Near-infrared (NIR) fluorescence tomography of multiple fluorophores has previously been limited by the bandwidth of the NIR spectral regime and the broad emission spectra of most NIR fluorophores. We describe in vivo tomography of three spectrally overlapping fluorophores using fluorescence lifetime-based separation. Time-domain images are acquired using a voltage-gated, intensified charge-coupled device (CCD) in free-space transmission geometry with 750 nm Ti:sapphire laser excitation. Lifetime components are fit from the asymptotic portion of fluorescence decay curve and reconstructed separately with a lifetime-adjusted forward model. We use this system to test the in vivo lifetime multiplexing suitability of commercially available fluorophores, and demonstrate lifetime multiplexing in solution mixtures and in nude mice. All of the fluorophores tested exhibit nearly monoexponential decays, with narrow in vivo lifetime distributions suitable for lifetime multiplexing. Quantitative separation of two fluorophores with lifetimes of 1.1 and 1.37 ns is demonstrated for relative concentrations of 1:5. Finally, we demonstrate tomographic imaging of two and three fluorophores in nude mice with fluorophores that localize to distinct organ systems. This technique should be widely applicable to imaging multiple NIR fluorophores in 3-D.
A theoretical framework is presented that allows a lifetime based analysis of the entire temporal diffuse
fluorescence response curve from a turbid medium. Optimization studies using singular value decomposition
analysis show that direct time domain fluorescence reconstructions are optimally performed
using a few points near the peak and rise portions of the temporal response. It is also shown that the
initial portion of the fluorescent response curve offers superior contrast-to-noise performance, while the
late decay portions offer minimal cross-talk between multiple lifetime components.
Noninvasive molecular imaging of amyloid plaques in murine Alzheimer's disease models would accelerate drug
development and basic Alzheimer's research. Amyloid plaques differ from traditional fluorescent targets in size and
spatial distribution and therefore present a unique challenge for biomarker development and tomography. To study
imaging feasibility and establish biomarker criteria, we developed a digital mouse head model from a 100 &mgr;m-resolution,
digital, segmented mouse atlas1. The cortical region of the brain was filled with a spatially uniform distribution of
plaques that had different fluorescent properties from the surrounding brain tissue, similar to current transgenic mouse
models of Alzheimer's disease. Fluorescence was simulated with a Monte Carlo algorithm using different plaque
densities, detection geometries, and background fluorescence. Our preliminary results demonstrated that shielding
effects might require nonlinear reconstruction algorithms and that background fluorescence would seriously hinder
quantitative burden estimation. The Monte Carlo based approach presented here offers a powerful way to study the
feasibility of non-invasive imaging in murine Alzheimer's models and to optimize experimental conditions.
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