作者希望感谢里奥哈·萨卢德基金会（Fondos FEDER）和西班牙科学部长，创新与大学（MCIU）（Grant PGC2018-094276-B-I00）的支持。克里斯蒂娜·罗梅罗·阿兰达（Cristina Romero Aranda）由AECC（Asociación Española Contra el Cáncer）的奖学金资助。

1. 在培养皿上种植秀丽隐杆线虫
<ol><li>准备NGM板并用 大肠杆菌 OP50作为食物来源播种（图1）。生长和维持 秀丽隐杆线虫， 如17所述。将种子板在4°C下储存长达一个月。</li>
	<li>在添加蠕虫之前，将板调整到所需的温度。<ol>
<li>要转移蠕虫，请从旧盘子中取出一块琼脂并将其放在新鲜盘子上。</li>
		<li>或者，用无菌蠕虫拾取器（一根1英寸的32号铂丝，带有扁平的尖端，安装在巴斯德移液器的尖端上）捕获单个动物，并将其放在新板上。</li>
	</ol>
</li>
	<li>在所需的温度下生长蠕虫。<ol>
<li>在20°C，标准温度下生长 秀丽隐杆线虫 。然而，为了分析热敏突变体的发展，通常在25°C下进行过夜孵育。 根据需要调整孵育的持续时间和温度，具体取决于特定的突变体。</li>
	</ol>
</li>
</ol>2.在安装胚胎之前准备4D显微镜记录（图2）
<ol><li>在准备胚胎之前设置显微镜和温度控制。 秀丽隐杆线虫 胚胎分裂得非常快。准备好在安装胚胎后立即开始记录。</li>
	<li>将记录温度调节至 15 °C、20 °C 或 25 °C。<ol>
<li>常规记录25°C下的胚胎。 在限制性温度下记录热敏突变体以显示其表型。在与突变体相同的温度下记录WT对照（如果在不同的制备中完成）。 注意:WT胚胎在25°C下发育更快，在15°C下发育较慢，细胞谱系没有额外的差异。将显微镜放在温控室中。通过冷却或加热载玻片进行额外控制是非常可取的。这可以通过在特定温度下通过显微镜物镜和聚光镜周围的金属环循环水来实现。物镜和制剂通过浸油直接接触，温度传递高效。该系统允许精确控制记录温度，并在胚胎发育过程中进行温度变化。</li>
	</ol>
</li>
	<li>在显微镜软件中定义记录参数。<ol>
<li>对于标准的 秀丽隐杆线虫 记录（无荧光扫描），请选择: z-堆叠由 30 个焦平面组成，每个焦平面的距离为 1 微米。 每个 z 堆栈开始之间的间隔为 30 秒。 1500 个 z 堆栈（12.5 小时的记录）。 注意:商业和开源显微镜控制程序都可用于定义捕获图像的工作流程。现在显微镜已经准备好记录了。</li>
	</ol>
</li>
</ol>3. 准备和安装胚胎
<ol><li>准备一个薄而均匀的琼脂垫，作为获得漂亮图像的第一步（图3）。<ol>
<li>在去离子水中制备50ml4.5%琼脂溶液。在微波炉中加热至沸腾，将0.5 - 1毫升倒入3毫升玻璃管中。</li>
		<li>用蜡膜小心地密封管子，以避免干燥。密封的琼脂管可以在室温下储存长达两个月。</li>
		<li>在实验室工作台上，在80°C下加热块: 一个纯凡士林（熔化）的试管，里面有一把细油漆刷。 装有蒸馏水的试管，其中含有巴斯德移液器。 注意:这确保了所有必需的材料都是热的，琼脂在制作垫的过程中不会凝固。</li>
		<li>从其中一个琼脂管的顶部取下蜡膜，并在酒精燃烧器上小心地加热以融化琼脂。小心谨慎，因为从玻璃管中排出的热琼脂可能会导致灼伤。</li>
		<li>琼脂熔化后，将管子放在加热块中以保持琼脂呈液体形式。</li>
		<li>或者，在实验前1h将琼脂管放入加热块中以熔化它们而无需使用燃烧器。融化的琼脂应在一天后丢弃。</li>
		<li>将显微镜载玻片（载玻片A）放在一块塑料上的另外两张之间。</li>
		<li>拿另一张幻灯片（幻灯片B），用一只手用手指握住它。</li>
		<li>另一只手，使用温暖的巴斯德移液器将一小滴融化的琼脂放在幻灯片A的中心。</li>
		<li>立即将载玻片B按在琼脂滴上，在载玻片A和B之间形成一个非常薄的垫子，将这些载玻片夹在一起，直到步骤3.2.3。</li>
	</ol>
</li>
	<li>安装胚胎。<ol>
<li>用采摘器收集5-10个怀孕雌雄同体，并将它们放入装满水的制表玻璃杯中。</li>
		<li>使用手术刀切开雌雄同体线虫，并在立体显微镜下从子宫中提取早期卵子（1-4个细胞）。</li>
		<li>取下步骤3.1.10中的载玻片，轻轻滑出载玻片B以暴露载玻片A上的琼脂垫。</li>
		<li>将一个早熟的卵子放在琼脂垫的中心，用毛细管移液。</li>
		<li>或者，将含有一组胚胎的液滴移液到琼脂垫上，然后寻找早期胚胎。在立体显微镜下执行此步骤。</li>
		<li>如有必要，用粘在牙签末端的睫毛推动鸡蛋来移动鸡蛋。</li>
		<li>用毛细管移液管除去多余的水分。 注意:通过在酒精燃烧器上加热巴斯德移液器并从两端拉出，可以很容易地制备毛细管移液器。</li>
		<li>用盖玻片小心地覆盖制剂。为避免气泡，将盖玻片的一个边缘放在载玻片上，然后沿着相邻边缘轻轻滑动手术刀，以缓慢而倾斜地将盖玻片垂到制剂上。</li>
		<li>使用移液器用水填充琼脂垫周围3/4的空间。将1/4的空间留给空气。</li>
		<li>用凡士林密封盖玻片，以避免在长时间记录期间干燥。</li>
		<li>使用细刷在盖玻片边缘周围延伸一层薄薄的融化的凡士林。 注意:现在准备工作已准备好进行录制。</li>
	</ol>
</li>
</ol>4. 调整DIC并开始4D显微镜记录
<ol><li>将载玻片放在显微镜载物台上。使用低倍率物镜（5x或10x）聚焦胚胎。</li>
	<li>更改为 100 倍浸入式物镜。</li>
	<li>调整显微镜的光学元件以获得Nomarski图像。<ol>
<li>聚焦聚光镜。</li>
		<li>完全打开聚光镜的孔径并关闭场膜片（这将提供更高的数值孔径，从而提供更高的分辨率）。</li>
		<li>检查显微镜上的两个偏振片的方向是否达到最大消光。</li>
		<li>转动Wollaston棱镜以获得胚胎的漂亮三维图像，一侧照亮。将棱镜转向另一个方向，以获得在另一侧照亮胚胎的效果。 注意:这些步骤可以在记录之前对测试样品执行，因此只需要对被分析的样品进行微调。</li>
	</ol>
</li>
	<li>在显微镜中启动图像捕获。</li>
</ol>5. 分析 4D 影片（图 4）。
注意:记录完成后，使用细胞谱系示踪软件重建和分析细胞谱系。
细胞谱系示踪软件是一种强大的工具，用于对细胞培养物或组织片段中的胚胎发育或动力学进行详细分析。该程序提取并量化有关样品细胞动力学的几个数据集，包括生成每个记录细胞的完整细胞谱系，包括细胞分裂，细胞周期长度，迁移或凋亡及其动力学。此外，细胞分化可以通过细胞的形态变化或特定标记物的表达来评分。基本上，软件屏幕显示两个窗口:在左侧窗口中，4D电影可以向前和向后播放，也可以上下播放到顶部或底部级别，以便在整个录制过程中可以按时间和空间跟踪每个单元格。在右边的寡妇身上，细胞谱系被生成。单击4D电影中的细胞核会在谱系窗口中生成一个点，该点存储细胞名称，命运和空间坐标的信息。特定细胞的细胞谱系是通过向前播放4D电影并定期单击细胞核以标记该特定细胞随时间推移的有丝分裂而产生的。对每个记录的细胞重复此过程会产生胚胎或样品的完整细胞谱系。每个细胞的空间坐标的存储信息随后用于重建3D胚胎模型和细胞迁移路径。
<ol><li>打开沿袭追踪软件并创建一个新项目，方法是转到上方的栏菜单并选择: 文件|新建项目。</li>
	<li>根据记录温度选择细胞谱系模板:DB08用于在25°C下记录，DB10用于在20°C下记录，DB12用于在15°C下记录。</li>
	<li>在新兴窗口中设置记录参数:扫描计数（通常为1500），扫描间隔时间（30秒），液位计数（30）和液位间距（1微米）。</li>
	<li>选择图像文件和格式。<ol>
<li>选择保存图像的映像目录。</li>
		<li>选择是应将图像另存为单个图像（每个级别和时间一个图像）还是将图像另存为多图像 z 堆栈。</li>
		<li>确定文件命名和图像格式。通常，单个图像以以下名称保存: X0000L00C1（用于扫描 0，级别 0，通道 1） X0000L01C1（用于扫描 0，级别 1，通道 1） X0000L02C1（用于扫描 0，级别 2，通道 1） ... X0001L00C1（用于扫描 1，级别 0，通道 1） X0001L01C1（用于扫描 1，级别 1，通道 1） ... X0300L04C1（用于扫描 300，级别 4，通道 1） ... 注意:以压缩格式保存图像可节省硬盘上的空间。</li>
	</ol>
</li>
	<li>定义光通道:1 个用于 DIC 光学器件，2 个用于 GFP，3 个用于 RFP 等。添加在 4D 录制中使用的那些。单击"已启用通道处理"以检测它们。</li>
	<li>开始追踪胚胎的细胞谱系。屏幕现在包含两个主要窗口:视频窗口和细胞谱系窗口。<ol>
<li>在谱系窗口中，选择一个谱系分支，然后使用鼠标在视频窗口中单击与此细胞对应的细胞核。</li>
		<li>使用光标键，通过向前、向后、向上或向下播放 4D 影片，在空间上和随时间推移跟随单元格。</li>
		<li>定期点击细胞核。这将在谱系分支中生成一个点，并注册此时单元格的空间坐标。结果，细胞谱系进展，胚胎的3D重建是可能的。</li>
		<li>通过单击返回键标记有丝分裂。然后选择其中一个子单元格并像以前一样跟随它。</li>
	</ol>
</li>
	<li>对其余的胚胎细胞重复该过程（步骤5.6.1至5.6.4），以追踪完整的细胞谱系，或跟踪感兴趣的特定细胞，例如正在经历细胞凋亡的细胞。</li>
	<li>将突变谱系与刻板的WT 秀丽隐杆线虫 细胞谱系进行比较。</li>
</ol>

<ol>
	<li>Hardin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+embryonic+morphogenesis+in+C.+elegans.">Imaging embryonic morphogenesis in C. elegans.</a> Methods in Cell Biology. 106, 377-412 (2011).</li><li>Mace, D. L., Weisdepp, P., Gevirtzman, L., Boyle, T., Waterston, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-fidelity+cell+lineage+tracing+method+for+obtaining+systematic+spatiotemporal+gene+expression+patterns+in+Caenorhabditis+elegans.">A high-fidelity cell lineage tracing method for obtaining systematic spatiotemporal gene expression patterns in Caenorhabditis elegans.</a> G3: Genes, Genomes, Genetics (Bethesda). 3 (5), 851-863 (2013).</li><li>Nieto, 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=ccz-1+mediates+the+digestion+of+apoptotic+corpses+in+C.+elegans.">ccz-1 mediates the digestion of apoptotic corpses in C. elegans.</a> Journal of Cell Science. 123 (12), 2001-2007 (2010).</li><li>Cabello, J., 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=PDR-1/hParkin+negatively+regulates+the+phagocytosis+of+apoptotic+cell+corpses+in+Caenorhabditis+elegans.">PDR-1/hParkin negatively regulates the phagocytosis of apoptotic cell corpses in Caenorhabditis elegans.</a> Cell Death &amp; Disease. 5, 1120 (2014).</li><li>Pinto, S. M., Almendinger, J., Cabello, J., Hengartner, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Acetylcholine+Signaling+Reduces+Cell+Clearance+Deficiencies+in+Caenorhabditis+elegans.">Loss of Acetylcholine Signaling Reduces Cell Clearance Deficiencies in Caenorhabditis elegans.</a> PLOS One. 11 (2), 0149274 (2016).</li><li>S&#225;enz-Narciso, B., G&#243;mez-Orte, E., Zheleva, A., Gastaca, I., Cabello, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+developmental+networks+by+Rac/Rho+small+GTPases:+How+cytoskeletal+changes+during+embryogenesis+are+orchestrated.">Control of developmental networks by Rac/Rho small GTPases: How cytoskeletal changes during embryogenesis are orchestrated.</a> Bioessays. 38 (12), 1246-1254 (2016).</li><li>Zheleva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+mRNA+export+unmasks+different+tissue+sensitivities+to+low+mRNA+levels+during+Caenorhabditis+elegans+development.">Reduction of mRNA export unmasks different tissue sensitivities to low mRNA levels during Caenorhabditis elegans development.</a> PLOS Genetics. 15 (9), 1008338 (2019).</li><li>Schnabel, R., Hutter, H., Moerman, D., Schnabel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+normal+embryogenesis+in+Caenorhabditis+elegans+using+a+4D+microscope:+variability+of+development+and+regional+specification.">Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: variability of development and regional specification.</a> Developmental Biology. 184 (2), 234-265 (1997).</li><li>Verbrugghe, K. J. C., Chan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+C.+elegans+Embryos+using+an+Epifluorescent+Microscope+and+Open+Source+Software.">Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software.</a> Journal of Visualized Experiments. (49), e2625 (2011).</li><li>Boyd, L., Hajjar, C., O'Connell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+Microscopy+of+Early+Embryogenesis+in+Caenorhabditis+elegans.">Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans.</a> Journal of Visualized Experiments. (54), e2852 (2011).</li><li>Urbach, R., Schnabel, R., Technau, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pattern+of+neuroblast+formation,+mitotic+domains+and+proneural+gene+expression+during+early+brain+development+in+Drosophila.">The pattern of neuroblast formation, mitotic domains and proneural gene expression during early brain development in Drosophila.</a> Development. 130 (16), 3589-3606 (2003).</li><li>Dolinski, C., Borgonie, G., Schnabel, R., Baldwin, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buccal+capsule+development+as+a+consideration+for+phylogenetic+analysis+of+Rhabditida+(Nemata).">Buccal capsule development as a consideration for phylogenetic analysis of Rhabditida (Nemata).</a> Development Genes and Evolution. 208 (9), 495-503 (1998).</li><li>Houthoofd, W., Jacobsen, K., Mertens, C., Vangestel, S., Coomans, A., Borgonie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+cell+lineage+of+the+marine+nematode+Pellioditis+marina.">Embryonic cell lineage of the marine nematode Pellioditis marina.</a> Developmental Biology. 258 (1), 57-69 (2003).</li><li>Hejnol, A., Schnabel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+eutardigrade+Thulinia+stephaniae+has+an+indeterminate+development+and+the+potential+to+regulate+early+blastomere+ablations.">The eutardigrade Thulinia stephaniae has an indeterminate development and the potential to regulate early blastomere ablations.</a> Development. 132 (6), 1349-1361 (2005).</li><li>Hejnol, A., Schnabel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+a+couple+of+dimensions+can+do+for+you:+Comparative+developmental+studies+using+4D+microscopy-examples+from+tardigrade+development.">What a couple of dimensions can do for you: Comparative developmental studies using 4D microscopy-examples from tardigrade development.</a> Integrative and Comparative Biology. 46 (2), 151-161 (2006).</li><li>Bischoff, M., Parfitt, D. E., Zernicka-Goetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+embryonic-abembryonic+axis+of+the+mouse+blastocyst:+relationships+between+orientation+of+early+cleavage+divisions+and+pattern+of+symmetric/asymmetric+divisions.">Formation of the embryonic-abembryonic axis of the mouse blastocyst: relationships between orientation of early cleavage divisions and pattern of symmetric/asymmetric divisions.</a> Development. 135 (5), 953-962 (2008).</li><li>Mora-Lorca, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+reductase+gsr-1+is+an+essential+gene+required+for+Caenorhabditis+elegans+early+embryonic+development.">Glutathione reductase gsr-1 is an essential gene required for Caenorhabditis elegans early embryonic development.</a> Free Radical Biology and Medicine. 96, 446-461 (2016).</li></ol>

作者没有什么可透露的。

现代生物学的主要挑战之一是了解多细胞生物的发育。秀丽隐杆线虫已成为研究发育中胚胎中细胞增殖和细胞分化之间精细协调的最合适模型之一。从光学角度来看，其透明的体和小尺寸使这种线虫成为DIC显微镜的理想标本。具有类似特征的其他生物体也进行了4D显微镜分析11，12，13，14，15，16。
对于这些发育研究，正向或反向遗传学的基因失活为其参与胚胎发生提供了线索。一旦一个基因被证明在发育中发挥作用，下一步就是确定它在建立正确的身体计划方面的确切作用。免疫染色是大多数模型的首选方法。该技术阐明了细胞分化或特定标记物表达中的问题。然而，这种方法的一个主要局限性是，它只提供在开发过程中的固定点上单个或多个标记的表达式的静态视图。这些标记物在整个发育过程中的动态视图只能通过在不同时间点染色不同的胚胎来获得。此外，在这种固定样品中不可能进行细胞谱系重建。
4D显微镜是研究胚胎发育的补充方法。该技术以细胞级分辨率揭示发育动态。胚胎中的任何缺陷，例如纺锤体取向，细胞迁移，凋亡，细胞命运规范等方面的问题都将出现在4D电影中，该电影可以向前和向后可视化，由研究人员进行量化和评分。使用这种技术，胚胎中的几乎每个细胞都可以被跟踪到胚胎开始移动的那一刻。仅用可见光和Nomarski光学器件进行4D显微镜检查的胚胎不会引起光损伤。荧光扫描也可以在记录中插入，以检测基因表达的时间和位置。与标准WT谱系胚胎相比，遭受显着光损伤的胚胎通过细胞周期延长来识别，该细胞周期延长导致强烈的紫外线照射。在这种情况下，可以通过降低紫外灯强度和增加相机灵敏度或曝光时间来减少光损伤。形态学特征和分子标记可以帮助阐明任何突变体的胚胎发育。
在实验室中设置4D显微镜系统很容易实现，经过一些实践，可以在显微镜领域每个细胞的分辨率水平上对细胞动力学和细胞培养物和活透明标本的谱系进行无与伦比的分析。DIC图像上的细胞谱系示踪仍然手工处理。这很耗时，尽管该软件可以检测谱系错误，例如标记同一单元格的不同谱系分支，但错误是可能的。虽然GFP标记细胞的自动检测已经发展良好2，但基于未标记细胞和可见光图像的补充谱系示踪软件仍处于早期阶段，对于完整的胚胎分析并不真正有用。毫无疑问，图像识别系统在可见光显微镜领域的应用将带来这一领域的长足进步。

4D显微镜是一种多焦点延时记录系统，允许研究人员在空间上和随时间变化地记录和量化生物样品的细胞动力学。细胞培养物，酵母或活组织可以进行4D分析，但该技术特别适用于分析活胚胎的发育。该分析的分辨率达到胚胎每个细胞的水平。可以检测到每个细胞分裂，并且可以随着时间的推移跟踪细胞运动。根据细胞获得的位置和形状评估细胞的命运。Nomarski光学元件的使用增强了未染色透明样品的对比度，使用干涉焦平面的正交偏振光束。生成的图像显示为三维，一侧发光。
已经开发了基于使用共聚焦显微镜和GFP转基因动物自动检测细胞核和生成细胞谱系的其他方法1，2。这些系统的优势是显而易见的:该软件大大覆盖了在一段时间内手动标记每个原子核的需求（尽管在后期阶段需要一些手动监督）。然而，涉及细胞形状或膜动力学变化的细胞过程，例如在细胞分化，迁移，凋亡或尸体吞噬期间发生的过程，仍然隐藏在荧光标记的细胞核图像中作为黑色背景。
相比之下，4D Nomarski显微镜（也称为DIC显微镜，微分干涉对比显微镜）显示了野生型或突变动物发育过程中发生的细胞核和细胞形状变化。这允许使用标准显微镜进行细胞谱系追踪，仅使用透射光。除了显示特定的表达模式之外，一般不需要使用转基因动物，在这种情况下，荧光扫描可以插入。因此，对于许多从事动态细胞过程（例如胚胎发生或细胞凋亡）的实验室来说，这可能是最佳方法，可以在DIC显微镜下突出显示3，4，5，6，7。
几种灵活且用户友好的程序可用于捕获微观图像并重建记录样品中的细胞谱系，3D模型，细胞迁移路径等。在标准实验中，在一系列焦平面上以恒定的距离获取图像，其数量取决于样品厚度。可以通过增加扫描频率来优化分析的时间分辨率。除了计算机存储容量之外，录制的持续时间几乎没有限制。例如，对于 秀丽隐杆线虫 胚胎发育分析，我们通常每30秒在30个焦平面上采集图像（每个焦平面1微米步长），持续12小时。
这些系统已应用于几种动物胚胎的分析，例如 秀丽隐杆线虫8，9，10， 黑腹果蝇11， 其他线虫胚胎12，13，缓步动物14，15 甚至早期小鼠胚胎16。唯一的要求是具有能够在显微镜下在载玻片上制备的透明胚胎。
综上所述，基于DIC的4D显微镜对于1）分析小型透明动物的胚胎发育特别有用:追踪细胞谱系，细胞迁移路径，生成3D模型等;2）定义基因表达模式;3）研究细胞培养动力学，从酵母到人类细胞;4）分析组织动力学或胚胎片段;5）量化细胞死亡动力学和尸体吞噬;6）基于胚胎发育特征进行比较系统发育分析。如果对这些主题（或类似主题）中的任何一个感兴趣，则可以使用4D显微镜。

<table>
	<tbody>
		<tr>
			<td>Caenorhabditis elegans (N2)</td>
			<td>GCG (Caenorhabditis Genetics Center)</td>
			<td>N2</td>
			<td>WT C. elegans strain. Can be requested at GCG (Caenorhabditis Genetics Center): https://cgc.umn.edu/</td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans (VZ454)</td>
			<td>GCG (Caenorhabditis Genetics Center)</td>
			<td>VZ454</td>
			<td>gsr-1(tm3574) C. elegans mutant strain. Can be requested at GCG (Caenorhabditis Genetics Center): https://cgc.umn.edu/</td>
		</tr>
		<tr>
			<td>Cell Lineage Tracing software</td>
			<td>SIMI</td>
			<td>Simi BioCell</td>
			<td>This is the software to reconstruct the embryo cell lineage. For a detailed explanation check at: http://www.simi.com/en/products/cell-research/simi-biocell.html</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Hamamatsu</td>
			<td>Orca-R2</td>
			<td>Miscroscope camera for both transmitted and UV light</td>
		</tr>
		<tr>
			<td>Microscope control software</td>
			<td>Caenotec</td>
			<td>Time to Live</td>
			<td>This software controls the microscope to perform the 4D image capture. Can be requested at: Caenotec Prof. Ralf Schnabel 
			Kleine Dorfstr. 9 38312 B&#246;r&#223;um, Germany, Ph: ++49 151 11653356 r.schnabel(at)tu-bs.de</td>
		</tr>
		<tr>
			<td>Microscope control software</td>
			<td>Micro-manager</td>
			<td>Micro-manager</td>
			<td>This software controls the microscope to perform the 4D image capture. Can be downloaded at: https://micro-manager.org/</td>
		</tr>
		<tr>
			<td>Motorized microscope</td>
			<td>Leica</td>
			<td>Leica DM6000</td>
			<td>Motorized upright microscope to perform 4D microscopy</td>
		</tr>
		<tr>
			<td>Standard equipment in a Molecular Biology lab.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ16FA</td>
			<td>Steromicroscope to manipulate nematodes and prepare embryos.</td>
		</tr>
	</tbody>
</table>

4d microscopy: unraveling caenorhabditis elegans embryonic development using nomarski microscopy

4D显微镜是解开不同动物胚胎发育过程的宝贵工具。在过去的几十年里， 秀丽隐杆线虫 已成为研究发育的最佳模型之一。从光学角度来看，其大小和透明体使这种线虫成为DIC（微分干涉对比或Nomarski）显微镜的理想标本。本文说明了生长 秀丽隐杆线 虫，准备和安装其胚胎，进行4D显微镜和细胞谱系示踪的方案。该方法基于Nomarski图像的多焦点延时记录和使用特定软件进行分析。该技术揭示了细胞水平的胚胎发育动力学。突变体中的任何胚胎缺陷，例如纺锤体取向，细胞迁移，凋亡或细胞命运规范方面的问题，都可以有效地检测和评分。实际上，胚胎的每个细胞都可以被跟踪到胚胎开始移动的那一刻。通过4D DIC显微镜追踪 秀丽隐杆线虫 胚胎的完整细胞谱系是费力的，但使用特定软件极大地促进了这项任务。此外，该技术易于在实验室中实现。4D显微镜是一种多功能工具，为胚胎发育进行无与伦比的分析开辟了可能性。

为了表征基因gsr-1的秀丽隐杆线虫突变体的胚胎发育，该基因编码谷胱甘肽还原酶，再生还原谷胱甘肽（GSH）并参与维持线虫中的氧化还原稳态，我们对gsr-1（tm3574）缺失突变体进行了4D显微镜检查，该突变体是功能等位基因丧失，导致早期胚胎停滞表型18。WT和平衡gsr-1（tm3574）突变体秀丽隐杆线虫均生长在以大肠杆菌OP50为食物来源的NGM平板上17。gsr-1（tm3574）蠕虫在20°C下以杂合子形式生长两代，然后将分离纯合子蠕虫（由于母体负荷而能够长到成年期）转移到25°C进行过夜孵育，然后进行胚胎分析。将蜗杆板在纸板箱内孵育以避免冷凝（图1）。将Gravid线虫切开以提取年轻胚胎。
为了在相同条件下比较突变体与刻板WT的胚胎发育，将WT（作为对照）和 gsr-1 （tm3574）胚胎放在彼此相邻的同一制剂上。4D显微镜工作流程在配备DIC光学元件的标准电动正置显微镜上运行。显微镜控制程序上选择的记录参数为:30个焦平面的z-stack，每个1微米距离，每个z-stack的开始之间的30秒间隔和1500个z-stack（12.5小时的记录）。将记录温度调节至25°C（在室内和显微镜载物台上）（图2）。
记录完成后，打开图像文件，并通过单击视频窗口中显示的细胞核，使用谱系示踪软件重建细胞谱系（图4）。将追踪的 gsr-1（tm3574） 突变胚胎细胞谱系与背景中描绘的 秀丽隐杆线虫 WT谱系进行比较。一个主要结果是在胚胎发育过程中检测到细胞周期的渐进性延迟。结果，突变胚胎在中间阶段被阻止，而WT胚胎进展并最终孵化为幼虫。
在显微镜下制备和直接观察胚胎或用针对晚期胚胎标志物的抗体进行免疫染色可以揭示与WT相比，突变体中存在高比例的年轻胚胎。然而，细胞周期延迟的直接证明和精确定量只能通过4D显微镜实验优雅而轻松地显示和量化。胚胎发育的其他重要特征，如细胞分化或凋亡（图5），也可以使用4D显微镜以动态方式可视化，该显微镜在单个实验中提供了对发育的多个方面的详细分析。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61736/61736fig01.jpg"> 图1:在实验室条件下生长 的秀丽隐杆线 虫。线虫生长在 大肠杆菌种子的NGM板上，储存在纸板箱中，并在15°C，20°C或25°C下孵育 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61736/61736fig01large.jpg" target="_blank">。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61736/61736fig02.jpg"> 图2:4D显微镜记录软件的屏幕截图。 两种不同的显微镜控制软件程序（A 和 B）的示例。这些程序创建工作流程，以在4D显微镜记录期间控制显微镜和图像捕获。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61736/61736fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61736/61736fig03.jpg"> 图3:琼脂垫制备和 秀丽隐杆线虫 胚胎安装的连续照片显示。A. 准备好的琼脂管。 B-C. 准备琼脂垫。 四. 滑梯部分装满水。 E. 用凡士林密封载玻片。 六. 最后的准备。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61736/61736fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61736/61736fig04.jpg"> 图4:细胞系示踪软件的串行屏幕截图。 该程序允许重建细胞周期延迟突变体（左）和WT（右） 秀丽隐杆线虫胚胎的 胚胎细胞谱系。一个。开发的早期步骤。B-C.两个胚胎的发育都会随着时间的推移而发展。D.WT胚胎正常发育并开始伸长，而突变体停滞。在所有情况下，程序都会显示视频窗口和沿袭窗口。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61736/61736fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61736/61736fig05v2.jpg"> 图5: 秀丽隐杆线虫 WT胚胎中凋亡细胞的扁豆折射形状。由形态学特征定义的细胞命运可以通过4D显微镜评估。该图像显示了豆子阶段 的秀丽隐杆线虫 胚胎。活细胞显示光滑形状的细胞核，周围环绕着颗粒状细胞质。相反，凋亡细胞（黄色箭头）凝结并采用扁豆状的折射形状。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61736/61736fig05large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about 4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy at JoVE.com

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

在这里，我们提出了一种制备和安装 秀丽隐杆线虫 胚胎的方案，在4D显微镜下记录发育并示踪细胞谱系。

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 ...

4d-microscopy-unraveling-caenorhabditis-elegans-embryonic-development

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

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

2.5K 次观看.  CIBIR (Center for Biomedical Research of La Rioja), La Rioja, Spain. 在这里，我们提出了一种制备和安装 秀丽隐杆线虫 胚胎的方案，在4D显微镜下记录发育并示踪细胞谱系。

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

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

科研

发育生物学

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

转录组分析]<em&gt;活体</em&gt;生产牛胚胎植入前胚胎采用双色微阵列平台

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

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 ...

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.

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.

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

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

用于在大容量中查找特定细胞目标的串行剖面的多模式分层成像

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

评估溶酶体碱化在活的秀丽线虫中的肠道

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

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 ...

4D显微镜:使用Nomarski显微镜揭示 秀丽隐杆线虫 胚胎发育

4D显微镜:使用Nomarski显微镜揭示秀丽隐杆线虫胚胎发育