作者感谢罗伯特Kelsh的好心分享斑马鱼SOX10促进剂。

1。胚胎收集的光转化<ol><li>设置至少十对SOX10的：晚上5至下午6时枫转基因斑马鱼。 </li><li>第二天早上，拉分频器和收集中午前后鸡蛋。他们转移到培养皿中，并把它们放到28.5℃培养箱。 </li><li>在大约24小时受精后，通过去除死胚清洁的培养皿。检查是否使用了光场显微镜和任何荧光显微镜与增强型绿色荧光蛋白（EGFP）过滤胚胎13的发展阶段。枫将是可见的，通过这个过滤器。 </li><li>当胚胎达到60 HPF转移5明亮的荧光的胚胎成一个单独的培养皿中，并在必要时dechorionate他们。 </li></ol> 2。安装胚胎的光转化的<ol><li>使用普通荧光显微镜安装胚胎。安装在激光共聚焦显微镜的胚胎是非常有挑战性的。 </li><li>使用支架ARD E3水或使用下面的食谱准备新鲜E3： 添加每升蒸馏水DH 2 O以下添加以下做出1X E3L： 0.292克5.0 mM氯化钠 0.013克0.17MM氯化钾 0.044克0.33MM 氯化钙 （干燥剂，96％，ACS试剂） 0.081克0.33毫米硫酸镁4（anhydrated） 可选：添加200微升0.05％亚甲蓝到1X E3作为杀真菌剂，搅拌。 介质保持在室温下放置1周。 </li><li>使用标准的三卡因或使用下面的食谱准备三卡因： 5升0.2％三卡因混合下列成分： 45毫升三 - 氯（1M pH9.5的） 5 LH 2 O 10克三卡因</li><li>准备“电影果汁”（50毫升E3，0.015％三卡因溶液），加入3.75毫升0.2％三卡因至46.25毫升E3。 </li><li>通过混合46.25毫升E3水在一个50毫升玻璃烧瓶46.35毫克低熔点（LMP）琼脂糖制备琼脂糖和由微波溶解的固体颗粒。然后加3.73毫升0.2％三卡因琼脂糖，把琼脂糖玻璃烧瓶至50℃水浴中，直到使用。 </li></ol> 3。准备和胚胎的安装<ol><li>麻醉胚胎与步骤1.1.2和转移胚胎到腔幻灯片制作E3/0.015％三卡因溶液。 </li><li>吸收所有多余的E3与纸巾及认沽琼脂糖到胚胎。 </li><li>位置胚胎完全与背微加载的提示。确保在整个胚胎不浮于琼脂糖的顶部，但整个身体向下推。然后倒入E3/0.015％三卡因溶液在琼脂糖嵌入式胚胎，直到腔被完全充满液体。 </li></ol> 4。调整软件设置上的共聚焦显微镜<ol><li>使用20X空气浸入式物镜（数值孔径0.75;针孔大小20）为重点的胚胎。然后按下显微镜L100光按钮并选择软件的渠道。打开图标查看/收购controls/A1settings与集团公司k是共聚焦设置按钮。接下来，单击“自动”，然后再次关闭该窗口之前选择以下途径： CH1：DAPI通道403.4纳米（可用于350-410纳米之间的波长） CH2：488.0纳米 CH3：561.9纳米</li></ol> 5。光转化<ol><li>关闭通道1和3，然后点击删除按钮联锁和扫描之前打开通道2。 </li><li>专注于感兴趣的光转化，细胞是最好的开始1frame/sec和高压（HV）200，然后往上走在帧/秒，并降低相应的高压，直至达到1/32帧/秒和HV左右完成100-120，根据背景噪声。 </li><li>当胚胎在焦点上，看在屏幕的右侧边缘，抓住绿色边缘，使其更小，因此对于光转化，然后右击鼠标适合感兴趣的区域。越小越好，因为光转化为不可逆的。 </li><li>打开帧/秒，以1和HV 200和打扫描。光转化的区域的放大图像现在是可见的。 </li><li> Photoconvert细胞通过打开通道1（403.4纳米）。暴露在photoconverting激光随时随地从5-60秒，这取决于区域的大小，直到通过GFP的通道看到绿枫几乎是缺席的。 </li><li>当绿枫信号几乎不存在，请关闭通道1。打开通道2和3上。然后单击右侧窗口上A1上的扫描区场和打“回归原始大小”来检查，如果需要的细胞进行光转换。 </li></ol> 6。制作一个Z堆栈<ol><li>查找图标菜单栏中“捕捉Z系列&#39;和启动之前设置Z堆栈的上限和下限。设置的LUT。 </li></ol> 7。软件设置定时<ol><li>去A1Simple界面框中，勾选以下设置： 1/32帧/秒 大小1024 平均4个或8倍速取决于Z-堆栈具有要采取的一个循环的数目</li><li>转到：查看/采集控制/ ND序列的获取和设置时间范围（8〜30分钟），连续单击，然后单击Z堆栈。之前，设置边框作为测试，并开始电影。 </li><li>不断增加E3/0.015％三卡因液腔滑全天。到了晚上，完全填补了腔。这将持续到第二天早上。 </li><li>第二天早上，笔芯E3/0.015％三卡因溶液，并检查胚胎是否还活着。荧光的损失表示死亡。继续笔芯，并检查了一整天。 </li><li>当结构已经完成形成，或胚胎已经死亡，完成电影。 </li></ol> 8。后期制作加工<ol><li>点击工具栏中“显示最大密度投影”，然后用鼠标右键单击该图像，并创建新的文档。视频现在是在可能的最佳质量可见。去删除不需要的框架和调整的LUT。最后保存为。AVI。这种格式将被罚款的Windows。对于Mac下载软件TØ转换为。或MOV，WMV。 </li></ol>

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	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+at+Birth+of+Cleft+Lip+With+or+Without+Cleft+Palate:+Data+From+the+International+Perinatal+Database+of+Typical+Oral+Clefts+(IPDTOC).">Prevalence at Birth of Cleft Lip With or Without Cleft Palate: Data From the International Perinatal Database of Typical Oral Clefts (IPDTOC).</a> Cleft Palate Craniofac. J. 48 (1), 66-81 (2011).</li><li>Dixon, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleft+lip+and+palate:+understanding+genetic+and+environmental+influences.">Cleft lip and palate: understanding genetic and environmental influences.</a> Nat. Rev. Genet. 12 (3), 167-178 (2011).</li><li>Mangold, E., Ludwig, K. U., Nothen, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakthroughs+in+the+genetics+of+orofacial+clefting.">Breakthroughs in the genetics of orofacial clefting.</a> Trends Mol. Med. 17 (12), 725-733 (2011).</li><li>Kimmel, C. B., Miller, C. T., Moens, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+and+morphogenesis+of+the+zebrafish+larval+head+skeleton.">Specification and morphogenesis of the zebrafish larval head skeleton.</a> Dev. Biol. 233 (2), 239-257 (2001).</li><li>Dougherty, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Fate+Map+of+First+Pharyngeal+Arch+Structures+in+the+sox10:+kaede+Zebrafish+Transgenic+Model.">Embryonic Fate Map of First Pharyngeal Arch Structures in the sox10: kaede Zebrafish Transgenic Model.</a> J. Craniofac. Surg. 23 (5), 1333-1337 (2012).</li><li>Trainor, P. A., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hox+genes,+neural+crest+cells+and+branchial+arch+patterning.">Hox genes, neural crest cells and branchial arch patterning.</a> Curr. Opin. Cell Biol. 13 (6), 698-705 (2001).</li><li>Schilling, T. F., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segment+and+cell+type+lineage+restrictions+during+pharyngeal+arch+development+in+the+zebrafish+embryo.">Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo.</a> Development. 120 (3), 483-494 (1994).</li><li>Swartz, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+a+palatogenic+gene+program+in+zebrafish.">Examination of a palatogenic gene program in zebrafish.</a> Dev. Dyn. 240 (9), 2204-2220 (2011).</li><li>McCollum, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+toxicity+screening+in+zebrafish.+Birth+Defects+Res.">Developmental toxicity screening in zebrafish. Birth Defects Res.</a> C. Embryo Today. 93 (2), 67-114 (2011).</li><li>Wada, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+is+required+for+cranial+neural+crest+morphogenesis+and+chondrogenesis+at+the+midline+in+the+zebrafish+skull.">Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.</a> Development. 132 (17), 3977-3988 (2005).</li><li>Eberhart, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Hedgehog+signaling+from+neural+to+oral+epithelium+organizes+anterior+craniofacial+development.">Early Hedgehog signaling from neural to oral epithelium organizes anterior craniofacial development.</a> Development. 133 (6), 1069-1077 (2006).</li><li>Ando, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc. Natl. Acad. Sci. U S A. 99 (20), 12651-12656 (2002).</li><li>Kimmel, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203 (3), 253-310 (1993).</li><li>Kawakami, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish-secreted+matrix+protein+you/scube2+is+implicated+in+long-range+regulation+of+hedgehog+signaling.">The zebrafish-secreted matrix protein you/scube2 is implicated in long-range regulation of hedgehog signaling.</a> Curr. Biol. 15 (5), 480-488 (2005).</li><li>Lombardo, V. A., Sporbert, A., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+tracking+using+photoconvertible+proteins+during+zebrafish+development.">Cell tracking using photoconvertible proteins during zebrafish development.</a> J. Vis. Exp. (67), e4350 (2012).</li></ol>

作者宣称，他们有没有竞争的财务权益。

这里会显示在斑马鱼模型颅面部发育的可视化的新方法。的SOX10：枫转基因斑马鱼线已经被成功地用于描述中集的详细的迁移模式被用来作为模式生物5。 以前的研究已经用毛地标，如眼靶细胞，并依赖于枫的mRNA注射，光转化试验或笼荧光葡聚糖光转化为10，11，14，15红袜：相比于这10枫转基因株系具有很多优点方法。神经嵴细胞已经贴有枫，符合一个非常特?...

口面裂代表了最普遍的颅面畸形，与1/700-1，000分娩的影响1。早期胚胎颅面部发育受阻可导致形成唇腭裂（CL / P）的。虽然原因综合征型唇裂已经在很大程度上表明，中颜面部的clefting的非综合征形式的遗传和表观遗传基础仍需要发现2-4。为了了解这些畸形的病因和发病机制，有必要阐明颅面结构的细胞基础上的发展。 在所有的脊椎动物物种颅神经嵴细胞（CNCC）从背神经管迁移到填充咽弓，这将有助于形成颜面部结构。早期胚胎神经嵴发育受阻可导致形成颅面畸形包括CL / P 5-7。 在广告DITION以斑马鱼和哺乳动物颅面部发育（CNCCs位于同源区域）之间的结构相似性，基因调控网络是高度保守的。还已经表明，CNCCs开发在脊椎动物物种和斑马鱼8之间以相同的方式，使斑马鱼的强大有机体为CL / P的发育和遗传基础的研究它有许多优点，包括体积小，快速，前宫内胚胎发育，以及高繁殖率。此外，胚胎是光学透明的，使得它适合于观察的显微镜9根据复杂的发育事件。这是一个理想的动物模型进行迁移和颅神经嵴细胞分化的研究。 枫转基因模型5：扩大先前发表的作品8，10，11，国家会议中心的迁徙模式是使用SOX10中详细描述。枫是一个照片兑换蛋白TU从绿色到红色RNS光活化后，使之能够追踪CNCCs精确。在此变换的肽骨架被裂解，这表明转化是稳定的，这意味着细胞可以被跟踪到其最终目的地12。标记下SOX10的转录调控枫转基因株系表明，脊椎动物味觉和斑马鱼筛片是同源融合双侧上颌骨突起（MXP）的额鼻突出（FNP），而形成的Y字型融合缝之间的相似种。 在其他应用中，SOX10：枫转基因斑马鱼模型被用来生成斑马鱼胚胎的视频在不同的发育阶段，以显示形成的正常和异常颅面结构。细胞的特定群体的光转化使得能够跟踪他们的发展。使用这种方法的方法来创建开发craniofac的实时成像IAL结构在斑马鱼被引入，因此很容易直观地展现这个复杂的发展过程。 该协议旨在共享使用筛窦板的SOX10的正常发展产生这些视频的经验：枫转基因斑马鱼作为一个例子。此协议可进一步应用于制造从颅神经嵴细胞在斑马鱼产生的任何结构的时间推移录像。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>sox10: kaede transgenic zebrafish line</td>
			<td>MGH</td>
			<td></td>
			<td>Available via the Liao lab</td>
		</tr>
		<tr>
			<td>Petri dishes 100x15 mm</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 35 mmx10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Low melting point (LMP) Agarose</td>
			<td>Invitrogen</td>
			<td>15517022</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Tek 2 Chamber SlideSystem</td>
			<td>LabTek</td>
			<td>154453</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloaders 200/pk</td>
			<td>Fisher</td>
			<td>E5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R Si Confocal Ti series</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements Software AR3.2 64-bit</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy

脊椎动物palatogenesis是一个非常精心设计和复杂的发展过程，它涉及颅神经嵴（CNC）细胞，收敛性和扩展面部日珥，以及颅面骨骼成熟的迁移。研究颅神经嵴向斑马鱼口感的特定区域一个SOX10的贡献：生成枫转基因斑马鱼线。 SOX10提供枫报告蛋白的谱系限制性的神经嵴，从而使细胞标记更精确的处理比传统的染料或报告mRNA注射。枫是光转换蛋白，由绿色变为红色光激活后，使人们有可能跟随细胞精确。该SOX10：转基因枫线被用来执行谱系分析，划定数控细胞群产生上颌下颌与元素，并说明同源性面部日珥到脊椎动物。本协议描述了一步s到产生SOX10的生存时间推移视频：枫斑马鱼胚胎。筛窦板的发展将成为一个实际的例子。这个协议可以应用于制作转基因斑马鱼任何枫或类似photoconvertible报告蛋白的时间推移共聚焦记录。此外，它可以被用来捕捉颅面结构在斑马鱼突变体不仅正常，而且还发育异常。

在SOX10：枫转基因株系，迁徙，后迁徙CNCCs被标记荧光绿色。国家会议中心的细胞标记绿色荧光枫复述内源性SOX10表达5。 在其他应用中，这种动物模型被用来更好地可视化中集依赖颅面结构的发展。具体的结构，也颅面畸形的病理发展，特别是唇腭裂的正常发育已被抓获。已经产生了一系列的斑马鱼的生存时间推移录像在不同的发育时间点。 作为...

Watch this Scientific Journal Video about Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy at JoVE.com

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

实验数据的可视化已成为展示结果给科学界的一个关键因素。越来越多的胚胎活延时录制的产生有助于复杂的发育过程中更好地介绍和了解。该协议是一步一步的通过枫蛋白在斑马鱼光转化指南细胞标记。

颅面部发育中的SOX10可视化：使用枫转基因斑马鱼线时间推移共聚焦显微镜

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

visualization-craniofacial-development-sox10-kaede-transgenic

Research

JoVE Journal

Biology

12.7K 次观看.  Massachusetts General Hospital. 实验数据的可视化已成为展示结果给科学界的一个关键因素。越来越多的胚胎活延时录制的产生有助于复杂的发育过程中更好地介绍和了解。该协议是一步一步的通过枫蛋白在斑马鱼光转化指南细胞标记。 

视频: Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Monitoring Actin Disassembly with Time-lapse Microscopy

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.  ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

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 ...

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 ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

颅面间质细胞的电

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

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 ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...