作者承认从ZonMW-VICI资助918.11.635（荷兰）的资金支持。笔者想感谢阿姆斯特丹自由大学的工具店的实际建议和制造安装组件和卡尔蔡司荷兰亚诺Voortman对显微镜有益的建议。 Marjolein Blaauboer在对手稿几易其稿批判评论一个很大的支持。

所有实验程序均按照关于动物法荷兰实验进行。 注:浸没滤纸夹心法是从1修改。 1.淹没滤纸三明治准备<ol><li> Pannett -康普顿（PC）-saline 27,28 <ol><li>准备储备溶液A（1升超纯h及2 O 121克氯化钠，15.5克氯化钾，10.42克CaCl 2·H 2 O，并12.7克氯化镁2·6H 2 O）和B（1升超纯H 2 0，2.365克的Na 2 HPO 4·2H 2 O和0.188克的NaH 2 PO 4·2H 2 O）在清洁1升的瓶子。在4℃下保存。 </li><li>通过混合900毫升超纯的 H 2的O用40ml原液A和60ml库存溶液B（在此，以避免沉淀）制备1升的PC-盐水。准备PC盐水新鲜FROM股票方案A和B每星期。 注:储备溶液可以储存在4℃下几个月。高压灭菌和/或添加抗生素或抗真菌剂是没有必要的。 </li></ol></li><li>受精鸡蛋孵化<ol><li>在湿润的培养箱中，放置在其一侧的鸡蛋，并在37.5℃下孵育，直到它们达到希望的年龄。 注意:超过两周储存鸡蛋不再（优选在12 - 15℃）之前的实验中，作为孵化率将显著下降</li></ol></li><li>滤纸携带者（改编自1） <ol><li>如果可能，使用激光切割机从粗过滤纸（28毫米×22毫米）削减沙漏形滤纸载体。锥度在中间的宽度从22毫米至10.4毫米。切出的中央椭圆形开口（10.6毫米×5.5毫米），具有平行于滤纸载体的较大侧（ 图1-M中的椭圆取向的长轴）。 <ol><li>任选地，准备一个纸板模版具有正确的尺寸和使用铅笔其中心孔的轮廓，并且转移到厚过滤纸。剪下用细剪刀滤纸载体。 </li></ol></li><li>切两个相同的滤纸载体被取出的每个胚。作为准备的情况下，备份期间explanting胚胎丢失额外的过滤纸载体。 </li></ol></li><li>温度控制的油浴（在实验当天） <ol><li>放置温度控制烧杯中直立放大显微镜的电动XY阶段的中心和烧杯连接到其温度控制器。 </li><li>放四个大型不锈钢环（30毫米外径的4毫米高，1.5壁厚）到一个6cm培养皿中，作为权重，并将培养皿在温度控制烧杯的中部。 注:此培养皿只是作为一个占位符来调整油LEVE湖</li><li>填用130ml轻质矿物油的温度控制烧杯中。调节油位，如果必要，以便它结束6cm培养皿中的上边缘低于约3毫米。 </li><li>除去从温度控制烧杯中的6cm培养皿中，并设置温度至40℃。 </li></ol></li></ol> 2.淹没滤纸夹层生产<ol><li>培养基（在实验当天） <ol><li>倾30毫升PC的生理盐水到50毫升离心管中（如在步骤1.1.1至1.1.2的方法制备）。制备填充用30ml的PC-盐水之一50ml离心管中被取出的每两个胚胎。 </li><li>小心裂纹非孵育卵成10cm培养皿。 </li><li>通过沿培养皿的壁的塑料移液管，并在薄蛋白吸吮收获薄蛋白。收集30毫升薄蛋白在50毫升离心管为要取出的每两个胚胎。确保只获得第t欣蛋白。 注:通常3 - 4个鸡蛋允许约30ml瘦蛋白的收获。 </li><li>倾20毫升薄蛋清到每一个的PC-盐水填充的50ml离心管（如在步骤2.1.1的方法制备）。 </li><li>通过几次轻轻颠倒试管混合的PC-盐水与薄蛋白。让它沉淀几分钟，检查混合物，被称为培养基从这里开始，是显而易见的。如果培养基不明确，从原液准备新鲜的PC-和生理盐水收获的新鲜瘦蛋白。 </li></ol></li><li>放置两个小的不锈钢环（20毫米外径的4毫米高，1.5壁厚，6.5 mm空隙环）尽可能远离可能在一个新鲜6厘米塑料培养皿并用22培养毫升填写介质用25毫升的塑料吸管（ 图1-H）（如在步骤1.4.1至1.4.4的方法制备）。确保累积在介质的顶部的碎屑保留在离心管中。 </li><li>取出所有碎片Øř从使用塑料移液管的介质的气泡。 </li><li>填充10cm培养皿用75毫升的PC-盐水（在步骤2.15中使用）。 </li><li>从孵化器中取出一个鸡蛋，让它休息的长椅上侧1分钟，让胚胎来到蛋黄的顶部。 </li><li>小心敲碎鸡蛋倒入培养皿中（ 图1-A）。泡在厚蛋白一块细薄纸的边缘和通过拉，如果需要居中胚。 </li><li>从使用塑料移液管（带切断，以便于厚蛋白的摄取尖端）培养皿除去大部分薄和厚蛋白的。 </li><li>轻轻抹着厚厚的蛋白用一块纸巾，从中心（ 图1-B）搬走创建围绕胚胎的粗蛋白的窗口。附着到卵黄膜防止薄纸。 </li><li>用镊子小心地将滤纸载体上的维生素Elline膜周围的胚，以平行于胚胎（ 图1-C）的前-后轴线椭圆孔径的长轴。 </li><li>捏指甲剪之一前端通过卵黄膜靠近滤纸载体和围绕整个滤纸载体（ 图1-D）的快速切割。 </li><li>使用镊子，提起滤纸载体从蛋黄之外的方向平行的斜对胚胎（ 图1-E）的前-后轴线。 </li><li>转动滤纸载体周围具有在顶部的胚胎的腹侧和在PC-盐水淹没它。沉浸沿着胚胎的前后轴的滤纸载体在一个倾斜方向。 </li><li>摆脱所有的蛋黄仍然附着卵黄膜，并轻轻地用PC-盐水从一个塑料移液管冲洗它的胚盘。保持在浸没的滤纸载体的整体LL倍（ 图1-F）。 </li><li>在倾斜方向中删除从沿着胚胎的前后轴对PC-盐水滤纸载体并获得由涂抹滤纸载体边缘上的纸巾清除过剩的液体。 </li><li>水平握住滤纸，面朝胚胎的腹侧，并将另一滤纸载体上的第一个顶部，使用第二镊子。其孔应完全匹配，从而夹持滤纸的两个层（ 图1-G）之间的胚胎。 </li><li>立即浸没滤纸载体三明治到培养基中沿胚胎的前 - 后轴线的倾斜方向。将其放置在跨越两个钢环（ 图1-H）之间的空间的面积。 </li><li>通过放置在另外两个的顶部两个钢环，确保固定滤纸三明治与电子光圈mbryo保持完全未覆盖（ 图1-I）。 </li><li>检查中等水平，并确保上间隔刚好浸在培养基中。如果有必要，添加或删除少量介质。 </li><li>小心地将培养皿中的温度控制的油浴中的中心与通过将三个大的不锈钢环（30毫米外径的4毫米高，1.5壁厚）旁边的培养皿中的油横向稳定的菜。确保该钢环触摸盘（ 图1-J）的两侧。 </li><li>慢慢地吸取上用塑料移液管的介质的顶部约6毫升轻矿物油的，这样，介质覆盖有矿物油的连续层（ 图1-K）。 </li><li>设置时间推移采集。 注:成像胚胎作为五个重叠子图像（瓷砖）的水平全景允许详细成像（5X物镜），与一般overvi形态29-30的EW。 3和10分钟之间的成像时间间隔允许形态的详细跟踪变化31和采集的小的z栈（五到七个与30μm的距离不同的平面）用来补偿多胚胎30的增厚和转动的。 Z-飞机最高的对比度可以单帧进一步处理前32可以选择到定时短片（见图像采集的详细信息代表结果部分）。 </li></ol><img alt="图1" src="/files/ftp_upload/54636/54636fig1.jpg" /> 图1:设置淹没滤纸三明治。 （A）一个鸡蛋裂解成培养皿。 （B）的最厚和薄蛋白的从培养皿用塑料移液管除去（未示出）。然后，在厚蛋白的窗口创建一个轮用纸巾纸轻轻抹着厚厚的蛋白胚胎。 （C）滤纸载体周围放置胚上的蛋黄的顶部。 （D）将滤纸载体松散从蛋黄的顶部除去周边卵黄膜和（E）切割。 （F）剩下的蛋黄小心在PC盐水浴冲走。 （G）的胚胎内夹有一第二个相同的滤纸载体。 （H）将滤纸三明治浸没在所述介质和位于两个金属不锈钢环（胚胎面对背或腹侧）。 （Ⅰ）的两个环稳定从顶部三明治。 （J）含有胚胎被转移到温度控制的油浴中的培养皿。不锈钢戒指横向稳定培养皿。 （K）的培养基覆盖有矿物油的层。淹没滤膜夹层的（L）示意图。 （M），用于浸没滤纸夹持滤纸载体的尺寸。 <a href="http://ecsource.jove.com/files/ftp_upload/54636/54636fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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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=Spatially+and+temporally+controlled+electroporation+of+early+chick+embryos.">Spatially and temporally controlled electroporation of early chick embryos.</a> Nat. Protoc. 3 (3), 419-426 (2008).</li><li>. GmbH ZEN 2 - (blue edition) - Software Guide Available from: <a target="_blank" href="https://www.unige.ch/medecine/bioimaging/files/3914/3765/2064/ZEN_2_blue_edition_-_Software_Guide.pdf">https://www.unige.ch/medecine/bioimaging/files/3914/3765/2064/ZEN_2_blue_edition_-_Software_Guide.pdf</a> (2014)</li><li>Darnell, D. K., Schoenwolf, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+Avian+Embryos.">Culture of Avian Embryos.</a> Dev. Biol. Protoc. Vol. I. 135, 31-38 (2000).</li></ol>

作者什么都没有透露。

作为成熟的内皮细胞培养1的新变种，淹没滤纸三明治通过周围温度和湿度控制的孵化器有利于收购的早期鸡胚前的长期明场和暗场时间推移电影大毛显微镜可有可无。而不是在半固体琼脂 - 蛋白底部被培养后，将胚保持完全在由PC的盐水和薄蛋白的一个简单的培养基淹没。蒸发和热损失是由矿物油浮在培养基顶部的层最小化。胚胎可以很容易地培养，无论是背或腹侧，无质量明显差异。如这里所示，胚胎在浸没滤纸三明治是显微干预访问，等形态发生浸泡珠的应用程序。未来的应用包括实时荧光成像，显微注射和电穿孔和基因沉默马nipulate和腹侧（如早期的心脏和体节形成）和背侧（ 例如，在原肠胚后期，神经管闭合和早期大脑）发育过程中研究了各种形态的事件。如果包含滤纸夹持培养皿被替换为灌注腔，使矿物油层下的培养基的交换的时间 - 依赖性药物治疗是可行的。浸没滤纸三明治将开发结合其全部潜力的成像与基于激光的成像（包括光片镜）和浸泡的目标。 与浸没滤纸三明治的制备一些困难将在下面的段落来解决。虽然它仅需要的训练适量从蛋黄转移胚胎成滤纸三明治，几个步骤仍可显著影响在培养的胚胎的质量。之前滤纸载体放置在蛋黄，围绕胚胎卵黄膜应从任何厚蛋白被释放。只有这样，滤纸和卵黄膜之间的连接是强大到足以承受PC-盐水洗涤。携带胚胎应越过液体/空气界面尽可能小的滤纸以最小化在膜上的张力。一旦带入PC-盐水洗涤，整个滤纸承运人必须完全保持在任何时候都淹没了。在PC-盐水浴中的时间必须限制在约30 - 60秒，如卵黄膜将开始从滤纸释放。仍然，胚胎的清洁应非常彻底执行，如蛋黄残余稍后将掩盖胚胎的图。洗涤时，从移液管流从来没有直接点到胚胎，但总是从其径向离开。从PC-盐水取出胚胎后，确保横握滤纸，以尽量减少紧张的胚胎膜。然后，稳定与第二滤纸载体的胚胎。一旦第二滤纸载体放置，避免产生任何横向张力，这可能会改变相对于彼此的滤纸载体和撕裂胚胎膜。当滤纸三明治被带入培养基，也应该越过空气/液体界面只有一次，以尽量减少在膜上的张力。第二对用于稳定从顶部滤纸夹层不锈钢环的应非常缓慢放置并没有任何压力，以最大限度地减少了胚胎膜的压缩。胚胎的面积opaca的小破裂培养的第一个小时期间通常愈合，但受损的胚胎可以并且应该总是由新的样本替换的培养基的顶部吸油层之前。 要像一个完整的胚胎或高分辨率定时短片多个样本，麦克风roscope必须配备一个摩托化的xy阶段，通过在显微镜软件控制。它是至关重要的，以与一最小速度在xy阶段移动，以避免在培养基中和油中的胚胎和紊流，这将降低图像质量的损害。用于采集这里介绍的时间推移的电影，机动化的xy级大约1.75毫米/秒的速度移动。在未来，成像可以进一步由第一移动台到所需位置和短间歇周期之后获取图像让振动和湍流消逝改善。 作为限制，如果使用没有浸没目标直立显微镜潜在用户应该知道必要的相对长的工作距离（约1厘米）对于该培养方法。这个工作距离的结果从介质层和矿物油的对胚胎的顶部。在6cm培养皿中，油层具有thicknes约1秒 - 1.5毫米，和胚胎浸没约4mm深的培养基中。根据物镜的直径，6cm培养皿中的上缘也可以限制进入胚胎。这可以通过使用一个更大的盘被规避。 从顶部的工作距离可通过最小化液体层的厚度会稍微减少。另外，从下面的工作距离可通过使胚胎进一步下降到培养皿的底部，并降低了加热烧杯的玻璃窗的厚度被最小化。虽然该协议进行了优化，以正直的长工作距离变倍显微镜的工作，它可以被安装到一个倒置显微镜，以及。未来用户应记住，在培养液中浸没胚胎太深可能导致O 2 -diffusion问题，尽管初步的实验表明，有限的扩散似乎并没有成为一个概率LEM，只要胚胎被充分大型贮存培养基并夹在滤纸包围未被按压到培养皿的底部。 另一个限制是在温度控制。通过调整控制器的温度下加热的烧杯至40℃，在培养基中的温度平衡到当介质覆盖有矿物油的胚胎周围37.5℃。这表明，一些能量被烧杯中，其中，在加热元件和温度传感器都位于底部，和胚胎的位置之间丢失。温度控制可以通过重新定位传感器和重新分配的加热元件被优化。

由于历史的开端胚着迷的男人。怎样的胚的结构和形态在这种定义明确的时间和空间的方式发展？鸡胚已经成为经典脊椎动物模型胚胎发生有几个原因之一:这是容易获得的，价格低廉，在其早期阶段透明的，并且可访问的实验操作，因为它的发展母亲以外。此外，它具有在早期形态发生的事件，如心脏和大脑的形成，原肠胚形成，神经胚和体节形成15大致平坦的几何形状。 形态发生过程可以当它们被动态研究，如通过采集时间推移的图像得到最好的理解。鸡胚在卵 16但交通不便的实验操作和透射光显微成像有限的可见性可视化。因此，断绝人的技术已经被提出来培养鸡胚前卵 ，无论是在整个蛋黄培养系统13,17 - 19或作为外植体培养1,20 - 23。在整个蛋黄培养系统，胚胎保持在完好蛋黄的顶部，和蛋的整个内容转移到孵化另一个容器。此容器可以是一个替代蛋壳17，培养皿19中 ，或由塑料保鲜13,18的吊床。在整个蛋黄培养胚胎可以理想地，直到孵化培养，对微操作访问。这些技术可用于研究胚胎的发育血液循环（这增加了对比度）开始后，而年轻胚胎仍难以显现特别有用。如果它们从蛋黄切除并作为体外外植体培养的这些早期胚胎可以成像最好。该外植体实验方便人操作，并且需要用于培养更少的空间。多年来，通过新的丹尼斯在1955年及其变化首创的技术是对鸡胚植培养方法的黄金标准，从而使时间推移显微镜和显微外科干预20,21胚胎进行访问。尽管它的巨大成功，新的技术相对苛刻且费时。当卵黄膜，携带胚盘，围绕玻璃环拉伸以获得适当的张力1胚盘常分开。此外，胚胎只能用其腹侧培养。欧共体（早期小鸡）培养，通过查普曼和COLLIGNON 1开发了一种较近期的技术，通过使用滤纸载体具有中心孔绕过卵黄膜的拉伸。滤纸附着到卵黄膜，从而保持其自然的张力，并包围像画框1胚胎。胚胎然后培养在琼脂-蛋白底，通常与他们的腹侧。培养背侧在HH8 15岁以上的胚胎似乎是唯一可行的。许多团体已经适应滤纸载体自己的需要，例如通过与从外科缝合线的纤维24或涂覆的膜25胶原制成的支撑取代琼脂-蛋白底部。通常情况下，用新的技术或与欧盟文化培养的胚胎暴露，用自己的背或腹面空气，促进氧扩散1,20。这些外植体培养必须保持在湿润的气氛中，以避免介质的蒸发并防止胚胎干燥。这是通过孵育在含有湿润薄纸1较大培养皿外植体培养的培养皿来实现的。这些胚胎的时间推移图像的获取要求要么整个显微镜或脾气周围使用一个气候控制培养箱ATURE控制容器与加热盖以防止冷凝在光路14上 。然而，鸡胚还可以开发前卵在培养基中完全浸没滤纸培养25，夹在重矿物油和蛋白的一个层之间的新的技术2,21的adaptions，或如在"康沃尔粘稠培养折叠胚的外植体"23,26，不与空气直接接触。 浸没滤纸夹层是滤纸载体技术的新变体。通过对培养鸡胚前OVO结合前面的知识，它使气候控制孵化器和热盖可有可无。胚胎被夹在两个相同的（激光切割）滤纸载体24和培养的完全在一个简单的培养基（Pannett康普顿盐水27,28和薄蛋白在一个3:2的比例）浸没之间在控制含一温度呃。轻质矿物油浮在介质的顶部的层防止蒸发2,21和热量损失最小化到环境中。容器的底部有一个玻璃窗口，整个安装是在直立的长工作距离变倍显微镜进行。这种设置允许收购约30小时的胚胎发育的高分辨率明场和暗场时间推移电影，从早期的原条期至28体节期（相当于汉堡汉密尔顿阶段5至16日，HH5-16 ）15。胚胎可以与他们的腹侧或背侧进行培养同样出色。因此，胚胎观察和腹侧（ 例如，早期心脏7,8和体节形成6）和背侧的访问操作（ 例如，在原肠胚后期2，神经管闭合3 - 5，和早期大脑9 - 13）发展。浸没滤纸三明治provides为显微珠注入，显微注射，基因沉默，和电穿孔研究的简单和稳定的环境。它的成像潜力可以通过使用基于激光的成像（包括光片镜）和浸泡的目标更进一步推。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Milli-Q&#160;Reference Water Purification System</td>
			<td>Millipore</td>
			<td>Z00QSV0WW&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Duran&#160;laboratory bottles, with caps, 1L</td>
			<td>Sigma-Aldrich/Duran</td>
			<td>Z305200-10EA&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solution A</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Merck Millipore</td>
			<td>1064041000</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck Millipore</td>
			<td>1049361000</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 H2O</td>
			<td>Merck Millipore</td>
			<td>1023821000</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</td>
			<td>Merck/Sigma-Aldrich</td>
			<td>13152-1KG-D&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solution B</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4.2H2O</td>
			<td>Merck</td>
			<td>1065801000</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4.2H2O</td>
			<td>Merck</td>
			<td>1063420250</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Whatman&#160;qualitative filter paper, Grade 595</td>
			<td>Sigma-Aldrich</td>
			<td>10311611</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser cutting machine MLS-90120-C130, CO2-laser, 130W, wavelength 10.6 &#181;m</td>
			<td>Micro Lasersystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tork Extra Soft Facial Tissues</td>
			<td>Tork</td>
			<td>140280</td>
			<td></td>
		</tr>
		<tr>
			<td>Latex gloves</td>
			<td>Sigma-Aldrich</td>
			<td>Z645613</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>EMDAMED</td>
			<td>EMDA 300.131</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Sigma-Aldrich</td>
			<td>Z350613</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>VWR</td>
			<td>WITG5.480.003</td>
			<td></td>
		</tr>
		<tr>
			<td>accu-jet pro Pipette Controller</td>
			<td>BrandTech Scientific</td>
			<td>26332</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological plastic pipettes, 25mL</td>
			<td>Sigma-Aldrich</td>
			<td>CLS4251</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic document sleeves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Egg incubator - Incubator mod. Cip Cip 40</td>
			<td>Fiem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fertilized chicken eggs (Gallus gallus)</td>
			<td>Drost BV (Loosdrecht, NL)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Tissue culture dishes</td>
			<td>Sigma-Aldrich</td>
			<td>P5731</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Greiner Bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm Petri dishes</td>
			<td>Sigma-Aldrich</td>
			<td>P5481</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Centrifuge tubes , Greiner centrifuge tubes&#160; or Cellstar tubes</td>
			<td>Sigma-Aldrich</td>
			<td>T2318</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>greiner bio-one</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Stainless steel rings, custom-made</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Large rings, 30 mm outer diameter, 4 mm in height, 1.5 mm wall thickness</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small rings, 20 mm outer diameter, 4 mm in height, 1.5 mm wall thickness, 6.5 mm gap in the ring</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nail scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, Dumont #5, Inox</td>
			<td>Fine Science Tools , F.S.T.</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine, really sharp scissors for cutting filter paper</td>
			<td>VWR</td>
			<td>21-102</td>
			<td></td>
		</tr>
		<tr>
			<td>M3516&#160;Sigma-Aldrich Mineral Oil</td>
			<td>Sigma-Aldrich</td>
			<td>CAS Number&#160;8042-47-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Agar bottoms for EC culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539&#160;SIGMA</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653&#160;SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microbead implantation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polybead Sampler Kit III (45 &#181;m beads were used)</td>
			<td>Polysciences, Inc.</td>
			<td>16905-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Accupuncture needles: type J, No.02, 0.12x30mm</td>
			<td>SEIRIN</td>
			<td>&#160;type J, No.02, 0.12x30mm</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010023&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Temperature controlled beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RTD Temperature Sensor with M4 Threaded Housing RTD-850M</td>
			<td>Omega</td>
			<td>RTD-850M</td>
			<td></td>
		</tr>
		<tr>
			<td>Hermetically Sealed RTD Sensors HSRTD-3-100-A-3M</td>
			<td>Omega</td>
			<td>HSRTD-3-100-A-1M</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermistor and RTD Extension Wire EXTT-3CU-26S-50</td>
			<td>Omega</td>
			<td>EXTT-3CU-26S-50</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Pin Mini Connectors for Thermocouples, 3-Wire RTD&#39;s and Thermistors MTP-U-M</td>
			<td>Omega</td>
			<td>MTP-U-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Kapton (Polyimide Film) Insulated Flexible Heaters KHLV-103/(10)-P</td>
			<td>Omega</td>
			<td>KHLV-103/(10)-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature/Process Controller CNi16D24-C4EIT-DC</td>
			<td>Omega</td>
			<td>CNi16D24-C4EIT-DC</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom-made aluminium beaker, isolated with polyacetal (drawings see sheet 2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscope setup</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axio Zoom.V16 with ApoTome.2 ZEN</td>
			<td>Carl Zeiss AG</td>
			<td>495010-0009-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective PlanNeoFluar Z 1.0x/0.25 FWD 56mm</td>
			<td>Carl Zeiss AG</td>
			<td>435281-9100-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Rotary Control MARC</td>
			<td>Carl Zeiss AG</td>
			<td>435604-0000-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Transillumination top 450 mot</td>
			<td>Carl Zeiss AG</td>
			<td>435500-9000-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized measuring stage S 150x100 mot; CAN (D)</td>
			<td>Carl Zeiss AG</td>
			<td>435465-9020-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Insert plate S, glass 237x157x3mm (D)</td>
			<td>Carl Zeiss AG</td>
			<td>435465-9053-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Controller EMS 3</td>
			<td>Carl Zeiss AG</td>
			<td>435610-9010-000</td>
			<td></td>
		</tr>
		<tr>
			<td>System Control Panel SYCOP 3</td>
			<td>Carl Zeiss AG</td>
			<td>435611-9010-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamamatsu ORCA Flash 4.0 camera</td>
			<td>Hamamatsu</td>
			<td>C11440-22CU</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopy High-End Workstation Xeon Quad-Core mulitlingual</td>
			<td>Carl Zeiss AG</td>
			<td>490004-0025-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Language Package Windows 7 x64 Ultimate English US</td>
			<td>Carl Zeiss AG</td>
			<td>410373-0200-000</td>
			<td></td>
		</tr>
		<tr>
			<td>LCD TFT Monitor HP ZR2440w 24&#34;</td>
			<td>Carl Zeiss AG</td>
			<td>410350-2403-000</td>
			<td>not available anymore</td>
		</tr>
		<tr>
			<td>ZEN pro 2012 Hardware License key</td>
			<td>Carl Zeiss AG</td>
			<td>410135-1002-120</td>
			<td>not available anymore</td>
		</tr>
		<tr>
			<td>ZEN Software Driver Hamamatsu Cameras Hardware License Key</td>
			<td>Carl Zeiss AG</td>
			<td>410136-1045-110</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN module Z Stack</td>
			<td>Carl Zeiss AG</td>
			<td>410136-0030-110</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN Module Tiles/ Positions</td>
			<td>Carl Zeiss AG</td>
			<td>410136-1025-110</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN Module Time Lapse</td>
			<td>Carl Zeiss AG</td>
			<td>410136-1031-110</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN Module Experiment Designer</td>
			<td>Carl Zeiss AG</td>
			<td>410136-1022-110</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN Desk 2012 Hardware License Key</td>
			<td>Carl Zeiss AG</td>
			<td>410135-1004-120</td>
			<td>not available anymore</td>
		</tr>
	</tbody>
</table>

a submerged filter paper sandwich for long-term ex ovo time-lapse imaging of early chick embryos

由于其可用性，费用低，平坦几何形状和透明度， 前卵鸡胚已成为形态发生的事件，例如原肠胚形成2的研究的主要脊椎动物动物模型，神经胚3 - 5，体节形成6，心脏弯曲7， 8，和大脑的形成9 - 13，早期胚胎发育过程中。关键是理解形态发生过程是通过时间推移成像动态地跟随他们。的小鸡胚胎发生前卵的时间推移电影捕获已被限于任一短的时间窗或需要进行培养箱来控制温度和湿度的周围的胚14。在这里，我们提出了一种新的技术来培养鸡胚使用透射光显微术高分辨率时间推移成像前OVO。浸没滤纸夹层是行之有效的滤纸载体TECHN的变体神游（EC培养）1，并允许鸡胚而不需要一个气候室中培养。胚胎被夹在两个相同的滤纸载体之间，并且在由轻质矿物油的层覆盖的简单，温度控制介质保持完全浸没。从原条阶段（汉堡汉密尔顿阶段5，HH5）15开始直到至少28体节阶段（HH16）15，胚胎可以是与它们的腹或背侧进行培养。这使得收购占地约30小时的胚胎发育时间推移电影。代表时间推移框架和电影中。胚形态相比，在标准EC-培养培养的胚胎。 浸没滤纸三明治提供一个稳定的环境，以研究早期背部和腹部的形态发生过程。它也允许对实时荧光成像和微操作，如显微珠小鬼lantation，显微注射，基因沉默，和电穿孔，并有很大的潜力，以与基于激光的成像（包括光片显微镜）浸入目标相结合。

<img alt="图2" src="/files/ftp_upload/54636/54636fig2.jpg" /> 图2:EC文化 1 和深层过滤纸三明治文化 之间的胚胎形态比较 。选择时间推移框架图显示在3小时的时间间隔的胚胎。帧被调整为充分胚胎形态的概述。胚胎年龄匹配的7-体节阶段（HH9）15。 0小时表示每个实验的开始。前总是向左。图像用暗场照明获得的。 （一）在胚胎EC文化1，培养腹侧在琼脂，蛋白底1,33。高质量的图像可以被获取，作为胚胎在z方向密闭。 （B和C）两胚浸没滤纸夹层培养:（B）的腹侧和（C）的背侧。在滤纸夹层胚胎发育不为至少27小时的质的差异。他们开发功能的血液循环和颅颈及弯曲和转动成功。 <a href="http://ecsource.jove.com/files/ftp_upload/54636/54636fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 时间推移收购取决于可用的显微镜​​系统。在这项研究中，工作距离长，采用直立变倍显微镜。缩放系数设置为5X。在与目镜的16X放大率结合，这导致在80X总放大倍数，以1.3微米/像素的理论分辨率（如在显微镜软件说明）30。为了增加现场的视图，胚胎成像为五个子图像（砖）水平全景。因此，小片区域，包括五个重叠瓦片，被定义29。这片区域被定位成完全覆盖滤纸载体的椭圆形开口在其水平延伸（ 图2B - 0小时）。每一瓦片有大小2,048点¯x2,048像素，覆盖大约270毫米的场的图，并且分别获得具有10％的重叠瓦片，导致总场的视图的大约12毫米×2.7毫米全景。电动XY平台与采集单独瓦片29之间大约1.75毫米/秒的速度相对移动的目标。实时图像集中于前presomitic中胚层和最近成立的体节（我们组的研究重点）。为了补偿在Z方向上的胚胎的增厚和卷曲，小的z栈（五至七个不同的平面与30μm的距离）在每一个时间点30被收购。这些被围绕presomitic胚层的前末端的焦平面选中。时延ACQ的持续时间uisition设定为50小时，并分别选择31的3,4，或10分钟的成像时间间隔。 10小时，并重新定义围绕新的最佳焦平面的Z堆叠 - 时间推移采集的质量可以通过重新聚焦每5得到改善。在实验结束时，整个时间序列被手动编辑的，并分别创建并保存到一个单独的文件夹32包含的z平面与最佳聚焦图像的子集。图像的这些子集是时间缝制创建具有最佳聚焦的完整时间序列图像的。这个完整的时间序列的各图像的砖是缝合和没有阴影校正30熔合在一起，和时间推移帧被输出为100％的尺寸和95％压缩32的JPEG图像。时间推移帧被导入为图像序列到专业的视频编辑软件。详细的100％的缩放被稳定，且整体对比度调节到我们的喜好。最后时的失误是编码Ð与H.264编解码器和MPEG-4中的容器出口。电影以15帧/秒（fps）的速度和1920×1080像素的分辨率的帧速率显示。 图2示出了在EC培养1（ 图2A）的胚胎和两个胚胎在浸没滤纸三明治培养，其中之一是培养的腹侧（ 图2B），而另一个背侧（ 图2C）之间的比较。所有胚胎年龄匹配的七个体节阶段（HH9）15并用4分钟（ 图2A和B）或10分钟（ 图2C）的时间分辨率的成像。的3小时的间隔选择的帧被描绘。欧盟文化（ 图2A）允许非常稳定的成像，由于胚胎搁在一个稳定的琼脂，蛋白底1,33。整个胚胎仍然留在一个焦平面整个Ť他整个时间推移。这可能是对于较短实验（几个小时），并非常早期胚胎是有益的，但它与在z方向上的胚，可能妨碍其成功立体发展在长期的强约束。在开始时，该限制仅导致胚胎的头的轻微的横向压扁相比在其腹侧（在0小时图2A和图2B进行比较）浸没滤纸三明治培养的胚胎。以后，头的转动被削弱（ 图2A - 21小时）和心脏跳动减慢。活血化瘀几乎停止。见补充电影1对内皮细胞培养的胚胎完整的延时。在这部电影上面板显示的胚胎完整的概述，而在左下面板在可视化全分辨率的心脏发育。在presomitic中胚层的分割成体节可以SE烯在右下方的细节。 在浸没滤纸三明治胚胎，在另一方面，是在它们的三维膨胀仅由它们周围的膜的自然张力限制。它们可以是具有它们的腹（ 图2B）或背侧（ 图2C）中培养任一。无论哪种方式，胚胎表明连续体节形成，和他们的头转动。他们开发颅颈和弯曲和功能的血液循环。在滤纸三明治既胚胎的焦点上的分段中胚层进行成像，使头部的部分逐渐移出焦点作为胚胎生长，加厚，并开始转动，而中胚层的分割部分留在焦点（对比图2B和2C在21小时至27小时）。 补充电影2显示了胚胎的完整时间推移这部电影描述在图2B中。摄像间隔是4分钟。这部电影的顶部面板显示胚胎的完整概述。胚胎的发展是连续拍摄超过46小时，但经过大约30小时，将重点放在分割中胚层迷路了由于整体增厚和胚胎组织的转折点。左下面板显示的心脏从收购时间推移帧全分辨率的早期发展。通过在中线两侧的成对的心脏原基的融合，一个几乎直的管状结构形成，这以后开始跳动和弯曲到右侧。右下方的面板显示最近形成的体节和presomitic中胚层的全分辨率前尖随着时间的推移跟踪新的体节的形成。 补充电影3示出了另一胚胎培养，并在浸没滤纸夹层上成像的腹侧。摄像间隔是4分钟。电影占地面积日胚胎从舞台HH5-6大约电子发展阶段HH14 15，比约占27小时的培养时间。头折叠形式和向后的进展。从双边原基的心脏形态，开始跳动，俯下身去的权利。第一个三到四体节同时形成。附加体节从presomitic胚层的前末端出芽顺序。下部面板显示全分辨率胚胎的头部区域，从而允许头部折叠和早期心脏形成在高细节的观察。由于胚胎的透明性，神经管的封闭可以在将来头部区域被观察到。 补充电影4显示体节形成在同一个胚胎在全分辨率到下面的面板全部成像时间。 补充电影5示出了胚胎培养的背侧（ 图2C）的完整的时间推移。摄像间隔为10分钟。而UPP器面板显示从七体节阶段（HH9）胚胎的约阶段HH16-17的发展，下面板被裁剪在全分辨率该阶段，以显示在早期脑形态学的改变。神经管的局部肿胀可以观察，导致前脑，中脑和后脑的区域化，这将在稍后阶段13分成胚胎大脑的五个子区域。此外，光学囊泡的生长可以清楚地看到。在培养大约30小时后，将胚胎死亡。 显示与显微操作浸没滤纸三明治的兼容性，多个聚苯乙烯微珠（〜40微米的直径）植入或在浸没滤纸三明治培养鸡胚胎的中胚层presomitic的附近。微珠在PBS中洗涤，以除去储存缓冲器和IMPLAN泰德之前覆盖用轻质矿物油的层培养基（比较步骤的协议部分的2.18）。的玻璃巴斯德移液管用于通过显微镜的目镜珠粒转移到胚胎表面，下观察。五个洞的内胚层通过用银针轻轻地刮它创造。一个特制的J形工具，通过拉伸和在火焰弯曲的玻璃巴斯德吸管创建的，用于操纵所述珠和小心推动他们入孔，直到它们变得不动。三孔充满了一个珠子每个（ 图3A - 0小时和3B *）和两个洞有两个小珠（ 图3A - 0小时）。 图3A所示，在选择的时间推移帧，鸡胚的珠的显微植入后的发展。三个珠成功掺入组织在所希望的位置，和他们的运动被成像在当然在高细节（ 图3B）实验。胚胎的发育似乎没有由操纵或珠的存在下被削弱。见补充电影6全时间推移电影。摄像间隔是4分钟。 <img alt="图3" src="/files/ftp_upload/54636/54636fig3.jpg" /> 图3: 微珠植入术后时间推移成像。验证的原理实验示出与微珠注入浸没滤纸三明治的兼容性。胚胎的头部的左侧，但此实验过程中未进行成像。图像用暗场照明获得的。 （一）选择延时帧显示七微珠植入后3小时间隔操纵胚胎（〜40微米直径）。拉长不断胚胎形成额外SOMIT上课。三珠似乎已经正确连接，并拥有超过文化的18小时组织流动内侧移动。其他四个球弹出，6和9之间的孔文化的人力资源和向后漂移。 （B）的三个单珠详细视图（B1至B3）在实验开始时（0小时，B *）和后孵育12小时（B **）注入到presomitic胚层。刚刚成型体节的数量被指示（S9中B *和S18 B中**）。 <a href="http://ecsource.jove.com/files/ftp_upload/54636/54636fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="电影1" src="/files/ftp_upload/54636/54636movie1.jpg" /> 补充电影1:EC- 文化腹侧。胚胎内皮细胞培养1，培养的腹侧，从七体节阶段开始在琼脂，蛋白博特（HH9）15米1,33。摄像间隔是4分钟。的相对时间被显示在左上角。高质量的图像可以被获取，作为胚胎是在z方向上封闭。 21小时后，心跳和血流几乎被捕，胚胎已经开始瓦解。上图显示的胚胎调整大小的概述，而较低的面板可视化心脏发育（左图），并在全分辨率（100％缩放）的presomitic中胚层（右图）的分割。 <a href="https://www.jove.com/files/ftp_upload/54636/Movie_1_EC_culture-Ventral_up.mp4" target="_blank">（点击下载）。</a> <img alt="电影2" src="/files/ftp_upload/54636/54636movie2.jpg" /> 补充电影2: 淹没滤纸三明治腹侧。胚胎从七体节期（HH9）15日起，腹面SID在淹没纸夹层培养Ë朝上。摄像间隔是4分钟。相对时间显示在小时和分钟左上角，24小时后重新启动。上面板显示的胚胎在46小时的发展已调整大小的概述，连续。左下方的面板在全分辨率（100％缩放）图像采集的第一个10小时期间可视化早期心脏发育。右下图描述在全分辨率（100％缩放），中胚层分割经过大约30小时的发展，直到焦点失去了应有的整体增厚和胚胎组织的转折点。 <a href="https://www.jove.com/files/ftp_upload/54636/Movie_2_Sandwich-Ventral_up.mp4" target="_blank">（点击下载）。</a> <img alt="电影3" src="/files/ftp_upload/54636/54636movie3.jpg" /> 补充电影3: 头折叠形成。胚胎培养的腹侧从水下滤纸三明治，他广告程序/头折阶段（HH5-6）15至约22体节期（HH14）的培养时间超过15大约27小时。摄像间隔是4分钟。相对时间显示在小时，分钟左上角，24小时后重新启动。上面板显示胚胎的发展已调整大小的概述。下面板描绘了全分辨率（100％缩放）胚胎的头部区域。头部折叠和早期心脏和神经管的封闭物的形成可被观察到。 <a href="https://www.jove.com/files/ftp_upload/54636/Movie_3_Sandwich-Ventral_up-Head.mp4" target="_blank">（点击下载）。</a> <img alt="电影4" src="/files/ftp_upload/54636/54636movie4.jpg" /> 补充电影4: 体节形成。胚胎培养的腹侧中从首过程/头倍阶段（HH5-6）浸没滤纸夹层15约22体节期（HH14）15的培养时间超过大约27小时。摄像间隔是4分钟。相对时间显示在小时，分钟左上角，24小时后重新启动。上面板显示胚胎的发展已调整大小的概述。较低的面板描绘了全分辨率（100％缩放）的分割旁轴中胚层。 <a href="https://www.jove.com/files/ftp_upload/54636/Movie_4_Sandwich-Ventral_up-Somitogenesis.mp4" target="_blank">（点击下载）。</a> <img alt="电影5" src="/files/ftp_upload/54636/54636movie5.jpg" /> 补充电影5: 淹没滤纸三明治背侧。胚胎培养的背侧从七体节期（HH9）15淹没滤纸三明治大约HH16-17阶段15。摄像间隔为10分钟。将显示相对时间在小时和分钟的左上角，24小时后重新启动。上面板显示胚胎的发展已调整大小的概述。下图描述在全分辨率（100％缩放）早期大脑形态学变化。胚胎后在培养大约30小时死亡。 <a href="https://www.jove.com/files/ftp_upload/54636/Movie_5_Sandwich-Dorsal_up-Head.mp4" target="_blank">（点击下载）。</a> <img alt="电影6" src="/files/ftp_upload/54636/54636movie6.jpg" /> 补充电影6: 微球植入。胚胎培养的腹侧在淹没滤纸夹层约19小时，从九个体节期（HH9-10）15日开始。摄像间隔是4分钟。相对时间显示在小时，分钟左上角。上图显示尾部区域的调整大小概述七微珠植入公关esomitic中胚层。较低的面板显示，在全分辨率（100％缩放）植入微球的区域。 <a href="https://www.jove.com/files/ftp_upload/54636/Movie_6_Sandwich-Ventral_up-Microbeads.mp4" target="_blank">（点击下载）。</a>

Watch this Scientific Journal Video about A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos at JoVE.com

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-submerged-filter-paper-sandwich-for-long-term-ex-ovo-time-lapse

Research

JoVE Journal

Developmental Biology

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

22.1K 次观看.  MOVE Research Institute Amsterdam.   

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

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

在小鸡视网膜有效的基因转移的原代细胞培养研究:<em&gt;前OVO</em&gt;电穿孔法

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

科研

发育生物学

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

优化<em&gt;前OVO</em&gt;鸡胚培养，以发展的高级阶段

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

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 ...

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

一个<em&gt;离体</em&gt;方法培养大鼠肠系膜微血管网络的时间推移成像

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects 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 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 ...

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 ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

多光子时间推移成像实时可视化发展: 迁移神经嵴细胞在斑马鱼胚胎中的可视化

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

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 ...

水下滤纸三明治长期

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在小鸡视网膜有效的基因转移的原代细胞培养研究:

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捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

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斑马鱼发展长期成像的多功能安装方法

长期共焦成像根和下胚轴发育的简单室

长期的实时成像

多光子时间推移成像实时可视化发展: 迁移神经嵴细胞在斑马鱼胚胎中的可视化

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法