Cellular Mitosis Microbead Rheology Laser Tracking Initiation Video-Based Spot Tracking CISMM: Computer Integrated Systems for Microscopy and Manipulation Collaborators: UIUC – Paul Selvin, Cystic Fibrosis: C. William Davis, Richard Boucher; Biology: Edward Salmon, Lisa Cameron Project Lead: Russell M. Taylor II Investigators: Richard Superfine 12/5/2003 Driving Applications Approach and Evaluation The study of the rheology (viscoelasticity) is important for understanding intracellular transport and structure, and the flow of biological fluids such as mucus. We are developing tools for microbead rheology for the immediate application of mucus flow in human lung cell cultures, for both healthy and CF cultures. Detailed study of the motion of beads under Brownian motion and under magnetic forces will explore the biochemical mechanism that regulate viscoelasticity. Biology Edward Salmon Lisa Cameron In Cellular Mitosis, each pair of chromo- somes lines up at the metaphase plate, connected to a pair of kinetochores. The kinetochores are pulled apart by interactions with the microtubule spindle. Unknown are the applied forces and the signaling mechanism that starts the pulling. We hope to learn about the periodicity and stability of these motions, and whether groups of kinetochores move in concert. Kinetochore Pair Cystic Fibrosis C. W. Davis, R. Boucher Potential Collaborator: John Crocker, U. Penn. 3DFM Core Richard Superfine Gary Bishop Controlled placement of a bead within a laser’s focus in 3 dimensions requires delicate and tedious user interaction. As the viscosity of the specimen fluid decreases, this process becomes difficult and time consuming. By coupling rapid 2D video tracking with user-controlled focal depth, we aim to provide robust and rapid initiation of laser tracking in a wide range of samples. The eventual goal is automatic capture of beads on cilia that are beating at 15+ Hz. Requirements: Tracking multiple beads in brightfield Tracking multiple beads in fluorescence Tracking changing-focus beads Requirements: Rapid online tracking Integration with instrument Requirements: Tracking small spots in fluorescence Insensitivity to image noise Evaluation We are using four mechanisms to measure the accuracy and precision of this approach: Comparison with different tracking approaches Laser tracking compared to video tracking Comparison with calibrated stage motion Fixed bead in Mad City Labs stage Comparison with other validated algorithms Running on data from Selvin lab Validation with synthetic test data Compute Airy pattern Add pixel boundaries Add background noise Add photon shot noise Move spot location Track with algorithm Compare to actual motion Ahmet Yildiz, Joseph N. Forkey, Sean A. McKinney, Taekjip Ha, Yale E. Goldman, Paul R. Selvin, “Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization,” Science 300 (27). Model-Based Tracking This project takes a model-based approach to tracking spots, where a model of the intensity distribution within the spot is compared against the image to find the location at which the best match is found. This position can be found to sub-pixel accuracy, and is robust to image noise that is uncorrelated with the spot cross-section. Three Models A Disk model is used to track spots that have an evenly dark or bright central area with a sharp transition to a contrasting surround (Brightfield imaging of beads). (Runs at 60+ frames/second.) A Cone model is used to track spots that have high intensity in the center that becomes uniformly lower away from the center (fluoresence of single fluorophores), or low intensity going to high. A Circularly-Symmetric model is used to track spots whose cross section changes over time (as they move in and out of focus). This slower, more general model minimizes the variance in concentric rings surrounding the bead center. Another Approach: The project Texture- Based Motion Tracking takes a pixel- matching approach between successive images as described in a separate poster. New Collaborator: Paul Selvin (UIUC) Tracking of molecular motors with single molecule fluorescence tags Video-based Spot tracking provides high spatial resolution position information, rivaling that of laser based techniques. It is simple from an instrumentation standpoint, requires little operator input and can track multiple objects simultaneously. Software is available to outside users for download. Available for outside users to download at