我们要感谢的雷蒙德和富赛克勒基金会为他们在我们的研究支持。

1，三维球体瘤形成，药物治疗和图像采集是在我们以前的文章15进行描述。 2，软件安装<ol><li>安装许可MATLAB软件到用于图像分析的计算机。还需要在MATLAB下工具箱将会安装 - 信号处理工具箱，图像处理工具箱，和并行计算工具箱*（*所需的并行计算模式）。 注：许多大学购买和维护组许可证的，这样的软件是免费下载和使用的附属科学家。 </li><li>从SpheroidSizer.zip文件安装SpheroidSizer程序（http://pleiad.rwjms.rutgers.edu/CBII/downloads/SpheroidSizer.zip）： <ol><li>将zip文件保存在本地文件系统。 </li><li>解压缩SpheroidSizer文件。 </li><li>保存文件到指定的目录/文件夹，随后将被简称为T他“安装目录”。 注：SpheroidSizer已经在Windows 7操作系统被广泛的测试。它预计将有最低限度的调整（未测试）工作的替代操作系统。 </li></ol></li></ol> 3，准备通过SpheroidSizer影像分析<ol><li>确定成像系统（图像的绝对规模在每像素（微米/像素）微米）的图像缩放/分辨率。 注：如果每个像素的摄像头芯片上的尺寸是已知的，在图像规模可以被计算为目标放 ​​大倍数×像素大小 （微米/像素）。这个值可以从配备有显微镜作为嵌入的元数据或从成像系统供应商的帮助成像软件来获得。这个值将被要求在步骤4.6。 </li><li>转换任何专有的图像文件格式为接受的文件格式 - TIFF，JPEG等常见图像文件格式。 </li><li>命名的图像文件，并安排directoriES（ 图5A）。 注意：该软件依赖于目录结构和文件名的正确布局的结果格式化成片格式： <ol><li>名称以下列格式的图像文件：[板块名称] _ [行] [列] [扩展]或[板块名称] [空格] [行] [列] [扩展]。 [行]遵循字母顺序[专栏]如下的数字顺序。 注：可自由查看的自动批量重命名软件，可以发现，以帮助用户在这一步。 </li><li>安排基于该实验以下面的方式在目录/文件夹：每个实验都应该有一个目录。在每个实验的目录，应该有子目录，每个时间点。在每个时间点的子目录，应该有来自所有板块全部图像。 注：为了使分析结果进行优化排序，格式化的结果，我们建议保持对每个标识符位数相同的号码通过在L填充0的EFT， 例如 ，时间点被命名为000H，072H，144H和。 </li></ol></li></ol> 4，图像的球体通过SpheroidSizer分析<ol><li>打开MATLAB，然后打开“命令窗口”，键入cd&#39;[安装目录]“，然后按[返回]。 </li><li>在“命令窗口”，然后按[返回]输入“SpheroidSizer1_0”启动SpheroidSizer程序。 </li><li>单击SpheroidSizer1.0窗口中的“浏览”按钮，选择包含所有影像实验目录。 </li><li>选择“包含子文件夹”中的“文件夹”文本字段中指定的目录下处理多个嵌套的图像文件夹下的切换。 注意：如果“包括子文件夹”切换没有被选中，不仅直接目录下的图像进行处理，并且所有子文件夹都将被忽略。 </li><li>选择“上的即时显示”选项disp已与正在执行的计算闪每个分割图像，其质量控制源图像的顶部。 注：计算速度更快，如果没有选择“上的即时显示”选项。 </li><li>请在框中分析图像的“决议”（图像缩放/分辨率在微米/像素），使程序正确转换球体的测量，从像素到微米。 注意：所有在同一个文件夹或图像一起分析应在显微镜一样具有相同的目标下服用，以使图像缩放/分辨率保持不变，每个实验。 </li><li> （可选）用户可以按照步骤5的高级用户自定义设置。 </li><li>点击“计算”开始计算。 注意：该软件在进行计算之前进行自动的文件名检查。如果一个对话框显示，指示 - “存在于文件名错误”，单击“退出并显示错误列表”和修复中列出的文件名错误（请参阅步骤3.3）。然后，单击“计算”重新开始计算。 </li><li>点击“暂停”按钮暂停计算;和计算可以通过点击相同的按钮，显示“继续”继续进行。 注意：“成绩表”显示“文件夹”，“文件”，“音量”（以mm 3），“长度”（单位为μm），“宽度”（单位为μm），和“有效”（复选框）所有被分析的球体（ 图5C）的。量是基于测量的长轴（长度）和短轴（宽度）计算（V = 0.5 *长*宽2）。 “有效”复选框是一个选项供用户选择，如果图像的分析是有效的还是无效的质量控制后，见第6步。 </li></ol> 5，先进的用户自定义设置<ol><li>点击“高级”按钮，在SpheroidSizer1.0窗口，弹出高级配置窗口，以调整用户自定义设置（ 图5B）。 </li><li> “输入”下的高级配置窗口中输入在“输出格式”利息的文件名和“列表输出”复选框。 </li><li>从“2-10”中的“减持”框中输入一个数字。这是为软件，以减少为了提高计算速度，计算所述图像的大小的系数。较大的数目，更快的运算速度。默认的“缩小”设置为10。 </li><li>请在“包含类型”框中输入将要处理的图像文件的扩展名。 </li><li>输入图像文件的扩展或者结局并非将要由程序中的“排除式”对话框进行如下处理：“_crude.jpg”。 </li><li>选择“无”为“特殊颜色”来处理8位和16位山坳或图像正常;选择“12位”为“特殊颜色”正确处理12位彩色图像</li><li>勾选“使用并行计算”，如果用于图像分析的计算机配有多个CPU和/或多核CPU。如果这是真的，然后转到步骤5.7.1;如果没有，那么跳过步骤5.7.1和5.7.2。 注意：将发生错误，如果正在使用的计算机不支持所选择的配置。 <ol><li>检查在高级配置窗口中的“使用并行计算”选项。 注意：只有使用并行计算模式，当4个或更多的内核可用于计算机。 </li><li>从“＃工”4-12输入一个数字（运算核心）框。 注意：这个数目必须等于或小于计算的内核在用户的计算机的数量。最多12是由MATLAB并行计算工具箱强加支持12个内核的最大值。当并行计算是ING执行，一个小对话框，询问用户等待并行计算来完成;计算不能被暂停，并且也不是并行计算模式正在执行的“关于的即时显示”功能。 </li></ol></li></ol> 6，质量控制<ol><li>单击“结果表”对应的单元格，以确认一个球体，在分析图像的精确边界轮廓， 注：原和质量控制的图像将显示在右侧进行审查。用户可以检查所有图像顺序使用键盘上的向下箭头。 </li><li>使用以下两种工具，如果需要缩小旋转椭圆体的选择的图像上的边界： <ol><li>点击“手动初始化”按钮，显示原始图像。然后单击并按住鼠标右键球体外面并拖动椭圆工具来覆盖原来的图像上的球体。 注：A莫如轮廓算法启动使用用户提交的轮廓，并执行收敛于期望的球体轮廓。 “成绩表”将被自动更新为新的结果。 “手动初始化”工具允许用户手动主动轮廓提供初始化。 </li><li>点击“手绘图”按钮，显示原始图像。然后用鼠标或触摸屏启用精确绘制球体的边界。 注：本大纲直接测量产生长轴和短轴，这在“成绩表”进行更新。 “手绘图”工具仅用于当“手动初始化”工具不能收敛的球体所需的边界上。 </li></ol></li><li>取消选中在“有效”列中的“成绩表”的相应行中，当图像不包含经检查任何有效的球体的复选框。一个“;无效“的标签出现在质量控制图像的左上角。如果“有效”未选中，所有的测量值是空在出口格式化和输出结果文件的球体。 注：以下键盘快捷键都可以使用在“结果表”：“向下箭头”的下一张图片; “V”的有效/无效; “m”为“手动初始化”工具和“h”为“手绘图”工具。 </li></ol> 7，保存和导出数据<ol><li>单击SpheroidSizer1.0窗口中的“导出学习”按钮导出分析的中间状态，如果用户需要完成项目之前退出软件。指定要保存的文件名和目录。 </li><li>点击“导入学习”按钮，从“出口研究”带回上述中间状态的结果，并继续WORK对了。 注意：中间状态文件在本机的MATLAB格式（垫），不直接读取任何其他软件程序。内置了该软件的安全功能，使的情况下打开的项目程序退出时无意中自动出口。当需要时，用户可以找到这个文件，名称开头为“〜tmp目录”，其中包含在[安装目录]相应的时间戳。 </li><li>单击SpheroidSizer1.0窗口“结果格式”保存结果。 注：两种形式的结果保存在实验中的目录。导出的文件名可以在高级配置窗口中进行设置（见协议步骤5.2）。该格式输出文件是一个制表符分隔的表，组织音量值到在每个时间点的上升车牌号码的顺序原板格式;和所有的时间点被组织为升序（ 图5D）中。该名单O安输出文件是包含所有在有序列表（ 图5E）的形式测量的制表符分隔的表。 </li></ol>

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没有利益冲突的声明。

这项研究提出了一种快速，灵活，有效和自动化的程序 - SpheroidSizer为准确测定的3D球体瘤的大小。 SpheroidSizer是易于使用，不需要用户输入。对于SpheroidSizer准确，顺利和成功运行的最关键步骤包括：该球体被成像在场地中央不接触井的边缘;所有被作为一个项目一起分析这些文件应在显微镜一样具有相同的目标下进行成像;所有被分析的文件是正确命名和布置如在协议指示;和之前的计算正确的用户自定义设置输入。 SpheroidSizer的优点包括其容忍的图像中逐渐变化的背景，以及产生对应于利用主动轮廓算法的球状体的一般球形光滑轮廓的能力。的活跃表现初始化不佳，或其他地方的边缘从所需的轮廓分心的存在：轮廓可以在两种情况下受到损害。特别是在我们的测试的情况下，第二种情况有时发生时，一个大的旋转椭圆体的坏死核心吸引导致较小的轮廓被报告的活动轮廓。值得一提的是，其他自动门限为基础的方法也遭受在这种情况下，除非阈值是由手专门设置。因此，该软件提出了努力来帮助用户检测并通过提供易于质量控制功能弥补损害的分割。如果细分误差从初始化发生不佳，用途可以使用“手动初始化”工具来覆盖自动初始化。当图像质量太差的主动轮廓线，用户可以轻松地“手画”即送入量化的轮廓。现有软件如CellProfiler可以在半自动化的追求时尚的适于这种应用Ñ​​。工作流程可以很累赘，当大量不同的成像条件的影像呈现，或当图像的一个子集需要更多的人为干预，正确测量。 SpheroidSizer提供了一个全功能于一身的套件，用于计算和质量控制，以管理高通量图像分析工作流程。 SpheroidSizer目前仅限于检测每幅图像的球体，只测量球体的轴向长度。该程序可以被扩展，以支持在球体与坏死核心需要研究诸如量化进一步定量，检测在一个图像中的多个球体或监视球体的形状。此外，该程序可以被修改，以检测并测量来自动物或人的切除肿瘤，其在体内的临床前或临床研究进行时肯定会有利于研究者的大小。后处理所检测的球体，也可以调查目的ing在减少所需的质量控制人的努力，进一步提高吞吐量。 SpheroidSizer是一个广义的图像分析中的应用用于从任何类型的细胞产生的，并因此可用于广泛的癌症研究界三维肿瘤球状体。

三维（3D）肿瘤球状体是“球对称肿瘤细胞类似于组织的聚集体，没有人工基质细胞附着”1-3。肿瘤的细胞学和形态学球状体更好地模拟了体内的肿瘤组织的组织与微环境比单层的二维（2D）细胞。 3D球体瘤已成为一种实用的体外模型用于抗癌治疗药物高通量筛查或研究的候选药物的疗效在动物体内或临床试验前4。在临床上，任何抗癌药物治疗的疗效是基于减少的肿瘤生长进行评估。类似地，球体体积可作为疗效的体外抗癌药物研究的一种度量。球体体积（V = 0.5 *长*宽2）乃根据主要和次要轴向长度决定（通常称为长度和宽度）该球状体的6,7。大多数研究者必须手动绘制每个球体的长度和宽度，往往采用由显微镜公司提供的，并与成像仪器一起销售的软件。这种技术成为问题时，高通量药物屏幕执行和超过数百个图像的产生。最近的一些研究报道了使用开源的图像分析软件工具箱，如CellProfiler 8-10和ImageJ的11开发简陋分割程序/宏涉及光量校正和简单的阈值。这些程序通常需要进行重新调整，用于根据所述照明条件和图像对比度变化不同的图像的批次;因此，这些软件包不能满足高通量的图像分析的鲁棒性要求。弗里德里希和合作者（2009）使用专有软件来测量球体的体积半AUTOM胶东东部5。在Monazzam和他的同事的纸张10中描述的方法是一种半自动化的方法来测量球体的大小只有一个小的数字图像。因此，存在一个明显需要强大的，灵活的，自动化的，并准备使用的图像分析工具，为3D肿瘤球体。 在这项研究中，我们描述SpheroidSizer - 一个基于MATLAB的和开源软件应用程序自动，准确地测量肿瘤球体的大小。 SpheroidSizer被设计来处理的3D球体图像的许多不同批次在同一会话中。利用主动轮廓算法12-14，SpheroidSizer可以容忍图像的对比度变化，有力忽略背景照明逐渐变化，并认识球体的形象。它也可以容忍许多平常的文物， 如碎片，源于标本。该工作流被设计为使得用户能够执行质量控器升过程中或经过计算。的分析结果，手动重写可以很容易地在现场进行。利用并行计算工具箱，分析速度，可以进一步通过协调多个计算内核上的计算同时在用户的计算机上工作的升压。此外，SpheroidSizer结果输出有两种不同的形式，以方便接口与下游分析工具。

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			<td height="51" style="height:51px;width:123px;">Axiovert 200M inverted microscope</td>
			<td style="width:176px;">Carl Zeiss Microscopy, LLC</td>
			<td style="width:88px;"></td>
			<td style="width:183px;">microscope for imaging</td>
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		<tr height="17">
			<td height="17" style="height:17px;width:123px;">Vostro 1720</td>
			<td style="width:176px;">Dell Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:183px;">single-core regular laptop</td>
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			<td height="35" style="height:35px;">HP Z820</td>
			<td style="width:176px;">HP Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:183px;">multi-core performance workstation</td>
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			<td height="35" style="height:35px;width:123px;">Matlab and Simulink R2013a</td>
			<td>Mathworks, Inc, Natick, MA</td>
			<td style="width:88px;"></td>
			<td style="width:183px;">Matlab software&#160;</td>
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high-throughput image analysis of tumor spheroids: a user-friendly software application to measure the size of spheroids automatically and accurately

越来越多的三维（3D）肿瘤球体作为体外模型用于药物开发的应用程序需要他们适应大规模筛选格式在药物筛选中的每一步，包括大型图像分析。目前还没有现成的使用和免费的图像分析软件，以满足这一大规模的格式。大多数现有方法涉及手动绘制成像的三维球状体，这是一种单调乏味和耗时的过程的长度和宽度。这项研究提出了一种高通量的图像分析软件的应用 - SpheroidSizer，是衡量成像的3D肿瘤自动精确球体的长轴和短轴长度;计算每个个体的3D球体瘤的体积;然后将结果在电子表格中两种不同的形式，方便操作在随后的数据分析输出。该软件的主要优势是其强大的图像分析应用程序，适合大量图像。它提供了高吞吐量计算和质量控制流程。估计的时间来处理1000张照片大约是15分钟的最小配置的笔记本电脑，或约1分钟的多核性能的工作站上。图形用户界面（GUI）的设计也便于质量控制，并且用户可以手动覆盖计算机结果。本软件使用的关键方法是改编自主动轮廓算法，又称蛇，这是特别适合与光照不均和嘈杂的背景下，经常困扰着自动化成像处理在高通量筛选的图像。免费赠送的“手动初始化”和“手绘图”工具提供了在处理不同类型的球体和多样化的高质量图像的灵活性SpheroidSizer。此高通量图像分析软件显着降低劳力和加快了分析过程。实施这一软件为beneficial三维肿瘤球体成为体外模型在工业界和学术界药物屏幕的例程。

SpheroidSizer设计，生产自动化检测，划定和3D球体的测量，劳动和显着降低急性提高效率，大量的图像。 图1A显示SpheroidSizer的工作流程。核心计算步骤包括自动初始化，主动轮廓算法和轮廓量化。自动计算后，质量控制功能使用“手动初始化”和“手绘图”工具的组合来挽救任何不完美的分割。 图1B说明了详细的自动化的主动轮廓算法。在初始化步骤（第0次迭代）使用基本的图像处理步骤生成的旋转椭球体的近似大小和位置，并生成一个球形起始轮廓，估计大小。起始轮廓送入主动轮廓算法。反过来它遍历根据局部图像进行调整梯度和形状曲度。主动轮廓算法结束时的轮廓稳定（收敛）， 即 477次迭代为这个图象时，或者当执行迭代的预先定义的最大数。在这个例子中，初始化轮廓被故意放大，以更好地展示的算法。在现实中，在初始化通常是非常接近实际的边界并且需要少得多的迭代算法收敛。随后，该算法将检测到的球体边界的形态测量。球体的长轴和短轴采用MATLAB图像处理工具箱（ 图1C）测量。长轴被定义为线段连接的单个对轮廓，其被称为长度（L）上的最远点。短轴被定义为最长的线垂直于主轴线，其被称为宽度（W）。在这种情况下，L和W的值是由于非常接近球体是球形的。旋转椭圆体的体积计算为V = 0.5 * L * W 2。 其中SpheroidSizer的特点是它的自动化即使在不平或嘈杂的背景利用主动轮廓算法（ 图2B-D）图像的球体的边界检测。亮场图像计算处理往往是不均衡的背景下，这误导自适应阈值为基础的方法来产生不必要的阈值结果所困扰。这个问题是特别明显的，当多孔板的使用和井的壁可在其上创建图像的阴影效应。然而，因为主动轮廓算法不是在背景逐渐着色变化敏感，则能够确定在这些亮场图像与正确的初始化球体分割， 图2示出了具有不平坦的或有噪声的背景图像的几个例子，如不平照明（ 图2B </str翁&gt;），碎片（ 图2C）或坏死核心（ 图2D）。用自动化的主动轮廓算法，SpheroidSizer描绘这些球状体准确地在所有这些图像所示的红色轮廓在每个图的下面板。 SpheroidSizer的质量控制功能的关键是一种高通量的工作流程。 “手动初始化”和“手绘图”工具是此应用程序的有价值的免费工具。其中数百幅图像或数千，这是无可避免的自动化算法是不能够正确地检测出球体的一些图片。 如图3A所示 ，当不当检测的旋转椭圆体因在初始化步骤引起的， 即不正确的尺寸或图像（上图）中的起始轮廓的位置，“手动初始化”工具的工作原理是使用户能够适当地定义spher的位置和大小手动OID（下图）。它触发的主动轮廓算法，主动与手动定义轮廓和执行收敛所需的轮廓。对于这些艰难的图像类似于图3B中的原始图像，球体位于一个分散和嘈杂的背景。 SpheroidSizer无法通过自动化的方法（上图）或“手动初始化”工具，正确的初始化（中间面板）正确识别的球体。在这种情况下，“手绘图”工具可用于手动绘制如图所示在下部面板的旋转椭球体的轮廓。该程序使用用户定义的边界来测量球体的长轴和短轴和计算量。所有的校正结果立即并入“结果表”，并可以相应地导出。 以确定SpheroidSizer在更大的数据集的性能，我们首先通过比较操作时间分析同一组使用1）手动测量显微镜供应商提供的软件288图像; 2）SpheroidSizer与单核笔记本电脑经常;和3）SpheroidSizer具有多核并行计算性能的工作站。手工测量请跟随我们的典型协议开发软件之前：每个球体的长度和宽度都是由手工绘制，并使用供应商的程序（如见图4A的顶部面板上的红色线）计量;然后用户拷贝下来测量的值。 SpheroidSizer通过产生的旋转椭球体的边界（如所示的红色轮廓在图4A中的下面板），测量轴向主要和次要的长度，并输出其结果在电子表格中处理每个图像。如表1所示，根据来自288的图像的计算，平均需要31.67秒手动测量每幅图像的球体;而只需要SpheroidSizer少于2秒＆<html> ＃160;在单核普通笔记本电脑上运行时;和小于1秒的12芯性能的工作站上运行时。因此，图像分析为每幅图像超过18倍更快的使用SpheroidSizer比手动测量。它极大地降低了劳动当超过数千张图片进行了分析。接着，我们的可变性，在图4A中所示的球状体24的手动测量和SpheroidSizer之间的测量值进行比较。该球状体24被测量三次通过两种方法;与每个单独的旋转椭球体的标准差的计算。就像在图4B中 ，从SpheroidSizer（绿线和量子点）的标准偏差是惟在质量控制步骤，它仍显示出比那些从手动测量方法较小的标准偏差校正三个球体接近于零。这些都表明SpheroidSizer更有效，更准确地进行图像分析。 e_content“&gt;我们使用人类BON-1 3D肿瘤球体，以找出哪些化合物与HSP90抑制剂组合用于测试在体内的抗肿瘤作用的潜在候选人。人力BON-1 3D球体瘤生长进行药物筛选在如前面的纸张15中所述的琼脂糖包被的96孔板中。八种不同的化合物与6系列稀释液加媒体和车辆分别筛选其用10nM和在重复的20nM的HSP90抑制剂单一和组合的效果。两个球体分别用于单独的化合物或组合的化合物的各浓度。四个96孔板中，总384的球状体中使用，所有的球状体进行成像，在0，72，144，168，和192小时。总共1920图像制作从这个实验，花了SpheroidSizer仅30分钟即可完成1,920图像的计算分析与另外50分钟的质量控制和数据导出。SPHEROidSizer加快图像分析过程中的巨大。 图5A显示了一个屏幕截图的文件夹的安排和文件名 ​​这个实验作为协议步骤3.3一个例子。 图5B-E显示的图像分析弹出窗口和结果的屏幕截图。使用SpheroidSizer作为插图协议步骤4，5和7以三维球体的体积从格式化的结果表从SpheroidSizer出口，我们做了图表 - 在复合疗法的3D球体瘤生长与治疗时间。从这个实验中两个有代表性的图形示于图5F和5G。图5F显示了HSP90抑制剂和克拉屈滨（绿线）的组合治疗抑制的三维球状体以上的单一治疗HSP90抑制剂（紫线）或克拉屈滨（橙色线），这表明增长了HSP90抑制剂的联合治疗和克拉屈滨可能具有抗肿瘤EF体内 fects。 图5G显示了HSP90抑制剂和阿霉素（绿线）的组合疗法不抑制三维成长球状体以上的单一治疗阿霉素（橙色线）或HSP90抑制剂（紫线），这表明HSP90抑制剂和阿霉素的组合治疗方法可能不具有体内抗肿瘤效果。这个实验有助于我们更好地选择化合物，以测试其体内抗肿瘤作用和SpheroidSizer的关键是迅速实验数据分析。 <img alt="表1" fo:content-width="6in" src="/files/ftp_upload/51639/51639table1.jpg" width="600" /> 表1人工测量和SpheroidSizer在分析同一组288的图像。之间的图像分析。手术时间比较 <a href="https://www.jove.com/files/ftp_upload/51639/51639table1.jpg" target="_blank">请Ç</a>舔这里查看此表的放大版。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51639/51639fig1highres.jpg" src="/files/ftp_upload/51639/51639fig1.jpg" /> 图1 SpheroidSizer - 。的主动轮廓算法用于测量球体的大小一个开放源代码的软件应用程序A）应用程序B中的核心工作流程）的插图在迭代中的不同阶段。请注意，初始化轮廓（迭代0）被故意放大，以展示该算法。C）的主要和次要的眼轴长度测量，并通过SpheroidSizer体积的计算。 L - 长轴：线段连接一对轮廓（简称长）的最远点; W - 短轴：最长的线垂直于主轴线（称为宽度）。 <p<html>类=“jove_content”FO：保持together.within页=“总是”&gt; <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51639/51639fig2highres.jpg" src="/files/ftp_upload/51639/51639fig2.jpg" /> 球体与坏死的核心图2。从SpheroidSizer的自动分割，表现出对各种图像条件下的鲁棒性代表性的结果。A）典型的图像质量好B）具有不同的亮度和对比度的图像。C）和分散注意力的碎片图像D）图片。在各图的顶部面板的图像是源/原始图像;在每个图的下面板的图像是质量控制的图像;而红色轮廓是由自动化计算得出的球体分割。 hres.jpg“SRC =”/ files/ftp_upload/51639/51639fig3.jpg“/&gt; 图3插图的“手动初始化”和“手绘图”工具。一）“手动初始化”工具使整个球体一个合适的椭圆形初始化的绘制，当不准确的球体分割的自动初始化后出现：B ）的“手绘图”工具可以让球体边界的准确手绘图，当不准确的球体分割发生自动和手动初始化。围绕球体的蓝线显示的初始化轮廓;红色轮廓是确定球体的边界。请注意，在“手动初始化”中A）和B中“手绘图”球体）椭球体是故意放大，以更好地展示工具。 <img alt="图4" fo:content宽度=“5英寸”FO：SRC =“/ files/ftp_upload/51639/51639fig4highres.jpg”SRC =“/ files/ftp_upload/51639/51639fig4.jpg”/&gt; 图4：在分析同一套24幅图像SpheroidSizer和手动测量之间的图像分析性能的比较。 A）代表性的球状体，以显示如何球状体的长度和宽度是由人工测量和SpheroidSizer确定。前24图像包含在使用手动测量红线每个球体的手绘长/宽;较低的24幅图像（同24幅图像）包含使用SpheroidSizer B）的长度或宽度从各个球体三次测量的标准偏差在红色轮廓的计算机绘制球体边界。 <img alt="图5" fo:content-width="6in" src="/files/ftp_upload/51639/51639fig5highres.jpg" width="600" /> 图5：利用球体的一个典型的例子粒度仪在药物筛选-从药物屏幕采用BON-1三维肿瘤球体A）屏幕截图的文件夹的安排和文件名 ​​这个项目B）屏幕截图先进的收集，该球体“图像的图像分析在SpheroidSizer C）的配置窗口的屏幕快照SpheroidSizer1.0窗口，并显示结果表。D）屏幕截图SpheroidSizer E）导出的格式输出文件的屏幕截图SpheroidSizer，F出口列表输出文件）后与HSP90抑制剂和克拉屈滨在与HSP90抑制剂和阿霉素的治疗肿瘤的三维球体。G）增长治疗肿瘤的三维球体生长。 <a href="https://www.jove.com/files/ftp_upload/51639/51639fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> &lt;/ P&gt;

Watch this Scientific Journal Video about High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately at JoVE.

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

我们提出了一种高通量的图像分析软件应用程序来测量成像的明视场显微术的三维肿瘤球状体的尺寸。此应用程序提供了快速和有效的方式来研究治疗药物的球体，这有利于研究人员谁希望使用球体在药物屏幕的效果。

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 ...

high-throughput-image-analysis-tumor-spheroids-user-friendly-software

Research

JoVE Journal

Biology

24.8K 次观看.  Raymond and Beverly Sackler Foundation, New Jersey. 我们提出了一种高通量的图像分析软件应用程序来测量成像的明视场显微术的三维肿瘤球状体的尺寸。此应用程序提供了快速和有效的方式来研究治疗药物的球体，这有利于研究人员谁希望使用球体在药物屏幕的效果。 

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

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

科研

生物学

Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects

Measuring fruit morphology and color traits of vegetable and fruit crops in an objective and reproducible way is important for detailed phenotypic analyses of these traits. Tomato Analyzer (TA) is a software program that measures 37 attributes related to two-dimensional shape in a semi-automatic and reproducible manner1,2. Many of these attributes, such as angles at the distal and proximal ends of the fruit and areas of indentation, are difficult to quantify manually. The attributes are organized in ten categories within the software: Basic Measurement, Fruit Shape Index, Blockiness, Homogeneity, Proximal Fruit End Shape, Distal Fruit End Shape, Asymmetry, Internal Eccentricity, Latitudinal Section and Morphometrics. The last category requires neither prior knowledge nor predetermined notions of the shape attributes, so morphometric analysis offers an unbiased option that may be better adapted to high-throughput analyses than attribute analysis. TA also offers the Color Test application that was designed to collect color measurements from scanned images and allow scanning devices to be calibrated using color standards3. 
TA provides several options to export and analyze shape attribute, morphometric, and color data. The data may be exported to an excel file in batch mode (more than 100 images at one time) or exported as individual images. The user can choose between output that displays the average for each attribute for the objects in each image (including standard deviation), or an output that displays the attribute values for each object on the image. TA has been a valuable and effective tool for indentifying and confirming tomato fruit shape Quantitative Trait Loci (QTL), as well as performing in-depth analyses of the effect of key fruit shape genes on plant morphology. Also, TA can be used to objectively classify fruit into various shape categories. Lastly, fruit shape and color traits in other plant species as well as other plant organs such as leaves and seeds can be evaluated with TA.

Tomato Analyzer (TA) quantifies attributes of two dimensional shapes and color in a reproducible and accurate manner. A step-by-step procedure for obtaining high quality digitalized images of tomato fruit, morphological and color analyses of these images and several applications using the data generated through this software are described.

番茄分析器：一个有用​​的软件应用，收集准确和详细的二维物体的形态和色度数据

Measuring fruit morphology and color traits of vegetable and fruit crops in an objective and reproducible way is important for detailed phenotypic ...

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 ...

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 ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

细菌生长的精确、高通量分析

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

Diagonal Method to Measure Synergy Among Any Number of Drugs

A synergistic drug combination has a higher efficacy compared to the effects of individual drugs. Checkerboard assays, where drugs are combined in many doses, allow sensitive measurement of drug interactions. However, these assays are costly and do not scale well for measuring interaction among many drugs. Several recent studies have reported drug interaction measurements using a diagonal sampling of the traditional checkerboard assay. This alternative methodology greatly decreases the cost of drug interaction experiments and allows interaction measurement for combinations with many drugs. Here, we describe a protocol to measure the three pairwise interactions and one three-way interaction among three antibiotics in duplicate, in five days, using only three 96-well microplates and standard laboratory equipment. We present representative results showing that the three-antibiotic combination of Levofloxacin + Nalidixic Acid + Penicillin G is synergistic. Our protocol scales up to measure interactions among many drugs and in other biological contexts, allowing for efficient screens for multi-drug synergies against pathogens and tumors.

In this protocol, we describe how to make Loewe additivity-based drug interaction measurements for pairwise and three-way drug combinations.

测量任意数量药物协同作用的对角方法

A synergistic drug combination has a higher efficacy compared to the effects of individual drugs. Checkerboard assays, where drugs are combined in many ...

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

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 studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

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 ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

微殖器的高通量实时成像，测量生长和基因表达中的异质性

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...