由一个从美国国家科学基金会授予MF

OMT的是一个适应的设计整合了一个虚拟的羽模拟器2 1磁性的系绳系统。以下协议将解释如何优化所获得的视频图像（第一部分）和运行两种类型的基本visuo嗅觉实验（第2和3）。 第1部分：视频采集优化视频图像采集到的数据的质量是至关重要的。按照这些步骤清洁图像。 <ol><li>放置在舞台上使用一双镊子的新鲜拴飞。 </li><li>位置火线板相机（火我1394store.com）下明确的丙烯酸相机的看法是通过直接向上通过商会的玻璃管，真空室，腹栓飞方面。要确保视频系统的镜头，没有红外过滤器涂层。 </li><li>照亮飞，用圆阵的红外发光二极管（小零件公司）定位下的LED显示屏。聚焦均匀照明飞的身体上的LED分别。 </li><li>为了减少眩光面板系统和动态观察，以尽量减少视觉线索，它是很有用的相机视图与平坦的黑色烤漆涂层内所有的反射表面的。客房灯应关闭，以减少眩光和亮度调制蝇眼检测。 </li><li>目视检查摄像机的图像。飞应在黑色背景明亮的白色。如果图像是模糊的镜头，可能需要集中。如果飞行是黑暗的，LED阵列可重点不突出，或不够适当的照明供电。如果背景含有明亮的物体，装入略高于飞黑色绒纸的磁盘，以尽量减少背景反射。 </li><li>我们收购视频图像采集工具箱在Matlab中使用自定义的子程序。 </li></ol>第2部分：找到气味羽的飞在OMT的一个简单的实验是观察一个饿（饿死）飞行定位，然后积极跟踪一缕缕诱人的食物的气味。操作的视觉环境，影响飞行的能力，有力跟踪食欲的食物的气味。请注意，实验通常是在一个随机块格式运行在实验为了尽量减少任何的偏见。定制软件在Matlab编写的例程（Mathworks公司）控制所有外部硬件（气体多路复用器，LED舞台上，视频采集）。 <ol><li>放置在舞台上使用一双镊子的新鲜拴飞。 </li><li>在每次实验中，使用可视化显示，旋转飞 3几个革命，以确保整个360偏航轴周围的平滑运动。如果不旋转飞行，顺利将它丢弃。如果问题仍然存在，可能的原因是对齐的磁铁。注意：如果舞台上的分布在旋转试验中数轨迹的标题显示了显着的山峰（即不超过360度的单位），有可能由于对齐磁铁定位偏差。 </li><li>目前与下面的示例中随机顺序的实验条件下的飞行：我水蒸气产生的气味端口）高对比度的条纹图案;二）高对比度的条纹图案有吸引力的气味蒸气;三）单一的竖条纹偏移90度从一个单一的90度的水蒸汽喷嘴;及iv）抵消气味蒸汽的条纹。根据我们的经验，30秒的时间间隔以及苍蝇的挑战是要找到自己的气味源的实验。我们收购的每一个实验条件的持续时间为30 Hz的视频和分析图像脱机使用定制软件在Matlab编写的例程（其中最有用的内置MATLAB函数regionprops.m分析椭圆的几何性质。 </li><li>使用气味浓度高时，残留气味可能释放出的气味端口，关闭后的气味。为了尽量减少任何影响，这可能是非常有用的一个旋转的全景旋转的飞了几秒钟，让系统冲洗掉任何残留气味。 </li><li>这也有利于直观地旋转飞之间的试验性治疗（一）保持积极从事实验（二）继续留在一个地方苍蝇的苍蝇。一个示例实验，可能有以下顺序：I）第二十第二初步诊断自旋，II）五秒钟中间旋转;三）21第二个条件对＃1;四）5个第二中间旋转; V）21第二个条件对＃2。 </li></ol>第3部分：内气味羽剩余飞基本在OMT的第二个实验是直观地拖动到气味羽飞，瞬间改变的视觉条件，然后衡量其能否保持在羽。此外，请注意，所有的实验都是在做一个随机块备考吨，以尽量减少实验为了在任何偏见。 <ol><li>放置在舞台上使用一双镊子的新鲜拴飞。 </li><li>视觉旋转（旋转）作为诊断检查，以确保动物在各个方向的能力，以东方（ 见2.2部分）的每一个实验前的舞台周围飞几次。 </li><li>每个条件前，打开实验的气味（水或醋）和视觉上的“拖放”到羽羽的位置，一个小竖条纹的振荡飞。这需要强大的条纹固定观察到的行为[4]在果蝇的优势。 </li><li>现在飞是面向羽流的，删除的振荡条纹，目前下面的示例Visual条件：一）高对比度与水蒸气（ 图1A）全景条纹;二）统一的背景与水蒸气（ 图1B）; III）的高对比度的全景条纹与气味蒸汽（ 图 1C）;四） 气味蒸汽（图 1D）统一的背景。 2.3收购的视频。一个示例实验可能有流动的顺序为：I）20的第二个初步诊断自旋;二）五秒钟羽状条纹振荡;三）21第二个条件对＃1;四）五秒钟羽状条纹振荡; V）21第二个条件对＃2 。 </li></ol>代表性的成果： <img src="/files/ftp_upload/old/1110_2009_1_22_1/Fig 1.png" alt="图1" width="600" height="195" /> 图1 第1部分介绍了如何优化OMT的视频图像。视频图像，应该有一个暗黑色的背景照明飞明确的说法。过于明亮的图像，降低背景干扰，降低红外灯的强度，或关闭室内灯光，可以提高。如果飞行是不当的照明，调整红外灯或调节红外灯的强度的重点可能会解决这个问题。2和第3部分介绍了一些简单的实验来进行验证的OMT的正常运作。在所有情况下，一只苍蝇应直观地“被迫”（通过optomotor反应）在舞台上旋转的几个革命的运动，以确保顺利和完整范围。一只苍蝇在第2部分中面临的挑战是找到自己的气味羽。为了保持飞行从事实验，以减少残留气味释放的影响，并继续从维护整个实验的单标题的苍蝇，它是非常有用的视觉旋转飞行试验之间的几秒钟。一只苍蝇不应该本地化水蒸气羽显着程度，并应始终本地化气味羽中存在丰富的全景视觉线索。一只苍蝇在第3部分在视觉上直接拖动到气味羽和挑战，以保持其在前往羽对各种固定视觉背景。如果拖成水汽羽飞，它应迅速转身离开。如果飞拖入气味柱和有足够的视觉线索调解鲁棒跟踪，飞将保持在羽。

<ol>
	<li>Bender, J. A., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+stimulation+of+saccades+in+magnetically+tethered+Drosophila.">Visual stimulation of saccades in magnetically tethered Drosophila.</a> J Exp Biol. 209, 3170-3182 (2006).</li><li>Duistermars, B. J., Frye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossmodal+visual+input+for+odor+tracking+during+fly+flight.">Crossmodal visual input for odor tracking during fly flight.</a> Curr Biol. 18, 270-275 (2008).</li><li>Reiser, M. B., Dickinson, 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+modular+display+system+for+insect+behavioral+neuroscience.">A modular display system for insect behavioral neuroscience.</a> J Neurosci Methods. 167, 127-139 (2008).</li><li>G&#246;tz, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Course-control,+metabolism+and+wing+interference+during+ultralong+tethered+flight+in+Drosophila+melanogaster.">Course-control, metabolism and wing interference during ultralong tethered flight in Drosophila melanogaster.</a> J. Exp. Biol. 128, 35-46 (1987).</li></ol>

该系统的图像采集利用一种廉价的FireWire板相机和Matlab中编写的软件。清晰的图像，是为获得准确的数据痕迹的关键。上面所描述的策略是清理在这个系统中的图像非常有用。这个系统的其他视频跟踪程序已被描述 1 。此外，这个系统可以很容易地进行修改，包括实时视频跟踪。这里所描述的例子实验协议的意思是一个起点，以确保正常运作的OMT。这是可以试验不同的视觉显示，甚至不同...

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visually mediated odor tracking during flight in drosophila

飞虫使用视觉线索，以稳定其在风中的标题。此外，许多动物跟踪在风中进行气味。因此，迎风视觉稳定跟踪直接气味追踪艾滋病。但嗅觉信号直接影响视觉跟踪的行为独立于风的线索呢？此外，嗅觉的分子遗传学和神经生理学的最新进展，有上进心新型定量的行为分析，评估行为的影响（如）基因失活特定的嗅觉激活电路。我们修改了一个磁性的系绳系统原本是为视觉实验设计，配备狭窄的层流气味羽状竞技场。在这里，我们就可以执行后飞是拴能够在磁场舞台导航的实验重点。我们将展示如何获取视频图像测量身体的角度进行了优化，如何判断稳定的气味追踪，并说明了两个实验研究的追踪气味的视觉线索影响。

Watch this Scientific Journal Video about Visually Mediated Odor Tracking During Flight in Drosophila at JoVE.com

Visually Mediated Odor Tracking During Flight in Drosophila

在这里，我们介绍了如何优化嗅觉磁系绳（OMT）仪器获得的视频图像。我们还描述了两个样本研究visuo的嗅觉融合的实验协议。

在果蝇的飞行过程中可视介导的气味追踪

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

visually-mediated-odor-tracking-during-flight-in-drosophila

Research

JoVE Journal

Biology

9.8K 次观看.  University of California, Los Angeles. 在这里，我们介绍了如何优化嗅觉磁系绳（OMT）仪器获得的视频图像。我们还描述了两个样本研究visuo的嗅觉融合的实验协议。

视频: Visually Mediated Odor Tracking During Flight in Drosophila

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

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

一个磁性的系绳系统，以调查在果蝇的视觉和嗅觉介导的飞行控制

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

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 ...

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

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  ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

在对飞行性能的准确，快速测定方法的改进<em&gt;果蝇</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Methods Article

在果蝇的飞行过程中可视介导的气味追踪