这项工作是由美国癌症协会（PF - 07 - 103 - 01 - CSM），美国国立卫生研究院（R01 GM61275和P30 CA042014），白血病和淋巴瘤协会，Huntsman癌症基金会的支持。

siRNA转染和细胞制备<ol><li>准备加入的纤维连接蛋白（10μg/mL）0.5ML，以及每个将用于4以及LabTek II腔玻璃罩。孵育10-15分钟室温。 </li><li>根据制造商的指示，准备反向转染的siRNA /脂质体RNAiMAX（Invitrogen）的配合。使用推荐量为每室24道菜要使用（100μL总）。一个类似的产品/转染的协议可以被替换。 </li><li>取出从腔玻璃罩井中的纤维连接蛋白。以及每个将用于添加的siRNA /脂质体RNAiMAX的复合物100μL。 </li><li>分装20000组蛋白H2B - mCherry表达细胞，每孔含转染混合物（500μL）。 </li><li>允许细胞孵化前转染混合物取代常规的细胞培养液1毫升〜24小时。 </li><li>孵育细胞图像采集前24小时（转染后48小时内）。 </li></ol>显微镜设置和图像采集<ol start="7"><li>图像采集设置有三个主要部分：1）一个细胞的孵化器，适合在显微镜舞台上和潮湿的环境中保持恒定的37℃，5％的CO 2; 2）倒置显微镜配备有一个自动化的阶段，自动荧光和明帘，自动滤光轮; 3）软件包，集成和控制的阶段，百叶窗，和滤光轮。在我们的成像设置，我们使用一个定制OKO实验室细胞培养箱LabTek二腔玻璃罩可以使用。控制的阶段，百叶窗，滤光轮MetaMorph软件提示：OKO实验室阶段顶部的孵化器系统包括了各种不同的盘，碟，或盖玻片大小的适配器。 </li><li>位置LabTek第二腔玻璃罩在孵化器打开盖子的预热和平衡的孵化器，适配器放置在墓室玻璃罩，并在与橡皮泥的地方举行。所以留在腔玻璃罩盖细胞培养液中不会干出来的。盖上盖子，整个孵化器和显微镜舞台上的地位，确保孵化器是牢固地举行。 </li><li>使用Metamorph，成立了自己的实验，在软件的菜单栏选择“应用程序/多维收购”。这将弹出一个窗口，在其中你可以选择的频率和长度的timelapse的，每种颜色的曝光时间（如果做多个设置），数量和舞台位置的位置，和Z -节数（如需要的话）。选择在哪里保存数据文件提示的功能，如果使用其他软件比Metamorph，可能会略有不同，但整体的概念是相同的。事无巨细，一个开源的基于ImageJ收购与大多数硬件设置兼容的软件，是一个很好的替代。 </li><li>对于本议定书的目的，我们将在两个波长（明和mCherry）为8小时，在15级职位提示每15分钟获取图像：如果需要更好的时间分辨率，那么你可以收购之间的时间间隔缩短，但，较少的阶段职位，可以使用，从而较少的有丝分裂细胞将可用于分析。 时，考虑的时间间隔要考虑到过滤器轮之间切换过滤器设置所需的时间，这一点很重要 。 </li><li>确定最佳曝光时间为每个波长的第一重点领域的使用明照明细胞。在软件中，调整自动对焦功能的唯一的明通道（这将允许该软件在每次收购之前，每个阶段的位置进行自动对焦） 提示：我们用一个40X干相衬的目标，给出色的分辨率没有对石油的需求。视光学所需的分辨率，可用于其他目标 。 </li><li>切换到mCherry波长和捕捉图像，使用50毫秒的曝光。调整的曝光时间，看到了良好的形象所需的最低。关键是要限制的光的照射，以确保持续的细胞活力量。作为一般规则，这最能实现一个中性密度滤光镜（减少激发光通量）和一个较长的相机的曝光时间，而不是通过增加光照强度。重复此过程要使用的任何额外的波长提示：要确定一个长期的细胞活力为您的实验给予曝光，你可以预览图像所需timelapse的总人数，一个细胞可以生存，暂时调整时间点间距从几分钟到秒（即10分钟变成10秒）。如果你的细胞存活100-200风险（或适当数量），那么您的曝光罚款。如果不是，减少您的激发光，曝光时间，或总数的图像模拟您完整的实验。 </li><li>下一步，选择和适当数量标志着一个阶段岗位，让您可以实现细胞的人口有足够的采样。 Metamorph将记录的位置，将返回每个收购提示：一般，我试图找到岗位，其中有大量的细胞图像，但是没有那么多，他们过于密集 。 此外，重要的是选择足够的位置，所以，你将有一个很好的机会观察，通过有丝分裂进展的细胞大量。 </li><li>毕竟timelapse，波长的曝光时间，和舞台上的地位的信息已被记录，选择“预览”屏幕上的按钮，以确定是否该软件是适当控制设备。 </li><li>一旦你感到满意，选择“获取”按钮在屏幕上进行收购。然后坐下来暗示 ，使计算机和显微镜做的工作：通过观察至少有一个收购的丰满圆润的自动化，以确保一切正常工作 。 </li></ol>图像处理和分析<ol start="16"><li>收购完成后，下一步就是转移到一个形式，是比较容易处理的图像数据。对于本议定书的目的，我将展示如何使用Metamorph电影，那么如何使用ImageJ程序生成图像的蒙太奇。两种格式将被用于量化的目的。 </li><li>第一步是审查数据。要做到这一点，选择菜单栏中的“应用程序/审查多维数据”。选择文件的位置，然后选择“查看”。这将允许你从每个舞台上的地位的图像的堆栈单独审查。选择所需的舞台上的地位，然后选择“载入图像”。您将能提前通过数据帧帧。 </li><li>对于那些阶段职位中，细胞通过有丝分裂（DNA和细胞形态确定），您可以使电影的蒙太奇使用Metamorph，或与其他程序，如ImageJ（通过美国国立卫生研究院免费提供）。 </li><li>要在Metamorph电影中，选择“过程/保存电影”，在菜单栏中。您将可以选择使用帧的速度，和文件类型的电影。然后选择“保存电影”。 </li><li>为了使蒙太奇，我。STK文件保存为图像的堆栈，它可以用于其他程序，如ImageJ。 </li><li>在ImageJ打开该文件，并提前帧，直到找到一个细胞通过有丝分裂。作物的面积和周围的细胞选择“图像/重复” 提示：确保所有帧重复。 </li><li>为了蒙太奇，选择“图像/垛/蒙太奇”在菜单栏中。在这里您可以指定您要包括，蒙太奇的大小和形状的帧，以及是否要帧之间的边界。选择这些热播的“好”。 。保存为TIF文件的蒙太奇，并做进一步处理ImageJ或Adobe Photoshop中的任何提示：更改Metamorph使用更方便的图像处理，以8位格式的 16位格式的文件蒙太奇。 </li><li>要计算出在不同阶段的有丝分裂的时间，确定T = 0（DNA凝结或细胞四舍五入的第一个迹象），计数染色体在赤道（在前期/前中期的时间），帧帧的数量，直到对齐，直到染色体开始隔离（在中期的时间），帧，直到细胞开始变平（后期/末期/胞质）。在每个有丝分裂阶段的计算时间取决于每帧之间的时间间隔。 </li></ol>代表性的成果<img src="/files/ftp_upload/1878/1878fig1tmb.jpg" border="0" /> 图1说明了一个典型的对照siRNA转染实验中使用的HeLa细胞系表达组蛋白H2B - mCherry结果。这种方法已被用来有效地量化的有丝分裂的时机，揭示了一个意想不到的作用nucleoporin Nup153在有丝分裂1月底的时间。请<a href="http://www.jove.com/files/ftp_upload/1878/1878fig1.jpg">点击这里</a>看到图1的放大版本。

<ol>
	<li>Mackay, D. R., Elgort, S. W., Ullman, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleoporin+Nup153+has+separable+roles+in+both+early+mitotic+progression+and+the+resolution+of+mitosis.">The nucleoporin Nup153 has separable roles in both early mitotic progression and the resolution of mitosis.</a> Mol Biol Cell. 20, 1652-1660 (2009).</li><li>Khodjakov, A., Rieder, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+division+process+in+living+tissue+culture+cells.">Imaging the division process in living tissue culture cells.</a> Methods. 38, 2-16 (2006).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat Methods. 2, 905-909 (2005).</li><li>Meraldi, P., Draviam, V. M., Sorger, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+and+checkpoints+in+the+regulation+of+mitotic+progression.">Timing and checkpoints in the regulation of mitotic progression.</a> Dev Cell. 7, 45-60 (2004).</li><li>Mora-Bermudez, F., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+structural+dynamics+of+chromosomes+in+living+cells+by+fluorescence+microscopy.">Measuring structural dynamics of chromosomes in living cells by fluorescence microscopy.</a> Methods. 41, 158-167 (2007).</li><li>Prosser, S. L., Fry, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+imaging+of+the+centrosome+cycle+in+mammalian+cells.">Fluorescence imaging of the centrosome cycle in mammalian cells.</a> Methods Mol Biol. 545, 165-183 (2009).</li><li>Malureanu, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BubR1+N+terminus+acts+as+a+soluble+inhibitor+of+cyclin+B+degradation+by+APC/C(Cdc20)+in+interphase.">BubR1 N terminus acts as a soluble inhibitor of cyclin B degradation by APC/C(Cdc20) in interphase.</a> Dev Cell. 16, 118-131 (2009).</li><li>Anderson, D. J., Hetzer, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reshaping+of+the+endoplasmic+reticulum+limits+the+rate+for+nuclear+envelope+formation.">Reshaping of the endoplasmic reticulum limits the rate for nuclear envelope formation.</a> J Cell Biol. 182, 911-924 (2008).</li><li>Beaudouin, J., Gerlich, D., Daigle, N., Eils, R., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+envelope+breakdown+proceeds+by+microtubule-induced+tearing+of+the+lamina.">Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina.</a> Cell. 108, 83-96 (2002).</li><li>Marcus, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+spindle+behavior+using+confocal+microscopy.">Visualization of spindle behavior using confocal microscopy.</a> Methods Mol Med. 137, 125-137 (2007).</li><li>Steigemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aurora+B-mediated+abscission+checkpoint+protects+against+tetraploidization.">Aurora B-mediated abscission checkpoint protects against tetraploidization.</a> Cell. 136, 473-484 (2009).</li><li>Draviam, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+genomic+screen+identifies+a+role+for+TAO1+kinase+in+spindle-checkpoint+signalling.">A functional genomic screen identifies a role for TAO1 kinase in spindle-checkpoint signalling.</a> Nat Cell Biol. 9, 556-564 (2007).</li><li>Neumann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNAi+screening+by+time-lapse+imaging+of+live+human+cells.">High-throughput RNAi screening by time-lapse imaging of live human cells.</a> Nat Methods. 3, 385-390 (2006).</li><li>Stegmeier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaphase+initiation+is+regulated+by+antagonistic+ubiquitination+and+deubiquitination+activities.">Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities.</a> Nature. 446, 876-881 (2007).</li></ol>

在成像和荧光蛋白技术的最新进展，实时成像已经成为常规的细胞分析2方面。一个各种绿色荧光蛋白（和其他颜色的变种3）标签蛋白被广泛使用或相对容易构造的，可以用来追踪一个细胞分裂的标志，包括DNA / 染色体的动态4，5，中心体复制，细胞周期蛋白B动态7，核信封细分和重组8，9，10有丝分裂纺锤体的形成， 胞质 11的各个阶段。标签蛋白，可单独或联合使用时的激发/发射光谱，荧光标记是兼容的，允许特定事件之间的协调，以进行评估。如果更大的空间和/或时间分辨率/所需，是否可以使用激光扫描，旋转盘，或共振扫描共聚焦显微镜，并与日益增加的分辨率复杂的显微镜选项列表中继续增长。在哺乳动物细胞的有丝分裂的实时成像也已适应在高通量的RNAi和小分子屏幕12-14使用。因此，一个S最初的实验中，使用这种技术，而可能是相对简单的，这一战略的基础上的可能性是多方面的。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Lipofectamine RNAiMAX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13778-075</td>
<td>&nbsp;</td>
<tr>
<td>Lab Tek II Chambered Coverglass (4-well)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>12-565-337</td>
<td>Additional chamber sizes are available</td>
</tr>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F1141</td>
<td>&nbsp;</td>
<tr>
<td>Stage-top cell incubator</td>
<td>&nbsp;</td>
<td>OKO Lab</td>
<td>&nbsp;</td>
<td>Can use any appropriate chamber</td>
</tr>
<tr>
<td>Automated inverted fluorescence microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Can use any appropriate microscope</td>
</tr>
<tr>
<td>Software package</td>
<td>&nbsp;</td>
<td>Metamorph</td>
<td>&nbsp;</td>
<td>Can use any appropriate software</td>
</tr>		</tbody>
		</table>
		</div>

time-lapse imaging of mitosis after sirna transfection

蜂窝组织和染色体在有丝分裂过程中发生的动态变化是紧密协调，以确保准确的基因和细胞内容继承。有丝分裂，如染色体运动的标志性赛事，可以很容易地跟踪单个单元格，使用时间推移，哺乳动物细胞表达特定的蛋白GFP标记线的荧光显微镜的基础上。结合RNAi为基础的枯竭，这可能是一个功能强大的方法，针对后一种特定的蛋白质的水平已经降低缺陷发生有丝分裂阶段（S）。在这个协议中，我们提出了一个消耗上有丝分裂的时机的一个潜在的有丝分裂调控蛋白的效果评估的基本方法。是与siRNA的转染细胞，放置在舞台上方的孵化室，和使用自动荧光显微镜成像。我们介绍了如何使用软件设置一个失效时间的实验中，如何处理图像序列，使静止图像的蒙太奇或电影，以及如何量化和分析有丝分裂阶段使用一个细胞系表达的时序mCherry标记组蛋白H2B。最后，我们讨论设计一个时间推移实验的重要因素。这种策略是其他方法的补充和提供的优势1）在寻找作为一个人口和2）分析有丝分裂的细胞，而不需要使用药物治疗的细胞周期同步时，可能无法观察到的动力学变​​化的敏感性。从这样的成像实验的视觉信息不仅可以使有丝分裂的子阶段进行评估，而且还可以提供意想不到的洞察力，不会明显流式细胞仪细胞周期分析。

Watch this Scientific Journal Video about Time-lapse Imaging of Mitosis After siRNA Transfection at JoVE.com

Time-lapse Imaging of Mitosis After siRNA Transfection

在这里，我们描述了一个基本的协议，以图像和量化现场后，转染哺乳动物组织培养细胞的有丝分裂的时机。

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 ...

time-lapse-imaging-of-mitosis-after-sirna-transfection

Research

JoVE Journal

Biology

16.9K 次观看.  University of Utah. 在这里，我们描述了一个基本的协议，以图像和量化现场后，转染哺乳动物组织培养细胞的有丝分裂的时机。

视频: Time-lapse Imaging of Mitosis After siRNA Transfection

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

拆装时间推移显微镜监测肌动蛋白

科研

生物学

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

多色成像定时转基因斑马鱼：可视化由针对性的神经细胞消融激活视网膜干细胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

一种用于阴南芥叶片安装的通用方法

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

延时结构照明显微镜线粒体核素的特定标签

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

LarvaSPA，一种用于长期延时成像的安装果蝇幼虫的方法

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...