Two-photon fluctuation correlation spectroscopy: Method and applications to protein aggregation and intracellular diffusion

Fluctuation correlation spectroscopy (FCS) is an experimental technique for the study of hydrodynamics and molecular interactions of microscopic systems. The method monitors the statistical behavior of spontaneous equilibrium fluctuations in number occupation of a fluorescent species in a subvolume of a sample. Microscopic models have been developed which relate observed statistical fluctuations in fluorescence observables to physical quantities of interest. This thesis presents the first application of two-photon molecular excitation to FCS. Two-photon excitation offers several advantages over one-photon excitation for FCS experiments. These advantages are discussed, and model systems are studied to calibrate and characterize the capabilities of the technique. The theoretical framework necessary to recover physical parameters from observed fluctuation spectra is also presented.There are two biophysical applications proposed and discussed in this thesis. The first is the study of particle mobilities and intracellular diffusion. Measurements of particle diffusion in the cytoplasm of live cells are presented. The second application is the detection of protein aggregation in solution using two-photon scanning FCS. Measurements are shown demonstrating the potential to study aggregation equilibria at low protein concentrations, and the dissociation reactions of several oligomeric proteins are studied. The possibility to measure dissociation kinetics is also demonstrated.U of I OnlyETDs are only available to UIUC Users without author permissio