In Vivo fluorescence correlation spectroscopy

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DESCRIPTION (provided by applicant): The purpose of the proposed research is to develop fluorescence correlation spectroscopy into an effective, reliable tool for in vivo characterization of dynamic processes. This technology has enormous potential for biomedical applications, such as in vivo drug screening. Fluorescence correlation spectroscopy (FCS) studies the fluorescence fluctuations of a small number of molecules inside the focal spot of a laser with statistical analysis tools. Among the unique information provided by FCS is the absolute fluorophore concentration and kinetic parameters, such as the diffusion coefficient and chemical reaction coefficients. FCS delivers dynamic information with submicron spatial resolution and without the need to apply an external perturbation rendering it especially attractive for in vivo measurements. Another recently developed analysis technique based on the photon counting histogram will be employed to extract additional information on brightness heterogeneity from FCS data. A careful experimental examination of the in vivo properties of green fluorescent protein (GFP) will be conducted, while explicitly considering the influence of the cellular environment on FCS measurements. In addition, GFP tagged retinoid receptors will be used to perform an in vivo study to resolve their molecular oligomerization state in the presence and absence of ligands.