Fluorescence Lifetimes and Spectra of RPE and Sub-RPE Deposits in Histology of Control and AMD Eyes

PURPOSE: To investigate fluorescence lifetimes as well as spectral characteristics of drusen and RPE autofluorescence in AMD. METHODS: Fluorescence 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 to 647 nm. Lifetimes were measured using time-correlated single photon counting in two spectral channels: 500 to 550 nm and 550 to 700 nm. Fluorescence decays over time were approximated by a series of three exponential functions. The amplitude-weighted mean fluorescence lifetime was determined. RESULTS: We identified 196 sub-RPE deposits (AMD, n = 76; control, n = 120) and recorded 241 RPE sites. The peak emission wavelength of sub-RPE deposits was significantly green shifted compared with RPE (peak at 570 nm vs. 610 nm), but did not differ between AMD and control donors. Sub-RPE deposits showed considerably longer mean fluorescence lifetimes than RPE (ch1, 581 ± 163 ps vs. 177 ± 25 ps; ch2, 541 ± 125 ps vs. 285 ± 31 ps; P < 0.001). Sub-RPE deposits found in AMD eyes had longer lifetimes than deposits of controls (ch1, 650 ± 167 ps vs. 537 ± 145 ps; ch2, 600 ± 125 ps vs. 504 ± 111 ps; P < 0.001). In AMD eyes, sub-RPE deposits showed a more homogenous autofluorescence distribution and more deposits were larger than 63 µm than in control eyes. CONCLUSIONS: Ex vivo fluorescence imaging of sub-RPE deposits in cross-sections enables the separation of their autofluorescence from that of over- or underlying structures. Our analysis showed considerable variability of sub-RPE deposit lifetimes but not spectra. This indicates that sub-RPE deposits either consist of a variety of different fluorophores or expose the same fluorophores to different microenvironments.