Photooxidation Dynamics of Lipofuscin in the Presence of Carotenoid-Binding Protein AstaP: Fluorescence Lifetime Imaging and Pigment Composition Analysis

To introduce noninvasive optical diagnostic methods based on detection of tissue autofluorescence signals into medical practice, it is necessary to correlate the optical response with the functional state of a particular chromophore. Diagnostics of the state of lipofuscin granule chromophores is an important task to assess pathologic changes in the visual system in various diseases, primarily age-related macular degeneration. A key feature of lipofuscin granules (LGs) is the diversity of the chromophores and their susceptibility to oxidation. Here, we used time-resolved emission spectroscopy, confocal fluorescence lifetime imaging microscopy, and high-performance liquid chromatography (HPLC) to follow changes of LG chromophore composition upon photooxidation. In both isolated LGs and LG-loaded retinal pigment epithelium cells (ARPE-19), the distributions of the short lifetime component and its amplitude of LG fluorescence shifted, and the mean lifetime increased upon photooxidation, indicating depletion of population of rapidly relaxing bisretinoids, including A2E, and accumulation of their oxidized species, as confirmed by HPLC data. We applied differential evolution algorithms to decompose LG autofluorescence parameters and identified distinct photooxidation patterns in the presence and absence of antioxidant protection. Delivery of zeaxanthin by water-soluble carotenoprotein ΔNC-AstaP attenuated photooxidation-induced changes in the decay kinetics of both isolated and intracellular LGs, suppressed the accumulation of oxidized bisretinoids, and prevented complete photooxidation. Taken together, our results establish that time-resolved lipofuscin autofluorescence provides a quantitative, label-free readout of chromophore composition and oxidative status that is compatible with functional imaging. We propose that such lifetime-based measurements can be employed for early assessment of retinal pathology and for monitoring antioxidant interventions targeting lipofuscin-induced oxidative stress in emerging clinical techniques such as fluorescence lifetime imaging ophthalmoscopy.