests of Rodent Bones

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care costs. The ability to understand the mechanisms underlying bone disease and the response to treatment is not only desired, but critical. Mechanical testing of bone serves as a valuable technique for understanding and quantifying a bone&#39;s susceptibility to fracture. While this method appears simple to perform, inappropriate and inaccurate conclusions may be reached if governing assumptions and key steps are disregarded by the user. This has been observed across disciplines as studies continue to be published with misuse of methods and incorrect interpretation of results. This protocol will serve as a primer for the principles associated with mechanical testing along with the application of these techniques-from considerations of sample size through tissue harvesting and storage, to data analysis and interpretation. With this in hand, valuable information regarding a bone&#39;s susceptibility to fracture may be obtained, furthering understanding for both academic research and clinical solutions.

作者聚焦：提高全骨弯曲测试的准确性和可重复性 

Mechanical testing of rodent bones is a valuable method for extracting information regarding a bone's susceptibility to fracture. Lacking proper practical understanding, the results may be overinterpreted or lack validity. This protocol will serve as a guide to ensure mechanical tests are performed accurately to provide valid and functional data.

设计、执行和解释涉及啮齿动物骨骼全骨弯曲试验的研究的实际考虑因素

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care ...

同步加速器微CT高通量皮质骨样品采购和分析的剖面、科林和图像处理指南

摘要

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此视频中的章节

高分辨率三维成像 EX - VIVO生物样品显微CT

造血干细胞的自动定量 - 在脱钙骨的组织学图像间质细胞的相互作用

使用双光束扫描电子显微镜从玻璃体生物样品制备薄片进行冷冻电子断层扫描

用 Imagej 插件对小梁骨的外边界辅助分割和量化

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

一种基于微 ct 的小动物脑损伤和定位电极表征方法

神经科学中透射电镜切片采样的一种无偏方法

设计、执行和解释涉及啮齿动物骨骼全骨弯曲试验的研究的实际考虑因素

设计、执行和解释涉及啮齿动物骨骼全骨弯曲试验的研究的实际考虑因素

设计、执行和解释涉及啮齿动物骨骼全骨弯曲试验的研究的实际考虑因素