Name: use of micro x-ray computed tomography with phosphotungstic acid preparation to visualize human fibromuscular tissue
Uploaded: 2019-09-05T14:30:00
Description: Watch this Scientific Journal Video about Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue at JoVE.com

这项研究得到了延世大学医学院（6-2018-0099）的教师研究资助。作者感谢那些慷慨地将遗体捐献给延世大学医学院的人们。我们感谢金正恩和钟浩邦（延世大学医学院外科解剖教育中心的工作人员）的技术支持。我们也感谢吉诺斯有限公司在这项研究中使用的高质量微CT扫描系统。

这项研究中使用的所有尸体都合法地捐赠给延世大学医学院的外科解剖教育中心。
1. 获取样品
<ol><li>用彩色铅笔在尸体上画一条切口线，以指示样品采集的切割区域。检查绘制的切口线是否从中直延伸到中侧，横向延伸到侧眼睑，优于下眼睑的上边缘，下至轨道边缘线下方 1 厘米。 注：根据微型CT设备的最大扫描尺寸考虑样本大小（我们的设备可以获取最大对象尺寸为7×...

<ol>
	<li>Nierenberger, M., Remond, Y., Ahzi, S., Choquet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+three-dimensional+collagen+network+in+soft+tissues+using+contrast+agents+and+high+resolution+micro-CT:+Application+to+porcine+iliac+veins.">Assessing the three-dimensional collagen network in soft tissues using contrast agents and high resolution micro-CT: Application to porcine iliac veins.</a> Comptes Rendus Biologies. 338 (7), 425-433 (2015).</li><li>Vymazalov&#225;, K., Vargov&#225;, L., Zikmund, T., Kaiser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+possibilities+of+studying+human+embryos+and+foetuses+using+micro-CT:+a+technical+note.">The possibilities of studying human embryos and foetuses using micro-CT: a technical note.</a> Anatomical Science International. 92 (2), 299-303 (2017).</li><li>Tesa&#345;ov&#225;, M., 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=Use+of+micro+computed-tomography+and+3D+printing+for+reverse+engineering+of+mouse+embryo+nasal+capsule.">Use of micro computed-tomography and 3D printing for reverse engineering of mouse embryo nasal capsule.</a> Journal of Instrumentation. 11 (3), 1-11 (2016).</li><li>Nemetschek, T., Riedl, H., Jonak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topochemistry+of+the+binding+of+phosphotungstic+acid+to+collagen.">Topochemistry of the binding of phosphotungstic acid to collagen.</a> Journal of Molecular Biology. 133 (1), 67-83 (1979).</li><li>Rao, R. N., Fallman, P. M., Falls, D. G., Meloan, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+PAS-phosphotungstic+acid-Diamine+Supra+Blue+FGL+and+immunological+reactions+for+type+I+collagen.">A comparative study of PAS-phosphotungstic acid-Diamine Supra Blue FGL and immunological reactions for type I collagen.</a> Histochemistry. 91 (4), 283-289 (1989).</li><li>Metscher, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+for+comparative+morphology:+simple+staining+methods+allow+high-contrast+3D+imaging+of+diverse+non-mineralized+animal+tissues.">MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues.</a> BMC Physiology. 9 (11), (2009).</li><li>Metscher, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+for+Developmental+Biology:+A+Versatile+Tool+for+High-Contrast+3D+Imaging+at+Histological+Resolutions.">MicroCT for Developmental Biology: A Versatile Tool for High-Contrast 3D Imaging at Histological Resolutions.</a> Developmental Dynamics. 238 (3), 632-640 (2009).</li><li>Nieminen, H. 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=Determining+collagen+distribution+in+articular+cartilage+using+contrastenhanced+micro-computed+tomography.">Determining collagen distribution in articular cartilage using contrastenhanced micro-computed tomography.</a> Osteoarthritis Cartilage. 23 (9), 1613-1621 (2015).</li><li>Kwon, O. J., Kwon, H., Choi, Y., Cho, T., Yang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structure+of+the+orbicularis+retaining+ligament:+an+anatomical+study+using+micro+computed+tomography.">Three-dimensional structure of the orbicularis retaining ligament: an anatomical study using micro computed tomography.</a> Scientific Reports. 8 (1), 17042 (2018).</li><li>Dullin, C., 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=&#956;CT+of+ex-vivo+stained+mouse+hearts+and+embryos+enables+a+precise+match+between+3D+virtual+histology,+classical+histology+and+immunochemistry.">&#956;CT of ex-vivo stained mouse hearts and embryos enables a precise match between 3D virtual histology, classical histology and immunochemistry.</a> PLoS One. 12 (2), e0170597 (2017).</li><li>Zikmund, T., 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=High-contrast+differentiation+resolution+3D+imaging+of+rodent+brain+by+X-ray+computed+microtomography.">High-contrast differentiation resolution 3D imaging of rodent brain by X-ray computed microtomography.</a> Journal of Instrumentation. 13 (2), 1-12 (2018).</li><li>Anderson, R., Maga, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+procedure+for+rapid+imaging+of+adult+mouse+brains+with+MicroCT+using+iodine-based+contrast.">A novel procedure for rapid imaging of adult mouse brains with MicroCT using iodine-based contrast.</a> PLoS One. 10 (11), e0142974 (2015).</li><li>Nieminen, H. 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=3D+histopathological+grading+of+osteochondral+tissue+using+contrast-enhanced+micro-computed+tomography.">3D histopathological grading of osteochondral tissue using contrast-enhanced micro-computed tomography.</a> Osteoarthritis Cartilage. 26 (8), 1118-1126 (2018).</li><li>Greef, D. D., Buytaert, J. A. N., Aerts, J. R. M., Hoorebeke, L. V., Dierick, M., Dirckx, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Details+of+Human+Middle+Ear+Morphology+Based+on+Micro-CT+Imaging+of+Phosphotungstic+Acid+Stained+Samples.">Details of Human Middle Ear Morphology Based on Micro-CT Imaging of Phosphotungstic Acid Stained Samples.</a> Journal of Morphology. 276 (9), 1025-1046 (2015).</li><li>Sutter, S., 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=Contrast-Enhanced+Microtomographic+Characterisation+of+Vessels+in+Native+Bone+and+Engineered+Vascularised+Grafts+Using+Ink-Gelatin+Perfusion+and+Phosphotungstic+Acid.">Contrast-Enhanced Microtomographic Characterisation of Vessels in Native Bone and Engineered Vascularised Grafts Using Ink-Gelatin Perfusion and Phosphotungstic Acid.</a> Contrast Media &amp; Molecular Imaging. 2017, (2017).</li></ol>

我们在人体软组织的检查中采用PTA制剂实施微CT。简单地说，标本被收获并固定在形式上几天，随后在连续乙醇溶液中脱水。在正式固定后将样品直接放入PTA溶液中，由于快速脱水，可能导致一些组织破裂。因此，在PTA染色之前需要连续脱水。接下来，使用 PTA 溶液对样品进行大约一周的染色。然后可以执行微CT扫描、3D重建和分析。我们的目标是使用此方法观察 ORL 和相邻结构。我们成功地将组织...

人工解剖和组织学观察通常用于检查人体组织，如肌肉和结缔组织。然而，人工解剖很容易损坏精细的结构，组织学观察提供了关于平面横截面表面1，2的信息有限。因此，需要改进的方法，以更精确和有效地检查组织。
传统的计算机断层扫描（CT）一般用于临床实践，但它缺乏区分小结构2，3的能力。微X射线CT（微CT）是一种有效的工具，用于在不破坏小结构的情况下获取小结构的三维（3D）信息。然而，微CT的应用有限，因为只有密集的组织才能清晰地可视化;它不能用于区分软组织。为了克服这一限制，可以使用染色剂。在扫描4、5期间，磷酸（PTA）、磷酸、磷酸和卢戈尔碘等增反剂可提高软组织对比度。比较这些制剂的几项研究表明，PTA表现出良好的性能，并且易于处理6，7，8。
食道保持韧带（ORL）是轨道周围的一个微妙的结构，在常规观测9中很容易损坏。我们使用带有PTA作为对比剂的微CT，检查并成功检索了该结构的3D信息。该方法可应用于其他人体组织的研究，如心脏和肝脏，并适当修改10，11，12。

<table>
	<tbody>
		<tr>
			<td>12 Tungsto(&#8549;)phosphoric acid n-hydrate 
			Phosphotungstic acid</td>
			<td>Junsei</td>
			<td>84220-0410</td>
			<td>PTA powder</td>
		</tr>
		<tr>
			<td>CTvox</td>
			<td>Bruker</td>
			<td>ver 2.7</td>
			<td>3D recon software</td>
		</tr>
		<tr>
			<td>Nrecon</td>
			<td>Bruker</td>
			<td>ver 1.7.0.4</td>
			<td>Reconstruction software</td>
		</tr>
		<tr>
			<td>Skyscan</td>
			<td>Bruker</td>
			<td>1173</td>
			<td>MicroCT scanner</td>
		</tr>
		<tr>
			<td>Tconv</td>
			<td>Bruker</td>
			<td>ver 2.0</td>
			<td>File resizing software</td>
		</tr>
	</tbody>
</table>

use of micro x-ray computed tomography with phosphotungstic acid preparation to visualize human fibromuscular tissue

人工解剖和组织学观察是研究人体组织的常见方法。然而，人工解剖会损坏精细结构，而处理和组织学观察通过横截面成像提供有限的信息。微 X 射线计算机断层扫描 （microCT） 是一种在不损坏标本的情况下获取三维信息的有效工具。然而，它显示区分软组织部分的效率有限。使用抗对比度剂，如磷酸（PTA），可以通过改善软组织对比度来解决这个问题。我们使用PTA实施微CT来研究人类双目体保持韧带（ORL），这是轨道区域的微妙结构。在这种方法中，收获的试样固定在形式素中，在串醇溶液中脱水，并沾染PTA溶液。染色后，进行微CT扫描、3D重建和分析。使用这种方法可以清楚地显示皮肤、韧带和肌肉。试样大小和染色持续时间是该方法的基本特征。合适的试样厚度约为5~7毫米，超过其工艺速度减慢，最佳持续时间为5~7天，低于该厚度时，中心区域偶尔会出现空孔。为了保持切割过程中小件的位置和方向，建议在每个零件的相同区域进行缝纫。此外，需要对解剖结构进行初步分析，以正确识别每一块。帕拉膜可用于防止干燥，但应注意防止样品变形。我们的多向观察表明，ORL是由连续板的多层网状结构，而不是如前所述的线状纤维组成。这些结果表明，使用PTA进行微CT扫描有助于检查人体组织复杂结构内的特定隔间。它可能有助于分析癌症组织、神经组织和各种器官，如心脏和肝脏。

ORL的详细重建是通过使用PTA制备的微CT实现的（图4）。明显观察到了皮肤和骨膜之间倾斜的韧带纤维肌结构（图4A）。在日冕视图中（图4B），纤维的数量和复杂性横向增加。在水平视图（图4C）中，观察到了具有树形的精细网状物。我们观察到一种形状，其特征是连续板，而不是螺纹状纤维，如前所述。在?...

Watch this Scientific Journal Video about Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue at JoVE.com

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

微型 X 射线计算机断层扫描能有效地从未受损的人体标本中获取三维信息，但在观察软组织方面成效有限。使用磷酸对比剂可以解决此问题。我们实施了这种造影剂来检查人类脆弱的纤维肌组织（双眼保留韧带）。

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 ...

我们实施带磷酸的微CT，以增强人体球状保持韧带的非侵入性研究中的软组织对比度，这是轨道区域内的一种微妙结构。该协议经过优化，有助于研究人员进行人体软组织的非侵入性研究，并且操作简单，费用合理。要获取样品，请先使用彩色铅笔指示样品采集时皮肤上的切割区域。确认标记线在医疗上延伸到中层校园，横向延伸至横向校园，优于下眼睑的优越边界，低于轨道边缘线以下一厘米。接下来，使用刀片沿标记线切割面部组织，确保切口足够深，使刀片尖端接触骨骼。收获的样品必须包括皮肤、皮下组织、肌肉、脂肪和周分。采集后，立即将样品在室温下固定为10%甲醛5至7天。在固定期结束时，将样品切成三块五到七毫米厚。使用针和黑线缝制每个样品的超边，以便可以在以后的点确定样品的方向。将样品在上升乙醇浓度系列中脱水一天，将样品留在70%乙醇溶液中，直到染色。在微机断层扫描计划前一周，将 PTA 加入 2.1 克至 210 毫升 70%乙醇，并将溶液以每分钟 55 至 60 次旋转的速度放在摇床上。准备三个70毫升的塑料容器，每个切片片。当染料溶解到酒精溶液中时，用溶液填充容器。将一个样品放入每个 1%PTA 的容器中，每分钟 55 至 60 次旋转摇动样品 5 到 7 天。染色完成后，将样品储存在新鲜 70%乙醇中一周，以准备扫描。对于 microCT 扫描，请将样品包装在 Parafilm 中以防止干燥，然后将样品放在扫描仪托盘上。将扫描仪的源电压设置为70千伏，源电流设置为114微安，铝滤片设置为0.5毫米，图像像素大小设置为20平方微米，像素设置为2，240乘2，曝光到500毫秒，旋转步长为0.3度。然后，启动扫描。扫描完成后，打开重建软件并选择"操作"和"打开数据集"以启动扫描的文件。选择"设置"选项卡，将环伪影缩减为 7 和光束硬化校正。要开始重建，请选择"开始"。最终数据将存储在指定的文件夹中。在重建结束时，打开文件调整大小软件，然后选择源数据集以启动重建的文件。在"目标数据集"选项卡上选择 jpg，然后使用"无插值"选项选择 1/2 调整大小选项，然后在"图像压缩"选项卡中将幻灯片栏调整为 100 并开始转换。对于 3D 重建，打开 3D 卷渲染软件，然后选择"操作"和"加载音量数据"。要调整亮度和对比度级别，请修改"传输功能编辑器"选项卡中的直方图中的形状传输功能。接下来选择"选项"和"照明"，然后选择"阴影"和"曲面照明"图标，以实现逼真的建模色调。单击并拖动，右键单击和拖动以移动和旋转 3D 图像以查找最佳视图，滚动以根据需要放大或缩小。单击"移位"并拖动以滑动位置以查看剖面图像。打开"灯光"图标并调整照明指示栏以查找最佳照明以进行查看，然后关闭"灯光"图标并关闭"照明"选项卡。在"选项"选项卡中，选择"显示"和"剪切框"以隐藏最终图像的框。然后，选择"操作"和"保存图像"以存储图像。ORL 的这种详细重建是通过微CT实现的，如证明的PTA制剂，允许可视化韧带纤维肌肉结构斜向于真皮和周密之间延伸。在日冕视图中，纤维的数量和复杂性横向增加。在水平视图中，观察到与树化地层精心设计的网格。在下垂视图中，ORL 纤维的厚度减小率低。总体而言，这种多向观测证明，ORL是由连续板的多层网格组成，根据光纤的位置，其数量和厚度的变化。与试样体积相比，染色持续时间不够时，最终图像可能包括试样中心区域的空孔。PTA 旁边的其他代理可以使用，不同的代理具有不同的优点或染色功能的兴趣。因此，根据实验优化染色区域是很有用的。

Research

JoVE Journal

Bioengineering

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Micropatterned表面透明质酸与肿瘤细胞相互作用的研究

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

科研

生物工程

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

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 ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

人类心脏静脉系统使用对比计算机断层扫描灌注固定的标本解剖重建

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

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

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

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 ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

计算机断层扫描和成骨血管生成耦合光学成像评估头盖骨自体移植和移植物的整合

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

在心肌结构，功能的体内定量评估，灌注与活力使用心脏微型计算机断层扫描

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

应用活体显微计算机断层成像技术对骨微结构高分辨成像大鼠后肢的定位与抑制

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

微流控装置中组织模型的 Perfusable 血管网

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

含脂肪酸脂质体的制备、纯化及应用

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...