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 border="0" cellpadding="0" cellspacing="0" width="675">
	<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 (&#955; = 540 &#177; 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

15.4K 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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

阿的Cre - LOX P为检测细胞融合重组方法在体内</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

在体外合成的原住民，纤维长间距“和”分段长间距胶原蛋白

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

支持的脂双层云母制备的高分辨率光学显微成像

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

含有宏观结构的光学组织幻影的制备与表征

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

光片荧光显微术在小鼠心脏研究中的应用

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...