Figure 3. Panel A) Example of 4D tracking.

With the advances in computer performance, ever larger 4D data sets can be analyzed rapidly. Colored surfaces represent watershed-segmented objects and the tracks shown in red color connect the center of gravity of these surfaces over time. B) Analysis of in vitro motility data. Here, actin filaments were segmented using the “in vitro motility assay” particle production mode (time along tracks is color-coded from blue to red). The inset demonstrates in this situation a relatively narrow distribution of actin filament speed. C) Diatrack can also track objects segmented by third party methods. Here, bacteria were segmented as described in²³ and then tracked within our software using the “pre-segmented” mode. On the basis of this type of data, flow movies may be generated automatically that show locally coherent streaming of bacteria in the biofilm (see supplementary movie 2 and inset). D) Automating identification of mRNA export events. PP7-labelled mRNA particles (green channel) in yeast were tracked over time using both manual tracking (white trace), and using Diatrack (blue trace) -showing excellent overlap. Together with custom image analysis to detect the nuclear envelope (red channel), this high-throughput tool now routinely allows the identification of rare mRNA export events as well as their kinetic characterization (e.g. export time, nuclear envelope scanning time etc.). E) Characterizing fluid flows. Top view images of a planar cross-section of a liquid drop captured using a high-speed video camera. 5 μm fluorescent microparticles are observed to either follow smooth concentric trajectories associated with the acoustic streaming flow in a high viscosity drop (top), or exponentially divergent trajectories associated with chaotic acoustic streaming flow in a low viscosity drop (bottom). Each particle trajectory is labelled in a different color⁹². F) Tracking GFP-Rab5 endosomes in RPE cells. A subpopulation of these organelles is transported rapidly and efficiently along microtubules. In the representation used here, time is coded as the vertical axis. The insets characterize the effectiveness of the displacements i.e. the ratio of straight-line trajectory length to total trajectory length, and the total track lengths, respectively. Original movie was obtained from Flores-Rodriguez et al.¹⁰⁸