Extraction of rotational correlation times from noisy single molecule fluorescence trajectories

Monitoring single molecule probe rotations is an increasingly common approach to studying dynamics of complex systems, including supercooled liquids. Even with advances in fluorophore design and detector sensitivity, such measurements typically exhibit low signal to noise and signal to background ratios. Here, we simulated and analyzed orthogonally decomposed fluorescence signals of single molecules undergoing rotational diffusion in a manner that mimics experimentally collected data of probes in small molecule supercooled liquids. The effects of noise, background, and trajectory length were explicitly considered, as were the effects of data processing approaches that may limit the impact of noise and background on assessment of environmental dynamics. In many cases, data treatment that attempts to remove noise and background were found to be deleterious. However, for short trajectories below a critical signal to background threshold, a thresholding approach that successfully removed data points associated with noise and spared those associated with signal allowed for assessment of environmental dynamics that was as accurate and precise as would be achieved in the absence of noise.