Fluorescence Lifetimes and Spectra of RPE and sub-RPE Deposits in Histology of Normal and AMD Eyes

Abstract

PurposeTo investigate autofluorescence lifetimes as well as spectral characteristics of drusen and retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). MethodFluorescence lifetimes and spectra of five eyes with AMD and nine control eyes were analyzed in cryosections by means of two-photon excited fluorescence at 960 nm. Spectra were detected at 490 - 647nm. Lifetime was measured using time-correlated single photon counting in two spectral channels: 500-550nm and 550-700nm. The fluorescence decays over time were approximated by a series of three exponential functions and the amplitude-weighted mean fluorescence lifetime {tau}m was determined. Results196 sub-RPE deposits were identified (AMD n=76, healthy n=120) and 230 RPE sites recorded. The peak emission wavelength of drusen was significantly green-shifted compared to RPE (peak at 570nm vs. 610nm), but not different between patients and controls. Drusen showed considerably longer {tau}m than RPE: (ch1: 581 {+/-} 163 ps vs. 177 {+/-} 25 ps, ch2: 541 {+/-} 125 ps vs. 285 {+/-} 31 ps, p < 0.001). Drusen found in AMD eyes had longer lifetimes than drusen of controls (ch1: 650 {+/-} 167 ps vs. 537 {+/-} 145 ps, ch2: 600 {+/-} 125 ps vs. 504 {+/-} 111 ps, p < 0.001). In addition, drusen in AMD eyes showed a more homogenous fluorescence distribution and more drusen were larger than 63{micro}m than in control eyes. ConclusionsEx vivo fluorescence imaging of drusen in cross-sections enables the separation of their autofluorescence from that of over- or underlying structures. Our analysis showed considerable variability of drusen lifetimes but not spectra. This indicates that drusen consist of a variety of different fluorophores or expose the same fluorophores to different microenvironments. Changes in drusen lifetimes could be related to AMD progression.