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

摘要

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.