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Updated: Feb 27, 2026
Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases
Published on: March 11, 2016
Near-Infrared Fluorescence Lifetime Imaging Microscopy Reveals Loss of RPE Cell Body Polarity in Age-Related Macular
Katharina Wall1, Sebastian Fritsch1, Marc Vaisband2
1Department of Ophthalmology, University Hospital Bonn, Bonn, Germany.
Purpose:
Retinal pigment epithelium (RPE) cells exhibit a markedly polarized distribution of organelles and membrane specializations. We aimed to determine whether fluorescence lifetime imaging microscopy (FLIM) using different excitation wavelengths reveals altered polarity in age-related macular degeneration (AMD).
Methods:
Retinal cross-sections from human donors (11 with AMD; 87.9 ± 4.5 years, and 9 healthy controls; 83.4 ± 2.7 years) were imaged at the fovea, perifovea, near-, mid-periphery, and ora serrata using FLIM (excitation = 488, 780 nm; 5 regions of interest [ROI; 50 µm width] per location). At each location, individual RPE cells were classified as healthy, non-uniform, or pathological. Cell bodies were divided into four equal apical-to-basal compartments to determine the fluorescence lifetime (FLT) gradient. Post-processing included multiexponential decay modeling and phasor analysis. Ordinal logistic regression (LR) and linear mixed-effects models (LMMs) compared healthy and diseased cells.
Results:
In 477 ROIs (3-5 cells/ROI), healthy RPE at 780 nm displayed a distinct intracellular FLT gradient. In 52 pathological ROIs, this was significantly reduced (regression coefficient = -0.029, P = 0.007). Ordinal LR confirmed an association between polarity loss and dysmorphia (log cumulative odds ratio = -6.50, P = 0.033). Mean FLT was shorter in non-uniform (-0.009 nanoseconds [ns], P = 0.03) and pathological RPE (-0.036 ns, P < 0.001) than in healthy RPE. At 488 nm, pathological RPE exhibited prolonged FLT without an apical/basal gradient.
Conclusions:
At 780 nm, FLIM detects a loss of RPE cell polarity that likely reflects intracellular reorganization and metabolic change. Disruption in NIR-autofluorescence lifetimes may provide an early biomarker for pathological RPE alterations in AMD.
Insights
Fluorescence lifetime imaging microscopy (FLIM) reveals loss of retinal pigment epithelium (RPE) cell polarity in age-related macular degeneration (AMD). This polarity loss, detected using near-infrared autofluorescence, may serve as an early biomarker for AMD progression.
Area of Science:
- Ophthalmology
- Cell Biology
- Biophysics
Background:
- Retinal pigment epithelium (RPE) cells are crucial for retinal health and exhibit distinct polarity.
- Age-related macular degeneration (AMD) is a leading cause of vision loss associated with RPE dysfunction.
- Understanding RPE cell alterations in AMD is vital for developing early diagnostic tools.
Purpose of the Study:
- To investigate RPE cell polarity using fluorescence lifetime imaging microscopy (FLIM) with varying excitation wavelengths.
- To determine if FLIM can detect altered RPE polarity in human donors with AMD compared to healthy controls.
Main Methods:
- Human retinal cross-sections from AMD patients and healthy controls were analyzed using FLIM at 488 nm and 780 nm excitation.
- RPE cells were assessed across five retinal locations, and their apical-to-basal fluorescence lifetime (FLT) gradient was analyzed.
- Statistical models, including ordinal logistic regression and linear mixed-effects models, were used to compare healthy and diseased RPE cells.
Main Results:
- Healthy RPE cells at 780 nm showed a distinct FLT gradient, which was significantly reduced in pathological RPE cells from AMD donors.
- Polarity loss in RPE cells was strongly associated with AMD.
- Pathological RPE cells exhibited shorter mean FLT at 780 nm and prolonged FLT without a gradient at 488 nm.
Conclusions:
- FLIM at 780 nm effectively detects loss of RPE cell polarity, indicative of intracellular reorganization and metabolic changes in AMD.
- Disruptions in near-infrared (NIR) autofluorescence lifetimes represent a potential early biomarker for pathological RPE alterations in AMD.
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