Three-dimensional imaging of Förster 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 Förster 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 Förster 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 Förster 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.
McGinty
James
J
Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, United Kingdom.
Soloviev
Vadim Y
VY
Tahir
Khadija B
KB
Laine
Romain
R
Stuckey
Daniel W
DW
Hajnal
Joseph V
JV
Sardini
Alessandro
A
French
Paul M W
PM
Arridge
Simon R
SR
D002122:Calcium Chloride / Q000494:pharmacology; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D055095:Optics and Photonics / Q000379:methods*; D019047:Phantoms, Imaging; D011487:Protein Conformation; D012542:Scattering, Radiation; D012828:Silicones / Q000737:chemistry; D013997:Time Factors
D012828:Silicones; D002122:Calcium Chloride