title: Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion.
abstract: Analysis of particle trajectories in images obtained by fluorescence microscopy reveals biophysical properties such as diffusion coefficient or rates of association and dissociation. Particle tracking and lifetime measurement is often limited by noise, large mobilities, image inhomogeneities, and path crossings. We present Speckle TrackerJ, a tool that addresses some of these challenges using computer-assisted techniques for finding positions and tracking particles in different situations. A dynamic user interface assists in the creation, editing, and refining of particle tracks. The following are results from application of this program: 1), Tracking single molecule diffusion in simulated images. The shape of the diffusing marker on the image changes from speckle to cloud, depending on the relationship of the diffusion coefficient to the camera exposure time. We use these images to illustrate the range of diffusion coefficients that can be measured. 2), We used the program to measure the diffusion coefficient of capping proteins in the lamellipodium. We found values ∼0.5 μm(2)/s, suggesting capping protein association with protein complexes or the membrane. 3), We demonstrate efficient measuring of appearance and disappearance of EGFP-actin speckles within the lamellipodium of motile cells that indicate actin monomer incorporation into the actin filament network. 4), We marked appearance and disappearance events of fluorescently labeled vesicles to supported lipid bilayers and tracked single lipids from the fused vesicle on the bilayer. This is the first time, to our knowledge, that vesicle fusion has been detected with single molecule sensitivity and the program allowed us to perform a quantitative analysis. 5), By discriminating between undocking and fusion events, dwell times for vesicle fusion after vesicle docking to membranes can be measured.
authors.affiliation: Department of Physics, Lehigh University, Bethlehem, Pennsylvania, USA.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Smith
authors.forename: Erdem
authors.initials: E
authors.lastname: Karatekin
authors.forename: Andrea
authors.initials: A
authors.lastname: Gohlke
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mizuno
authors.forename: Naoki
authors.initials: N
authors.lastname: Watanabe
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Vavylonis
mesh_terms: D051344:Actin Capping Proteins / Q000378:metabolism; D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002465:Cell Movement; D004058:Diffusion; D005456:Fluorescent Dyes / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence / Q000379:methods*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011554:Pseudopodia / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism; D014584:User-Computer Interface
chemical_list: D051344:Actin Capping Proteins; D000199:Actins; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008081:Liposomes; D050600:SNARE Proteins