这项工作是支持由格兰特GM096076从NIH区计划。

1。准备苍蝇解剖<ol><li>麻醉健康所需基因型（例如，表达的GFP-标记的蛋白质）是小于一周龄的苍蝇。选择10-15大的女性，圆形的奶油色的腹部。 </li><li>传输所选的苍蝇和几个（3-5）的男性的小瓶中含有新的轻yeasted食品，并允许在25℃下的苍蝇催肥两天的果蝇准备的详细信息，请参阅威尔等6。育肥苍蝇确保各种阶段，尤其是阶段8-12，将可用于成像。不善的育肥或unfattened的，苍蝇通常只包含非常初期的阶段（1-6）和成熟（14期）卵母细胞。 </li></ol> 2。使用一个直立的复合式显微镜的成像卵母细胞的制备<ol><li>准备一个标准的玻璃贴上两个盖玻片相距约1厘米的幻灯片，，使用硅greas的幻灯片E。仅使用足够的润滑油，以确保良好的密封效果的盖玻片和幻灯片之间。用力按下每个盖玻片上传播的油脂薄且均匀。添加一些卤化烃油27（Sigma公司）的盖玻片和幻灯片之间的交界处。他们将作为衬垫，防止伤害孤立的卵母细胞在随后的步骤中。号1.5盖玻片很好地工作，与匹配的后期阶段的卵母细胞（0.16-0.18毫米）的平均厚度。 </li><li>将2-3滴卤烃油27的表面上的一个22毫米2盖玻片。为了获得健康的卵母细胞，个别女从食品瓶用口吸液，并轻轻地敷的卤化烃油不先麻醉。 </li><li>使用锋利的解剖镊子（如杜蒙第5），针对盖玻片飞和拉断尖的腹部。通过拖动它们沿盖玻片的表面，在油中取出的卵巢。这将促进表面粘附N的卵母细胞的盖玻片。 </li><li>轻轻挑逗除了卵巢，寻找阶段10B的卵子发生的卵母细胞。在此阶段的卵母细胞，可以识别通过寻找蛋室，其中的卵母细胞占据的总体积的至少50％-60％的蛋室。在卵子发生在这一点上，覆盖在卵母细胞的卵泡细胞已成为柱状，和环绕护士细胞仍然连接到一个小区域以外的所有侧上的卵母细胞。使用钳删除单个卵母细胞从卵巢管套。 </li><li>继续使用1-3女性，在盖玻片上，直到5-8完好的卵母细胞已被隔离。删除任何不能运行组织。 </li><li>小心反转含有使预先准备好的滑动定位的两个隔离件之间的卵母细胞的卵母细胞上的盖玻片。不要按上倒盖玻片，因为这将损坏的卵母细胞。如有必要，添加少量卤烃油吨他边缘的“三明治”，以确保空间被填满。 （可选：让幻灯片休息的几分钟，以便解决盖玻片）。幻灯片已经准备好为成像。 </li></ol> 3。卵母细胞的制备用倒置的复合式显微镜的成像<ol><li>卵母细胞解剖基本上在第2条中所描述的，除了一个玻璃的的底部插入（MatTek，35毫米），小培养皿中的盖玻片上，而不是使用。这是不必要的覆盖标本，解剖后，组织可以直接成像的菜，但必须小心，以确保卵母细胞仍然满身油污，且不得干出来的。 </li></ol> 4。实时成像<ol><li>带来DIC或相差光学成为关注的焦点和研究的卵母细胞的卵母细胞的损害，如漏水的毛囊细胞的细胞质中，或膨出的任何迹象。图片健康和完好的卵母细胞出现。 </li><li> STREA明可以直接监控，而不需要为GFP标记，因为蛋黄囊泡在细胞质中自身荧光7。这个信号可以被捕获在FITC标记的范围的范围内，但可能会需要比那些用于GFP-标记的蛋白质更高的增益设置。 </li><li>得到的流的最佳图像从光学部分的正下方的卵母细胞的表面，靠在玻璃盖玻片/的Petri玻璃。在这里，稍扁的卵母细胞，从而为更好地平面的焦点。可以拍摄的影像200X - 1000 X.使用这里描述的布置，空气的目标应可以使用，因为盖玻片形成的卵母细胞和目标之间的势垒。这是最佳的，因为它极大地减少样品的漂移和运动。 </li><li>一旦一个地区的利益为重点，关闭brighfield的光学和切换到共聚焦成像。使用该软件的快速扫描/预览功能，调节焦距和获得是必要的。 </li><li>用于捕获的设置会有所不同从显微镜显微镜，但一般的参数如下：使用〜488纳米的激发波长和发射波长为〜515 nm。设置Z轴捕获，使得Z轴保持恒定，10秒的帧与帧之间具有滞后。捕获的测试序列的5-10帧，以确保的卵母细胞是静止的，焦点的平面的是适当的。 </li><li>一个典型的时间流逝的顺序将20 - 50之间的帧捕捉，并立即完成后，以评估视频质量可以评论。幻灯片中，该过程可重复进行其他细胞，或根据需要额外女性可以解剖。 </li></ol> 5。故障排除和常见问题<ol><li>漂移 - 当使用正置显微镜，细胞有时会漂移由于石油传播。这可以通过仅使用足够的油以覆盖盖玻片下的面积最小化。如果问题仍然存在，请使用较少的硅克rease贴上垫片幻灯片的。 </li></ol> 注：有些聚焦软件包允许跟踪的细胞，但一般来说，最好是放弃漂流的卵母细胞的成像，而使用另一个标本。 <ol start="2"><li>无流的迹象 - 如果流并不明显，这可能是由于两个原因之一，无论是在焦平面图像采集是不正确的（通常也深入到内部的卵母细胞），或已损坏的卵母细胞。时间和实践都可以最大限度地减少错误。 </li></ol>

<ol>
	<li>Becalska, A. N., Gavis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lighting+up+mRNA+localization+in+Drosophila+oogenesis.">Lighting up mRNA localization in Drosophila oogenesis.</a> Dev. 136, 2493-2503 (2009).</li><li>He, L., Wang, X., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shining+light+on+Drosophila+oogenesis:+Live+imaging+of+egg+development.">Shining light on Drosophila oogenesis: Live imaging of egg development.</a> Curr. Opin. Gen. Dev. 21, 612-619 (2011).</li><li>King, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ovarian development Drosophila melanogaster. , (1970).</li><li>Spradling, A. C., Bate, M., Martinez-Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+genetics+of+oogenesis.">Developmental genetics of oogenesis.</a> The Development of Drosophila melanogaster. 1, 1-70 (1993).</li><li>Serbus, L. R., Cha, B. -. J., Theurkauf, W. E., Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynein+and+the+actin+cytoskeleton+control+kinesin-driven+cytoplasmic+streaming+in+Drosophila+oocytes.">Dynein and the actin cytoskeleton control kinesin-driven cytoplasmic streaming in Drosophila oocytes.</a> Dev. 132, 3743-3752 (2005).</li><li>Weil, T. T., Parton, R. M., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+Individual+Drosophila+Egg+Chambers+for+Live+Imaging.">Preparing Individual Drosophila Egg Chambers for Live Imaging.</a> J. Vis. Exp. (60), e3679 (2012).</li><li>Theurkauf, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+microtubule-dependent+cytoplasmic+streaming+in+cappuccino+and+spire+mutant+oocytes.">Premature microtubule-dependent cytoplasmic streaming in cappuccino and spire mutant oocytes.</a> Science. 265, 2093-2096 (1998).</li><li>Morin, X., Daneman, R., Zavortink, M., Chia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+trap+strategy+to+detect+GFP-tagged+proteins+expressed+from+their+endogenous+loci+in+Drosophila.">A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila.</a> PNAS. 98, 15050-15055 (2001).</li><li>R&#246;per, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rtnl1+is+enriched+in+a+specialized+germline+ER+that+associates+with+ribonucleoprotein+granule+components.">Rtnl1 is enriched in a specialized germline ER that associates with ribonucleoprotein granule components.</a> J. Cell Sci. 120, 1081-1092 (2007).</li><li>Pokrywka, N. J., Payne-Tobin, A., Raley-Susman, K. M., Swartzman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules,+the+ER+and+Exu:+New+associations+revealed+by+analysis+of+mini+spindles+mutations.">Microtubules, the ER and Exu: New associations revealed by analysis of mini spindles mutations.</a> Mech. Dev. 126, 289-300 (2009).</li><li>Petri, W. H., Mindrinos, M. N., Lombard, M. F., Margaritis, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+development+of+the+Drosophila+chorion.ih+a+chemically+defined+organ+culture+medium.">In vitro development of the Drosophila chorion.ih a chemically defined organ culture medium.</a> Wilhelm Roux's Arch., Devl. Biol. 186, 351-362 (1979).</li></ol>

我什么都没有透露。

果蝇的卵母细胞是一种多功能的模型系统的功能和组织细胞和亚细胞成分有关解决各种问题。蛋白质的功能的一个完整的理解，需要的空间和时间方面的蛋白质行为的监测。成像系统和遗传工具提供给Drosophilists的进展有可能使即使是小规模的实验室，以进行高品质的成像实验，在生活的卵母细胞。在这里，我勾勒出一个协议分析GFP标记的自体荧光蛋白和结构的行为在中期到后期卵子发生与品种的遗传背景相结合，可用于探测蛋白质的功能。 在这个协议中，我描述的过程，卵母细胞成像的基本聚焦设置，使采集的视频经过时间的荧光蛋白和结构准备。额外的德泰韦伊等人所描述的6 LS对卵母细胞制备各种各样的飞菌株表达内源性蛋白质已被通过外显 ​​子捕获标签8，无限多的蛋白质变体可设计，并重新引入苍蝇。 在与生物体组织时，试样的健康是非常重要的。蛋室从舞台10B的11基本上是自主的方式，完成卵子使他们短期成像研究的理想选择。必须采取的几个关键步骤，获得高品质的数据。在清扫期间，它是重要的操作尽可能少的卵巢和卵母细胞，并尽量减少解剖和成像的完成之间的时间量。理想的情况下，一个小夹层站应位于容纳共聚焦范围在同一房间内，并且应当被成像蛋室在一个时间只有少数。这个数字在这里推荐（5-8）确保清扫时间最小化，同时最大限度地获得良好的图像质量的可能性。我建议短的系列图像捕捉图像质量评估和确认，捕捉设置进行了优化，拍摄前一个较长的时间间隔序列。大多数软件允许单个帧被保存，然后，这些图像可以在各种使用ImageJ 12或其他软件的方式进行分析。

<table border="1"><tbody><tr><td>名称</td><td>公司</td><td>目录号</td><td>评论</td></tr><tr><td>卤烃油27 </td><td> Sigma-Aldrich公司</td><td> H8773-100ML </td><td></td></tr><tr><td> ＃5SF钳</td><td>精细科学工具</td><td> 11252-00 </td><td></td></tr><tr><td>硅脂</td><td> Sigma Aldrich公司</td><td> Z273554-1EA </td><td></td></tr><tr><td>玻璃底培养皿</td><td> MatTek </td><td> P35G-0-20-C </td><td></td></tr></tbody></table>

live imaging of gfp-labeled proteins in drosophila oocytes

果蝇的卵母细胞已被确立为一个多功能的系统调查的基本问题，如细 ​​胞骨架功能，细胞组织和细胞器的结构和功能。各种GFP标记的蛋白质的可用性装置超过几分钟或几小时内的过程中，在活细胞中，使用这种技术可以监测许多细胞过程，如RNP运输，上皮形态和组织重塑过程中已经描述了非常详细在果蝇卵母细胞1,2。 的能力，在视频影像结合多样化的突变使一个巨大的各种基因和程序进行审查，令人难以置信的细节。这方面的一个例子是在卵子发生3,4中的过程的卵胞质流，从而启动。这种剧烈运动的空泡是微动蛋白依赖的，并提供了一个有用的系统在这几个阶段的调查骨架功能的温度。 在这里，我提出一个协议时间的推移生活卵母细胞成像的使用几乎任何激光共聚焦显微镜设置。

GFP-标记的蛋白质的成像会有所不同，这取决于如何强烈的标签的蛋白的表达。一个中期阶段的卵母细胞表达的reticulon的如1（Rtnl-1），外显子8,9 GFP标记产生的强烈信号。 rtnl-1出现共同定位期间的卵胞质串流10（ 图1A）与的长微管本。 Rtnl-1是一个很好的观察数据流标记，也是中后期卵母细胞的微管组织的一个指标。 野生型蛋室卵胞质流中明显运动的自身荧光的蛋黄囊泡是显而易见的，因此，当使用一帧的捕获速率每10秒（ 图1B）。五帧可以产生的最大投影标记的路径移动泡。 <img alt="图1" src="/files/ftp_upload/50044/50044fig1.jpg" /> 图1。实时成像都autofluorescent和GFP标记的结构。A）单时间推移图像的一个阶段11个卵母细胞表达Rtnl1-GFP 400倍。 Rtnl1-GFP纤维移动流卵母细胞的波浪状运动。五架B）最大的一个阶段10B蛋室的自体荧光黄泡，每10秒拍摄50秒的总，在放大400倍的预测。比例尺= 30微米

Watch this Scientific Journal Video about Live Imaging of GFP-labeled Proteins in Drosophila Oocytes at JoVE.com

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

GFP标记的蛋白质或个人的自体荧光结构的一种协议，用于实时成像<em&gt;果蝇</em描述&gt;的卵母细胞。

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 ...

live-imaging-of-gfp-labeled-proteins-in-drosophila-oocytes

Research

JoVE Journal

Biology

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

11.2K 次观看.  Vassar College. GFP标记的蛋白质或个人的自体荧光结构的一种协议，用于实时成像果蝇的卵母细胞。

视频: Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

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 ...

科研

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 ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

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 ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

准备个人果蝇蛋钱伯斯实时成像

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...