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。

<ol>
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ImageJ macros [software] Available from: <a target="_blank" href="https://github.com/nicolasloyer?tab=repositories">https://github.com/nicolasloyer?tab=repositories</a> (2020)</li></ol>

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&#39;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 ...

applications-immobilization-drosophila-tissues-with-fibrin-clots-for

Research

JoVE Journal

Developmental Biology

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

3.0K 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

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

长期的实时成像<em&gt;果蝇</em&gt;眼睛光盘

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

成像核内作用棒在活热应力 果蝇 胚胎

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

晚期胚胎 果蝇 性腺的解剖和活体成像

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

使用实时成像可视化氧化损伤对果蝇卵室的影响

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

使用纤维素凝块固定 的果蝇 组织用于实时成像

使用纤维素凝块固定的果蝇组织用于实时成像