改善斑马鱼幼虫实验操作的长期活体成像装置
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Fabricate molds | |||
| Solidworks Professional Accedemic Research 3D modeling software | Dassault Systemes | SPX0117-01 | Fisher Unitech |
| Viper Si2 SLA 3D printer | 3D Systems Inc. | 23200-902 | 3D Systems Inc. |
| Accura 60 photopolymer resin | 3D Systems Inc. | 24075-902 | 3D Systems Inc. |
| denatured alcohol | Sunnyside | 5613735 | Menards |
| UV post cure apparatus | 3D Systems Inc. | 23363-101-00 | 3D Systems Inc. |
| TouchNTuff nitrile gloves | Ansell | 92-600 | McMaster Carr |
| 220B, 400B, 600 grit T414 blue-bak sandpaper | Norton | 66261139359, 54, 52 | MSC |
| borosilicate glass disc, 2" diameter | McMaster-Carr | MIL-G-47033 | McMaster-Carr |
| ultrasonicator cleaner | Branson | 1510R-MTH | |
| isopropyl rubbing alcohol 70% | Hydrox | 54845T43 | McMaster-Carr |
| 10oz clear plastic cup | WNA Masterpiece | 557405 | Amazon |
| 6"craft stick | Perfect Stix | Craft WTD-500 | Amazon |
| Name | Company | Catalog Number | Comments |
| Fabricate zWEDGI PDMS device | |||
| Sylgard 184 silicon elastomeric kit | Dow-Corning | 4019862 | Ellworth Adhesives |
| 10mL syringe | Becton Dickinson | 305219 | Vitality Medical Inc |
| desiccator | Bel-Art Scienceware | F42027-0000 | Amazon |
| 4 in ratcheting bar clamp | Pittsburgh | 68974 | Harbor Freight |
| lab oven | Quincy Lab Inc. | 20GC | Global Industrial |
| tweezer set | Aven | 549825 | McMaster-Carr |
| compressed air filtered nozzle | Innotech | TA-N2-2000FT | Cleanroom Supply |
| vacuum bench vise | Wilton Tool Group | 63500 | MSC Industrial |
| 55mm glass bottom dish; 30mm micro-well #1.5 cover glass | Cellvis | D60-30-1.5-N | Cellvis |
| plasma cleaner | Harrick Plasma | PDC-001 | Harrick Plasma |
| Name | Company | Catalog Number | Comments |
| Loading Larvae | |||
| Pipetteman, P200 | Gilson | F123601 | |
| 100% ethanol (diluted to 70% with water prior to use) | Pharmco-aaper | 111000200 | |
| Transfer pipette | Fisherbrand | 13-711-5A | Fisher Scientific |
| powdered skim milk | 2902887 | MP Biomedicals | |
| double distilled water | |||
| N-phenylthiorurea | Sigma-Aldrich | P7629 | Sigma-Aldrich |
| tricaine (ethyl 3-aminobenzoate) | C-FINQ-UE | Western Chemical | |
| low melting point agarose | Sigma-Aldrich | A0701 | Sigma-Aldrich |
| heat block (dry bath incubator) | Fisher Scientific | 11-718-2 | Fisher Scientific |
| E3 buffer | |||
| large orifice pipette tip, 200 uL | Fisherbrand | 02-707-134 | Fisher Scientific |
| General purpose pipette tip, 200 uL | Fisherbrand | 21-197-8E | Fisher Scientific |
| #15 scalpel blade | Feather | 2976 | Amazon |
| 25G syringe needle | BD | BD305122 | Fisher Scientific |
| Name | Company | Catalog Number | Comments |
| Imaging | |||
| inverted microscope | |||
| Imaris imaging software | Bitplane |
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