我们感谢安德烈亚斯Schönle，马克斯 - 普兰克学会国立精神卫生研究所，生物物理化学，德国和大卫J.桑德斯卓姆，美国马里兰州贝塞斯达，国家卫生研究院提供技术咨询。我们感谢弗兰克Kötting，欧洲神经科学研究所，哥廷根构造成像室和麻醉设备。由老年痴呆症Forschung主动TMRYZ赠款支持这项工作是由细胞和分子神经科学，蒂宾根大学的研究生院的奖学金，奖学金由中国国家留学基金管理委员会，SBH的支持。

一）大会的成像室<ol><li>选择的选择阶段的幼虫（如早期的第三龄幼虫离开约24小时的观测时间间隔在25 °，直到徘徊阶段彗星）。 </li><li>幼虫用清水冲洗干净的培养基中，民建联干。 </li><li>大衣中心室底部的元素，该地区面临的幼虫，用的卤烃油的薄膜。 </li><li>忍受的腹侧方在会议厅内的显微镜物镜面临的幼虫（使神经肌肉接头（NMJ）26日和27日进行成像）（图1 AE）。 </li><li>广场上一层油的塑料垫片，电网朝上（图1）通风槽。请确认在这一步:间隔的高度大约有一半的幼虫直径，缝隙宽度应幼虫直径的两倍左右。 </li><li>放置在显微镜的成像室</li></ol>二）幼虫麻醉<ol start="7"><li>用适当的麻醉设备/蒸发器，连接香港总商会的两个进气口。 请验证的前端: 有效地氟醚在房间内的空气浓度不应超过安全法规规定的！只有一个很小的地氟醚的总量是需要麻醉的幼虫。地氟醚1（V / V）15％的应用程序已被证明是一个很好的出发点，确定要在实验中使用的理想浓度。出于安全原因，我们建议，蒸发器，不应该包含更多地氟醚，比需要2-4小时 ，在体内成像。 </li><li>浸入一杯水室出口管的另一端，然后打开阀门控制流室约五秒钟的麻醉。 请确认在这一步: 气泡在水中升天？如果没有商会是漏水。 </li><li>关闭阀门，约三秒钟。 </li><li>在显微镜的监视残余幼虫运​​动。检查的心跳和肌肉的运动。如果有必要打开和关闭阀门，在步骤8和9所述，直到完成幼虫麻醉，然后关闭所有阀门，并开始成像。 请确认在这一步: 剩余的肌肉运动或心跳表明幼虫是不正确麻醉的。完整的麻醉是至关重要的高分辨率图像。幼虫一般应不长于15-20分钟，麻醉在一个给定的的时间。 </li></ol>三）成像<ol start="11"><li>确定正确的地位和形象的利益格局（图2-4）。 </li></ol>四）从麻醉恢复<ol start="12"><li>成像完成后，让空气进入墓室。 请确认在这一步: 检查发送的卤素灯幼虫的心跳和肌肉收缩。当肌肉开始收缩，它是安全的删除从成像室的幼虫。 </li><li>从麻醉设备分离室，从显微镜中取出。仔细拆解的商会，并放置在泥飞栽培介质的幼虫。 </li><li>存放在适当的温度在孵化器的菜。 </li></ol>五）时间序列<ol start="15"><li>重复步骤2-14，直到已获得足够的时间点。 替代协议: 如果30分钟的时间间隔内超过一次成像幼虫，它可能是实际离开它，直到下一次麻醉时间点在成像室。重复步骤7-14，直到已获得足够的时间点。 请确认在这一步: 幼虫不保存时间超过两小时的影像室，也不是它是麻醉时间长于15-20分钟。 </li></ol><img src="/files/ftp_upload/2249/2249fig1.jpg" alt="图1" /> 图1成像室大会 。 （a）将上一层油的幼虫和塑料垫片。 （b）将垫片上一个22 × 22毫米盖玻片和插入室有机玻璃导环，未来（三）修复的金属环的幼虫位置，和（D）关闭室。 （e）现在是随时可以安装在显微镜室。 <img src="/files/ftp_upload/2249/2249fig2.jpg" alt="图2" /> 图2在果蝇幼虫体壁肌肉 。肌肉和NMJs一个CD8 + GFP - SH表达融合蛋白8的可视化。作为观察对焦时成幼虫通过角质层腹侧的肌肉显示。在最浅肌层，27，（AC）显示，在（吉隆坡）最内部的肌肉，第6和7，显示的。比例尺:100微米，片之间的ΔZ是2微米。 <img src="/files/ftp_upload/2249/2249fig3.jpg" alt="图3" /> 图3在果蝇幼虫体壁肌肉的身份。表明身份，是A3，段L3 果蝇幼虫的肌肉。肌肉和NMJs一个CD8 + GFP - SH表达融合蛋白8的可视化。作为观察对焦时通过角质层腹侧到幼虫的肌肉显示。最浅肌层，27岁，在AC显示，在吉隆坡的最内部的肌肉，6日和7所示。比例尺:100微米，ΔZ之间切片是2微米。 <img src="/files/ftp_upload/2249/2249fig4.jpg" alt="图4" /> 图4 果蝇幼虫神经肌肉接头的身份。 （AD）NMJs的一个DGluRIIA MRFP融合蛋白 1表达的可视化。 NMJs是作为观察重点是通过角质层腹侧成幼虫。在（AB），最肤浅的NMJ，27岁，是所示，在CD最内部的NMJs，6日和7所示。比例尺:100微米，ΔZ之间切片为5μm。 （E）和（F）的肤浅的（E）和更大的内部（F）的NMJs的身份，以供参考。

<ol>
	<li>Rasse, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+receptor+dynamics+organizing+synapse+formation+in+vivo.">Glutamate receptor dynamics organizing synapse formation in vivo.</a> Nat Neurosci. 8, 898-905 (2005).</li><li>Rasse, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In vivo imaging of long-term changes in the Drosophila neuromuscular system [dissertation]. , (1988).</li><li>Fuger, P., Behrends, L. B., Mertel, S., Sigrist, S. J., Rasse, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+synapse+development+and+measuring+protein+dynamics+using+two-color+fluorescence+recovery+after+photo-bleaching+at+Drosophila+synapses.">Live imaging of synapse development and measuring protein dynamics using two-color fluorescence recovery after photo-bleaching at Drosophila synapses.</a> Nat Protoc. 2, 3285-3298 (2007).</li><li>Wang, X., Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+axonal+transport+of+mitochondria.">Imaging axonal transport of mitochondria.</a> Methods in enzymology. 457, 319-333 (2009).</li><li>Vinegoni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoscopic+fluorescence+tomography+for+in-vivo+imaging+of+developing+Drosophila.">Mesoscopic fluorescence tomography for in-vivo imaging of developing Drosophila.</a> J Vis Exp. , (2009).</li><li>Lin, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+imaging+of+Drosophila+larva+by+multiphoton+autofluorescence+and+second+harmonic+generation+microscopy.">Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy.</a> Journal of biomedical optics. 13, 050502-050502 (2008).</li><li>Levitan, E. S., Lanni, F., Shakiryanova, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+vesicle+motion+and+release+at+the+Drosophila+neuromuscular+junction.">In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction.</a> Nat Protoc. 2, 1117-1125 (2007).</li><li>Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y. &. a. m. p. ;. a. m. p., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+a+synapse+grow:+noninvasive+confocal+imaging+of+synaptic+growth+in+Drosophila.">Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.</a> Neuron. 22, 719-729 (1999).</li></ol>

最初，该方法的开发研究谷氨酸对果蝇幼虫的体壁肌肉的突触。 果蝇神经肌肉接头（NMJ）的特点是一个定型的肌肉和神经细胞的细胞质架构，因而非常适合在体内成像。然而，描述的麻醉协议是不仅限于成像NMJ果蝇幼虫的透明度，有利于适应所描述的协议的研究器官发育，细胞迁移，细胞内的亚细胞的轴突货物和重组运输。

试剂: <ul><li>地氟醚（Suprane，巴克斯特，Unterschleißheim，德国） </li><li>卤烃油（如Voltalef H10S油/法国阿托菲纳，皮托，） </li><li>飞培养基</li></ul>设备: <ul><li>倒共聚焦显微镜</li><li>双目显微镜（如STEMI 2000年，卡尔蔡司耶拿，德国） </li><li>小画笔，用来排序苍蝇</li><li>孵化器</li><li>定制成像室（对于机械制图见参考文献3） </li><li>汽化器/麻醉设备（细节见参考文献3） </li></ul>

in vivo imaging of intact drosophila larvae at sub-cellular resolution

在光学成像，基因编码的荧光基团和遗传工具最近已经启用了改进，使所需的转基因动物线的建立有效的生物过程的研究，并在生活中甚至在某些情况下的行为，有机体。在这个协议中，我们将描述如何麻醉完整的果蝇幼虫，使用挥发性麻醉剂氟烷，跟随在亚细胞决议1-3突触人口的发展和可塑性。虽然其他有用的方法来麻醉果蝇幼虫以前曾描述4,5,6,7,8，本文介绍的协议表明由于以下组合键功能显着改善:（1）麻醉的程度非常高，甚至在被逮捕的心脏跳动允许横向分辨率可达150纳米1，（2）&gt; 90％，每麻醉周期的成活率高，允许在一个小时内的五年多时间点的记录天2（ 3）高灵敏度，使我们在2个实例研究的动态表达的蛋白质在生理水平。在细节上，我们能够以可视化的突触后的谷氨酸受体亚基GluR - IIA表示，通过在稳定的转基因株系的内源性启动子1和外显子陷阱行FasII - GFP 1。 （4）与其他方法相比4,7幼虫可成像不仅活着，而且还完整无缺（即非解剖）观察到发生在若干天1。影随行的细节功能的各个部分在体内成像室2,3，幼虫，麻醉过程，如何重新确定在幼虫的具体岗位和幼虫安全拆除的正确安装，从成像会议厅。

Watch this Scientific Journal Video about In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution at JoVE.com

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

本协议描述了一种可靠的方法，麻醉和完整的成像<em&gt;果蝇</em&gt;幼虫。我们利用挥发性麻醉药氟烷允许重复成像，在亚细胞的分辨率和结构的重新鉴定几天<sup&gt; 1</sup&gt;。

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-imaging-of-intact-drosophila-larvae-at-sub-cellular-resolution

Research

JoVE Journal

Biology

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

14.5K 次观看.  University of Tübingen. 本协议描述了一种可靠的方法，麻醉和完整的成像果蝇幼虫。我们利用挥发性麻醉药氟烷允许重复成像，在亚细胞的分辨率和结构的重新鉴定几天 1。

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

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

高分辨率测量的气味驱动在果蝇幼虫的行为

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

科研

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

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 ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

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 ...

Microgavage of Zebrafish Larvae

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe interactions17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.

We present a novel method for microgavage of larval zebrafish utilizing standard embryo microinjection and stereomicroscopy equipment. We demonstrate that microgavage is a safe and efficient technique useful for delivering controlled amounts of diverse materials specifically into the larval zebrafish intestinal lumen.

Microgavage斑马鱼幼虫

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe ...

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  ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

在啮齿类动物呼吸道上皮纤毛运动功能的高分辨率成像的体外方法

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...