作者想感谢梅奥诊所的材料和结构测试核心, 他们在进行骨折测试时的技术支持。此外, 我们还要感谢拉梅什 Raghupathy 和 Ian Gerstel 在梅奥诊所任职期间协助编写 dic 方案的剧本和具体细节, 以及明尼苏达大学的维克多 Barocas 研究小组底层开源软件, 它执行数字图像相关应变计算的核心11。这项研究由 Grainger 基金会的 Grainger 创新基金资助。

所有实验都是通过机构审查委员会的批准进行的。这些样本是从解剖学研究实验室合作获得的. 
 1. 准备用于测试的标本 <ol> <li> 在 RT 中解冻股骨 24 h. </li>
	<li> 当股骨在队列中进行测试时, 取出在冻结前应用的任何包装, 用干毛巾擦拭股骨, 去除残余水分、脂肪堆积或软组织。将较大的大转子放入一个带有骨水泥的预制铝杯中. </li>
	<li> 用一个盒子尽可能多地容纳微粒, 用白色的塑料底漆喷涂骨头, 以达到一种薄而均匀的涂层。注意用一层均匀的油漆覆盖骨, 以获得最佳的对比度, 并与股骨表面有很强的附着力. 
	注意: 未测量厚度. </li>
	<li> 让油漆干燥至少5分钟。这是很重要的, 以避免与斑点滴的意外混合2节. </li>
	<li> 用湿布包裹骨头以避免组织干燥. </li>
</ol> 2。斑点进程 <ol> <li> 近似地, 将1部分水添加到2部分的油漆中, 以便在骨骼上进行最佳斑点。将水逐渐 (为更好的混合物) 添加到丙烯酸漆中使其成为黑色. </li>
	<li> 在黑色颜料的调色板上蘸一把干净的牙刷, 以吸收颜色并轻拂画笔, 使白色涂层上的黑色斑点. </li>
	<li> 让油漆干5分钟, 然后再继续. </li>
</ol> 3。图像采集 <ol> <li> 在机械试验机上插入准备好的有斑点的股骨, 方法是将远端装入夹具, 并拧紧两个螺钉以固定试样. </li>
	<li> 调整两个高强度放电光反射器, 使股骨表面达到尽可能高的光照, 同时避免相机图像中的反射。请在3节中快速执行以下步骤, 以避免在测试之前对样品进行不必要的加热. </li>
	<li> 减少前、后视图的光圈高速摄像机镜头, 使整个区域的股骨在视野中的兴趣集中. </li>
	<li> 调整光线反射器以进一步改善光照, 同时减少眩光. </li>
	<li> 将图像采集软件设置为捕获6000帧/秒和 #160; 分辨率为 1024年 x 512 像素。设置接收到12288的触发信号后要获取的帧总数。当从测试系统接收到触发信号时, Arm 高速视频软件用于图像采集。测试完成后, 视频驻留在照相机和 #39 的缓冲区内存中. </li>
	<li> 使用图像采集软件, 通过指定所需的路径和文件名并单击和 #34, 将视频保存到磁盘中; 保存和 #34;。准备在 5-40 分钟之间等待此过程的完成, 这取决于要保存的帧数. </li>
</ol> 4。图像准备 <ol> <li> 为股骨的正面和背面视图创建单独的工作目录 </li>
	<li> 使用视频分析软件打开适当的高速视频录制, 并记下关键帧参考号码在 1) 开始 加载帧执行器移动, 和 2) 在 断开事件后立即帧 . </li>
	<li> 要从高速视频缩减未压缩的 TIFF 图像序列, 请打开并运行 #34; mov_frames. #34; 在相关的股骨侧的工作目录中编写脚本. 
	<ol> <li> 在结果对话框中, 输入步骤4.2 中标识的结束帧编号, 步骤大小为 25-40。单击并 #34; 提取帧和 #34; 检查工作目录以确保正确提取了 tiff 文件. </li>
	</ol> </li> </ol> 5。有限元网格创建 <ol> <li> 使用外部有限元啮合程序创建有限元网格。用有限元法计算了差分位移矢量的2D 应变。将初始提取的 *. tiff 图像导入有限元软件预处理作为样条创建的模板. </li>
	<li> 查找两个容易识别位于帧的相反角的图像中的信讬点, 并记录它们的 X 和 Y 坐标 (这些将最终用于步骤 6.1)。根据有限元软件用于导入 *. tiff 图像的约定, 这些坐标是任意的。这些点的坐标将用于在步骤6.2 中将有限元网格的节点与视频图像的相应像素进行注册. </li>
	<li> 在图像编辑软件中, 打开导入到有限元软件预处理器中的同一图像, 并记录与步骤5.2 中标识的点相关联的像素的 X 和 Y 方向值。这些最终将在步骤6.1 中使用. </li>
	<li> 在 #34; 草图和 #34; 有限元啮合程序的模块, 使用样条工具勾勒出表示感兴趣区域的闭合剖面。验证区域是不是太大, 这样, 骨骼的表面会在骨折前由于旋转而移动到区域之外. </li>
	<li> 准备在步骤5.4 中创建的闭合截面, 通过在菜单和 #34 下播种全局网格大小为 1 mm 的边缘进行啮合; 种子部件实例和 #34;. </li>
	<li> 下 #34; 分配网格控件和 #34;, 将元素形状设置为四边形. </li>
	<li> 将已关闭的分区网格. </li>
	<li> 将网格导出为由节点坐标和元素定义组成的网格数据库的 ASCII 文件. </li>
	<li> 在文本编辑器中打开所生成的有限元素输入文件, 将包含节点编号的节点块复制到一个新的文本文件中, 并将其另存为和 #34; nodes.txt 和 #34;。重复执行元素块并将新的文本文件保存为和 #34; elements.txt 和 #34;. </li>
</ol> 6。将 FE 网格与高速视频图像进行注册, 并进行数字图像相关分析 <ol> <li> 在新会话内, 创建2元素行向量, 称为 ab1 和 ab2 , 并使用步骤5.2 中标识的值. px1 和 px2步骤5.3 中标识的值, 方法是在命令行上键入这些向量名称。将工作区保存为和 #34;p oints #34;... </li>
	<li> 运行脚本和 #34; convert_imagesize #34; 用提取的高速视频图像对有限元网格中的点进行注册. </li>
	<li> 运行脚本和 #34; rrImageTrackGui #34;。加载第一个图像 (和 #34;p 01.tif 和 #34;) 并输入作为要处理的图像总数提取的最后一个. tiff 文件的编号. </li>
	<li> 加载在步骤5.7 中创建的网格, 方法是确保将网格选项设置为 #34; 从文件和 #34 中读取; 然后单击并 #34; 接受和 #34;。有限元网格应出现在骨骼图像上. </li>
	<li> 根据跟踪参数的下列准则指定跟踪值, 并单击和 #34;P roceed 和 #34; (请记住, 参数值是图像大小、纹理和变形量, 需要进行测试仔细的个案基础上). 
	<ol> <li> 使用起始内核大小21。内核大小, n, 是大小 of n x n 窗口 (其中 n 是奇数) 的像素, 用于互相关, 并确定该区域的变形向量将用于应变计算. </li>
		<li> 使用起始子像素大小4。亚像素的大小, m, 是大小的 (2 米 + 1) x (2 m + 1) 子的亚像素变形计算通过假设均匀应变在该子. </li>
		<li> 使用起始平滑系数2。平滑系数是在计算应变之前, 在履带位置的位移场上平滑应用的量. </li>
		<li> 使用起始 maxMove 系数 10. maxMove 因子是任何节点相对于其相邻和 #39 的轨迹, 可以远离其轨迹的最大像素数。这有助于避免严重的变形跟踪. </li>
		<li> 使用起始 smoothGrid 系数15。smoothGrid 因子是用于平滑处理的网格大小 (比跟踪节点的网格略粗). </li>
	</ol> </li> <li> 选择一个具有显著对比度的参考线, 同时避免带有眩光或模糊的区域。通过单击和 #34 检查此点; 检查导轨和 #34; 并验证相关峰值是否强 (至少是振幅的两倍) 与它的邻居相比。单击和 #34; 接受和 #34; #34;P erform 跟踪和 #34; 当满足时。这可以是一个冗长的计算过程, 其中差分位移计算的时序图像序列. </li>
	<li> 步骤6.6 完成后, 单击并 #34; 动画和 #34;。动画完成后, 单击并 #34; 写入应变 (后处理软件) 和 #34;, 输入 * exe, 然后选择 writeStrainRR_simple 11 。这将计算菌株。关闭 GUI. </li>
</ol> 7。位移和应变数据的后处理 <ol> <li> 获取应变 与 帧数、运行和 #34; analyzeFailurePrecursor 和 #34; 从命令行到步长大小的输入参数 (选择 20-30)。峰值将意味着骨损伤, 最大的峰值将对应于接近全球骨衰竭的框架. </li>
	<li> 创建应变、运行和 #34 的影片文件; makeMovies 和 #34; 从命令窗口中使用参数 (numVars、endstep、标志). 
	注意: 参数 numVars 定义为 1-3 是位移, 4-6 是 xx , yy , 和 xy 应变分量, 7 和 #38; 8 是两个主要和冯米塞斯应变, 9 是应变能。参数 endstop 是要包含在影片中的最后一个框架。
	<ol> <li> 将可选参数标志设置为 1, 以便仅为 numVars 参数指定的实体和0创建所有变量的影片. </li>
	</ol> </li> </ol> 8。对结果进行微调和细化 <ol> <li> 如果 DIC 跟踪给出了糟糕的结果, 如不连续的应变场, 这是在连续体力学假设之外, 确定发生了什么以及为什么跟踪失败。重复6节, 特别注意跟踪参数的调整。第二个选项可能是返回到有限元软件, 并创建一个更均匀和可能更细的网格. </li>
	<li> 如果 dic 跟踪给出了合理的结果, 则为 dic 创建更精细的图像系列。使用步骤4.2 中的关键帧参考编号和视频的相应帧速率, 在断裂试验中确定三不同的感兴趣制度的帧间距, 并记住这些点应移动不超过6帧之间的像素. 
	注: 对于测试的初始段, 当应变在股骨缓慢上升时, 帧间距将相对较大 (例如, 对于 100 mm/秒的位移率, 此部分的帧间距为3333和 #956; s)。对于更接近于断裂框架的中间部分, 应变增加得更快, 需要更小的帧间距 (1667 和 #956; s 用于 100 mm/秒的位移率)。在断裂前的最后部分, 帧间距在最小 (16.7 和 #956; s 在100毫米/秒的位移率). </li>
	仅用于文档用途的 <li> 可选: 使用步骤8.2 中的信息, 在名为 "和 #34 的 ASCII 文件中创建格式化数据项; steps.txt 和 #34; 包含每个帧间距的一行数据。每行的格式将是该制度的起始帧, 该框架由要跳过的帧数 (基于步骤 8.2) 分隔, 然后是该制度的最终框架 ( 即 和 #160; #34 的格式; 1:20:200 和 #34; 将指示提取软件提取帧1到帧200的步骤 20). 
	<ol> <li> 紧接该指定之后, 插入一个选项卡并指定图像提取号码范围 (用于和 #34; 1:20:200 和 #34; 例如, 完整的行指定将是和 #34; 1:20:200 和 #60;P ress 选项卡和 #62; 1:11 和 #34; 没有引文)。对其他两个测试机制重复, 以便在 #34 中有三行信息; steps.txt 和 #34; 文件。此文件用作记录如何从原始的高速视频中提取图像. </li>
	</ol> </li> <li> 在该对话框中指定多个帧间距制度时, 再次运行 mov_Frames. m 代码。输入步骤8.2 中标识的帧号和步长, 以确定在 "工具和 #39" 对话框中预期的开始、完成和帧跳过参数。请确保在新目录中执行此项, 否则将覆盖原始图像. </li>
	<li> 重复6和7节, 并查看改进结果。每个股骨可能需要不同的额外的迭代, 这取决于骨折事件的性质, 斑点图案和照明。重复步骤6.5 时, 将设置保持不变, 除非将 maxMove 减少到 6 (从 10). </li>
</ol>

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作者没有相关的披露。

我们介绍了一个协议, 以一致准备在骨折试验期间的高对比度成像的股骨样本, 然后用于估计全场应变分布的 DIC。这个协议确保了适当的对比纹理黑色跟踪斑点对坚实的白色背景的骨骼表面。根据该协议, 我们成功地复制了八十九股骨的 DIC 分析的应变估计。
DIC 是一种光学方法, 它涉及在高速摄像机拍摄的一系列图像上放置一个网格, 并使用互相关算法跟踪帧之间的像素亮度变化。在实验过程中, 我们发现了一些需要考虑的考虑因素的准确性和鲁棒性的方法, 这些细节都反映在所提出的协议详细。首先, 我们发现相机的灵敏度和分辨率对空间应变测量有重要意义。第二, 应避免在白色表面上形成一个非常精细的对比黑色标记的纹理, 因为它们可能对摄像机不可见。第三, 相机和照明应设置在适当的距离, 以确保最佳孔径大小的景深和图像的质量和对比度。过多的光照可能导致图像饱和, 导致对比度较差。最后, 需要设置图像之间的时间间隔, 这样, 表面斑点不会在帧之间移动超过6像素, 以便在互相关时准确地捕获跟踪。
正如在这项工作中所证明的, DIC 有能力提供完整的现场时序应变估计股骨骨折试验, 这是不容易获得应变仪的实验技术。虽然应变计测量已经被一些研究人员所采用, 但这种测量可能会由于对骨骼表面的附着性不足、测量条件和有限的空间分布1213而受阻。相比之下, 全场应变数据是非常有用的验证 QCT/有限元模型的骨强度通过比较应变场之间的模型和试验, 它也有临床应用, 以关联型的应变模式股骨表面的发育在这个生理的秋季负荷情况下5,9。虽然夹具符合性可能是一个问题时, 测试非常僵硬的股骨, DIC 绕过这个问题, 直接从骨局部变形, 从而消除夹具的依从性, 作为一个错误的来源估计股骨僵硬.这些图像相关性的结果可能有助于开发更好的 QCT/有限元模型, 包括材料失效和损伤和断裂的量度。这些最终可以帮助指导治疗的决定, 特别是对骨质疏松症患者。
但是, 该方法确实有几个缺点。骨标本表面必须均匀地覆盖有与背景高度对比的随机散斑图样。偶尔反射从照明或大变形可能改变算法的能力精确地跟踪模式从框架到框架 (图 4)。第二个限制是, 当使用单摄像机 (2D) DIC, 应变计算可能会受到影响, 当骨表面平面偏离与相机图像传感器平面14平行。这可能发生在骨折试验时, 股骨表面向或远离相机。我们正在探索在这方面的未来工作, 添加第二个摄像头和利用 3D DIC 方法, 以提高精确度。直到最近, 这种方法在研究环境中已经无法实现, 但现在变得越来越容易获得。对生物组织的方法的另一个限制是对股骨表面的油漆粘附的不确定性。根据我们的观察, 这不是一个问题, 在我们的测试, 但任何滑动的股骨组织和油漆将影响的结果。此外, 任何非组织留在骨准备可能会干扰皮质应变测量。最后, 图像跟踪设置和网格密度是影响 DIC 分析结果质量的因素, 需要认真考虑。
目前的协议提出了一种方法, 有效和一致地准备股骨标本的数字图像相关分析和估计的相应应变场的高速相机成像在骨折测试。在我们的实验室中已经证明了在一个6年的时间内, 在多个测试时限和不同的研究人员和操作员之间产生一致性。在这里提出的用于股骨准备和检测的 DIC 的程序可以很容易地扩展到其他骨骼类型。

数字图像相关 (DIC) 是一种图像后处理方法, 用于在目前的协议中估计的全场表面应变的尸体股骨试验标本从时序图像中获得的机械断裂试验。该技术在1980的实验应力分析中首次被开发和应用, 并在近几年的使用中迅速增加1,2,3。它有几个关键的优势超过传统的方法安装应变计的结构, 包括增加空间分布的应变场, 更精细的测量长度通过增加相机分辨率, 并避免问题的应变计粘合或合规性。生物组织 DIC 的一个主要优势, 如骨骼, 是它可以应用于不规则的几何图形, 包括高度异构的材料属性4,5。它的主要缺点是传统的应变采集方法是, 它需要昂贵的高速摄像机的足够分辨率的测量区域的利益, 以实现足够的空间和时间采样准确估计应变场。
从骨折 DIC 分析得到的时间应变场的初步应用是验证 QCT/有限元模型中的应变估计5。这种验证是许多骨科研究小组的重点, 主要利用远程测量的力量和位移从负载细胞和位移传感器6,7,8。此外, 骨折图像分析的断裂模式已与这些远程测量结合起来, 作为进一步的模型验证9。最近, 应用 DIC 法对股骨近端断裂和裂纹扩展的有限元模型进行了验证, 并对其进行了10。利用模型与实验之间的应变相关性, 对近股骨的计算模型的有效性有了更深入的把握, 进一步提高了 QCT/有限元诊断方法在临床上的应用效果。
这项工作解释了详细的协议, 以纳入必要的步骤, DIC 分析在断裂试验的近端股骨。该程序包括在骨表面喷涂白色涂料的骨准备步骤, 然后在骨骼干白表面斑点黑点, 利用高速视频获取具有足够空间和时间分辨率的图像的方法摄像机, 以及我们用于计算这些图像中的应变场的过程和工具。我们还解释了一些可能影响测量质量的警告。

<table border="0" cellpadding="0" cellspacing="0" width="1549">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Krylon plastic primer white</td>
			<td style="width:360px;">Krylon, Peoria, AZ, USA</td>
			<td style="width:425px;">N/A</td>
			<td style="width:479px;">Used as a base coat for a smooth white finish on bone surface</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Water-based acrylic white and black paint&#160;</td>
			<td style="width:360px;">Plaid Enterprises (Ceramcoat), Norcross, GA, USA</td>
			<td style="width:425px;">N/A</td>
			<td style="width:479px;">Paint source for white and black colors</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Mixing bowl</td>
			<td style="width:360px;">Not specific (generic)</td>
			<td style="width:425px;">N/A</td>
			<td style="width:479px;">Used to mix and prepare paint</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Foam brush</td>
			<td style="width:360px;">Linzer Products, Wyandanch, NY, USA</td>
			<td style="width:425px;">N/A</td>
			<td style="width:479px;">Used to apply paint on bone surface</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Toothbrush</td>
			<td style="width:360px;">Colgate-Palmolive, New York, NY, USA</td>
			<td style="width:425px;">Firm bristle</td>
			<td style="width:479px;">Used to apply appropriate size and distribution of speckling pattern</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Hygenic Orthodontic Resin (PMMA)</td>
			<td style="width:360px;">Patterson Dental, St Paul, MN, USA</td>
			<td style="width:425px;">H02252</td>
			<td>Controlled substance and can be purchased with proper approval</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Kenmore Freezer</td>
			<td style="width:360px;">Sears Holdings, Hoffman Estates, IL, USA</td>
			<td style="width:425px;">N/A</td>
			<td>Used to maintain a -20oC storage enviroment for bone specimens</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Physiologic Saline (0.9% Sodium Chloride)</td>
			<td style="width:360px;">Baxter Healthcare, Deerfield, IL, USA</td>
			<td style="width:425px;">NDC 0338-0048-04</td>
			<td>Used for keeping specimens hydrated</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Scalpels and scrapers</td>
			<td style="width:360px;">Aspen Surgical (Bard-Parker), Caledonia, MI, USA&#160;</td>
			<td style="width:425px;">N/A</td>
			<td>Used to remove soft tissue from bone specimens</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Fume Hood</td>
			<td style="width:360px;">Hamilton Laboratory Solutions, Manitowoc, WI, USA</td>
			<td style="width:425px;">70532</td>
			<td>Used for ventilation when preparing PMMA for potting of specimens</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Lighting units</td>
			<td style="width:360px;">ARRI, Munich, Germany</td>
			<td style="width:425px;">N/A</td>
			<td>Needed for illumination of target for image capture</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">High-speed video camera</td>
			<td style="width:360px;">Photron Inc., San Diego, CA, USA</td>
			<td style="width:425px;">Photron Fastcam APX-RS&#160;</td>
			<td>Used to capture the high speed video recordings of the fracture events</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Photron FASTCAM Imager and Viewer</td>
			<td style="width:360px;">Photron Inc., San Diego, CA, USA</td>
			<td style="width:425px;">Ver.3392(x64)</td>
			<td>Used to record and view the high speed video recordings</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Camera lens</td>
			<td style="width:360px;">Zeiss, Oberkochen, Germany</td>
			<td style="width:425px;">Zeiss Planar L4/50 ZF Lens</td>
			<td>Needed for appropriate image resolution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">ABAQUS CAE</td>
			<td style="width:360px;">Dassault System&#232;s, Waltham, MA, USA</td>
			<td style="width:425px;">Versions 6.13-4</td>
			<td>Used for defining region of interest and creating finite element mesh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">MATLAB</td>
			<td style="width:360px;">Mathworks, Natick, MA, USA</td>
			<td style="width:425px;">Version 2015b</td>
			<td>Used for image processing and DIC analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">TecPlot</td>
			<td style="width:360px;">TecPlot Inc., Bellevue, WA</td>
			<td style="width:425px;"></td>
			<td>Used for post processing of strain fields</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Strain Calculator Software</td>
			<td style="width:360px;">Victor Barocas Research Group, University of Minnesota, Minneapolis, MN, USA</td>
			<td style="width:425px;">http://license.umn.edu/technologies/20130022_robust-image-correlation-based-strain-calculator-for-tissue-systems</td>
			<td>Used to calculate strain field</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:285px;">mov_frames.m</td>
			<td style="width:360px;">Matlab script, Mayo Clinic, Rochester, MN,USA</td>
			<td style="width:425px;">N/A</td>
			<td>Used to downsample uncompressed images from high speed video files</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:285px;">convert_imagesize.m</td>
			<td style="width:360px;">Matlab script, Mayo Clinic, Rochester, MN,USA</td>
			<td style="width:425px;">N/A</td>
			<td>Used to register image pixel coordinates with mesh coordinates</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">rrImageTrackGui.m</td>
			<td style="width:360px;">Matlab script, Mayo Clinic, Rochester, MN,USA</td>
			<td style="width:425px;">N/A</td>
			<td>Used to perform the image cross-correlation to obtain deformations and run Strain Calculator</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:285px;">analyzeFailurePrecursor.m</td>
			<td style="width:360px;">Matlab script, Mayo Clinic, Rochester, MN,USA</td>
			<td style="width:425px;">N/A</td>
			<td>Used to track the peak strain components temporally</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:285px;">makeMovies.m</td>
			<td style="width:360px;">Matlab script, Mayo Clinic, Rochester, MN,USA</td>
			<td style="width:425px;">N/A</td>
			<td>Used to create portable *.avi movies of the deformation components, strain components, principal strains, von Mises strain, and strain energy</td>
		</tr>
	</tbody>
</table>

a method to estimate cadaveric femur cortical strains during fracture testing using digital image correlation

本协议描述了利用数字图像相关技术对从机械测试中获得的尸体股骨表面的高速视频图像中的皮层应变进行估计的方法。这种光学方法要求在一个坚实的白色背景上有许多对比的信讬标记的纹理, 以准确地跟踪表面变形, 因为载荷被应用到试样上。在测试之前, 在相机视图的兴趣表面被涂上了一个水性白色底漆, 并允许干燥几分钟。然后, 黑色的油漆在白色的背景上仔细地斑点, 特别考虑到水滴的均匀大小和形状。照明是精心设计和设置这样, 有最好的对比, 这些标记, 同时尽量减少反射通过使用过滤器。图像是通过高速视频捕获1.2万帧/秒获得的。提取之前和包括骨折事件的关键图像, 并在经过特定区域的详细询问窗口中的连续帧之间进行变形估计。这些变形然后被用来计算表面应变在断裂试验期间。应变数据是非常有用的识别骨折开始在股骨, 并最终验证的股骨近端骨折强度模型从定量计算 Tomography-based 有限元分析 (QCT)。

在斑点过程之前, 股骨是从多余的脂肪和软组织清洗, 和大转子是盆栽在一个铝杯。在酯 (PMMA) 凝固过程中, 骨头被用盐水浸泡过的布料包裹, 以避免组织干燥。一旦 PMMA 固化后, 在喷涂(图 1)之前, 再次对骨骼进行清洗。然后, 用水性塑料白颜色喷涂或刷骨表面。干燥后, 白色表面上有黑色斑点, 在白色背景上有一个随机的黑点图案(图 2)。一旦将骨骼放置在测试夹具中, 就会设置灯和高速摄像机, 并在测试(图 3)之前检查模式和照相机焦点的最佳对比度。DIC 方法需要高对比度的斑点模式和足够的光照。否则, 结果可能会受到一些问题的影响, 如曲面的会、对比度差和图像暗淡(图 4)。高速视频中的未压缩图像能够在多个时序采样机制中提取, 并且可以通过图形用户界面(图 5)来操作 DIC 跟踪算法。股骨样本的轮廓用于确定应变场估计的感兴趣区域(图 6A) , 并用于创建应变计算的有限元网格(图 6B)。在检测过程中, 通过监测应变偏差的程度来检测骨折的发生, 用峰值表示骨骼损伤和骨折的时间框架(图 6C)。最后, 2D 应变场叠加到未经测试的骨骼图像上, 以增强可视化效果(图 6D)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54942/54942fig1.jpg"> 
图 1: 在绘制骨骼前准备骨骼.(A) 解冻后从脂肪和湿气中清除骨骼;(B) 灌封大转子;(C) 在喷涂过程前清洗<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54942/54942fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54942/54942fig2.jpg"> 
图 2: 绘制过程.(A) DIC 工作区和必要的工具;(B) 用白色底漆喷涂骨;(C) 在骨骼表面上刷白颜色;(D) 斑点白色骨表面上的黑点;(E) 骨骼的最后斑点的表面准备好测试<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54942/54942fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/54942/54942fig3.jpg"> 
图 3: 照明和照相机设置.(A) 设置灯具和护盾;(B) 设置高速摄像机;(C) 将一个骨骼样本加载到测试机器上, 并准备好用于检查和测试的灯和照相机;(D) 检查图像中是否有摄像头的功能;(E) 检查有兴趣的区域, 在股骨颈, 为聚焦区域, 景深, 缺乏模糊, 和一般的图像质量 DIC<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54942/54942fig3large.jpg" target="_blank">请点击这里查看这个数字的更大版本</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/54942/54942fig4.jpg"> 
图 4: 骨骼 DIC 警告.(A) 会在头部区域;(B) 当白色表面不干燥时, 黑白相间的混合和流动;(C) 对比度差, 本地会, 图像清晰度较差<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54942/54942fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/54942/54942fig5.jpg"> 
图 5: 在 DIC 处理中使用的自定义脚本对话框.(A) mov_frames, (B) rrImageTrackGui. m, (C) 生成的2D 网格<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54942/54942fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/54942/54942fig6.jpg"> 
图 6: 中间 DIC 结果的示例.(A) 样条绘制以突出显示感兴趣区域, (B) 生成的网格覆盖在骨骼图像上, (C) 应变偏差作为高速视频帧的功能, (D) 计算出的应变轮廓图与2相关。在骨折前测试图像<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54942/54942fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation at JoVE.com

A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation

在该协议中, 使用数字图像相关技术对骨折试验中的股骨表面应变进行估计。该方法的新颖性包括在股骨表面上应用高对比度随机散斑图、精心指定的光照、高速视频捕捉以及应变计算的数字图像相关分析。

基于数字图像相关的骨折检测中股骨皮质应变的估算方法

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral ...

a-method-to-estimate-cadaveric-femur-cortical-strains-during-fracture

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.3K 次观看.  Mayo Clinic. 在该协议中, 使用数字图像相关技术对骨折试验中的股骨表面应变进行估计。该方法的新颖性包括在股骨表面上应用高对比度随机散斑图、精心指定的光照、高速视频捕捉以及应变计算的数字图像相关分析。

视频: A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

光频域成像<em&gt;体外</em肺切除标本:获得一对一的图像病理组织学的相关性

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

科研

生物工程

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

基质贴壁细胞的微管吸吮的估计单元刚度

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

一种改进的机械性能测试方法来评估骨种植体支抗

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density distribution and geometry on the femoral mechanical properties. Currently, there is no standard protocol for mechanical testing of such geometrically complex bones to measure strength, and stiffness. To address this gap we have developed a protocol to test cadaveric femora to fracture and to measure their biomechanical parameters. This protocol describes a set of adaptable fixtures to accommodate the various load magnitudes and directions accounting for possible bone orientations in a fall on the hip configuration, test speed, bone size, and left leg-right leg variations. The femora were prepared for testing by cleaning, cutting, scanning, and potting the distal end and greater trochanter contact surfaces in poly(methyl methacrylate) (PMMA) as presented in a different protocol. The prepared specimens were placed in the testing fixture in a position mimicking a sideways fall on the hip and loaded to fracture. During testing, two load cells measured vertical forces applied to the femoral head and greater trochanter, a six-axis load cell measured forces and moments at the distal femoral shaft, and a displacement sensor measured differential displacement between the femoral head and trochanter contact supports. High speed video cameras were used to synchronously record the sequence of fracture events during testing. The reduction of this data allowed us to characterize the strength, stiffness, and fracture energy for nearly 200 osteoporotic, osteopenic, and normal cadaveric femora for further development of engineering-based diagnostic tools for osteoporosis research.

In this manuscript, we present a protocol to fracture test cadaveric proximal femora in a sideways fall on the hip configuration using instrumented fixtures mounted on a standard servo hydraulic frame. Nine digitized signals comprising forces, moments, and displacement along with two high speed video streams are acquired during testing.

方法和股骨骨折测试在横盘整理的秋天在臀部位置检测的夹具

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density ...

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count.

用数字全息显微镜 (DHM) 定量化低浓度微生物

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

大鼠牵张成骨的高效可再生性协议

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the fragility of bone, making it fracture-prone. Owing to the large socio-economic impact of bone fracture in our society, there is a need for novel ways to assess the mechanical performance of each hierarchical level of bone. Although stiffness and strength can be probed at all scales &#8211; nano-, micro-, meso-, and macroscopic &#8211; fracture assessment has so far been confined to macroscopic testing. This limitation restricts our understanding of bone fracture and constrains the scope of laboratory and clinical studies. In this research, we investigate the fracture resistance of bone from the microscopic to the mesoscopic length scales using micro scratch tests combined with nonlinear fracture mechanics. The tests are performed in the short longitudinal orientation on bovine cortical bone specimens. A meticulous experimental protocol is developed and a large number (102) of tests are conducted to assess the fracture toughness of cortical bone specimens while accounting for the heterogeneity associated with bone microstructure.

This study assesses the fracture toughness of bovine cortical bone at the sub-meso levels using microscopic scratch tests. This is an original, objective, rigorous, and reproducible method proposed to probe fracture toughness below the macroscopic scale. Potential applications are studying changes in bone fragility due to diseases like osteoporosis.

牛皮质骨脆性评估的划痕试验

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

一种基于变形虫的快速图像细菌毒力检测方法

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...