作者要感谢亚历山大欧派提供技术援助和支持，詹姆斯·巴顿提供视频旁白和雷蒙娜Flatz和Joellen斯威尼对于出现在影随行。这项工作是由MJ默多克慈善信托基金（批准号:2012205，以JED）的支持。

1，体视显微镜系统注意:尽管该协议是很容易适应地耦合到一台数码相机以获取视频的能力几乎任何立体显微镜系统中，提供了在我们的实验中使用的（表的材料/设备）的系统上的信息。 <ol><li>使用三目体视显微镜连接到一个商业的数码相机获取数字视频。 </li><li>为了夫妻商用数码相机的立体显微镜的三目口，取出立体显微镜的光电管口的½xC型并用1X C型取代。 </li><li>安装一台数码相机连接器（43毫米螺纹）的1X C型安装。 </li><li>装入两个降压型环，58毫米至48毫米，和48毫米至43毫米，到相机成色弥合从数字照相机耦合器的连接到透镜适配器套件，用于数字照相机。 </li><li>安装数码相机镜头转接器套件。 </li><li>收购与显微镜的放大倍率和数码相机集光学变焦为12倍左右组合的放大倍率（每秒30帧，640×480像素）的视频。注意:在立体显微镜的放大倍数，必须按照三目端口的新重新1X C接口来补偿。 </li></ol> 2，成像果蝇三龄幼虫<ol><li>磁带上的​​永久性标记的黑色舞台板立体显微镜连接到一台数码相机，这样的标志帽的侧占地约的⅓至¼看在相机液晶监视器中观察到的垂直领域。使用标记的顶部作为阶段执行幼虫成像，因为它们都在一个颜色分类，可用于对颜色代码和分化幼虫的基因型被成像。 </li><li>标定的图中的数字照相机的液晶显示器中观察到的标记物顶部的表面上的场用细点标记。 </li><li>选择三龄幼虫的形象。的准则选择第三龄幼虫体长，在生命周期的幼虫阶段，前部和后部气孔的存在从食物源的出现和口装置11的下颌骨钩的结构。保证幼虫的清洁，用水彻底清洗它。 </li><li>从上面照亮永久性标记阶段性顶部光从光纤照明系统。调整入射光的角度，以提供最佳的照明。 </li><li>聚焦显微镜上的永久性标记上方的边缘。开始采集数字视频。 </li><li>将幼虫上的标记帽的侧约75°远离纵轴，只是外的视场，以朝向视场朝向幼虫（ 图1）的前部。注意:放置在标志帽的侧幼虫可让相机届的运动纪录Ë幼虫从横向的角度来看。它有助于保持幼虫浸有水，这样他们不脱落标记帽的侧面。必须谨慎行使但是，不要使用太多的水过量将坚持以幼虫，因为它爬行穿过田野。 </li><li>轻轻知己知彼幼虫用小画笔，以强制其在整个视场爬行。要有耐心，因为幼虫很少合作，经常要返回到起点很多时候他们直接穿过田野爬之前。 </li><li>记录约10-15分钟不间断的数字视频资料和作物，并删除所有不必要的镜头收购后的数字视频编辑软件。 </li></ol> 3，影像学成人果蝇 <ol><li>将一个成年果蝇的一次性1.5毫升光谱聚苯乙烯比色皿。 注: 二氧化碳 anaesthetization成年果蝇立即behav前ioral分析协议可以妥协的结果12。建议在成年果蝇给予24小时期间的CO 2 anaesthetization执行的行为测试13日之前恢复。 </li><li>塞用小棉球反应杯的末端。确保棉花球被包装得足够紧，以占据较大的薪金空间，并且其限制飞的比色皿的减少量仓。 </li><li>放置反应杯上的白色舞台板立体显微镜，并正确对准的图中的数字照相机的液晶显示器中观察到的字段的反应杯的容积减少隔室。 </li><li>从上面照射比色皿光从光纤照明系统。调整入射光的角度，以提供最佳的照明。 </li><li>聚焦显微镜，并开始采集数字视频。 </li><li>不间断的数字视频资料和作物记录30-45分钟，去除一切不必要的镜头后收购数字视频编辑软件。 </li></ol>

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作者宣称，不存在利益冲突。

果蝇的实力作为研究神经系统的功能模型系统主要来自强大的遗传工具可用收敛性和强大的行为分析的广泛的发展。在这里，我们提出了从横向的角度获得高品质的数字果蝇成虫和幼虫的表型机车视频的简单和普及显微技术。我们已经成功地使用这种方法来表征和量化后麻痹的严重程度"尾巴翻转&#39;通过直接测量，该尾部从水平轴在爬行周期14所提出的最大角度在神经系统?...

共同果蝇黑腹果蝇是用于研究神经系统1-3的功能的有力的实验模型系统。结构和神经系统与人类的功能进化保守性，以及易于遗传操作和遗传工具繁多，使果蝇的首映有机体来模拟人类神经退行性疾病4。引起的神经系统的机能障碍的基因突变往往导致活突变体的幼虫和成虫的果蝇与受损的运动。在神经系统有缺陷的突变体观察到的表型包括降低速率运动，异常协调，和痉挛性运动中的成年人，以及在体壁肌肉的蠕动收缩缺陷，和幼虫局部麻痹。这些表型都被利用于高通量遗传筛选和突变体幼虫5的运动测定法的发展，6和成人7-10 果蝇旨在量化运动障碍和识别所必需的神经系统的功能的基因。而，这些方法是用于定量幼虫和成虫机车行为极为有用，它们不能传达关于每个特定的异常行为的定性信息。例如，尽管突变体三龄幼虫可在行为分析表现出改变的运动参数，它可能是不清楚的，如果这是在爬行周期改变在节奏蠕动收缩，一般缺乏协调，或者在后体的局部麻痹的结果墙肌肉。在这里，我们描述了从横向的角度获得高品质的数字果蝇成虫和幼虫的表型机车视频的简单和普及显微技术。从横向的角度来看收购数字视频可以直接观察locomotiv微妙的区别和分析Ë行为的更翔实的侧面视图方向。

<table><tbody><tr><td>Trinocular Stereozoom Microscope</td><td>Olympus Corporation</td><td>SZ6145TR</td><td>&amp;frac12; C-mount was removed and replaced with 1X C-mount</td></tr><tr><td>1X C-mount</td><td>Leeds Precision Instruments</td><td>LSZ-1XCMT2</td><td></td></tr><tr><td>Digital Camera Coupler (43 mm thread)</td><td>Qioptiq Imaging Solutions</td><td>25-70-10-02</td><td></td></tr><tr><td>58 mm to 48 mm Step Down Ring</td><td>B&amp;amp;H Video</td><td>GBSDR5848</td><td></td></tr><tr><td>48 mm to 43 mm Step Down Ring</td><td>B&amp;amp;H Video</td><td>GBSDR4843</td><td></td></tr><tr><td>Lensmate Adapter Kit for Canon G10</td><td>LensMateOnline.com</td><td></td><td></td></tr><tr><td>Canon PowerShot G10 Digital Camera</td><td>Canon U.S.A., Inc.</td><td></td><td></td></tr><tr><td>1.5 ml Spectroscopic Polysterene Cuvette</td><td>Denville Scientific</td><td>U8650-4</td><td></td></tr></tbody></table>

acquisition of high-quality digital video of drosophila larval and adult behaviors from a lateral perspective

黑尾果蝇是用于研究神经系统的功能的有力的实验模型系统。造成神经系统功能紊乱的基因突变往往产生可行的幼虫，并有运动缺陷表型，很难用文字充分说明，或用一个摄影图像完全代表成年人。科学出版的电流模式，但是，支持提交的数字视频媒体作为补充材料陪手稿。在这里，我们描述了从横向的角度获得高品质的数字均果蝇幼虫和成虫的表型视频的简单和普及显微技术。影片从一个侧面视图幼虫和成虫的运动是有利的，因为它允许观察和微妙的区别和变化的分析，异常机车的行为。我们已经成功地使用该技术来可视化和量化aberran吨爬行在三龄幼虫的行为，除了成人突变表型和行为，包括梳理。

我们已经成功地使用这种技术来获得和量化与损失的stathmin基因的功能（ 图2）14相关联的幼虫行为表型。在中stathmin基因编码划分的微管蛋白二聚体的可溶性微管蛋白的游泳池的微管调节蛋白和微管结合，并促进其分解15,16。中stathmin功能是必需的，以保持在周围神经14的轴突微管的完整性。中断在果蝇三龄幼虫的结果在表型中stathmin活性，?...

Watch this Scientific Journal Video about Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective at JoVE.com

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

在这里，我们描述了一个简单而普及显微镜技术来获得高品质的数字果蝇成虫和幼虫突变表型的视频从横向的角度来看。

收购高品质的数字视频<em&gt;果蝇</em&gt;从横向角度看幼虫及成虫行为

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

acquisition-high-quality-digital-video-drosophila-larval-adult

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

8.8K 次观看.  Willamette University. 在这里，我们描述了一个简单而普及显微镜技术来获得高品质的数字果蝇成虫和幼虫突变表型的视频从横向的角度来看。 

视频: Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

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

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 ...

生物学

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but effective method to observe and document the behavior or morphology of flies. Flies were placed in a translucent observation chamber c.a. &#216;15 x 5mm (no food inside) or &#216;15 x 12 mm (with an 8 mm-high piece of food inside). After covering with an ultraviolet (UV)/clear filter with high light transmittance, the chamber was placed under a 5-50x zoom stereo microscope, and mini light-emitting diode (LED) video lights were placed on both sides of the microscope to illuminate the chamber to obtain uniform, soft, bright, and nearly shadow-free light. Then, a compact digital camera with 3-5x optical zoom, which can record 1080 P high-definition or higher resolution video (at a frame rate of &#8805;30 fps), was connected to the eyepiece of microscope through a bracket, and photographs or videos were taken through the eyepiece. By adjusting the zoom knob of the zoom stereo microscope, it was very easy to track the flies and take panoramic or detailed close-up images as needed, while the camera recorded everything under the microscope. Because the flies can stay at any position in the chamber, they can be observed and recorded from all directions. The photographs or videos taken are of good image quality. This method can be used both for scientific research and teaching.

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

This protocol describes how to make a simple adult Drosophila behavior observation chamber, and how to take high-definition photographs/videos of the morphology or behavior of different types of fruit flies in the observation chamber through relatively simple and affordable methods.

可调高清成像系统，用于 果蝇 成人的行为研究

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but ...

行为学

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

一种测定果蝇运动活性的简单技术

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

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

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

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

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

在完整的果蝇幼虫体内的亚细胞分辨率成像

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

<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 ...

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

发展果蝇神经元的快速丹德林分体动力学的延时实时成像和定量

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

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实时成像

高分辨率视频跟踪移动的，在成年果蝇。

可调高清成像系统，用于果蝇成人的行为研究

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

一种测定果蝇运动活性的简单技术

在完整的果蝇幼虫体内的亚细胞分辨率成像

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

发展果蝇神经元的快速丹德林分体动力学的延时实时成像和定量

收购高品质的数字视频

摘要

探索更多视频

实时成像

高分辨率视频跟踪移动的，在成年果蝇。

可调高清成像系统，用于 果蝇 成人的行为研究

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

一种测定果蝇运动活性的简单技术

在完整的果蝇幼虫体内的亚细胞分辨率成像

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

发展果蝇神经元的快速丹德林分体动力学的延时实时成像和定量

可调高清成像系统，用于果蝇成人的行为研究

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