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>
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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。检查所有气泡在脱气后是否已被消除, 并注意玻璃盘的应用。玻璃盘必须在一个斜面上应用, 以确保空气不会被困在和内部。在过程的大多数步骤中, 灰尘可能是一个问题。为了防止灰尘, 彻底完成每个清洁步骤, 并在步骤之间的一个干净的遮光罩存储部件。检查模具的过剩树脂, 可能会创建一个粗糙表面之前, 紫外线固化。当打磨时, 请确保砂纸接触所有的几何图形, 以确保所有曲面都是齐平的 (图 2, 步骤 1.3)。在用异丙醇填充模具前, 应先将酒精和空气清洗干净。当挤出模具中的额外的硅橡胶时, 确保夹具紧紧地握住模具和玻璃罩 (图 2, 步骤 2.3)。
从模具中卸下设备 (图 5B)
确保烤箱至少 100oC 和固化时间至少为 1-1.5 小时。如果没有足够的固化, 它可能很难从模具中删除。其他的可能性, 不删除包括不混合的时间不够长, 使用不正确的基础和硬化剂的比例和残留在模具后, 3D 印刷, 然后污染的硅橡胶。从烤箱中取出后, 设备和模具之间的空气袋 (图 5B) 表明该设备将很容易被移除。
对玻璃底碟的粘附 (图 5C)
由于灰尘、不同心度或等离子处理的长度不足, 对玻璃盘的粘附可能会很差。当把设备放到玻璃上时, 把盘子放在一个角度上, 然后把它放在玻璃圈上, 确保它不会碰到井边。如果设备不粘附, 请小心地将设备放到镊子后端的玻璃滑块上, 从精致的隧道区域开始, 向外向边缘按压。如果设备仍然不坚持, 实验通过增加时间在血浆清洁剂以一分钟的增量。
虽然幼虫可以在设备上成像一段短暂的时间 (分钟), 而不使用琼脂, 为了长期活成像尾鳍区域, 琼脂被放置在头部在装载室后, 鱼已经加载。虽然这种琼脂的应用似乎不会影响伤口愈合的反应或生存能力的幼虫5, 这可能会影响研究更多的前区域的幼虫。虽然我们已经能够图像幼虫多达60小时, 我们还没有检查是否生存能力会受到影响, 以更长的成像时间。此外, 这里提出的特殊设计是优化的年龄2至4天后受精 (dpf) 幼虫趴在他们的两侧的尾部横断和伤口愈合成像。其他发展阶段, 方向和实验协议可能需要改变设计。然而, 故意的功能模块化设计, 使它容易服从这种改变。
此外, 该设备的制造需要访问3D 打印机和其他微流体材料和设备, 例如, 硅橡胶和等离子清洗器, 这可能限制了对某些用户的设备的可访问性。然而, 这种模具制造方法的选择, 因为3D 印刷能力, 连同微制作, 是越来越普遍的学术校园。此外, 随着技术的迅速发展和打印机的成本降低, 还有3D 打印服务提供商。
鉴于其分割的设计和功能, zWEDGI 提供了改进的时间推移图像收集使用琼脂嵌入技术。首先, 琼脂糖不包围尾巴区域在 zWEDGI, 允许伤口愈合和再生的进展不被限制, 而生存的幼虫在 zWEDGI 是类似于嵌入式和嵌入控制5。其次, 使用高分辨率3D 打印制造模具, 使 zWEDGI 具有独特的倾斜几何, 使幼虫在三维度中相对于成像平面。这消除了时间依赖性的操作幼虫到适当的方向, 因为琼脂硬化。zWEDGI 设计的第三个重要好处是开放式腔室设计, 允许尾部和头部区域可用于实验操作。这是对伤口愈合研究的具体兴趣, 但这也使 zWEDGI 一个潜在的有用的工具, 其他操纵。此外, 该装置的室设计为在试验过程中化合物的区域应用提供了机会。由于缓冲 (在伤室) 和琼脂糖 (在装载室)5中的扩散微分, 使 semi-isolates 在研究伤口愈合或其他生物过程.另外, 由于幼虫被安装在分开的渠道, 个体幼虫可以被辨认和被检索后为另外的处理例如 RNA 或蛋白质纯化或抗体标记。最后, 由于该装置是由已粘合到玻璃底盘的硅橡胶制成, 一旦幼虫被移除, 该装置就可重复使用。
在前进中, 模块化的设计和易于制造的 zWEDGI 将很好地为修改的功能。目前, 室的几何是专门为受伤和成像实验所描述, 使其广泛应用于实时成像伤口愈合。但是, 幼虫直接位于玻璃上, 因此没有任何材料 (即), 这可能干扰成像, 存在于幼虫和成像表面之间。因此, 其他地区的幼虫, 如树干,ld 可用于成像。此外, zWEDGI 的3D 印制模具的设计, 可以随时修改, 以适应其他发展阶段, 不同的方向的幼虫和各种处理。因此, 这些属性使它成为一个有价值的工具, 以捕捉时间推移显微镜的事件在各种时间尺度。使用其他的玻璃底碟格式可以允许更大的硅橡胶设备和空间的更多渠道。考虑到3D 印刷工艺技术的日益普及, 以及随后对各种实验协议的修改容易, zWEDGI 可能被证明是高分辨率活成像显微镜领域的有力工具。

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 border="0" cellpadding="0" cellspacing="0" width="920">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Fabricate molds</td>
			<td style="width:127px;"></td>
			<td style="width:145px;"></td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Solidworks Professional Accedemic Research 3D modeling software</td>
			<td>Dassault Systemes</td>
			<td>SPX0117-01</td>
			<td>Fisher Unitech</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Viper Si2 SLA 3D printer</td>
			<td>3D Systems Inc.</td>
			<td>23200-902</td>
			<td>3D Systems Inc.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accura 60 photopolymer resin</td>
			<td>3D Systems Inc.</td>
			<td>24075-902</td>
			<td>3D Systems Inc.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">denatured alcohol</td>
			<td>Sunnyside</td>
			<td>5613735</td>
			<td>Menards</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV post cure apparatus</td>
			<td>3D Systems Inc.</td>
			<td>23363-101-00</td>
			<td>3D Systems Inc.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TouchNTuff nitrile gloves</td>
			<td>Ansell</td>
			<td>92-600</td>
			<td>McMaster Carr</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">220B, 400B, 600 grit T414 blue-bak sandpaper&#160;</td>
			<td>Norton</td>
			<td>66261139359, 54, 52</td>
			<td>MSC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">borosilicate glass disc, 2&#34; diameter</td>
			<td>McMaster-Carr</td>
			<td>MIL-G-47033</td>
			<td>McMaster-Carr</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ultrasonicator cleaner</td>
			<td>Branson</td>
			<td>1510R-MTH</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">isopropyl rubbing alcohol 70%</td>
			<td>Hydrox</td>
			<td>54845T43</td>
			<td>McMaster-Carr</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10oz clear plastic cup</td>
			<td>WNA Masterpiece</td>
			<td>557405</td>
			<td>Amazon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6&#34;craft stick</td>
			<td>Perfect Stix</td>
			<td>Craft WTD-500</td>
			<td>Amazon</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fabricate zWEDGI PDMS device</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sylgard 184 silicon elastomeric kit&#160;</td>
			<td>Dow-Corning</td>
			<td>4019862</td>
			<td>Ellworth Adhesives&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10mL syringe</td>
			<td>Becton Dickinson</td>
			<td>305219</td>
			<td>Vitality Medical Inc</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">desiccator</td>
			<td>Bel-Art Scienceware</td>
			<td>F42027-0000</td>
			<td>Amazon</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 in ratcheting bar clamp</td>
			<td>Pittsburgh</td>
			<td>68974</td>
			<td>Harbor Freight</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">lab oven</td>
			<td>Quincy Lab Inc.</td>
			<td>20GC</td>
			<td>Global Industrial</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tweezer set</td>
			<td>Aven</td>
			<td>549825</td>
			<td>McMaster-Carr</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">compressed air filtered nozzle</td>
			<td>Innotech</td>
			<td>TA-N2-2000FT</td>
			<td>Cleanroom Supply</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">vacuum bench vise</td>
			<td>Wilton Tool Group</td>
			<td>63500</td>
			<td>MSC Industrial</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td>Harrick Plasma</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Loading Larvae</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipetteman, P200</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% ethanol (diluted to 70% with water prior to use)</td>
			<td>Pharmco-aaper</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Fisherbrand</td>
			<td>13-711-5A</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">powdered skim milk</td>
			<td></td>
			<td>2902887</td>
			<td>MP Biomedicals</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">double distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-phenylthiorurea</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tricaine (ethyl 3-aminobenzoate)</td>
			<td></td>
			<td>C-FINQ-UE</td>
			<td>Western Chemical</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">low melting point agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A0701</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">heat block (dry bath incubator)</td>
			<td>Fisher Scientific</td>
			<td>11-718-2</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E3 buffer&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">large orifice pipette tip, 200 uL</td>
			<td>Fisherbrand</td>
			<td>02-707-134</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">General purpose pipette tip, 200 uL</td>
			<td>Fisherbrand</td>
			<td>21-197-8E</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">#15 scalpel blade&#160;</td>
			<td>Feather</td>
			<td>2976</td>
			<td>Amazon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25G syringe needle</td>
			<td>BD&#160;</td>
			<td>BD305122</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">inverted microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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, 详细描述如下。
加载
zWEDGI 设计由3个通道组成, 每个信道都设计用于抑制一个幼虫 (图 1B)。对于幼虫的装填和初始方向, 打开的装货室是3毫米宽 (图 1A) 容纳一个大孔吸管末端为存放幼虫 (图 3A)。此外, 长度和宽度提供了足够的空间, 允许后续操作的幼虫进入正确的方向, 与尾部向限制隧道和背部向顶端 (图 3B)。
克制
一旦幼虫被装入海峡, 它可以被画尾巴首先入克制的隧道通过去除液体从受伤的房间或轻轻地微移入地方与吸管尖或相似的工具 (图 3C)。装载室的漏斗形状的几何 (图 1A), 从3毫米到0.45 毫米, 引导幼虫进入期望的侧向方向 (图 3C)。幼虫的方向在它的边近似地克制尾巴与盘子的玻璃底部平行为改善的成像。与使用硅片的微流体器件不同, 3D 印制模具的几何形状在 z 方向上不需要一致。因此, 限制隧道的覆盖和锥形在 z-axis, 使入口高度大于出口, 从0.5 毫米到0.3 毫米的高度 (图 1B, 隧道的等距视图)。这逐渐变细促进幼虫的教导和铺平尾巴往盖子玻璃成像表面。这种功能消除了用户对样品进行定位的需要, 而琼脂糖是硬化。
流体从装载室 (图 3D) 中移除, 并用低熔点琼脂糖代替, 以稳定通道内的头部, 而尾部则在伤室 (图 3E) 的缓冲中保持无限制。最小的琼脂糖, 可能会泄漏通过限制隧道可以很容易地从伤人室 (图 3F) 中删除。缺乏琼脂糖在伤人的房间允许无限制的再生和发展尾鳍鳍5。
伤
一旦幼虫通过隧道和头部被琼脂糖限制, 尾鳍可用于横断, 因为它凸出到受伤的房间。半圆伤室从水平对称偏移1.9 毫米。这允许手术刀刀片插入略高于尾鳍, 允许足够的空间, 使刀片向下绘制横跨尾巴, transecting 尾鳍 (图 3G)。7毫米直径半圆最宽的部分发生在尾巴受伤, 以适应这个议案。除了允许用户对幼虫进行缠绕外, 这种条块分割的设计还为区域应用化合物提供了一个在尾部区域5的机会。semi-isolation 的这一独特特性将允许对各种药物、化学物质或生物制剂对伤口愈合过程的影响进行局部测试。
成像
zWEDGI 装置的目标是允许高分辨率的斑马鱼尾鳍的光学显微成像, 不受琼脂糖嵌入的阻碍。由于这个原因, 该设备是保税的商业可用的玻璃底盘, 提供一个光学质量表面的显微镜使用高 NA 透镜。在这里, 我们目前的图像使用多显微镜的第二次谐波 (倍) 成像的胶原纤维在尾鳍, 以说明成像能力的 zWEDGI。然而, zWEDGI 可以应用于其他光学显微镜方法, 如共聚焦显微镜, 利用倒置显微镜阶段的配置。与 zWEDGI, 不像琼脂糖嵌入与后续去除的方法, 尾鳍可以很容易地成像在设备前伤 (图 4A), 在设备内受伤, 然后立即返回显微镜为 post-wound成像 (图 4B-E)。以前的工作发现胶原纤维组织在伤口愈合过程中使用倍的变化。13倍可用于检测某些类型的有序结构, 包括纤维胶原蛋白, 而不使用外生标签。14在 zWEDGI 中成像的活体尾鳍鳍的图像质量类似于在固定组织中获得的5 , 但 zWEDGI 提供了许多优点。最重要的是, 尾鳍的愈合和再生可以不受阻碍的琼脂糖嵌入与幼体生存类似的幼虫生长无拘束的5。此外, 由于可以在幼虫在装置中时进行伤人, 因此可以在同一尾鳍 (图 4A) 上采集前 post-wounding 图像。zWEDGI 允许收集3维的数据随着时间的推移, 提供了一个更完整的看法, 动态变化发生在细胞外基质结构 (图 4B)。这里说明的是倍纤维缠绕松弛, 在3维, 在 zWEDGI 内伤后。在伤人之前, 倍检测到的胶原纤维从脊索向外辐射到鳍尖, (图 4A)。受伤后不久, 合同鳍的尖端和 th 中心之间的距离e 翅增加与创伤放松。数据收集的3D 性质允许空间重建, 使用渲染软件, 如 Imaris。这些重建, 以及随后的旋转选项, 说明了鳍的收缩和松弛不仅发生在图像集合的 x y 平面 (图 4 B, C, D), 而且在 z 轴 (图4 G-J, l-O, Q t;补充电影 1, 2)当尾巴从收缩的状态的向上卷曲铺平。zWEDGI 可与其他成像模式在较长的时间段, 如使用共聚焦显微镜的图像神经元在尾鳍发展超过24小时5。由于设备的约束单元是平行排列的, 因此无需在定位幼虫时旋转设备, 设置显微镜并在多个标本间移动以进行成像是简单明了的, 可以自动收集多幼虫的延时图像数据。为了扩大可以成像的样品的数量, zWEDGI 设备可以放在其他的玻璃底格式, 如6井板 (图 2C)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56340/56340fig1.jpg"> 
图 1: 最终设计示意图 (A)示意图显示了 zWEDGI 装置的总体布局和测量, 突出了功能性分隔室的术语。(B)从模具中取出的硅橡胶设备的等角视图。(C)插页显示隧道, 突出入口和出口高度的变化。(D)在设备中限制的幼虫模型。图从以前发布的文章5中修改, 并具有来自斑马鱼日志的重印权限。<a href="//ecsource.jove.com/files/ftp_upload/56340/56340fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56340/56340fig2.jpg"> 
图 2: 制造示意图 (A)步骤1以3D 建模软件 (1.1) 中的设备模具设计开始, 然后3D 打印 (1.2)。模具是 UV 光固化, 然后打磨和清洁, 使凸起的几何有均匀的高度 (1.3)。步骤2涉及填充模具与混合和脱的硅橡胶 (2.1) 和应用在模具的玻璃盘在一个斜面, 以防止捕获气泡在设备的硅橡胶 (2.2)。玻璃和模具被夹在一起 (2.3), 以固化在烤箱在 100oC 至少一个小时 (2.4)。步骤3使用过滤空气和平尖镊子从模具 (3.1) 中取出该设备。该装置然后放在成像盘盖子 (3.2) 的顶部, 是等离子处理 (3.3), 连同玻璃底部, 让该公司坚持到玻璃底碟 (3.4)。(B)已完成的 zWEDGI 设备粘合在玻璃底盘中, 可供成像使用。(C)多个 zWEDGI 设备可以放置在一个玻璃底部 6-井板中, 以便在同一实验中对许多幼虫进行图像处理。图从以前发布的文章5中修改, 并具有来自斑马鱼日志的重印权限。<a href="//ecsource.jove.com/files/ftp_upload/56340/56340fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56340/56340fig3.jpg"> 
图 3: zWEDGI (A) 中幼虫的装载和伤人幼虫被装入装载室用宽孔吸管。(B)幼虫朝向以背侧向上在装载房间漏斗和尾巴的平的部分往克制的隧道。(C)使用从在伤室入口处举行的吸管尖端吸入的吸力, 将幼虫引入隧道, 将其置于适当的成像方向。(D)流体从装载腔中移除, 以允许(E)在头部区域周围添加琼脂糖以稳定幼虫。琼脂糖是红色的在这里为示范目的。最小的琼脂糖泄漏到伤室, 可以很容易地删除, 如(F) 所示。( G )一旦幼虫在通道内受到限制 , 手术刀刀片就会入到幼虫上方 , 并在尾鳍的后面切片 , 脊索。(H)幼虫现在可以 post-wound 成像了。刻度条 (A-H) = 1 毫米;刻度条 (F) = 1 mm;缩放条形图 (G) = 1 mm 从以前发布的文章5中修改, 并从斑马鱼日志中获得重印权限。<a href="//ecsource.jove.com/files/ftp_upload/56340/56340fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56340/56340fig4.jpg"> 
图 4: 3D 多倍纤维在斑马鱼尾鳍、前 post-wounding 和 zWEDGI 中的时间推移成像.zWEDGI 设计提供高分辨率成像的能力, 在这种情况下, 倍纤维组织前 (pre-wound) 和跟踪尾鳍横断 (post-wound)。数据以4分钟的间隔收集为 z 堆栈。(A-E)显示尾鳍中纤维的倍作为 z 栈的投影, 在四间隔之前和 post-transection。Z 投影是使用 ImageJ 软件进行的。15 t = 0 是成像的开始, 大约20分钟 post-transection。双箭头表示伤口边缘 (短白线) 与脊索 (长白线) 的位置相对于最初收缩后的伤口松弛时的距离增加。(F-J)。倾斜3D 重建原始数据。(k-O)。在 F J 中显示的倾斜3D 重构的表面渲染。(P-t)。3D 重建表面渲染的侧面图, 说明了3维空间中的收缩和松弛是如何发生的, 这可以通过使用 zWEDGI 的数据来评估, 而在这里, 尾鳍不受琼脂糖的限制。刻度条 (A-E) = 100 微米;刻度线 (F t) =100微米3D 渲染是使用成像软件进行的。另请参阅补充电影1和2。图从以前发布的文章5中修改, 并具有来自斑马鱼日志的重印权限。<a href="//ecsource.jove.com/files/ftp_upload/56340/56340fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56340/56340fig5.jpg"> 
图 5: zWEDGI 制造的关键步骤 (A)在玻璃盘夹紧时, 为确保没有气泡形成或被困在设备中, 在将其应用到模具中时, 在将其装入模具并将夹紧玻璃放在其上时, 加气将被填满。此外, 为了防止在加载和伤人的房间 "闪烁", 确保对设备的顶部彻底砂, 以确保几何的表面是冲洗时, 夹在玻璃。(B)当设备在烤箱中充分固化时, 在该装置和模具之间的空气袋表明该设备很容易拆卸。如果设备不易拆卸, 很可能是由于固化时间或温度不够, 或模具本身没有得到充分的紫外光固化, 导致了硅橡胶残留的树脂污染。(C)当将设备应用于等离子处理后的玻璃底盘时, 用镊子后端的光压力, 从隧道区域开始向外向设备边缘工作。如果设备没有良好的粘结, 如设备与玻璃之间的气穴所示, 在等离子焊机中延长了时间, 确保了在液晶显示器和玻璃表面之间没有灰尘。<a href="//ecsource.jove.com/files/ftp_upload/56340/56340fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Movie 1" src="/files/ftp_upload/56340/56340mov1.jpg"> 
补充影片 1: 在松弛 post-wounding 时尾鳍倍纤维多时间推移图像的倾斜3D 表面渲染.倍的纤维在尾鳍中的影像为 zstacks, 在受伤后的一段时间内 (电影开始大约20分钟后) 在 zWEDGI。Z 栈的重建和表面渲染使用成像软件。尾部倾斜显示三维。前面是左边。影片与图 4 (k-O) 中的静止图像相对应。刻度条 = 50 微米。<a href="//ecsource.jove.com/files/ftp_upload/56340/Suppl_movie_1_SHG_tilt.mov" target="_blank">请单击此处下载影片.</a>
<img alt="Movie 2" src="/files/ftp_upload/56340/56340mov2.jpg"> 
补充电影 2: 3D 的侧面视图在松弛 post-wounding 时尾鳍倍纤维的多时间推移图像渲染.倍的纤维在尾鳍中的影像为 zstacks, 在受伤后的一段时间内 (电影开始大约20分钟后) 在 zWEDGI。Z 栈的重建和表面渲染使用成像软件。显示的侧面视图强调捕获 z-axis 中的动态更改。前面是左边。影片与图 4 (P-t) 中的静止图像相对应。刻度条 = 40 微米。<a href="//ecsource.jove.com/files/ftp_upload/56340/Suppl_movie_2_SHG_side.mov" target="_blank">请单击此处下载影片.</a>

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

Nanyang Technological University

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

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

时间推移荧光成像拟南芥根系生长的根环境的快速操作使用RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

科研

生物工程

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

多器官芯片 - 一种微流体平台的长期多组织共培养

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

在机械拉伸活细胞成像

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

混合μCT-FMT成像和图像分析

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

单轴拉伸应变下活细胞核动力学的高分辨率成像

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...

Targeting the Rat's Small Bowel: Long-Term Infusion into the Superior Mesenteric Artery

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA cannulation is more difficult in rats due to small anatomical dimensions. The aim of the study is to describe a surgical technique for cannulation of the SMA in rats to perform long-term infusion of drugs into the SMA vascular bed in unrestricted animals, which will result in a high rate of catheter patency after the post surgical recovery for 24 hours. 
To avoid the risk of SMA thrombosis or bleeding from direct access, a proximal branch of the SMA is isolated, ligated distally and cannulated with a 0.25 mm polyurethane capillary tube whose tip is advanced close to the origin of the SMA from the aorta. The cannula is then tunnelled subcutaneously to the back of the animal's neck and through the skin via an artificial valve. The external portion of the cannula is inserted in a semi-rigid support system and connected to the continuous infusion pump outside the cage where the rat is free to move. 
Correct positioning of the cannula was demonstrated by post-surgical angiography and autopsy findings. Catheter patency after 24 hours of saline infusion into the SMA region was assured in most rats by the total discharge of the pump and recognition of a functional cannula for blood sampling or saline infusion.

Access for long-term infusion in the superior mesenteric artery (SMA) of rats is a surgical procedure that consists of cannulation of a proximal branch of the SMA. The cannula exits from the abdominal wound and is tunneled through the subcutaneous space back to the interscapular fold.

靶向大鼠的小肠:长期输注到肠系膜上动脉

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...