C.elegans Tracking and Analysis
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Presentation on the quantification of locomotory behaviors in C. elegans
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C.elegans Tracking and Analysis
- 1. Quantification of locomotory behaviors in C. elegans Julie Korich, Ph.D. Staff Scientist/Research Liaison
- 2. C. elegans as a Model Organism Various disciplines employ C. elegans nematode as a model organism: • Biology: Cell Differentiation, Meiosis, and RNAi studies • Neuroscience: Neuronal function, differentiation, behavior • Toxicology: Adverse effects of chemicals on organisms • Ecology/Soil Science: Environmental impact on soil quality mbfbioscience.com
- 3. Modeling Locomotory Behaviors • Locomotory assays are used in many areas of research including neurodegenerative disease, aging, and toxicology • It is a common screening tool for genetic assays • How do you quantify locomotory behaviors? mbfbioscience.com
- 4. Challenges with Manual Marking • Tedious and laborious – makes large scale studies difficult • Lack of Precision • Observer variability mbfbioscience.com
- 5. Available Tracking Software • Single worm trackers • WormTracker 2.0 (Shafer Lab) • Nemo (Tavernarakis Lab) • Multimodal Illumination and Tracking System (Lu Lab) • CoLBeRT (Samual Lab) • Opto-mechanical system for virtual environments (Lockery Lab) • Multi Worm Trackers • The Parallel Worm Tracker (Goodman Lab) • OptoTracker (Gottschalk Lab) • The Multi Worm Tracker (Kerr Lab) Husson, S. J. et al. Keeping track of worm trackers (September 10, 2012), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.156.1, http://www.wormbook.org.
- 6. Challenges of Automatic Tracking • Background clutter and existing worm tracks • Juvenile worms and eggs on medium • Interactions • Head/Tail identification • Entanglement • Tracking large # of worms • Velocity of movement • Swimming/thrashing Video provided by Dr. Kate Harwood
- 7. How WormLab Tracks • Supports high mag and low mag (whole plate) tracking • Composed of 2 parts: • Detection (finds new worms as the enter the movie) • Tracking (determining changes in worm position and shape from frame to frame) • Thresholding tools to refine background and improve detection despite moderate background clutter • Uses geometric model, worm motion model, backtracking and Multiple Hypothesis Tracking for accurate detection mbfbioscience.com
- 8. Worm Detection • The image is inverted and segmented to identify potential worm objects • The algorithm measures 2 points of high curvature from a closed planar B-spline curve around the boundary of the worm object mbfbioscience.com
- 9. Head and Tail Determination • Identification based on the worm’s shape and frequency of movement • We apply the same spatial and temporal cues used by human observers: • The worm’s tail area is lighter than the head • The worm’s tail area is thinner than the head • The head moves more frequently than the tail • Head/tail identification can be swapped for entire track by user mbfbioscience.com Detected Head
- 10. Geometric Model • Based on the center line of the worm and boundary • Modeled on a spline basis to allow easy scaling and resampling at different resolutions • User can determine the # of points along the center line used in the analysis • 3 pts: head, tail, center • 17-19 pts: bending analysis • 59 pts: full resolution (default) mbfbioscience.com
- 11. Worm Motion Model • ɳ = movement along centerline (peristaltic progression factor) • Δα = Displacement orthogonal to the trajectory • Also use elongation and contraction to model motion mbfbioscience.com
- 12. Tracking Across Frames • A deformable model estimation algorithm fits the geometric model from the previous frame to the current frame • Backtracking is performed to re-establish worms with their previous tracks if lost • Backtracking used if video starts with entangled worms mbfbioscience.com
- 13. Multiple Hypothesis Tracking • Apply a set of hypothesized worm locations across time, thus building a hypothesis tree • Resolve conflicts by finding the path of Maximum Fitness (best fit across frames) mbfbioscience.com
- 14. Detection of Complex Behaviors • The geometric model, worm motion model, and MHT help identify worms in ambiguous conformations: • Coiled worms, • Overlapping worms • Omega bends • Reversing worms mbfbioscience.com
- 16. Editing Functions • Manually draw a worm that is not detected prior to tracking • Swap head and tail across a track • Join tracks • Split tracks • Delete worms per frame or across all frames mbfbioscience.com
- 17. Metrics and Analyses • Length: Distance between head and tail along central axis • Width is calculated from N points along the worm • Direction is the direction of travel • Postion is the center of the median axis • Instaneous speed: Velocity along the central axis from one frame to the next • Moving Average Speed: Instantaneous speed averaged over multiple frames • Amplitude: Amplitude of the sine wave that best fits the worm posture • Wavelength: Period of the sine wave the best fits the worm’s posture • Bend Angle: Bending angle at the midpoint mbfbioscience.com
- 18. Detection of Omega Bends mbfbioscience.com • Begins when the bending angle between head-midpoint and tail-midpoint drops below 1.57 radians ( 90 ) and continues until the angle exceeds 1.57 radians
- 19. Detection of Reversals mbfbioscience.com • Reversal is defined as worm moving backwards for user defined set of frames
- 20. Head Bending Analysis mbfbioscience.com • Indicates foraging • Worm sampled with 19pts • Bending angle is 3pt from head
- 21. Imaging Suggestions • Contrast: dark solid worms on light background • Lawn: replate worms to minimize tracks • Frame Rate: 5-10fps is adequate, faster for swimming worms • Cameras: • Industrial machine vision cameras (CCD) work • Webcams (low cost CMOS not so much) • Recommend monochrome cameras • Image size: • Whole plate: 2500x2500 resolution (5 Megapixels) • Single worms: 800x600, 1200x1024 and faster frame rates mbfbioscience.com
- 22. Video provided by Dr. Kate Harwood
- 23. WormLab Overview • PC & MAC compatible • Accepts video files in numerous formats • Includes data and video export (with tracking overlay) • Workflow based – easy to train and use • Export metrics to Matlab and Excel mbfbioscience.com
- 24. • Control camera hardware to record videos from stereoscopes, inverted microscopes, or macro photography setup • Automatic Save • Variable Frame Rate • Scaling Tool: Calculate the pixel size • Scaling and frame rate are saved within the video file, and automatically read by WormLab for analysis • Support DCAM/IIDC compliant cameras (Point Grey, Allied Vision and Sony) Camera Control mbfbioscience.com
- 25. • Track swimming, thrashing worms: • Use a modified worm motion model to map the oscillation of the center point radially • Quantify pharyngeal pumping • Synchronization of stimulation and tracking • New analyses for bending and shape interpretation • Development of different assays – chemotaxis studies, etc. WormLab – Future Directions mbfbioscience.com
- 26. Summary • WormLab for automatic detection and tracking of worms • Provide metrics including size, speed, direction • Track in complex backgrounds, entanglements, and shapes • Capture video sequences or open previously acquired sequences mbfbioscience.com
- 27. • I would like to thank Tony Cooke for organizing the seminar and the University of Washington • Email questions to: • Julie Korich at julie@mbfbioscience.com • View our website for additional information, videos, and instructions to download a tree trial (http://www.mbfbioscience.com/wormlab) mbfbioscience.com Acknowledgments
- 28. • Email questions to Julie Korich at julie@mbfbioscience.com • Download a free trial www.mbfbioscience.com/wormlab • Watch a webinar that gives an overview of WormLab www.mbfbioscience.com/webinars mbfbioscience.com Learn More
- 29. • I would like to thank Tony Cooke for organizing the seminar and the University of Washington mbfbioscience.com Acknowledgments
Hinweis der Redaktion
- Good afternoon. Welcome to the next webinar in our continuing series from MBF Bioscience. My name is Susan Hendricks. As a staff scientist, I am grants manager for our federally funded research activities at MBF.I’m joined today by Jeff Sprenger…Today we’ll be demonstrating the latest advancements for WormLab, software to track and analyze nematode locomotory behavior.
- In today’s webinar, we will demonstrate the capabilities of WormLab software. Before the walkthrough, we will spend just a minute to become acquainted with our company’s history to give you a sense of how we came to be creating this type of software. Medium-throughput behavioral analysis of C. elegans requires sophisticated automated methods to quantify measurements of worm morphology and dynamic movement. The software can track worms of diverse sizes and conformations in the presence of common artifacts and clutter, even when worms are overlapping with others. Multiple hypothesis testing and backtracking methods permits analysis of worms that are entangled and/or interacting with other worms. An intuitive workflow provides a series of steps to guide the user to acquire or load a video sequence, set sampling parameters to detect worms and run the trackingalgorithm across the sequence. The software also provides the investigator with advanced tools for manual worm detection and editing. Results, which can be exportedto Excel, can be viewed per frame or as summarized totals per metric of interest.Controlled image acquisition overcomes a key hurdle to increase throughput for behavioral studies which require video capture at a fast enough frame rate to permit extraction of behavioral features of interest at an appropriate magnification. Behavioral analyses can be performed immediately following capture, or the data can be archived for later analysis. We will pause after each of these topics to address specific questions. Feel free to submit questions at any time via the questions window and we’ll address them as we go.If you have additional questions that were not addressed by the conclusion of the presentation, please send your questions to info@mbfbioscience.com and we’ll respond to you directly.Let’s get started…As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- WormLab quantifies worms moving on agar plates, with or without a lawn. It has been designed to keep track of worms even after interaction events so that the identity of worms throughout an entire experiment can be known (rather than returning information about the entire population of plated worms). The software uses algorithms that fit the worms– which provides many individual details about the worm including length, width, wavelength, among others. WormLab is able to detect and track a number of species of nematodes, and at different developmental stages as well.(No whole plate analysis as yet, but is planned.)
- As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- As the first organism with a completely sequenced genome, C. elegans is a powerful research tool. This small multicellular organism shares genetic similarities to many vertebrates including 38% homology with the human genome. The developmental fate of each of the approximately 1000 cells is known. Further, the identity and connectivity of all 302 neurons have been mapped. C. elegans are small (size) and have a short generation time ideally suiting them for medium and high throughput analyses.Currently, most methods to assess C. elegans phenotypic behavior are manual and laborious to perform which precludes medium-throughput studies. We wanted to increase the throughput of these detailed studies. WormLab is a commercial product that allows investigators to perform automated, quantitative analyses of locomotory and other complex behaviors of freely moving C. elegans nematodes. Locomotion assays are used in many areas of research including those focused on neurodegenerative disease, aging, and toxicology . Additionally, locomotion is a common screening tool for genetic assays.
- Smoothed Speed uses a locally weighted polynomial regression. The smoothing implements ‘loess’ method, the locally weighted polynomial (2nd degree) regression developed by Cleveland and Devlin [1]. The basic idea is to use locally weighted quadratic regression to smooth data. The smoothing process is local because each smoothed value is determined by neighboring data points defined within the span. The regression is a weighted regression where a weight function is defined for data points contained within the span.In addition to the regression weight function, a robust weight function is also incorporated to make the process resistant to outliers.
- Smoothed Speed uses a locally weighted polynomial regression. The smoothing implements ‘loess’ method, the locally weighted polynomial (2nd degree) regression developed by Cleveland and Devlin [1]. The basic idea is to use locally weighted quadratic regression to smooth data. The smoothing process is local because each smoothed value is determined by neighboring data points defined within the span. The regression is a weighted regression where a weight function is defined for data points contained within the span.In addition to the regression weight function, a robust weight function is also incorporated to make the process resistant to outliers.
- Smoothed Speed uses a locally weighted polynomial regression. The smoothing implements ‘loess’ method, the locally weighted polynomial (2nd degree) regression developed by Cleveland and Devlin [1]. The basic idea is to use locally weighted quadratic regression to smooth data. The smoothing process is local because each smoothed value is determined by neighboring data points defined within the span. The regression is a weighted regression where a weight function is defined for data points contained within the span.In addition to the regression weight function, a robust weight function is also incorporated to make the process resistant to outliers.
- WormLab will continue to grow as users provide feedback and input about the analyses needed most by researchers. We envision a long