Systematic Error in Fluorescence Correlation Measurements Identified by a Simple Saturation Model of Fluorescence

Abstract

The distortion of the fluorescence correlation function of a dye solution becomes larger with the increase in the excitation power, and eventually the parameters, such as the number of molecules and the diffusion time, obtained by the fluorescence correlation function systematically change. The most fundamental reason for this change is thought to be the distortion of the Gaussian excitation-detection field due to the saturation of the photocycle of the chromophore. The deviation from linearity of the fluorescence intensity causes the distortion of the fluorescence correlation function. Consequently, a smaller excitation power reduces the distortion and ensures an accurate measurement of the absolute value of these parameters. At the same time, the measurements at a fixed excitation power can be used to quantitatively determine the relative value of concentration and of the diffusion time. The deviation in the linearity of the fluorescence intensity and the deviation of the parameters show a high degree of correlation, and a 10% deviation of the intensity results in a prediction of a approximately 10% deviation in the number of molecules and a approximately 5% in the diffusion time.