Development and measurement of two-photon-base fluorescence correlation spectroscopy

We presented the fluorescence correlation spectroscopy (FCS) based on two-photon microscopy system to access physical parameters that give rise to fluctuations in fluorescence signal. As given excitation volume, the diffusion coefficient of molecules due to Brownian Movement into or out of excitation volume is determined. Further, depended on autocorrelation analysis based on diffusion coefficients of molecules, the fraction of bound species from measured samples containing the dye labeled anti-rabbit IgG and rabbit IgG can be separated. The mean standard deviation of diffusion coefficient measurement in our two-photon based system is about 0.064. The changes of diffusion coefficient with 15 nM concentration solution of fluorescein are 246.173 um^2/s to 35.1626 um^2/s with different concentration solution of glycerol ranging from 2.5% to 40%. Moreover, the maximal binding fraction of kinetics mode and end mode between Anti-Rabbit IgG and Rabbit IgG is 60% and 73%, respectively.