Age, lipofuscin and melanin oxidation affect fundus near-infrared autofluorescence

BACKGROUND: Fundus autofluorescence is a non-invasive imaging technique in ophthalmology. Conventionally, short-wavelength autofluorescence (SW-AF) is used for detection of lipofuscin, a byproduct of the visual cycle which accumulates with age or disease in the retinal pigment epithelium (RPE). Furthermore, near-infrared autofluorescence (NIR-AF) is used as a marker for RPE and choroidal melanin, but contribution of lipofuscin to the NIR-AF signal is unclear. METHODS: We employed fluorescence microscopy to investigate NIR-AF properties of melanosomes, lipofuscin and melanolipofuscin granules in histologic sections of wildtype and Abca4(−/−) mouse eyes, the latter having increased lipofuscin, as well as aged human donor eyes. Differentiation between these pigments was verified by analytical electron microscopy. To investigate the influence of oxidative and photic stress we used an in vitro model with isolated ocular melanosomes and an in vivo phototoxicity mouse model. FINDINGS: We show that NIR-AF is not an intrinsic property of melanin, but rather increases with age and after photic or oxidative stress in mice and isolated melanosomes. Furthermore, when lipofuscin levels are high, lipofuscin granules also show NIR-AF, as confirmed by correlative fluorescence and electron microscopy in human tissue. However, lipofuscin in albino Abca4(−/−) mice lacks NIR-AF signals. INTERPRETATION: We suggest that NIR-AF is derived from melanin degradation products that accumulate with time in lipofuscin granules. These findings can help to improve the interpretation of patient fundus autofluorescence data. FUNDING: This work was supported by Bundesministerium für Bildung und Forschung, Deutsche Forschungsgemeinschaft and Chinese Scholarship Council. Major instrumentation used in this work was supported by Deutsche Forschungsgemeinschaft, the European Fund for Regional Development and the state of Baden-Württemberg.