Name: microstructured devices for optimized microinjection and imaging of zebrafish larvae
Uploaded: 2017-12-08T13:00:00.000+00:00
Duration: 7 min 40 s
Description: Watch this Scientific Journal Video about Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae at JoVE.com

作者要感谢大卫 Langenau 慷慨地提供水族馆空间;埃里克石头, 约翰 c. 摩尔和秦唐帮助与斑马鱼的维护和试剂, 和安妮罗伯逊和艾略特 Hagedorn 从伦纳德区的实验室购买的斑马鱼菌株在这里使用。他们还要感谢奥克塔维奥 Hurtado 关于光刻技术的建议。FE 的经费来自实力的儿童医院和美国澳大利亚协会的研究金。这项工作由 NIH 资助 GM92804。

根据《2011N000127 议定书》, 马萨诸塞州总医院研究动物保育小组委员会批准了微注射幼虫。
1. 设备制造
注: 此处描述的用于设计光刻掩模的所有计算机辅助绘图 (CAD) 文件 (图 1) 均可供下载。查看链接的材料表。
<ol><li>使用标准光刻技术7, 在1000级洁净室中制造主模具晶片。对于微结构阵列和通道, 按照制造商的说明, 在3层中依次使用3个单独的掩码对环氧基负光刻胶进行模式设计: 100 µm 为浅特征, 200 µm 为中等特征, 400 µm的深层特征。对于成像设备, 使用400µm 为浅层特征和600µm 的深部特征模式两个图层。</li>
	<li>将完成的晶片带到 15 cm 培养皿的底座上 (图 2)。</li>
</ol>2. 微表面阵列的制备
<ol><li>制作可用的设备, 铸造烷 (矽), 使用主晶片作为模具。将50克的硅烷单体与5克的引发剂结合在一起, 在塑料称重皿中彻底混合。</li>
	<li>把混合物仔细地浇在模子上。</li>
	<li>德加在真空干燥在 16-25 inHg (54-85 帕斯卡) 为 1 h。</li>
	<li>为了治疗, 在65° c 烘烤至少3小时。该公司应治疗的坚定, 但灵活的固体。</li>
	<li>小心地用手术刀切割主晶片上的装置, 确保手术刀尖端与晶片表面保持接触。使用镊子, 剥离的硅烷副本远离硅片和转移到一个干净的 10 cm 培养皿, 功能侧向上 (图 3)。</li>
	<li>三十五年代用等离子炉对该装置进行氧等离子体处理, 以减少憎水性。</li>
	<li>盖有斑马鱼胚胎培养基 (E3), 含168毫克/升的乙基 3-aminobenoate 甲磺酸 (MS-222, pH 7.5)。使用转移吸管从设备表面流出的水, 从更深的特征中除去任何气泡。</li>
</ol>3. 微表面阵列的制备-琼脂糖
<ol><li>要制作一个负的硅橡胶模具, 首先要处理的是一个具有氧等离子体的硅橡胶装置。</li>
	<li>
使用 1h、1h、2h、2hPerfluorooctyltriethoxysilane (PFDTS) 硅烷蒸气来创建一个惰性表面8。简要:
	<ol>
<li>在通风柜内的真空室中, 将待处理的硅橡胶置于特征侧。</li>
		<li>在µL 旁边, 放置一个小的开放的玻璃或铝盘, 小心翼翼地将200的 PFDTS (98% 硅烷) 移入盘中。 注意: PFDTS 硅烷是高度挥发性的, 与水剧烈反应, 是易燃的, 在接触时会引起严重的皮肤和眼部损伤。使用前请仔细阅读 MSDS。</li>
		<li>关闭真空室, 并应用真空15-30 分钟, 或直到 PFDTS 硅烷完全蒸发。</li>
	</ol>
</li>
	<li>将该设备放置在培养皿中, 并将其用作新的硅橡胶的模具, 如步骤 2.1-2.3 所述。</li>
	<li>在65° c 烘烤至少3小时, 然后从处理过的装置和新鲜的培养皿中剥离整个新鲜的硅氧烷层。这可以用来作为一个负模铸造琼脂糖。</li>
	<li>准备琼脂糖, 煮沸2% 溶液的低凝胶温度琼脂糖在 E3 在微波炉, 直到琼脂糖完全溶解。</li>
	<li>倒入热的琼脂糖的消极模具和删除任何气泡的琼脂糖覆盖设备的流动热琼脂的功能与转移吸管。</li>
	<li>一旦气泡被去除, 冷藏在4° c 设置琼脂糖。</li>
	<li>通过手工将 E3 从下方变形, 将琼脂糖块转移到新的培养皿中, 然后用含有 MS-222 的含水的湿剂把琼脂糖块从底片上取下。</li>
</ol>4. 制备硅橡胶成像器件
<ol><li>制作可使用的设备, 铸造硅晶片, 用主晶圆作为模具。将50克的硅烷单体与5克的引发剂 (与步骤2.1 相同) 结合在一起, 用塑料叉将塑料称量盘彻底混合。</li>
	<li>把混合物仔细地浇在模子上。</li>
	<li>德加在真空干燥在 16-25 inHg (54-85 帕斯卡) 为 1 h。</li>
	<li>为了治疗, 在65° c 烘烤至少3小时。该公司应治疗的坚定, 但灵活的固体。</li>
	<li>从主晶片切割设备, 用 1.5 mm 冲床冲出端口。</li>
	<li>如步骤2.6 所述, 三十五年代用氧等离子体处理设备和6井玻璃底井板。</li>
	<li>债券一设备的特点-侧向下到每个玻璃片的 6-井板通过放置在一个85° c 板10分钟。 注: 为了确认粘合成功, 使用镊子将牢固的压力施加到粘合装置的一侧。设备应保持在玻璃片。</li>
</ol>5. 斑马鱼文化
<ol><li>养殖斑马鱼胚胎到 2 dpf 使用标准技术3。</li>
	<li>Dechorionate 胚胎使用钳或1毫克/毫升蛋白酶混合 (见材料表)3至少30-60 分钟之前加载到设备, 让他们伸直。</li>
	<li>麻醉胚胎使用 (168 毫克/升) MS-222 (pH 7.5), 加入1毫升的25X 库存解决方案的 E3 在其培养皿。</li>
</ol>6. 斑马鱼对微表面-草皮阵列的定位
<ol><li>在其培养皿中准备2.7 步 (图 3A) 中的硅橡胶块, 使其覆盖一层薄薄的 (1-2 毫米) 的 E3, 从而更容易操作胚胎。</li>
	<li>使用转移吸管将所需的胚胎数量 (一般为10-20 每个条件) 转移到硅橡胶块的表面。</li>
	<li>使用微工具 (发圈或类似的), 将胚胎推入微草皮 (图 3A)。草皮的使用特别适合背部和腹侧方向。 注: 对于草皮与更紧的适合 (背部和腹), 尝试定位的胚胎在块的表面平行的草皮, 然后滚动到位置使用头发循环。把他们推向更深的草皮将有助于保持他们的稳定。</li>
</ol>7. 斑马鱼在微表面-微通道上的定位
<ol><li>使用转移吸管绘制 E3 的微通道 (图 3B), 直到 E3 的水平低于该块的边缘, 但仍存在于储层和通道中, 并位于该层的一个薄薄的图层上。</li>
	<li>使用转移吸管将10胚胎从步骤5.3 转移到水库, 注意不要溢出边缘。</li>
	<li>使用头发循环, 操作每个胚胎进入漏斗在适当的渠道入口和东方, 以便胚胎的头朝向渠道, 并且胚胎有适当的取向, 当它进入渠道。</li>
	<li>使用毛发循环将每个胚胎向下滑动, 直到它到达通道壁上的定向 microfeatures, 帮助保持它的位置。使用微通道是特别推荐的横向方向。 注: 为了减少微注射过程中的胚胎滑动, 请通过从储层中提取 E3 来调整表面张力的大小。</li>
</ol>8. 斑马鱼的显微注射
<ol><li>
准备注射针头。
	<ol>
<li>使用微拉拔器制作硼硅酸盐玻璃 microcapillary 针, 如前所述4。所产生的针应该是锥形到一个点在一端, 与锥形长度约10毫米。</li>
		<li>准备注射的溶液, 在这种情况下, 100 nm 趋化剂 (n-Formylmethionine-亮-苯丙氨酸 (fMLP) 或烯 B4 (LTB4)) 和 100 nm 70 kDa 罗丹明葡聚糖在 PBS 中的示踪物。</li>
		<li>使用一个细锥形吸管尖端 (见材料表) 将5µL 的溶液装入针中。</li>
		<li>将针安装在磁性支架上的微上, 并连接到 picopump 微量。</li>
	</ol>
</li>
	<li>用镊子捏锥形尖端, 在45°角处折断针尖。</li>
	<li>通过调整 picopump 控制器上的注入时间, 调整注塑丸大小为 1 nL。</li>
	<li>调整注射针头的角度。在微控制器上, 45 °的针角通常是一个好的起点, 针与胚胎的长度平行。一个较陡的角度可以用来最小化胚胎在显微注射过程中的横向运动。</li>
	<li>用左手和针微控制培养皿, 使针尽可能靠近注射的目的部位, 在这种情况下, 耳囊泡。 注: 耳囊可被确定为眼睛后部的长方形器官, 有两个较小的不同的暗长方形结构 (耳)。</li>
	<li>使用微控制器, 穿透靶组织 (在这种情况下, 耳泡), 使针尖的针头是在囊泡, 并注入预设的音量使用 picopump 脚开关。 注意: 如果组织抵抗渗透, 试着轻轻地敲击微控制器旋钮。</li>
	<li>为了释放胚胎, 通过转移吸管从上到下的表面流动 E3, 或者旋转盘状, 使胚胎被释放到周围的 E3。</li>
	<li>使用转移吸管将胚胎移植到 E3 无 MS-222 的恢复盘中。</li>
</ol>9. 使用硅橡胶成像装置对胚胎进行筛选
<ol><li>从步骤4.7 中的 E3 (+ MS-222) 通过端口来进行设备的质数。这可以用1000µL 吸管或窄尖端转移吸管来完成。</li>
	<li>用 E3 + MS-222 填充, 直到设备盖好。</li>
	<li>使用转移吸管从步骤8.7 向每个井输送4注入的胚胎。胚胎可以预先麻醉, 如果需要孵化在 E3 + MS-222 在1-2 分钟之前加载 (步骤 5.3)。</li>
	<li>使用微工具 (发圈或类似的), 将胚胎置于成像通道入口的方向 (根据所使用的设备), 将其定位到所需的位置 (尾部首先或头部), 并将它们部分推入设备 (图 4A)。这将有助于他们均匀地进入设备。</li>
	<li>使用1000µL 吸管或转移吸管从端口绘制 E3, 直到将胚胎以正确的成像方向绘制到通道中 (图 4B)。 注意: 小心不要把胚胎从诱捕点吸出来, 这样会破坏胚胎并改变它们的方向。</li>
	<li>将井板转移到显微镜进行成像 (图 4C)。</li>
	<li>
图像使用倒置荧光显微镜。
	<ol>
<li>通过选择适当的镜头来调整放大倍数。10X 通常是一个有用的放大率成像斑马鱼中性粒细胞迁移。</li>
		<li>调整每个通道的光源强度和曝光时间, 使每个信号清晰, 但不饱和。</li>
		<li>捕获每个通道的图像。在这里, 我们使用绿色荧光蛋白 (GFP, 前: 470/22, Em: 525/50), 得克萨斯州红 (TxRed, 前: 585/29, Em: 628/32) 和传输通道。在后处理过程中可以组合多通道图像 (图 4B-I)。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat Rev Genet. 8 (5), 353-367 (2007).</li><li>Dang, M., Fogley, R., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+Novel+Cancer+Therapies+Using+Chemical+Genetics+and+Zebrafish.">Identifying Novel Cancer Therapies Using Chemical Genetics and Zebrafish.</a> Adv Exp Med Biol. 916, 103-124 (2016).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book: a guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Benard, E. L., 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=Infection+of+zebrafish+embryos+with+intracellular+bacterial+pathogens.">Infection of zebrafish embryos with intracellular bacterial pathogens.</a> J Vis Exp. (61), (2012).</li><li>Ellett, F., Irimia, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructured+Surface+Arrays+for+Injection+of+Zebrafish+Larvae.">Microstructured Surface Arrays for Injection of Zebrafish Larvae.</a> Zebrafish. 14 (2), 140-145 (2017).</li><li>Bischel, L. L., Mader, B. R., Green, J. M., Huttenlocher, A., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Entrapment+By+Restriction+Array+(ZEBRA)+device:+a+low-cost,+agarose-free+zebrafish+mounting+technique+for+automated+imaging.">Zebrafish Entrapment By Restriction Array (ZEBRA) device: a low-cost, agarose-free zebrafish mounting technique for automated imaging.</a> Lab Chip. 13 (9), 1732-1736 (2013).</li><li>Brower, K., White, A. K., Fordyce, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-step+Variable+Height+Photolithography+for+Valved+Multilayer+Microfluidic+Devices.">Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices.</a> J Vis Exp. (119), (2017).</li><li>Shao, G., Wu, J., Cai, Z., Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+elastomeric+high-aspect-ratio+microstructures+using+polydimethylsiloxane+(PDMS)+double+casting+technique.">Fabrication of elastomeric high-aspect-ratio microstructures using polydimethylsiloxane (PDMS) double casting technique.</a> Sens Actuators A Phys. 178, 230-236 (2012).</li><li>Bhuiyan, M. S., 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=Acinetobacter+baumannii+phenylacetic+acid+metabolism+influences+infection+outcome+through+a+direct+effect+on+neutrophil+chemotaxis.">Acinetobacter baumannii phenylacetic acid metabolism influences infection outcome through a direct effect on neutrophil chemotaxis.</a> Proc Natl Acad Sci U S A. 113 (34), 9599-9604 (2016).</li><li>Henry, K. 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=PhagoSight:+an+open-source+MATLAB&#174;+package+for+the+analysis+of+fluorescent+neutrophil+and+macrophage+migration+in+a+zebrafish+model.">PhagoSight: an open-source MATLAB&#174; package for the analysis of fluorescent neutrophil and macrophage migration in a zebrafish model.</a> PloS one. 8 (8), e72636 (2013).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dynam. 203 (3), 253-310 (1995).</li><li>Hemmil&#228;, S., Cauich-Rodr&#237;guez, J. V., Kreutzer, J., Kallio, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+simple,+and+cost-effective+treatments+to+achieve+long-term+hydrophilic+PDMS+surfaces.">Rapid, simple, and cost-effective treatments to achieve long-term hydrophilic PDMS surfaces.</a> Applied Surface Science. 258 (24), 9864-9875 (2012).</li><li>Masselink, W., Wong, J. C., Liu, B., Fu, J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+silicone+imaging+casts+for+zebrafish+embryos+and+larvae.">Low-cost silicone imaging casts for zebrafish embryos and larvae.</a> Zebrafish. 11 (1), 26-31 (2014).</li><li>Yang, F., Gao, C., Wang, P., Zhang, G. J., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish-on-a-chip:+microfluidics+for+zebrafish+research.">Fish-on-a-chip: microfluidics for zebrafish research.</a> Lab Chip. 16 (7), 1106-1125 (2016).</li><li>Wielhouwer, E. 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=Zebrafish+embryo+development+in+a+microfluidic+flow-through+system.">Zebrafish embryo development in a microfluidic flow-through system.</a> Lab Chip. 11 (10), 1815-1824 (2011).</li><li>Shen, Y. 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=A+student+team+in+a+University+of+Michigan+biomedical+engineering+design+course+constructs+a+microfluidic+bioreactor+for+studies+of+zebrafish+development.">A student team in a University of Michigan biomedical engineering design course constructs a microfluidic bioreactor for studies of zebrafish development.</a> Zebrafish. 6 (2), 201-213 (2009).</li><li>Li, Y., 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=Zebrafish+on+a+chip:+a+novel+platform+for+real-time+monitoring+of+drug-induced+developmental+toxicity.">Zebrafish on a chip: a novel platform for real-time monitoring of drug-induced developmental toxicity.</a> PLoS One. 9 (4), e94792 (2014).</li><li>Akagi, 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=Miniaturized+embryo+array+for+automated+trapping,+immobilization+and+microperfusion+of+zebrafish+embryos.">Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos.</a> PLoS One. 7 (5), e36630 (2012).</li><li>Akagi, 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=Fish+on+chips:+Microfluidic+living+embryo+array+for+accelerated+in+vivo+angiogenesis+assays.">Fish on chips: Microfluidic living embryo array for accelerated in vivo angiogenesis assays.</a> Sens Actuators B Chem. 189, 11-20 (2013).</li><li>Lin, X., 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=High-throughput+mapping+of+brain-wide+activity+in+awake+and+drug-responsive+vertebrates.">High-throughput mapping of brain-wide activity in awake and drug-responsive vertebrates.</a> Lab Chip. 15 (3), 680-689 (2015).</li><li>Noori, A., Selvaganapathy, P. R., Wilson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+in+a+microfluidic+format+using+flexible+and+compliant+channels+and+electroosmotic+dosage+control.">Microinjection in a microfluidic format using flexible and compliant channels and electroosmotic dosage control.</a> Lab Chip. 9 (22), 3202-3211 (2009).</li></ol>

作者声明没有利益冲突。

在这里, 我们描述了使用的设备, 我们最近开发, 以方便 2 dpf 斑马鱼微注射5, 并介绍了一个简单的琼脂糖免费安装装置, 方便成像的胚胎。这些工具突出了光刻技术的效用, 用于制作适用于斑马鱼技术的设备。
我们发现 MSA 装置特别适用于注射针内的细胞或微粒, 如真菌孢子或人类癌细胞, 这需要使用更大的钻孔针来运送。注射到耳泡 (图 4) 提出了一个特殊的挑战, 因为胚胎经常在与针头接触时滚动。我们发现, 使用微通道, 定向和稳定斑马鱼的横向方向大大提高了这一技术的吞吐量和准确性, 在我们手中。
快速成像的横向定向斑马鱼在不同的时间点, 如评估中性粒细胞的招募或异种转移, 避免琼脂糖增加了胚胎之间的一致性和减少的机会损害后成像救援。这些设备也显示了延长的时间推移成像的承诺, 并已被用来成功捕获多通道, 多图像与最小的鱼移动超过12小时。因为它们不受琼脂糖的约束, 所以在这段期间幼虫的活性伸长 (300 µm 从 54-78 hpf)11是无限制的, 可以正常进行。
在这些工具的准备和使用中, 最关键的步骤是使用大多数基于芯片的微流控设备: 设备必须彻底润湿和避免气泡。为避免此类问题, 在制造 E3 后立即将设备浸湿, 而氧等离子体处理所赋予的表面亲水性仍然存在。用氧等离子体重新处理, 也可用于瞬时恢复对老化的硅橡胶器件的亲水性。
这里使用的精确几何图形利用了光刻技术的高分辨率, 但是目前的设计在第二天的受精过程中限制了斑马鱼的使用。基于当前平台的几何结构重新设计将使 1 dpf 和 3 dpf 的成功定位成为微注射。更具浮力的 4 dpf 幼虫与充气的鱼鳔的定位可能更具挑战性, 并要求与更高的水张力相结合的舒适的几何图形。集成流或真空的复杂设备可能提供适用于更广泛发展阶段的替代方法, 但也需要使用泵和油管, 增加成本和设备故障的风险。
这是一种成本效益高、有用的材料, 用于制造类似于此处所描述的设备, 提供生物相容性、透明性和可重用。对斑马鱼使用 E3 的一个缺点是憎水性, 这可以使设备表面的浅层维护具有挑战性。这一问题可以通过在琼脂糖中铸造注射装置来避免, 虽然这限制了可重用性, 并增加了设备的脆性。优选的替代方法是识别生物相容性的表面处理, 从而提高了硅氧烷12的亲水性, 或使用适当的塑料基板。
目前的方法, 在 2 dpf 注射斑马鱼使用要么水紧张4或简单的渠道铸造琼脂糖使用商业模具固定和定位的幼虫。这些方法提供了有限的方向控制, 可以复杂的快速干燥的胚胎。这些微结构集成到草皮和通道中, 用于定位和固定幼虫, 而不完全依赖于水的张力, 从而降低了干燥的风险。使用3D 印制模具的草皮以前曾被用于成像目的的胚胎定位13, 尽管这些草皮的深度使它们无法进行显微注射。
使用微流控系统的斑马鱼成像是越来越常见的14。 多流系统已经开发, 允许观察的斑马鱼的发展随着时间的能力引进化合物的需求15,16,17。更复杂的设备已被设计成阵列的胚胎, 而仍然在他们的绒毛膜, 这提供了一个简单的球状几何, 可以很容易地操作在这些系统的吞吐量相对较高的18,19。几个小组开发了平台为排和成像幼虫阶段斑马鱼在各种各样的方向6,20, 最方便用户的是被动微泵驱动 "斑马" 系统开发的毕比实验室6.与斑马系统不同的是, 我们在这里描述的设备使用吸入, 用标准的转移吸管产生, 将幼虫引到通道中, 而平行而不是序贯的幼虫, 可以拯救单个幼虫后成像通过同一端口应用正流。此外, 我们的方法需要更小的足迹, 因此, 可以结合到传统的玻璃底部 6-井板的图像。
目前的实验设计主要利用单独的显微注射和成像装置, 使它们可以与常规的微注射设备和标准倒置显微镜一起使用。微流控方法自动注射斑马鱼卵已经存在21, 并与 "鱼陷阱" 类型的方法相结合, 用于高度精确的排和幼虫的方位20, 可以预见, 设备集成自动幼虫注射和成像也可能有一天成为现实。

斑马鱼已经成为一个强大的模型, 许多领域, 从基础发展生物学研究到大规模的遗传和化学屏幕1,2。常规的基因操作, 如基因过度表达、击倒、CRISPR/Cas9 突变和转基因依赖于单细胞受精卵的微注射, 这导致了简单易用的商业用于定向和稳定注射的卵子的可用工具3。其他方法, 如移植和感染, 往往需要注射到后期胚胎和幼虫使用较大的规范毛细管针头4。然而, 使用更大的规格针提出了重大的技术挑战, 因为它是更难穿透目标组织不推或滚动的胚胎。在这些条件下, 获得稳定胚胎所需的适当的水张力, 同时避免在手术过程中干燥是困难的, 而且胚胎可能不是理想的定向注射到靶组织。
在注射后, 筛出的胚胎选择那些已成功注射的胚, 并捕捉初始时间点的图像, 通常是有用的。为了应对这些挑战, 我们开发了一系列的微设备, 帮助稳定 2 dpf 胚胎的各种方向, 既用于微注射5, 也用于快速基于图像的扫描后注射。
为了在这些装置中获得足够的结构分辨率, 我们利用了光刻技术。这些方法通常用于微电子工业, 并且最近被外推用于微流控芯片的制造, 因此可以实现垂直结构, 范围从1-1、000µm, 这是一种适合于斑马鱼胚胎和幼虫操作的鳞片。所有的设备都是用烷 (硅氧烷) 制造的, 它价格低廉, 身体健壮, 生物惰性, 而且透明。
微表面阵列 (协议) 被格式化成块的, 具有图案的顶面, 类似于简单的渠道琼脂糖块通常用于卵注射。对于后注射筛选, 6 成像设备可以排列在一个标准的玻璃底 6-井板。这些装置是为易于装载的胚胎而设计的, 而卸载程序方便地允许对特定的胚胎进行抢救, 比以前由毕比实验室6。

<table><tbody><tr><td>Dow Corning Sylgard 184 Polydimethylsiloxane (PDMS)</td><td>Ellsworth Adhsives</td><td>184 SIL ELAST KIT 0.5KG</td><td>For casting the devices. Kit includes PDMS monomer and Initiator</td></tr><tr><td>Low gelling temperature agarose</td><td>Sigma Aldrich</td><td>A9414-10G</td><td>For casting agarose devices</td></tr><tr><td>PFDTS silane</td><td>Sigma Aldrich</td><td>448931-10G</td><td>For casting of negative PDMS molds</td></tr><tr><td>Tricaine (MS-222)</td><td>Sigma Aldrich</td><td>E10521-10G</td><td>To anesthetize zebrafish</td></tr><tr><td>Rhodamine Dextran 70,000 Da</td><td>ThermoFisher</td><td>D1818</td><td>To trace microinjections</td></tr><tr><td>Leukotriene B4 (LTB4)</td><td>Cayman Chemicals</td><td>20110</td><td>Neutrophil chemoattractant</td></tr><tr><td>N-Formylmethionine-leucyl-phenylalanine (fMLP)</td><td>Sigma Aldrich</td><td>F3506-50MG</td><td>Neutrophil chemoattractant</td></tr><tr><td>15 cm Petri dish</td><td>Fisher scientific</td><td>08-757-148</td><td>For Casting from the master wafer</td></tr><tr><td>Glass-bottom 6-well plates</td><td>MatTek</td><td>P06G-0-20-F</td><td>For imaging devices</td></tr><tr><td>Borosilicate glass microcapillaries</td><td>World Scientific Instruments</td><td>TW-100-4</td><td>For microinjection needles</td></tr><tr><td>Transfer pipettes</td><td>Sigma Aldrich</td><td>Z350796</td><td>For transferring zebrafish embryos</td></tr><tr><td>Microloader tips</td><td>Fisher scientific</td><td>E5242956003</td><td>For loading the microinjection needles</td></tr><tr><td>Harris Uni-Core 1.5 mm punch</td><td>Ted Pella Inc.</td><td>15111-15</td><td>To punch ports in PDMS imaging devices</td></tr><tr><td>No. 11 Scalpel</td><td>Fine Science Tools</td><td>10011-00</td><td>For cutting PDMS</td></tr><tr><td>Dumont No. 5 Forceps</td><td>Fine Science Tools</td><td>11252-10</td><td>For dechorionating embryos and breaking microinjection needle tips</td></tr><tr><td>Marzhauser Micromanipulator</td><td>ASI</td><td>MM33-R</td><td>For manipulating microinjection needle</td></tr><tr><td>Magnetic stand</td><td>MSC</td><td>SPI - 87242624</td><td>For mounting micromanipulator</td></tr><tr><td>MPPI-3 Picopump controller</td><td>ASI</td><td>MPPI-3</td><td>To control microinjection volume and timing</td></tr><tr><td>EVOS inverted fluorescent microscope</td><td>ThermoFisher</td><td>EVOS FL</td><td>To image injected embryos</td></tr><tr><td>Dissecting microscope</td><td>Nikon</td><td>SMZ745</td><td>For visualizing microinjecion</td></tr><tr><td>AutoCAD software</td><td>Autodesk</td><td></td><td>Download AutoCAD files from: https://dx-doi-org.remotexs.ntu.edu.sg/10.6084/m9.figshare.4282853 and on the ZFIN community protocols wiki page: https://wiki.zfin.org/display/prot/ZFIN+ Protocol+Wiki &amp;nbsp;</td></tr></tbody></table>

microstructured devices for optimized microinjection and imaging of zebrafish larvae

斑马鱼已经成为各种人类疾病的强有力的模型, 并成为越来越多的实验研究的有用工具, 通过大规模的遗传和化学屏障跨越了基础发育生物学。然而, 许多实验, 特别是那些与感染和异种模型有关的试验, 依赖于胚胎和幼虫的显微注射和成像, 这些都是需要技能和专长的费力技术。为了提高目前显微注射技术的精确度和吞吐量, 我们开发了一系列微装置, 在2天后, 在腹侧、背部或侧面定向后, 对斑马鱼胚胎进行定位和稳定。程序.为了帮助对胚胎进行成像, 我们还设计了一个简单的装置, 使4只斑马鱼在平行的方向上与一个玻璃覆盖层滑动。在这里, 我们展示了光刻方法的有效性, 为斑马鱼技术的优化创造了有用的设备。

此处介绍的方法演示了设计 (图 1) 和用于 2 dpf 斑马鱼的设备的制造, 使用光刻 (图 2) 和软光刻 (图 3) 技术。此方法允许对许多设计迭代和修改进行快速测试, 并且在其他开发阶段与斑马鱼一起使用的微观结构尺寸的改变和优化可能会延长它们的应用范围。
我们示范使用这些设备的挑战显微注射技术和方便成像。耳囊泡 (图 4D, 白色虚线轮廓) 提供了一个有用的, 免疫特权和孤立的网站, 以测试中性粒细胞招募在斑马鱼9。为了测试斑马鱼中性粒细胞对标准化的反应能力, 100 nM 的 fMLP 和 LTB4 被微量到 2 dpf 斑马鱼胚胎的耳囊中 (图 4F, J), 使用微通道设备 (图 1G) 在侧向方向稳定胚胎。注射用高分子量罗丹明葡聚糖追踪, 并在Tg (mpx: EGFP)胚胎中进行, 以促进中性粒细胞招募的可视化。Uninjected 胚胎 (图 4D, H) 和单独注射罗丹明 (图 4E, I) 的胚胎被用作阴性对照。在注射后, 胚胎被转移到成像通道设备 (图 1A, B), 并使用 EVOS 荧光显微镜 (图 4C) 在30分钟和 5 h 后注入 (图 4D-G和图 4H-K分别)。如预期, 在早期 timepoint (图 4I) 中, 少量的荧光中性粒细胞被吸收到模拟注射的耳囊中, 可能是由损伤介导的招募所致。有趣的是, 在实验中观察到了罗丹明葡聚糖示踪剂在耳中的积累。对于两个化 (图 4F, J), 嗜中性粒细胞都是以更高的数量来招募的, 特别是响应 LTB4 (图 4F)。
本研究的代表性结果表明该方法在斑马鱼显微注射和成像中的应用是成功的。在斑马鱼中性粒细胞招募分析的背景下, 这些工具的应用结合了详细的实时成像技术和复杂的细胞迁移研究10可以为今后在该领域的实验提供一个有用的平台。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56498/56498fig1.jpg"> 图 1: 计算机辅助绘图 (CAD) 设计光刻面具.(A-c)用于微表面草皮的 CAD 设计, 用于在侧面 (A)、腹侧 (B) 和背 (C) 方向定位。不同的颜色线代表 CAD 面具设计为不同的特征, 与绿色100µm, 蓝色200µm 和红色400µm 特征高度。缩放条形图 = 500 µm (d-f) 微通道的 CAD 设计, 用于定位在侧面 (D)、腹 (E) 和背 (F) 方向。不同颜色的线条代表不同功能的掩码设计, 绿色100µm、蓝色200µm 和红色400µm 特征高度。标尺: 500 µm. (g I) 设计用于加载头 (g) 或尾部第一 (H 和 I) 的成像设备的 CAD。蓝线代表400µm 特征, 红线代表600µm 特征。箭头显示了幼虫的加载方向。缩放条 = 200 µm。此图已从埃利特et al.中修改5<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56498/56498fig1large.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/56498/56498fig2.jpg"> 图 2: 硅胶主晶片.(A) 主晶片的顶部显示了将单个幼虫定向到草皮的特性。(B) 主晶圆的底部部分, 显示微通道的设计。此图已从埃利特 et al.中修改5<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56498/56498fig2large.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/56498/56498fig3.jpg"> 图 3: 微的硅橡胶设备.(A) 从主晶片上投出的晶圆块, 显示草皮用于定向单个幼虫。(B) 从主晶片上投出的晶圆块, 显示微通道。此图已从埃利特et al.中修改5<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56498/56498fig3large.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/56498/56498fig4.jpg"> 图 4: 代表性结果: 对斑马鱼中性粒细胞进行显微注射后, 化注入耳泡.(A-b)用于成像的加载和安装胚胎。(A) 头第一个设备与胚胎准备好装货, 与口岸指示 (黑箭头)。(B) 已加载的胚胎在它们被绘制到位后 (黄色箭头)。(C) 6-井玻璃底板与成像设备显微镜。这种格式允许成像4侧向定向胚胎每井 (24 胚胎总数), 使用标准倒置显微镜。(D-G)嗜中性粒细胞 (EGFP-绿色) 在 uninjected (D) 胚和耳囊内注射罗丹明葡聚糖 (红, 打开白色箭头) 单独 (E), 100 nm LTB4 (F), 和 100 nm fMLP (G) 30 分钟后注入 (mpi)。(H-K)中性粒细胞 (EGFP-绿色) 在 uninjected (H) 胚和 5 H 以下耳囊泡注射罗丹明葡聚糖 (红, 打开白色箭头) 单独 (I)、100 nm LTB4 (J) 和 100 nm fMLP (K) 5 H post 注入 (hpi)。缩放栏 = 200 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56498/56498fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae at JoVE.com

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

斑马鱼胚胎和幼虫的显微注射是许多斑马鱼模型中的关键但具有挑战性的技术。在这里, 我们提出了一系列的微型工具, 以帮助稳定和定位斑马鱼的显微注射和成像。

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 ...

microstructured-devices-for-optimized-microinjection-imaging

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.5K 次观看.  Massachusetts General Hospital–Harvard Medical School–Shriners Burns Hospital. 斑马鱼胚胎和幼虫的显微注射是许多斑马鱼模型中的关键但具有挑战性的技术。在这里, 我们提出了一系列的微型工具, 以帮助稳定和定位斑马鱼的显微注射和成像。

视频: Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

科研

生物工程

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 ...

发育生物学

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

生物学

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

神经科学

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

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

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 ...

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

斑马鱼光学杯形态形成的四维成像

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...

Low-cost Polyethylene Terephthalate Lamination Microfluidics Designs for Multiplexed Zebrafish Imaging

Zebrafish embryos are transparent and thus uniquely suited for noninvasive intravital imaging of fundamental processes, such as wound healing and immune cell migration. Microfluidic devices are used for entrapment to support long-term imaging of multicellular organisms, including zebrafish. However, the fabrication of these devices using soft lithography requires specialized facilities and competency in 3D printing, which may not be accessible to every lab. Our adaptation of a previously developed low-cost polyethylene terephthalate lamination method for constructing microfluidic devices increases accessibility by enabling design fabrication and iteration for a fraction of the technical investment of conventional techniques. We use a device made with this method, the Rotational Assistant for Danio Imaging of Subsequent Healing (RADISH), to accommodate drug treatment, manual wounding, and long-term imaging of up to four embryos in the same field of view. With this new design, we successfully capture gross morphological characteristics of the calcium&#160;signal around laser ablation and manual transection wounds for multiple embryos in the 2 h immediately following injury, as well as neutrophil recruitment to the wound edge for 24 h.

An innovative method for fabricating microfluidic devices using polyethylene terephthalate (PET) lamination significantly reduces the cost and complexity of entrapping and imaging multiple live zebrafish embryos.

Zebrafish embryos are transparent and thus uniquely suited for noninvasive intravital imaging of fundamental processes, such as wound healing and immune ...