Funded by a grant from the National Science Foundation to MF

The OMT is an adaptation of a magnetic tether system 1 designed to incorporate a virtual plume simulator 2. The following protocols will explain how to optimize the acquired video images (Part 1) and run two types of basic visuo-olfactory experiments (Part 2 &amp; 3).
Part 1: Video Acquisition
Optimizing the video image is crucial for the quality of the acquired data. Follow these steps for cleaner images.
<ol>
<li>Place a freshly tethered fly in the arena using a pair of forceps.</li>
<li>Position the firewire board camera (Fire-I 1394store.com) under the clear acrylic vacuum chamber such that the camera view is directly upward through the chamber, through a glass tube, to the ventral aspect of the tethered fly. Be sure that there is no IR filter coating on the lens of the video system.</li>
<li>To illuminate the fly, use a circular array of infrared LEDs (Small-Parts Inc) positioned just under the LED panels. Focus each LED on the fly&rsquo;s body individually for uniform illumination.</li>
<li>To reduce glare from the panel system and to minimize visual cues observed by the fly, it is useful to coat all reflective surfaces within the camera view with flat black paint. Room lights should be switched off to reduce glare and brightness modulations detectable by the fly&rsquo;s eye.</li>
<li>Visually inspect the camera image. The fly should be bright white against a black background. If the image is blurred the lens may need to be focused. If the fly is dark, the LED array may be unfocused or insufficiently powered for proper illumination. If the background contains bright objects, mount a disk of black flock paper just above the fly to minimize background reflectance.</li>
<li>We acquire video with custom subroutines written in Matlab using the Image Acquisition toolbox.</li>
</ol>
Part 2: The fly finding the odor plume
One simple experiment in the OMT is observing a hungry (starved) fly locating and then actively tracking a plume of attractive food odor. Manipulating the visual surroundings influences the fly&rsquo;s ability to robustly track an appetitive food odor. Note that experiments are typically run in a random block format to minimize any bias in experimental order. All external hardware (gas multiplexers, LED arena, video acquisition) are controlled by custom software routines written in Matlab (Mathworks).
<ol>
<li>Place a freshly tethered fly in the arena using a pair of forceps.</li>
<li>Before every experiment, using the visual display, rotate the fly for several revolutions 3 to ensure smooth motion around the entire 360 yaw axis. If the fly does not spin smoothly discard it. If the problem persists, the likely cause is misaligned magnets. Note: If the distribution of arena heading for several trajectories during the spin trial shows significant peaks (i.e. is not flat over 360 degrees), there is likely an orientation bias due to misaligned magnets.</li>
<li>Present the fly with the following sample experimental conditions in random order: i) high-contrast striped pattern with water vapor emanating from the odor port; ii) high-contrast striped pattern with attractive odor vapor; iii) a single vertical stripe offset 90 degrees from the water vapor nozzle; and iv) a single 90 degree offset stripe with odor vapor. In our experience, 30 second intervals work well for experiments where flies are challenged to locate the odor source on their own. We acquire video at 30 Hz for the duration of each experimental condition and analyze the images offline using custom software routines written in Matlab (one of the most useful built-in Matlab functions is regionprops.m which analyzes the geometric properties of an ellipse.</li>
<li>When using high odor concentrations, residual odor may be released from the odor port after the odor is switched off. To minimize any effects this may have it is useful to spin the fly for a few seconds with a rotating panorama to allow the system to flush out any residual odor.</li>
<li>It is also beneficial to visually rotate the fly between experimental treatments to (i) keep the flies actively engaged in the experiment and to (ii) keep flies from remaining in one spot. An example experiment may have the following order: i) twenty second initial diagnostic spin; ii) five second intermediate spin; iii) twenty second condition pair #1; iv) five second intermediate spin; v) twenty second condition pair #2.</li>
</ol>
Part 3: The fly remaining within the odor plume
A second basic experiment in the OMT is to visually drag a fly into an odor plume, instantaneously change the visual conditions, and then measure its ability to remain in the plume. Again, note that all experiments are done in a random block format to minimize any bias in experimental order.
<ol>
<li>Place a freshly tethered fly in the arena using a pair of forceps.</li>
<li>Visually rotate (spin) the fly several times around the arena before every experiment as a diagnostic check to ensure that the animal has the capacity to orient in every direction (see Part 2.2).</li>
<li>Before each condition, turn on the experimental odor (water or vinegar) and visually &ldquo;drag&rdquo; the fly into the plume by oscillating a small vertical stripe at the position of the plume. This takes advantage of the powerful stripe fixation behavior observed in flies [4].</li>
<li>Now that the fly is oriented toward the plume, remove the oscillating stripe and present the following sample visual conditions: i) high contrast panoramic stripes with water vapor (Fig. 1A); ii) uniform background with water vapor (Fig. 1B); iii) high contrast panoramic stripes with odor vapor (Fig. 1C); iv) uniform background with odor vapor (Fig. 1D). Acquire video as in 2.3. An example experiment may have the flowing order: i) twenty second initial diagnostic spin; ii) five second plume stripe oscillation; iii) twenty second condition pair #1; iv) five second plume stripe oscillation; v) twenty second condition pair #2.</li>
</ol>
Representative Results:
<img src="/files/ftp_upload/old/1110_2009_1_22_1/Fig 1.png" alt="Figure 1" width="600" height="195" />
Figure 1
Part 1 explains how to optimize the OMT video images. The video image should have a clear view of the illuminated fly on a dark black background. Overly bright images can be improved by reducing background interference, reducing the intensity of the IR LEDs, or by turning off the room lights. If the fly is improperly illuminated, adjusting the focus of the IR LEDs or modulating the intensity of the IR LEDs may fix the problem. Parts 2 &amp; 3 describe simple experiments to conduct to verify the proper operation of an OMT. In all cases, a fly should be visually &ldquo;forced&rdquo; (via optomotor responses) to rotate several revolutions in the arena to ensure a smooth and complete range of motion. In Part 2 a fly is challenged to find an odor plume on its own. To keep the fly engaged in the experiment, to reduce the effect of residual odor release, and to keep flies from maintaining a single heading throughout the experiment, it is useful to visually rotate the fly for a few seconds between trials. A fly should never localize a water vapor plume to a significant degree, and should always localize an odor plume in the presence of rich panoramic visual cues. In Part 3 a fly is visually dragged directly into an odor plume and challenged to maintain its heading within the plume against a variety of stationary visual backgrounds. If the fly is dragged into a water vapor plume, it should quickly turn away. If the fly is dragged into an odor plume and there are sufficient visual cues to mediate robust tracking, the fly will remain in the plume.

<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>

The image acquisition for this system utilizes an inexpensive firewire board camera and software written in Matlab. Clear images are crucial for obtaining accurate data traces. The strategies described above are useful for cleaning up images in this system. Other video tracking procedures for this system have been described 1. Furthermore, this system could easily be modified to include real-time video tracking. The example experimental protocols described here meant to be a starting point to ensure the proper...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Firewire camera</td>
<td>&nbsp;</td>
<td>1394store.com</td>
<td>Fire-I board camera BW</td>
<td>4.3mm lens no IR coating</td>
</tr>
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<td>IR LEDs</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Black spray paint</td>
<td>&nbsp;</td>
<td>Rustoleum</td>
<td>&nbsp;</td>
<td>Flat black</td>
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<td>Black flock paper</td>
<td>&nbsp;</td>
<td>Edmund Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Panel system</td>
<td>&nbsp;</td>
<td>Caltech</td>
<td>&nbsp;</td>
<td>3</td>
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<td>Matlab 2006a</td>
<td>&nbsp;</td>
<td>Mathworks</td>
<td>&nbsp;</td>
<td>Image acquisition toolbox</td>
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visually mediated odor tracking during flight in drosophila

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 stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

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

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

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

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9.8K Views.  University of California, Los Angeles. Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Video: 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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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