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Proceedings Paper

Tomographic imaging of flourescence resonance energy transfer in highly light scattering media
Author(s): Vadim Y. Soloviev; James McGinty; Khadija B. Tahir; Romain Laine; Daniel W. Stuckey; P. Surya Mohan; Joseph V. Hajnal; Alessandro Sardini; Paul M. W. French; Simon R. Arridge
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Paper Abstract

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.

Paper Details

Date Published: 26 February 2010
PDF: 10 pages
Proc. SPIE 7573, Biomedical Applications of Light Scattering IV, 75730G (26 February 2010); doi: 10.1117/12.840522
Show Author Affiliations
Vadim Y. Soloviev, Univ. College London (United Kingdom)
James McGinty, Imperial College London (United Kingdom)
Khadija B. Tahir, Imperial College London (United Kingdom)
Romain Laine, Imperial College London (United Kingdom)
Daniel W. Stuckey, Imperial College London (United Kingdom)
P. Surya Mohan, Univ. College London (United Kingdom)
Joseph V. Hajnal, Imperial College London (United Kingdom)
Alessandro Sardini, Imperial College London (United Kingdom)
Paul M. W. French, Imperial College London (United Kingdom)
Simon R. Arridge, Univ. College London (United Kingdom)


Published in SPIE Proceedings Vol. 7573:
Biomedical Applications of Light Scattering IV
Adam P. Wax; Vadim Backman, Editor(s)

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