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?...

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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		<col />
		<col />
		<col />
	</colgroup>
	<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?...

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

Research

JoVE Journal

Biology

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

12.1K 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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

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 ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.