我们感谢马克寺崎初步意见，对利用双光子显微镜，创造局部组织损伤，凯西茹班安装时间推移成像的意见，并讨论Sagasti和Portera - Cailliau实验室。最初的实验中，只要在中，马伍兹霍尔海洋生物实验室的草基金会研究员。在Sagasti实验室的工作是支持由白厅基金会，该Klingenstein基金会和巴勒斯惠康生物科学基金事业奖补助。

第1部分：安装斑马鱼的胚胎长期成像<ol><li>准备嵌入1％低熔点琼脂糖溶液。离子水在微波炉，分装成小管和储存在摄氏42度加热块加热溶解琼脂糖。 </li><li>选择成像的胚胎，并用镊子轻轻拉出绒毛膜除了删除chorions。胚胎可以被放置在受精后22-24小时（HPF），抑制色素形成到5％的机动部队铃声的解决方案。这提高了成像的清晰度和表演的感觉神经轴突的双光子axotomies至关重要。如果允许形成的天然色素是，自体荧光掩盖轴突上的双光子显微镜。林格对斑马鱼的解决方案包含了116毫米氯化钠，氯化钾2.9毫米，1.8毫米氯化钙 2，和5mm的HEPES，pH值7.2 。 </li><li>加入tricaine〜0.02％的机动部队林格麻醉胚胎。确保动物没有反应，然后再继续触摸。 </li><li>准备长期的成像室，采用真空润滑脂玻璃或聚四氟乙烯环一侧固定到玻璃盖玻片。 </li><li>一个胚胎转移到42度的琼脂糖溶液用玻璃吸管，照顾不转让的机动部队林格，然后转移到事先准备好的盖玻片一滴琼脂糖胚胎。 </li><li>需要琼脂糖变硬胚胎的位置。请记住，成像室，将翻转当您完成，使盖玻片将排在前面。 </li><li>如果你有一个机动的阶段，可以一次图像的多个地点，重复步骤每个胚胎1.4-1.6。 </li><li>允许琼脂糖完全巩固，然后填写与0.02％tricaine的机动部队林格环。 </li><li>应用真空润滑脂环（S）的另一侧，然后盖上载玻片上环（S）。翻转密封腔（S）。这些商会可用于直立或倒置显微镜成像。 </li></ol>第2部分：两个光子干切断变色龙钛蓝宝石激光使用一个定制的双光子显微镜<ol><li>准备用于成像。在显微镜下的幻灯片固定地点安装胚胎（S）。与40X（0.8 NA）的水目标聚焦在一个胚胎。打开激光。我们已经能够再现可视化和损害的GFP表达神经元使用下列参数。要在一个非破坏性的力量可视化的轴突，设置激光的波长910纳米（nm）和一个功率为30毫瓦（mW的上市是权力的样品量）。打开图像处理软件。我们使用的ScanImage软件开发卡雷尔斯沃博达的实验室，2，3 。 </li><li>新闻焦点的双光子激光扫描胚胎，找到你想axotomize轴突，捕获这个轴突的形象。马克的第一个和最后一个Z轴位置，获得的图像，并做出最大的Z堆栈投影。 </li><li>在70X放大你想axotomize和停止扫描轴突的分支。增加兆瓦样品破坏电力180兆瓦，并执行与激光单次扫描。为此，我们设置的Z切片数量为1，按下“抓图”。切断轴突，这应该是足够的。方法的讨论，请参阅您的显微镜和实验的目标，以优化此过程。 </li><li>缩小，降低功率30兆瓦，并拍摄图像。 </li></ol>第3部分：双光子干切断蔡司510焦/双光子显微镜<ol><li>将安装在胚胎阶段，使用25X水的目标或其他合适的目标，把重点。 </li><li>打开双光子（910纳米）和氩（488 nm）的激光多轨设置，以便它可以从一个切换到其他。虽然两个激光器用于检测绿色荧光蛋白，两个光子发射是用红色显示，和氩激光与绿色排放区分这两个。 </li><li>使用氩激光，以确定一个轴突伤害。在z设置标志的第一个和最后一个光学部分。共聚焦图像，并创建一个最大的Z堆栈投影。 </li><li>选择地区要受伤的轴突，使这一地区成为关注的焦点。氩激光关闭和开启双光子激光。 〜9％的传输，以确保轴突仍然集中在激光强度在双光子扫描标本。 </li><li>点击“停止”按钮，使裁剪工具将提供。使用作物放大感兴趣的领域。通常我们放大〜70X（可变焦“模式”标签下检查）。在“渠道”标签变化的双光子传输到15-20％的传输〜9％的强度。 </li><li>切断轴突激活“快速的XY”按钮约1秒钟，然后按下停止按钮，以避免过量的损害。轴突应该被看作是分散的碎片，如果程序工作。 </li><li>为了确保轴突被损坏切换到488纳米氩激光，采取另一种共聚焦图像，并创建一个最大的投影。 </li></ol>第4部分：共焦成像时间推移蔡司LSM 510 <ol><li>准备用于成像。如果您使用了激烈的阶段，一定要关闭前至少30分钟，成立时间推移电影。在显微镜下的幻灯片固定地点安装胚胎（S）。聚焦在一个理想的空中目标的胚胎。我们使用20倍，0.5 NA的目标。打开蔡司LSM 510成像软件。打开相应的激光器，并成立所需的配置。现在，我们将描述一个长期长期成像蔡司LSM的多时间软件的详细协议。这些方法可以适用于在自己的显微镜成像软件的使用。 </li><li>定义你的第一个胚胎多的时间窗口的位置和配置。打开扫描，舞台，和多的时间窗口。启动快速扫描的扫描窗口上。移动阶段所需的XY位置。马克在扫描窗口的第一个和最后的Z轴位置。按停止，然后月中旬，在扫描窗口。等待中间ž片完成扫描，然后按标记的位置，在舞台窗口。如果你只成像一个胚胎，单击“单位置”选项卡上多的时间窗口。点击“替换XYZ”，然后“保存配置。同意当记者问到覆盖以前的配置。继续到步骤4.4。如果你有一个机械化的阶段，你可以设置多个地点的多个胚胎形象。在这种情况下，你应该定义你的第一个位置的XYZ和配置之前选择“多个位置”选项卡。此外，请务必位置1，略低于“多个位置”选项卡的下拉菜单，在下拉菜单中的配置节说多禄1，在你点击保存配置。 </li><li>定义您剩余的胚胎多的时间窗口的位置和配置。找到你的下一个胚胎，然后按扫描速度快，移动到所需的XY位置的阶段，并标记你的第一个和最后一个扫描窗口的Z位置。按停止，然后月中旬，在扫描窗口。等待中间ž片完成扫描，然后按标记的位置，在舞台窗口。点击“替换XYZ”。检查略低于“多个位置”选项卡的下拉菜单上的位置2，在下拉菜单中的配置节，多禄2说，然后点击“保存配置”。同意当记者问到覆盖以前的配置。重复这个过程中剩余的胚胎。 </li><li>图像采集多禄窗口的设置参数。选择在“块列表”一节中的GR - gl选项，然后输入所需的号码组重复。这是共聚焦将获得的图像在每个位置的次数。在“参数”部分中输入所需的等待时间间隔。这是从一个成像重复开始的下一个重复的启动时间量。选择的配置节“ZStackXY”。输入您的基本文件名，在底部的部分。点击“选择图片DB”，并选择你的MDB。点击“选项”按钮。选择你的MDB文件夹来保存临时文件。检查“保持最终​​图像打开”，“最终的图像保存”，和“中间的Z堆栈”。选择等待的时间间隔。点击确定关闭选项窗口。点击“开始时间”。离开多时间窗口的成像时间的开放。 </li><li>分析您的数据。当时间推移成像已经完成，多时间软件将编译所有到为每个位置的摘要文件的临时文件。如果你想停止电影完成之前，请务必按“完成”而不是“停止”，这样的总结文件将被创建。临时文件和摘要文件应被自动保存到指定的MDB文件夹。从LSM软件菜单，点击“3D视图”，然后“投影”。摘要在下拉菜单中选定的文件，点击“应用”。这将产生最大的Z堆栈的预测，每个共聚焦图像。在LSM软件菜单中，单击“文件”，然后“出口”。一系列tif文件保存最高的预测。然后，您可以创造出的TIF文件使用ImageJ或QuickTime Pro的系列电影。在LSM图像的轴突可以追溯到三个尺寸，使用图像分析软件（如MicroBrightfield NeuroLucida），生成详细的有关轴突形态的定量信息。 </li></ol>第5部分：代表结果一个成功的实验，将导致蜂窝动态准确地表述的轴突从伤病中恢复。你的胚胎，将健康的成像后，无明显的变性和强烈的心跳。干切断应导致精确的损害，只有切断轴突的分支。不应该有任何损伤周围神经轴突和最小的细胞死亡。我们相信，我们看到的干切断〜50％的实验的网站直接在一个单一的表皮细胞死亡。如果你观察到更多的伤害，你应该优化你的两个光子的协议，如在讨论中所述。

<ol>
	<li>Sagasti, A., Guido, M. R., Raible, D. W., Schier, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repulsive+interactions+shape+the+morphologies+and+functional+arrangement+of+zebrafish+peripheral+sensory+arbors.">Repulsive interactions shape the morphologies and functional arrangement of zebrafish peripheral sensory arbors.</a> Curr Biol. 15, 804-814 (2005).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomed Eng Online. 2 (13), (2003).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li><li>Sacconi, L. <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+multiphoton+nanosurgery+on+cortical+neurons.">In vivo multiphoton nanosurgery on cortical neurons.</a> J Biomed Opt. 12, 050502-050502 (2007).</li><li>Galbraith, J. A., Terasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+damage+in+thick+specimens+by+multiphoton+excitation.">Controlled damage in thick specimens by multiphoton excitation.</a> Mol Biol Cell. 14, 1808-1817 (2003).</li><li>Yanik, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosurgery:+functional+regeneration+after+laser+axotomy.">Neurosurgery: functional regeneration after laser axotomy.</a> Nature. 432, 822-822 (2004).</li><li>Quinto-Su, P. A., Venugopalan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+laser+cellular+microsurgery.">Mechanisms of laser cellular microsurgery.</a> Methods Cell Biol. 82, 113-151 (2007).</li></ol>

我们用描述的正是axotomize外周感觉神经轴突和生活斑马鱼的胚胎中直接观察到再生的方法。长期时间推移在斑马鱼的共焦成像可用于观察在体内的发育过程。双光子干切断过程可以修改许多不同的实验目标。我们使用相同的一般程序消融整个三叉神经的感觉神经元胞体，放大细胞体内，而不是一个分支的末梢神经轴突。荧光识别任何类型的细胞，可以精确的损坏或集中功率飞秒激光烧蚀。?...

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

斑马鱼长有被利用来研究时间推移成像的生活透明的胚胎发展的细胞和分子机制。在这里，我们描述的方法安装长期成像斑马鱼的胚胎，并演示如何自动捕获的时间推移，使用共聚焦显微镜图像。我们还描述了一种方法来创建控制，精确地破坏个人使用集中安装在双光子显微镜飞秒激光功率在斑马鱼的外周感觉轴突分支。成功的双光子干切断参数必须为每个显微镜进行了优化。定制的双光子显微镜和蔡司510焦/双光子提供​​了两个例子，我们将展示双光子干切断。 斑马鱼三叉神经感觉神经元1感官神经元特异性启动子驱动的转基因表达的GFP可以可视化。我们已经适应了这种斑马鱼三叉模式，以直接观察活的斑马鱼胚胎的感觉轴突再生。胚胎与tricaine麻醉和定位，因为它巩固了在琼脂糖下降。固定的胚胎被密封在一个充满成像与苯基硫脲嘧啶（PTU）林格室。我们已经发现胚胎可以在这些商会不断成像为12-48小时。一个单一的共聚焦图像，然后捕获，以确定所需的干切断的网站。双光子显微镜成像低，非破坏性的权力下的感觉轴突位于该地区的利益。放大所需的站点上的干切断后，功率增加，单一的，定义区域扫描足以切断轴突。然后设置多个位置的时间推移成像共聚焦显微镜直接观察到，从性轴索损伤恢复。

Watch this Scientific Journal Video about Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos at JoVE.com

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

在这里，我们描述了一个用于安装长期成像，双光子成像和组织损伤的技术，和时间推移共焦成像的斑马鱼胚胎的方法。

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.  ...

two-photon-axotomy-time-lapse-confocal-imaging-live-zebrafish

Research

JoVE Journal

Biology

14.0K 次观看.  University of California, Los Angeles. 在这里，我们描述了一个用于安装长期成像，双光子成像和组织损伤的技术，和时间推移共焦成像的斑马鱼胚胎的方法。

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

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 ...

科研

生物学

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

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 ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

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 ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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 ...

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 ...

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, ...