Towards Single Molecule Imaging of Fluorescence Anisotropy

Given the complexity of biological systems, information on a single molecule basis is crucial to accurately probe molecular properties. In fact, single molecule imaging provides real time conformational dynamics, which can underline heterogeneity in molecular distribution in terms of dipole orientations, spectra or intramolecular distances, in both stable and unstable systems. In addition to these structural data, functional information, in real time, can also be accessed and reaction kinetics determined.Fluorescence anisotropy is a method of contrast, which by quantifying the fluorescence depolarization, can access the fluorophore's rotational correlation times or quantify energy transfer (e.g. FRET). This is another technique to probe the fluorophore's environment in terms of viscosity, interactions between molecules and ligand-substrate binding.Here, we present an adapted and optimized Total Internal Reflection Fluorescence (TIRF) microscope combined with steady state fluorescence anisotropy detection for single molecule imaging. Our set-up is used for FRET imaging of GFP/mRFP FRET biosensors by measuring the depolarisation of the acceptor upon donor excitation. Preliminary data aiming towards single molecule imaging with a fluorescence anisotropy read-out, made by measurements on purified Immunoglobilin E (IgE) FRET biosensors, are presented and future prospects discussed.