Three-dimensional imaging of Forster resonance energy transfer in heterogeneous turbid media by tomographic fluorescent lifetime imaging

We report a three-dimensional time-resolved tomographic imaging technique for localizing protein-protein interaction and protein conformational changes in turbid media based on Forster resonant energy-transfer read out using fluorescence lifetime. This application of tomoFRET employs an inverse scattering algorithm utilizing the diffusion approximation to the radiative-transfer equation applied to a large tomographic data set of time-gated images. The approach is demonstrated by imaging a highly scattering cylindrical phantom within which are two thin wells containing cytosol preparations of HEK293 cells expressing TN-L15, a cytosolic genetically encoded calcium Forster resonant energy-transfer sensor. A 10 mM calcium chloride solution was added to one of the wells, inducing a protein conformation change upon binding to TN-L15, resulting in Forster resonant energy transfer and a corresponding decrease in the donor fluorescence lifetime. We successfully reconstruct spatially resolved maps of the resulting fluorescence lifetime distribution as well as of the quantum efficiency, absorption, and scattering coefficients. (C) 2009 Optical Society of America