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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.
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