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Inferring diffusion in single live cells at the single molecule level

The movement of molecules inside living cells is a fundamental feature of biological processes. The ability to both observe and analyse the details of molecular diffusion in vivo at the single molecule and single cell level can add significant insight into understanding molecular architectures of diffusing molecules and the nanoscale environment in which the molecules diffuse. The tool of choice for monitoring dynamic molecular localization in live cells is fluorescence microscopy, especially so combining total internal reflection fluorescence (TIRF) with the use of fluorescent protein (FP) reporters in offering exceptional imaging contrast for dynamic processes in the cell membrane under relatively physiological conditions compared to competing single molecule techniques. There exist several different complex modes of diffusion, and discriminating these from each other is challenging at the molecular level due to underlying stochastic behaviour. Analysis is traditionally performed using mean square displacements of tracked particles, however, this generally requires more data points than is typical for single FP tracks due to photophysical instability. Presented here is a novel approach allowing robust Bayesian ranking of diffusion processes (BARD) to discriminate multiple complex modes probabilistically. It is a computational approach which biologists can use to understand single molecule features in live cells.