The standard deviation in fluorescence correlation spectroscopy

The standard deviation (SD) in fluorescence correlation spectroscopy (FCS) has been mostly neglected in applications. However, the knowledge of the co rrect SD is necessary for an accurate data evaluation, especially when fitt ing theoretical models to experimental data. In this work, an algorithm is presented that considers the essential features of FCS. It allows predictio n of the performance of FCS measurements in Various cases, which is importa nt for finding optimal experimental conditions. The program calculates the SD of the experimental autocorrelation function online. This procedure lead s to improved parameter estimation, compared to currently used theoretical approximations for the SD. Three methods for the calculation of the SD are presented and compared to earlier analytical solutions (D. E. Koppel. 1974. Phys. Rev. A. 10:1938-1945.), calculation directly from fluorescence inten sity values, by averaging several FCS measurements, or by dividing one meas urement into a set of shorter data packages. Although the averaging over se veral measurements yields accurate estimates for the SD, the other two meth ods are considerably less time consuming, can be run online, and yield comp arable results.