Three-dimensional imaging of Förster 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–proteinninteraction and protein conformational changes in turbid media based on Förster resonant energy-transfernread out using fluorescence lifetime. This application of “tomoFRET” employs an inverse scattering algo-nrithm utilizing the diffusion approximation to the radiative-transfer equation applied to a large tomographicndata set of time-gated images. The approach is demonstrated by imaging a highly scattering cylindricalnphantom within which are two thin wells containing cytosol preparations of HEK293 cells expressingnTN-L15, a cytosolic genetically encoded calcium Förster resonant energy-transfer sensor. A 10 mM calciumnchloride solution was added to one of the wells, inducing a protein conformation change upon bindingnto TN-L15, resulting in Förster resonant energy transfer and a corresponding decrease in the donornfluorescence lifetime. We successfully reconstruct spatially resolved maps of the resulting fluorescencenlifetime distribution as well as of the quantum efficiency, absorption, and scattering coefficients.n© 2009 Optical Society of America