We consider the problem of optical tomographic imaging in a weakly scattering medium in the presence of highly
scattering inclusions. The approach is based on the assumption that scattering media consist of weakly and
highly scattering regions, whose transport coefficients differ by an order of magnitude. The image reconstruction
algorithm is based on the variational framework and employs angularly selective intensity measurements. The
methodology is verified by reconstruction of optical and fluorescent parameters from numerically simulated
Three-dimensional localization of protein conformation changes in turbid media using Förster Resonance Energy Transfer (FRET) was investigated by tomographic fluorescence lifetime imaging (FLIM). FRET occurs when a donor fluorophore, initially in its electronic excited state, transfers energy to an acceptor fluorophore in close proximity through non-radiative dipole-dipole coupling. An acceptor effectively behaves as a quencher of the donor's fluorescence. The quenching process is accompanied by a reduction in the quantum yield and lifetime of the donor fluorophore. Therefore, FRET can be localized by imaging changes in the quantum yield and the fluorescence lifetime of the donor fluorophore. Extending FRET to diffuse optical tomography has potentially important applications such as in vivo studies in small animal. We show that FRET can be localized by reconstructing the quantum yield and lifetime distribution from time-resolved non-invasive boundary measurements of fluorescence and transmitted excitation radiation. Image reconstruction was obtained by an inverse scattering algorithm. Thus we report, to the best of our knowledge, the first tomographic FLIM-FRET imaging in turbid media. The approach is demonstrated by imaging a highly scattering cylindrical phantom concealing two thin wells containing cytosol preparations of HEK293 cells expressing TN-L15, a cytosolic genetically-encoded calcium FRET sensor. A 10mM calcium chloride solution was added to one of the wells to induce a protein conformation change upon binding to TN-L15, resulting in FRET and a corresponding decrease in the donor fluorescence lifetime. The resulting fluorescence lifetime distribution, the quantum efficiency, absorption and scattering coefficients were reconstructed.
We report a novel technique to reconstruct fluorescence lifetime distributions in turbid media by using Fourier domain
reconstruction of time gated imaging data. The time gating provides sufficient temporal resolution to determine short
fluorescence lifetimes while the use of the Fourier transform, which is essential for the time de-convolution of the system
of the integral equations employed in the reconstruction, permits a relatively rapid reconstruction of 3-D tomographic
data. This approach has been applied experimentally to reconstruct fluorescent lifetime distributions corresponding to
phantoms with wells filled with fluorescent dyes embedded inside highly scattering slabs. In practice, the scattering
medium can itself be fluorescent and we also suggest a simple iterative technique to account for background autofluorescence, which we have also tested experimentally.