Three-Channel Fluorescence Lifetime Imaging of Endogenous Fluorophores

Abstract

Autofluorescence of endogeneous fluorophores, such as nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and tryptophan can provide valuable information about metabolic processes and states of cells and tissues. When combined with time-correlated single photon counting (TCSPC), the amount of bound and free NADH and relative amount of NADPH can be readily extracted from the fluorescence decay data. These three FLIM detectors allow us to monitor the three endogenous molecules simultaneously in living cells.One of the challenges in analyzing endogenous fluorescence is the spectral overlap of generally broad emission spectra of the fluorophores. To separate individual components of multi-photon excited autofluorescence (three-photon excited tryptophan and two-photon excited NADH and FAD) we have employed three independent FLIM channels with spectral ranges centered around 360 nm, 480 nm and 540 nm.The calculation of lifetimes of individual components is complicated due to the inevitable emission spectral overlap between NADH and FAD and the inherent instability of multiple-exponential fitting problem. Because TCSPC decay curves can be easily transformed into phasor plots at various harmonic frequencies, we attempt to solve this problem using global analysis methods based both on exponential fitting with global parameters and phasor plot analyses. This work summarizes the main properties and the performance of both methods, and suggests optimal parameters (such as excitation wavelengths and collection times) during image acquisition.