In vivo lipofuscin quantification by visible-light optical coherence tomography-based fundus autofluorescence imaging

We developed visible-light optical coherence tomography-based fundus autofluorescence (VIS-OCT-FAF) for simultaneous FAF and OCT imaging. We previously showed that the ratio of quantified FAF to quantified OCT of the retinal pigment epithelium (RPE), qAF/qOCT, correlates with the A2E amount measured <i>ex vivo</i>. In this study, we further demonstrated that qAF/qOCT linearly correlates with the total RPE lipofuscin fluorophore concentration, confirming it as a true representation of total RPE fluorophores. Fluorescence intensities of lipids, extracted from the RPE-choroid of albino and pigmented rats after VIS-OCT-FAF imaging, were measured and compared with a fluorescence calibration curve of synthetic A2E to establish an intensity equivalence (INEQ) between total RPE fluorophores and A2E concentration. Pigmented rats exhibited higher qAF/qOCT and thus higher lipofuscin content than albinos, which can be revealed <i>in vivo</i> only when the true FAF intensity is quantified. These findings demonstrate that VIS-OCT-FAF enables quantitative <i>in vivo</i> assessment of RPE lipofuscin, expanding FAF clinical applications.