We want to thank Tobias Pietzsch for providing his powerful open source software BigDataViewer. We thank the Light Microscopy Facility of the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), namely Jan Peychl, Sebastian Bundschuh and Davide Accardi for technical assistance, perfect maintenance of the microscopes used in the study and for comments on the manuscript and H. Moon (MPI-CBG Scientific Computing Facility) for the BigDataServer. We thank Julia Eichhorn for assembling the Movie 1. The Norden lab members and Svea Grieb provided many helpful comments on the manuscript. Jaroslav Icha, Christopher Schmied and Jaydeep Sidhaye are members of the International Max Planck Research School for Cell, Developmental and Systems Biology and doctoral students at TU Dresden. Pavel Tomancak is supported by the ERC Starting Grant: Quantitative Analysis of the Hourglass Model of Evolution of Development and Human Frontier Science Program Young Investigator grant RGY0093/2012. Caren Norden is supported by the Human Frontier Science Program (CDA-00007/2011) and the German Research Foundation (DFG) [SFB 655, A25].

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形态是塑造胚胎和生长和分化驱动器一起从一个受精卵的个体发育成为成熟的多细胞生物体的过程。动物发育过程中形态方法可最好通过完整活标本1-3的成像进行分析。这是因为这种全胚胎成像保留所有驱动和调节发展包括信号分子的梯度，细胞外基质，脉管，神经支配以及周围组织的机械性能的部件。为了弥合的秤，在该形态出现时，快速亚细胞事件对整个组织在数小时或数天的发展的背景下一分钟的时间尺度被捕获。满足所有这些要求，正交平面照明显微镜5的现代执行4被开发。最初，它被命名为选择性平面照明显微镜（SPIM），4;现在是一个无所不包的长期轻质板材荧光显微镜（LSFM）通常使用。 LSFM使成像的高时间分辨率，同时诱发比激光扫描或旋转盘共焦显微镜6,7光毒性较少。如今，已经有基本光片照射原理许多实现，它已被用来对图像进行了大量的各种标本和过程以前无法进入的研究人员8-11。 我们首先要强调LSFM比传统激光共聚焦显微镜方法的几个关键优势: 获得从实时成像显微实验有意义的结果，重要的是观察仅最低限度地影响了标本。然而，许多生物，包括斑马鱼是激光照射非常敏感，使得它具有挑战性的图像它们与高时间分辨率共聚焦显微镜的光毒性无EFfects像停滞或延迟发育6,7。 LSFM目前与在样品7中的至少破坏性影响的荧光成像技术。由于薄激光光片照射是在特定的时间点拍摄的试样的仅一部分，光片显微镜是非常有效地利用光子。因此，低曝光允许健康样品的较长时间推移观察， 如 12-17。此外，由于LSFM的微创，获取的图像的数量不再受样品可以多少光耐受支配，而是由多少数据可以被处理和存储。 沿保持试样在接近生理条件的同一线，LSFM带有适合用于活胚胎替代样品放置策略。在LSFM技术中，将胚通常嵌入式低百分比琼脂糖的薄柱内。该mountin克到琼脂糖气缸允许旋转的完全自由，因此，可以从完美的角度被观察的样本（在LSFM称为视图）和从几个视图同时。多视点成像和随后的多视角的融合是大的，散射的标本特别有益，并允许高，各向同性分辨率捕捉它们。其他可能的LSFM安装策略汇总可以在官方显微镜的操作手册中找到，由E.雷诺的书面实验室样品制备节。它是一个推荐的读取，特别是如果我们的目标是图像不同的样品与此处所述。 在LSFM图像采集是宽视场，摄像为主，而不是激光扫描共聚焦显微镜。这导致更高的信号对用于采集的图像的信噪比（SNR），并且可以非常快速（几十到几百帧每秒）。 LSFM的高灵敏度进一步使弱荧光SAMP成像莱斯，像在内源水平18，或在不久的将来表达的转录因子，使用CRISPR / Cas9内源性蛋白标记。高信噪比也是成功的下游图像分析非常重要。高速不仅需要捕捉快速内进程，同时也从图像速度不够快多视图整个胚胎。的多个视图的无缝融合才能实现，如果所观察到的现象不会获得从单独的视图来这些数Z图库的过程中改变。 LSFM的优点通常不会来在图像质量为代价。 LSFM的横向分辨率比共聚焦显微镜的分辨率略差。这是因为在LSFM使用的检测目标具有较低的数值孔径相比，在标准共焦设置水或硅浸入目标1.2-1.3（通常为1.0或更小）。另外，由于在LSFM宽场检测（艾比森针孔的CE），有相比共焦显微镜更失焦光。失焦光的量由光片厚度来确定。尽管如此，这些缺点是由在LSFM的更高的SNR补偿。在实践中，这导致相比例如纺丝圆盘共焦采集15类似质量的图像。因此，这使得能够像细胞膜或细胞核， 例如功能可靠提取，用于细胞谱系追踪15,19。 LSFM的轴向分辨率被确定，除了上述被检测，由光片的厚度。可以LSFM在某些情况下，轴向分辨率优于共焦显微镜的分辨率。首先，在分辨率的改善来当光片是比检测目标，这通常发生于具有低放大倍数的物镜成像大试样的轴向分辨率更薄。第二种方式，该LSFM如何ACHieve更好的轴向分辨率，是多视角的融合，其中，从不同的角度高XY分辨率的信息组合成一个图像堆栈。由此产生的合并协议栈具有各向同性分辨率接近分辨率的值在横向方向20,21。用于注册到对方这篇文章中所描述的多个视图的策略是基于使用荧光聚苯乙烯珠镶嵌在样品20,21周围的琼脂糖受托标记。 作为LSFM商品化的结果，该技术现在可以科学家22的一个广泛的社区。因此，写这个协议的动机是为了使这项技术访问缺乏LSFM实践经验发育生物学家，并得到这些科学家使用这种技术，他们的样品开始。我们的协议使用商用光片显微镜，这就构成了概念上简单的显微镜Ť帽子是操作方便。我们将另外要提到最近的其他协议与家庭建造LSFM设置，这可能是合适的回答具体问题23-25 ​​成像斑马鱼。另一个进入选项LSFM是开放平台26,27，它们使用开放式访问原则，使轻质板材镜到更广泛的社区。在硬件和软件方面两者的文档可在http://openspim.org和https://sites.google.com/site/openspinmicroscopy/找到。 在这个协议中，我们使用了硬骨鱼斑马鱼作为模型系统来研究与LSFM发育过程。斑马鱼眼的形态是，强调许多LSFM的好处的例子。 LSFM已经过去用于研究眼球发育在青鳉28和在斑马鱼29,30。在眼睛发育的早期阶段它是复杂的定位正确胚胎传统显微镜，作为膨松蛋黄不允许胚胎趴在侧以其眼睛朝向目标。然而，随着LSFM安装到琼脂糖柱中，样品可以被再现地定位。此外，从视泡到视杯阶段的过渡过程中，眼睛经历伴随着增长，这需要捕捉大Z堆栈和视大领域的主要形态发生重排。也对这些挑战LSFM优于常规共焦成像。视杯的形成过程是三维的，因此它是很难理解，并从一个视图通过单独成像可视化。这使得具有各向同性分辨率成像多视点有利。视杯形成之后，视网膜变得激光曝光越来越敏感。因此，与LSFM相关联的低光毒性为长期成像的主要优点。 在这里，我们提出了一个优化的协议为一到三天的成像老斑马鱼胚胎一个第二幼虫集中在眼睛的发育。我们的方法可以覆盖多达12-14小时的高时空分辨率时间推移电影录音。重要的是，我们还显示了数据处理，这是在LSFM的必要步骤的流水线，因为这种技术必然产生大的数据集，常在TB的范围。

<table border="0" cellpadding="0" cellspacing="0" width="908">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Lightsheet Z.1 microscope</td>
			<td style="width:184px;">Carl Zeiss Microscopy</td>
			<td style="width:119px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low melting point agarose</td>
			<td>Roth</td>
			<td>6351.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low melting point agarose</td>
			<td>Sigma</td>
			<td>A4018 or A9414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl 3-aminobenzoate methanesulfonate (MESAB/MS-222/Tricaine)</td>
			<td>Sigma</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Phenylthiourea (PTU)</td>
			<td>Sigma</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">500 nm red fluorescent beads F-Y 050</td>
			<td>Millipore (Estapor)</td>
			<td>80380495</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20 &#956;l (1mm inner diameter, marked black) capilllaries</td>
			<td>Brand</td>
			<td>701904</td>
			<td>sold as spare part for transferpettor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Teflon tip plungers for 20 &#956;l capillaries</td>
			<td>Brand</td>
			<td>701932</td>
			<td>sold as spare part for transferpettor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Circular glass coverslips diameter 18 mm, selected thickness 0.17 mm</td>
			<td>Thermo scientific (Menzel-Glaser)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O-rings for chamber windows 17&#215;1.5 mm</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tweezers, style 55</td>
			<td>Dumont</td>
			<td>0209-55-PO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml Luer-Lock syringes</td>
			<td>Becton Dickinson</td>
			<td>300865</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 cm extension cable for infusion compatible with Luer-Lock syringes</td>
			<td>Becton Dickinson</td>
			<td>397400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Links</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Link 1 Multiview reconstruction application</td>
			<td></td>
			<td></td>
			<td>https://github.com/bigdataviewer/SPIM_Registration http://fiji.sc/Multiview-Reconstruction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Link 2 BigDataViewer</td>
			<td></td>
			<td></td>
			<td>https://github.com/bigdataviewer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Link 3 BigDataViewer</td>
			<td></td>
			<td></td>
			<td>http://fiji.sc/BigDataViewer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Link 4 Multiview reconstruction application-issues</td>
			<td></td>
			<td></td>
			<td>https://github.com/bigdataviewer/SPIM_Registration/issues</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Link 5 BigDataViewer-issues</td>
			<td></td>
			<td></td>
			<td>https://github.com/bigdataviewer/bigdataviewer_fiji/issues&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Link 6 BigDataServer</td>
			<td></td>
			<td></td>
			<td>http://fiji.sc/BigDataServer.</td>
		</tr>
	</tbody>
</table>

using light sheet fluorescence microscopy to image zebrafish eye development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

LSFM是跨尺度的成像发育过程的理想方法。几个应用程序此处编译示出细胞内的结构，以及细胞和整个组织的短期和长期的成像。这些实施例还表明，LSFM在从视杯形成眼发育的各阶段中的视网膜的有用的工具来神经发生。 电影1作为一般LSFM方法的图示，首先示出了一个完整的胚胎的缩小视图嵌入在明琼脂糖气缸和后表示在荧光通道中的视网膜的详图。 <img alt="电影1" src="/files/ftp_upload/53966/53966movie1.jpg" /> 电影1:斑马鱼视网膜LSFM要说明LSFM方法，这部电影第一次显示了在胚胎成像完好一个内部的明切换到荧光之前garose缸。以后它表明视大字段使整个视网膜的观察。接着，将动画显示视网膜的放大小区域以突出的良好亚细胞的分辨率。用于成像间隙-GFP 37转基因斑马鱼胚胎:一个Ath5。此转基因在视网膜（主要神经节细胞和光感受器的前体）的标签不同的神经元。影片的荧光部分被捕获为单一视图使用40X /为1.0W目标用5分钟的间隔用双面光照记录。一个30微米厚卷的最大强度投影显示， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53966/Movie1.mp4" target="_blank">请点击这里下载此文件。</a> 电影2演示了如何非常快的细胞内事件能够以高分辨率捕获;在这种情况下，微管在生长其加在视网膜神经元祖细胞的结束。中所包含的电影信息允许微管加末端生长的跟踪和定量。 <img alt="电影2" src="/files/ftp_upload/53966/53966movie2.jpg" /> 电影2:在单个细胞中微管动力学这部电影撷日益增长的加尖标记蛋白EB3-GFP标记38微管外加小费。该蛋白质是在一个单一的视网膜祖细胞中表达。的微管主要在方向从根尖生长到基底（从上到下）。的EB3彗星的平均速度测量为0.28±0.05微米/秒。在细胞具有高微核活性的顶侧的亮点是中心体。野生型胚胎注射HSP70:EB3-GFP质粒DNA。电影是在约28 hrpost fertiliz获得热休克（37℃15分钟）后4小时通货膨胀（HPF）作为一个单一的视图与单面记录照明使用63X /为1.0W客观，1秒的时间间隔。单细胞被从视图覆盖大多数视网膜的场裁剪。显示两片投影的最大强度。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53966/Movie2.mp4" target="_blank">请点击此处下载此文件。</a> 如图3所示，如何胞内结构可以跟随在许多小时。这里，内转运的视网膜神经节细胞（RGC）的中心体被捕获。 <img alt="图3" src="/files/ftp_upload/53966/53966fig3.jpg" /> 图3:视网膜神经节细胞易位期间中心体定位的时间推移实验的这蒙太奇显示了中心体的整个视网膜神经节细胞（RGC）的成熟的位置。在神经祖细胞中心体在心尖过程（1时00分）的最顶端本地化。在细胞分裂过程中，两个中心体作为灯杆为有丝分裂纺锤体（2时25分）。这种划分的结果在于，分化成一个RGC与成为感光细胞的前体的第二子小区中的一个子细胞。分割后，对RGC的细胞体转位到视网膜的基部侧，而顶进程仍然在顶侧附连。曾经的RGC达到基底面，它的顶端突起分离和中心体与它（6点15）移动。中心体可以在逐渐心尖过程（6点50，7:20 8:10）一起缩回执行。在最后一帧（8点55）的神经节细胞生长从其基部侧的轴突，而中心体仍在顶部本地化。马赛克表达，质粒DNA注射一个细胞阶段实现到野生型胚胎。的细胞被Ath5可视:GFP-CAAX（绿色）常量构作，其标签和视网膜神经节细胞神经元等。所述中心体（箭头）由中心蛋白-tdTomato 29表达（品红色）标记的。视网膜的顶面是在图像的顶部和基底侧在底部。一个30微米厚卷的最大强度投影被示出。这些照片是从电影覆盖整个视网膜裁剪。影片开始于大约34小时受精后（HPF）。为Z堆栈被收购每隔5分钟用40X /为1.0W目标。时间显示在HH:MM。比例尺代表10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53966/53966fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 在图4中显示，细胞如何单个行为可以从数据中捕获整个组织中提取，如电影1。一个RGC的易位可以很容易地追踪和根尖和基底处理ES紧随其后。 <img alt="图4" src="/files/ftp_upload/53966/53966fig4.jpg" /> 图4:单视网膜神经节细胞的易位从心尖的RGC易位到视网膜的基底侧如图3中所描述的终端有丝分裂后出现的RGC由Ath5表达标记:间隙-GFP 37转基因。一个30微米厚卷的最大强度投影被示出。这些照片是从电影覆盖整个视网膜裁剪。影片开始于大约34高倍视野。为Z堆栈被收购每隔5分钟用40X /为1.0W目标。时间显示在HH:MM。比例尺代表5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53966/53966fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图5显示多视点LSFM的捕获组织SC的能力强麦形态发生过程与视杯形态的例子细胞的分辨率，在此期间，视泡转变成一个视杯。图像质量可以通过多视点摄像，当最终图像被从信息组合出的5个不同的视图（在这种情况下）与各向同性分辨率一个Z堆栈大幅提高。该图中示出了多视点去卷积后加权平均融合和图像对比度进一步增益和分辨率后的图像质量的改善。图中示出了在通过数据集不同方位的两个光学片。此外，从反褶积数据集的蒙太奇显示随着时间的推移视杯形态。所有的时间点，然后在电影3所示。 <img alt="图5" src="/files/ftp_upload/53966/53966fig5.jpg" /> 图5:单一视图和multiv的两种方法之间的图像质量比较IEW融合。（A）从侧面观和（B）背视图中显示单一视图数据的一个光片。条纹伪像和信号样本内更深的退化是显而易见的。也图像中的稠合数据（CF）可见的一部分在该特定视图中不捕获。 （C）相同的光学切片，现在从侧面观和（D）背视图中显示多视角融合的数据。需要注意的是条纹伪像被抑制和结构深样品中更好的解决。 （E）相同的光切片今为多视角从去卷积侧位片和（F）背视图中显示的数据。注意提高对比度和分辨率，以便各个细胞膜和细胞核可很好区分。该决议不会降低样本内显着更深。该数据集与双面照明相距约20度收购了5次。 AB的z栈出100微米与1.5微米的步长分别在每一个视图中10分钟的间隔获得10小时用20X /为1.0W目标。对多视点融合和反卷积输入图像进行向下采样2×加快图像处理。多视点反褶积的15次迭代中运行。 （G）的蒙太奇显示了从反褶积数据的背视眼初期发展从视泡到视杯阶段期间要突出形态发生事件的裁剪区域。视神经囊泡的两层，其最初是类似柱状上皮，分化成不同的细胞群。接近表皮远侧层成为视网膜神经上皮（RN）和近端层靠近神经管成为视网膜色素上皮细胞（RP）。在RN长和内陷的细胞形成视杯（1:40至5:00）;在同一时间RP的细胞变平。表面外胚层被诱导以形成透镜（1:40），WHICH内陷后（5:00）。影片开始于17 HPF。时间显示在HH:MM。所有的细胞膜由β肌动蛋白标记:RAS-GFP转基因和所有的核通过该HSP70标记:H2B-RFP转基因。比例尺代表30微米。 FB前脑，LE镜头，OP嗅placode，RN视网膜神经上皮，RP视网膜色素上皮细胞。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53966/53966fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="电影3" src="/files/ftp_upload/53966/53966movie3.jpg" /> 电影3:视杯形态显示为单一视图和多视图融合两种方法的定时短片说明从视泡到视杯阶段视杯形态的完整过程。它示出了从侧视图（上）和从背面观单个光片（底）的单一光学片。发展中视泡的细胞发生复杂的重排，最终形成与内层视网膜神经上皮和外视网膜色素上皮细胞的半球形视杯。透镜从表面外胚层形成。它与神经上皮内陷沿着并视杯坐。所有细胞膜由β肌动蛋白的表达标记:RAS-GFP（绿色）转基因和细胞核都标有HSP70:H2B-RFP（品红色）。影片开始于约17高倍视野。数据集与来自5次相距大约20度的双面照明和AZ的约100μm栈被收购，每10分钟用20X /为1.0W目标获得的。时间显示在HH:MM。比例尺代表50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53966/Movie3-final.mp4" target="_blank">请点击此处下载此文件。</a>

Watch this Scientific Journal Video about Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development at JoVE.com

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

using-light-sheet-fluorescence-microscopy-to-image-zebrafish-eye

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

33.8K 次观看.  Max Planck Institute of Molecular Cell Biology and Genetics. Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

视频: Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

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 ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

光片为主的生活的荧光显微镜或固定和染色<em&gt;赤拟谷盗</em&gt;胚胎

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

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 ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

4D显微镜:使用Nomarski显微镜揭示 秀丽隐杆线虫 胚胎发育

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...

Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, proliferation, and differentiation. Time-lapse imagining offers unique insight to the behavior of cells during eye development because it allows us to visualize oculogenesis in vivo. Zebrafish are an excellent model to visualize this process due to their highly conserved vertebrate eye and their ability to develop rapidly and externally while remaining optically transparent. Time-lapse imaging studies of zebrafish eye development are greatly facilitated by use of the transgenic zebrafish line Tg(rx3:GFP). In the developing forebrain, rx3:GFP expression marks the cells of the single eye field, and GFP continues to be expressed as the eye field evaginates to form an optic vesicle, which then invaginates to form an optic cup. High resolution time lapse imaging of rx3:GFP expression, therefore, allows us to track the eye primordium through time as it develops into the retina. Lightsheet microscopy is an ideal method to image ocular morphogenesis over time due to its ability to penetrate thicker samples for fluorescent imaging, minimize photobleaching and phototoxicity, and image at a high speed. Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software. The resulting dataset can provide valuable insights into the process of ocular morphogenesis, as well as perturbations to this process as a result of genetic mutation, exposure to pharmacological agents, or other experimental manipulations.

Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software.&#160;

使用rx3:GFP转基因斑马鱼通过光片显微镜可视化眼部形态发生

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, ...