We are thankful to S. Thomas who has initiated this project, J. Hermann and U. Burgbacher for help in the construction of the FIM setup. This work was funded by the DFG (SFB 629 B6).

NOTE: Here, the use of FIM is presented for handy high throughput analysis of larval locomotion for screening in free-moving conditions and under influence of a heat stimulus. Other applications such as analysis of olfactory stimuli dependent locomotion or high resolution imaging of rolling or other behaviors might need subtle changes to the protocol, which are provided upon request.
1. Set-up of Experiments
<ol style="margin-left: 40px;">
	<li>Use about 100 larvae per genotype in total (1 hr time required) to obtain statistically meaningful values. NOTE: In case several genotypes need to be compared and recorded on different days, record the same number of larvae per genotype per day.</li>
	<li>For most applications, record at 10 Hz for parameter analysis. For high resolution imaging applications, vary imaging speed if needed.</li>
	<li>For tracking of third instar larvae, conduct experiments about 120 hr after egg laying. Make sure that cultures yield enough larvae in the experimental period (see 4.3).</li>
	<li>For non-stimulus applications, prepare a crawling surface containing translucent agar (see section 2). To contain larvae in the stimulus range for heat gradient application, surround the field of view with an aversive salt agar barrier (see section 3). 
	NOTE: Other applications may require different surfaces.</li>
	<li>Make sure to use a room with constant environmental conditions (temperature, light, air flow, air humidity etc.).</li>
</ol>
2. Crawling Surface Preparation (Translucent Agar)
NOTE: In the FIM setup an agar surface is added to provide a moist crawling surface. In addition, it also improves illumination properties.
<ol style="margin-left: 40px;">
	<li>Boil 0.8% food grade agar in deionized ultrapure water. For screening application prepare 400 ml.</li>
	<li>Pour agar at 50 &#176;C (hand-hot) on a separate 33 cm x 33 cm acrylic-glass plate. The surface tension of the agar and thus the temperature will define the thickness of the agar slab. At 50 &#176;C, 2 mm thick agar slabs are obtained. Do not agitate after boiling and pour constantly to avoid bubbles. Prepare fresh agar slabs for every imaging period of about 4 hr.</li>
	<li>Fill standard 6 cm petri dishes with the remaining 0.8% agar solution to sort, clean and habituate larvae prior to the experiment (1 dish per genotype).</li>
	<li>In case an aversive agar barrier is needed for the application, proceed to section 3.</li>
	<li>Trim off approximately 2 cm of the perimeter of the agar slab to obtain a plane square surface for recording. Remove the excess agar. 
	NOTE: The size is dependent on the application.</li>
	<li>Transfer the agar slab to the FIM setup directly after cooling by gently pushing the gliding agar over the edge of the acrylic glass plate.</li>
</ol>
3. Optional: Adding an Aversive Agar Barrier to the Crawling Surface (Salt Agar)
<ol style="margin-left: 40px;">
	<li>Boil 2.5% food grade agar with 3 M NaCl in deionized ultrapure water. For screening in a heat gradient prepare 200 ml. 
	NOTE: The volume depends on the application.</li>
	<li>Cut a 2 cm wide notch in the previously poured crawling surface surrounding the field of view (22 x 22 cm). 
	NOTE: Different barrier forms and fields of view may be required depending on the application.</li>
	<li>Fill the notch with salt agar 0.1-0.3 cm higher than the tracking surface.</li>
</ol>
4. Fly Handling
<ol style="margin-left: 40px;">
	<li>Rear flies in 25 &#176;C incubator with 12 hr light/dark cycle adjusted to the experiment&#8217;s time on standard fly food at 65% air humidity.</li>
	<li>For crosses, anesthetize 30 virgin female flies and 8 males with CO2 and cross them in 130 ml culture vials with 35 ml food. 
	NOTE: One 130 ml culture vial with 35 ml food will yield about 20-30 late third instar larvae within the imaging span of 4 hr. Imaging and tracking works for all larval instar stages.</li>
	<li>Drop a little water in culture vials to drive late third instar larval movement and collect the largest larvae from vial walls using a small paintbrush.</li>
	<li>2-5 min before recording, transfer larvae for one video to a Petri-dish containing 0.8% food grade agar in deionized ultra pure water to habituate and clean them.</li>
</ol>
5. Adjusting the FIM Imaging Setup for Recordings (Non Stimulus Conditions)
<ol style="margin-left: 40px;">
	<li>Adjust camera lens focus and aperture if needed. Set the exposure time for the respective camera. 
	NOTE: These settings do not need to be changed during an experiment.</li>
	<li>Adjust illumination intensity to obtain good contrast by imaging one larva.</li>
	<li>Keep environmental conditions in the room constant during recordings. Darken the room to not disturb larvae with directional light.</li>
</ol>
6. Optional: Adjusting the FIM Imaging Setup for Recordings (Gradual Temperature Stimulus Conditions)
NOTE: The heat gradient device is a 42 x 42 x 0.2 cm3 aluminum plate with a matt black paint and isolating material on the top site. The plate is perfused with water from two different circuits connected to calorifiers/coolers and pumps at both ends (see Figure 1B). The temperatures can be regulated from -5 to +50 &#176;C.
<ol style="margin-left: 40px;">
	<li>Turn on the heat gradient device 1 hr prior to the experiments and place it above the setup to allow establishing the desired temperature profile and components to equilibrate.</li>
	<li>Transfer the crawling surface agar with a salt barrier to the setup.</li>
	<li>Place the radiator plate over the agar and adjust the spacing between the plate and the crawling surface to 2 mm (~1 mm above larvae).</li>
	<li>Establish a linear gradient of at least 0.8&#176;C/cm from 34 &#176;C (approx. 2 cm from the barrier in the field of view) to 18 &#176;C (approx. 2 cm from the barrier at the opposite site in the field of view) by adjusting the temperature of the water circuits to 1 &#176;C and 45 &#176;C. 
	NOTE: Different gradient properties of the metal plate require different temperatures which need to be ascertained empirically. These settings do not need to be changed during the experiments.</li>
	<li>Adjust settings as described in section 5.</li>
	<li>Allow the crawling surface to equilibrate in the gradient before experiments for 20 min. Test the temperature gradient with a pyrometer.</li>
	<li>Proceed with section 8.</li>
</ol>
7. FIM Imaging (Non Stimulus Conditions)
<ol style="margin-left: 40px;">
	<li>Use a small wet paintbrush to gently gather 15 larvae from the habituation petri dishes and transfer them to the center of the tracking surface. Don&#8217;t transfer food remains or too much water (see 7.8).</li>
	<li>Record 1 - 50 larvae on a 22 cm x 22 cm tracking area. Use 15 animals per video for most screening applications (for statistical reasons use at least 100 individuals per genotype).</li>
	<li>Gently separate larvae and wait for about 10-20 sec until all larvae started to move straight before recording.</li>
	<li>Record larval locomotion for 2 min at 10 frames per sec for non stimulus conditions. Use uncompressed tif images as the output format.</li>
	<li>During recording, collect larvae for the next video from vials to habituate them. Critical: Do not disturb recorded larvae with light.</li>
	<li>After recording, remove larvae with a large paintbrush and discard them in accordance with local safety and legislation rules.</li>
	<li>After removing larvae, use the paintbrush to clean and moisturize the crawling surface with ultra pure deionized water.</li>
	<li>Critical: Keep the surface moist at all times but avoid excessive moisture, which can be seen as halos or drops surrounding larvae in recordings and disturb tracking.</li>
</ol>
8. Optional: FIM Imaging (Gradual Temperature Stimulus Conditions)
<ol style="margin-left: 40px;">
	<li>After the temperature gradient is established (see section 6), prepare the recording software for instant recording within 20 sec after placing the larvae on the crawling surface (see 8.2) e.g., set the number of frames per video and define the saving path.</li>
	<li>Lift the radiator plate slightly and place the larvae at 33 &#176;C 2 cm from the salt barrier. Refer to 6. and 7. for further general instructions. Lower the radiator plate again and start recording for 3-4 min directly after larvae started to move straight.</li>
	<li>After recording, remove larvae directly, clean and moisturize the surface. Do not touch the salt agar to avoid spreading of NaCl.</li>
	<li>Critical: Keep the surface moist at all times but avoid excessive moisture, which can be seen as halos or drops surrounding larvae in recordings and disturb tracking.</li>
	<li>Allow the agar surface to equilibrate again after moisturizing for 1-2 min, while saving images and gathering new animals before preparing for next video. Control the temperature gradient using a pyrometer every 5 videos and adjust temperature device if needed (water temperature, height, and x-y orientation in respect to the tracking surface).</li>
</ol>
9. Tracking of Larval Locomotion
NOTE: For more details refer to the attached manual for FIMTrack (supplement). For a program flow chart see Figure 2.
<ol style="margin-left: 40px;">
	<li>Adjust tracking parameters for the respective experiments using the preview option.
	<ol>
		<li>Adjust pixel per cm according to camera and field of view.</li>
		<li>Adjust frames per sec based on the camera settings.</li>
		<li>Adjust brightness thresholds so that all animals are detected correctly (feedback is given in the preview).</li>
		<li>Adjust larval area size thresholds. Note the feedback option in the preview: single animals are highlighted in yellow, colliding larvae are highlighted in red and the area of each animal is given in blue.</li>
	</ol>
	</li>
	<li>Start tracking by using the button in the bottom right. 
	NOTE: After successful tracking, an image featuring larval tracks and a csv file containing the calculated locomotion and posture features is stored in the image directory.</li>
	<li>Use the FIM Results viewer module (Edit &#62;Results Viewer&#8230;) to review and manually adjust the tracking results. If required, define stimulus regions to evaluate data in respect to the stimulus regime e.g., crawling orientation in respect to the heat gradient or distance to an odorant source. For more detailed description please use the manual (supplement).</li>
</ol>
10. Evaluation of the Data
<ol style="margin-left: 40px;">
	<li>Import the csv file into Excel, Matlab or any other program for further statistical analysis.</li>
</ol>

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The authors have nothing to disclose

In behavioral neuroscience it is mandatory to quantitatively decipher complex behavioral traits. Thus, large numbers of individuals must be observed at high resolution and automated procedures are needed for statistical analysis. Here, FIM imaging is described, a novel, simple and robust imaging setup, which provides the means to monitor locomotion of a wide range of animals. The efficacy of the FIM imaging setup was tested using Drosophila larvae, planarian flatworms and C. elegans worms. The FIM technology provides intrinsically high contrast to detect even internal structures of the animals, such as the brain, trachea, the gut or the proventriculus. Importantly, these internal structures are robustly identified so that they can serve to automatically identify the orientation of the animal19.
The quality of the movies can be influenced by excessive amount of water on the crawling surface. Thus, it is critical to control the moisture of the agar. Too old agar or too much water on the surface can cause artifacts. Likewise, ensure that no air bubbles are included in the crawling surface. In general, a well-prepared agar surface allows recording movies for 4 hr.
Due to the underlying physical principles FIM imaging generates almost&#160;noise free image recordings, resulting in a superb image quality. This in turn facilitates subsequent computer-based image analysis and enables high throughput. However, the methodology is restricted to analyzing animals that directly contact the agar surface. The tracking software is challenged by animals forming a donut shape. Although a binary indicator recognizes the donut shape, a wrong spine might be calculated.
Due to the modular construction of the tracking table dual and triple color imaging is in reach. Moreover, additional stimuli (light, odorants, electrical or mechanical stimuli) can easily be delivered from above. The FIMTrack program designed to match the power of FIM imaging can be easily adopted to track Drosophila larvae, C. elegans or planarians. Thus and due to its straightforward and cheap construction (see http://FIM.uni-muenster.de), FIM imaging is feasible for a wide range of biomedical applications and in particular allows urgently needed high throughput studies.

Most animals have the ability to move in a highly sophisticated and controlled manner. To decipher the genetic basis underlying locomotion control it is mandatory to quantitatively assess different behavioral patterns. In this respect, Drosophila can serve as an ideal model. Tracking of freely flying Drosophila is tantalizing 1-4 but crawling of Drosophila larvae occurs in two dimensions at relatively low speed and can thus be monitored easily. Camera-based setups combined with appropriate illumination are used to acquire images 5. Both incident or transmitted light is employed in behavioral experiments 6,7. However, due to the semi-translucent body of the larvae and possible light reflections of the crawling surface faithful recording of larval movements can be challenging. To overcome such problems, some complex methods have been devised. Recently, dark field illumination was introduced to enhance the foreground/background contrast 8. As an alternative to camera-based recording, lens-less optical imaging and image-sensor-less on-chip acquisition techniques have been introduced 9-11.
Several tracking programs have been introduced recently, including commercially available software 12 and custom solutions. Examples for high-throughput tracking programs are the Multi Worm Tracker (MWT) 13 and Multianimal Gait And Track (MAGAT) 8. Both have in common, that multiple animals can be tracked in a single open-field arena so that colliding animals lead to multiple new animal identities. To overcome this limitation, a multi-well setup was introduced separating 12 animals into individual wells 14. Precise quantification of locomotion of single individuals can be achieved by using a movable tracking stage in combination with a microscope 15. However, all these approaches are either cost inefficient, lack sufficient resolution or too time consuming for high throughput phenotyping.
To overcome the above mentioned limitations, we have developed FIM (FTIR-based Imaging Method) based on Frustrated Total Internal Reflection (FTIR) 16 (Figure 1). This new imaging approach provides an unprecedented high contrast and even allows multi-color recording of crawling animals 16. The underlying principle of this handy and effective method is easy. An acrylic glass plate is flooded with light (e.g., 875 nm infrared). Due to different refractive indices of acrylic glass and air, the light is totally reflected at the glass/air boundary. No heating of the acrylic glass is noted 16. Only if objects with a higher refractive index touch the light-flooded table, can light enter these objects. If the animals touch the surface, light is reflected and can be captured from below (Figure 1). In consequence, only the contact area of the animals appears as a bright spot, which allows detailed imaging with an overall black background. Thus, FIM-imaging allows to record perfect movies for computer vision algorithms. The simple and robust use of FIM now brings detailed high throughput analysis of complex animal behavior into reach and can be used for studying information processing: e.g., olfaction 8, 16; vision 17 or thermosensation 18.
<img alt="Figure 1" fo:content-width="6.5in" src="/files/ftp_upload/52207/52207fig1highres.jpg" width="650" /> 
Figure 1.&#160;FIM setup with heat-stimulus integration and underlying physical principles. (A) The FIM setup. Illumination intensity can be regulated at front panel. (B) To deliver a heat stimulus, a black painted aluminum plate, perfused with hot and cold water at both sides, is placed 2 mm above the agar surface which itself is 2 mm thick. The gradient is established on the heat radiator plate and the agar by the temperature differences. (C) The physical principle of frustrated total internal reflection: An acrylic glass plate is illuminated by infrared light. &#952;1, &#952;2, and &#952;3 indicate the light reflection angles. nA, n1, n2 and n3 denote the refractive indices of air, acrylic glass, agar and the larva respectively and fulfill the inequality nA &#60;n1 &#60;n2 &#60;n3. Due to refraction, the reflection angle changes during transition. If the angle is below the critical angle, light is not reflected anymore, can pass through the layers and can be captured from below. <a href="https://www.jove.com/files/ftp_upload/52207/52207fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The spectrum of processes that can be analyzed by FIM is broad. Without any further adjustments, FIM imaging can be used to monitor all larval stages of Drosophila (Figure 5B) or can be used to follow the foot-prints of adult Drosophila 19. Likewise, the trajectories of C. elegans or the movement of planarian flatworms can be easily recorded (Figure 5C). Even the analysis of fungal hypha or root hair growth appears feasible 19. In our current FIM setup, 4 x 16 infrared light emitting diodes (IR- LEDs) are integrated into a 32 x 32 cm2 acrylic glass plate, called the tracking table (Figure 1). The intensity of the IR-LEDs is adjusted depending on the weight of the objects on the tracking table, which can be easily done by a micro controller connected to the circuit via pulse-width modulation (PWM). FIM yields very high contrast images over a broad range of illumination intensities. Importantly, it generates excellent results at already low overall infrared irridation.
A camera with an infrared filter is placed below the tracking table, which allows integration of additional stimuli into the setup. Heat stimuli can be easily applied by a heat radiator plate and light stimuli are applied by an LCD projector. Also odorants can be contained in gradients by simple lids 8. For heat gradient experiments, the heat radiator plate is perfused with hot and cold water on both sides respectively and placed 2 mm above the larvae (Figure 1B).
The generation of high contrast, high quality movies opens the possibility for sophisticated computer based image analysis, thus we implemented the FIMTrack software to extract a large set of features from images (Figure 2). First six primary features were defined from the contour of the animal (Figure 3A). These features provide the baseline for further calculation of six secondary features which describe the animals shape and its position at certain stimuli at a given time point (Figure 3B). Currently, nine tertiary features are calculated that are integrating temporal aspects and thus characterize the locomotion of the animal together with the primary and secondary features (Figure 3C).
<img alt="Figure 2" fo:content-width="6.5in" src="/files/ftp_upload/52207/52207fig2highres.jpg" width="650" /> 
Figure 2.&#160;FIMTrack overview, algorithmic workflow and larval representation. (A) How to use FIMTrack. The images are loaded. Gray value threshold and larval size thresholds defining single larvae must be set. The larval area must be in [min-size, max-size]. Tracking is started by the highlighted button. (B) Tracking workflow. After the start button is clicked, the background image is calculated (minimal intensities over time). As long as there are frames left, the larvae are segmented based on the gray threshold and the min- and max-size threshold. For all segmentations the larval representations are calculated (compare to (C)). Each new model is associated to a given trajectory if a valid track is available. If the last frame is reached, finalizing post processing is done followed by the output generation. (C) Larval representation. The animal consists of a head and a tail point (h and t). Between these points an arbitrary odd number of spine points si can be set with a radius ri. In addition, the center of mass m and the main body bending angle &#947; are calculated. Several motion related parameters are sketched by purple lines. <a href="https://www.jove.com/files/ftp_upload/52207/52207fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="6.5in" src="/files/ftp_upload/52207/52207fig3highres.jpg" width="650" /> 
Figure 3.&#160;Features calculated by FIMTrack. (A) Primary features based on the contour of the animals. (B) Secondary features, based on primary features. (C) Tertiary features, based on primary features in consecutive frames and additional inputs <a href="https://www.jove.com/files/ftp_upload/52207/52207fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="823">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">Name of the Material/Equipment</td>
			<td style="width:191px;">Source</td>
			<td style="width:191px;">Catalog Number</td>
			<td style="width:251px;">Comments</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">FIM setup</td>
			<td style="width:191px;">Custom&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:251px;">details for construction or purchase of setups is available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acrylic glass plate</td>
			<td style="width:191px;">Custom&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:251px;">Additional for agar pouring</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Heat radiator plate</td>
			<td style="width:191px;">Custom&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:251px;">Aluminum plate (paintet in matt black) perfusable on opposing sites with adjustable mounting</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:191px;">Water calorifier/cooling pumps and hoses</td>
			<td style="width:191px;">Custom&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:251px;">based on GE healthcare MultiTempIII (No.: 18-1102-78) and Dr Bruno Lange GmBH (Typ: LTG013)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Standard Camera (4 MP)</td>
			<td style="width:191px;">Basler</td>
			<td style="width:191px;">acA2040-25gm</td>
			<td style="width:251px;">Camera defaultly used for the FIM setup</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Test Camera (1.4 MP)</td>
			<td style="width:191px;">QImaging</td>
			<td style="width:191px;">&#160;1394 firewire (01- QIC-F-M-12 MONO)</td>
			<td style="width:251px;">Camera used for comparison</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Test Camera (0.8 MP)</td>
			<td style="width:191px;">Point Grey</td>
			<td style="width:191px;">Dragonfly 2 (DR2-13S2M/C-CS)</td>
			<td style="width:251px;">Camera used for comparison</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Test Camera (0.3 MP)</td>
			<td style="width:191px;">Sony</td>
			<td style="width:191px;">PS Eye USB2.0 camera</td>
			<td style="width:251px;">Camera used for comparison</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:191px;">Computer</td>
			<td style="width:191px;">Custom&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:251px;">equipped with at least i5 Intel processor or better, 16 GB RAM and sufficient HDD storage space [&#62;1TB]</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Standard Fly food</td>
			<td style="width:191px;">Custom&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">Standard Fly vials 135 ml</td>
			<td style="width:191px;">Sarstedt AG&#38;Co, N&#252;mbrecht, Germany</td>
			<td style="width:191px;">78,895</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Petri dishes 9cm</td>
			<td style="width:191px;">Sarstedt AG&#38;Co, N&#252;mbrecht, Germany</td>
			<td style="width:191px;">821,473</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ultrapure deionized water</td>
			<td style="width:191px;">Merck Millipore, Darmstadt, Germany</td>
			<td style="width:191px;">Synergy&#160;</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NaCl</td>
			<td style="width:191px;">Carl Roth GmbH, Karlsruhe, Germany</td>
			<td style="width:191px;">3957.2</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Food grade agar</td>
			<td style="width:191px;">AppliChem GmbH, Darmstadt, Germany</td>
			<td style="width:191px;">A0917,5000</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paintbrush (small and large)</td>
			<td style="width:191px;">Milan</td>
			<td style="width:191px;">Aquarell 310 Size 0 and 2</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Pyrometer</td>
			<td style="width:191px;">Trotec</td>
			<td style="width:191px;">BP20</td>
			<td style="width:251px;"></td>
		</tr>
	</tbody>
</table>

fim imaging and fimtrack: two new tools allowing high-throughput and cost effective locomotion analysis

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to a certain degree, many animals have to be analyzed, to obtain statistically significant data. This in turn requires a computer assisted automated quantification of locomotion patterns. To obtain high contrast images of almost translucent and small moving objects, a novel imaging technique based on frustrated total internal reflection called FIM was developed. In this setup, animals are only illuminated with infrared light at the very specific position of contact with the underlying crawling surface. This methodology results in very high contrast images. Subsequently, these high contrast images are processed using established contour tracking algorithms. Based on this, we developed the FIMTrack software, which serves to extract a number of features needed to quantitatively describe a large variety of locomotion characteristics. During the development of this software package, we focused our efforts on an open source architecture allowing the easy addition of further modules. The program operates platform independent and is accompanied by an intuitive GUI guiding the user through data analysis. All locomotion parameter values are given in form of csv files allowing further data analyses. In addition, a Results Viewer integrated into the tracking software provides the opportunity to interactively review and adjust the output, as might be needed during stimulus integration. The power of FIM and FIMTrack is demonstrated by studying the locomotion of Drosophila larvae.

For imaging several different cameras with different resolution properties were tested (Figure 4). All cameras where equipped with an appropriate IR filter. According to the low resolution of the lowest priced camera in this test, the field of view is limited to 10 cm x 10 cm. Best results were obtained using a 4 megapixel (MP) camera. This leads to a resolution of 100 pixels per third instar larval length and allowed to easily recognize internal structures. Additionally, the peristalsis of the animal can be easily extracted (Figure 4A). However, one can still obtain high contrast movies using less expensive cameras, which can also be analyzed by FIMTrack. Using a 1.4 MP camera with a depth of 8 bit and a resolution of 1,392 x 1,040 pixels is about half the price and allows a resolution of 45 pixels per third instar larval length in the field of view. The head but no other internal structures can be recognized (Figure 4B). Tracking and detection of peristalsis is possible but accuracy is reduced (Figure 4B).
With an even cheaper 0.8 MP camera with a spatial resolution comparable to the 1.4 MP camera, the larval head cannot be recognized faithfully anymore (Figure 4C). Tracking and peristalsis analysis is possible but including more jitter based on the increased noise. Surprisingly, even a low resolution USB webcam provides sufficient quality movies to calculate larval trajectories (0.3 MP camera, below 20&#8364;, Figure 4D). The peristalsis can be calculated from the area but measurements are very noisy.
In our setup we routinely use the 4 MP camera. For screening, this camera allows monitoring a 22 cm x 22 cm arena, which obviously provides the opportunity to analyze large numbers of animals simultaneously so that high throughput analysis is feasible. Using this setting, larval length is represented by 40 pixels which still allows recording and analysis of peristalsis. An exemplary image of 15 larval trajectories in a heat gradient is given in Figure 5A. In addition, the use of a macro lens allows to image larvae with very high resolution where many internal organs become visible and head recognition is further improved (Figure 5B). Moreover these can be used for further more detailed analysis of behaviors of a wide range20. The same setup can be easily used to image crawling C. elegans worms (Figure 5C).
<img alt="Figure 4" fo:content-width="6.5in" src="/files/ftp_upload/52207/52207fig4highres.jpg" width="650" /> 
Figure 4.&#160;FIM imaging and tracking results for different cameras. (A) Left: FIM imaging of three larvae crawling on a 10 cm x 10 cm tracking stage captured using a 4 MP camera with 10 fps. Mid: Clipping and center of mass trajectory of a single larva. The area of the animal is indicated. Right: Area of the larva plotted over 100 frames. The red arrow indicates the time point of the clipped image. (B) Equivalent to (A) but captured using a 1.4 MP camera. (C) Equivalent to (A) but captured using a 0.8 MP camera. (D) Equivalent to (A) but captured using a 0.3 MP camera. <a href="https://www.jove.com/files/ftp_upload/52207/52207fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="6.5in" src="/files/ftp_upload/52207/52207fig5highres.jpg" width="650" /> 
Figure 5.&#160;Heat stimulus, and high resolution applications. (A) Heat stimulus application (compare to Figure 1). Trajectories calculated via FIMTrack. (B) High resolution application image of a third, second and first instar larva using a macro lens. The third instar larval length is represented by 400 pixels in a field of view of 2.5 x 2.5 cm. (C) A C. elegans worm captured using FIM imaging. Scale bars are indicated. <a href="https://www.jove.com/files/ftp_upload/52207/52207fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis at JoVE.com

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

FIM is a novel, cost effective imaging system designed to track small moving objects such as C. elegans, planaria or Drosophila larvae. The accompanying FIMTrack program is designed to deliver fast and efficient data analysis. Together, these tools allow high-throughput analysis of behavioral traits.

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

fim-imaging-fimtrack-two-new-tools-allowing-high-throughput-cost

Research

JoVE Journal

Neuroscience

11.6K Views.  Westfälische Wilhelms-Universität Münster. FIM is a novel, cost effective imaging system designed to track small moving objects such as C. elegans, planaria or Drosophila larvae. The accompanying FIMTrack program is designed to deliver fast and efficient data analysis. Together, these tools allow high-throughput analysis of behavioral traits.

Video: FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Here, we present a reproducible in vitro electroporation protocol for genetic manipulation of primary cerebellar granule cell precursors (GCPs) that is cost-effective, efficient, and viable. Moreover, this protocol also demonstrates a straightforward method for the molecular study of primary cilium-dependent Hedgehog signaling pathways in primary GCP cells.

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...

Viewing Microtubule Networks in &lt;em&gt;Drosophila&lt;/em&gt; Neuromuscular Junctions and Muscle Cells

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Author Spotlight: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.