Khadija B. Tahir

Fluorescence lifetime imaging by using time-gated data acquisition

The use of the time gating technique for lifetime reconstruction in the Fourier domain is a novel technique. Time gating provides sufficient data points in the time domain for reliable application of the Fourier transform, which is essential for the time deconvolution of the system of the integral equations employed in the reconstruction. The Fourier domain telegraph equation is employed to model the light transport, which allows a sufficiently broad interval of frequencies to be covered. Reconstructed images contain enough information needed for recovering the lifetime distribution in a sample for any given frequency within the megahertz-gigahertz band. The use of this technique is essential for recovering time-dependent information in fluorescence imaging. This technique was applied in reconstruction of the lifetime distribution of four tubes filled with Rhodamine 6G embedded inside a highly scattering slab. Relatively accurate fluorescence lifetime reconstruction demonstrates the effectiveness and the potential of the proposed technique.

Fluorescence lifetime optical projection tomography

We describe a quantitative fluorescence projection tomography technique which measures the 3-D fluorescence lifetime distribution in optically cleared specimens up 1 cm in diameter. This is achieved by acquiring a series of wide-field time-gated images at different relative time delays with respect to a train of excitation pulses, at a number of projection angles. For each time delay, the 3-D time-gated intensity distribution is reconstructed using a filtered back projection algorithm and the fluorescence lifetime subsequently determined for each reconstructed horizontal plane by iterative fitting to a mono-exponential decay. Due to its inherently ratiometric nature, fluorescence lifetime is robust against intensity based artefacts as well as producing a quantitative measure of the fluorescence signal. We present a 3-D fluorescence lifetime reconstruction of a mouse embryo labelled with an alexa-488 conjugated antibody targeted to the neurofilament, which clearly differentiates between the extrinsic label and the autofluorescence, particularly from the heart and dorsal aorta.

Fluorescence lifetime tomography of live cells expressing enhanced green fluorescent protein embedded in a scattering medium exhibiting background autofluorescence

We present a novel fluorescence lifetime tomography system applied to a highly scattering autofluorescent phantom containing live cells expressing the fluorophore enhanced green fluorescent protein (EGFP). The fluorescence signal was excited using a fiber-laser-pumped supercontinuum source and detected using wide-field time gating imaging. To facilitate rapid 3D reconstruction of the fluorescence lifetime distribution, the time-resolved data were Fourier-transformed in time to give complex functions that formed a data set for the Fourier domain reconstruction. Initially the presence of an unspecified background autofluorescence signal impeded reconstruction of the lifetime distribution, but we show that this problem can be addressed using a simple iterative technique.

Experimental measurements of light scattering from samples with specified optical properties

We have measured the photon intensity transmitted through cylindrical and slab phantoms of different concentration and size scattering particles. Our continuous wave (CW) measurements show quantitatively that the mean cosine of the scattering angle (g) must be taken into account unless the phantoms have very high scattering coefficient. We have compared the results for phantoms made of titanium dioxide in resin, which is commonly used to simulate tissue despite its low g value (0.56), with those for silica in resin phantoms of high g value (0.95). The comparison has shown different angular scattered intensity, both in form and magnitude, for phantoms that have the same reduced scattering coefficient but different g value. The results show that, in some cases, using the reduced scattering coefficient and absorption alone for comparing phantoms or for reconstruction using inverse methods based, for example, on diffusion theory would lead to incorrect interpretations.

Tomographic imaging of flourescence resonance energy transfer in highly light scattering media

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 donors 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.

Higher-order transport approximations for optical tomography applications

In this paper we discuss the application of higher-order transport approximations using finite element-spherical harmonics methods (FE-PN) to multidimensional photon propagation problems. The combined methodology offers fast and accurate modeling of photon propagation in multidimensional diffusive and non-diffusive media. This is of great importance to the practical solution of the inverse scattering problems which characterize optical tomography.

tomoFLIM - fluorescence lifetime projection tomography

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.

Computer-aided design of photomultiplier tubes using a 3D program

The major design goals of photomultiplier tubes are: to maximize the collection between individual stages, thereby optimizing the tube sensitivity; and to minimize the transit time spread of electrons between individual emission and collection surfaces, thereby optimizing the time resolution. The numerical modelling involves computing the electrostatic fields and electron trajectories for various electrode structures. A fully three dimensional (3D) program is used to give a better representation of the tubes design, and also allows the freedom of using accelerating electrodes and focusing rings of different shapes and heights, which can be modelled accurately in 3D.

Time-Domain Fluorescence Lifetime Tomography

We present a platform for fluorescence lifetime tomography utilising tuneable supercontinuum excitation and wide-field time-gated technology. Applied to optical projection and diffuse fluorescence tomography, we demonstrate 3-D time-resolved fluorescence reconstruction in transparent and scattering phantoms.

Fluorescence lifetime imaging through turbid media reconstructed in the Fourier domain using time-gated imaging data

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.