Studies On Fluorescence Correlation Spectroscopy And Its Applications

Fluorescence Correlation Spectroscopy (FCS) is a fast-advancing experimental method. In FCS, fluctuating fluorescence by sparse molecules in a tiny volume is treated by an autocorrelation calculation to obtain information on concentration and diffusion coefficient of the fluorescing particles. FCS is highly sensitive and non perturbative to fragile biological samples and is suitable for real time detection. Therefore, FCS is becoming an increasingly popular tool for chemical, biological and medical applications.The thesis is organized as follow: First, we give a brief introduction on FCS, including its principle, theoretical framework and experiments, followed by a comprehensive review of its history and recent progresses in methodology and applications.Saturated excitation by a strong excitation beam will alter the spatial distribution function of the laser induced fluorescence, therefore affect the results of FCS measurements. Using a revised theoretical model which takes into account of saturated excitation, we derived a general expression for the effective detection volume as a function of the excitation laser intensity. Quantitative relations between the particle number and diffusion time, both are obtained from FCS measurements, and the excitation laser intensity are established and used to remove the systematic error due to saturated excitation. The success of this technique is demonstrated in both Monte Carlo simulations and experiments.FCS possesses many remarkable advantages over conventional laser induces fluorescence measurement. It is capable of a sensitivity of single molecular detection and sub femto-litter focal volume. It also gives both macro concentration of the fluorescing particles and their characteristic diffusion time. These features make FCS a powerful detection tool for applications in Microfluidic Analysis. In collaboration with Analysis Center of Tsinghua University, we used FCS in micro channel system and realized detection of IgG immune reaction with reduce analysis time.