Name: long-term live imaging device for improved experimental manipulation of zebrafish larvae
Uploaded: 2017-10-27T15:00:00.000+00:00
Duration: 14 min 5 s
Description: Watch this Scientific Journal Video about Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae at JoVE.com

作者希望承认 Morgridge 研究所和光学和计算仪器实验室的主要项目资金。我们也承认来自 NIH 的 R01GM102924 (AH 和 KWE) 的资金。KWE 构思并设计了这项研究。在 DL、KP 和 RS 的支持下进行了所有的实验。KWE、JS、RS、AH 和对手稿的撰写贡献良多。

注意: 根据威斯康星大学-麦迪逊分校研究动物资源中心的指导原则, 为2到4天胚乳 (dpf) 的斑马鱼幼虫制定了基本的 zWEDGI 设计. 
 1. 模具的设计和3D 打印 <ol> <li> 模型在3D 建模软件 5 中具有所需几何图形和属性的设备的该组件创建一个毛坯模具和该零件的组件, 并通过在与 PDSM 部件对应的模具中创建一个空腔来生成该零件的负模。将模具保存为用于3D 打印的. stl 文件 ( 图 2 , 步骤 1.1). 
	注意: 此处提供的模具设计的光格式 (. stl) 文件 ( 图 1 ) 可在 https://morgridge.org/designs/上下载. </li>
	<li> 使用光3D 打印机打印模具 ( 图 2 , 步骤 1.2)。在一个单一的印刷, 如果可能的话, 使多个模具, 因此, 一个以上的设备可以塑造一个单一的一批硅橡胶. 
	注: 所示的例子是用光树脂 5 在高分辨率模式下打印的, 其直径为 0.075 mm、0.05 mm 的层厚。
	<ol> <li> 使用细刷子、喷雾瓶中的变性酒精和压缩空气轻轻擦洗和除去未树脂的清洁模具。从微通道区域中取出任何材料. </li>
		<li> 未辞职对斑马鱼幼虫是有毒的, 在每边60分钟的紫外线后固化装置中, 将霉菌贴在每个侧面. 10 . </li>
	</ol> </li> <li> 在平坦的表面上用200砂砾砂纸将模具的凹槽侧砂, 直到所有密封面与砂纸 (加载通道几何和模具周长) 接触。轻砂与400和600砂砂纸, 逐步, 以产生平滑的冲洗表面, 在所有几何面 ( 图 2 , 步骤 1.3)。用拨号指示器测量砂光后的凹槽深度, 以验证其是否接近设计深度。
	<ol> <li> 清洁模具和盖盘 (1 #190; 英寸直径 x 和 #188; 英寸厚的硼硅酸盐玻璃或丙烯酸; 每模一玻璃罩) 放置在一个超声波清洁器, 充满水30分钟或冲洗下自来水. </li>
		<li> 用压缩空气吹干, 用异丙醇和过滤压缩空气清洗模具和盖子。使用干净的工作台作为制造设备的地方, 以最小化空气碎片的污染。在需要的时候, 把清洁的模具和玻璃盖放在干净的长凳或盖的培养皿里. </li>
	</ol> </li> </ol> 2。zWEDGI 设备的制造 <ol> <li> 将184硅胶弹性体烷 (聚硅氧烷) 以 5:1 (基到活化剂) 的比例浇注到塑料杯中。用木制工艺棒拌匀2分钟, 将凝胶搅拌到自己身上, 如揉捏面包。对于5模具, 使用10克的基地和2克的活化剂。
	<ol> <li> 在真空中的气体混合物中干燥 25-45 分钟, 直到所有气泡都消失. </li>
		<li> 用10mL 注射器将每一个3D 印刷模具的腔体填充到大约0.75 毫升的硅橡胶上, 直到模具略微溢出半月板 ( 图 2 , 步骤 2.1). </li>
		<li> 消除气体填充的模具45分钟, 以清除填充时可能形成的额外气泡. </li>
	</ol> </li> <li> 将玻璃盖板应用于填充的充满了硅橡胶的模具的顶部, 将光盘按角度向下按以防止气泡被捕获 ( 图 2 , 步骤 2.2)。在应用玻璃盘盖时, 允许多余的硅橡胶被排出. </li>
	<li> 使用小棘轮钳将阀盖紧紧地固定在模具上. 
	注意: 这将创建一个平坦的表面, 一旦该公司被治愈, 并通过孔, 3D 印刷的几何图形是冲洗与玻璃覆盖滑。或者, 要增加一次可以固化的设备的数量, 请构建一个 multi-clamp 设备 ( e. g 。 图 2 , 步骤 2.3). </li>
	<li> 在固定的模具中固化 100 o C 在烤箱中为 90 min ( 图 2 , 步骤 2.4)。从烤箱中取出模具, 并允许冷却, 直到它们可以很容易地处理。如果夹紧装置为金属, 则从夹具中取出模具和盖盘总成, 防止金属因余热而继续固化. 
	注意: 该设备是最容易从模具中删除, 而仍然略有温暖, 并没有完全治愈。然而, 一旦模具从烤箱中取出, 并删除了盖盘, 该设备可以坐几天, 然后继续脱模。如果设备是 demolded 后不久, 从固化炉, 把它放在一个覆盖的培养皿, 以减少污染物, 同时允许它完全治愈. </li>
</ol> 3。等离子键 zWEDGI 到玻璃盘上 <ol> <li> 在工作台副中夹住包含该设备的模具, 使模具和 #39 的几何图形朝向上, 与工作台工作台平行。
	<ol> <li> 通过使用平尖镊子从模具中松开 "硅橡胶拉" 标签, 开始卸下该设备。使用镊子在模具的周长周围工作 (如从平底锅中取出蛋糕 ( 图 2 , 步骤 3.1). </li>
		<li> 使用过滤过的压缩空气和镊子, 通过按住拉舌并在设备下吹气, 轻轻地将设备拉出模具。慢慢地工作, 让空气帮助从模具中分离出的硅橡胶, 采取特殊的护理与镊子周围的薄隧道部分的技巧. </li>
	</ol> </li> <li> 将该设备倒置到玻璃底盘盖的内侧, 以便限制隧道楔接触塑料 ( 图 2 , 步骤 3.2). </li>
	<li> 将盘盖与倒置的 zWEDGI 和相应的玻璃底盘放到等离子清洗器中, 内杯面朝上 ( 图 2 , 步骤 3.3)。
	<ol> <li> 疏散等离子清洗室直到压力达到 500 mTorr. </li>
		<li> 将射频 (RF) 电源设置为高。将设备和天线暴露于射频频率约2分钟, 慢慢 de-pressurize 房间, 并将设备和菜放回干净的房罩. </li>
	</ol> </li> <li> 从盘盖中卸下该设备。翻转 zWEDGI 到正确的方向上的玻璃, 通过把它小心翼翼地放置在玻璃底盘的中心井 ( 图 2 , 步骤 3.4) <ol> <li> 使用镊子的后端, 轻轻按下确保气泡不被夹在下面的装置。在微通道的微小几何图形周围施加压力, 并将其平滑到边缘 ( 图 5C ). 
		注: 完全接触与玻璃, 确保更好地坚持从等离子键。该 zWEDGI 装置可以等离子粘合到其他玻璃底碟格式, 如玻璃底 6-井平台e ( 图 2C ). </li>
		<li> 将盖子盖在设备盘上, 然后从干净的房间盖上卸下. 
		注意: 一旦设备被固定到玻璃, 协议可以暂停, 直到它们准备好使用. </li>
	</ol> </li> </ol> 4。通道准备和加载幼虫 注意: 一般斑马鱼养殖是根据斑马鱼的书, 在网上提供 http://zfin.org/zf_info/zfbook/zfbk.html。成年斑马鱼和胚胎的维持, 如前所述 1 。采用了野生型 AB 菌株。遵循机构和 #8217 的动物保育协议, 以了解关于活体幼虫的成像要求. 
<ol> <li> 用最小的和 #160 冲洗通道; 100 和 #181; L 70% 乙醇每通道, 使用微冲洗通过限制隧道。除去乙醇, 用双蒸馏水冲洗2或3次。允许风干. </li>
	<li> 用脱脂牛奶 (浓度1% 稀释在水中) 填充通道, 室温下10分钟, 以最小化幼虫对玻璃片的粘附。然后, 用两次蒸馏水轻轻地将设备浸入水中冲洗。让空气干燥倒置. 
	注意: 这里可以暂停协议。这种 zWEDGI 装置的制备可以在使用前数天完成。贮存在室温下. </li>
	<li> 1% 低熔点 (LMP) 琼脂糖的结合100和 #181; l 2% 熔融 LMP 琼脂糖与100和 #181; l 2x 和 #160; Tricaine (0.4 毫克/毫升 Tricaine-乙基 3-氨基) 在 E3 缓冲区 11 5ml 到 38 o c. 维护1%琼脂糖/tricaine 溶液在 38 o C 在热块中以防止胶凝. 
	注: 对于多显微镜 11 , 使用 un-pigmented 斑马鱼变种 (如 精灵 12 ) 或维持 E3 中含有0.2 毫米 n-phenylthiourea 的幼虫, 以防止色素形成 11 以最小化颜料对近红外波长的吸收. 
	注意: n-phenylthiourea 是有毒的。遵循制度和 #39 的处置规则. </li>
	<li> E3 缓冲区中的麻醉幼虫, 包含0.2 毫克/毫升 tricaine (Tricaine/E3) 11 。等到幼虫不动, 响应到触碰刺激. </li>
	<li> 预湿通道的升 Tricaine/E3。
	<ol> <li> 使用宽孔吸管尖端拾取单个幼虫。将幼虫存入加载通道 ( 图 3A )。使用吸管尖端或类似的工具, 使幼虫在装载室, 使背侧面临的更直的边缘的加载室和尾部面临的限制隧道 ( 图 3B ). </li>
		<li> 小心地从伤室中提取液体, 允许幼虫流入限制通道 ( 图 3C )。去除大部分液体, 同时保持幼虫周围的水分 ( 图 3D )。这一过程可以通过倾斜的盘子轻微向受伤的房间. </li>
	</ol> </li> <li> 将 1% LMP 琼脂糖放在 Tricaine/E3 (〜 38 o C) 上的幼虫和 #39 的头部, 填充装载室 ( 图 3E )。允许琼脂糖凝固与幼虫在适当的位置。根据需要, 将 Tricaine/E3 添加到伤人室以保持水化。对其余2通道重复此加载过程. </li>
	<li> 使用注射器针, 小心地删除通过限制隧道渗透到伤人室的任何琼脂糖 ( 图 3F )。添加额外的 tricaine/E3, 或者只是在琼脂糖 (用于伤人, 短期成像, 或伤口治疗隔离) 或填补文化盘 (长期成像)。更换养殖盘盖以防止蒸发。幼虫可以在这一点 (受伤) 或受伤的影像. </li>
</ol> 5。伤人和成像幼虫 <ol> <li> 使用无菌手术刀刀片, 在伤室中脊索 11 后的尾鳍上横断面 ( 图 3G , H)。添加额外的 tricaine/E3, 如果需要, 并更换文化盘盖. 
	注意: 另外, 根据兴趣的发展时间窗口, 幼虫可能受伤, 允许恢复在 E3 和维护在一个孵化器 (28.5 o C) 直到期望的成像时间, 在那点他们可以被装载入渠道作为如上所述. </li>
	<li> 将 zWEDGI 装置与麻醉幼虫一起安装在一个倒置显微镜下的一个舞台插入, 将容纳60毫米玻璃底盘。在最上层的通道中找到幼虫的尾巴, 在需要的地方旋转盘子, 以获得所需位置的尾部。特定实验所需的图像. 
	注: zWEDGI 广泛适用于高分辨率光学显微镜, 包括 widefield 荧光和激光扫描显微镜。在对斑马鱼幼虫进行成像时, 有许多参数的考虑, 但具体的成像参数是仪器、样品和实验依赖。在这里, 幼虫尾巴被映像在一个自定义修造多显微镜 5 , 11 使用以下参数: 40X 长工作距离水浸泡物镜, 890 nm 激光励磁,445/20 nm 发射过滤器和 512 x 512 分辨率. </li>
</ol> 6。实验结束 <ol> <li> 从显微镜中取出 zWEDGI 盘。安乐的幼虫通过放置在冰水浴或在 4 o C 的 zWEDGI 至少20分钟, 并评估没有心跳和循环. 
	注意: 由于幼虫是在单独的舱室中维持的, 幼虫可以通过用镊子或吸管轻轻地在琼脂上提取来单独地恢复。琼脂可以从头部区域和用于其他程序的幼虫中除去, 如用于抗体染色的固定或用于 RNA 或蛋白质提取的处理. </li>
	<li> 在去除幼虫和琼脂后, 用乙醇和蒸馏水清洗 zWEDGI, 如步骤4.1 所述, 将其倒置至风干。存放在阴凉, 干燥的位置。根据需要在下次使用前重新涂上脱脂牛奶 (步骤 4.2). 
	注: zWEDGIs 可以多次重复, 直到该公司开始远离玻璃. </li>
</ol>

<ol>
	<li>Yoo, S. K., Freisinger, C. M., LeBert, D. C., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+redox,+Src+family+kinase,+and+calcium+signaling+integrate+wound+responses+and+tissue+regeneration+in+zebrafish.">Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish.</a> J. Cell Biology. 199 (2), 225-234 (2012).</li><li>Kawakami, A., Fukazawa, T., Takeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+fin+primordia+of+zebrafish+larvae+regenerate+by+a+similar+growth+control+mechanism+with+adult+regeneration.">Early fin primordia of zebrafish larvae regenerate by a similar growth control mechanism with adult regeneration.</a> Dev. Dynam. 231 (4), 693-699 (2004).</li><li>Konantz, J., Antos, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+genetic+morpholino+approach+using+cardiac+ventricular+injection+to+transfect+multiple+difficult-to-target+tissues+in+the+zebrafish+larva.">Reverse genetic morpholino approach using cardiac ventricular injection to transfect multiple difficult-to-target tissues in the zebrafish larva.</a> JoVE. (88), (2014).</li><li>Hall, C., Flores, M. F., Kamei, M., Crosier, K., Crosier, P., Sampath, K., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+Innate+Immune+Cell+Behavior+During+Normal+Development,+Wound+Healing+and+Infection.">Live Imaging Innate Immune Cell Behavior During Normal Development, Wound Healing and Infection.</a> Live Imaging in Zebrafish: Insights into Development and Disease. , (2010).</li><li>Huemer, K., Squirrell, J. M., Swader, R., LeBert, D. C., Huttenlocher, A., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=zWEDGI:+Wounding+and+Entrapment+Device+for+Imaging+Live+Zebrafish+Larvae.">zWEDGI: Wounding and Entrapment Device for Imaging Live Zebrafish Larvae.</a> Zebrafish. , (2016).</li><li>Lisse, T. S., Brochu, E. A., Rieger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+tissue+repair+in+zebrafish+larvae+with+time-lapse+brightfield+stereomicroscopy.">Capturing tissue repair in zebrafish larvae with time-lapse brightfield stereomicroscopy.</a> JoVE. (95), (2015).</li><li>Kamei, M., Isogai, S., Pan, W., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+blood+vessels+in+the+zebrafish.">Imaging blood vessels in the zebrafish.</a> Methods Cell Biol. 100, 27-54 (2010).</li><li>Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+zebrafish+embryonic+brain+by+confocal+microscopy.">Live imaging of the zebrafish embryonic brain by confocal microscopy.</a> JoVE. (26), (2009).</li><li>Petzold, A. M., Bedell, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCORE+imaging:+specimen+in+a+corrected+optical+rotational+enclosure.">SCORE imaging: specimen in a corrected optical rotational enclosure.</a> Zebrafish. 7 (2), 149-154 (2010).</li><li>Macdonald, N. P., Zhu, F., 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=Assessment+of+biocompatibility+of+3D+printed+photopolymers+using+zebrafish+embryo+toxicity+assays.">Assessment of biocompatibility of 3D printed photopolymers using zebrafish embryo toxicity assays.</a> Lab Chip. 16 (2), 291-297 (2016).</li><li>LeBert, D. C., Squirrell, J. M., Huttenlocher, A., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+harmonic+generation+microscopy+in+zebrafish.">Second harmonic generation microscopy in zebrafish.</a> Methods Cell Biol. 133, 55-68 (2016).</li><li>White, R. M., Sessa, A., 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=Transparent+Adult+Zebrafish+as+a+Tool+for+In+Vivo+Transplantation+Analysis.">Transparent Adult Zebrafish as a Tool for In Vivo Transplantation Analysis.</a> Cell Stem Cell. 2 (2), 183-189 (2008).</li><li>LeBert, D. C., Squirrell, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+9+modulates+collagen+matrices+and+wound+repair.">Matrix metalloproteinase 9 modulates collagen matrices and wound repair.</a> Development. 142 (12), 2136-2146 (2015).</li><li>Campagnola, P. J., Millard, A. C., Terasaki, M., Hoppe, P. E., Malone, C. J., Mohler, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+high-resolution+second-harmonic+generation+imaging+of+endogenous+structural+proteins+in+biological+tissues.">Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues.</a> Biophys. J. 82 (1 Pt 1), 493-508 (2002).</li><li>Schindelin, J., Arganda-Carreras, I., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat. Methods. 9 (7), 676-682 (2012).</li></ol>

作者没有什么可透露的。

zWEDGI 装置的目的是通过在高分辨率显微镜物镜的小工作距离内稳定和定位鱼来捕捉3D 的延时成像。在满足这些设计规范的同时, 它也是对传统 agar-based 的实时成像准备的一种改进。在制造 zWEDGI 的过程中有三关键步骤 (以下), 如果不能正确完成, 可能会导致设备出现故障:
准备工作 (图 5A)
为了防止气泡, 德加德的模具后, 已增加。检查?...

斑马鱼幼虫的再生能力斑马鱼使它们成为检查伤口反应的理想模型生物体以及愈合和再生1、2、3、4。进入一系列的转基因斑马鱼线和斑马鱼的解剖透明度进一步提高他们的效用在体内伤口反应事件的研究以及 longer-term 再生过程4。对这些生物过程的研究使用高分辨率的延时荧光显微镜, 因此需要一个活成像斑马鱼的设备, 允许高稳定性和最小的斑马鱼幼虫的运动, 同时保持生存能力。这是关键, 该设备允许有效的伤人, 而愈合和再生发生不受设备的影响。
活体成像中植入琼脂糖中幼虫的标准活成像稳定方法限制生长和伤口再生5 , 并且可能会增加死亡率, 因为幼虫开始显示四后的心脏应激和组织坏死的迹象小时4。因此, 从感兴趣的区域去除琼脂糖通常是必要的, 以允许正常的发育和再生6, 当琼脂糖被切断时, 将幼虫暴露在潜在的损伤中。此外, 使用琼脂糖嵌入技术, 用户必须在琼脂糖固化5、6、7之前的短时间内定位幼虫。快速操控幼虫不仅需要使用者的技能, 它还会危害幼虫。虽然已经描述了稳定活体成像的幼虫的方法, 以绕过这些缺点, 如脊琼脂井3或 divets8, 使用硅胶真空油脂创建一个成像室与 PVC 管道或其他材料6, 和旋转油管9, 这些方法中的许多是劳动密集型, 凌乱, 经常不可, 不允许环境操作 (药物治疗, 伤人等) 后, 鱼已经安装。
因此, zWEDGI 设备 (图 1) 旨在克服在允许操作标本的同时, 对斑马鱼幼虫进行长期实时成像的琼脂安装的一些缺点。该 zWEDGI 包括三半分隔商会 (图 1A), 以允许加载, 克制, 伤人和成像2至4天胚乳斑马鱼幼虫。该装置是由烷 (硅氧烷) 制成, 放在 60 mm 玻璃底成像盘的盖子上。这里提出的设计是为了伤口愈合研究, 但使用模块化设计和标准制造技术使 zWEDGI 设计可修改和服从各种实验程序, 特别是对于程序需要对实验操作和长期成像进行最小的约束。

<table><tbody><tr><td>&lt;strong&gt;Fabricate molds&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Solidworks Professional Accedemic Research 3D modeling software</td><td>Dassault Systemes</td><td>SPX0117-01</td><td>Fisher Unitech</td></tr><tr><td>Viper Si2 SLA 3D printer</td><td>3D Systems Inc.</td><td>23200-902</td><td>3D Systems Inc.</td></tr><tr><td>Accura 60 photopolymer resin</td><td>3D Systems Inc.</td><td>24075-902</td><td>3D Systems Inc.</td></tr><tr><td>denatured alcohol</td><td>Sunnyside</td><td>5613735</td><td>Menards</td></tr><tr><td>UV post cure apparatus</td><td>3D Systems Inc.</td><td>23363-101-00</td><td>3D Systems Inc.</td></tr><tr><td>TouchNTuff nitrile gloves</td><td>Ansell</td><td>92-600</td><td>McMaster Carr</td></tr><tr><td>220B, 400B, 600 grit T414 blue-bak sandpaper&amp;nbsp;</td><td>Norton</td><td>66261139359, 54, 52</td><td>MSC</td></tr><tr><td>borosilicate glass disc, 2&quot; diameter</td><td>McMaster-Carr</td><td>MIL-G-47033</td><td>McMaster-Carr</td></tr><tr><td>ultrasonicator cleaner</td><td>Branson</td><td>1510R-MTH</td><td></td></tr><tr><td>isopropyl rubbing alcohol 70%</td><td>Hydrox</td><td>54845T43</td><td>McMaster-Carr</td></tr><tr><td>10oz clear plastic cup</td><td>WNA Masterpiece</td><td>557405</td><td>Amazon</td></tr><tr><td>6&quot;craft stick</td><td>Perfect Stix</td><td>Craft WTD-500</td><td>Amazon</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Fabricate zWEDGI PDMS device&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sylgard 184 silicon elastomeric kit&amp;nbsp;</td><td>Dow-Corning</td><td>4019862</td><td>Ellworth Adhesives&amp;nbsp;</td></tr><tr><td>10mL syringe</td><td>Becton Dickinson</td><td>305219</td><td>Vitality Medical Inc</td></tr><tr><td>desiccator</td><td>Bel-Art Scienceware</td><td>F42027-0000</td><td>Amazon</td></tr><tr><td>4 in ratcheting bar clamp</td><td>Pittsburgh</td><td>68974</td><td>Harbor Freight</td></tr><tr><td>lab oven</td><td>Quincy Lab Inc.</td><td>20GC</td><td>Global Industrial</td></tr><tr><td>tweezer set</td><td>Aven</td><td>549825</td><td>McMaster-Carr</td></tr><tr><td>compressed air filtered nozzle</td><td>Innotech</td><td>TA-N2-2000FT</td><td>Cleanroom Supply</td></tr><tr><td>vacuum bench vise</td><td>Wilton Tool Group</td><td>63500</td><td>MSC Industrial</td></tr><tr><td>55mm glass bottom dish; 30mm micro-well #1.5 cover glass</td><td>Cellvis</td><td>D60-30-1.5-N</td><td>Cellvis</td></tr><tr><td>plasma cleaner</td><td>Harrick Plasma</td><td>PDC-001</td><td>Harrick Plasma</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Loading Larvae&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pipetteman, P200</td><td>Gilson</td><td>F123601</td><td></td></tr><tr><td>100% ethanol (diluted to 70% with water prior to use)</td><td>Pharmco-aaper</td><td>111000200</td><td></td></tr><tr><td>Transfer pipette</td><td>Fisherbrand</td><td>13-711-5A</td><td>Fisher Scientific</td></tr><tr><td>powdered skim milk</td><td></td><td>2902887</td><td>MP Biomedicals</td></tr><tr><td>double distilled water</td><td></td><td></td><td></td></tr><tr><td>N-phenylthiorurea</td><td>Sigma-Aldrich</td><td>P7629</td><td>Sigma-Aldrich</td></tr><tr><td>tricaine (ethyl 3-aminobenzoate)</td><td></td><td>C-FINQ-UE</td><td>Western Chemical</td></tr><tr><td>low melting point agarose</td><td>Sigma-Aldrich</td><td>A0701</td><td>Sigma-Aldrich</td></tr><tr><td>heat block (dry bath incubator)</td><td>Fisher Scientific</td><td>11-718-2</td><td>Fisher Scientific</td></tr><tr><td>E3 buffer&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>large orifice pipette tip, 200 uL</td><td>Fisherbrand</td><td>02-707-134</td><td>Fisher Scientific</td></tr><tr><td>General purpose pipette tip, 200 uL</td><td>Fisherbrand</td><td>21-197-8E</td><td>Fisher Scientific</td></tr><tr><td>#15 scalpel blade&amp;nbsp;</td><td>Feather</td><td>2976</td><td>Amazon</td></tr><tr><td>25G syringe needle</td><td>BD&amp;nbsp;</td><td>BD305122</td><td>Fisher Scientific</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>inverted microscope</td><td></td><td></td><td></td></tr><tr><td>Imaris imaging software</td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

long-term live imaging device for improved experimental manipulation of zebrafish larvae

斑马鱼幼虫是发育生物学和创面愈合的重要模型机体。此外, 斑马鱼幼虫是一个有价值的系统, 用于实时高分辨率显微成像的动态生物现象在空间和时间与细胞分辨率。然而, 传统的琼脂糖封装的实时成像方法可能会阻碍幼虫的发育和组织再生。因此, 这份手稿描述了 zWEDGI (斑马鱼伤人和诱捕装置的增长和成像), 这是设计和制作的功能分割装置, 以定位幼虫的高分辨率显微镜, 同时允许尾鳍横断在装置内, 随后无节制的尾部发展和再生。这种装置允许受伤和长期成像, 同时保持生存能力。鉴于 zWEDGI 模具是3D 打印, 其几何的可, 使其易于修改, 为不同的斑马鱼成像应用。此外, zWEDGI 提供了许多好处, 例如, 在实验中对幼虫的伤害或试剂的应用, 多个幼虫的平行定位, 以简化成像和设备的可重用性。

zWEDGI 微流控芯片是一种功能性的分割装置, 旨在容纳四主要功能 (如下所示) 与活成像的尾鳍伤愈合和再生在斑马鱼幼虫。由于它不仅是现成的, 而且是生物相容性的行业标准, 而且在模具中也很管用, 因此选择了 zWEDGI 制造。此外, 在设备形成后, 该设备可可重复使用并使其失效。zWEDGI 具体地允许 1) 幼虫的装载入设备, 2) 克制在适当的方向为成像, 3) 损伤尾鳍和 4) 显微成像, ...

Watch this Scientific Journal Video about Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae at JoVE.com

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

这份手稿描述了 zWEDGI (斑马鱼伤人和诱捕装置的增长和成像), 这是一个条块分割的装置, 旨在定位和抑制斑马鱼幼虫。该设计允许尾部横断面和长期收集高分辨率的荧光显微图像的伤口愈合和再生。

改善斑马鱼幼虫实验操作的长期活体成像装置

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

long-term-live-imaging-device-for-improved-experimental-manipulation

Research

JoVE Journal

Bioengineering

8.7K 次观看.  University of Wisconsin-Madison. 这份手稿描述了 zWEDGI (斑马鱼伤人和诱捕装置的增长和成像), 这是一个条块分割的装置, 旨在定位和抑制斑马鱼幼虫。该设计允许尾部横断面和长期收集高分辨率的荧光显微图像的伤口愈合和再生。

视频: Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

科研

发育生物学

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. ...

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

生物学

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

斑马鱼幼虫显微注射和成像的微装置

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

在斑马鱼幼虫的高通量自动化的行为分析

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

神经科学

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 ...

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

实时和重复测量电活动改变的个体活斑马鱼的骨骼肌生长

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

发展中国家斑马鱼的表皮细胞挤压实时成像

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

虚拟现实交互分析 Zebrafish 心脏收缩

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

作者聚焦:斑马鱼中的高分辨率 4D 光片成像和虚拟现实用于心脏功能的单细胞分析

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

斑马鱼心脏收缩的 4D 光片成像

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...

改善斑马鱼幼虫实验操作的长期活体成像装置

摘要

探索更多视频

此视频中的章节

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

斑马鱼发展长期成像的多功能安装方法

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

斑马鱼幼虫显微注射和成像的微装置

在斑马鱼幼虫的高通量自动化的行为分析

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

实时和重复测量电活动改变的个体活斑马鱼的骨骼肌生长

发展中国家斑马鱼的表皮细胞挤压实时成像

斑马鱼心脏收缩的 4D 光片成像

斑马鱼心脏收缩的 4D 光片成像