Journal of biomedical optics · 2026
Spectral dependence of lipofuscin fluorescence lifetimes revealed by FLIM with a superconducting nanowire single-photon detector.
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Morozov Pavel, Andreev Vladislav, Yakovleva Marina, Kostyukov Alex, Feldman Tatiana, Ryabova Anastasia, Romanishkin Igor, Shirmanova Marina, Goltsman Gregory, Kuzmin Vladimir, Ostrovsky Mikhail, Arkhipchenko Alexandra, Maksimov Eugene, Becker Wolfgang, Shcheslavskiy Vladislav
Аннотация
SIGNIFICANCE: The noninvasive assessment of oxidative stress in the retinal pigment epithelium (RPE), a key factor in the pathogenesis of age-related macular degeneration (AMD), is an important aspect in ophthalmic diagnostics. Although fundus autofluorescence (FAF) is clinically used, its diagnostic power is limited. Fluorescence lifetime imaging ophthalmoscopy shows promise, but the photophysical underpinnings of lifetime changes in lipofuscin-the dominant RPE fluorophore-remain poorly understood. Lipofuscin granules (LGs) in the RPE are a primary source of fundus autofluorescence and are critically involved in the pathogenesis of AMD. This study characterizes the spectral and lifetime properties of LGs using a fluorescence lifetime imaging microscopy (FLIM) system integrated with a wide-range sensitivity, large-active-area superconducting nanowire single-photon detector (SNSPD) coupled with a standard 50 μ m multimode fiber.
AIM: We present experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of LGs in the RPE cells to probe the molecular oxidative state of RPE lipofuscin, aiming to improve AMD understanding and propose a method for early diagnosis of retinal pathologies.
APPROACH: We use a cutting-edge FLIM system integrated via a multimode fiber with a large-active-area SNSPD. Unlike previous studies, our approach provides component-resolved and spatially resolved fluorescence decay kinetics across a broad spectral range (500 to 1000 nm), which is typically inaccessible to conventional detectors. LGs were isolated from 100 human (aged 50 to 75 years) donor RPE cells and subjected to in vitro photo-oxidation to simulate AMD-like conditions. Steady-state spectroscopy revealed a blue shift in emission following photo-oxidation.
RESULTS: FLIM analysis, employing a tri-exponential decay model, showed a significant increase in the mean fluorescence lifetime τ m from ∼ 247 to ∼ 587 ps after photo-oxidation. Crucially, we identified a pronounced direct spectral dependence of fluorescence lifetimes τ m versus wavelength: for native LGs, τ m increased from 136 to 319 ps at registration range 500 to 650 nm; for photo-oxidized LGs, it increased from 174 to 623 ps over the same region. High-performance liquid chromatography confirmed the degradation of the bisretinoid A2E and accumulation of oxidized derivatives, correlating with the observed photophysical changes.
CONCLUSIONS: These findings demonstrate that component-resolved visible-near infrared spectral FLIM provides a sensitive, noninvasive approach to probe the molecular oxidative state of RPE lipofuscin, offering potential for early diagnosis of retinal pathologies.
AIM: We present experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of LGs in the RPE cells to probe the molecular oxidative state of RPE lipofuscin, aiming to improve AMD understanding and propose a method for early diagnosis of retinal pathologies.
APPROACH: We use a cutting-edge FLIM system integrated via a multimode fiber with a large-active-area SNSPD. Unlike previous studies, our approach provides component-resolved and spatially resolved fluorescence decay kinetics across a broad spectral range (500 to 1000 nm), which is typically inaccessible to conventional detectors. LGs were isolated from 100 human (aged 50 to 75 years) donor RPE cells and subjected to in vitro photo-oxidation to simulate AMD-like conditions. Steady-state spectroscopy revealed a blue shift in emission following photo-oxidation.
RESULTS: FLIM analysis, employing a tri-exponential decay model, showed a significant increase in the mean fluorescence lifetime τ m from ∼ 247 to ∼ 587 ps after photo-oxidation. Crucially, we identified a pronounced direct spectral dependence of fluorescence lifetimes τ m versus wavelength: for native LGs, τ m increased from 136 to 319 ps at registration range 500 to 650 nm; for photo-oxidized LGs, it increased from 174 to 623 ps over the same region. High-performance liquid chromatography confirmed the degradation of the bisretinoid A2E and accumulation of oxidized derivatives, correlating with the observed photophysical changes.
CONCLUSIONS: These findings demonstrate that component-resolved visible-near infrared spectral FLIM provides a sensitive, noninvasive approach to probe the molecular oxidative state of RPE lipofuscin, offering potential for early diagnosis of retinal pathologies.