The authors thank David Larson for developing the original protocol for imaging the live Drosophila pupal eye. We thank our colleagues Richard Carthew, Shoichiro Tsukita and Matthew Freeman, who developed the transgenic flies that we combined to generate our live-imaging fly lines. Brittany Baldwin-Hunter gave helpful comments on the manuscript. This work was supported by start-up funds awarded to Ruth Johnson by Wesleyan University.

图3示出的实验程序的概要。 1.组织准备<ol><li>要确定目标基因的表达修饰的后果，双份以下的交叉，在25℃保持C:UAS-转基因 （男性）X GMR-GAL4; UAS-α-猫:GFP，UBI-DE-加元:GFP; + / SM5-TM6b（处女女）注:要获得控制组织交叉UAS-lacZ的男性为GMR-GAL4; UAS-α-猫:GFP，UBI-DE-加元:女性GFP。 β半乳糖苷酶产生在视网膜中的细胞行为没有缺陷。 </li><li>为了提高转基因的RNAi的疗效，跨UAS-RNA干扰雄性GMR-GAL4，UAS-DCR-2; UAS-α-猫:GFP，UBI-DE-加元:GFP; + / SM5-TM6b处女的女性。 注:在细胞行为表达异位DCR-2已经观察到偶尔的缺陷在视网膜（数据未示出）。警惕，建议如果使用这种方法。 </li><li> S选白色预蛹适当的基因型和地点1.5 ml离心管。如果蛹表达DCR-2，选择男或女蛹:UAS-DCR-2，X染色体上的插入，表达是男性更强。蛹应在0小时的APF蛹壳的色素前绝育手术 - 男性性腺如在蛹（大约三分之一从后）的侧面大半透明地球仪可见。 </li><li>孵育含蛹，在一个干净的塑料提示框，在25℃的离心管。为了防止蛹的地方干燥10 平方厘米片组织，浸泡在蒸馏水中，将尖端盒。 注意:如果湿度在尖部 - 框变得太高蛹壳可能会变得潮湿和难以在后续步骤中进行解剖。 </li><li>孵育适当的时间。为了捕捉构图眼有关的细胞行为，准备蛹成像约17小时APF，20小时APF或24小时APF.查看时，特定的细胞行为是最好的观察作进一步的讨论代表性的成果。 </li><li>将一块新鲜的双面胶带上一个黑色的SYLGARD解剖盘。定位蛹，背侧，与坚持双面胶带（ 图1A）蛹的头。尝试不坚持蛹的胸部和腹部地区的双面胶带。用钳子小心地提起并取出鳃盖，露出蛹头。沿蛹壳的侧撕到区域露出围绕一只眼睛。 </li><li>轻轻地取下胶带蛹。如果蛹仍粘附的，施加一个小体积的蒸馏水，以结蛹壳和带之间，以削弱的粘合。 </li></ol> 2.安装<ol><li>构造从吸墨纸小15毫米2帧和冲头在middlethat一个孔为5.5毫米的直径（ 图1B）。浸泡在蒸馏水中并将其放置在中心的显微镜载玻片。使用30毫升注射器，挤压出凡士林的均匀环以围绕吸墨纸帧。确保凡士林环稍高于蛹的宽度和该环的直径略微大于盖玻片的宽度小于高。 </li><li>添加一个小珠凡士林直接到幻灯片，在冲压成的吸水纸（ 图1C）.Carefully安排蛹，在其一侧洞口，由凡士林珠（ 图1D）的支持。 </li><li>放置盖玻片上凡士林环的顶部，使得其接触上述眼球神经上皮（ 图1D）的上皮。轻轻压缩编制以密封的凡士林盖玻片并生成蛹眼睛区域和盖玻片之间的小平面的接触表面。 </li></ol> 3.荧光成像<ol><li>图像使用蛹准备荧光显微镜。每7分钟捕获通过眼睛神经上皮的心尖域，在粘着路口的区域连续切片。 注:a）适当的串行部分通常是4-5元的z栈包括0.2微米的Z-步骤。 b）在上皮细胞的拓扑结构，包括轻度颠簸和不规则的褶皱，可能使其具有挑战性的收购聚焦图像组织大型的区域。而成像，人们可以发现感兴趣的区域的一部分，以在聚焦，和相邻区域是失焦。包括蒸馏水蛹眼睛和盖玻片之间的小液滴可以消除一些急性组织褶皱而不是蛹眼形态的早期阶段的轻度不均匀拓扑特征。在这些情况下，过采样的组织通过扩展Z堆叠直到在焦切片获取整个字段。三）捕获连续切片，每7分钟应使活成像为3至4个小时没有显著热镀ction在GFP荧光可能危害图像质量的强度。较短的时间间隔可以降低成像的总时间。 </li><li>继续成像为3至4个小时。不要使用自动对焦和时间的函数，是许多荧光显微镜，因为蛹增长和血淋巴抽移动视网膜的位置警惕再聚焦，需要每14-21分钟。注:该成像超过4时间可能会减缓或停滞眼睛的形态。 </li><li>使用适当的反卷积软件，以减少背景和增强连续切片图像的对比度。对于每一个Z堆栈文件，请在LAS AF软件以下（见表材料）:导航到工具面板中，选择3D反卷积，然后单击应用。 </li><li>生成每个卷积堆栈文件的最大投影（MP）影像:导航到工具面板中，选择3D投影，然后单击应用。 注:最大投影的算法可能无法生成一个统一的LY聚焦图像用于组织已经被过取样。一个技术磨砺出焦地区在步骤5.2概述。 </li></ol> 4.成像后蛹救援和表型验证<ol><li>为了解决文物是否引进或成像过程中损害了蛹，小心地取出从成像钻机蛹:使用镊子取出盖玻片和蛹转移到食物的干净的小瓶。储存在室温或25℃，直到成年果蝇出现。 </li><li>仔细检查成人眼睛的表型，并比较成蝇新兴从原始蝇交叉的眼睛。 </li></ol> 5.图像处理<ol><li>自动图像对齐方式: 注:本次现场果蝇组织将转向和整个成像过程成长。因此，图像的中心聚集在连续的时间点可能不对应于同一点，在果蝇的组织。在另外的整个视网膜盘片旋转21〜23小时之间APF约30°。为了突出单个细胞的行为和减少因有机体的生长杂念，调整每个MP image.While这可以手动执行，这里使用的图像编辑软件（ 见表材料 ）有内置的算法，可以加快这一进程。 <ol><li>从主菜单，打开脚本，选择将文件加载到堆栈的文件选项卡。 </li><li>导入MP图像文件转换加载图层面板中，选择确定。确保尝试自动对齐源图像选项没有被选中。 注意:如果选择此选项，该软件可使用比对算法的扭曲的图像数据。 </li><li>一旦所有的MP文件已加载到图层面板中，确保所有的图像都按时间顺序，例如，最早的时间点是在层堆栈的顶部。如果层不按正确的顺序，拖放他们，直到他们被命令正确。 </li><li>从主菜单中的编辑选项卡中选择自动对齐图层。选择重新定位，然后单击确定。 </li><li>如果自动对齐算法不能帧定位到相关的焦点，让使用移动工具小幅调整。 </li></ol></li><li>复合锐化: 注:离焦的初始的MP图像的区域可以通过"裁剪"在LAS AF软件的相应解卷积堆栈进行锐化。裁剪一叠文件允许一个手动限制经受最大投影算法的z堆栈的间隔。 <ol><li>找到在工具面板中的裁剪功能（进程选项卡下）。手动限制的最初和最后的片，使得它们仅跨越最初失焦区域内的粘合带。单击应用生成针对该区域优化的新的堆栈文件。 </li><li>生成一个新的投影最大的本地优化堆栈和出口为TIFF文件。 </li><li>在日Ë图像编辑软件（见表材料），开放式脚本（位于主菜单中的文件选项卡），然后选择将文件加载到堆栈。 </li><li>导入特定时间点进入加载图层面板中的初始MP图像文件，并在本地优化的MP文件，并选择确定。确保尝试自动对齐源图像选项没有被选中。 </li><li>选择这两个图层的图层窗格，然后选择自动混合图层（位于主菜单中的编辑选项卡）来自动屏蔽两个图像的适当区域，产生一个统一的，集中的视网膜领域。保存此合成图像为TIFF文件。 </li><li>重复步骤5.2.1到5.2.5的所有不理想的MP图像。 </li></ol></li><li>进一步的调整:旋转，裁剪和调整图层电影的水平: <ol><li>与所有层的选择，使用变换工具（编辑&gt;自由变换），旋转图像，使视网膜的背腹轴一致到电影帧的y轴。 </li><li>如果需要的话，可使用裁剪工具，以减少视场成所需的尺寸和形状。 </li><li>使用电平调整（的各个帧），以帮助提高细胞膜和细胞体之间的对比。 </li><li>为文体的目的，并强调特定的细胞的行为，引入假色突出显示的每个帧中的兴趣点。 </li></ol></li><li>动画:转换图像成电影: <ol><li>从主菜单中的Windows选项卡，打开动画面板。选择做自层帧从位于动画面板的右上角的菜单。 </li><li>注意，这些层被添加到动画窗格按顺序从堆栈的底部开始。为了扭转帧的顺序，首先选择所有的帧，然后从动画面板菜单中选择逆向框架。 </li><li>使用帧拇指下方的下拉菜单中选择用于每个帧一帧的延迟时间钉子。 注:帧延迟为电影1至4本文介绍的是0.8秒。 </li><li>在主菜单中，打开导出的文件选项卡，然后选择渲染视​​频。选择QuickTime导出下文件选项，然后选择所需的视频格式。在渲染选项设置帧速率自定义，进入15帧速率，并单击渲染。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

野生型的发展（上图）的描述形成的基础，在RNAi的图案事件或过表达基因型比较。精确确定哪些细胞行为是由感兴趣的蛋白质调节的时活组织发育的比较是非常宝贵的。此外，并没有在这里描述的，活细胞成像使得人们能够使所关注的基因的作用的定性和/或定量描述的事件模式的眼睛。经30-32小时APF最色素细胞已经获得稳定的位置和凋亡已经从视网膜现场修剪多余的细胞。在此之后，仅微妙的变...

化合物果蝇眼睛的特征在于它的〜750小眼由一个蜂窝状晶格附件色素细胞4,5的隔开的定型装置。局部细胞运动，细胞生长，改变细胞形状和细胞凋亡:这些色素细胞是由事件的协调组合图案化。这种上皮现场可视化允许一个研究在生理学相关和泰然自若的三维环境支撑这些事件的分子机制。 相较于以前的协议6,7，这里概述的技术结合用于稳定外部组织的运动，不能从成像过程耦合的有效方法。这种方法提高了在显影果蝇蛹眼上皮细胞的行为的研究- ，生长，旋转的组织，并移过成像的过程。此外，运动稳定技术去这里划线将是在无关的运动发生其他上下文研究细胞是有用的。 可视化细胞界限在果蝇视网膜领域，产生了转基因飞线表达UBI-DE-钙粘蛋白:GFP和UAS-α-catenin的:GFP在眼部专用的驱动程序GMR-GAL4 1-3的控制权。使用两个GFP标记膜标记允许小区边界的在较低强度的光的可视化。本上重复暴露于高能量波长的光，使增加的帧速率和电影的持续时间最小化组织损伤和光漂白。为了提高转基因的RNAi，UAS-DCR-2也被纳入到第二飞线8疗效。 在以前协议的第三次更新，这是很容易组装在大多数实验室一个简单的成像钻机描述。该装置避免了要求有一个专门的成像řIG一所大学的"加工车间"或类似的服务产生。该成像钻机类似于用于其它图像蛹组织9,10。 这里介绍的是可以用来直接评估，经过17至42小时puparium形成（小时APF）有助于眼睛的图案来自〜的形态发生事件的简单的活细胞成像协议。具体地，该协议使得人们能够确定蛹发育过程中修饰基因表达的后果。

<table><tbody><tr><td>GMR-GAL4; UAS-a-cat:GFP, ubi-DE-Cadherin:GFP ; + / SM5-TM6b</td><td>Available on request from authors</td><td></td><td></td></tr><tr><td>UAS-dcr-2; UAS-a-cat:GFP, ubi-DE-Cadherin:GFP; + / SM5-TM6b&amp;nbsp;</td><td>Available on request from authors</td><td></td><td></td></tr><tr><td>Scotch double stick tape</td><td>3M</td><td>665</td><td></td></tr><tr><td>Black Sylgard dissection dish</td><td></td><td></td><td>Fill glass petri dish to rim with Sylgard (colored black with finely-ground charcoal powder) or Sylgard 170; leave at room temperature for 24-48 hr to polymerize</td></tr><tr><td>Sylgard</td><td>Dow Corning</td><td>SYLG184</td><td></td></tr><tr><td>Sylgard (Black)</td><td>Dow Corning</td><td>SYLG170</td><td></td></tr><tr><td>Glass petri dish</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>25 x 75 x 1 mm glass microscope slide</td><td>Fisher Scientific</td><td>12-550-413</td><td></td></tr><tr><td>22 x 40 mm glass coverslip</td><td>VWR</td><td>48393-172</td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>91150-20</td><td></td></tr><tr><td>Whatman 3mm Chromatography Paper</td><td>Fisher Scientific</td><td>05-713-336</td><td></td></tr><tr><td>Vaseline</td><td>Fisher Scientific</td><td>19-086-291</td><td>Or purchase at local pharmacy.</td></tr><tr><td>30 ml syringe</td><td>Fisher Scientific</td><td>S7510-30</td><td></td></tr><tr><td>Leica TCS SP5 DM microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>LAS AF Version 2.6.0.7266 microscope software</td><td>Leica Microsystems</td><td></td><td></td></tr></tbody></table>

live-imaging of the drosophila pupal eye

果蝇蛹发展的内在过程可以转移和扭曲眼球上皮细胞的方式，使各个细胞的行为很难在活细胞成像跟踪。这些方法包括:视网膜旋转，细胞生长和有机体的运动。此外，在上皮的拓扑，包括细微凸块和凹凸褶皱经常引入作为蛹被用于成像准备，使其具有挑战性获得在单个焦平面在焦像以上几个小眼。这里的工作流程概述了补救这些问题，让细胞过程中果蝇蛹眼睛的发育很容易分析。适当上演蛹被布置在可容易地装配在大多数实验室成像钻机泛素- DE-钙粘蛋白:GFP和GMR-GAL4驱动的UAS-α连环蛋白:GFP被用于可视化小区边界在眼上皮1 -3。反褶积之后是施加到在多个焦平面捕获荧光图像，产生用于每个时间点的最大的投影图像，并使用图像编辑软件增强。比对算法用于快速稳定多余的动作，使得单个细胞的行为更容易追踪。

实时成像蛹眼睛是一个成功的策略，以观察细胞的行为，有助于神经上皮的图案。特定蛋白质的作用，可以通过表达眼睛的发育过程中修改的蛋白质水平的转基因可以容易地评估。要做到这一点的GMR-GAL4用于驱动所述形态发生沟后面的转基因表达。这提供了不扰动早期事件，在头两个幼虫龄建立眼场的优点。此外，许多的RNAi的转基因需要显著的时间来有效地降低蛋白质的水平（数据未示出?...

Watch this Scientific Journal Video about Live-imaging of the Drosophila Pupal Eye at JoVE.com

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 ...

Research

JoVE Journal

Developmental Biology

Live-imaging of the Drosophila Pupal Eye

9.4K 次观看.  Wesleyan University. 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.

视频: Live-imaging of the Drosophila Pupal Eye

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

科研

神经科学

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 ...

发育生物学

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

住在果蝇眼睛成虫光盘胶质细胞迁移成像

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

生物学

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

在体内果蝇蛹肌肌腱形态的影像学研究

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

为介观荧光体层摄影术在体内成像的发展果蝇</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

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 ...

<meta charset="utf8"></head><body>无蛹的 果蝇 Pupal 腿部形态发生的扩展实时成像

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which fate-determined cells form three-dimensional organismal shapes remains unclear. Recent advances in confocal microscopy have facilitated efforts to observe cell dynamics during development through live imaging. The Drosophila melanogaster pupa is ideal for live imaging due to its immobility, transparent pupal cuticle, and the availability of fluorescent reporter lines. A primary challenge for imaging is the puparium, the cuticle surrounding the pupa, which obstructs optical imaging. While previous methods involved either partial or complete removal of the puparium, maintaining pupal viability for extended periods after this procedure has remained challenging. Here, a simple method is presented for days-long live imaging of the Drosophila leg during the pupal stage, involving complete puparium removal. The method includes removing the puparium from a pupa adhered to double-sided tape, followed by assembling a small chamber on a glass-bottom dish to enclose the pupa and a drop of water. This setup is straightforward, reliable, and supports extended pupal survival by preventing desiccation. Long-term live imaging of the Drosophila pupa has significantly contributed to capturing how the adult leg undergoes dramatic three-dimensional structural changes over 2-3 days. These changes include the transient formation of an intriguing structure (the Parthenon-like structure) by epithelial cells, rapid tissue narrowing, joint formation, and bristle elongation. This method is applicable to the observation of various tissues and can potentially be combined with other techniques, such as optical gene induction, to advance the understanding of cell dynamics during the final shape formation of tissues in the pupal stage.

<meta charset="utf8"></head><body>作者聚焦：揭示 果蝇 发育过程中的动态上皮结构

Here, a protocol is presented for long-term live imaging of the Drosophila leg during the pupal stage, involving complete removal of the puparium. This protocol can also be applied to other tissues.

果 蝇 蛹切除后蛹腿部发育的长期实时成像

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which ...

实时成像

摘要

探索更多视频

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

长期的实时成像

住在果蝇眼睛成虫光盘胶质细胞迁移成像

在体内果蝇蛹肌肌腱形态的影像学研究

为介观荧光体层摄影术在体内成像的发展果蝇

的实时成像

发展和成人的制备果蝇大脑和视网膜实时成像

果蝇蛹切除后蛹腿部发育的长期实时成像

果蝇蛹切除后蛹腿部发育的长期实时成像

果蝇蛹切除后蛹腿部发育的长期实时成像

实时成像

摘要

探索更多视频

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

长期的实时成像

住在果蝇眼睛成虫光盘胶质细胞迁移成像

在体内果蝇蛹肌肌腱形态的影像学研究

为介观荧光体层摄影术在体内成像的发展果蝇

的实时成像

发展和成人的制备果蝇大脑和视网膜实时成像

果 蝇 蛹切除后蛹腿部发育的长期实时成像

果 蝇 蛹切除后蛹腿部发育的长期实时成像

果 蝇 蛹切除后蛹腿部发育的长期实时成像

果蝇蛹切除后蛹腿部发育的长期实时成像

果蝇蛹切除后蛹腿部发育的长期实时成像

果蝇蛹切除后蛹腿部发育的长期实时成像