这项研究是由美国国家科学基金会职业奖（MCB - 0746066，转录激活，规范应急行为表征）RDW 我们非常感谢LJ希姆库斯，BS高盛，G.孙明扬，M.歌手，LG韦尔奇，KA的墨菲，有益的讨论和对稿件的意见和HG泰勒。

用品需要： <ul><li> Klett米</li><li>移液器和提示</li><li> 2.5毫升离心管</li><li>离心</li><li> CTTYE媒体： <ul><li> Casitone 1.0％（DIFCO实验室），0.5％的酵母提取物（DIFCO实验室）， </li><li> 10.0毫米的Tris - HCl（pH值8.0），1.0毫米的KH 2 PO 4，硫酸镁 8.0毫米</li></ul></li><li> TPM的媒体： <ul><li> 10.0毫米的Tris - HCl（pH值8.0），1.0毫米的KH 2 PO 4，硫酸镁 8.0毫米</li></ul></li><li>琼脂糖</li><li>置水浴至55 ° C </li><li>显微镜幻灯片</li><li>大型盖玻片</li><li> 2 × 2厘米，0.5毫米厚的硅橡胶垫片（格雷斯生物实验室公司） </li><li>封口膜（或磁带标签） </li><li>镊子</li><li>小长尾夹</li><li>微取样吸管（费舍尔） </li><li> 100μL玻璃一次性提示（费舍尔） </li><li> Kimwipes </li></ul>第1部分：细胞制备开始创造一个无菌的环境。 <ul><li>清洁工作区，唐手套，光刻录机。 </li></ul><ol><li>开始接种到含有CTTYE肉汤瓶和大力纷飞在黑暗中32℃孵育过夜培养的细胞。 </li><li>文化一旦达到了5 × 10 8个细胞/ ml成2.5 ml离心管，吸管1毫升细胞的密度。 </li><li> 16,000 XG（或最大速度）2分钟离心沉淀细胞。 </li><li>倒出并丢弃上清液。 </li><li>洗细胞沉淀用1 ml TPM（盐平衡，营养的自由媒体）。 <ul><li>重新挂起和涡</li></ul></li><li>重新颗粒细胞为16,000 XG（或最大速度）2分钟旋转。 </li><li>倒出并丢弃上清液。 关键的一步 </li><li>完全重新悬浮颗粒在100μLTPM的使用相结合的吹打和涡旋媒体。 重要提示：此步骤可确保有没有留在管细胞团块。这可能需要一段时间。 </li><li>设置在室温下的细胞，预留而准备的跟踪检测板。 </li></ol>第2部分：琼脂的制备<ol><li>准备50毫升TPM（化验媒体）和CTTYE（营养磁盘）媒体。 </li><li>添加0.5克琼脂糖（琼脂糖低于琼脂衍射）。 </li><li>高压灭菌消毒。 </li><li>灭菌后，媒体保持在55 ° C在孵化器（或水浴），而建设的跟踪检测板配合。 </li></ol>第3部分：营养磁盘建设<ol><li>火焰灭菌的玻璃显微镜玻片上放置一个0.5毫米厚的无菌的硅橡胶垫片，形成一个小的好。 </li><li>移液器〜300μL的55 °彗星CTTYE媒体/琼脂糖到以及创建幻灯片上的垫圈和包含的营养磁盘。 CTTYE媒体/琼脂糖应土堆。 </li><li>将火焰消毒CTTYE媒体/琼脂糖上的幻灯片（无垫片）。 重要说明：这张幻灯片必须设置的角度，以防止形成气泡。 </li><li>一旦幻灯片到位，钳复杂连同小长尾夹 - 每边一个。 </li><li>裁剪复杂放置在4 ° C和允许CTTYE媒体/琼脂糖设置。这通常需要大约5分钟。 </li></ol>第4部分：跟踪检测的准备 - 设置板配合物装配组件，准备幻灯片的复合物，放置营养磁盘，倒入TPM的媒体/琼脂糖，单独和干板复合物，板细胞，并组装跟踪检测板复合物。 复杂的部件组装板<ol><li>对于每一个复杂的，火焰灭菌玻璃显微镜幻灯片</li><li>将幻灯片上的蒸压垫片和预留（消毒端升级）。这将形成检测的底部。 </li><li>火焰消毒的玻璃盖滑，并将其放置在顶部用标签胶带（或封口膜）包裹的第二个玻璃显微镜幻灯片，并预留（消毒端升级）。这是作为一个支持幻灯片，以防止开裂盖玻片。这将形成检测的顶部。 </li><li>火焰消毒第三玻璃显微镜玻片上，并预留（消毒端升级）。这将用于扁平化琼脂糖。 重要信息：确保垫片形式按用镊子与玻璃密封，否则媒体/琼脂糖干出来的。 </li></ol> P<html>花边营养磁盘重要事项：步骤5至10必须做一个幻灯片复杂一次;否则，媒体/琼脂糖开始凝固造成影片质量差。 <ol start="5"><li>删除营养磁盘复杂的形式4℃（第5步，从第3部分）和撬除了揭露现在凝固CTTYE /琼脂糖幻灯片。 </li><li>删除CTTYE媒体/琼脂糖从1毫米直径的“营养磁盘”远高于使用100μL玻璃一次性尖端的微型采样吸管放置在垫片上创建的盖玻片（上述第3步） 。 </li></ol>倒板 关键的一步 <ol start="7"><li>移液器〜300μL的55 °彗星TPM媒体/琼脂糖到盖玻片和包含的营养磁盘上的垫片创建。 TPM媒体/琼脂糖应土堆。 关键的一步 </li><li>将火焰消毒幻灯片没有TPM媒体/琼脂糖上的垫片（从第3步）。 重要说明：这张幻灯片必须设置的角度，以防止形成气泡。 </li><li>幻灯片（从第5步）一旦到位，钳复杂连同小长尾夹 - 每边一个。 </li><li>裁剪复杂放置在4 ° C和允许媒体/琼脂糖设置。这通常需要大约5分钟。 </li></ol>单独和干板重要信息：要防止干燥媒体/琼脂糖，11到20步，只应在一个幻灯片复杂执行一次。 重要提示：为了获得最佳结果，11到13步，应在4 ° C。 <ol start="11"><li>一旦媒体/琼脂糖设置，删除长尾夹，挤压复杂松开胶带包裹的幻灯片。 </li><li>取下胶带包裹的幻灯片，并进一步使用的长凳上。 关键的一步</li><li>使用镊子作为一个楔子，单独从支持幻灯片（无垫片）盖玻片/垫片/媒体/琼脂糖复杂和丢弃的支持幻灯片。 重要事项：不要使用撬议案，独立的盖玻片/垫片复杂。这可能导致在盖玻片打破和/或媒体/琼脂糖坚持支持幻灯片。 </li><li>放置盖玻片上的胶带包裹的幻灯片/垫片/媒体/琼脂糖复杂（垫圈朝上），删除4 ° C。将这个复杂的刻录机，让所有可见的新暴露的媒体/琼脂糖表面水分蒸发 - 不超过1分钟。 要点：不要让媒体/琼脂糖干太长，因为这可能会影响M。 xanthus群的行为。 </li></ol>板细胞关键的一步<ol><li>一旦多余的水分蒸发，吸管集中细胞上的媒体/琼脂糖约1毫米，从营养上（第1部分中的步骤8）0.5μL。 重要提示：这是极为重要，以确保细胞存入移动吸管，直降，然后是直线上升。这将确保群将是圆形的。 重要事项：不要触摸枪头向媒体/琼脂糖这是极为重要的。这将使得表面上的抑郁症和改变行为的 M. xanthus群。 提示：压低在接近媒体/琼脂糖的枪头。这将使细胞下降，出现在吸头的底部，并使其更易于存款细胞。 </li><li>一旦细胞的沉积，将复杂的燃烧器，让干细胞现场 - 不超过20秒。 </li></ol>组装检测<ol><li>一旦干细胞当场封面上的防滑垫片/垫圈/媒体/琼脂糖/细胞复合物（从第13步），配合幻灯片/垫片复杂（从第1步），并轻轻按压在一起，形成一个密封。 关键的一步</li><li>清洁表面的幻灯片和盖滑与Kimwipe删除左边的封口膜的残留。完成检测应该类似于图1。 </li><li> （滑下放在保持在32℃的加热阶段完成的幻灯片复杂，盖玻片）后立即擦拭（15步），以避免水汽凝结形成形成。如果形成结露，让几秒钟之前开始的图像采集软件幻灯片上加热阶段的复杂坐。 </li><li>选择适当的目标（2X，4X，或10X）。 </li></ol>第5部分：电影的制备T“的关键一步<ol><li>打开相机和显微镜，并检查灯光亮度（确保光线不是太激烈）。启动电脑，然后打开图像采集程序。 </li><li>点击实时图像窗口和聚焦显微镜。图像应该出现类似如图3所示的。请注意，琼脂表面均匀。 </li><li>开始获取图像。 关键的一步</li><li>定期检查的重点，在第一个小时，媒体/琼脂往往解决造成焦点漂移。 </li><li>干净的工作区域。 </li><li>一旦完成图像采集，传输收购存储图像，并打破90％的乙醇浸泡过夜幻灯片复杂。 </li><li>高压灭菌器垫片重用。 </li></ol><img src="/files/ftp_upload/2038/2038fig1.jpg" alt="图1" /> 图1。TM板复杂的卡通插图。 （A）给出了在爆炸视图和断面的基本TM板复杂。 （b）显示了较大的垫圈使用。 <img src="/files/ftp_upload/2038/2038fig2.jpg" alt="图2" /> 图2。显微镜集群。每个显微镜节点（插图）由尼康E400显微镜，目标，激烈的阶段，透视相机，笔记本电脑。每个节点通过网络连接在一起，并链接到主控制器的计算机。两个节点的设立与EXFO光源和两个Uniblitz百叶窗组成的的荧光功能。 <img src="/files/ftp_upload/2038/2038fig3.jpg" alt="图3" /> 图3。20X的跟踪检测仪器的图像在时间= 0 。比例尺，1毫米。 <img src="/files/ftp_upload/2038/2038fig4.jpg" alt="图4" /> 图4。TM板复杂的适应性。 （一）M. 100X形象xanthus滑翔运动的CTTYE在1.0％的琼脂。 （二）P. 20X形象假单胞菌抽搐蠕动。 （三）20倍影像S.沙雷氏菌蜂拥蠕动。 （二）及（C）测定在1.0％琼脂的LB。 （四）M. 40X形象耻垢的LB滑动在0.5％的琼脂蠕动。使用替代实验配置图1B看到此图片被抓获。 视频1。一个时间推移视频跟踪检测群。 视频2。一个M.时间推移视频xanthus群，其中1％的细胞荧光标记。被抓获的交替阶段，对比和荧光图像和覆盖澄清荧光细胞群内的位置。 CTTYE在1.0％的琼脂，该视频被抓获。 视频3。M 的时间推移视频xanthus滑翔运动的。 CTTYE在1.0％的琼脂，该视频被抓获。 一个P 的时间推移视频视频4。 假单胞菌抽搐蠕动。该视频被抓获就在1.0％琼脂LB。 S 的 ​​时间推移视频视频5。 沙雷氏菌蜂拥蠕动。该视频被抓获就在1.0％琼脂LB。 M 的时间推移视频录象6。 耻垢滑动蠕动。该视频被抓获使用替代实验配置图1B看到LB在0.5％的琼脂。

<ol>
	<li>Taylor, R. G., Welch, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+as+an+emergent+property+of+a+swarm.">Chemotaxis as an emergent property of a swarm.</a> J Bacteriol. 190, 6811-6816 (2008).</li><li>Curtis, P. D., Taylor, R. G., Welch, R. D., Shimkets, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Organization+of+Myxococcus+xanthus+During+Fruiting+Body+Formation.">Spatial Organization of Myxococcus xanthus During Fruiting Body Formation.</a> J Bacteriol. 189, 9126-9130 (2007).</li><li>Mignot, T., Merlie, J. P., Zusman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+pole-to-pole+oscillations+of+a+bacterial+gliding+motility+protein.">Regulated pole-to-pole oscillations of a bacterial gliding motility protein.</a> Science. 310, 855-857 (2005).</li><li>Stoodley, P., Hall-Stoodley, L., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detachment+surface+migration,+and+other+dynamic+behavior+in+bacterial+biofilms+revealed+by+digital+time-lapse+imaging.">Detachment surface migration, and other dynamic behavior in bacterial biofilms revealed by digital time-lapse imaging.</a> Methods Enzymol. 337, 306-319 (2001).</li><li>O'Toole, G., Kaplan, H. B., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+as+microbial+development.">Biofilm formation as microbial development.</a> Annu Rev Microbiol. 54, 49-79 (2000).</li><li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellar+and+twitching+motility+are+necessary+for+Pseudomonas+aeruginosa+biofilm+development.">Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development.</a> Mol Microbiol. 30, 295-304 (1998).</li><li>Dalton, H. M., Poulsen, L. K., Halasz, P., Angles, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substratum-induced+morphological+changes+in+a+marine+bacterium+and+their+relevance+to+biofilm+structure.">Substratum-induced morphological changes in a marine bacterium and their relevance to biofilm structure.</a> J Bacteriol. 176, 6900-6906 (1994).</li><li>Dworkin, M., Eide, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myxococcus+xanthus+does+not+respond+chemotactically+to+moderate+concentration+gradients.">Myxococcus xanthus does not respond chemotactically to moderate concentration gradients.</a> J Bacteriol. 154, 437-442 (1983).</li><li>Dworkin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactic+behavior+of+Myxococcus+xanthus.">Tactic behavior of Myxococcus xanthus.</a> J Bacteriol. 154, 452-459 (1983).</li><li>Wu, S. S., Kaiser, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+expression+of+the+pilA+gene+in+Myxococcus+xanthus.">Regulation of expression of the pilA gene in Myxococcus xanthus.</a> J Bacteriol. 179, 7748-7758 (1997).</li><li>Bustamante, V. H., Mart&#237;nez-Flores, I., Vlamakis, H. C., Zusman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Frz+signal+transduction+system+of+Myxococcus+xanthus+shows+the+importance+of+the+conserved+C-terminal+region+of+the+cytoplasmic+chemoreceptor+FrzCD+in+sensing+signals.">Analysis of the Frz signal transduction system of Myxococcus xanthus shows the importance of the conserved C-terminal region of the cytoplasmic chemoreceptor FrzCD in sensing signals.</a> Mol Microbiol. 53, 1501-1513 (2004).</li></ol>

定时microcinematography（TM）已经成为一个标准的方法来研究原核蠕动2-7。传统上，商标是由基板8-11使用滤纸芯，薄琼脂垫，或琼脂砖。这些方法是适当和符合成本效益的，使用时为一般细菌运动插图产生的图像序列。但是，如果图像序列的结果必须产生重复性好，定量严谨的数据，这些方法都是费时又有点不可靠。例如，人为错误造成的这些技术的变化可能导致各种各样的不准确?...

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<td>1.0% Casitone</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
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<td>0.5% yeast extract</td>
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<td>Difco Laboratories</td>
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<td>Micro-sampling pipette</td>
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<td>Fisher</td>
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<td>100 &mu;l glass disposable tip</td>
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<td>Fisher</td>
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<td>2 x 2 cm, 0.5-mm-thick silicone rubber gasket</td>
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<td>Grace Bio-Lab Inc.</td>
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recording multicellular behavior in myxococcus xanthus biofilms using time-lapse microcinematography

δ- proteobacterium 黏细菌群包含数以百万计的细胞作为一个集体，协调运动通过一系列信号来创建复杂的，动态的模式，为应对环境线索。这些模式是自我组织和应急，他们无法通过观察单个细胞的行为的预测。使用时间推移microcinematography跟踪检测，我们确定了一个鲜明的新兴模式，在M. xanthus所谓的趋化作用，作为一个群的定向运动了对营养源 1梯度定义。 为了有效地表征microcinematography趋化通过时间推移，我们开发了一个高度修改板复杂（图1）和建造的8显微镜（图2），每一个能够捕捉时间推移视频集群。检测是严格的，足以让一致的，可量化的数据复制，产生的视频，让我们来观察和跟踪群行为的微妙变化。一旦捕捉到的视频转移到分析/存储计算机与足够的内存来处理和存储成千上万的视频。此设置的灵活性，已证明是有益的几个成员的M. xanthus社会。

Watch this Scientific Journal Video about Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography at JoVE.com

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

研究<em&gt;黏细菌</em&gt;群的行为，我们设计了一个时间推移microcinematography协议，可用于不同的检测修改。它采用的是标准的显微镜适应增长的条件下，使用价格低廉，可重复使用的硅胶垫片，并产生可重复的结果。我们已经用这种方法来量化的多细胞的趋化。

在录制多细胞行为黏细菌生物膜，使用定时Microcinematography

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of ...

recording-multicellular-behavior-myxococcus-xanthus-biofilms-using

Research

JoVE Journal

Biology

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

12.2K 次观看.  University of South Carolina (USC). 研究黏细菌群的行为，我们设计了一个时间推移microcinematography协议，可用于不同的检测修改。它采用的是标准的显微镜适应增长的条件下，使用价格低廉，可重复使用的硅胶垫片，并产生可重复的结果。我们已经用这种方法来量化的多细胞的趋化。

视频: Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

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.  ...

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 ...

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 ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

使用时间的推移显微镜可视化缺氧诱导假死<em&gt; C.线虫</em&gt;胚胎

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

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 Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

在录制多细胞行为黏细菌生物膜，使用定时Microcinematography

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此视频中的章节

拆装时间推移显微镜监测肌动蛋白

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

有丝分裂的siRNA转染后的时间推移成像

时间推移显微镜早期胚胎发育线虫

随机使用时移视频显微镜细胞迁移的定量分析

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

使用时间的推移显微镜可视化缺氧诱导假死

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

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

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism