Estelle Hirsinger: Core funding from the Institut Pasteur and Agence Nationale de la Recherche (ANR-10-BLAN-121801 DEVPROCESS). Estelle Hirsinger is from the Centre National de la Recherche Scientifique (CNRS). Benjamin Steventon was funded by the Agence Nationale de la Recherche (ANR- 10-BLAN-121801 DEVPROCESS), then a Roux fellowship (Institut Pasteur) then an AFM-Te&#769;le&#769;thon fellowship (number 16829). He is now supported by a Wellcome Trust/Royal Society Sir Henry Dale Fellowship.

1.溶液的制备和拉玻璃针<ol><li>在4毫克/毫升使三卡因（3-氨基苯甲酸乙酯，也称为3-氨基苯甲酸乙酯）的25倍原液在20mM Tris pH值8.8和使该溶液的pH为7.在分装用4mL的和存储-20℃。 注:麻醉三卡因优先作用于神经电压门控钠通道从而阻断肌肉抽搐和运动6。 </li><li>使在0.17毫克/毫升在E3的胚胎培养基的最终浓度三卡因的工作溶液。 7 注:使三卡因即兴工作溶液作为该溶液的pH值漂移。 </li><li>通过加热，在微波的溶液溶解该低熔点琼脂糖到的在一个50毫升管的E3胚胎培养基1.5％的最终浓度。让这个解决方案平衡至42 - 45°C在任何一个水浴或台式孵化器。如果准备大型立式菜光片成像（步骤4），使一个附加的25毫升1％的标准熔融琼脂糖在E3的胚胎培养基。 </li><li>用硼硅玻璃长丝毛细管的外径1.20毫米，ID0.69毫米，长10 mm尺寸。 </li><li>如果毛细血管被拉在设备和试剂 ，使用以下设置的表说明的加热丝针拉马类型:热火600，120拉，速度50，时间225，压力500。一对尖锐的钳，断线针刚刚过去的点，它弯曲创造一个干净和锋利的针定向胚胎和去除多余的琼脂糖。的微型手术刀也可使用以除去过量的琼脂糖。 </li></ol> 2.嵌入胚胎的倒立或直立显微镜<ol><li>提高胚胎高达E3胚胎中期的适当阶段。 7 </li><li>在所需的阶段，dechorionate胚胎一双锋利的钳的双眼dissec下方婷显微镜。 </li><li>孵育dechorionated胚胎在三卡因工作溶液至少5分钟。 </li><li>一旦熔化的琼脂糖溶液冷却到45℃，使用玻璃巴斯德吸管向dechorionated胚胎直接与E3介质的最小转移转移到50ml管中。 </li><li>用约1ml安装介质一起取出胚胎，并添加约。 100微升的胚胎与10毫米微孔35毫米的玻璃底培养皿的中心圆一起安装培养基。 </li><li>作为安装介质是设置，将胚胎琼脂糖的朝外（ 图1A）的尾部的圆的边缘。使用毛细管针以保持胚胎在所需的横向取向直至凝胶被完全地设置。图像后身体发育，应注意在定向作为横向的方向尽可能胚胎。因此，在保持通过仔细调适这个位置的胚胎TS与毛细管针。 注意:多个胚胎可以在此时加入到安装盘，如果需要的话。如果是这种情况，传送琼脂糖低熔入盘的第一和胚胎添加到琼脂糖滴的中心。然后，推胚胎出到玻璃环的边缘，并且定向到所需的位置。多达六个胚胎可以安装这种方式之前琼脂糖变得固化。 </li></ol> 3.去除多余琼脂糖围绕车身后部注意:这部分描述由琼脂糖从围绕后部身体区域去除的步骤。在后体伸长的情况下，重要的是要确保尾可以生长出正常。通过除去琼脂糖之后的胚胎已通过琼脂糖被完全包含，胚胎为左括由头部区和大约一半的卵黄囊。 <ol><li>一旦琼脂糖下降已成立，淹没培养皿迪与三卡因工作液sh的。 </li><li>下用透射光碱解剖双目显微镜，调整反射镜位置与入射光的角度，使得强烈的对比允许在琼脂糖削减要通过操作清楚地看到。 </li><li>执行切割1（ 图1），使用毛细管针或微型手术刀切割沿蛋黄琼脂糖中途，从一个位置只是后侧到成形心脏字段的体节5（ 第 5的位置前体节）。 </li><li>开始如图所示相邻的成形心脏初始切割，并通过琼脂糖到玻璃的充分的方法。 </li><li>保持毛细管或微型手术刀琼脂糖深处，慢慢地看到上下，而开始进行切长在胚胎， 如图1所示。切尽量靠近胚胎尽可能无穿刺胚胎或蛋黄。 </li><li>接着，使削减2和3（ 图1 </strong&gt;），从对胚胎的位置，并用切去一路下跌到玻璃开始见底菜。使切割2，使得它相切的前5体节，允许充分dorsalward展开后的身体。使在90°角与蛋黄表面切割3并从HEARTFIELD开始。 注:切割琼脂糖直到玻璃圆的边缘（ 如图1）助剂中除去琼脂糖作为一个完整的块（步骤3.6）。然而，这是没有必要的。 </li><li>在切口1和3的交叉点开始，朝着切口2的端部缓慢对角线切割而慢慢以撞出周围后体琼脂糖的平方向上抬起。 注意:在某些情况下，这将在一个块中被释放，并在操作在一气呵成完成。在其他情况下，可能需要多次尝试DIS-小屋周围胚胎的所有琼脂糖。 </li><li>有一对锋利的钳子，去除脱落b从胚胎中琼脂糖的锁。为了帮助在此过程中，将琼脂糖件移动到盘的一侧，并使用培养皿的壁作为支撑翻开出琼脂糖片。 </li></ol><img alt="图1" src="/files/ftp_upload/55210/55210fig1.jpg" /> 图1:安装调校的图。 （A）图显示了培养皿的中央玻璃环内的安装胚胎的位置。右边是通过周围琼脂糖与dottted红线所示每个连续砍胚胎的变焦。 （ 二 ）安装的胚胎是在图解侧位片显示易于为倒置和直立目标的访问。 （C），显示胚胎如何安装垂直光片影像调校类似的双字母组合。 <a href="http://ecsource.jove.com/files/ftp_upload/55210/55210fig1large.jpg" target="_blank">请CL</a>ICK这里查看该图的放大版本。 <ol start="9"><li>如果肌肉抽搐不完全阻塞在该点，从4毫克/毫升，pH为7原液添加三卡因滴。 </li></ol> 4.胚胎安装立式灯表显微镜注:这是上面列出的方法的变化可以让众多的目标，通过垂直导向SPIM访问样本成像。该变化背后的想法是解除样品比盘底部略高，以允许两个成像目标容易获得。 <ol><li>样品安装前，涂层100毫米的塑料陪替氏培养皿，在E3的培养基1％琼脂糖至5毫米的高度，并允许设置。 </li><li>地点1 mL的低融点琼脂糖一滴的菜的中心，并允许设置。 </li><li>在低熔点琼脂糖胚胎嵌入在部分二，然而，这一次，从50毫升管取出胚胎用溶液（0.5毫升）墨滴较小的琼脂糖并放置在1之上这一小滴的菜的中心毫升下降。 </li><li>使用毛细管针放置胚胎的小滴的中心，并且直到凝胶被设置保持其正确的方位。 </li><li>洪水与三卡因工作液的培养皿去除多余的琼脂糖在第3节，但没有通过1％的标准琼脂糖垫切割。 </li></ol>

<ol>
	<li>Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+the+Zebrafish+Embryonic+Brain+by+Confocal+Microscopy.">Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy.</a> J Vis Exp. (26), e1-e2 (2009).</li><li>Delaune, E. A., Fran&#231;ois, P., Shih, N. P., Amacher, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell-Resolution+Imaging+of+the+Impact+of+Notch+Signaling+and+Mitosis+on+Segmentation+Clock+Dynamics.">Single-Cell-Resolution Imaging of the Impact of Notch Signaling and Mitosis on Segmentation Clock Dynamics.</a> Dev Cell. 23 (5), 995-1005 (2012).</li><li>Lawton, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+tissue+fluidity+steers+zebrafish+body+elongation.">Regulated tissue fluidity steers zebrafish body elongation.</a> Development. 140 (3), 573-582 (2013).</li><li>Dray, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Fibronectin+Interactions+Propel+Vertebrate+Trunk+Elongation+via+Tissue+Mechanics.">Cell-Fibronectin Interactions Propel Vertebrate Trunk Elongation via Tissue Mechanics.</a> Curr Biol. 23 (14), 1335-1341 (2013).</li><li>Steventon, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+tailoured+contribution+of+volumetric+growth+and+tissue+convergence+to+posterior+body+elongation+in+vertebrates.">Species tailoured contribution of volumetric growth and tissue convergence to posterior body elongation in vertebrates.</a> Development. 143, 1732-1741 (2016).</li><li>Attili, S., Hughes, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetic+Tricaine+Acts+Preferentially+on+Neural+Voltage-Gated+Sodium+Channels+and+Fails+to+Block+Directly+Evoked+Muscle+Contraction.">Anaesthetic Tricaine Acts Preferentially on Neural Voltage-Gated Sodium Channels and Fails to Block Directly Evoked Muscle Contraction.</a> PLoS ONE. 9 (8), 103751-103756 (2014).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book, 5th Edition; A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Schr&#246;ter, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+zebrafish+somitogenesis.">Dynamics of zebrafish somitogenesis.</a> Dev Dyn. 237 (3), 545-553 (2008).</li><li>Megason, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+toto+imaging+of+embryogenesis+with+confocal+time-lapse+microscopy.">In toto imaging of embryogenesis with confocal time-lapse microscopy.</a> Methods Mol Biol (Clifton, N.J.). 546, 317-332 (2009).</li><li>Sugiyama, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+cell-cycle+progression+in+the+developing+zebrafish+embryo.">Illuminating cell-cycle progression in the developing zebrafish embryo.</a> PNAS. 106 (49), 20812-20817 (2009).</li></ol>

The authors have nothing to disclose.

这种安装技术使得胚胎转移到显微镜和目的是在多尺度下后身体伸长了长期的时间推移成像实验过程中仍然保持。此外，它是通用的，因为它允许在两个直立和倒置显微镜调校成像和一个建议是为如何可以进一步适于垂直取向SPIM制成。 在本协议中的一个关键步骤是仔细去除周围后身体即允许这种结构的正常发展的重要多余的琼脂糖。除去周围的蛋黄的顶部琼脂糖特别是当在?...

后体伸长是在胚胎发育的基本过程，其中胚胎延伸以形成体轴的很大一部分。它是一个复杂的形态发生过程，其中多细胞行为起作用协调以产生个体组织的水平的形态的一个例子。这些差分组织变形然后一起作用在整个结构水平，以产生后验体的延伸率。要了解这些过程是如何被控制和开发过程中的协调，我们一定能够按照这些流程在多尺度（ 即在分子，细胞，细胞群体和组织的水平），并直接将之与整个结构的形态。 斑马鱼的胚胎是理想的成像后的身体伸长其光学透明性和小尺寸允许微创光成像的应用方法很适合于LIVË成像。 1这已通过一系列最近的出版物中，在分子的水平已经阐明后身体发展光证明，2个单细胞，3和组织间的行为，4以及在细胞群体和整个器官的水平。 五 先进的成像技术如共焦，多光子显微术和选择性平面照明显微镜（SPIM）正在使发育过程的长期成像降低光毒性和光致漂白的效果。活样品的安装鲁棒技术，以实现三个目标是必需的:1）足够的支持，以转移到显微镜和采集期间过程中保持在正确的方向的样品，2）样品的运动的足够的自由度以允许的生长在不影响其正常DEVEL后部本体区域opment，最后3）在一定程度上的安装方法的通用性，允许在不同的影像调校影像。 此协议引入了成像斑马鱼D.鱼发展的安装技术。该技术包括安装胚胎，使得头部和卵黄囊区域几乎完全包含在琼脂糖，而留下的后体区伸长和正常发育。因此，它也是对显影体的其他区域的长期成像作为琼脂糖使得能够通过标准的光成像技术成像的适当的方法。这个协议演示安装在横向取向的胚胎的，虽然它也可以安装在变质取向胚胎。它将进一步展示如何适应直立，倒立和垂直光表镜的设置方法。

<table border="0" cellpadding="0" cellspacing="0" width="727">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">CONSUMABLES</td>
			<td style="width:236px;"></td>
			<td style="width:135px;"></td>
			<td style="width:125px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass-bottomed dishes</td>
			<td>Mattek</td>
			<td>P35-1.5-10-C</td>
			<td>35mm petri dish, 10mm microwell. No. 1.5 cover glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillaries for injection needles</td>
			<td>&#160;Sutter&#160;</td>
			<td>BF 120-94-10</td>
			<td>We use orosilicate glass with filament, OD 1.20 mm, ID 0.94 mm, length 10 cm. However, filament needles are not necessary and most injection standard needles should work.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-scalpel</td>
			<td>Feather</td>
			<td>P-715</td>
			<td>Micro Feather disposable opthalmic scalpel with plastic handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur Pipettes</td>
			<td></td>
			<td></td>
			<td>230 mm long</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">REAGENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low-melting point agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine forceps&#160;</td>
			<td>FINE SCIENCE TOOLS GMBH&#160;</td>
			<td>11252-30&#160;</td>
			<td>Dumont #5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle puller&#160;</td>
			<td>Sutter&#160;</td>
			<td>P97</td>
			<td>Heating-filament needle puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Binocular dissecting microscope</td>
			<td>Leica</td>
			<td>S8 Apo</td>
			<td></td>
		</tr>
	</tbody>
</table>

a versatile mounting method for long term imaging of zebrafish development

斑马鱼胚胎提供了一个理想的实验系统来研究复杂的形态发生过程，由于其方便可及性和光学透明性。特别是，后体伸长是在胚胎发育，使多个组织变形一起作用以引导体轴的一个大的部分形成一个基本过程。为了通过长期延时成像观察这一过程，有必要利用一个安装技术，允许足够的支撑，以传递到显微镜和采集过程中保持在正确的方向的样品。此外，安装也必须提供为后体区的生长移动的足够的自由度，而不影响它的正常发育。最后，必须有一定程度的安装方法，允许在不同的影像调校成像的多功能性。在这里，我们提出了一个安装技术成像后身体elongatio发展N的斑马鱼斑马鱼D.。该技术包括安装胚胎，使得头部和卵黄囊区域几乎完全包含在琼脂糖，而留下了后体区伸长和正常发育。我们将展示如何能够适应直立，倒立和垂直光表镜调校。虽然该协议的重点是安装胚胎的体内后成像，它可以很容易地适应的斑马鱼发育的多个方面的实时成像。

上面概述的协议细节斑马鱼胚胎的安装长期时间推移成像一个通用的技术。这样的一个例子示于图2A和在动画/视频图1。胚胎在mRNA的编码photoconvertible荧光蛋白kikumeGR 1细胞阶段注入。在15体节阶段如上所述并成像为12小时与10倍的物镜的倒置共聚焦显微镜它们封片。所得共焦堆栈被最大限度突出显示为所示。这个电影清楚地表明，后体被允许在整个?...

Watch this Scientific Journal Video about A Versatile Mounting Method for Long Term Imaging of Zebrafish Development at JoVE.com

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

斑马鱼发展长期成像的多功能安装方法

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

a-versatile-mounting-method-for-long-term-imaging-zebrafish

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

11.3K 次观看.  Institut Pasteur. Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

视频: A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

科研

发育生物学

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

水下滤纸三明治长期<em&gt;防爆大毛</em&gt;早期鸡胚时间推移成像

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

长期共焦成像根和下胚轴发育的简单室

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

长期的实时成像<em&gt;果蝇</em&gt;眼睛光盘

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

为梭状体开发拉瓦尔斑马鱼感染模型

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

斑马鱼发展长期成像的多功能安装方法

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