我们感谢梅丽莎格里芬和詹娜Rozacky为他们的专家鱼护。 PDM感谢EGN写​​的援助，慷慨和忍耐。这项工作是由美国国立卫生研究院/ NIDCR R01DE020884到JKE支持。

1。畜牧突变等位基因<ol><li>提高和斑马鱼的繁殖所描述的21。 </li><li>在本研究中使用的斑马鱼突变体等位基因PDGFRA b1059 16，SMAD5 B1100 22，和SMO B577 23。来源这些斑马鱼品系包括ZIRC。 </li></ol> 2。解决方案及器具的制备注：所有的解决方案，并实现可预先和存储以供将来使用。 <ol><li>使胚胎培养基（EM），如前所述21。 </li><li>使4克/升的MS-222（三卡因）。溶解4克磷酸二钠（的Na 2 HPO 4）在450ml的无菌水。加2克MS-222溶液中。内7.0-7.2调节pH至。加无菌水至500ml的总体积。 </li><li>可选：4克/升丁香油。权衡0.2克纯丁香油在50ml锥形管中。添加EM至50ml的总体积。贮存于4℃。 </li><li>使3％methylcellulo本身。把加入50ml的EM + 0.1 M羟乙基烧开。起锅。拌入1.5克甲基纤维素，直到溶液是均一的。贮存于4℃。 </li><li>使0.2％的琼脂糖。添加0.1克琼脂糖至50ml的EM。把EM烧开在微波炉或放在热板上并验证琼脂糖完全溶解。等分入微量离心管中并储存500μl的量在4℃下注意：根据需要体积可以进行调整。 </li><li>使毛细血管扑克。将下降0.9 mm内径的毛细管内的超级胶水。插入的6磅测试单丝钓线一个4-5厘米长的细管内，使得约3-4毫米线的延伸超过所述管的端部。 </li><li>使每个试样2桥接盖玻片。超级胶水22 X 22-1玻璃盖到24×60-1盖板玻璃中间留自由的两端。让单打（每1月底小盖玻片）胚胎不到一天老了，双打（在彼此顶部每端2小盖玻片）胚胎差一分四点Ð胚胎五天年长AYS老，或三元（对方每月底的前3名小盖玻片）。 </li><li>填充10毫升注射器用高真空润滑脂。注意：真空润滑脂用作密封剂，以防止试样过长时间脱水，并具有低电势的毒性相比更不稳定密封剂。 </li></ol> 3。安装斑马鱼胚胎的共聚焦显微镜（见图1） <ol><li>准备麻醉媒体。通过微波在20秒的间隔，直至完全融化液0.2％琼脂糖等分。拌8微升的MS-222的入192微升0.2％的琼脂糖或5微升4克/升丁香油入195微升0.2％的琼脂糖组成。保持在42℃加热块。注：长时间曝光到MS-222导致比丁香油强发育迟缓确实在斑马鱼胚胎24。 </li><li>如果需要的话：手动dechorionate未孵化的转基因胚胎将只是之前技术分析。使用镊子，慢慢德AR绒毛膜打开，直到胚胎被释放。 </li><li>麻醉斑马鱼胚胎。添加1ml的胚胎媒体的MS-222至24毫升4克/升的股票。或者，加390微升4克/升丁香油至24鱼水24毫升。注：丁香油行为慢慢等时间推移显微镜之前执行此步骤至少15-20分钟。 </li><li>放置大约一个朝上的桥接盖玻片的中央空间的真空润滑脂珠。慢慢地推动真空润滑脂出注射器，使得一个平滑，连续的珠的靠近和桥梁和盖玻片的边缘的内侧。 </li><li>传播4％的甲基纤维素中使用木敷贴桥接的盖玻片的中间的圆。 </li><li>转移的胚胎将被成像。当胚胎被麻醉绘制成一个玻璃吸管。握住吸管垂直向上，以允许胚胎沉到液体底部的吸液管。使移液管插入琼脂糖溶液，并允许胚胎降到琼脂糖。也不排除液体。 </li><li>放置标本。得出一些琼脂糖溶液和胚胎放入吸管。驱逐胚胎的琼脂糖溶液对甲基顶部的下降。使用毛细管扑克胚胎向下推到甲基纤维素和定位在所希望的成像胚胎。 </li><li>密封标本。 <ol><li>广场上安装的一个顶一个桥接盖玻片，朝下。直到桥直接接触和琼脂糖滴密封到两个盖玻片施加均匀压力的夹在盖玻片的两侧。确认胚胎的方向正确。 </li><li>擦沿玻璃的木制涂药，理顺在真空脂珠裂缝和缝隙。擦去边缘多余的油脂。 </li></ol></li></ol> 4。转基因斑马鱼的胚胎时移共聚焦成像 （该协议已被优化，蔡司LSM 710共聚焦显微镜我们ING ZEN成像软件，并且可以修改为与其他系统使用。） <ol><li>激活设备。打开激光共聚焦显微镜和任何外部激光设备。打开连接到共聚焦显微镜在电脑上。开放式共焦成像软件，并选择“启动系统”，以激活图像采集控制。 </li><li>准备加热阶段。降低共聚焦显微镜的工作平台和移动物镜出来的位置。配有电子加热阶段替换默认的阶段。旋转控制器，用于加热的阶段，并设定为29.5℃的温度</li><li>放置标本中的视场。放置在台上的装片。将20X物镜到位，提高了工作平台。激活可见光发射器，并期待通过目镜。围绕胚胎的头部在视场中。 </li><li>定义实验的设置。 <ol><li>通过选择激活荧光通道被检测和荧光波长选择颜色查找表（LUT）每个通道。如有必要，可使用手动控制，在软件中打开所需的激光（ 如 561纳米的RFP）。对于每个荧光通道，设置激光强度为10.0和600-700之间的光电倍增管电压（HV或收益）。设置平均到4和位深度为16位。 </li><li>激活的Z堆栈和时间系列模块。设置在针孔的5微米或更小的Z-的分辨率，并将Z-间隔对所建议的最佳距离。 注：一般情况下，5微米的Z-分辨率允许每个Z堆栈图像采集速度不够快的一个“快照”胚胎在特定的时间点，同时也解决单个细胞。降低了平均化也降低了每个图像的时间量。 </li><li>确定图像之间的实验的总长度所需的时间间隔（通常10-20分钟）和。 </li></ol></li><li>设置位置信息。 <ol><li>打开相机到现场模式。调整对焦和提高激光强度，如果要看到荧光。设置数码变焦0.6为更广泛的观点，如果需要的话。 </li><li>定位阶段，使得所有的利益结构是可见的，有房的视野的生长和背部向前。聚焦通过胚胎到荧光的上限和下限。设置在第一和最后Z堆叠切片位置至少100μm以上超出这些限制。 </li></ol></li><li>优化的荧光强度。 <ol><li>设置显示LUT到的范围的指标。荧光结构现在应出现白色，和视场的其余部分应该是蓝色的（无检测）或黑色。 </li><li>增加高压/增益略低于饱和地方的水平（红色）像素，显示效果。如果需要，调整偏移，因此大部分的背景没有检测到（蓝色）。注：小针孔直径，增加激光强度既提高了图像清晰度从头，但激光的强度必须保持低以保持活体组织。增加高压/增益和针孔直径（&lt;5微米），一般最好增加激光强度。 </li><li>保持针孔直径宽，足以采集图像及时，平均套4。的Z间隔应始终设置为针孔直径所建议的最佳距离。进行短扫描（平均= 1）不同的设置，并使用最小的激光强度，实现所需的分辨率。 </li></ol></li><li>运行该实验所需的时间长度。随后，从加热阶段的胚胎取出，慢慢地分开盖玻片。淹没在电磁盖玻片和胚胎在30毫米培养皿。使用玻璃移液管轻轻地清洗胚胎关闭盖玻片并从培养皿取出盖玻片。将胚胎成28.5℃培养箱中继续发展。 </li><li>比较增长。在实验结束时，采取个别Zs的即进行类似上演到试样在实验的开始，但琼脂糖安装同级胚胎钉图像，在28.5℃培养箱中提出的EM。 </li><li>测量咽弓，如需要。测量可以在一些共聚焦软件套件来执行。发现这里测量了使用Fiji25通过导入和Z-投射个别帧。LSM文件执行。 </li></ol>

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+derive+from+the+more+apical+daughter+in+asymmetric+divisions+in+the+zebrafish+neural+tube.">Neurons derive from the more apical daughter in asymmetric divisions in the zebrafish neural tube.</a> Nat. Neurosci. 13, 673-679 (2010).</li></ol>

作者宣称，他们有没有竞争的财务权益。

时间推移共聚焦显微镜是一个强大的工具，用于发展的分析。在这里，我们验证了该方法的有效性学习咽弓形态中的斑马鱼突变体是采用转基因的标记的神经嵴细胞的重要信号通路。除了 ​​组织水平进行分析，时间流逝的分析也适用于分析在细胞尺度28。许多广泛使用的斑马鱼的方法也可以被掺入到时间推移显微镜实验，包括显微注射吗啉代的，mRNA表达，或转基因构建体，以及胚胎之?...

颅面形态发生是一个复杂的多步骤的过程，需要多种细胞类型之间协调的相互作用。大部分颅面骨骼是由神经嵴细胞来源，其中许多必须从背神经管迁移到被称为咽弓1瞬态结构。与许多组织中，颅面骨骼的形态是不是可以通过胚胎在发育的特定时间点的静态图像理解更复杂。虽然它是耗时的执行， 在体内时间推移显微镜提供了一个连续的外观在发育中的胚胎的细胞和组织。在延时系列中的每个图像借给上下文别人，并帮助调查员走向推断，为什么出现一种现象，而不是演绎什么是发生在那个时候。 体内成像因此是一个功能强大的工具，描述实验方法解构，引导形态的途径。斑马鱼斑马鱼是脊椎动物胚胎发育的一个流行的遗传模式，并特别适于在体内成像形态的。现代，便利的方法转基因和基因组修饰突飞猛进提供给斑马鱼研究工具的数量。这些工具提升已经非常强大的方法进行基因操纵和显微镜。在几乎任何所需的遗传背景在体内几乎所有组织的成像更接近现实比幻想。 咽弓的形态发生运动是由信号的神经嵴和邻近的上皮细胞，两者外胚层和内胚层之间的交互引导。但是也有一些必要的驱动的颅面骨骼元素的形态由上皮细胞表达大量的信号分子。在这些信号分子，刺猬（嘘）是非常重要的f或颅面部发育2-8。嘘是由口腔外胚层和内胚层咽2,6,9,10均表示。 Shh的中内胚层表达调节拱门10，神经嵴的拱门10内的图案，和颅面骨骼11的生长形态发生运动。 BMP信号也是颅面部发育12极为重要，可能会改变咽弓的形态。 BMP信号调节波峰的咽弓13,14内背/腹图案。 SMAD5在斑马鱼的破坏会导致严重的腭缺陷和麦氏软骨的破坏在15中线适当地融合。此外，该突变体也显示出降低和融合在腹侧软骨的元素，与第2 次 ，第3 次 ，有时稠的中线15第 4 次咽弓元素。这些融合强烈建议Bmp信号指示这些咽元素的形态。 PDGF信号是必要的颅面发育，但在咽弓形态发生未知的角色。小鼠和斑马鱼突变体PDGFRA有深刻的面中部的clefting 16-18。至少在斑马鱼这面中部的clefting是由于适当的神经嵴细胞迁移16的故障。神经嵴细胞继续表达PDGFRA他们已经进入了咽弓后。此外，PDGF配体是由面部上皮细胞表达和咽弓16,19,20内，从而PDGF信号也可以在下面的迁移咽弓的形态发生中发挥作用。然而，分析，并没有被执行在PDGFRA突变咽弓的形态发生。 在这里，我们展示 pharyngul的活体共焦显微镜一个阶段的转基因斑马鱼，并描述此期间内，咽弓的形态。我们进一步表明，受扰乱的BMP，PDGF和Shh信号通路的突变组织行为。

<table border="0" cellpadding="0" cellspacing="0" width="837">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">6 lb. test monofilament line</td>
			<td style="width:189px;">Cortland Line Company</td>
			<td style="width:185px;">SLB16</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Agarose I</td>
			<td style="width:189px;">Amresco</td>
			<td style="width:185px;">0710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Argon laser</td>
			<td style="width:189px;">LASOS Lasertechnik GmbH</td>
			<td style="width:185px;">LGN 3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Calcium chloride</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:185px;">C8106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Capillary tubing, 100 mm, 0.9 mm ID</td>
			<td style="width:189px;">FHC</td>
			<td style="width:185px;">30-31-0</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Clove oil</td>
			<td style="width:189px;">Hilltech Canada, Inc.</td>
			<td style="width:185px;">HB-102</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">High vacuum grease</td>
			<td style="width:189px;">Dow Corning</td>
			<td style="width:185px;">2021846-0807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Isotemp dry-bath incubator</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:185px;">2050FS</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Laser scanning microscope</td>
			<td style="width:189px;">Carl Zeiss AG</td>
			<td style="width:185px;">LSM 710</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Magnesium sulfate hexahydrate</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:185px;">230391</td>
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			<td height="21" style="height:21px;width:243px;">Microscope cover glass, 22x22-1</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:185px;">12-542-B</td>
			<td></td>
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			<td height="21" style="height:21px;width:243px;">Microscope cover glass, 24x60-1</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:185px;">12-545-M</td>
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			<td height="21" style="height:21px;width:243px;">Potassium chloride</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:185px;">M-11321</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Potassium phosphate dibasic</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:185px;">P3786</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium chloride</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:185px;">M-11624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium phosphate dibasic</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:185px;">S7907</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">TempController 2000-2</td>
			<td style="width:189px;">PeCon GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Tricaine-S</td>
			<td style="width:189px;">Western Chemical, Inc.</td>
			<td style="width:185px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing craniofacial morphogenesis in zebrafish using 4d confocal microscopy

延时成像是一种技术，它允许直接观察形态发生的过程中，或形状的生成。由于它们的光学清晰度和顺从遗传操作，斑马鱼的胚胎已经成为一种流行的模式生物与在活胚胎进行形态的延时分析。一个活斑马鱼胚胎的共焦成像要求的感兴趣组织的持续标记有荧光标记物，如转基因或注射染料。该过程要求的胚胎被麻醉并在这里以这样一种方式，健康发展继续正常进行。成像参数必须设置为占三维增长和平衡解决单个细胞，而得到发展的快速快照的需求。我们的研究结果表明，以荧光标记的斑马鱼胚胎的体内成像进行长期检测在不同组织行为的能力颅神经嵴造成颅面畸形。造成麻醉和安装发育迟缓是微乎其微的，而且胚胎是安然无恙的过程。时移成像胚胎可以在以后的发展点返回到液体培养基中，随后成像或固定。随着转基因斑马鱼线和良好的特点命运映射和移植技术，成像越来越丰盈任何需要的组织是可能的。因此，时间推移的体内成像结合有力地与斑马鱼基因的方法，包括突变体和显微注射的胚胎的分析。

在野生型胚胎中，神经嵴人口下面，沿前/后和背/腹轴咽弓伸长移动时在一个方向喙（ 电影1）。在30小时后，受精（HPF），第一咽弓的前/后长度为1.8-1.9倍，其背/腹高度之间。背/腹伸长率稳步前进，比前/后延长至36.5 HPF更快。从这里，背/腹高高原各地的104微米至48 HPF。前/后继续延长至48个高倍视野，从低于1.5倍的背/腹高度36.5 HPF至1.95倍的背/腹高度在48 HPF整体前/后长度增加。 <p c...

Watch this Scientific Journal Video about Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy at JoVE.com

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

时间推移共焦成像是一个强大的技术表征胚胎发育有益。在这里，我们描述的方法和表征颅面形态发生在野生型，以及PDGFRA，Smad5和与SMO突变体胚胎。

使用4D共聚焦显微镜分析颅面形态的斑马鱼

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

analyzing-craniofacial-morphogenesis-zebrafish-using-4d-confocal

Research

JoVE Journal

Biology

10.9K 次观看.  The University of Texas at Austin. 时间推移共焦成像是一个强大的技术表征胚胎发育有益。在这里，我们描述的方法和表征颅面形态发生在野生型，以及PDGFRA，Smad5和与SMO突变体胚胎。 

视频: Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

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.  ...

科研

生物学

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 ...

Electroporation of Craniofacial Mesenchyme

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

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

颅面间质细胞的电

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

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 ...

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, ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

同时进行pH测量的内吞和胞车厢中使用共焦显微镜活细胞

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

酵母的4D显微镜

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

使用前 Vivo 共聚焦显微镜可视化淋巴节点结构和细胞定位

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &amp;#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

利用单分子定位显微镜分析人类 iPSC 衍生肌细胞中的α-Actinin 网络

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...