Following protein association in vivo with fluorescence fluctuation spectroscopy

The combination of fluorescence correlation spectroscopy and two-photon excitation provides us with a powerful spectroscopic technique. Its submicron resolution and single molecule sensitivity make it an attractive technique for in vivo applications. Experiments have demonstrated that quantitative in vivo fluorescence fluctuation measurements are feasible, despite the presence of autofluorescence and the heterogeneity of the cellular environment. I will demonstrate that molecular brightness of proteins tagged with green fluorescent protein (GFP) is a useful and robust parameter for in vivo studies. Knowledge of photon statistics is crucial for the interpretation of fluorescence fluctuation experiments. I will describe photon counting histogram (PCH) analysis, which determines the molecular brightness and complements autocorrelation analysis. Non-ideal detector effects and their influence on the photon statistics will be discussed. The goal of in vivo fluorescence fluctuation experiments is to address functional properties of biomolecules. We will focus on retinoid X receptor (RXR), a nuclear receptor, which is crucial for the regulation of gene expression. The fluorescence brightness of RXR tagged with EGFP will be used to probe the oligomerization state of RXR.