我们协助开发时间的推移协议和熊黛林Pillitteri建设的POLAR :: POLAR-EGFP感谢Rychel阿曼达。我们也感谢ABRC提供PM-RB建设的。这个协议是一个支持PRESTO奖来自日本的科学技术和代理的发展。极地研究还得到了大学，华盛顿版权研究基金（RRF，4098）和美国国家科学基金会（MCB-0855659）。 KMP是一个NSF研究所研究员（DGE-0718124），吉是HHMI的GBMF的研究者。

1。种子消毒<ol><li>准备种子消毒液：33％的家用漂白剂，0.1％的Triton X-100。 </li><li>广场种子携带所需的荧光记者的结构（S）和1.7 ml管型（S）和适用于1毫升灭菌水溶液。孵育15分钟nutator。 </li><li>在无菌罩，使用移液器删除解决方案，从管消毒，背后留下的种子。用1ml无菌水冲洗。重复四次。 </li><li>在4℃下孵育2天或以上。 </li></ol> 2。商会的媒体的制备<ol><li> 20毫升0.5％Bacto琼脂（不是纯粹的琼脂糖），在水中在200毫升的烧瓶中，和微波溶液混合直至溶解。沸腾的解决方案时一定要小心。 </li><li>该溶液冷却至约60℃。 </li><li>收集1毫升的琼脂溶液用移液管，慢慢地适用于玻璃盖内的Lab-Tek II双腔室幻灯片（德国盖玻片＃1.5系统）。解w高血压脑出血是太热或太快，可能会熔化，胶水或裂纹玻璃。 </li><li>立即添加第二个琼脂溶液的毫升。琼脂培养基上，现在应该完全覆盖滑动室的底部。这个最小的媒体足够的支持子叶的植物，其中包含储存的营养物质，保持水分，使气体扩散为四到五天的发展。 </li><li>与台式酷溜室覆盖。如果烧瓶盖紧，溶液可能被保存和再加热，以备将来使用。 </li></ol> 3。种子解剖<ol><li>从4°C存储，删除不育种子管。 </li><li>放置在舞台上，在解剖显微镜和纸巾与水滋润。调整组织来创建一个平滑的表面。组织被用于稳定种子解剖。 </li><li>吸取20-30的种子从管组织，小心翼翼地将它们放置在解剖范围的视野。 </li><li>使用锋利的钳子（Roboz，＃5生物学尖端），小心地取出种子，从幼苗内。两外层和内珠被必须被移除。 </li><li>当成像子叶，这是有利于除去的下胚轴和胚根。胚子含有足够的营养，在此协议下发展了好几天。如果完好，下胚轴将整顿和延长戏剧性的是，移动子叶时间的推移视野。用手术刀切子叶下胚轴。 </li><li>维持解剖子叶浸渍在水中，在微管或类似。 </li><li>重复,3.4-3 .6，直至达到所需数量的子叶。 15-20成功的解剖之前，建议安装。 </li></ol> 4。在会议厅内的幻灯片安装胚子<ol><li>使用一个小的（18毫米）盖玻片，穿过琼脂培养基室幻灯片的玻璃基板整层。 </li><li>移液管解剖子叶从保持的最低水管进入腔滑动，根据解除的琼脂层。 </li><li>轻轻地放下琼脂上的子叶。如果存在过量的水，浸泡用纸巾，小心不要打扰子叶。样品应不再移动时，容易安装完成。 </li><li>将盖室幻灯片和显微镜载物台移动幻灯片。 </li></ol> 5。时间的推移成像<ol><li>倒激光共聚焦显微镜图像所需的荧光（S）。 LSM700使用的参数：GFP的激发波长为488 nm和收集在440-530 nm的带通滤波器; RFP，在555nm处和收集在570-610 nm的带通滤波器的激励。 </li><li>检查是否有损坏的标本。编程的位置，完整，正确安装的子叶到显微镜的软件。 2009年在禅宗：聚焦一个子叶与20倍的目标，移动目标为中心的室内滑梯。在采集模式 ，改变缩放到0.5和帧大小&lt;/ em&gt;的48x48像素。下位置，选择位置 “复选框和扫描概述图片...”按钮，然后增加水平和垂直的瓦数为30-35。当扫描完成后，单击该的位置按钮下的“ 尺寸 ”选项卡和地点十字线，在每个子叶观察。 </li><li>设置z系列，围绕这些的子叶表皮的2009年所有samples.In禅：Z-Stack的复选框。下位置列表中选择每个位置，然后单击“ 移动 ”按钮。专注于子叶表皮的最上面的平面，单击“ 设置 ”下的第一个按钮的Z-Stack。重点在表皮是有益的可见至最低位置， 最后单击“ 设置 ”按钮。单击该按钮旁边的优化 ，这显示出最佳的Z-层厚，设置。检查每个子叶，以确认这些设置包括所有样品的Z参数不能设置为个人，禅2009年idual位置。 </li><li>设置所需的分辨率和长度的时间序列。在表皮细胞分裂的蛋白质动力学间隔为15-30分钟三天有效。此时间间隔可以根据需要改变。 2009年在禅宗：单击“ 时间序列 ”复选框。在时间序列中 ，设置循环使用滑块或在文本字段中键入所需的图像的数量。设置时间间隔为30，并使用下拉菜单选择分 。 </li><li>开始XYZT多位置扫描。请注意，由扫描规格的位置的数目必须限制，如果总的扫描时间是大于所需的时间间隔，将不正确的时间点。成像时，GFP和RFP在59 Z-片在30分钟的时间间隔使用我们的系统，最多同时六的位置是可能的。 2009年在禅宗：改变帧尺寸 ，所需的分辨率，然后点击开始实验 。 </li></ol> 6。视频编辑<ol><li>从的共聚焦数据文件，使Z-投影每个时间/位置组合和出口为无损格式，如TIFF图像文件的最大强度。文件名 ​​应该是在每一个系列的位置（ 例如 ，软件将它们组合成一部电影的POLAR-GFP_pos1_0001，POLAR-GFP_pos1_0002等，而不是POLAR-GFP_0001_pos1）。 2009年在禅宗：使用复制子集下的“ 处理 ”选项卡，分裂成单独的文件，然后最大强度投影的位置。最后，导出为TIFF 文件：出口，全分辨率图像窗口- （系列 ）。 </li><li>使用FIJI 7,8使连续运行的bUnwarpJ宏（ 插件：注册：bUnwarpJ）的图像。变更登记模式为单声道。所有的高级选项可以使用默认值。长的时间间隔序列，下载并安装宏“仿射+一致的弹性2D图像配准”，这将申请bUnwarpJ的一系列图像，自动，和开放Ÿ我们的数据文件：导入：图像序列中使用它。取消选中MOPS地标提取和运行。 </li><li> FIJI / ImageJ的或QuickTime Pro打开的顺序排列的，并保存在需要的视频格式（AVI是一个不错的选择）。 </li></ol>

<ol>
	<li>Sulston, J. E., Schierenberg, E., White, J. G., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Embryonic+Cell+Lineage+of+the+Nematode+Caenorhabditis+elegans.">The Embryonic Cell Lineage of the Nematode Caenorhabditis elegans.</a> Developmental Biology. 100, 64 (1983).</li><li>Foe, V., Odell, G., Edgar, B. A., Bate, M., Martinez Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3+Mitosis+and+Morphogenesis:+Point+and+Counterpoint.">Chapter 3 Mitosis and Morphogenesis: Point and Counterpoint.</a> Development of Drosophila melanogaster. , (1993).</li><li>Vincent, C. A., Carpenter, R., Coen, E. 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+Lineage+Patterns+and+Homeotic+Gene+Activity+During+Antirrhinum+Flower+Development.">Cell Lineage Patterns and Homeotic Gene Activity During Antirrhinum Flower Development.</a> Current Biology. 5, 1449 (1995).</li><li>Beemster, G. T. S., De Vusser, K., De Tavernier, E., De Bock, K., Inz&#233;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Variation in Growth Rate between Arabidopsis Ecotypes Is Correlated with Cell Division and A-Type Cyclin-Dependent Kinase Activity. Plant Physiology. 129 (2), 854 (2002).</li><li>Grandjean, O., Vernoux, T., Laufs, P., Belcram, K., Mizukami, Y., Traas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Analysis+of+Cell+Division,+Cell+Growth,+and+Differentiation+at+the+Shoot+Apical+Meristem+in+Arabidopsis.">In Vivo Analysis of Cell Division, Cell Growth, and Differentiation at the Shoot Apical Meristem in Arabidopsis.</a> Plant Cell. 16 (1), 74 (2004).</li><li>Pillitteri, L. J., Peterson, K. M., Horst, R. J., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Profiling+of+Stomatal+Meristemoids+Reveals+New+Component+of+Asymmetric+Cell+Division+and+Commonalities+among+Stem+Cell+Populations+in+Arabidopsis.">Molecular Profiling of Stomatal Meristemoids Reveals New Component of Asymmetric Cell Division and Commonalities among Stem Cell Populations in Arabidopsis.</a> Plant Cell. 23 (9), 3260 (2011).</li><li>Abramoff, M. D., Magalhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11 (7), 36 (2004).</li><li>Arganda-Carreras, I., Sorzano, S. &. #. 2. 2. 5. ;. n. c. h. e. z., Marabini, C. O., Carazo, R., de Solorzano, J. M. O. r. t. i. z. -., C, J., Kybic, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consistent+and+Elastic+Registration+of+Histological+Sections+Using+Vector-Spline+Regularization.">Consistent and Elastic Registration of Histological Sections Using Vector-Spline Regularization.</a> Computer Vision Approaches to Medical Image Analysis, ser. Lecture Notes in Computer Science. 4241, 85-95 (2006).</li><li>Peterson, K. M., Rychel, A. L., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Out+of+the+Mouths+of+Plants:+The+Molecular+Basis+of+the+Evolution+and+Diversity+of+Stomatal+Development.">Out of the Mouths of Plants: The Molecular Basis of the Evolution and Diversity of Stomatal Development.</a> Plant Cell. 22 (2), 296 (2010).</li><li>Czymmek, K. J., Fogg, M., Powell, D. H., Sweigard, J., Park, S. -. Y., Kang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Time-lapse+Documentation+Using+Confocal+and+Multi-Photon+Microscopy+Reveals+the+Mechanisms+of+Invasion+into+the+Arabidopsis+Root+Vascular+System+by+Fusarium+oxysporum.">In vivo Time-lapse Documentation Using Confocal and Multi-Photon Microscopy Reveals the Mechanisms of Invasion into the Arabidopsis Root Vascular System by Fusarium oxysporum.</a> Fungal Genetics and Biology. 44 (10), 1011 (2007).</li><li>Campilho, A., Garcia, B., Toorn, H. V., Wijk, H. 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没有利益冲突的声明。

这个时间的推移激光共聚焦技术使荧光标记的蛋白质在拟南芥的子叶表皮，极性和动态变化的蛋白质的情况下，其功能是至关重要的一个正确的认识，单个细胞中的表达和定位的纵向研究。此前，一直持续时间延时成像用于检查拟南芥根真菌感染11和分生组织的生长5,12，但添加子叶表皮扩大这种技术的多功能性，并允许其使用额外的蛋白质，特别是协助气孔发展领域...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Bacto琼脂</td><td> BD </td><td> 214010 </td><td></td></tr><tr><td>一室幻灯片</td><td> Nunc公司（Thermo Scientific的） </td><td> 15536​​0 </td><td>或两个腔室（155379） </td></tr><tr><td>激光扫描共聚焦显微镜</td><td>蔡司</td><td> LSM700 </td><td>禅2009软件</td></tr><tr><td> 20倍物镜</td><td>蔡司</td><td> 420650-9901 </td><td> NA 0.8，规划复消色差透镜</td></tr><tr><td>解剖显微镜</td><td>奔驰（国家） </td><td> 431TBL </td><td>从下面点亮</td></tr><tr><td> ＃5镊子，生物学尖端</td><td> Roboz手术器械</td><td> RS-4978 </td><td>非常细的小秘诀是关键</td></tr></tbody></table>

long-term, high-resolution confocal time lapse imaging of arabidopsis cotyledon epidermis during germination

在整个发育序列体内的细胞行为的动态影像可以是一个强大的技术了解组织图案的机制。在动物发育，的关键细胞增殖和图案的事件发生得非常快。例如，在秀丽隐杆线虫的幼虫体内所有细胞分裂计划完成后6小时内受精，与七个有丝分裂周期1; 果蝇胚胎发育的16个或更多的有丝分裂发生在不到24小时2。与此相反，在植物发育的细胞分裂是缓慢的，通常在一天3,4,5的顺序。这会带来了独特的挑战和记录植物器官的细胞分裂和分化过程中的动态行为，需要进行长期实时成像调查信令，细胞命运和发展，在植物拟南芥表皮是一个很好的模型系统。。在子叶，这种组织是由空气和抗水的路面细胞穿插均匀分布的气孔，阀的打开和关闭控制的气体交换和水分损失。这些气孔的适当的间距，它们的功能是至关重要的，和它们的发展如下不对称分裂和细胞分化的步骤来产生有组织的表皮（ 图1）的序列。 该协议允许在表皮细胞和蛋白质的观察超过几天发展。这个时间框架的干细胞分裂和分化的表皮细胞，包括气孔和表皮路面细胞可以精确的文档。荧光蛋白可以稠合到感兴趣的蛋白的细胞分裂和分化过程期间，以评估它们的动态。这项技术使我们了解了一种新的蛋白质，POLAR 6的国产化，在气孔系细胞的增殖阶段，在日Ê的拟南芥子叶的表皮，它是在不对称分裂事件和细胞皮层不久之前分裂发生的一特征区移动到前面的细胞中表达。图片可以注册和流线型的视频轻松使用公共域软件的可视化动态和蛋白定位的细胞类型，因为它们随着时间的推移而改变。

<html>用这种方法收集的信息的时间点的一组是在图3中所示。细胞膜被标记为与RFP（PM-RB）和GFP融合POLAR蛋白质在其本地子（的POLAR :: POLAR-GFP）在30分钟的时间尺度，我们可以看到细胞分裂蛋白定位的变化他们前面。非对称细胞分裂在气孔血统形式的干细胞，如气孔，它保留接受进一步的不对称分裂的能力，meristemoids和他们的姐妹细胞的前体，称为气孔的血统地细胞（SLGCs）。的SL...

Watch this Scientific Journal Video about Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination at JoVE.com

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

我们描述了一个协议，用玻片和媒体的固定几天的发展，记录植物子叶共聚焦成像的表皮气孔分化。萤光标记的蛋白质，可以动态跟踪的表达和亚细胞定位，细胞分裂和细胞分化过程中增进了解其可能的作用。

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 ...

long-term-high-resolution-confocal-time-lapse-imaging-arabidopsis

Research

JoVE Journal

Biology

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

13.6K 次观看.  University of Washington. 我们描述了一个协议，用玻片和媒体的固定几天的发展，记录植物子叶共聚焦成像的表皮气孔分化。萤光标记的蛋白质，可以动态跟踪的表达和亚细胞定位，细胞分裂和细胞分化过程中增进了解其可能的作用。

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

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

长期使用宽视场显微镜的成像哺乳动物细胞

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.  ...

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 ...

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

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

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, ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

使用平板扫描仪，以收集拟南芥根向地反应的高分辨率时间失效图片

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

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 ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

有丝分裂细胞同步与高分辨共聚焦显微镜联合应用研究细胞周期蛋白在有丝分裂过程中的作用

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

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 ...