Tomographic fluorescence lifetime multiplexing in the spatial frequency domain

The ability to simultaneously recover multiple fluorophores within biological tissue (multiplexing) can have important applications for tracking parallel disease processes in vivo . Here we present a novel method for rapid and quantitative multiplexing within a scattering medium, such as biological tissue, based on fluorescence lifetime contrast. This method employs a tomographic inversion of the asymptotic (late) portion of time-resolved spatial frequency (SF) domain measurements. Using Monte Carlo simulations and phantom experiments, we show that the SF-asymptotic time domain (SF-ATD) approach provides a several-fold improvement in relative quantitation and localization accuracy over conventional SF-time domain inversion. We also show that the SF-ATD approach can exploit selective filtering of high spatial frequencies to dramatically improve reconstruction accuracy for fluorophores with subnanosecond lifetimes, which is typical of most near-infrared fluorophores. These results suggest that the SF-ATD approach will serve as a powerful new tool for whole-body lifetime multiplexing.