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Fluorescent techniques developed for probing molecular events in vitro have been translated to small animal in-vivo fluorescence imaging using narrow-band, high-power laser sources, fast photon detection electronics and sophisticated image reconstruction algorithms. Targeted near-infrared fluorescent probes are being developed to increase image contrast and functional information collected by the in-vivo optical images. Besides fluorescence intensity, fluorescence lifetime can also be utilized to probe tissue physiology. Fluorescence lifetime, the average time that a fluorophore remains in the excited state, is specific to the molecule and its environment, but not necessarily to its concentration. Therefore, fluorescence lifetime imaging can be used to probe microenvironments within tissues to differentiate, for example, cancer from surrounding tissue by physiological differences. In the current study we demonstrate the ability to distinguish between two molecular probes in mice using fluorescence lifetime while using single excitation and emission wavelengths. Fluorescence detection was accomplished by measuring diffuse emission at 3 mm distance from excitation and raster-scanning whole-body regions of interest using time-correlated single photon counting technique. The resulting temporal point-spread function data was collected for grid points 1.5 mm apart. Fluorescence decays were de-convolved to determine measured fluorescence lifetimes by least-squares fit with associated chi-square error values. Relative signal from each fluorophore was then determined for specific tissues including liver, kidney and tumor areas. The results demonstrate that relative biodistributions of individual fluorophores with similar photonic characteristics can be simultaneously monitored in vivo using fluorescence lifetime imaging.
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