作者感谢琳达·特纳与微生物制剂的援助。 RZ和LGW是由罗兰大学，哈佛大学和CGB资金资助的短斗篷​​基金会的学者，科学无国界计划，巴西（进程＃7340-11-7）

1。设置和数据采集<ol><li>从冻结的股票增长22 链球菌菌株V4051-197（平滑游泳）细胞在KTY介质的文化。孵育在旋转摇床上过夜，在35℃和150rpm下，以饱和。 </li><li>接种10ml新鲜KTY培养基用500μl的饱和培养物。孵育另外的3.5小时，在35℃和150rpm下，直到细胞在λ= 600纳米（约5×10 8个细胞/ ml），达到约1.0的光密度。 </li><li>稀释1:400在新鲜培养基中，得到游动细胞的最终浓度。 </li><li>在显微镜载玻片上，使润滑脂的环（ 例如，从填充有凡士林注射器）约1毫米的高度，然后将一滴在中心的样品溶液。放置在顶部，轻压玻璃盖的边缘密封，确保液与两个滑动盖和玻璃接触。由此产生的样品室应该是阿柔ND 100-200微米的深度。 </li><li>将样品室中的盖玻璃朝下显微镜，并小心，以防止气泡的形成在油玻璃和透镜之间。 </li><li>重点放在样品室的底部表面的显微镜，并带来冷凝器进行对焦。 </li><li>关掉标准照明体和放置在LED头显微镜的聚光器孔径的后面。设置LED电源的最大输出。 </li><li>关闭冷凝器光圈到最大程度，并且如果需要，轻推在其安装的LED，直到照明的中心在上述物镜的数值孔径。 </li><li>切换电脑和相机，以及所有的光重定向到显微镜的相机端口。调整帧速率和图像尺寸的图像采集软件。 </li><li>使画面曝光时间越短越好，同时仍保持良好的对比度。检查图像亮度直方图，以保证图像不饱和特德或曝光不足。 </li><li>如果有必要，重新聚焦显微镜，让感兴趣的对象稍微散焦（通常为10-30微米）。在物体与焦平面应该在相同的培养基中（ 即焦平面应位于样品室室内）。 </li></ol> 2。重建处理数据的第一步骤是将数值重新调整视频帧在一系列不同的深度，产生一个堆栈的图像。用户友好的软件，这样做可以在这里找到: <a href="http://www.rowland.harvard.edu/rjf/wilson/Downloads.html">http://www.rowland.harvard.edu/rjf/wilson/Downloads.html</a>与示例图像（使用倒置显微镜获得的，和60X油浸物镜）和光线场景文件追踪渲染。 <ol><li>利益输入和帧其各自的背景为单独的图像文件，在该接口上的有关空格。背景图像是一帧使得fairly代表视频的背景下，在没有全息图，并且将用于抑制任何固定图案噪声可能干扰全息图的定位和分析。 </li><li>在运行程序之前将值输入到屏幕左侧的全局设置框。前三个是输出栈参数:第一帧的轴向位置（&#39;开始注重&#39;）;在重建切片堆叠数（&#39;步数&#39;）;各片之间在堆栈中的轴向距离（&#39;步长&#39;） 。 </li><li>为了重建与正确的比例的目的，步长应为相同的尺寸的横向的像素间距。在"像素/微米"框，光照波长和介质的折射率在过去两年框中输入摄像机的横向取样频率（1/pixel间距）。该程序的默认值适合重建的示例数据。 </li><li>按"翻转的Z-梯度"按钮，如果OBJE中心克拉是黑暗的全息图（见示例帧2005和讨论更多的细节部分）。 </li><li>检查带通滤波器开/关状态（默认为开启）。这个可选的带通滤波器，位于下方的渐变，翻转开关盒，负责抑制由噪声点的贡献。带通滤波器生成后立即应用到每一个图像切片。 </li><li>检查开/关状态（默认为上）中间输出开关。中间分析步骤都写在两个输出视频，未压缩的avi文件:第一是重新调整堆栈（文件名&#39;_stack.avi&#39;结尾），第二个是堆栈中的轴向梯度操作后（文件名以&#39; _gradient.avi&#39;）。理想情况下，这第二个堆栈将包含感兴趣的对象突出显示为对一个黑暗的背景明亮的物体。 </li><li>设置完成后在主窗口中的所有参数，按"运行"。选定的帧将出现在软件的主箱。 </li><li>使用工具栏将图像放大（左键）和缩小（按shift +左键）的左侧找到放大镜工具。使用工具栏上的矩形工具，选择感兴趣区域（ROI）的区域。捕捉尽可能多的全息图的边缘尽可能的矩形内，因为这将优化改造。 </li><li>按"进程"按钮，生成两个图像堆栈。检查使用ImageJ（可免费在获得由此产生的堆栈<a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</a> ）。如果感兴趣的对象可以清楚地看出，在梯度图像的堆栈，请继续下一步要提取的对象的坐标。 </li></ol> 3。翻译<ol><li>除了框架重新调整，这个程序可以定位在X，Y，Z中感兴趣的对象的坐标为每个体素。按"特征提取"按钮来启用该功能。 </li><li>输入一个路径，包括扩展名（例如:c:家 output.inc），为输出坐标文件。在"输出坐标式"对话框中选择"POV-Ray的风格"。这将导致程序编写可以使用免费的POV-Ray的光线跟踪软件包（可从以下地址获得可视化的对象文件<a href="http://www.povray.org/">http://www.povray.org/</a> ）。 </li><li>重新处理图像作为上文第2节。该方案将提供一系列的（X，Y，Z）在选定的ROI坐标，在"输出坐标"框写入的文件名。物体坐标的提取需要显著长于堆栈生成。 </li><li>确保样品POV-Ray的文件（与重构代码）是在同一文件夹中刚刚生成的坐标文件。编辑范例。POV文件，并在引号替换文件名就行了 ＃包括"MYFILE.inc" 与由重建代码所产生的数据文件的名称。 </li><li>点击POV-Ray的的"运行"按钮，呈现一个位图图像。 Ŧ他的相机位置，灯光和纹理选项只是一些在POV-Ray的可自定义的，详情参见联机文档。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

在这个实验方案中最重要的步骤是从一个稳定的实验装置的图像的精确捕捉。差的背景资料，高保真重建几乎是不可能的。同样重要的是，避免物镜与内部相位对比元件（如通过物镜的背面看时暗环形可见的），因为这样可以降低重建图像。感兴趣的对象应该是远远不够的焦平面那几双衍射条纹的可见其形象（一个合适的启发是，散焦图案应具有焦点的对象的10倍线性尺寸 - 见示例数据）。物体太近焦平面从双图像和抽样文物遭殃，如前所述。同样重要的是有相机的像素间距的良好特性，因为这是在正确地确定再聚焦距离的一个关键因素。通常情况下，相机说明文档离子会列出"像素大小"中的规范，这可有点暧昧，因为某些类型的摄像机（CMOS摄像头尤其是）可以有不属于光敏感像素之间的空间显著的地区。最可靠的方法来获得该信息是用一个标准的校准目标，如一个USAF 1951分辨率图表，并直接从图像测量的采样频率（每微米的像素数）。 在单色照明的限制，一个DIHM系统的分辨率由物镜（NA）和光照（λ）23,24的波长的数值孔径被最终设置。横向分离点应位于至少距离Δ 纬度 =&#39;λ&#39;/ 2NA分开，如果他们单独解决。类似地，在理想的情况下，轴向分辨率的限制是由"Δ" 斧头 =λ/ 2（NA）2给出。当使用在DIHM的LED，是有一定限度的深度可成像的体积，这是由该光学系统和照明的相干性最终被确定。这个"敏感量"的典型深度大约是100-200微米用于LED，但这种类型的效果的详细说明可以在其他地方25被发现。在敏感体积中的其它两个方向的图像的大小的限制。虽然软件是相当强劲，某些操作是相当的内存大户。重建图像堆栈并获得强度梯度都相当保守，但提取从梯度堆栈关注特征的坐标可以消耗大量的内存（几百兆到几千兆字节）。为此，最好是感兴趣的重构体限制为在每个维度100-150像素。这种限制可以在一定程度得到缓解，如果代码运行在64位操作系统上有超过4 GB的RAM（软件代码的编写一台机器有12 GB的），虽然计算时间会变得令人望而却步。 容易达毫米 - - 和在较低的放大倍率来检查一个较大的系统中，LED光源可以用激光，其允许重建的物体处在一更大的深度从焦平面的更换。用这样的设置初步实验中，使用激光二极管耦合到单模光纤，允许重构的深度达使用10X显微镜物镜10毫米。这增加的景深是有代价的，但是，激光的大型相干长度会导致图像中的噪声，从眼镜直通车各种表面尤其是反射这些影响所抵消（样品室，镜片表面等的墙壁。）当该装置是足够稳定以获得良好的背景图像。不过，多余的反射有一个未知分布的阶段，因此，如果其规模足以媲美参考波（未散射光），reconstru的CTION可以成为不可能。其他作者已经修改的激光光源的相干性，例如，通过调制激光驱动电流26或利用一个旋转的毛玻璃屏幕，以减少相干性27。 从一个故障点，大多数问题在使用全息重建的软件最好通过检查数据分析的中期阶段，解决遇到的问题。在重建的图像栈的情况下，该物体应"去模糊"对称，因为它们成为焦点。如果衍射条纹看起来非常不对称的，一个贫穷或偏移的背景图像往往是罪魁祸首。如果对重建的图像已被取自堆叠相似的图像，例如，从一个移动物体的一个视频序列的帧，背景图像，有时可以通过平均所有的视频​​获得（通过平均或中值）的像素值帧。如果拍摄对象的视频序列时大幅度移动时，其任何一个像素值的贡献较小，主要贡献将来自于不变的背景框架，其中的主题是缺席。当一个对象的坐标不正确返回的情况下，梯度图像堆栈可以成为一个有用的诊断。如果该对象被看作是由一个亮环的梯度图像堆栈包围的空隙，梯度过滤器应翻转和图像后处理。正如引言中所述，瑞利 - 索末菲重构方法是不敏感的，从焦平面的物体的距离的标志。如果两个弱散射的对象是从焦平面但在相对侧上的距离相同，它们将成为焦点，在重建图像中的堆叠中的相同位置上。然而，他们的轴向强度模式将得到扭转。原本以上焦平面（相对于在倒置显微镜几何形状）中发现的对象的中心变化，从浅到深上穿过在焦婆sition，而下面由暗焦平面改变物体点燃。因此，梯度翻转选项提取上述焦平面（默认）或低于焦平面或者对象（具有梯度翻转设置为"开"），给一个明确的轴向坐标。请注意，这绝对持有只对弱散射物体，该方法非常适用于聚苯乙烯微球可达1微米，直径，但不很适合较大的聚苯乙烯颗粒。生物样品通常表现出更弱散射，因为它们的折射率接近周围介质（聚苯乙烯的折射率接近1.5）的，所以较大的物体，可以研究。 在今后的方向而言，该软件可以被扩展以处理多个帧中的视频，以使游泳微生物或胶体颗粒的跟踪在三个方面。该技术能提供高帧速率非常适合于跟踪即使是最快的游泳聚体，如海洋中居住的溶藻弧菌 ，其游泳速度可达150微米/秒28。单个细胞的显微游泳轨迹进行跟踪的第一前一段时间29，但是这通常需要专门的设备。古埃相位异常接近，不仅本身借给单细胞长距离的简单的跟踪，但提供了机会，研究不同个体的游泳行为之间的相关性。这具体取决于立体几何，数据迄今一直无法进入出于技术原因。 正如引言中所述，用荧光共聚焦显微镜的比较不理想，但共聚焦系统的普及使得三维成像方面有价值的比较。事实上，DIHM互补的共聚焦显微镜;有两者的比较优势和劣势。 DIHM允许更快的数据习得施氮水平:每秒几千册是家常便饭，给定一个足够快的相机。这允许（例如）多个游动细菌，这是超越较慢的共聚焦系统的性能跟踪。即使有一个快速照相机，DIHM是幅度比共聚焦显微镜便宜的顺序，和一个三维体积中的数据可以被存储在一个单一的二维图像，减少了对数据的存储和备份需求。 DIHM更容易扩展的，即具有较低的放大倍数，较大的样本和更长的工作距离工作是微不足道的。共聚焦显微镜仍占上风密集或复杂样品中，但是。例如，在一个致密的胶态悬浮体，多个散射使得全息重建几乎是不可能的;共 ​​焦系统中任一荧光发射或反射模式会更适合于研究这种系统9。此外，共聚焦显微镜是用于实验的系统其中，f在更自然的选择luorescent标签是至关重要。虽然荧光全息术已被证明与一系列的测试工作科目30目前收集效率远远低于一个良好的共焦系统。此外，这种实验系统目前需要专业的光学仪器和经验来操作，希望他们会变得更加普及与发展。 总之，DIHM是无与伦比的其获取高分辨率的三维微观对象的图像，在高速行驶时的能力。我们提供用户友好的软件，使这项技术进入到以最小的改动现有显微镜的非专业。此外，重建软件与自由查看的计算机渲染软件轻松集成。这使得直观的可视化微观主体的，从任何角度观看。鉴于涉及。许多快速微观过程的立体性ING弱散射物体（ 如跳动的真核生物的鞭毛和纤毛，细菌游泳，或扩散胶体），这种技术应该是利益在广泛的领域。

内嵌数字全息显微镜（DIHM）允许显微镜的样品，如游泳微生物1,2和软物质系统3,4，以最小的修改一个标准的显微镜装置的快速三维成像。本文DIHM的教学演示提供，围绕在我们的实验室开发的软件中心。本文包括如何设置显微镜，优化数据采集，处理和记录的图像重建三维数据的描述。该软件（部分基于软件由DG格里尔和其他5开发）例如图像，且可自由查看我们的网站上。下面说明提供的配置显微镜，从全息图重建三维体积和使用一个免费射线跟踪软件包呈现所得的感兴趣体积所必需的步骤。本文总结了影响重建质量因素的讨论和DIHM与竞争方法进行了比较。 虽然DIHM被描述前一段时间（对于其原则和发展进行全面检讨，见金6），所需要的计算能力和图像处理专业技术迄今在很大程度上限制了它的使用，以专门研究小组，重点放在仪器的发展。这种局面正在改变在计算机和摄像头技术的最新进展的光。现代桌面电脑可以轻松应对处理和数据存储需求; CCD或CMOS摄像头存在于大多数显微镜实验室，以及必要的软件被免费提供在互联网上通过谁在开发该技术投入时间组。 各种方案已经被提出用于成像微观对象的配置中的三维样品体积。许多这些扫描技术7,8，其中一个堆栈的图像的记录由米通过样品echanically平移所述图像平面。激光扫描共聚焦荧光显微镜也许是最熟悉的例子。通常，荧光染料被添加到相位物体，以便实现样品的对比可以接受的水平，并在共聚焦装置用于空间定位的荧光发射。这种方法导致了显著的进步，例如在胶体科学它允许访问拥挤的系统9-11的三维动态。使用标签是荧光共聚焦显微镜和DIHM之间的一个重要差别，但两种技术的其他功能都值得比较。 DIHM具有显著速度上的优势在于，该装置没有移动部件。在共聚焦系统的机械扫描镜放置一个上限的数据采集率 - 通常大约30帧/秒的512×512像素的图像。可以通过物理译获得一摞这样的图像从不同的焦平面阿婷帧之间的样品台或物镜，导致每秒大约一体积为30帧堆栈的最终捕获率。在比较的基础上，现代CMOS相机的全息系统可以捕获2000帧/秒在相同的图像尺寸和分辨率，每个帧被`离线&#39;处理，得到样品体积的独立快照。重申一下:不需要荧光样品DIHM，虽然系统已经开发出来，进行荧光主体12的全息重建。以及三维体积信息，DIHM也可以被用来提供定量相衬图像13，但是这超出了本文的讨论范围之内。 原始DIHM数据图像是二维的，并且在某些方面看起来像标准的显微镜图像，虽然失焦。 DIHM和标准亮视野显微镜之间的主要区别在于，围绕物体的衍射环我n个视场，这些都是因照明的性质。 DIHM需要比亮场更连贯源 - 通常是LED或激光。在全息图上的衍射环含有重构的三维图像所需的信息。有两种主要的方法来解释DIHM数据，直接拟合和数值重新调整。第一种办法是适用的地方衍射花纹的数学形式事先已知3,4案件;这种情况被通过极少数样球体，缸，一半平面障碍物的简单对象遇见。直接拟合也可以适用于该对象的轴向位置是已知的情况下，图像可以使用的图像模板14中的查找表被安装。 第二种方法（数字重聚焦）是较为普遍，并依赖于使用的衍射环在二维全息图图像进行数值重构光场在一些（任意间隔）焦平面整个样品体积。这样做的6几个相关的方法存在，这项工作采用瑞利-索末菲反向传播技术所描述的李和格里尔5。这个程序的结果是一个栈，复制手动更改显微镜焦平面（因此得名&#39;数值重新调整&#39;）的效果的图像。一旦已经生成了一个堆栈的图像，必须获得在焦量被摄物体的位置。一个数字图像分析的启发式，如局部强度方差或空间频率内容的，已经呈现给量化聚焦的锐度在不同点的样品15中。在每一种情况下，当一个特定的图象量度被最大化（或最小化），该对象被认为是在焦点上。 不像其他的计划，寻求以识别特定的焦平面其中一个对象是"焦点"，在这项工作中的方法挑选输出Points的谎言感兴趣的对象内部，从而在广泛焦平面延伸。这种方法可以适用于广泛的主题，并且特别适合于延伸，弱散射样品（相位物体），如杆状胶体，细菌的链条或真核生物的鞭毛。在这样的样品，当对象穿过焦平面的图像的对比度改变;散焦图像具有光中心，如果该对象是在焦平面与暗中心的一侧上，如果它是在另一。纯相的对象几乎没有任何相反，当他们躺在正是在焦平面。这种现象对比反转的已经由其他作者16,17讨论，并最终由于的Gouy相位异常18。这已被放置在全息的上下文中更严格的立足点别处，其中所述技术的限制进行评估;在位置的典型的不确定性是150纳米（约一个像素）在EAC的顺序H方向19。古埃相位异常的方法是用于确定在三维空间中扩展的对象的结构的几个定义良好DIHM方案之一，但尽管如此某些对象是有问题的重构。直接位于沿着光轴（指着相机）的对象是难以精确地重建，在该对象的长度和位置误差变大。这种限制是部分由于受限制的位深度的记录全息图（不同的灰度级，该相机可以记录的数量）的像素。当感兴趣的对象是非常接近焦平面另一个有问题的配置发生。在这种情况下，对象的实际和虚拟图像重建接近，到复杂的光学领域都是难以解释而产生。第二，稍不那么重要这里关注的是，所得的衍射条纹占用更少的图像传感器，而这种粗粒INFormation导致较差质量的重建。 在实践中，一个简单的梯度滤波器被应用到三维重构的体积来检测强度强反转沿照射方向。地区，强度变化很快从亮到暗，或反之亦然，然后与散射区域相关联。弱散射物体有很好的描述为这样的元素20的非相互作用集合，这些个人缴费之和为总的散射场是利用瑞利-索末菲反向传播方法容易倒。在本文中，轴向强度梯度技术被应用到一个链链球菌细胞。细胞体是相位对象（物种大肠杆菌具有测量21为1.384在波长λ= 589nm的折射率;的链球菌菌株很可能是类似的），并在显示为连接小圆块的高强度链日渐变滤镜后É样品体积得到了应用。适用于该过滤体积标准阈值和特征提取方法允许容积像素（体素）相对应的区域内的细胞内的提取。这种方法的一个特别的优点是它允许在轴向方向上的物体的位置的明确的重建。类似的方法（至少，那些记录的全息图邻近的对象，通过显微镜的物镜成像）患有无法确定此位移的符号 。虽然瑞利 - 索末菲重建方法还签署无关在此意义上，梯度操作允许我们上面和下面的焦平面弱相位物体之间的区分。

<table border="0" cellpadding="0" cellspacing="0" width="780">
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	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:180px;">Nikon Eclipse Ti-E inverted microscope</td>
			<td style="width:187px;">Nikon Corp.</td>
			<td style="width:147px;"> </td>
			<td style="width:267px;"> </td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:180px;">LED</td>
			<td style="width:187px;">Thorlabs</td>
			<td style="width:147px;">M660L3</td>
			<td style="width:267px;">emission wavelength &#955;=660 nm, linewidth approximately 20 nm </td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:180px;">LED power supply</td>
			<td style="width:187px;">Thorlabs</td>
			<td style="width:147px;">LEDD1B</td>
			<td style="width:267px;"> </td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:180px;">Thread Adapter</td>
			<td style="width:187px;">Thorlabs</td>
			<td style="width:147px;">SM2T2</td>
			<td style="width:267px;"> </td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:180px;">Thread Adapter</td>
			<td style="width:187px;">Thorlabs</td>
			<td style="width:147px;">SM1A2</td>
			<td style="width:267px;"> </td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:180px;">Frame Grabber board</td>
			<td style="width:187px;">EPIX </td>
			<td style="width:147px;">PIXCI E4 </td>
			<td style="width:267px;"> </td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:180px;">High-Speed CMOS camera</td>
			<td style="width:187px;">Mikrotron</td>
			<td style="width:147px;">MC-1362</td>
			<td style="width:267px;"> </td>
		</tr>
	</tbody>
</table>

digital inline holographic microscopy (dihm) of weakly-scattering subjects

弱散射的物体，诸如小的胶体粒子和大多数生物细胞中，经常遇到的显微镜。的确，已经开发了一系列的技术，以便更好地可视化这些相位物体;相衬和DIC是最流行的方法用于增强对比度。但是，在超出成像平面的方向上的记录位置和形状仍然具有挑战性。本报告介绍了一种简单的实验方法精确地确定位置和在三维空间中的物体的几何形状，采用数字内嵌全息显微镜（DIHM）。从广义上讲，可访问的样品体积是由相机传感器尺寸在横向方向上，并且在轴向方向上的照明相干性来定义。典型样品体积范围从200 微米 ×200 微米 ×200微米采用LED照明，以5毫米 ×5 毫米 ×5毫米或更大的用激光照射。这样的照明光被配置成使得平面波入射到样品上。在样品体积的物体然后散射光，这与未散射的光干涉而形成的干涉条纹垂直于所述照明方向。该图中（全息图）包含所需的三维重建的深度信息，并且可以在标准的成像设备，诸如CMOS或CCD照相机上被捕获。瑞利 - 索末菲反向传播方法被用来数值重新聚焦显微镜图像，并基于所述的Gouy相位的简单成像启发式异常是用来识别散射重构的体积内的对象。这个简单而可靠的方法导致对象的位置和形状的显微样品中的一个明确的，无模型测量。

为了演示DIHM的能力，实验是在一个链链球菌细菌进行。链本身测量10.5毫米长，是由6-7球柱面单元（2所述细胞在链轮的接近分割），直径范围为0.6-1微米。 图1a和1b示出了主接口重建和渲染软件。数字重聚焦过程的例子如图2所示，其中一个空间带通滤波器已被应用， 图3示出了梯度滤波器对图2中的图像的效果。用于构造这两个图像的原始数据捆绑在一起下载的代码为例架108。最后， 图4显示了在重建的几何形状和良好的质量差的数据的效果。在这最后的数字这两个框架取自SAM的视频细胞电子链（不同链对1中的前两个数字）。一个很好的重建是可能的，在大多数情况下，但是，当链条导向端上重建失败，则在焦平面给予大的圆形物体。这种故障模式是沿着光轴面向对象的特性。对于规模，在计算机渲染图像，在地面上的检查是1微米的一侧。对于这种方法的精密度和准确度的详细讨论，读者应咨询威尔逊和19张。 <img alt="图1" fo:content-width="4in" fo:src="/files/ftp_upload/50488/50488fig1highres.jpg" src="/files/ftp_upload/50488/50488fig1.jpg" width="500px" /> 图1。软件接口。重建软件的主界面中显示面板（ 一 ），该文件输入框，全局设置参数和其他重要功能在文中提到的已被高亮显示。图（b）所示为POV-Ray的例子场景文件，在程序的默认场景编辑器。上的指示线引号之间的文件名应该从重建软件设置为输出文件。它可能需要改变，以便正确地可视化的重建相机的位置和方向（表示相关部分）。请参阅联机POV-Ray的文件作进一步的说明。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50488/50488fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/50488/50488fig2highres.jpg" src="/files/ftp_upload/50488/50488fig2.jpg" width="500px" /> 图2。数值重新调整图像的示例。图板在左栏显示数值重新聚焦（X，Y）平面在不同的重构体（如所示）内的高度。底部左侧图像显示了原始的全息图数据，通过由背景数据划分。大面板右侧显示通过相同的堆栈中（X，Z）片。沿z轴对比反转的点可以清楚地看到在从图像的顶部三分之一左右的方式。红线上的放大图像显示，其中该平面（X，Z）相交（X，Y）的飞机到左边。同样，在较小的面板蓝色线表示（X，Z）片的交集。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50488/50488fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/50488/50488fig3highres.jpg" src="/files/ftp_upload/50488/50488fig3.jpg" width="500px" /> 图3。梯度滤波的图像的例子。0;此图像显示应用梯度滤波器对图2中的数据的效果。需要注意的是感兴趣的对象突出显示为对称的亮点。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50488/50488fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/50488/50488fig4highres.jpg" src="/files/ftp_upload/50488/50488fig4.jpg" width="500px" /> 图4。这导致良好和差的渲染的图像数据的例子。在板（a）和（C）是从细胞的一个翻滚链的相同视频的两幅图像。在面板上的数据（ 一 ）是代表在该系列中最帧，其中，所述链不直接沿光轴取向。物体的形状和位置被忠实地再现于渲染图（b）。在面板（C）的情况下，该链一度导向端上，以在焦平面，在这种配置物体难以重构，并且典型地产生一种"二进制大对象"接近焦平面，如面板（四可见） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50488/50488fig4highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects at JoVE.com

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

弱散射物体的三维位置可被唯一地识别利用数字内嵌全息显微镜（DIHM），它涉及到一个小的修改，以一个标准的显微镜。我们的软件使用加上瑞利 - 索末菲反向传播，得到三维位置和微观相对象的几何形状的简单的成像启发式。

的弱散射主题内嵌数字全息显微镜（DIHM）

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

digital-inline-holographic-microscopy-dihm-weakly-scattering

Nanyang Technological University

Research

JoVE Journal

Biology

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

12.2K 次观看.  Harvard University. 弱散射物体的三维位置可被唯一地识别利用数字内嵌全息显微镜（DIHM），它涉及到一个小的修改，以一个标准的显微镜。我们的软件使用加上瑞利 - 索末菲反向传播，得到三维位置和微观相对象的几何形状的简单的成像启发式。 

视频: Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

Determining heat and mechanical pain threshold in inflamed skin of human subjects

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific nociceptive and inflammatory biochemicals in humans. In this article we will show pain-testing paradigms that are often used in tandem with microdialysis procedures. Combining pain tests with microdialysis provides the critical link between behavioral and biochemical data that allows identifying key biochemicals responsible for generating and propagating pain.
Two models of evoking pain in inflamed skin of human study participants are shown. The first model evokes pain with aid of heat stimuli. Heat evoked pain as described here is predominantly mediated by small, non-myelinated peripheral nociceptive nerve fibers (C-fibers). The second model evokes pain via punctuated pressure stimuli. Punctuated pressure evoked pain is predominantly mediated by small, myelinated peripheral nociceptive nerve fibers (A-delta fibers). The two models are mechanistically distinct and independently examine nociceptive processing by the two major peripheral nerve fiber populations involved in pain signaling.
Heat pain is evoked with aid of the TSA II, a commercially available thermo-sensory analyzer (Medoc Advanced Medical Systems, Durham, NC). Stimulus configuration and delivery is handled with aid of specific software. Thermodes vary in size and shape but in principle consist of a metal plate that can be heated or cooled at various rates and for different periods of time. Algorithms assessing heat-evoked pain are manifold. In the experiments shown here, study participants are asked to indicate at what point they start experiencing pain while the thermode in contact with skin is heated at a predetermined rate starting at a temperature that does not evoke pain. The thermode temperature at which a subject starts experiencing pain constitutes the heat pain threshold.
Mechanical pain is evoked with punctuated probes. Such probes are commercially available from several manufacturers (von Frey hairs). However, the accuracy of von Frey hairs has been criticized and many investigators use custom made punctuated pressure probes. In the experiments shown here eight custom-made punctuated probes of different weights are applied in consecutive order, a procedure called up-down algorithm, to identify perceptional deflection points, i.e., a change from feeling no pain to feeling pain or vice versa. The average weight causing a perceptional deflection constitutes the mechanical pain threshold.

Algorithms assessing heat and mechanical pain thresholds in experimentally inflamed skin of human study subjects are shown. The two pain testing paradigms independently examine nociceptive processing by the two major peripheral nerve fiber populations transmitting pain, i.e., non-myelinated C fibers and small myelinated A-delta fibers.

确定人类受试者皮肤发炎的热和机械痛阈

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific ...

科研

生物学

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

数字微流体进行自动蛋白质组学处理

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

数字核酸定量实验分析的简单堆积读数

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

酵母的4D显微镜

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...