C.elegans Tracking and Analysis

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Presentation on the quantification of locomotory behaviors in C. elegans

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  • 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
  • C.elegans Tracking and Analysis

    1. 1. Quantification of locomotorybehaviors in C. elegansJulie Korich, Ph.D.Staff Scientist/Research Liaison
    2. 2. C. elegans as a Model OrganismVarious disciplines employ C. elegans nematodeas 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 soilqualitymbfbioscience.com
    3. 3. Modeling Locomotory Behaviors• Locomotory assays are usedin many areas of researchincluding neurodegenerativedisease, aging, andtoxicology• It is a common screening toolfor genetic assays• How do you quantifylocomotory behaviors?mbfbioscience.com
    4. 4. Challenges with Manual Marking• Tedious andlaborious – makeslarge scale studiesdifficult• Lack of Precision• Observervariabilitymbfbioscience.com
    5. 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 (LockeryLab)• 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. 6. Challenges of Automatic Tracking• Background clutter andexisting worm tracks• Juvenile worms andeggs on medium• Interactions• Head/Tail identification• Entanglement• Tracking large # ofworms• Velocity of movement• Swimming/thrashingVideo provided by Dr. Kate Harwood
    7. 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 fromframe to frame)• Thresholding tools to refine background and improvedetection despite moderate background clutter• Uses geometric model, worm motion model, backtrackingand Multiple Hypothesis Tracking for accurate detectionmbfbioscience.com
    8. 8. Worm Detection• The image is inverted and segmented to identifypotential worm objects• The algorithm measures 2 points of high curvaturefrom a closed planar B-spline curve around theboundary of the worm objectmbfbioscience.com
    9. 9. Head and Tail Determination• Identification based on the worm’sshape and frequency of movement• We apply the same spatial andtemporal cues used by humanobservers:• The worm’s tail area is lighter than the head• The worm’s tail area is thinner than thehead• The head moves more frequently than thetail• Head/tail identification can be swappedfor entire track by usermbfbioscience.comDetected Head
    10. 10. Geometric Model• Based on the center line of theworm and boundary• Modeled on a spline basis toallow easy scaling andresampling at differentresolutions• User can determine the # ofpoints along the center lineused in the analysis• 3 pts: head, tail, center• 17-19 pts: bending analysis• 59 pts: full resolution (default)mbfbioscience.com
    11. 11. Worm Motion Model• ɳ = movement alongcenterline (peristalticprogression factor)• Δα = Displacementorthogonal to the trajectory• Also use elongation andcontraction to modelmotionmbfbioscience.com
    12. 12. Tracking Across Frames• A deformable modelestimation algorithm fitsthe geometric model fromthe previous frame to thecurrent frame• Backtracking isperformed to re-establishworms with their previoustracks if lost• Backtracking used ifvideo starts withentangled wormsmbfbioscience.com
    13. 13. Multiple Hypothesis Tracking• Apply a set ofhypothesized wormlocations across time,thus building ahypothesis tree• Resolve conflicts byfinding the path ofMaximum Fitness (bestfit across frames)mbfbioscience.com
    14. 14. Detection of Complex Behaviors• The geometric model, wormmotion model, and MHThelp identify worms inambiguous conformations:• Coiled worms,• Overlapping worms• Omega bends• Reversing wormsmbfbioscience.com
    15. 15. Editing Functions• Manually draw a worm that is not detected prior totracking• Swap head and tail across a track• Join tracks• Split tracks• Delete worms per frame or across all framesmbfbioscience.com
    16. 16. Metrics and Analyses• Length: Distance between head and tail along centralaxis• 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 fromone frame to the next• Moving Average Speed: Instantaneous speedaveraged over multiple frames• Amplitude: Amplitude of the sine wave that best fitsthe worm posture• Wavelength: Period of the sine wave the best fits theworm’s posture• Bend Angle: Bending angle at the midpointmbfbioscience.com
    17. 17. Detection of Omega Bendsmbfbioscience.com• Begins when the bendingangle between head-midpointand tail-midpoint drops below1.57 radians ( 90 ) andcontinues until the angleexceeds 1.57 radians
    18. 18. Detection of Reversalsmbfbioscience.com• Reversal is defined as wormmoving backwards for userdefined set of frames
    19. 19. Head Bending Analysismbfbioscience.com• Indicates foraging• Worm sampled with 19pts• Bending angle is 3pt fromhead
    20. 20. Imaging Suggestions• Contrast: dark solid worms on light background• Lawn: replate worms to minimize tracks• Frame Rate: 5-10fps is adequate, faster for swimmingworms• 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 ratesmbfbioscience.com
    21. 21. Video provided by Dr. Kate Harwood
    22. 22. WormLab Overview• PC & MACcompatible• Accepts video files innumerous formats• Includes data andvideo export (withtracking overlay)• Workflow based –easy to train and use• Export metrics toMatlab and Excelmbfbioscience.com
    23. 23. • Control camera hardware to record videos fromstereoscopes, inverted microscopes, or macrophotography setup• Automatic Save• Variable Frame Rate• Scaling Tool: Calculate the pixel size• Scaling and frame rate are saved within the video file, andautomatically read by WormLab for analysis• Support DCAM/IIDC compliant cameras (Point Grey, AlliedVision and Sony)Camera Controlmbfbioscience.com
    24. 24. • Track swimming, thrashing worms:• Use a modified worm motion model to map the oscillationof the center point radially• Quantify pharyngeal pumping• Synchronization of stimulation and tracking• New analyses for bending and shape interpretation• Development of different assays – chemotaxisstudies, etc.WormLab – Future Directionsmbfbioscience.com
    25. 25. Summary• WormLab for automatic detection and tracking ofworms• Provide metrics including size, speed, direction• Track in complex backgrounds, entanglements,and shapes• Capture video sequences or open previouslyacquired sequencesmbfbioscience.com
    26. 26. • I would like to thank Tony Cooke for organizingthe 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.comAcknowledgments
    27. 27. • Email questions to Julie Korich atjulie@mbfbioscience.com• Download a free trialwww.mbfbioscience.com/wormlab• Watch a webinar that gives an overview ofWormLab www.mbfbioscience.com/webinarsmbfbioscience.comLearn More
    28. 28. • I would like to thank Tony Cooke for organizingthe seminar and the University of Washingtonmbfbioscience.comAcknowledgments

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