biostudies-literatureKohler JMinnesota Supercomputing Institute, University of MinnesotaNIAID NIH HHSUniversity of MinnesotaNational Institutes of HealthNIGMS NIH HHS241-253https://www.ebi.ac.uk/biostudies/studies/S-EPMC9822791biostudies-literatureUnknown122(1)The experimental autocorrelation function of fluorescence correlation spectroscopy calculated from finite-length data is a biased estimator of the theoretical correlation function. This study presents a new theoretical framework that explicitly accounts for the data length to allow for unbiased analysis of experimental autocorrelation functions. To validate our theory, we applied it to experiments and simulations of diffusion and characterized the accuracy and precision of the resulting parameter estimates. Because measurements in living cells are often affected by instabilities of the fluorescence signal, autocorrelation functions are typically calculated on segmented data to improve their robustness. Our reformulated theory extends the range of usable segment times down to timescales appBiophysical journalAutocorrelation function of finite-length data in fluorescence correlation spectroscopy.PMC9822791AI150468T32 AI083196R01 AI150468R01 GM098550GM098550Mueller JDHur KHKohler JfalseAutocorrelation function of finite-length data in fluorescence correlation spectroscopy.The experimental autocorrelation function of fluorescence correlation spectroscopy calculated from finite-length data is a biased estimator of the theoretical correlation function. This study presents a new theoretical framework that explicitly accounts for the data length to allow for unbiased analysis of experimental autocorrelation functions. To validate our theory, we applied it to experiments and simulations of diffusion and characterized the accuracy and precision of the resulting parameter estimates. Because measurements in living cells are often affected by instabilities of the fluorescence signal, autocorrelation functions are typically calculated on segmented data to improve their robustness. Our reformulated theory extends the range of usable segment times down to timescales app2023-01-01T00:00:00Z2023 Jan2026-06-03T13:30:18.972Z2026-04-28T03:10:34.613ZS-EPMC98227913626697110.1016/j.bpj.2022.10.027