Measurement and model assessment of fluorescence lifetime sensing in multiply scattering media

The generation and propagation of fluorescence light within biological tissue offers the potential for biomedical diagnostics and analyte sensing. Arising from an exogenous fluorescent dye injected as a contrast agent or immobilized in a polymer implant, the fluorescent decay kinetics can be sensitive to the tissue??s biochemical environment, providing quantitative in vivo information of the confined tissue site. The impact of light propagation and decay kinetics upon the measured signals is important for consideration, simply because tissue scatters light, giving rise to nanosecond photon time-of-flights that are comparable to fluorescence relaxation kinetics. The goal of this study is to develop a time-dependent model describing (i) the generation of fluorescence from dyes exhibiting multi-exponential or more complex kinetics and (ii) its propagation in scattering media. In the preliminary study, fluorescence lifetime spectroscopy is investigated in tissue-like scattering solution. Two fluorescent dyes, 3,3-diethylthiatricarbocyanine iodide (DTTCI) and Indocynanine Green (ICG), which exhibit distinctly different lifetimes and each exhibits single-exponential decay kinetics, were employed. Measurements of phase-modulation as a function of modulation frequency were made at varying concentration ratios of the two dyes to experimentally simulate fluorescence multi-exponential decay kinetics in non-scattering and scattering solutions. The results suggest that frequency-domain measurements of fluorescent decay kinetics along with models of light propagation may be enhanced by scatter in order to probe kinetics more sensitively than in non-scattering solutions. The next study involved fluorescence lifetime sensing in scattering and non-scattering solutions with a pH sensitive dye, Carboxy Seminaphthofluorescein-1 (C-SNAFL-1), which is known to exhibit multi-exponential decay kinetics. The results demonstrate accurate pH sensing in scattering solution via fluorescence kinetics using a simplified propagation model incorporating an average lifetime. Finally, fluorescence lifetime sensing in immobilized systems were investigated. C-SNAFL-1 was immobilized in poly(ethylene glycol) (PEG) microparticles that were immersed in buffered polystyrene solutions. The results demonstrate the ability to perform pH sensing with fluorescence lifetime without the confounding effect of fluorophore loading or the use of 'reference' measurement within multiply scattering systems. In addition, the stability of the immobilized fluorescence sensor and the reliability of fluorescence lifetime measurement verify the prospect of this technology for implantable purposes.