我们感谢安永 H. K. Stelzer 有机会利用他的资源，以及他对手稿的宝贵评论，安妮塔·安德尔对 Tribolium 现场成像的支持，斯文·普拉斯提供技术支持，以及伊兰·戴维斯、妮可·格里德和杰罗德·舒比格通过布卢明顿股票中心分享他们的转基因 Drosophila 生产线。

1. 筹备工作
<ol><li>为适合科学问题的 LSFM 选择照明镜头/检测镜头/相机组合，并设置显微镜。视场的大小是相机芯片大小的商量和检测镜头的放大倍数。照明透镜应选择，以便整个视野被大致平面光片34覆盖。 表1中列出了三个推荐组合。</li>
	<li>要准备阿加罗斯别名和检索菜肴，请将 2 克低熔性蔗糖加入 200 mL 高压自来水中，并在 600-800 W 的微波炉中加热混合物，直到所有高糖颗粒溶解。在 1.5 mL 或 2 mL 反应管中准备几个 1 mL 的阿加罗斯阿加罗斯别名，然后用 agarose 填充几个 90 毫米的培养皿 3-5 毫米高。在 4 °C 时存储凝固的阿里报价和菜肴。</li>
	<li>对于 Drosophila: 要准备新鲜的饲养小瓶，请烹饪足够数量的定制或市售 的 Drosophila 介质，将 5-15 mL 传输到宽小瓶中，并将它们储存在 4 °C。 要准备产蛋菜肴，请将 1 克低熔性蔗糖加入 50 mL 的高压自来水中，并在 600-800 W 的微波炉中加热混合物，直到所有高糖颗粒溶解。让混合物冷却到45°C，然后加入50mL果汁（最好是苹果或红葡萄），并彻底混合。将混合物倒入 35 mm + Petri 菜肴中，并在 4 °C 时储存凝固的产蛋菜肴。</li>
	<li>对于 三叶草:要准备生长介质，通过710μm网状大小筛子传递全麦面粉以及非活性干酵母，然后用5%（w/w）筛酵母补充筛面粉。要准备产蛋介质，通过250μm的网状筛子将精制小麦粉和非活性干酵母通过，然后用5%（w/w）筛酵母补充筛面粉。</li>
</ol>2. 使用荧光微球校准基于样品室的 LSFM
注:校准的目的是对齐照明和检测镜头（图2A）的焦点，因为这是清晰图像的前提。LSFM 应定期校准，至少每 3-4 周校准一次。
<ol><li>在 80 °C 的干块加热器/搅拌机中重新液化 agarose 参考，然后让阿加罗斯阿利库特冷却到 35 °C。</li>
	<li>将 50μL 的糖液转移到 1.5 mL 反应管中，并添加 0.5 μL 的荧光微球溶液。混合在1，400转米1分钟。</li>
	<li>用10μL的糖/荧光微球溶液混合物填充蜘蛛网支架的槽孔，然后尽可能多地吸气，直到只剩下薄的糖膜。等待30-60s凝固。</li>
	<li>用高压自来水填充样品室。将蜘蛛网支架缓慢插入样品室，并在检测镜头前使用微翻译阶段移动槽孔。</li>
	<li>将带旋转阶段的蜘蛛网支架旋转到相对于照明 （x） 和检测 （z） 轴的 45° 位置。蜘蛛网支架不应在传输光通道中可见。</li>
	<li>切换到各自的荧光通道，调整激光功率和曝光时间，使荧光微球提供适当的信号。</li>
	<li>指定完全覆盖现在横向的阿加罗斯薄膜的视图量。通过计算各自照明镜头/检测镜头组合34的最大可能轴向分辨率来定义 z 间距。或者，横向分辨率的 4 倍可用作粗略近似。</li>
	<li>记录荧光微球的三维测试 z 堆栈，并将 x、y 和 z 最大投影与校准图（图 2）进行比较。如果微球出现模糊、模糊和/或失真（图2B，C），则调整照明和/或检测镜头的位置。</li>
</ol>3. 果 蝇 胚胎的收集
<ol><li>将 100-200 名 德罗索菲拉 系列的成人转移到新的养殖小瓶 2-3 天前的成像检测，以建立鸡蛋收集文化。如果尚不存在，请考虑使用古老的小瓶来启动后代文化。为了确保成年人不超过两周大，及时用后代培养取代胚胎采集培养。</li>
	<li>将产蛋盘加热至室温，并在蛋黄上加入一滴酵母酱。</li>
	<li>将成人从鸡蛋收集培养转移到一个空的窄小瓶，并将其放在产蛋盘的顶部。在室温下孵化鸡蛋收集设置15分钟。如果可能，在此步骤中避免麻醉（冷，CO2）。</li>
	<li>将成人送回饲养小瓶。以方便的温度/时间组合孵育产卵盘，然后用小画笔将10-20个胚胎从产卵盘转移到100μm网状细胞过滤器。丢弃产蛋盘。</li>
	<li>每个 德罗索菲拉 线重复步骤 3.1 到 3.4。</li>
</ol>4. 三宝胚 胎的集合
注:为方便起见，提供 三宝鸡 蛋收集程序方案（图3A），本步骤括号内的数字也参考。
<ol><li>将 200-300 名成人（约 400-700 毫克）的 Tribolium 选择线转移到一个空的 1 L 玻璃瓶 2-10 天前成像检测，以建立鸡蛋收集文化 （图 3A_01）。用50-100克的新鲜生长介质填充瓶子。如果尚不存在，请考虑使用可用的幼虫和幼虫开始后代文化。为确保成年人不超过3个月大，及时用后代文化取代卵子收集文化。</li>
	<li>通过800μm网状筛子（图3 A_02）传递鸡蛋收集培养。将含有非分阶段胚胎的生长介质返回初始瓶（图3 A_03），并将成人转移到空的1L玻璃瓶（图3 A_04）。加入10克产蛋介质（图3 A_05），在室温下孵化卵子收集设置1小时（图3 A_06，07）。</li>
	<li>通过 800μm 网状大小筛子（图 3A_08）传递鸡蛋收集设置。将成人返回到初始瓶（图3 A_09）。根据应成像的发育过程，孵育产卵介质，现在含有约30-100个胚胎，在方便的温度/时间组合（图3 A_10）。</li>
	<li>通过300μm网状筛子（图3 A_11）传递产卵介质，并将胚胎（图3 A_12）转移到100μm网状细胞过滤器。丢弃筛蛋介质（图3 A_13）。</li>
	<li>每个 三叶草 线重复步骤 4.1 到 4.4。</li>
</ol>5. 以次氯酸钠为基础的去皮
注:嗜 血杆菌 和 三叶草 胚胎都由胆汁覆盖，这是一种保护性且高度分散的蛋白质层，只要胚胎在去除后保持湿润，对正常发育就不重要。两种胚胎的分体协议是相同的。
<ol><li>用8 mL的高压自来水和7mL的高压自来水和1mL的次氯酸钠（NaOCl）溶液（图3B）填充A1、A2、A3和B3井，准备6井板。在立体显微镜下观察减压过程，最好是在透射光下。 注意:次氯酸钠具有腐蚀性。</li>
	<li>将含胚胎的细胞过滤器（步骤3.4和/或4.4）插入A1油井，在轻轻搅拌下清洗约30-60s的胚胎。</li>
	<li>将细胞过滤器移到B1井，大力摇动板30秒，然后将其转移到A2井，在轻轻搅拌下清洗胚胎1分钟。</li>
	<li>将细胞过滤器移到B2井，并大力摇动板，直到大多数胚胎完全减损（图3C），然后将其转移到A3井，在轻轻搅拌下清洗胚胎1分钟。</li>
	<li>在进行安装程序之前，将细胞过滤器存放在 B3 中。</li>
	<li>每行重复步骤 5.1 到 5.5。</li>
</ol>6. 使用蜘蛛网支架安装多个胚胎
<ol><li>在 80 °C 的干块加热器/搅拌机中重新液化 agarose 参考，然后让阿加罗斯阿利库特冷却到 35 °C。</li>
	<li>管子 10μL 的阿加罗斯在蜘蛛网支架的槽孔顶部。用移液器尖端，将阿加罗斯铺在槽上，然后尽可能多地吸气，直到只剩下薄的糖膜。等待30-60s凝固。</li>
	<li>对于每行，小心挑选一个或多个胚胎与一个小画笔，并把它们放在阿加罗斯薄膜。</li>
	<li>沿着槽孔的长轴排列胚胎，然后将胚胎前后轴与槽孔的长轴（图3D）对齐。</li>
	<li>通过将1-2μL的糖液输送到胚胎和糖膜之间的缝隙中，小心地稳定胚胎。等待30-60s凝固。</li>
	<li>将装有胚胎的蜘蛛网支架缓慢插入图像缓冲区样品室。</li>
</ol>7. 基于样品室的 LSFM 中的比较实时成像
<ol><li>在检测镜头前移动具有微透镜阶段的胚胎之一。确保蜘蛛网支架相对于照明 （x） 和检测 （z） 轴 （cf. 图 1C）处于 45° 位置。</li>
	<li>在传输光通道中，将胚胎移入视野中心。蜘蛛网支架不应可见。</li>
	<li>将胚胎与z中的微转解阶段移动，直到胚胎的中平面与焦点平面重叠，即直到轮廓看起来清晰。无需切换到荧光通道，则通过将 250 μm 从中平面移到两个方向来指定视野体积。</li>
	<li>可选地，如果需要沿多个方向成像，适当旋转胚胎并重复步骤 7.2 和 7.3。蜘蛛网支架支持在 90°的步骤中最多支持四个方向。</li>
	<li>对于安装在蜘蛛网支架上的所有其他胚胎（图 3E），重复步骤 7.1 到7.3（或 7.4）。确保最上层胚胎在检测镜头前时不会离开成像缓冲区。</li>
	<li>定义荧光通道（激光功率、曝光时间、检测滤光片）和时间推移（间隔、总持续时间）参数，并开始成像过程。有关指示性值，请参阅示例数据集的元数据表（补充表 1）。对于持续几天的检测，请考虑覆盖样品室打开至少部分，以减少蒸发。</li>
</ol>8. 检索和进一步培育成像胚胎
<ol><li>成像检测结束后，小心地将蜘蛛网支架从样品室中取出。</li>
	<li>用一把小画笔将胚胎从糖膜中分离出，并将其转移到贴有适当标签的显微镜滑梯上。将滑梯放入检索盘中，并在相应的标准饲养条件下孵化。</li>
	<li>关于实验方式，估计胚胎生成何时完成。随着孵化时间点的临近，经常检查检索菜肴，并将孵化的 果蝇 幼虫转移到单独饲养小瓶和 三宝幼 虫到24井板的单独井中。用生长介质填充油井的一半。在各自的标准饲养条件下孵化。</li>
	<li>一旦观察到的人是成年人，为他们提供一个合适的交配伙伴，并在几天后检查后代。</li>
</ol>9. 图像数据处理和元数据文档
注:对于图像数据处理，建议使用图像J衍生 FIJI35（imagej.net/Fiji/Downloads）。 FIJI 不需要安装，提供 32 个和 64 位版本。LSFM 数据最常用的格式之一是标记图像文件格式 （TIFF），它允许以 TIFF 容器的形式存储图像堆栈。
<ol><li>计算所有 z 堆栈的 z 最大投影（图像|堆栈|Z 项目，选择 最大强度）。最大的预测是数据简化方法，将空间维度从三个减少到两个。提供用于批处理的 FIJI 脚本（补充文件 1）。</li>
	<li>将各自的 z 最大投影串联起来，以创建时间堆栈（t 堆栈）。将这些保存在一个 TIFF 容器中。适用于所有记录的胚胎以及相应的方向和荧光通道（如果适用）。</li>
	<li>围绕 z 轴旋转 z 和 t 堆栈，使胚胎前后轴与 x 或 y 图像轴对齐（图像|转换|旋转）。沿所有三个图像轴裁剪 z 堆栈，并在 x 和 y 图像轴中裁剪 t 堆栈，以便胚胎周围仅保留最小缓冲空间（沿 x 和 y 轴 20-40 像素，沿 z 轴的 5-10 图像）保留（图像|调整| x 和 y 轴的画布大小， 图像|堆栈 | 工具| z 轴的切片守护者）。<ol>
<li>如果适用，针对所有记录的胚胎以及相应的方向单独进行此操作。荧光通道（如适用）应以相同的旋转和裁剪参数进行处理。</li>
	</ol>
</li>
	<li>尽可能详细地记录元数据。作为准则，可以使用示例数据集的元数据表，该表集在代表性结果部分中具有特色（补充表 1）。</li>
</ol>10. 数据可视化
注:此数据可视化指南主要侧重于创建 z 最大投影图像矩阵，该矩阵显示多个方向和/或随时间推一段时间内记录的多个胚胎。以下步骤描述了应用于创建所示数字的示例数据集的数据可视化过程，以及在"代表结果"部分链接的视频。
<ol><li>组合 t 多个方向的堆栈（图像|堆栈|工具|合并）和/或合并多个荧光通道 （图像|颜色|合并通道）可视化生物结构和/或感兴趣的过程。</li>
	<li>调整 t 堆栈的特性（图像|调整|亮度/对比度 ）根据需要使用"集"功能。应将显示的最低值设置在背景信号上方，最大显示值应产生方便的对比度。记录这两个值，因为它们可用于对各自的 z 堆栈进行一致的调整。使用"应用"功能进行印迹调整。根据实验模式，考虑处理所有具有相同值的记录胚胎。</li>
	<li>将强度调整的 t 堆栈保存为单独的文件，不要覆盖未调整的 t 堆栈。从具有专用图像选择功能（例如 ，图像|调整后的 t 堆栈中编译合适的子堆栈堆栈|工具|切片守护者）和使用蒙太奇工具 （图像|堆栈|使蒙太奇）创建图像网格，可用于图形设计。</li>
</ol>

作者没有什么可透露的。

LSFM 的独家应用领域之一是发育生物学。在这个学科中，观察活的标本很重要，否则无法动态地描述形态遗传过程。此处描述的基于样品室 LSFM 的同步实时成像实验框架很方便，原因有二。
环境方差是连续实时成像中不可避免的，可以主动解决。在与昆虫胚胎相关的活成像中，我们看到以下易感性。在胚胎采集时，卵子收集培养可能有不同的年龄。在 德罗索菲拉，已?...

荧光显微镜是生命科学中最重要的成像技术之一，特别是在细胞和发育生物学方面。在共焦荧光显微镜1中，这是自20世纪90年代中期以来最先进的三维荧光成像技术，同一镜头用于荧光激发和发射光检测。照明激光束激发照明/检测轴沿线的所有荧光素，在通过针孔检测之前，对各自的对焦信号进行区分。因此，对于每个二维图像，整个标本都会被照亮。因此，对于每个三维图像，即一叠空间连续的二维图像，整个标本被照亮几十到几百倍2，这促进光漂白和光毒性3。
近二十年前，光片技术4 成为三维荧光成像的有前途的替代品，成为发展生物学5的宝贵工具。在这种方法中，照明和检测是脱钩的。照明透镜用于在垂直排列的检测透镜的焦平面内生成深度只有几微米的光片。因此，对于每个二维图像，只有焦平面周围的细平面体积被照亮。因此，对于每个三维图像，整个标本只被照亮一次，这强烈地降低了光漂白和光毒性6。因此，光片荧光显微镜（LSFMs）为研究多个生物相关尺度的复杂过程提供了有效的解决方案，因此，在发育生物学中具有特殊价值，在亚细胞水平上必须分析高达几毫米的标本。
从历史上看，LSFM一直以样品室为基础，即7、8。在这些设置中，照明 （x） 和检测 （z） 轴通常垂直于重力轴 （y） 排列。样品室提供充足的实验自由。首先，它们提供大型成像缓冲能力，这反过来又便于使用灌注系统控制环境，例如维持特定温度9或应用生化应激器。此外，他们支持定制安装方法10，根据各自的实验需要量身定做，同时保持三维，在某些情况下动态11，标本的完整性。此外，基于样品室的设置通常配备了旋转功能，用于将标本围绕 y 轴旋转，从而沿两个、四个甚至多个方向对标本进行成像。由于常用模型生物的胚胎在显微镜中，沿腹腔多面、横向和/或前后体轴的连续成像规模相对较大，具有更全面的表示性。这允许长期跟踪沿着复杂的三维迁移路径12，13移动的细胞。
光片荧光显微镜已广泛应用于研究甲流的胚胎形态，系统地研究14、15，并特别注重生物物理方面的发展。例如，它被用来收集高分辨率的形态遗传学数据，以检测在细菌带拉长16期间内皮体入侵和轴延伸之间的生物力学联系，并进一步将复杂的细胞流与气喘17期间的力生成模式联系起来。它还与其他最先进的技术相结合，例如光遗传学，以研究在表皮18前后模式期间对Wnt信号的调节。
然而，只研究一个物种并不能提供对发展演变的见解。为了了解植物学背景下的胚胎生成，对替代昆虫模型生物进行了深入的研究。其中一个最全面调查的物种是红面粉甲虫三宝座，一种经济上相关的储存谷物害虫19，其胚胎形态也已经系统地与LSFM20成像。这两个物种的胚胎形态在几个方面差异显著，如分割模式21，以及胚胎外膜22的形成和降解。使用 LSFM 对后一个方面进行了广泛的分析。例如，已经证明，血清，一种胚胎外组织，包围和保护三叶草胚胎免受各种危害，其胚胎生成的更好部分23，24，也作为致命的遗传"驱动因素"，为自己的提取过程在回旋关闭25。此外，已经证明，在气喘期间，爆炸体的一个特定区域仍然锚定在玻璃膜上，以产生不对称的组织运动26，并在此观察之后，区域化组织流化允许细胞在塞罗萨窗口关闭27期间按顺序离开血清边缘。
在上述所有与嗜血杆菌和三叶草相关的研究中，都使用了基于样品室的LSFM。在大多数情况下，胚胎是沿着多个方向记录的，使用样品旋转功能。虽然没有明确说明，但可以假定它们是单独记录的，因此在连续的实时成像测定中彼此独立，类似于我们之前在Tribolium20，28上的工作。在某些情况下，这种方法是可以接受的，但在定量比较方法中，环境差异可能会扭曲结果。例如，人们早就知道昆虫的发育速度取决于温度，但最近的一项研究进一步表明，在德罗索菲拉，温度也可能影响30型摩尔磷的浓度。因此，如果胚胎生成的某些特征，例如细胞的动态比例、分裂率和迁移速度，应精确量化，则需要充分重复，而不需要环境方差。这最大限度地减少了标准偏差和标准错误，这反过来又有利于与其他，甚至只是略有不同的实验条件并列。
但是，基于样品室的 LSFM 主要针对高含量而非高吞吐量检测而设计。与共焦显微镜不同，该显微镜通常配备了用于显微镜滑梯、培养皿和井板的标准化夹紧机制，几乎所有基于样品室的 LSFM 都使用基于圆柱形的夹紧机制。这些机制是针对定制样品持有人，是旋转兼容的，以及非侵入性的10，但通常不设计为多个标本20，31，32。两个或两个以上胚胎同时进行活成像的框架，其中基于样品的室设置的优点不会受到损害，解决了环境方差问题，从而增加了LSFM用于比较研究的价值。
在我们的协议中，我们提出了一个实验框架，用于在基于样品室的LSFM（图1A）中进行比较活成像，其中y轴被用作"堆叠"胚胎的选项。首先，我们为基于样品室的 LSFM 提供荧光微球校准指南，对于缺乏校准辅助的仪器来说，这一点尤为重要。其次，我们描述了基于蜘蛛网支架28（图1B）的多个胚胎的安装方法，该方法与样本旋转兼容，从而允许沿多个方向同时成像多个样本（图1C）。几个胚胎在薄的糖膜上对齐，在插入样品室后，通过光片连续移动以获取三维图像。第三，我们为德罗索菲拉和三宝莲提供三个示范性的实时成像数据集。对于前者，我们将转基因线与荧光标记的核并列。对于后者，我们比较携带相同转基因的转基因子线的性能，但在不同的基因组位置。最后，我们讨论了平行化对比较活成像和环境方差33的重要性，讨论了我们的实验框架的吞吐量限制，并评估了我们对其他模型生物体方法的适应情况。

<table><tbody><tr><td>6-well plate</td><td>Orange Scientific</td><td>4430500</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>24-well plate</td><td>Orange Scientific</td><td>4430300</td><td>Only for live imaging involving &lt;em&gt;Tribolium&lt;/em&gt;</td></tr><tr><td>35-mm &amp;Oslash; Petri dish</td><td>Fisher Scientific</td><td>153066</td><td>Only for live imaging involving &lt;em&gt;Drosophila&lt;/em&gt;.</td></tr><tr><td>90-mm &amp;Oslash; Petri dish</td><td>Fisher Scientific</td><td>L9004575</td><td></td></tr><tr><td>100 &amp;micro;m mesh size cell strainer</td><td>BD Biosciences</td><td>352360</td><td></td></tr><tr><td>250 &amp;micro;m mesh size sieve</td><td>VWR International</td><td>200.025.222-038</td><td>Only for live imaging involving &lt;em&gt;Tribolium&lt;/em&gt;</td></tr><tr><td>300 &amp;micro;m mesh size sieve</td><td>VWR International</td><td>200.025.222-040</td><td>Only for live imaging involving &lt;em&gt;Tribolium&lt;/em&gt;</td></tr><tr><td>710 &amp;micro;m mesh size sieve</td><td>VWR International</td><td>200.025.222-050</td><td>Only for live imaging involving &lt;em&gt;Tribolium&lt;/em&gt;</td></tr><tr><td>800 &amp;micro;m mesh size sieve</td><td>VWR International</td><td>200.025.222-051</td><td>Only for live imaging involving &lt;em&gt;Tribolium&lt;/em&gt;</td></tr><tr><td>405 fine wheat flour</td><td>Demeter e.V.</td><td>SP061006</td><td>Only for live imaging involving &lt;em&gt;Tribolium&lt;/em&gt;</td></tr><tr><td>commercially available Drosophila medium</td><td>Genesee Scientific</td><td>66-115</td><td>Only for live imaging involving &lt;em&gt;Drosophila&lt;/em&gt; / Custom-made &lt;em&gt;Drosophila&lt;/em&gt; medium may also be used</td></tr><tr><td>fluorescent microspheres, 1.0 &amp;micro;m &amp;Oslash;</td><td>Thermo Fisher Scientific</td><td>T7282</td><td></td></tr><tr><td>inactive dry yeast</td><td>Genesee Scientific</td><td>62-108</td><td></td></tr><tr><td>low-melt agarose</td><td>Carl Roth</td><td>6351.2</td><td></td></tr><tr><td>narrow vials</td><td>Genesee Scientific</td><td>32-109</td><td>Only for live imaging involving &lt;em&gt;Drosophila&lt;/em&gt;</td></tr><tr><td>small paint brush</td><td>VWR International</td><td>149-2121</td><td></td></tr><tr><td>sodium hypochlorite (NaOCl), ~12% active Cl</td><td>Carl Roth</td><td>9062.3</td><td>Caution: sodium hypochlorite is corrosive</td></tr><tr><td>whole wheat flour</td><td>Demeter e.V.</td><td>SP061036</td><td>Only for live imaging involving &lt;em&gt;Tribolium&lt;/em&gt; / United Kingdom: wholemeal flour</td></tr><tr><td>wide vials</td><td>Genesee Scientific</td><td>32-110</td><td>Only for live imaging involving &lt;em&gt;Drosophila&lt;/em&gt;</td></tr></tbody></table>

simultaneous live imaging of multiple insect embryos in sample chamber-based light sheet fluorescence microscopes

基于光片的荧光显微镜为研究多个生物相关尺度上的复杂过程提供了有效的解决方案。样品室设置是发育生物学的最佳选择，这些设置是专门为保持标本的三维完整性而设计的，通常具有样品旋转功能。例如，它们被用来记录果蝇 德罗索菲拉褪黑麦加斯特 和红面粉甲虫 三宝座的整个胚胎形态。然而，许多可用的活成像协议只为单个胚胎提供实验框架。特别是对于比较研究，这种方法不方便，因为顺序成像标本受环境方差的影响。此外，这限制了在给定时间内可以检测的标本数量。我们为同步实时成像提供一个实验框架，以增加基于样品室的设置的吞吐量，从而确保所有标本的类似环境条件。首先，我们为光片荧光显微镜提供校准指南。其次，我们提出了一种与样本旋转相容的多个胚胎的安装方法。第三，我们提供示范性的三维D 罗索菲拉活成像数据集，为此，我们将三条转基因线与荧光标记的核以及 三聚氰胺并列，为此我们比较了携带相同转基因但在不同基因组位置的三条转基因子系的性能。我们的协议是专门为比较研究设计的，因为它主动解决环境方差，这总是存在于连续实时成像中。这对于定量分析和异常表型的定性尤其重要，例如，从淘汰实验中产生的表型。此外，它增加了总吞吐量，当光片荧光显微镜的获取有限时，这非常方便。最后，建议的安装方法可以适应其他昆虫物种和进一步的模型生物，如斑马鱼，基本上没有优化的努力。

我们的协议描述了基于样品室 LSFMs 的比较荧光实时成像的实验框架。例如，该框架可用于并列两个或两个以上物种的胚胎，（ii） 一个或多个基因被击倒的线状胚胎加上野生型对照组:（iii） 同一转基因系的多个胚胎:（iv） 来自不同转基因系的胚胎，或（v） 携带相同转基因基因的子线胚胎，但在不同的基因组位置。在本节中，我们为最后两个方案提供示例。
在我们的第一?...

Watch this Scientific Journal Video about Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes at JoVE.com

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

光片荧光显微镜是发育生物学中最有价值的工具。比较研究中的一个主要问题是环境差异。我们的协议描述了多个标本同时进行实时成像的实验框架，因此，主动解决这个问题。

样品室光片荧光显微镜中多个昆虫胚胎的同步实时成像

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample ...

simultaneous-live-imaging-multiple-insect-embryos-sample-chamber

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

2.9K 次观看.  Goethe-Universität. 光片荧光显微镜是发育生物学中最有价值的工具。比较研究中的一个主要问题是环境差异。我们的协议描述了多个标本同时进行实时成像的实验框架，因此，主动解决这个问题。

视频: Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

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 ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

光片为主的生活的荧光显微镜或固定和染色<em&gt;赤拟谷盗</em&gt;胚胎

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

生物学

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

使用自动高含量分析仪进行活细胞显微镜的果蝇胚胎制备和微注射

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, proliferation, and differentiation. Time-lapse imagining offers unique insight to the behavior of cells during eye development because it allows us to visualize oculogenesis in vivo. Zebrafish are an excellent model to visualize this process due to their highly conserved vertebrate eye and their ability to develop rapidly and externally while remaining optically transparent. Time-lapse imaging studies of zebrafish eye development are greatly facilitated by use of the transgenic zebrafish line Tg(rx3:GFP). In the developing forebrain, rx3:GFP expression marks the cells of the single eye field, and GFP continues to be expressed as the eye field evaginates to form an optic vesicle, which then invaginates to form an optic cup. High resolution time lapse imaging of rx3:GFP expression, therefore, allows us to track the eye primordium through time as it develops into the retina. Lightsheet microscopy is an ideal method to image ocular morphogenesis over time due to its ability to penetrate thicker samples for fluorescent imaging, minimize photobleaching and phototoxicity, and image at a high speed. Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software. The resulting dataset can provide valuable insights into the process of ocular morphogenesis, as well as perturbations to this process as a result of genetic mutation, exposure to pharmacological agents, or other experimental manipulations.

Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software.&#160;

使用rx3:GFP转基因斑马鱼通过光片显微镜可视化眼部形态发生

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

<meta charset="utf8"></head><body>斑马鱼胚胎的精细多视图光片显微镜实验方案

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

<meta charset="utf8"></head><body>作者聚焦:用于高分辨率多视图光片显微镜的斑马鱼胚胎安装策略 — 成像和图像重建技术

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

早期斑马鱼胚胎的光片显微镜成像和安装策略

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

样品室光片荧光显微镜中多个昆虫胚胎的同步实时成像

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在活斑马鱼胚胎成像的亚细胞结构

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

光片为主的生活的荧光显微镜或固定和染色

实时成像果蝇胚胎血细胞迁移

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

使用自动高含量分析仪进行活细胞显微镜的果蝇胚胎制备和微注射

使用rx3:GFP转基因斑马鱼通过光片显微镜可视化眼部形态发生

成像 C。线虫胚胎使用啶显微镜和开放源码软件

早期斑马鱼胚胎的光片显微镜成像和安装策略

早期斑马鱼胚胎的光片显微镜成像和安装策略