工作大部分已经在UMCG显微镜和影像中心（UMIC），这是由NWO 175-010-2009-023和ZonMW 91111006赞助被执行; STW"显微镜谷12718"来BNGG。这项工作是由一个ZonMW VENI授权，居里夫人的职业生涯集成补助（节省垂死的神经元）和老年痴呆症荷兰奖学金，以资助TJvH

与斑马鱼所有的实验都是由格罗宁根大学实验动物委员会根据国家和欧盟的指导方针批准。 1.样品制备<ol><li> 固定和嵌入 <ol><li> 固定术 <ol><li>麻醉在三卡因（0.003％）仔鱼加入到E3（5毫米氯化钠，0.17毫米氯化钾，0.33毫米氯化钙2，0.33mm的硫酸镁，10毫米的HEPES，pH值7.6），斑马鱼水和测试麻醉深度轻轻戳用解剖显微镜下一个200μl的移液管尖，直到运动不再检测（ 图1A）。 </li><li>当使用共焦或2光子成像在1.8％琼脂糖中所述的体内成像数据取得后的处理对相关的EM的样品，在琼脂糖5,15修复幼虫使用4％PFA的PBS中含有的Triton-X-100（0.05％）。 </li><li>从琼脂糖和工艺切割幼虫大型EM 5。 </li><li>固定在含的Triton-X-100（0.05％），用于在塑料管室温2小时的PBS中的新鲜的4％多聚甲醛的幼虫。 注:在培养物中生长的细胞（体外 ）可直接固定在戊二醛，如在下一步骤（1.1.1.5）中所述。 </li><li>除去溶液，加入100微升0.5％的煤灰，2％戊二醛（GA）和0.1M二甲胂酸钠（pH7.4）中。在4℃下存放过夜。冲洗样品中二甲胂0.1M的钠两次。 </li><li>吻侧切割头幼虫向后脑，方便锇的渗透。用细注射器/镊子。 </li><li>后缀幼虫在1％的四氧化锇（OSO 4）/1.5%亚铁氰化钾（K 4的[Fe（CN）6]）中在冰上的0.1M二甲胂酸钠2小时，并在双蒸H 2 O冲洗3×5分钟</li><li>通过在增加的乙醇浓度20分钟的孵育（50％，70％，3次100％）脱水乙醇。 </li></ol></li><li>嵌入 <ol><li>根据在EM中的实验室中使用的标准配方制备环氧树脂。 注意:我们使用环氧树脂如下:混合19.8克glycid醚100，用磁力搅拌器9.6克2-十二烯基琥珀酸酐和11.4克甲基纳迪克酸酐等。添加0.5克DMP-30（三 - （二甲氨基甲基）苯酚），并再次混合。 </li><li>孵育在50％环氧树脂幼虫过夜乙醇（体积/体积），同时旋转（RT）。 </li><li>与纯树脂代替稀释环氧树脂。孵育30分钟，再加入新鲜树脂和孵育30分钟。再次刷新树脂，然后孵育在室温3小时，随后在58℃15分钟，并在室温低压（200毫巴）下再过1小时。 </li><li>使用针定位在市售硅扁平包埋模具磁头解剖显微镜下或面向模具的或根据需要的一侧的前部牙签 （ 图1B）。 </li><li>聚合环氧水库在58℃过夜。如果环氧树脂仍然太软允许另一天进行聚合，并在58℃硬化。通过使用镊子施加压力测试刚度。如果此容易留下的压痕允许另一天进行聚合，并在58℃硬化。当试样完全硬化进行修剪和切片。 </li><li>修剪远离使用存根刀片（ 图1B）多余的树脂。 </li></ol></li></ol></li><li> 切片，对比和安装 <ol><li> 切片 <ol><li>使用超薄科环氧树脂块。 </li><li>用玻璃刀或者钻石histoknife削减为甲苯胺蓝/碱性品红半薄切片（500纳米）（ 图1D）染色，以检测正确的定位。 </li><li>通过拾取它们用玻璃巴斯德吸管，其前端已通过在火焰熔化关闭上显微镜载玻片转移半薄切片。干热板上üNTIL没有水离开了。染色10秒，在水中的1％甲苯胺蓝的热板上，并在1％的四硼酸钠与水，耐污漂洗10秒以0.05％碱性品红之后。与10X普通光学显微镜40X检查。 </li><li>当达到大脑内的适当的地点/取向，继续用金刚石刀切片环氧树脂块切割超薄切片（〜70纳米; 图1E） </li><li>用容易识别的解剖结构和周围的脑，包括嗅凹坑，眼睛，灰白质的边界，超薄切片期间识别感兴趣的区域，并当样品被倾斜，以调节切片角度。 </li><li>在单插槽L2×1铜网格（ 图1F），安装部分允许收购格栅条中断。使用的Formvar包被的网格支持部分。 </li></ol></li><li> 对比 <ol><li>网格染色超薄切片•为2％醋酸铀甲醇其次是雷诺2分钟柠檬酸铅另外2分钟16。 </li></ol></li></ol></li></ol> 2.图像采集<ol><li> 大型STEM 注意:我们使用的扫描与EM能够使用STEM检测高分辨率3,5,7,17 ​​获得多个视场大的外部扫描发生器。通常情况下，一个图像生成这种方式相当于视图〜100透射电子显微镜（TEM）图像的领域，显著降低缝合的量。 <ol><li> 在显微镜安装示例 <ol><li>放置网格在多个网格样本保持部分和转移到SEM的腔室。 </li></ol></li><li> 干探测器对准 <ol><li>把电网与样品梁下移动它，所以这将是约。从透镜杆5毫米。使用在20kV获取图像中透镜检测器。 </li><li>专注于网格边缘，调整工作距离为4.0毫米。 </li><li>移动样品架至安全位置，并在干检测器带来。 </li><li>通过用在透镜探测器转动调整螺钉而成像在最大速度居中在图像场的STEM检测器的孔。 </li><li>小心带回样品架，这将只是透镜杆和检测器之间适合。 </li><li>取得与该透镜内探测器的图像可以肯定的是在部分区域。 </li><li>切换到STEM检测（增益介质和&#39;减&#39;设置的所有象限），并获取图像，然后选择要扫描的区域。 </li><li>提高加速电压为21千伏及（又稳定的图像），等待梁的镇定，然后去29千伏2千伏步骤。 </li></ol></li><li> 采集图像 <ol><li>预照射样品（以防止由电子束的亮度的变化）通过放大出所以要扫描的整个区域适合图像窗口。 </li><li>改变孔径120微米（以保持适当的动态范围的检测器增益必须减少一个步骤）。 </li><li>德焦点，使图像模糊。 </li><li>使用面积减小扫描选项使得被扫描区域尽可能紧密。 </li><li>设定扫描速度，使得一帧中大约扫描。 1 - 2秒。 注意:根据大小和组织这通常需要半小时（100×100微米FOV）至最多3小时（1000×500微米）。 </li><li>至少100倍放大和扫描较小范围，持续10秒。 注意:当此区域不改变亮度相比其周围，预辐照是足够的。 </li><li>带回30微米孔径（STEM和中等增益） </li><li>重点样本。 </li><li>使用摇摆（在〜40000倍）对准孔。 </li><li>而在重点选择最亮区域/功能，设置扫描速度，这样的细节是可见的。 </li><li>在显微镜软件通过仔细观察直方图保持所有像素中的动态范围调整的亮度和对比度。做最黑暗的区域/功能相同。 </li><li>回到明亮的区域，并再次检查。在安全侧，从而存在对直方图的两侧一些空间。 </li><li>缩小所以要扫描的整个区域适合成像窗口，并开始大面积采集程序。 </li><li>使用向导选项从屏幕中选择一个地区设立了马赛克。使用像素大小2 - 取决于哪些细节是必要的5纳米。使用停留时间3微秒干。 </li><li>选择选项"在以前的瓷砖自动对焦"，可使用默认设置，公司大规模收购的软件支持自动对焦，复选框"验证在执行之前设置"并定义保存数据。 </li><li>按"优化"来检查显微镜设置。 注:现在所需要的时间将是SH拥有。这可能是高达72小时为1×0.5毫米区域。 </li><li>在窗口中的"最大分辨率瓦"式的20000个人这样瓷砖不会大于20K点¯x20K，防止钝边的效果。按"执行"。 </li><li>当被问及检查对焦和亮度/对比度（B / C），关闭外部扫描生成并仔细在显微镜软件调整对焦和散光。不要改变B / C。 注意:阶段可以移动和放大倍率改变，但放大率必须设置回所需的像素尺寸。 </li><li>再次接通外部扫描发生器，按"继续"。 </li></ol></li></ol></li></ol> 3.数据分析<ol style="margin-left: 40px;"><li>打开大型EM文件查看器程序，然后打开文件"马赛克信息"。这将打开所有的瓷砖TIF-文件。这最好另一个工作站上执行不妨碍新的收购。 </li><li>选择选项&#39;金缝合整个花叶（参数重叠半个模式，拼接的阈值0.90，降噪自动的。如果缝合的标准不能被满足，放大并手动将瓷砖的位置，然后点击"继续是&#39;）。 </li><li>导出为HTML文件，或者作为一个单一的TIF。 注意，对于TIF出口可能需要一降尺度。包括在文件名中启用测量后的最终像素大小。 </li><li>在图像编辑程序打开它执行的TIF文件的注释。同时，在web浏览器的最佳分辨率观测可缩放的HTML文件。 </li></ol>

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作者什么都没有透露。

EM允许与生物大分子方面的高分辨率成像细胞环境的分析。然而，这通常限制了视场。较大规模的3D EM是通过创造纳米级分辨率三维重建，需要复杂的数据处理10特别适合用于映射神经元的连接。相反，2D大规模的EM仅需要成像数据的单个部分和缝合，并且该数据的评估可以通过任何访问因特网浏览器。我们和其他人以前使用的大型EM分析组织和整体动物。对于大多数方法，透射电镜和扫描电镜的基础拼接也有自己的优势。这里，扫描透射的EM（STEM）被用来允许一代的大视场中的高的分辨率。通常，成像大FIEL当一个STEM图像相当于视图大约100 TEM图像，显著降低缝合的量的字段看在高分辨率DS。如果需要更高的分辨率，TEM可以是有利的。干具有优势的TEM非对比样品可以以良好的超微结构对比度6中使用。 此外，这里所描述的方法可以简单地调整为与背散射电子探测（BSD）上安装在二氧化硅晶片部分，拓宽了使用多个显微镜系统中使用。 HTML的可缩放组织EM文件是定量非常有用的，共享可能不被分析，以它的全部内容，在科研相结合LM和EM数据（CLEM）8，展示目的的数据，来分析病人数据，以及用于教育。可替代地，在一个扫描电镜大型EM，但不是在TEM，二氧化硅晶片，可以使用具有两个主要优点:BSD可以使用，这是更一般比STEM检测可在扫描电子显微镜。二，安装大段（&gt; 1毫米2区利息）很简单。安装在单槽电网是劳动密集型和技术挑战。透射电镜，扫描电镜和STEM之间详细的比较其他6详细说明。 BSD成像的缺点是，相比茎，图像增加噪音。这可以部分地通过增加像素停留时间，导致更长的采集时间来补偿。 虽然样品制备比较标准的EM处理（固定，嵌入和切片）需要5-7，这在技术上具有挑战性削减大型超薄切片完全没有文物。部分非常脆弱，容易断裂，褶皱或成像，这通常需要每个数据集多个小时期间被销毁。然而，由于原始偏数据的在线共享，它应该成为能够更容易地比较已公布的数据，并在开放域作为对照使用公布数据集。目前，一些新兴设备能够拉的RGE尺度分析都在使用，因此可访问该技术是比较有限的，但大多数成像中心欢迎合作努力。 对于大规模的部分的组织必须被适当地固定在整个样本。这就是为什么快，但温和的固定液PFA的混合物结合使用缓慢但强劲的固定液GA。切割和无人工捡了大截面是困难的。相比于单孔格栅收集区结合晶圆正与BSD更容易。相比于传统的EM重金属后染色更为关键。由于整个截面成像所有文物将是可见的。 TEM用户通常很难切换到扫描电镜，因为在显微镜的操作的差异。 量化和数据共享 -量化亚细胞的功能是单一EM图像困难。的可能性和出大规模的放大图像容易使所关注的细胞，其可以随后被细胞内的纳米级测量的鉴定。这些数据集表示特定的细胞类型，可以迅速地识别和在这些大的组织切片中的偏见地量化，是根据在不同的尺度可检测的特征。例如小胶质细胞可以根据它们的形态和密细胞质来鉴定。随后，当对单个细胞放大时，这些细胞的纳米级亚细胞和分子特征可以在相同的数据集内测量，如我们先前大规模的EM组织培养数据集2内表现出对雌激素受体 ​​的宽度。 nanotomy的另外一个优点是在线的大规模数据集的托管将允许其他人检查数据，也许对其他的功能，并汲取新假说的结论。 CLEM -除了便于量化EM，大型新兴市场更容易光microsco关联图片标签到EM 8级。在本实施例吞噬小胶质细胞在斑马鱼烧蚀模型的存在被示出。在神经科学中的一个主要问题是单个功能和贡献是小胶质细胞和吞噬细胞和免疫细胞的潜在的其他来源。早期的EM研究疾病18中的小胶质细胞的独特功能，亚细胞。不幸的是，很难选择性地标记在特别小胶质细胞在病理设置，因为它们显示出与基因表达，形态和功能的其他免疫细胞的大的重叠。因此，目前还不清楚是否以及何时在超微结构水平小胶质从免疫细胞的不同从其他来源包括衍生巨噬细胞浸润的单核细胞。理解是否有这些细胞之间的超微结构的差异将提供的功能差异分析的起点。结合选择性的转基因或表达的标记和CLEM允许DET超微结构特点挠度选择性的特定人群。 诊断与演示和教育 -通过可视化纳米单个数据集的电磁数据中的微观被大大方便了广大的观众。随着更多的可能性和工具，相关的和大规模的EM我们预计新兴市场的基础和医学研究的复兴。我们这里所表示的方法被施加到斑马鱼脑损伤模型5，但已被用于对人体组织7，大鼠脑3，在大鼠模型中对糖尿病4和在细胞培养物2，并且还可以在组合用TEM中使用基于方法1示出了该方法的多功能性。显微镜操作者不再录制高度选择，因此有偏见的图像，但所有无数的超​​微结构特征被记录和开放的全球分析。

最近的技术发展提高了EM的多功能性，适用性和定量的性质，导致超微结构分析的复兴。进展包括3D EM，大型2D EM及相关光学显微镜和电子显微镜（CLEM）改进的方法和试剂在内的微观分析的其它模式比较直接到EM水平8-10。大型二维EM是特别合适的量化或确定人体病理学（新颖）疾病特征，研究动物模型疾病和组织培养模型。由于视通常小字段是很难在高放大倍率的变化关联到组织大规模，以及以无偏和定量分析超微结构特征。 对于人体组织或病理动物模型评估，有色苏木和伊红（H＆E）的嵌入式甲醛固定，石蜡切片（FFPE）TIS的病理分析起诉是标准。除了简单也进行H＆E染色免疫标记，以确定病理异常。如果这些组织可以在对EM的水平，特定类型的细胞进行分析，和亚细胞的变化可确定。大型EM的公正性可以发现疾病的意想不到和新颖的特点。通过大规模的EM地区可达平方毫米，可以可视化。我们和其他先前应用nanotomy对大鼠胰岛4，细胞培养2，大鼠脑组织3，皮肤和粘膜7和整个斑马鱼1,5（www.nanotomy.org）。斑马鱼非常适用于体内成像，在特别是可视化这是很难在哺乳动物组织，包括脑的免疫细胞11访问的细胞类型。这里nanotomy过程进行详细说明，应用到由神经甲硝唑转换进行有条件神经烧蚀斑马鱼头冠状切片表达硝基还原酶（www.nanotomy.org）5,12-14。所有原始数据被呈现为可缩放的HTML文件，可视化分子到组织比例变化。原始数据的呈现允许被全球专家从其他角度的数据集的公正的分析。

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large-scale scanning transmission electron microscopy (nanotomy) of healthy and injured zebrafish brain

大型2D电子显微镜（EM），或nanotomy，是纳米级的高分辨电子显微学的组织范围内的应用。其他和我们以前应用的大规模EM对人体皮肤胰岛，组织培养和全斑马鱼1-7。在这里，我们描述了组织大规模扫描EM为亚细胞和分子特征公正的检测普遍适用的方法。 Nanotomy应用于探讨健康，神经退行性大脑的斑马鱼。我们的方法是基于标准化的EM样品制备协议:固定用戊二醛和锇，随后环氧树脂包埋，超薄切片，并在一个孔网格超薄切片，随后交染色铀和铅的安装。大型二维电磁马赛克图像是使用一个扫描EM使用扫描透射的EM（STEM）连接到外部大面积扫描发生器获得。大型EM的图像通常〜5 - 50 G像素我Ñ​​大小，以及使用可缩放的HTML文件，它可以在任何网络浏览器中打开，类似于在线地域的HTML地图最佳效果。这种方法可以适用于（人）的组织，整体动物的横截面，以及组织培养1-5。这里，斑马鱼的大脑中的非侵入性的神经元消融模型进行了分析。我们想象一个单一数据集组织，细胞和亚细胞的改变可以以各种细胞类型，包括神经元和小胶质细胞，大脑的巨噬细胞来定量内。此外，nanotomy便于EM的光显微镜（CLEM）8上的相同组织的相关性，如大的表面积先前成像的使用荧光显微镜，随后可以进行大面积的EM，产生的纳米解剖（nanotomy）组织。在所有的，nanotomy允许在组织的全计量的方式的EM水平偏检测的特征。

1周龄斑马鱼的头部的冠部的大型的EM数据集示出了在一个单一的大型图像许多组织和细胞的特征（www.nanotomy.org）。 的控制脑切片Nanotomy揭示延髓前脑包括嗅纤维束，神经元核和神经元的子隔室，包括突触（ 图2A，www.nanotomy.org）的神经组织的典型超微结构特征。在头部的细胞类型，可以区分包括在下颚大液泡，嗅觉神经细胞的嗜锇髓鞘，小血管的内皮细胞，结构包括脑膜（脑脊膜的斑马鱼对应的膜质层罩子软骨细胞哺乳动物大脑）。亚细胞结构包括表皮中不同CEL的多糖包被，不同核形态和灶<html>升类型和细胞器，包括高尔基体，内质网（ER），线粒体和突触小泡与突触后密度。 出乎意料细胞衬心室的细胞核相比在大脑中分散多个横向其它细胞是非常电子致密。此外，这些心室细胞核显示暗灶是在大脑中的其它细胞不存在，并显示在从异，这是在吞噬小胶质细胞（ 图2）的细胞核中发现的大小和结构不同。细胞内层心室发展中的斑马鱼主要包括放射状胶质细胞和神经祖细胞，这可能反映了比分化的细胞的染色质改变状态。如在组织干细胞通常是相当罕见的，nanotomy关联为与组织比例干细胞标记物因此EM可用于鉴定干细胞的超微结构特征组织标记。 ve_content"FO:保持-together.within页="1"&gt;大规模在斑马鱼幼体经历神经烧蚀冠部的EM（www.nanotomy.org）揭示了许多特定的组织，并且没有发现细胞和分子特征在对照动物。细胞功能包括吞噬小胶质细胞和空泡细胞的大小，有可能表示死亡的细胞（ 图2B，www.nanotomy.org）。前EM研究已在脊椎动物18,19确定的小胶质细胞。小胶质细胞的典型特征包括细长核，核膜，突出高尔基体，游离多核糖体，粗面内质网（ER）与狭长的囊泡，相对暗/致密细胞质，以及众多的夹杂物，如吞噬体，脂滴和溶酶体旁边片状染色质的团块。所有这些标志，在哺乳动物组织归因于小胶质细胞，这些细胞在斑马鱼的大脑（ 图2B，www.nanotomy.org）也被发现。 &lt;p系列="jove_content"FO: - together.within页保留="1"&gt; <img alt="图1" src="/files/ftp_upload/53635/53635fig1.jpg" /> 图1:斑马鱼脑大型组织电子显微镜的工作流程。 （A）7-日龄幼虫斑马鱼（B）在环氧树脂中嵌入并排斑马鱼幼体头。（C）玻璃刀半-变薄切片和定向标本。（D）代表半薄甲苯胺蓝染色切片显示两个并排端嵌入式斑马鱼。（E）钻石刀切割超薄（70纳米）的部分。（F）全斑马鱼幼体的大脑节上的一个孔电网（D）编辑的图像来表示的定位。（G）在这项研究中使用的大型2D EM显微系统。（H）图像处理的流程。 <a href="https://www.jove.com/files/ftp_upload/53635/53635fig1large.jpg" target="_blank">请点击此处</a>查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53635/53635fig2.jpg" /> 图2:Nanotomy结果:从高分子到组织 （A）的处理后7天受精控制和（B）大脑Nanotomy（神经烧蚀; 图1），显示出特有的神经元功能烧蚀退行性脑（B）中 ，这些包括吞噬小胶质细胞，黑暗的牢房发生细胞死亡和神经组织（（A）和（B））的海绵状外观。上图（改编自5）:区域的10倍放大显示在中间的面板显示。中东板:地区的10倍放大显示在较低的面板显示。 （A，中图），在（A，下图）神经纤维突触显示的高倍镜视野，突触小泡（SVS）和突触后密度（PD）。 （B，中间面板）示出典型的变形虫小胶质特征（下图），包括突出高尔基体（G），变形虫小胶质细胞或可能是巨噬细胞（中间面板，M）的高放大倍数图，夹杂物包括溶酶体空泡（L）。标尺:50微米（顶部），5微米（中间）和0.5微米（底部）。这个数字已经从5修改。 <a href="https://www.jove.com/files/ftp_upload/53635/53635fig2large.jpg" target="_blank">请点击此处查看本图的放大版本</a> ，或访问完整的数据在线（nanotomy.org）。

Watch this Scientific Journal Video about Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain at JoVE.com

Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution EM. Here we describe a universal method for nanotomy applied to investigate the zebrafish larval brain in health and upon non-invasive brain injury.

健康和受伤斑马鱼脑的大型扫描透射电子显微镜（Nanotomy）

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously ...

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Research

JoVE Journal

Developmental Biology

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

11.0K 次观看.  UMC Groningen. Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution EM. Here we describe a universal method for nanotomy applied to investigate the zebrafish larval brain in health and upon non-invasive brain injury. 

视频: Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

大规模的斑马鱼胚胎的心脏解剖的转录分析

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

科研

发育生物学

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

分离培养成年斑马鱼脑源性神经球

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

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 ...

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 ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

幼虫斑马鱼性腺组织的解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

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

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

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 ...

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

用于在大容量中查找特定细胞目标的串行剖面的多模式分层成像

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...