Januschke 实验室的工作由威康信托赠款 100031/Z/12/A 和 1000032/Z/12/A 支持。组织成像设施由威康的 WT101468 赠款支持。

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作者没有什么可透露的。

整个坐骑 D.黑色素加斯特 幼虫大脑的实时成像提供了在接近生理背景的条件下观察不对称神经干细胞分裂的机会。我们协议的第一部分介绍了我们解剖幼虫大脑的方法。正如已经说过的13，准备的一个关键方面是避免损害大脑。其中最具挑战性的方面是将大脑与邻近的想象磁盘分开，而不要过度拉扯大脑。我们的方法"切割不拉"是执行磨削运动与一对钳子的尖端，或滑动钳尖沿另外两个钳子提示持有组织之间的连接（图1A），而莱利特等人描述锯状运动与解剖针。我们建议实验者尝试所有方法，并采用最适合自己的方法。我们还建议使用BSA或FCS涂层的移液器提示，而不是解剖工具来转移孤立的大脑。涂层可防止组织粘附在塑料上，并允许组织安全转移，同时始终保持组织完全浸入，而不会使组织在转移过程中粘附在解剖工具上时出现可能的瞬态变形。涂层提示具有其他优势，允许同时转移多个大脑，这在控制特定介质中样本的驻留时间至关重要时特别有用:它们适合转移其他组织，即使是脆弱的组织，例如，脂肪体:它们可以通过使用较大的提示或切割尖端的四肢来容纳各种不同的样本大小。
接下来，此协议描述了使用纤维蛋白原凝固来固定文化盘盖上的幼虫大脑。大脑定向在一滴培养基 + 纤维蛋白原中， 之后血凝是由血栓的加入引起的。纤维素的形成是渐进的，提供了一个时间窗口，在此期间，样品的方向可以微调，如有必要（图2C）。如果需要稍微压缩样本（我们建议在幼虫大脑中不要压缩样本），也可以在步骤 3.4.4 和 3.5.2 期间按压血块或多或少地靠近样本进行微调。这种纤维蛋白原凝固的主要优点是能够在实时成像过程中取代培养介质。在我们的协议中使用气体透气膜的一个可能优势可能是，它提供了样品的最佳氧化，随着大脑被血块和某种介质从空气中分离出来，这种氧化可能更有限。因此，虽然我们没有评估文化中文化媒介的数量的影响，但我们建议限制血块顶部的文化介质的数量，同时保持血块完全浸入。
由于血块的操纵在实验上可能困难且耗时，我们建议实验者首先练习在没有样品的情况下操纵血块。调节覆盖唇上的培养介质 + 纤维蛋白原的下降量、纤维蛋白原的浓度或血栓的体积和浓度可以帮助使准备更加容易，并且在将协议调整到其他类型的组织时可能很重要。如果有足够的经验操纵血块，如果有必要，几个大脑可以固定在一个血块中，尽管它使方向的微调复杂化。在盖片上可以形成几个血块，例如，允许对不同基因型的样本进行成像。也可以将同一封面上的不同血块暴露给不同的文化媒体，尽管必须注意永远不要将覆盖不同血块的文化媒介的滴滴混合在一起。一旦幼虫大脑固定并准备好进行实时成像，我们建议遵循 Lerit 等人13 的建议，以避免过度的光损伤。我们只添加这些建议，不仅保持大脑在25°C的现场成像与舞台孵化器，而且还预热这个阶段至少30分钟之前，成像开始。在我们手中，如果不能系统地做到这一点，就会导致强烈的焦点漂移。
在某些情况下，能够在实时成像后进行相关显微镜检查、修复和免疫污渍示例是有利的。然而，使用纤维蛋白块的一个限制是，在不损坏大脑的情况下，机械地将大脑从血块中分离出几乎是不可能的。克服这一限制的一个可能的方法可能是开发方法，用普拉斯明降解纤维蛋白血块或通过添加肽模仿旋钮允许纤维蛋白聚合28诱导血块拆解。我们通常使用纤维蛋白凝块的样本范围从单一细胞到200 μM长组织，但我们也可以成功地固定在血块和图像大脑从成年大黄蜂超过3毫米宽。可以在血块中固定的样本量上限仍有待确定。同样，虽然我们通常图像固定样品4至5小时，我们成功地成像神经母细胞在初级培养长达3天，表明血块至少不干扰长期生存能力。相反，血块可以促进定期更换介质，这是长期成像的要求，而无需为此目的使用围网泵等设备。虽然我们尚未测量纤维蛋白块的渗透参数，但血块的纤维凝胶状性质似乎可以通过细胞渗透分子渗透。我们发现，在细胞生物学中使用的拉特伦库林A、科尔塞米德或1-NAPP1、HALO标签配体和其他标准染料到达纤维蛋白块中的细胞时没有任何问题，迄今尚未遇到凝块保留的分子，然而，这种可能性需要经验测试。
总之，使用纤维蛋白块提供了一个可靠和相对容易实现固定活组织活成像的方法。除了在限制横向漂移方面有用外，在活成像过程中改变培养介质的能力对于我们研究D.黑色素加斯特神经母细胞21的不对称细胞分裂的化学遗传学方法也被证明是无价的。我们预计，这项技术将有利于研究广泛的不同组织，特别是如果他们涉及化学遗传学，例如，蛋白质自我标记29。
我们的多倍堆栈宏依赖于涡轮Reg图像J插件，它对齐连续帧或堆栈的切片，以及多堆栈图像J插件，允许将转换应用于其他堆栈。MultiHyperStackReg 只需允许将这些转换应用于超堆栈（至少具有四个维度的堆栈）。由于使用多倍栈注册，正如我们描述的那样，需要大量的操作，并且可以非常耗时，我们还编写了自动HyperStackReg宏，它自动执行步骤 6.2 和 6.3 中描述的所有步骤。然而，与多倍堆栈相反，AutoHyperStackReg 目前缺乏根据该堆栈的子区域计算堆栈对齐的可能性，我们发现，这一选项有时对于令人满意的对齐至关重要。AutoHyperStackReg 的另一个限制是，其使用仅限于翻译，而不是 TurboReg 和 MultiStackReg 提出的其他转换，而 MultiHyperStackReg 可以适用于任何转换类型的超堆栈，如果用户需要它。未来的版本将实现这些功能，并将在GitHub30上提供。最后，我们的旋转线可以宏提供一个简单的方法来快速测量皮质信号的时间流逝，即使皮层改变其位置或方向随着时间的推移。其跟踪模式或非跟踪模式是否最适合分析取决于皮质信号的质量和一致性。跟踪模式更易于使用，因为用户不需要考虑每个时间点的皮层位置，并且可以适应位置和方向随着时间变化的巨大变化。然而，只有皮质信号保持足够强大，以便在整个电影中检测:未能检测皮层以外的任何东西（例如，靠近皮层的明亮的细胞质隔间）可能会危及每个后续时间点的检测（例如，细胞质隔间远离皮层，皮质线可以跟随它度过剩余时间）。非跟踪模式没有此陷阱，因为检测仅限于用户最初绘制的线（例如，明亮的细胞质隔间可能导致检测失败几个时间点，但随着细胞质隔间远离检测区域，皮层的正确检测会恢复），但如果皮层位置或方向随着时间的推移变化很大，则表现不佳。为跟踪模式开发的下一个功能（将在 GitHub30上可用）将仅用于分析指定时间点和定义参考时间点（而不是执行检测之前和之后的第一个时间点），这将允许用户至少避免有问题的时间点。

Drosophila仍然是研究各种生物问题的重要模型系统，几十年来在许多学科中擅长提高知识。使其特别突出的一个特点是，它特别适合现场成像。实时监测细胞子过程或细胞和组织行为的能力继续有助于生成与细胞和发育生物学相关的关键概念。许多成功的协议已经由不同的群体开发，以保持这种果蝇样品活着和健康，以研究标本和荧光分子的行为生活。来自苍蝇的器官和组织，如假想盘1，2，幼脑3，4，5，6，或卵室的成年女性7，8，9，或成人肠10例如可以提取和保存在培养成像与延时显微镜。实时成像的一个关键挑战是确保标本保持在成像表面附近，以获得最佳分辨率和不动，同时不影响对机械撞击敏感的样品完整性。例如，为了研究神经干细胞，称为神经母细胞或神经元在喉部大脑中，通过在密封的成像室11、12、13中用培养介质覆盖它们，可以使样本靠近成像表面。然而，这种方法不容易允许媒体交流，从而限制了实验室的机动性。或者，样品可以准备和放置在盖片上处理，以增加细胞的粘附性，并允许多点访问显微镜和扩展活细胞成像在开放培养皿，有可能允许媒体交换，但不能提供简单的手段，以防止样品漂移或损失期间媒体交换14，15。
最初为研究活鹤飞精子细胞16中的肌瘤而开发的纤维蛋白凝块方法特别适合克服这些问题。纤维蛋白原溶解在相关培养介质中，样品在加入Thrombin之前放置并定向在足够的玻璃表面成像室上，导致迅速形成不溶性纤维素凝块，粘性纤维网，粘附在玻璃表面和样品上，同时确保样品进入培养基。然后，在不干扰血块或样品位置的情况下交换介质。例如，当像原始方法一样添加抑制剂，或用于洗掉或脉冲追逐实验时，或在活细胞成像过程中添加荧光染料，同时保持细胞和组织到位时，培养介质交换是可取的。纤维蛋白块适合成像的嗜血杆菌组织以及个别细胞13，17。纤维蛋白块可以进一步用于帮助定向样品，由于其形状或其他特性，通常不会通过调节纤维蛋白块内的样品位置和血块本身的形状来以所需的方式定向。
在这里，我们提供最新的我们目前的纤维蛋白块为基础的成像方法，并提供工具，基于分割的图像分析，并专注于使用这种方法来研究神经母细胞分裂在发展中的苍蝇大脑。我们通常使用纤维素凝块方法，以遵循多个样品在不同的血块在同一培养皿。这允许 i） 成像子细胞事件，如中心体行为或 mRNA 本地化活18，19，ii） 监测样本的行为在药理处理不同基因型下使用多点访问 显微镜20，iii）研究酶急性抑制的影响21和iv）尽量减少漂移，以研究细胞在生理环境中的细胞分裂方向的变化，在有针对性的激光消融22。虽然血栓和纤维蛋白原和纤维蛋白块的不需要的影响需要对每个样本进行经验测试， 这种方法原则上适用于活细胞成像分析的任何类型的样本，在Ddrosophila中已成功地用于研究神经母细胞生物学的多个方面5，19，20，而且女性生殖系干细胞23的细胞传感器投影的动力学和极性的变化后，急性APKC抑制卵泡细胞的德索菲拉女性卵室21。
延时荧光成像导致生成复杂的时间解决的 3D 数据集，这些数据集需要提取此定量信息的方法。在这里，我们描述了我们基于 ImageJ 的24 宏的进一步发展，这些宏可用于纠正超堆栈和定制的 ImageJ 宏的漂移，从而允许对细胞皮层的多通道荧光进行半自动量化，以量化 Drosophila 幼虫大脑神经母细胞中的皮质蛋白质或初级细胞培养中的单个神经母细胞。

<table><tbody><tr><td>Albumin bovine serum, BSA</td><td>Merck</td><td>5470</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G7021</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Dumont #55 Forceps</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Fetal Bovie Serum, suitable for cell culture</td><td>Sigma</td><td>F7524</td><td></td></tr><tr><td>Fibrinogen from human plasma</td><td>Sigma</td><td>F3879</td><td></td></tr><tr><td>FluoroDish Cell Culture Dish - 35mm, 23 mm well</td><td>World Precision Instruments</td><td>FD35-100</td><td></td></tr><tr><td>PP1 Analog (1NA-PP1)</td><td>Merck Millipore</td><td>529579</td><td></td></tr><tr><td>Pyrex spot plate with nine depressions</td><td>Sigma</td><td>CLS722085-18EA</td><td></td></tr><tr><td>Schneider&#039;s Insect Medium</td><td>Sigma</td><td>S0146</td><td></td></tr><tr><td>Thrombin from bovine plasma</td><td>Merck</td><td>T4648</td><td></td></tr></tbody></table>

applications of immobilization of drosophila tissues with fibrin clots for live imaging

嗜血杆菌 是研究各种生物问题的重要模型系统。可以提取和保存来自苍蝇不同发育阶段的各种器官和组织，如假想盘、成年女性的幼脑或卵室或成年肠，用于使用延时显微镜成像，为细胞和发育生物学提供有价值的见解。在这里，我们详细描述了我们目前关于切除 喉 部的方案，然后介绍我们目前的方法，即使用纤维素血块在玻璃盖上固定和定位幼虫大脑和其他组织。这种固定方法只需要在文化媒介中加入纤维蛋白原和血栓。它适用于在同一培养皿中多个样品的倒显微镜上进行高分辨率时间延迟成像，最大限度地减少显微镜阶段多点访问显微镜运动中频繁造成的横向漂移，并允许在成像过程中添加和去除试剂。我们还提供定制宏，我们经常用来纠正漂移，并从延时分析中提取和处理特定的定量信息。

在现场成像期间，用纤维蛋白凝块和培养介质交换的果蝇组织固定。 在第2步（图1）中提出的程序被解剖后，并在第3步（图2）中提出的程序后，在同一盖片上的纤维蛋白血块中固定，幼虫大脑表示GFP标记的Bazooka（Baz::GFP），极性蛋白Par-3的飞行正交，在延时多位置对照成像中拍摄了30分钟。Baz::GFP 与apkcas4结合，这是一种等位基因，通过添加小型 ATP 模拟 1-NAPP120，可急性抑制非典型蛋白激酶 C （aPKC）。因此，在第 4 步中提出的程序之后，将 1-NAPP1 添加到培养介质中，并恢复 2.5 小时的实时成像。携带激酶 aPKC 的野生类型版本的控制大脑对 ATP 模拟没有反应，而携带 APKC（apkcas4）的模拟敏感突变的大脑在神经母细胞及其后代中显示巴兹神经肽和明亮集群的收缩（图 6，视频 1）。神经母细胞在整个延时期间不断分裂，表明组织保持健康，大脑尽管培养介质变化，但几乎没有漂移，表明它们已经很好地固定。同样，在27日被解剖后，表示巴兹的卵石:GFP在同一盖片上的菲布林血块中固定，并成像8分钟，之后1-NAPP1的应用导致apkc作为4突变卵泡细胞的收缩，而不是控制（图7，视频2）。
使用多倍堆栈注册纠正横向和对焦漂移。 显示横向和对焦漂移（视频 3，左面板）的超堆栈首先被更正为横向漂移（步骤 6.2，图 3，视频 3，中间面板），然后使用 MultiStackReg 插件和多堆栈注册宏对焦点漂移（步骤 6.3，图 4，视频 3，右面板）进行更正，从而大幅减少在原始超堆中观察到的运动。
使用旋转线扫描的皮质信号测量 表达巴兹的神经母细胞::GFP（绿色）和带有红外荧光蛋白（CD4::mIFP，红色）标记的跨膜蛋白CD4在一次迷雾中被成像。CD4::mIFP 信号用于用线卡（第 7 步、图 5、图 8A– C）检测神经母细胞不同部位的皮层，并在检测到的位置测量 Baz::GFP 信号（图 8D）。在分裂过程中，神经母细胞通过建立不同和相反的皮质域（肛门极和基底极）暂时两极分化。Baz 定义了 apical 极点，并且一致地，Baz::GFP 信号在神经母细胞的 apical 极中增加，并在分裂期间在基底极减少（图 8C，D）。皮层的位置在整个延时期间被令人满意地跟踪（图8C，视频4）。德罗索菲拉线和基因型在补充表1-2中引用。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61954/61954fig06.jpg"> 图6:在实时成像过程中，在固定幼虫大脑上应用ATP模拟。（A） 两个幼虫大脑的实时成像表达巴兹::GFP在同一盖片上固定。培养介质在 0 分钟内更改为 ATP 模拟 1-NAPP1 的 10 μM 浓度。控制大脑对ATP模拟没有明显反应，而携带APKC（aPKCAS4）模拟敏感突变的大脑显示神经细胞（箭头）收缩，神经母细胞及其后代中巴兹的明亮群落收缩。最大强度投影33片，深度为23微米。 比例杆:20μM.（B） 神经肽的放大。比例栏: 10 μM. <a href="https://www.jove.com/files/ftp_upload/61954/61954fig06large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61954/61954fig07.jpg"> 图7:在现场成像过程中，在固定的蛋室上应用ATP模拟。表达巴兹的两个卵子的实时成像: Gfp 在同一封面上固定。培养介质在 0 分钟内更改为 ATP 模拟 1-NAPP1 的 10 μM 浓度。控制卵室不会明显对ATP模拟反应，而携带APKC（aPKCAS4）模拟敏感突变的卵室显示上皮（箭头）收缩，最终导致粘附结点的明显分解。最大强度投影 33 片， 深度为 27μM 。比例栏: 20μM。 <a href="https://www.jove.com/files/ftp_upload/61954/61954fig07large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61954/61954fig08.jpg"> 图8:使用旋转线扫描皮质信号的测量。（A） 活 D. 梅拉诺加斯特 神经母细胞表达巴兹: : Gfp （绿色） 和 CD4:: mIFP （红色）.青色线:初始扫描线垂直跟踪到各种皮质区域进行测量。比例杆: 5 μM. （B） 使用皮质线和 CD4:mIFP （红色） 作为参考通道识别皮层 （深蓝色线） 。青色线:由"测量周围"参数定义的区域（此处为 2 像素），在皮层检测后测量信号。比例杆:2 μm. （C） 青色:在神经母细胞分裂过程中通过旋转线扫描方法从 （A） 中显示的初始扫描线识别皮质区域，使用 CD4::mIFP（红色）作为参考通道。比例杆: 5 μm. 箭头: 阿皮卡杆。箭头:基底杆。（D） 测量 Baz::GFP 信号强度随着时间的推移，在两个相应的区域，由旋转线卡显示在 （C） 中。在间歇期，Baz::GFP在神经母细胞的顶点极短暂地富集，从对面的基底极耗尽。 <a href="https://www.jove.com/files/ftp_upload/61954/61954fig08large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
视频 1: 在现场成像期间在固定幼虫大脑上应用媒体交换， 以添加抑制剂， 呈现在图 6中， 比例杆: 20μm.<a href="https://cloudflare.jove.com/files/ftp_upload/61954/MOV_1_Loyer_Januschke.mp4" target="_blank">请点击这里下载此视频。</a>
视频 2: 在现场成像期间在固定蛋室上应用媒体交换， 以添加抑制剂， 呈现在图 7中， 比例栏: 20 μm.<a href="https://cloudflare.jove.com/files/ftp_upload/61954/MOV_2_Loyer_Januschke.mp4" target="_blank">请点击这里下载此视频。</a>
视频 3: 使用多倍栈注册纠正横向和对焦漂移。 表达膜探头 PH::GFP 的神经母细胞的实时成像。Xy:单焦平面。Xz:正交遗物。左图:在修正漂移之前。中间:横向漂移修正后。右图:修正横向和对焦漂移后，比例栏:10μm。 <a href="https://cloudflare.jove.com/files/ftp_upload/61954/MOV_3_Loyer_Januschke.mp4" target="_blank">请单击此处下载此视频。</a>
视频 4: 使用旋转线扫描皮层的检测， 以图 8呈现， 比例栏: 5 μm.<a href="https://cloudflare.jove.com/files/ftp_upload/61954/MOV_4_Loyer_Januschke.mp4" target="_blank">请点击这里下载此视频。</a>
补充表 1: 成像的 德罗索菲拉 组织的基因型。 <a href="https://cloudflare.jove.com/files/ftp_upload/61954/Supplemental_Table_1_genotypes.xlsx" target="_blank">请单击此处下载此表。</a>
补充表 2: 使用的转基因基因的起源。 <a href="https://cloudflare.jove.com/files/ftp_upload/61954/Supplemental_Table_2_origine_of_transgenes.xlsx" target="_blank">请单击此处下载此表。</a>
补充编码文件 1:使用的转基因基因的起源。<a href="https://cloudflare.jove.com/files/ftp_upload/61954/Code1_MultiHyperstackReg.ijm" target="_blank">请单击此处下载此文件。</a>
补充编码文件 2:使用的转基因基因的起源。<a href="https://cloudflare.jove.com/files/ftp_upload/61954/Code2_AutoHyperstackReg.ijm" target="_blank">请单击此处下载此文件。</a>
补充编码文件 3:使用的转基因基因的起源。<a href="https://cloudflare.jove.com/files/ftp_upload/61954/Code3_RotatingLineScan.ijm" target="_blank">请单击此处下载此文件。</a>

Watch this Scientific Journal Video about Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging at JoVE.com

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

该协议描述了使用纤维蛋白块的样品固定应用，限制漂移，并允许在现场成像过程中添加和冲洗试剂。样品被转移到一滴含有培养基的纤维素中，然后通过添加血栓诱导聚合。

使用纤维素凝块固定 的果蝇 组织用于实时成像

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of ...

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Developmental Biology

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

3.1K 次观看.  University of Dundee. 该协议描述了使用纤维蛋白块的样品固定应用，限制漂移，并允许在现场成像过程中添加和冲洗试剂。样品被转移到一滴含有培养基的纤维素中，然后通过添加血栓诱导聚合。

视频: Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

科研

生物工程

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

生物学

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

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 ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

发展和成人的制备果蝇大脑和视网膜实时成像

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

在体内果蝇前运动神经末端的单分子跟踪

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

神经科学

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

LarvaSPA，一种用于长期延时成像的安装果蝇幼虫的方法

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...

使用纤维素凝块固定的果蝇组织用于实时成像

摘要

探索更多视频

使用微流体芯片实时成像和损伤研究的响应

可视化的Live果蝇胶质细胞神经肌肉交界处，与荧光染料

编制果蝇实时成像的胚胎使用悬滴协议

实时成像果蝇胚胎血细胞迁移

发展和成人的制备果蝇大脑和视网膜实时成像

在完整的果蝇幼虫体内的亚细胞分辨率成像

在体内果蝇前运动神经末端的单分子跟踪

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

GFP标记的蛋白质在实时成像

LarvaSPA，一种用于长期延时成像的安装果蝇幼虫的方法

使用纤维素凝块固定 的果蝇 组织用于实时成像

摘要

探索更多视频

使用微流体芯片实时成像和损伤研究的响应

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

编制果蝇实时成像的胚胎使用悬滴协议

实时成像果蝇胚胎血细胞迁移

发展和成人的制备果蝇大脑和视网膜实时成像

在完整的果蝇幼虫体内的亚细胞分辨率成像

在体内果蝇前运动神经末端的单分子跟踪

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

GFP标记的蛋白质在实时成像

LarvaSPA，一种用于长期延时成像的安装果蝇幼虫的方法

使用纤维素凝块固定的果蝇组织用于实时成像

可视化的Live果蝇胶质细胞神经肌肉交界处，与荧光染料