## Abstract

### Background

Single molecule tracking (SMT) analysis of fluorescently tagged lipid and protein probes is an attractive alternative to ensemble averaged methods such as fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP) for measuring diffusion in artificial and plasma membranes. The meaningful estimation of diffusion coefficients and their errors is however not straightforward, and is heavily dependent on sample type, acquisition method, and equipment used. Many approaches require advanced computing and programming skills for their implementation.

### Findings

Here we present TrackArt software, an accessible graphic interface for simulation and complex analysis of multiple particle paths. Imported trajectories can be filtered to eliminate spurious or corrupted tracks, and are then analyzed using several previously described methodologies, to yield single or multiple diffusion coefficients, their population fractions, and estimated errors. We use TrackArt to analyze the single-molecule diffusion behavior of a sphingolipid analog SM-Atto647N, in mica supported DOPC (1,2-dioleoyl-*sn*-glycero-3-phosphocholine) bilayers. Fitting with a two-component diffusion model confirms the existence of two separate populations of diffusing particles in these bilayers on mica. As a demonstration of the TrackArt workflow, we characterize and discuss the effective activation energies required to increase the diffusion rates of these populations, obtained from Arrhenius plots of temperature-dependent diffusion. Finally, TrackArt provides a simulation module, allowing the user to generate models with multiple particle trajectories, diffusing with different characteristics. Maps of domains, acting as impermeable or permeable obstacles for particles diffusing with given rate constants and diffusion coefficients, can be simulated or imported from an image. Importantly, this allows one to use simulated data with a known diffusion behavior as a comparison for results acquired using particular algorithms on actual, “natural” samples whose diffusion behavior is to be extracted. It can also serve as a tool for demonstrating diffusion principles.

### Conclusions

TrackArt is an open source, platform-independent, Matlab-based graphical user interface, and is easy to use even for those unfamiliar with the Matlab programming environment. TrackArt can be used for accurate simulation and analysis of complex diffusion data, such as diffusion in lipid bilayers, providing publication-quality formatted results.