biostudies-literatureUnknown116(5)Stolle MDNNatural Sciences and Engineering Research Council of CanadaUsing fluorescence correlation spectroscopy (FCS) to distinguish between different types of diffusion processes is often a perilous undertaking because the analysis of the resulting autocorrelation data is model dependant. Two recently introduced strategies, however, can help move toward a model-independent interpretation of FCS experiments: 1) the obtention of correlation data at different length scales and 2) their inversion to retrieve the mean-squared displacement associated with the process under study. We use computer simulations to examine the signature of several biologically relevant diffusion processes (simple diffusion, continuous-time random walk, caged diffusion, obstructed diffusion, two-state diffusion, and diffusing diffusivity) in variable-length-scale FCS. We show that, when used in concert, length-scale variation and data inversion permit us to identify non-Gaussian processes and, regardless of Gaussianity, to retrieve their mean-squared displacement over several orders of magnitude in time. This makes unbiased discrimination between different classes of diffusion models possible.Biophysical journal791-806https://www.ebi.ac.uk/biostudies/studies/S-EPMC6400862biostudies-literatureAnomalous Diffusion in Inverted Variable-Lengthscale Fluorescence Correlation Spectroscopy.PMC6400862Stolle MDNFradin CfalseAnomalous Diffusion in Inverted Variable-Lengthscale Fluorescence Correlation Spectroscopy.Using fluorescence correlation spectroscopy (FCS) to distinguish between different types of diffusion processes is often a perilous undertaking because the analysis of the resulting autocorrelation data is model dependant. Two recently introduced strategies, however, can help move toward a model-independent interpretation of FCS experiments: 1) the obtention of correlation data at different length scales and 2) their inversion to retrieve the mean-squared displacement associated with the process under study. We use computer simulations to examine the signature of several biologically relevant diffusion processes (simple diffusion, continuous-time random walk, caged diffusion, obstructed diffusion, two-state diffusion, and diffusing diffusivity) in variable-length-scale FCS. We show that, when used in concert, length-scale variation and data inversion permit us to identify non-Gaussian processes and, regardless of Gaussianity, to retrieve their mean-squared displacement over several orders of magnitude in time. This makes unbiased discrimination between different classes of diffusion models possible.2019-01-01T00:00:00Z2019 Mar2025-04-22T03:03:20.905Z2025-02-19T01:48:07.159ZS-EPMC64008623078239610.1016/j.bpj.2019.01.024