Matthew B. Smith

Segmentation and tracking of cytoskeletal filaments using open active contours

We use open active contours to quantify cytoskeletal structures imaged by fluorescence microscopy in two and three dimensions. We developed an interactive software tool for segmentation, tracking, and visualization of individual fibers. Open active contours are parametric curves that deform to minimize the sum of an external energy derived from the image and an internal bending and stretching energy. The external energy generates (i) forces that attract the contour toward the central bright line of a filament in the image, and (ii) forces that stretch the active contour toward the ends of bright ridges. Images of simulated semiflexible polymers with known bending and torsional rigidity are analyzed to validate the method. We apply our methods to quantify the conformations and dynamics of actin in two examples: actin filaments imaged by TIRF microscopy in vitro, and actin cables in fission yeast imaged by spinning disk confocal microscopy.

Automated actin filament segmentation, tracking and tip elongation measurements based on open active contour models

This paper presents an automated method for actin filament segmentation and tracking for measuring tip elongation rates in Total Internal Reflection Fluorescence Microscopy (TIRFM) images. The main contributions of the paper are: (i) we use a novel open active contour model for filament segmentation and tracking, which is fast and robust against noise; and (ii) different strategies are proposed to solve the filament intersection problem, which is shown to be the main difficulty in filament tracking. Application to experimental results demonstrated the robustness and effectiveness of this method.

Distributed Actin Turnover in the Lamellipodium and FRAP Kinetics

Studies of actin dynamics at the leading edge of motile cells with single-molecule speckle (SiMS) microscopy have shown a broad distribution of EGFP-actin speckle lifetimes and indicated actin polymerization and depolymerization over an extended region. Other experiments using FRAP with the same EGFP-actin as a probe have suggested, by contrast, that polymerization occurs exclusively at the leading edge. We performed FRAP experiments on XTC cells to compare SiMS to FRAP on the same cell type. We used speckle statistics obtained by SiMS to model the steady-state distribution and kinetics of actin in the lamellipodium. We demonstrate that a model with a single diffuse actin species is in good agreement with FRAP experiments. A model including two species of diffuse actin provides an even better agreement. The second species consists of slowly diffusing oligomers that associate to the F-actin network throughout the lamellipodium or break up into monomers after a characteristic time. Our work motivates studies to test the presence and composition of slowly diffusing actin species that may contribute to local remodeling of the actin network and increase the amount of soluble actin.

New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales

This paper introduces a new, easy-to-use method of fluorescence single-molecule speckle microscopy for actin with nanometer-scale accuracy. This new method reveals that actin flows in front of mature focal adhesions (FAs) are fast and biased toward FAs, suggesting that mature FAs are actively engaged in pulling and remodeling the local actin network.

Segmentation and Tracking of Cytoskeletal Filaments Using Open Active Contours

We developed an interactive software tool to quantify cytoskeletal filaments imaged by fluorescence microscopy in two and three dimensions. Our software allows users to visualize and record the position of filaments by implementing a robust algorithm in conjunction with user interaction. We use open active contours for segmentation and tracking of individual fibers. Open active contours are parametric curves that deform to minimize the sum of an external energy derived from the image and an internal bending and stretching energy. The external energy generates (i) forces that attract the contour towards the central bright line of a filament in the image, and (ii) forces that stretch the active contour towards the ends of bright ridges. Images of simulated semiflexible polymers with known bending and torsional rigidity are analyzed to validate the method. Analysis of simulated filaments illustrated the advantages and inherent limitations in representing filaments as a discrete series of points. We apply our methods to quantify the conformations of actin in two examples. We calculated the persistence length of actin filaments imaged by TIRF microscopy in vitro, and we segmented actin cables in fission yeast imaged by spinning disk confocal microscopy.