这项工作是由美国国立卫生研究院（U54CA143906到KGP，OJTM和R01HL101972到OJTM）和俄勒冈州医学研究基金会早期临床研究奖（KGP）资助。 OJTM是美国心脏协会研究者（13EIA12630000）。我们感谢奈特癌症研究所的埃里克·安德森博士准备在这项工作中使用的细胞样本。

1。显微镜规格开展成像在正确的时尚显微镜应具有以下规格: <ol><li>同时具有微分干涉对比（DIC）和明场（BF）的对比。 </li><li>有电脑控制Z轴移动。 </li><li>有一个可调节的光圈来改变聚光透镜的数值孔径。具有渐变的规则或电子读出的孔是需要知道的数值孔径的值。数值孔径应为0.1的NIQPM高达0.9（或更高版本）HTDIC。 </li><li>在显微镜应该有一个窄带滤色器被用于明场成像。此过滤器是必需的，以固定的折射率增量，用于将相位质量密度为NIQP一个波长相关的骨折。 </li></ol> 2。微分干涉对比（DIC）的Z堆栈收购<ol><li>打开SlideBook软件ND创建图像采集一张新的幻灯片。 </li><li>接下来，打开焦点的窗口。根据过滤器设置部分，选择DIC。在Scope选项卡，在冷凝段，调整光圈滑动条向右最远的位置（打开所有的方式）。这提供了高数值孔径的照明，提高了样品的光学切片。 </li><li>专注于利用DIC的目标样本。调整卤素灯的强度，直到所述样品是容易可见。为了确保相机不被饱和，选择聚焦窗口的摄像头选项卡，检查像素强度的直方图是相机的动态范围内。 </li><li>打开图像捕捉窗口。在捕获类型部分，选中3D方块。在3D拍摄部分，选择围绕中心范围 ，并返回到当前位置 </stron克&gt;框和指定的Z方向范围内，平面的数目，和步长大小。 Z方向的范围应包括整个样本。 </li><li>在图像捕获窗口的过滤器设置部分，选中DIC框中，指定曝光时间。在图像信息部分，命名图像（可选），然后选择Start开始成像。 </li></ol> 3。明场（BF）Z堆栈收购<ol><li>一旦显微镜完成收集DIC Z堆栈的样品，打开聚焦窗口，然后选择打开下的过滤器设置部分。调整光圈滑动条最左侧位置（关闭所有的方式），以提供低NA照明。 </li><li>改变显微镜光路滤波器，以绿色。调整卤素灯的强度，直到样品是可见的。确保有通过选择不饱和摄像机的<st荣&gt;摄像机选项卡中的焦点窗口和检查像素强度的直方图是在指定范围内。 </li><li>打开图像捕捉窗口。从DIC Z堆栈采集3D捕捉设置将被显示。 </li><li>在图像捕获窗口的过滤器设置部分，选中打开框中，指定曝光时间。在图像信息部分，命名图像（可选），然后选择Start开始 Z堆栈的图像采集。 </li></ol> 4。导出Z堆栈图片<ol><li>打开Z堆栈捕获。将视图改为100％。调整也可以在0显示的象素值的直方图和相机的最大象素值（4095为12位的相机，65535为16位）。 </li><li>选择视图 &gt; 导出 &gt; TIFF系列 。这将Z堆栈导出为一系列TIFF图像（每个Z平面）。保存为TIFF系列在一个单独的文件夹中，有一个底线末（ 即 「DIC堆栈1_」）的名称。每个DIC或BF Z堆栈重复此。 </li></ol> 5。容积测量<ol><li>打开名为"JoVE_HTDIC_v1.m"的HTDIC MATLAB程序。 </li><li>根据HTDIC程序段0，更新的依赖性目录变量。复制并粘贴含有以下"dependencies_directory ="的hilbert_transform_dic.m和sobel_edge_detect.m文件从资源管理器中的单引号之间（PC）的目录。该JoVE_HTDIC_v1.m程序ExecuteSection 0。 </li><li>在第1，更新了"images_directory"。再次，复制和粘贴包含在单引号之间英寸TIF形式，通过聚焦图像的目录。只运行第1节一次。 </li><li>校准和图像希尔伯特的旋转变换运行的代码段2。将出现一个对话框，标题为"定义HTDIC参数"。五NUMBERS都需要从用户:焦平面数，其中样品的DIC图像处于焦点，横向分辨率（微米/像素的图像），轴向分辨率（这是0.1图像采集在0.1微米轴向步骤），的DIC图像的旋转角度需要进行希尔伯特变换，典型值是45和-135。最后，输入的兴趣大小的区域，这个参数定义了随后将出现一个框的侧面的长度 - 典型值为400。 单击 OK。 </li><li>由焦平面号指定的DIC焦平面的图像会出现一个蓝色的盒子。定位框在感兴趣的功能， 例如一个细胞。盒子不一定是正方形。一旦盒子已被定位在期望的区域，双击箱内。 </li><li>另一个数字将会出现:这个数字包含在上一步中选择感兴趣的区域的裁剪和旋转图像。图像的对比度应该是这样的黑暗的特征出现在左边而明亮的功能出现在右边。拖动蓝色方块在该地区的利益，重塑是必要的。 </li><li>部3生成的掩模用于Z堆叠立方体。有两种类型的口罩可供选择:一个矩形遮罩来创建单元格周围的矩形和一个手绘工具，通过手工勾勒感兴趣的细胞。 </li><li>要生成一个矩形遮罩，取消线167和注释行170（通过在该行的开头放置一个"％"注释行了）。执行程序的第3节。在图像中单击并拖动鼠标以开始定义的矩形遮罩。双击复选框以接受它。 </li><li>要生成一个手绘面具，注释行和167行取消注释170，并执行第3节。单击并绘制所需的口罩用鼠标。双击口罩接受它。 </li><li>运行第4到构建希尔伯特变换的DIC图像堆栈和感兴趣的区域对应的DIC图像堆栈。在第3部分构成的掩模将被应用到希尔伯特变换DIC堆栈和当"maskON"被设定为1的行179的常规DIC堆栈。设置"maskON"为0将兴建图像堆栈不应用蒙版。 </li><li>执行第5优化的感兴趣区域的xz横截面图像的图像的分割。图500中，由程序生成时，会出现显示了三种不同类型的对比。该算法找到的单元格的边框的成功是依赖于所使用的面具和"门槛"在程序的229行中的值的组合。首先0.5的值。调整阈值，然后重新运行本节中的程序，直到正确的大纲是在其中一列来实现。 </li><li>如果大纲是最好的在第1列，使用6节使用的DIC图像分割，以确定该卷。如果第2列给出最佳的结果，执行第7节确定从希尔伯特变换的DIC图像细胞体积。如果列3 GAVe化效果，运行第8采用傅立叶过滤希尔伯特变换的DIC图像来确定音量。 </li><li>样品的测量量，报告在立方微米（FL）是在图600中，700或800的通过根据该程序在其上的测量量被选择制作的标题。 </li></ol> 6。质量测量<ol><li>打开名为"JoVE_NIQPM_v1.m"的NIQPM MATLAB程式。 </li><li>根据NIQPM程序段0，更新运行程序需要三个目录的位置。这些是"dependencies_directory，"该"brightfield_directory，"和"dic_directory"。 </li><li>接下来，运行第1节。这部分代码生成整个图像集满场的亮场图像立方体。只运行第1节一次。 </li><li>接下来，运行第2节。将出现一个对话框，标题为"定义NIQPM参数"。四个数字需要从用户:焦平面数，其中的亮场图像样品是在焦点，横向分辨率（微米/像素的图像），轴向分辨率（这是0.1进行图像采集在0.1微米轴向步骤），以及兴趣大小的区域中，这个参数定义的一个边的长度这也将随之出现对话框 - 一个典型的值是200。 单击 OK。 </li><li>由焦平面号指定的亮场焦平面的图像会出现一个蓝色的盒子。 <ol><li>如果图像不在焦点上，双击在蓝色方块并重新运行第2节时，一定要调整焦平面数字在对话框中。 </li><li>如果图像处于焦点，拖动蓝色方块中的图像四周，通过选择框的节点，并根据需要拖动调整它的大小。定位框周围感兴趣的功能， 例如一个细胞。盒子不一定是正方形。双击箱内接受它。 </li></ol></li><li>接下来，运行第3节构建一个栈的明场图像裁剪为在该地区票面​​。 </li><li>要生成相位图，伪DIC图像和比较，以明场图像和真实DIC图像，执行第4节。 </li><li>随着伪DIC和DIC的真实图像尽可能相似的质量密度图，总质量，而细胞密度的直方图可以通过运行第5A或5B来确定。第5A进行自动化边境检测领域 - 优化"门槛"行300 - 典型值0.1〜1之间变化。如需要优化的阈值重新​​运行本节。第5B开展群众性测定等用户勾勒感兴趣的细胞后。 </li></ol>

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Lett. 30, 2131-2133 (2005).</li><li>Wang, Z., Millet, L., Mir, M., Ding, H., Unarunotai, S., Rogers, J., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+light+interference+microscopy.">Spatial light interference microscopy.</a> SLIM). Opt. Exp. 19, 1016-1026 (2011).</li><li>Ikeda, T., Popescu, G., Dasari, R. R., Feld, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hilbert+phase+microscopy+for+investigating+fast+dynamics+in+transparent+systems.">Hilbert phase microscopy for investigating fast dynamics in transparent systems.</a> Opt. Lett. 30, 1165-1167 (2005).</li><li>Shaked, N. T., Rinehart, M. T., Wax, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-interference-channel+quantitative-phase+microscopy+of+live+cell+dynamics.">Dual-interference-channel quantitative-phase microscopy of live cell dynamics.</a> Opt. Lett. 34, 767-769 (2009).</li><li>Popescu, G., Park, Y., Lue, N., Best-Popescu, C., Deflores, L., Dasari, R. R., Badizadegan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+cell+mass+and+growth+dynamics.">Optical imaging of cell mass and growth dynamics.</a> Am. J. Physiol. Cell Physiol. 295, (2008).</li><li>Arnison, M. R., Cogswell, C. J., Smith, N. I., Fekete, P. W., Larkin, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+Hilbert+transform+for+3D+visualization+of+differential+interference+contrast+microscope+images.">Using the Hilbert transform for 3D visualization of differential interference contrast microscope images.</a> J. Microsc. 199, 79-84 (2001).</li><li>Baker, S. M., Phillips, K. G., McCarty, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+label-free+imaging+technique+for+the+quantification+of+thrombus+formation.">Development of a label-free imaging technique for the quantification of thrombus formation.</a> Cell. Mol. Bioeng. 5, 488-492 (2012).</li></ol>

作者在介绍的工作的任何财务权益。

在一般情况下，NIQPM是验证的光路长度从0.25-44.7受衍射限制的技术。验证了n个执行= 1.596聚苯乙烯球从0.11-9.8微米的悬浮在Fluoromount摹直径范围（数据未显示）。细胞具有光路长度，范围从近0-7。 当测量试样的密度分布人们可以发现，伪DIC图像看起来很好，而密度地图拥有多余的背景贡献。这是由于在作为输入到NIQPM算法的样本的亮场图像的噪声。两个可能的修改，NIQPM方法可?...

利用光学显微镜是目前普遍存在于细胞生物的调查。由于在可见光谱的低内源性吸光度和弱散射性质，细胞不强烈影响光波穿过它们的幅度，因此，当与标准的明视场显微镜成像的出现半透明。细胞标本做，但是，减慢光波通过他们行进在该线性关系的局部质量密度的空间，通过该光行进的特定区域的量的方式进行。这种异构的时间滞后或通过显微标本传输光波的"相"配置文件的利用率在1935年首次描述了弗里茨泽尼克1和Zernikein 1942年2实验实现。泽尼克被授予1953年诺贝尔经济学奖的这一成就。蔡司在1945年3商业化这种方式。 1955年，史密斯和Nomarsk我会介绍他们对使用微分干涉对比（DIC）显微镜，使用相位的空间梯度作为对比机制模式4和5的理论初步工作。 DIC是在1965in与诺马斯基6密切合作商业化蔡司。 1981年，两个实验室展示了第一个记录活细胞的DIC图像与视频摄像机纳入DIC显微镜7,8的光学列车。活细胞成像的时代诞生了。 因为这个时候，两个阶段的对比与DIC的商业显微镜的执行在很大程度上是不变的。这些方法由生物学家，主要用于生产细胞图像进行定性用途:监控形态，亚细胞结构的跟踪，动态膜9的调查。这些技术是定性在他们的"现成的，现成的"配置相位和DIÇ图像的光源强度的任意函数，该照明光学部件的设置，以及CCD摄像机增益，γ和曝光设置。 一个小军团物理学家和光学工程师一直在努力做商业成像方式定量。其中第一个努力是两个字母，以自然在1952年和1953年，其中医师出身的生物物理学家罗伯特更加光秃展示了使用相差显微镜的使用市售的相差显微镜通过这些细胞类型估计的相移，以确定细胞的细胞干重10，11。该领域已经开发出了众多围绕三个基本无标记的对比机制在随后几年的技术:相显微镜10,11，DIC显微镜12-17，和明视场18-22以确定光路长度，相位，质量密度，折射率，和细胞体积。 在第llel，收集了大量的自定义光学仪器也已自20世纪50年代开发的，并取得了深远的光学测量范围从虫体增长23应用程序，以记录细胞周期24到调查红细胞25膜动力学。特别是，在过去十年已经看到了大量的无标记定量显微镜的衍射相显微镜26的形式，断层相显微镜27，数字全息显微术28，相位敏感光学相干显微镜29，空间光干涉显微镜30，希尔伯特相差显微镜31，和定量相显微镜32。尽管他们的集体成就，这些仪器没有被分发给由于居多，他们复杂的仪器和计算要求的生物研究人员的大场。 在此我们ð问:描述使用标准光学显微镜通过明场和DIC图像的组合来进行质量，体积和密度对细胞样品的定量测量。两种主要的方法都:noninterferometric定量相显微镜（NIQPM），进行总细胞团和亚细胞密度分布的测量，以及希尔伯特变换微分干涉对比显微镜（HTDIC），以确定音量。 NIQPM和HTDIC的主要优势在于他们的技术可访问性。需要他们的成功执行的成像条件是大部分市售显微镜正常操作范围之内。此外，后处理算法是稳定的，快速的和健壮 - 用尽可能快速傅立叶变换（FFT）的算法在MATLAB已经实现。 NIQPM是一种方法来重构相和CEL的轴向集成的质量密度lular标本从明场图像。此轴向集成质量密度在样品的面积的总和给出了样品的总干质量的内容。该NIQPM协议是基于Paganin和纽金特18,19奠定了实验基础-在它被证明，细胞的相位分布可以从样品通过聚焦亮场图像重建-和弗兰克的理论工作，Altmeyer和韦尼克20 -解决了旁轴波动模型在一个高效的基于FFT的方式。的相位，以干质量密度的连接是通过更加光秃10,11和波佩斯库33基于该工作。 体积信息可以从通过聚焦DIC图像高NA的照明条件下，使试样的光学切片沿着光轴下获得。希尔伯特变换处理的DIC图像堆栈大大提高边缘检测算法来在三维空间中的样本边界，通过分离从背景的检体强度的灰度值的定位。与Arinson 等 34本作品源自虽然我们已经介绍了两种傅里叶滤波方法，以提高对比度和样品的体积自动分析Sobel算子的边缘检测方法。我们在聚苯乙烯球大小不等，从衍射极限可达20微米，直径36也验证HTDIC以前。 尽管这两个NIQPM和HTDIC是由于其在商业显微镜技术的发展方便，该方法是由显微镜本身的硬件配置根本的限制。这些技术的主要局限性是双重的:慢Z堆叠的采集时间在商业领域中，由于对整个样品台的翻译，而不是仅仅在物镜目前限制现象的调查比大约1帧/分钟速度更快，其次，衍射效应限制NIQPM和HTDIC的实用程序对象大小不等，从0.2上升到10和20微米，直径分别。因此，感兴趣的样品及其相关的动态时间必须达到一定的规模和时间上的限制，使典型的"现成的，现成的"工具使用这些方法。令人兴奋的是，大多数固定细胞标本很容易调查，这些方法。 该NIQPM和HTDIC协议的概述示于图1。在图2中 ，我们说明了最优和次优通过聚焦成像低和高NA的照明条件下为亮场和DIC图像。 图3和图4展示了参数NIQPM算法的依赖性突出成功的和不成功的实现。 <strong&gt;图5示出涉及HTDIC图像处理算法的步骤和说明了最佳实施的算法，以确定细胞的体积。

<table><tbody><tr><td>Zeiss Axio Imager 2 microscope</td><td>Carl Zeiss MicroImaging GmbH, Germany</td><td>Axio Imager D2</td><td>Microscope</td></tr><tr><td>Green filter (&amp;lambda; = 540 &amp;plusmn; 25 nm)</td><td>Chroma Technology Corp., Bellows Falls, Vermont</td><td>D540/25x</td><td>Green filter</td></tr><tr><td>SlideBook 5.5 software</td><td>Intelligent Imaging Innovations, Denver, Colorado</td><td></td><td>Image acquistion software</td></tr><tr><td>Polystyrene microspheres</td><td>Bangs Laboratory, Inc., Fishers, IN</td><td>PS06N</td><td>Polystyrene spheres</td></tr><tr><td>Fluoromount-G</td><td>SouthernBiotech, Birmingham, Alabama</td><td>0100-01</td><td>Mounting media</td></tr></tbody></table>

quantitative optical microscopy: measurement of cellular biophysical features with a standard optical microscope

我们描述了使用标准的光学显微镜通过明场和微分干涉对比意象的组合来执行质量，体积和密度对细胞样品的定量测量。两种主要的方法都:noninterferometric定量相显微镜（NIQPM），进行总细胞团和亚细胞密度分布的测量，以及希尔伯特变换微分干涉对比显微镜（HTDIC）来确定音量。 NIQPM是基于波传播的简化模型，称为傍轴近似，有三个基本假设:低数值孔径（NA）照明，弱散射光由标本弱吸收。幸运的是，未染色的细胞标本满足这些假设和NA低光照很容易在商业显微镜实现。 HTDIC是用来从下高NA光量的离焦的DIC图像获得体积信息振动性条件。高NA的照明使试样的增强切片沿着光轴。希尔伯特变换处理的DIC图像上叠加大大通过分离从背景的检体强度的灰度值提高边缘检测算法来在三维空间中的样本边界的定位。 NIQPM和HTDIC的主要优势在于他们的技术辅助使用"现成的，现成的"显微镜。有这些方法的两个基本限制:慢Z-堆栈采集时间在商业领域目前废除现象的调查比1帧/分钟的速度更快，其次，衍射效应限制NIQPM和HTDIC的实用程序对象从0.2上升到10 （NIQPM）和20（HTDIC）微米的直径，分别为。因此，感兴趣的试样和其相关联的时间动力学必须满足一定的大小和时间的限制，以便能够使用这些方法。令人兴奋的是，大多数固定cellulař标本很容易调查，这些方法。

通过聚焦图像采集过程中正确的样品照明是成功实施的NIQPM 一个次HTDIC算法的关键。在图2中 ，我们说明了低，高NA的照明既DIC和亮场对比度聚苯乙烯球和人的结肠腺癌细胞株SW620下。 图2A，2C，2I和2K展示的NIQPM最佳成像。 图2F，2H， 2N，而2P展示的HTDIC最佳成像。 图3和图4展示了参数的NIQPM...

Watch this Scientific Journal Video about Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope at JoVE.com

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

定量的光学显微镜:测量细胞生物物理特性与标准光学显微镜

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

quantitative-optical-microscopy-measurement-cellular-biophysical

Research

JoVE Journal

Bioengineering

15.5K 次观看.  Oregon Health & Science University, School of Medicine. We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

视频: Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

定量荧光测量全球本地化翻译

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

科研

生物化学

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

三维肿瘤球体的纵向形态和生理监测

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

癌症研究

Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living cells. The proposed instrument extends the sensitivity of label-free optical microscopy to nanoscale changes in organelle size and shape and can be used to accelerate the study of the structure-function relationship pertaining to organelle dynamics underlying fundamental biological processes, such as programmed cell death or cellular differentiation. The microscope can be easily implemented on existing microscopy platforms, and can therefore be disseminated to individual laboratories, where scientists can implement and use the proposed methods with unrestricted access.
The proposed technique is able to characterize subcellular structure by observing the cell through two-dimensional optical Gabor filters. These filters can be tuned to sense with nanoscale (10's of nm) sensitivity, specific morphological attributes pertaining to the size and orientation of non-spherical subcellular organelles. While based on contrast generated by elastic scattering, the technique does not rely on a detailed inverse scattering model or on Mie theory to extract morphometric measurements. This technique is therefore applicable to non-spherical organelles for which a precise theoretical scatter description is not easily given, and provides distinctive morphometric parameters that can be obtained within unstained living cells to assess their function. The technique is advantageous compared with digital image processing in that it operates directly on the object's field transform rather than the discretized object's intensity. It does not rely on high image sampling rates and can therefore be used to rapidly screen morphological activity within hundreds of cells at a time, thus greatly facilitating the study of organelle structure beyond individual organelle segmentation and reconstruction by fluorescence confocal microscopy of highly magnified digital images of limited fields of view.
In this demonstration we show data from a marine diatom to illustrate the methodology. We also show preliminary data collected from living cells to give an idea of how the method may be applied in a relevant biological context.

We demonstrate a dark-field microscopy method based on Gabor-like filtering to measure subcellular dynamics within single living cells. The technique is sensitive to alterations in the structure of organelles, such as mitochondrial fragmentation.

基于二维Gabor滤波器的光散射显微镜

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living ...

生物学

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

使用光学镊子测量禁闭中的亚细胞力学的直接力

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

发育生物学

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

生物工程

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

无透镜视频显微术在黏附细胞培养动态定量分析中的研究

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Quantitative Analysis of Viscoelastic Properties of Red Blood Cells Using Optical Tweezers and Defocusing Microscopy

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not only attributed to the normal variability of cells, but also to the differences in methods and models of cell response. Here, an integrated protocol using optical tweezers and defocusing microscopy is employed to obtain the rheological features of red blood cells in the frequency range of 1 Hz to 35 Hz. While optical tweezers are utilized to measure the erythrocyte-complex elastic constant, defocusing microscopy is able to obtain the cell height profile, volume, and its form factor a parameter that allows conversion of complex elastic constant into complex shear modulus. Moreover, applying a soft glassy rheology model, the scaling exponent for both moduli can be obtained. The developed methodology allows to explore the mechanical behavior of red blood cells, characterizing their viscoelastic parameters, obtained under well-defined experimental conditions, for several physiological and pathological conditions.

Here, an integrated protocol based on optical tweezers and defocusing microscopy is described to measure the rheological properties of cells. This protocol has wide applicability in studying the viscoelastic properties of erythrocytes under variable physio-pathological conditions.

使用光学镊子和散焦显微镜定量分析红细胞的粘弹性

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not ...

Imaging Biological Samples with Optical and Confocal Microscopy

Source: <a href="https://www.jove.com/author/Peiman_Shahbeigi-Roodposhti">Peiman Shahbeigi-Roodposhti</a> and Sina Shahbazmohamadi, Biomedical Engineering Department, University of Connecticut, Storrs, Connecticut
Optical microscopes have been around for centuries, and while they reached their theoretical limitation of resolution decades ago, new equipment and techniques, such as confocal and digital image processing, have created new niches within the field of optical imaging. The best optical microscopes will typically have a resolution down to 200 nm in ideal conditions. However, optical microscopes are limited by the diffraction of waves, a function of the wavelength, which is around 500 nm for visible light. While the resolution of optical microscopes does not reach that of electron microscopes, they are the most valuable tools in the imaging of biological macrostructures and are a staple in any biological lab.
In conventional light microscopes, the signal produced from the imaged object is from the full thickness of the specimen, which does not allow most of it to be in focus to the observer. This causes the image to have &#34;out of focus blur&#34;. The confocal microscope, on the other hand, illuminates the sample through a pin-hole, and is therefore able to filter out the out-of-focus light from above and below the point of focus in the object.
This demonstration provides an introduction to image acquisition using optical and confocal microscopy methods. Here, a sectioned piece of mouse brain will be studied.&#160; Image acquisition and analysis, including the tools to generate topographical maps and composite images, will be covered. The advantages and disadvantages of different imaging methods as they relate to resolution, depth of focus and sample type will also be discussed. The purpose of this demonstration is to provide more information on optical and confocal microscopes to determine if these microscopy modules are the best fit for a type of biological sample.

Biological Sample Imaging with Optical and Confocal Microscopy

使用光学和共聚焦显微镜成像生物样本

Source: Peiman Shahbeigi-Roodposhti and Sina Shahbazmohamadi, Biomedical Engineering Department, University of Connecticut, Storrs, Connecticut
Optical ...