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Proceedings Paper

tomoFLIM - fluorescence lifetime projection tomography
Author(s): James McGinty; Daniel W. Stuckey; Khadija B. Tahir; Romain Laine; Joseph V. Hajnal; Alessandro Sardini; Paul M. W. French
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Paper Abstract

Optical Projection Tomography (OPT) is a wide-field technique for measuring the threedimensional distribution of absorbing/fluorescing species in non-scattering (optically cleared) samples up to ~1cm in size, and as such is the optical analogue of X-ray computed tomography. We have extended the intensity-based OPT technique to measure the three-dimensional fluorescence lifetime distribution (tomoFLIM) in transparent samples. Due to its inherent ratiometric nature, fluorescence lifetime measurements are robust against intensity-based artifacts as well as producing a quantitative measure of the fluorescence signal, making it particularly suited to Förster Resonance Energy Transfer (FRET) measurements. We implement tomoFLIM via OPT by acquiring a series of wide-field time-gated images at different relative time delays with respect to a train of excitation pulses for a range of projection angles. For each time delay, the three-dimensional time-gated intensity distribution is reconstructed using a filtered back projection algorithm and the fluorescence lifetime is subsequently determined for each reconstructed horizontal plane by iterative fitting of an appropriate decay model. We present a tomographic reconstruction of a fluorescence lifetime resolved FRET calcium contruct, TN-L15 cytosol suspension, in a silicone phantom. This genetically encoded sensor, TN-L15, comprises the calcium-binding domain of Troponin C, flanked by the fluorophores cyan fluorescent protein and citrine. In the presence of calcium ions TN-L15 changes conformation bringing the two fluorophores into close proximity, resulting in FRET. We also present autofluorescence and fluorescently labelled tomoFLIM reconstructions of chick embryos, including a genetically encoded fluorophore TagRFP-T. The fluorophore was electroporated in ovo into the neural tube of the embryos, which were subsequently dissected two days post-electroporation, fixed in ethanol and optically cleared for OPT/tomoFLIM acquisition. The reconstructed 3-D fluorescence lifetime image provides contrast between the genetically labelled TagRFP-T and the emitted autofluorescence.

Paper Details

Date Published: 24 February 2010
PDF: 11 pages
Proc. SPIE 7570, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVII, 75700G (24 February 2010); doi: 10.1117/12.841753
Show Author Affiliations
James McGinty, Imperial College London (United Kingdom)
Daniel W. Stuckey, MRC Clinical Sciences Ctr., Imperial College Faculty of Medicine (United Kingdom)
Khadija B. Tahir, Imperial College London (United Kingdom)
Romain Laine, Imperial College London (United Kingdom)
Joseph V. Hajnal, MRC Clinical Sciences Ctr., Imperial College Faculty of Medicine (United Kingdom)
Alessandro Sardini, MRC Clinical Sciences Ctr., Imperial College Faculty of Medicine (United Kingdom)
Paul M. W. French, Imperial College London (United Kingdom)


Published in SPIE Proceedings Vol. 7570:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVII
Jose-Angel Conchello; Carol J. Cogswell; Tony Wilson; Thomas G. Brown, Editor(s)

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