资助离子和 RGB: 瑞士国家科学基金会 Ambizione-分数补助金 PZ00P3 142404/1 和 PZ00P3 163979。

所有实验都是按照帕特的声明进行的, 用于眼科和视力研究中的动物使用, 并得到地方当局的批准. 
 1. 在硅片上制备超薄切片 <ol> <li> 样品固定 <ol> <li> 准备含有0.1% 戊二醛的定影液, 4% 甲醛在0.1 米甲缓冲中. 
		注意: 小心! 在使用戊二醛、甲醛和甲缓冲液时要小心, 佩戴适当的个人防护设备, 并在通风罩中工作. </li>
		<li> 安乐5天后受精 (5 dpf) 斑马鱼幼虫与 tricaine (乙基 3-氨基甲基磺酸, 0.4% 瓦特/v 在 PBS (磷酸盐缓冲盐水, pH 7)), 如前所述 15 . </li>
		<li> 通过在冰上浸泡 pre-chilled 固定液来修复幼虫。取下头部, 在4和 #176 的夜间孵育; C 带轻柔摇摆. </li>
		通过1% 琼脂糖层板上的冷冻固定液, 用细钳和显微解剖刀 (在双目下) 修剪眼部周围的组织, <li> 解剖眼睛。用新鲜的 pre-chilled 固定剂把眼睛转到管子上. </li>
		<li> 在 PBS 中洗涤两次 (磷酸盐缓冲盐水, pH 值 7.4), 每次在室温下洗涤5分钟 (RT). </li>
	</ol> </li> <li> 明胶渗透和安装 <ol> <li> 将15毫升本地食品品牌明胶12% 瓦特/#160;(p hosphate 缓冲, 0.1 米 pH 7.4) 在40和 #176; c. 除去 PBS, 并在含有眼睛的管子上加入明胶溶液。轻轻拍打试管, 确保明胶渗入样品, 孵育 10-30 分钟, 在40和 #176; C 在 thermoblock 与温和晃动或在水浴. </li>
		<li> 填充 12 mm x 5 mm x 3 mm 硅或聚乙烯平面嵌入模具, 用温明胶在40和 #176; C 水浴。使用吸管在每个模具上加两只眼睛, 用解剖针在双目下正确对准, 让明胶在室温下冷却1分钟, 硬化4和 #176; C 为 20 min. </li>
		<li> 重新修剪在双目下的明胶块, 使用刀片式服务器每块用一只眼睛. </li>
		<li> 在冰上将明胶嵌入的眼睛转移到2.3 米的蔗糖中。在晚上4和 #176 孵育; C. </li>
		<li> 交换到新的2.3 米蔗糖溶液和存储在4和 #176; c 或-20 和 #176; c; 在这一步之后, 样品准备切片, 或者可以存储在-20 和 #176; c 几个星期到几个月. </li>
		<li> 将明胶块重新修整到几乎眼睛的大小, 然后再转到冷冻针。在液氮中冷冻并转移到低温 ultramicrotome. </li>
	</ol> </li> <li> Cryosectioning <ol> <li> 在-120 和 #176 处剪切 110 nm 厚剖面; C 在冷冻 ultramicrotome 中使用金刚石刀. </li>
		<li> 选取含有2% 纤维素 (在水中) 和2.3 米蔗糖溶液 (1:1) 的有线回路的截面。将截面转换为 7 mm x 7 mm 硅片。在4和 #176 中存储部分, 直到进一步处理. </li>
	</ol> </li> </ol> 2。Immunolabelling <ol> <li> 在0和 #176 上用 PBS 清洗硅片; C 为20分钟, 四和 #160;d 保护, 将晶片倒置放在水滴上。在室温下用 PBS 2x 冲洗2分钟. </li>
	<li> 在 PBS 中孵育3倍于0.15% 甘氨酸, 每1分钟。用 PBS 清洗 3x 1 分钟。前孵育与 PBG (PBS 与0.5% 牛血清白蛋白 BSA 和0.2% 明胶类型 B) 为 5 min. </li>
	<li> 在室温下用 PBG (4 和 #181、g/毫升) 的 anti-Tom20 (参见 材料表 ) 进行孵育30分钟. </li>
	<li> 洗 6x 1 分钟/PBG。孵育与 PBG 5 分钟在 RT. 孵育与兔 Alexa 647 F (ab 和 #39;) 2 (参见 材料目录 ) 在 PBG (7.5 和 #181; g/毫升) 为 30 min. </li>
	<li> 洗 6x 1 分钟/PBG。在 PBS 中洗 3x 2 分钟。在 pbs 中孵育 DAPI (4 和 #181; g/毫升), 十年代. 在 pbs 中冲洗 2x 2 分钟. </li>
</ol> 3。超分辨率显微术 <ol> <li> 通过将其放置在甘油 (80%) 的1:1 溶液的液滴和含有氧清除系统的成像缓冲器 (含10% 葡萄糖的200毫米磷酸缓冲液, 0.5 毫克/毫升glucoseoxidase, 40 和 #181; g/毫升过氧化氢酶, 15 毫米β-mercaptoethylamine 盐酸盐 (HCL), pH 值 8.0). </li>
	<li> 将晶圆片 (面朝下) 转移到玻璃底部 (厚度170和 #177; 5 和 #181; m) 培养皿放在甘油 (80%) 和成像缓冲器的1:1 混合物的新滴上 (如步骤 3.1). </li>
	<li> 从四面八方移除, 用吸管, 大部分的液体在晶片下面。使用硅胶条纹将晶片固定在培养皿的底部. 
	注: 硅胶条纹由双组分硅胶 (3 mm x 12 mm) 制成. </li>
	<li> 使用高数值孔径的倒置显微镜上的图像剖面 ( 如 160X 和 #160;/NA 1.43; 查看材料 表) 油浸超分辨率专用目标。在成像之前, 让样品平衡到显微镜温度, 以最小化/减少横向和轴向漂移. </li>
	<li> 将感兴趣的区域居中并获取第一个 widefield 荧光参考图像。更改为超分辨率操作模式。将相机的曝光时间调整为15毫秒, 并将电子倍增 (EM) 增益设置为最大值 300. </li>
	<li> 在荧光模式下用 642 nm 连续波激光照射最大激光功率 (对应于 ~ 2.8 kW/cm 2 ) 的样品。只要单分子闪烁在每个帧中都是很好的分离, 使单个信号重叠的概率较低, 则将激光功率设置为 ~ 0.7 kW/cm 2 。通过获得至少3万帧, 在荧光模式下记录原始图像. 
	注: 所有这些参数可以根据不同的试样和标记密度而变化. </li>
	<li> 从原始数据中, 生成一个重建事件列表 (定位在原始图像中闪烁的每个分子), 使用30光子的检测阈值 (需要根据样本进行调整), 方法是单击并 #34; 评估和 #34; 在和 #39; t 系列分析和 #39; #39; 工具和 #39;. </li>
	<li> 按高斯管接头显示超分辨率图像 16 通过单击和 #34 应用渲染像素大小; 创建图像和 #34; 在和 #39 中; 在和 #39; #39; 工具. #39; 
	注: 为了生成超分辨图像, 本研究采用了系统集成软件工具。但是, 所描述的超分辨率成像可以在任何其他单分子定位系统上执行, 并且可以使用开放源码软件工具处理数据, 如雷雨 17 . </li>
</ol> 4。白金阴影 <ol> <li> 移除硅片条纹并在晶片边缘附近添加一滴 PBS, 将其从培养皿中抬起。在 pbs 中清洗硅片 2x 2 分钟, 然后在 pbs 中用0.1% 戊二醛进行修复, 5 分钟洗涤 2x 2 分钟/洗涤. </li>
	<li> 在冰上的水中, 孵育5分钟/孵化1滴纤维素2% 的晶圆片两次。在离心管中插入硅片, 在 14100 x g 的九十年代离心机. 将其安装在扫描电镜下的铝制存根上, 导电碳水泥. </li>
	<li> 在样品上添加一层2到 10 nm 的铂/碳, 旋转阴影在8和 #176; 使用电子束蒸发装置设置: 1.55 伏, 55 毫安, 0.3 nm/秒, 角度8和 #176;, 旋转级别 4. </li>
</ol> 5。扫描电子显微镜 <ol> <li> 具有扫描电子 m 的图像剖面icroscope 在1.5 伏, 2 毫米工作距离和在透镜二次电子探测器. </li>
</ol> 6。光和电子显微镜的对准图像 <ol> <li> 通过单击和 #34 打开两种类型的图像, 并通过斐济 18 ; 文件 |开放和 #34;。通过单击和 #34 调整画布大小; 图像 |调整 |画布大小和 #34; 通过单击和 #34 将两个图像带到堆栈中; 图像 |栈 |图像堆叠和 #34;。将堆栈另存为 tiff 文件类型. </li>
	<li> 在斐济, 通过单击和 #34 打开一个新的 TrackEM2 19 接口; 文件 | 新建 |TrackEM2 (新) 和 #34;。通过右键单击黑窗口并选择和 #34 导入堆栈和两个图像; 导入栈和 #34;. </li>
	<li> 使用鼠标右键单击图像并选择和 #34, 将光学显微图像与电子显微镜图像手动对齐。手动将图层与地标和 #34 对齐;。
	<ol> <li> 选取 "选择" 工具 (黑色箭头) 以添加地标。使用核的形状作为参考, 以在两个图像中选择相同的边 ( 补充图 1 )。添加几个点 (至少需要选择三点)。使用鼠标右键单击仿射模型, 并选择和 #34; 应用转换 |仿射模型与 #34;. </li>
	</ol> </li> <li> 更改图层透明度 (参见 补充图 1 ) 以评估对齐的质量. </li> </ol>

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作者没有什么可透露的。

该方法结合构蛋白定位与上下文信息, 以确定蛋白质在一个细胞中的精确位置。我们在这里展示了实验的完成, 以可视化 Tom20 在线粒体外膜中的表达, 以及它与其他细胞器的关系, 比如在斑马鱼视网膜幼虫中的细胞核或感光细胞的外段。
得冷冻切片需要一些训练, 以获得保存完好的部分。但是, 这是在许多实验室中使用的方法, 并显示成功21。部分的转移到硅片是非常简单的, 不需要特别考虑。使用甘油在成像缓冲是一个非常关键的步骤, 以避免干燥的部分。对于超分辨率成像, 当晶片与培养皿的玻璃底部非常接近时, 获得最佳结果。硅胶条纹有助于保持晶片在这个位置。在去除条纹时要特别小心, 以避免损坏节。
冰层的厚度, 大约 100 nm, 比 Z 维度中的光学分辨率更薄, 此外, 由于超分辨率显微镜信号仅从这个薄薄的图层和扫描电子显微镜信号显示样品的形貌。这种方法也可以与多色成像相结合。然而, 必须特别小心, 样品可以在相同的条件下成像 (如图像缓冲) 和交叉通话应防止。
该方法的一个局限性是它的二维方法, 因为只有有限数量的串行部分 (大约3到7每个晶圆) 可以收集。因此, 处理体积分析的项目并不理想。然而, 它是一种方法, 可用于检测蛋白表达的任何类型的组织, 通过简单的切片样品。我们提供的方法不使用经典的造影剂, 如醋酸铀或柠檬酸铅。白金阴影的对比提供了非常翔实的地形对比, 但在某些情况下, 膜很难解决。这可能会给项目带来问题, 例如, 需要确定小泡中蛋白质的表达。
我们的协议, 基于得冰冻切片, 使用标准设备, 以获得超分辨率和扫描电子显微镜的相关结果。收集硅片上的切片和使用铂的对比是简单的步骤, 提供稳定性和重现性的样品制备。

方法, 以确定蛋白质的亚细胞定位和它们之间的关系, 以不同的单元, 是了解它们的功能和可能的相互作用的基本工具。超分辨率显微镜结合电子显微镜提供了这样的信息1。基态损耗显微镜后, 个别分子返回 (GSDIM) 是一个超分辨率显微镜技术兼容广泛的有机和遗传编码的荧光2和实现横向分辨率高达 20 nm3. 采用比标准衍射极限显微镜更高分辨率的方法, 提高了相关性4、56的准确性。为了达到与特定亚细胞室的蛋白质表达的最佳相关性, 并减少不确定度的体积7推荐使用相同的超薄切片进行光和电子显微镜。在不同的切片方法中, 得冰冻切片协议不需要脱水或树脂嵌入, 此外, 保留了许多表位抗原性, 并提供了良好的组织超微结构8。几种方法在相关光和电子显微镜 (克莱)4、5、9、10中显示了这些部分的适用性。
斑马鱼视网膜是一个有价值的模型, 研究视觉发展和人类疾病的机制, 鉴于其高度保守的结构和功能, 在脊椎动物。特别是, 视网膜光显示与哺乳动物光相同的结构, 与基底突触, apico 基部拉长核, 线粒体聚类在更顶端内段和外段组成的膜最顶端位置的磁盘11。蛋白质定位到不同的细胞隔间是保存在斑马鱼和人类之间, 允许调查的生物学功能的人类疾病相关的蛋白质12,13。
在这里, 我们提出了一个协议来准备斑马鱼视网膜标本, 以解决线粒体外膜蛋白 Tom20 的相关超分辨光和电子显微镜的定位。该方法是基于在硅片上收集低温切片, 并通过在应用了一层铂后产生的地形信息获得对比。这些步骤在易用性、重现性和完成实验的时间方面都有明显的技术改进。我们最近证明了该方法在小鼠组织中检测核孔和线粒体蛋白的适用性14。

<table><tbody><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>#158127</td><td></td></tr><tr><td>Glutaraldehyde EM Grade</td><td>EMS, USA</td><td>#16220</td><td></td></tr><tr><td>Cacodylate</td><td>Merck</td><td>#8.20670</td><td></td></tr><tr><td>Tricaine</td><td>Sigma-Aldrich</td><td>#886-86-2</td><td></td></tr><tr><td>Agarose, peqGOLD Universal</td><td>VWR International GmbH</td><td>35-1020</td><td></td></tr><tr><td>Flat embedding molds</td><td>BEEM Flat</td><td></td><td></td></tr><tr><td>Local food brand gelatin</td><td>Dr.Oetker</td><td>Extra Gold</td><td></td></tr><tr><td>Sucrose</td><td>Merck</td><td>#1.07687</td><td></td></tr><tr><td>Methylcellulose</td><td>Sigma</td><td>#M-6385</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>#G-7126</td><td></td></tr><tr><td>Gelatin type B</td><td>Sigma</td><td>#G-6650</td><td></td></tr><tr><td>BSA</td><td>Applichem</td><td>#A6588.0050</td><td></td></tr><tr><td>Silicon wafer</td><td>Si-Mat Silicon Materials</td><td></td><td>Type: P/Boron; Orientation &amp;lt;111&gt; ON; Growth method: CZ; Resistivity:1-30 ohm/cm; Surface: polished; Laser cut at 7 x7 mm</td></tr><tr><td>Cryo-pin</td><td>Baltic Preparation</td><td>#16701950</td><td></td></tr><tr><td>Wired loop &quot;Perfect loop&quot;</td><td></td><td></td><td></td></tr><tr><td>Silicone stripes</td><td>Picodent</td><td>Twinsil 22</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>#G8270</td><td></td></tr><tr><td>glucoseoxidase</td><td>Sigma-Aldrich</td><td>#G7141</td><td></td></tr><tr><td>catalase</td><td>Sigma-Aldrich</td><td>#C40</td><td></td></tr><tr><td>beta-Mercaptoethylamine hydrochloride</td><td>Sigma-Aldrich</td><td>#M6500</td><td></td></tr><tr><td>anti-Tom20</td><td>Santa Cruz Biotechnology</td><td>#sc - 11415</td><td></td></tr><tr><td>AlexaFluor 647 AffiniPure F(ab&#039;)2 fragment donkey anti rabbit IgG</td><td>Jackson Immuno-Research</td><td>#711-606-152</td><td></td></tr><tr><td>DAPI</td><td>ROCHE, Switzerland</td><td>#10236276001</td><td></td></tr><tr><td>Glycerol solution</td><td>Sigma-Aldrich</td><td>#49782</td><td></td></tr><tr><td>Glass bottom petri dish</td><td>Ibidi, Germany</td><td>u-Dish 35mm, high glass bottom, #81158</td><td></td></tr><tr><td>SEM aluminium stub</td><td>Agar Scientific</td><td>#G301F</td><td></td></tr><tr><td>Conducting carbon cement Leit-C</td><td>Plano, Germany</td><td>AG3300</td><td></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;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Diamond Knife (Cryo Immuno)</td><td>Diatome</td><td>DCIMM3520</td><td></td></tr><tr><td>Cryo-ultramicrotome</td><td>Leica Microsystems</td><td>Leica EM FCS</td><td></td></tr><tr><td>Widefield-TIRF microscope GSD Leica SR GSD 3D</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>160x 1.43 TIRF objective</td><td>Leica Microsystems</td><td>11523048</td><td></td></tr><tr><td>SEM - Zeiss Supra 50 VP</td><td>Zeiss</td><td>Supra 50 VP</td><td></td></tr><tr><td>FIB-SEM - Zeiss Auriga 40</td><td>Zeiss</td><td>Auriga 40</td><td></td></tr></tbody></table>

correlative super-resolution and electron microscopy to resolve protein localization in zebrafish retina

本文采用超分辨光学显微镜和扫描电镜相结合的方法来研究斑马鱼视网膜的亚细胞蛋白定位。超分辨率光显微镜的 sub-diffraction 极限分辨能力可以提高相关数据的准确性。简要地, 110 纳米厚的低温切片转移到一个硅片, 并在免疫荧光染色后, 图像的超分辨率光显微镜。随后, 在扫描电子显微镜 (SEM) 成像之前, 这些切片被保存在纤维素和铂阴影中。从这两个显微镜模式的图像很容易合并使用开放源码软件的组织地标。在这里, 我们描述了适应方法的幼虫斑马鱼视网膜。然而, 这种方法也适用于其他类型的组织和有机体。我们证明, 这种相关性获得的补充信息能够解决线粒体蛋白的表达与线粒体膜和嵴的关系, 以及细胞的其他舱室。

蛋白 Tom20 的表达, translocase 线粒体外膜复合体的亚基20, 是确定的, 在薄切片的斑马鱼视网膜, 通过超分辨率光显微镜 (图 1), 这一信息是补充与扫描电子显微镜获得的地形信号后, 铂阴影相同的部分。这些相关数据证实了蛋白质与某一特定的隔间、外线粒体膜的结合, 另外还提供了有关蛋白质与细胞的其他器官器的关系的信息。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56113/56113fig1.jpg"> 
图 1: 在斑马鱼视网膜上的克莱克.a.低放大率 widefield 图像 5 dpf 斑马鱼视网膜切片, 细胞核染色 DAPI (青色)。B.同一区域的扫描电子显微镜。C.高放大倍数 widefield 图像的帧在 b. DAPI (青色) 和 Tom20 线粒体染色的细胞核呈红色。D.在更高的放大倍数下 Widefield 相同剖面的图像。Tom20 表达的模式是线粒体的聚集。E. GSDIM 显微镜检测到的 Tom20 (红点) 的表达。核染色的 DAPI (青色)。F.与相关超分辨率和扫描电子显微镜相结合的 E 节。线粒体外膜上的线粒体簇 (M) 出现 Tom20 染色 (红点)。荧光 DAPI 信号在原子核 (N) 对应与 SEM 图象的地势。G. F 帧的高放大图像。扫描电子显微镜图像提供了 GSDIM 图像 (红点) 的上下文。线粒体嵴明显可见, Tom20 染色被定位于线粒体外膜。外段膜的光 (OS) 是明确解决。图像像素大小 5 nm。刻度栏: A、B 和 C:10 µm;D: 2 µm;E 和 F: 1 µm 和 G: 0.2 µm.<a href="http://ecsource.jove.com/files/ftp_upload/56113/56113fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充图 1: 光和电子显微图像的对准。TrackEM2 接口的. 屏幕截图, 带有 SEM 图像和不同核的编号地标 (黄色)。B. TrackEM2 界面上的屏幕截图与荧光图像和编号地标 (黄色) 沿不同 DAPI 染色核。要更改图层透明度, 可以使用菜单左上部分的滑块。缩放栏: 1 µm.<a href="http://ecsource.jove.com/files/ftp_upload/56113/Supplementary_Figure_1.tif">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina at JoVE.com

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

该协议描述了在斑马鱼视网膜上获得亚细胞蛋白定位结果的必要步骤, 方法是将超分辨光显微镜和扫描电子显微镜图像关联起来。

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

correlative-super-resolution-electron-microscopy-to-resolve-protein

Research

JoVE Journal

Developmental Biology

9.5K 次观看.  University of Zurich. 该协议描述了在斑马鱼视网膜上获得亚细胞蛋白定位结果的必要步骤, 方法是将超分辨光显微镜和扫描电子显微镜图像关联起来。

视频: Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

科研

发育生物学

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

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

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

神经科学

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

生物学

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

斑马鱼胚胎眼组织显微切割的

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

从成年斑马鱼中制备新鲜视网膜切片用于前体成像实验

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

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 ...

<meta charset="utf8"></head><body>小鼠视网膜中的突触回路分析和蛋白质定位

Investigating Retinal Circuits and Molecular Localization by Pre&#45;Embedding Immunoelectron Microscopy

The retina comprises numerous cells forming diverse neuronal circuits, which constitute the first stage of the visual pathway. Each circuit is characterized by unique features and distinct neurotransmitters, determining its role and functional significance. Given the intricate cell types within its structure, the complexity of neuronal circuits in the retina poses challenges for exploration. To better investigate retinal circuits and cross-talk, such as the link between cone and rod pathways, and precise molecular localization (neurotransmitters or neuropeptides), such as the presence of substance P-like immunoreactivity in the mouse retina, we employed a pre-embedding immunoelectron microscopy (immuno-EM) method to explore synaptic connections and organization. This approach enables us to pinpoint specific intercellular synaptic connections and precise molecular localization and could play a guiding role in exploring its function. This article describes the protocol, reagents used, and detailed steps, including (1) retina fixation preparation, (2) pre-embedding immunostaining, and (3) post-fixation and embedding.

<meta charset="utf8"></head><body>作者聚焦：了解小鼠视网膜突触连接和神经递质定位的超微结构基础

This protocol outlines the detailed steps of pre-embedding immunoelectron microscopy, with a focus on exploring synaptic circuits and protein localization in the retina.