Depth resolution and multiexponential lifetime analyses of reflectance-based time-domain fluorescence data.

Time-domain fluorescence imaging is a powerful new technique that adds a rich amount of information to conventional fluorescence imaging. Specifically, time-domain fluorescence can be used to remove autofluorescence from signals, resolve multiple fluorophore concentrations, provide information about tissue microenvironments, and, for reflectance-based imaging systems, resolve inclusion depth. The present study provides the theory behind an improved method of analyzing reflectance-based time-domain data that is capable of accurately recovering mixed concentration ratios of multiple fluorescent agents while also recovering the depth of the inclusion. The utility of the approach was demonstrated in a number of simulations and in tissuelike phantom experiments using a short source-detector separation system. The major findings of this study were (1) both depth of an inclusion and accurate ratios of two-fluorophore concentrations can be recovered accurately up to depths of approximately 1 cm with only the optical properties of the medium as prior knowledge, (2) resolving the depth and accounting for the dispersion effects on fluorescent lifetimes is crucial to the accuracy of recovered ratios, and (3) ratios of three-fluorophore concentrations can be resolved at depth but only if the lifetimes of the three fluorophores are used as prior knowledge. By accurately resolving the concentration ratios of two to three fluorophores, it may be possible to remove autofluorescence or carry out quantitative techniques, such as reference tracer kinetic modeling or ratiometric approaches, to determine receptor binding or microenvironment parameters in point-based time-domain fluorescence applications.