A nonparametric method for analysis of fluorescence emission in combined time and wavelength dimensions

We report a method for accurate recovery of tissue intrinsic fluorescence emission characteristics, including fluorescence lifetimes and spectral profiles, from complex two-dimensional (spectro-temporal) emission waveforms. Most algorithms for analysis of fluorescence data address separately the characteristics of either spectral emission or fluorescence relaxation time. We developed a novel nonparametric analytical method that allows for identification and estimation of the intrinsic Fluorescent Impulse Response Kernel (FIRK) simultaneously in time and wavelength dimensions. Modeling of FIRK was based on the characteristics of spectro-temporal fluorescence waveforms. Due to the decaying behavior of the fluorescence, a linear combination of discrete Laguerre functions was used to model the fluorescence response in time. To address the large variability of spectral profiles of distinct fluorophores, a discrete Fourier series expansion was used to model the variation of fluorescence intensity across wavelength. The proposed method was validated on synthetic fluorescence data and data measured from fluorescence lifetime standards and tissue endogenous fluorescent biomolecules. We determined that this method provides a direct recovery of the two-dimensional FIRK and accurate estimation (residual error < 6%) of a broad range of fluorescence lifetimes including the sub-nanosecond range. The FIRK retrieved using this method can further facilitate modeling and recognition of pathological and physiological conditions in tissues.