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 border="0" cellpadding="0" cellspacing="0" style="width:1367px;" width="1367">
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			<td height="42" style="height:42px;width:215px;">1-1/8&#34; plunge cutting carbide for composites</td>
			<td style="width:120px;">Warrior</td>
			<td style="width:133px;">61812</td>
			<td style="width:899px;">28.6mm plunge</td>
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			<td height="21" style="height:21px;width:215px;">70% Ethanol</td>
			<td style="width:120px;">Fisher Scientific</td>
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			<td height="21" style="height:21px;width:215px;">Blunt-tipped forceps</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:133px;">10-300</td>
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			<td height="21" style="height:21px;width:215px;">Centrifuge tubes</td>
			<td style="width:120px;">ThermoFisher</td>
			<td style="width:133px;">55398</td>
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			<td height="21" style="height:21px;width:215px;">Crystalbond 509-3 Epoxy</td>
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			<td style="width:133px;">821-3</td>
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			<td height="21" style="height:21px;width:215px;">CTAnalyser</td>
			<td style="width:120px;">Bruker microCT</td>
			<td style="width:133px;">v.1.15.4.0</td>
			<td>Download and install at https://www.bruker.com/products/microtomography/micro-ct-software/3dsuite.html</td>
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			<td height="21" style="height:21px;width:215px;">Dental Tool Kit</td>
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			<td height="42" style="height:42px;width:215px;">Diamond wafering saw blade for composite material</td>
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			<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>
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			<td height="42" style="height:42px;width:215px;">Fixturing clamps for XY machine table for mill/drill</td>
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			<td height="21" style="height:21px;width:215px;">Glass microscope slides</td>
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			<td style="width:133px;">26005</td>
			<td>75x50mm slides, 1mm thick</td>
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			<td height="21" style="height:21px;width:215px;">Glass slide chuck</td>
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			<td>Large enough to hold 75x50mm glass slides</td>
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			<td height="42" style="height:42px;width:215px;">Hot plate capable of reaching 140 &#176;C</td>
			<td style="width:120px;">ThermoScientific</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Incubator</td>
			<td style="width:120px;">NAPCO</td>
			<td>Model 4200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Isocut Fluid</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">111193032</td>
			<td>Lubricant; 30mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Jeweler&#39;s diamond coring drill bit</td>
			<td style="width:120px;">Otto Frei</td>
			<td style="width:133px;">#119.050</td>
			<td>2mm inner diameter hollow stem coring bit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NRecon</td>
			<td style="width:120px;">Bruker microCT</td>
			<td style="width:133px;">v.1.6.10.2</td>
			<td>Download and install at https://www.bruker.com/products/microtomography.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Oscillating saw</td>
			<td style="width:120px;">Harbor Freight</td>
			<td style="width:133px;">62866</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Oven-safe glass dishes</td>
			<td style="width:120px;">Pyrex</td>
			<td style="width:133px;">1117715</td>
			<td>Glass food storage container</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Precision slow-speed saw (Isomet 1000)</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">111280160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Razor blades</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">25181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Shallow aluminum tins</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">B01MRWLD0R</td>
			<td>~8cm diameter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Specimen cups</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">616784425436 885334344729</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Tergazyme detergent</td>
			<td style="width:120px;">Alconox</td>
			<td style="width:133px;">1304-1</td>
			<td>1.8kg box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ultrasonic cleaner</td>
			<td style="width:120px;">MTI Corporation</td>
			<td style="width:133px;">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

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

一个Biomining从宏基因组库的纤维素酶的活性高通量筛选

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

科研

生物学

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

成人的制备果蝇</em薄切片和显微镜分析>的眼睛

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

实用指南系统发育的非专家

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

肿瘤细胞球体的高通量图像分析:一个用户友好的软件应用来测量球体的大小自动准确

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

样品制备质谱术分析

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

高通量，矿化组织的多图像Cryohistology

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...