Tracking single molecules with the new open source tool Speckle TrackerJ

Advances in microscopic imaging continue to create unique demands for tracking multiple bright spots in time-lapse movies. We developed an open source, particle-tracking tool to obtain new results in single-molecule fluorescence microscopy.

HFSP Program Grant Award holders Dimitrios Vavylonis and Naoki Watanabe and colleagues
authored on Fri, 18 November 2011

HFSP Program Grant awardees Naoki Watanabe and Dimitrios Vavylonis combine mathematical modelling, image analysis, and single molecule fluorescence microscopy to quantify diffuse actin species at the leading edge of motile cells. Single molecule imaging of live cells yields important characteristics required for the development of mathematical models of actin turnover and for testing them against experiments.

Figure: Tracks of single actin molecules at the leading edge of an XTC cell. Speckle appearance and disappearance indicates polymerization into filaments and depolymerization. The speckles move away from the leading edge due to retrograde flow. The color scale shows the lifetime of each actin monomer in the filament.  

Image analysis of particle tracks in fluorescence microscopy images has a long history but up to now, there was no existing open source software tool with sufficient flexibility for the analysis of actin speckles in the images obtained by the Watanabe lab. This is in fact a common situation at the forefront of biological research, including images of single-vesicle fusion obtained by co-authors Erdem Karatekin and Andrea Gohlke.  In many similar situations, the signal to noise is very low, particle mobility is high during detection, a small sub-population needs to be selectively analyzed, and/or both the particles and the background have broad intensity variations. Careful supervision of all tracks is required in such challenging situations, especially if the experimental approaches are new.

To aid quantification of bright speckles in time-lapse movies, Smith et al. developed Speckle TrackerJ, a freely-available software suite to mark and track the appearance and disappearance of bright spots. The development of Speckle TrackerJ grew out of the need for a flexible tool combining supervised/assisted tracking with efficient automated algorithms. When imaging conditions are sufficiently good, Speckle TrackerJ allows unsupervised tracking with performance comparable to other existing tools. In extremely difficult situations, user input allows obtaining tracks where existing tools fail. Speckle TrackerJ was designed in such a way that it would have the flexibility to be applicable to many different biological imaging contexts.

Using this tool, the authors found that single capping protein speckles diffuse much slower than would be expected if they were free, suggesting capping protein association with protein complexes or the membrane. In the case of SNARE-mediated fusion, they marked appearance and disappearance events of fluorescently-labelled vesicles to supported lipid bilayers and tracked single lipids from the fused vesicle on the bilayer. This is the first time that vesicle fusion has been detected with single molecule sensitivity and the program allowed a quantitative analysis. By discriminating between undocking and fusion events, dwell times for vesicle fusion following vesicle docking to membranes was also measured.

Reference

Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion.  M. B. Smith, E. Karatekin, A. Gohlke, H. Mizuno, N. Watanabe and D. Vavylonis, Biophys J., 101:1794-1804 (2011).

Pubmed link

Cell link

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