FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis
December 24th, 2014
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 overall goal of the following methodology is to visualize and to quantify the locomotion of Drosophila Melan Gaster larvae, allowing high throughput screening in free moving conditions. This is achieved by creating a tracking surface on the FIM setup and using frustrated total internal reflection for indirect illumination. Larvae of different genotypes are then placed on the surface and crawling behavior in response to a heat gradient is recorded.
The videos are then analyzed by the FIM Track. Software and data obtained are used to generate plots describing differences in larva, locomotion patterns. The results show that precise, high throughput, flexible and easy to use quantifications of larva.
Locomotion can be done based on the FIM imaging technique and the associated FIM track program. The main advantage of this imaging technique of existing methods like using incident or transmitted light, is that only animals touching the surface are visible, resulting in a superb foreground background contrast with no disturbing Artifacts, though this method can provide insight into biology of oph lava. It can also be applied to study walking behavior flies and locomotion of other mono organisms such as sea elegance, plenaries, or even root growth of plants.
Visual demonstration of this method is critical to convince others that a complex technology is easy to use. The first step is to create a moist crawling surface for the FITR based imaging method or fim. To do this boil, 0.8%food grade auger in deionized ultrapure water.
Pour the hot auger onto an acrylic glass plate. Do not agitate the auger after boiling and pour constantly. To avoid bubbles at this temperature, a two millimeter thick slab should be created.
Use the remaining auger solution to fill standard six centimeter Petri dishes. These will be used to sort clean and habituate larvae prior to the experiment. Next, trim approximately two centimeters from the perimeter of the auger slab to obtain a plain square surface for recording.
Transfer the auger slab to the FIM setup directly after cooling. By gently pushing the auger over the edge of the acrylic glass plate, the auger should glide in place. You are now ready for imaging preparation to include an aversive auger barrier in the crawling surface.
Boil 2.5%food grade auger with three molar NACL in deionized ultrapure water. Next, cut a two centimeter wide notch surrounding the field of view into the previously poured crawling surface. When ready, fill the notch with salt auger 0.1 to 0.3 centimeters higher than the tracking surface.
Next, prepare the heat gradient device. The device is an aluminum plate covered with an insulating surface and perfused with water from pumps at both ends. To allow temperature control, turn on the heat gradient device one hour prior to use and place it above the setup to allow it to reach the desired temperature.
This time can be used for further preparations. When ready, transfer the crawling surface auger with a salt barrier to the setup. Place the radiator plate over the auger and adjust the spacing between the plate and crawling surface to two millimeters.
Establish a linear gradient from 34 degrees Celsius to 18 degrees Celsius by adjusting the temperature of the water circuits to one degree Celsius and 45 degrees Celsius, which is appropriate for this equipment. Allow the crawling surface to equilibrate in the gradient for 20 minutes. Before proceeding, test the temperature gradient with a TER to prepare the flies, retrieve them from a 25 degree Celsius incubator and drop a little water into the culture vials to drive late third in star larva movement.
Next, collect the largest larvae from the vial walls using a small paintbrush, two to five minutes before recording, transfer enough larvae for one video into a Petri dish that was prepared earlier. After the temperature gradient is established, set up the recording software. Specify the number of frames per video.
Define the saving path and set up for instant recording within 20 seconds. After placing the larvae on the crawling surface to begin image one of the larvae and adjust the illumination intensity to obtain good contrast. When ready, lift the radiator plate slightly and place the larvae at 34 degrees Celsius, two centimeters from the salt barrier.
Lower the radiator plate again and start recording in the dark to not disturb the animals. After recording, remove the larvae clean and moisturize the surface. To avoid spreading NACL, do not touch the salt auger while working, while the surface must be kept moist at all times, avoid excessive moisture, which can be seen as halos or drops surrounding larvae.
In recordings and disturbed tracking, allow the auger surface to equilibrate for one to two minutes before proceeding. During this time, save the collected images and gather new animals. To prepare for the next video, control the temperature gradient using a TER every five videos and adjust the temperature device if needed.
To begin tracking locomotion, adjust the tracking parameters using the preview option. Adjust millimeter per pixel according to camera and field of view. Next, adjust the frames per second based on the camera settings, use feedback given in the preview window to adjust the brightness thresholds so that all animals are detected correctly.
Next, adjust larval area size thresholds, single animals are highlighted by yellow circles colliding larvae are highlighted in red, and the area of each animal is given in blue. Start tracking by using the button in the bottom right after the tracking session, an image featuring larva tracks and a CSV file containing the calculated locomotion and posture features is stored in the image directory. Finally, use the FIM results viewer module to review and manually adjust the tracking results.
Define the crawling orientation in respect to the heat gradient to evaluate the data based on this stimulus. In the larva representation shown here, the head and tail point are marked between these points. An arbitrary odd number of spine points can be set with a radius.
In addition, the center of mass and the main body bending angle are calculated. Shown here are FI imaging and tracking results from different cameras. The images to the left capture three larvae crawling on a 10 centimeter by 10 centimeter tracking stage.
The mass trajectory and area of a single larva is shown to the right. The red arrow indicates the time point of the clipped image results from applying. A heat stimulus are shown here as different colored trajectories calculated via F track.
These high resolution application images of a third, second, and first in star larvae were obtained with a macro lens. Other organisms like sea can be imaged easily as well. Once mastered, about a hundred lab can be analyzed per hour per each tracking setup.
While attempting this procedure, it is important to remember to always record under the same conditions Following this procedure. Other methods like optogenetic stimulation or in vivo detection of neural activity could be included into the setup. After watching this video, you should have a good understanding of how easy it can be to efficiently analyze ular locomotion.
Overview
The FIM imaging system is designed for tracking small moving organisms like Drosophila larvae, enabling high-throughput behavioral analysis. The FIMTrack program facilitates efficient data analysis, allowing researchers to quantify locomotion patterns effectively.
Key Study Components
Area of Science
- Neuroscience
- Behavioral Analysis
- Imaging Techniques
Background
- The FIM system utilizes frustrated total internal reflection for indirect illumination.
- It provides superior contrast by only visualizing organisms in contact with the tracking surface.
- This method can be applied to various organisms, including C. elegans and planaria.
- Visual demonstrations are essential for showcasing the system's ease of use.
Purpose of Study
- To visualize and quantify the locomotion of Drosophila larvae.
- To enable high-throughput screening in free-moving conditions.
- To analyze behavioral responses to environmental stimuli, such as heat gradients.
Methods Used
- Preparation of a moist crawling surface using food-grade agar.
- Establishment of a heat gradient for experimental conditions.
- Recording of larval movement using the FIM imaging setup.
- Analysis of recorded videos with the FIMTrack software to quantify locomotion patterns.
Main Results
- The FIM system allows for precise tracking of larval locomotion.
- High-throughput analysis is achievable with minimal artifacts.
- Data generated can reveal significant differences in locomotion patterns across genotypes.
- The method is adaptable for studying various organisms and behaviors.
Conclusions
- The FIM imaging technique offers a cost-effective solution for behavioral analysis.
- It provides clear advantages over traditional imaging methods.
- The system is user-friendly, promoting its adoption in research settings.
Frequently Asked Questions
Executive Industry Relevance
High-throughput behavioral analysis of model organisms supports early-stage target validation by enabling quantitative assessment of phenotypic responses to genetic or environmental perturbations. The FIM imaging and FIMTrack workflow provides a cost-effective, reproducible platform for locomotor phenotyping, reducing variability in behavioral assays and increasing statistical power in discovery screens. This approach enhances predictive confidence in lead identification by linking genotype to measurable behavioral output in a scalable format.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses through quantitative measurement of larval locomotion as a functional readout of neuronal network activity.
- Operational Value: Supports biological de-risking by providing high-contrast imaging that minimizes artifacts and improves detection reliability in free-moving conditions.
- Predictive Value: Facilitates phenotypic screening across genotypes, allowing early discrimination of behavioral differences that may indicate target engagement or pathway modulation.
Screening & Assay Development
- Assay Readiness: Generates standardized CSV outputs containing locomotion and posture features, enabling automated data processing and cross-experiment comparison.
- Scalability: Allows analysis of up to 100 larvae per hour per tracking setup, supporting medium-throughput screening campaigns in discovery biology.
- Platform Reuse: Open-source architecture permits adaptation for other small organisms (e.g., C. elegans, planaria) and stimuli beyond heat gradients, increasing long-term utility.
Translational & Preclinical Research
- Translational Continuity: Provides a disease-relevant system for studying neuromotor phenotypes that may model aspects of neurological disorders, supporting preclinical validation.
- Mechanistic De-risking: Enables calculation of biomechanical parameters (e.g., center of mass, body bending angle) to clarify mechanistic links between genetic manipulation and motor output.
- Risk-Adjusted Advancement: Objective, quantifiable outputs help prioritize targets based on behavioral phenotype severity and consistency across replicates.
Pipeline & Workflow Integration
The FIM/FIMTrack system integrates into the discovery continuum from target validation through lead identification, where behavioral phenotyping informs compound screening and mechanistic follow-up.
- Discovery Biology: Supports hypothesis testing by quantifying locomotion patterns in response to stimuli, enabling pathway clarification and target confirmation.
- Screening: Delivers reproducible, quantitative readouts essential for assay standardization and hit confirmation in phenotypic screens.
- Analytics: Outputs include trajectory, area, and bending metrics that allow statistical comparison of conditions and genotypes.
- Translational Research: Connects larval behavioral phenotypes to conserved neurogenic mechanisms, supporting extrapolation to higher-order models.
- Enterprise Reuse: Modular, open-source design allows deployment across teams and model systems with minimal retraining.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing behavioral assay noise and improving signal detection.
- Operational Value: Standardized surface preparation and tracking parameters enhance reproducibility across users and sites.
- Strategic Value: Enables faster go/no-go decisions by providing early, high-resolution phenotypic data on target modulation.
- Portfolio Impact: Supports risk-adjusted prioritization through objective, scalable behavioral endpoints that reduce late-stage attrition due to lack of efficacy.
Implementation Considerations
- Requires expertise in behavioral neuroscience and basic image processing to optimize tracking parameters and interpret locomotion features.
- Dependent on consistent surface preparation (agar concentration, moisture control) and thermal gradient stability for reliable results.
- Necessitates cross-team agreement on feature extraction thresholds (e.g., larval area, brightness) to ensure data comparability.
- Adaptation to other models (e.g., zebrafish embryos, adult flies) may require surface and illumination adjustments.
- Practical limitation: performance may decrease with highly translucent or non-contacting organisms unless imaging parameters are tuned.
