Department of Ophthalmology, University Hospital Jena, Jena, Germany.
Department of Ophthalmology and Visual Sciences, School of medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States.
Invest Ophthalmol Vis Sci. 2020 Sep 1;61(11):9. doi: 10.1167/iovs.61.11.9.
To investigate fluorescence lifetimes as well as spectral characteristics of drusen and RPE autofluorescence in AMD.
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.
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.
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.
研究 AMD 中 drusen 和 RPE 自发荧光的荧光寿命和光谱特性。
通过在 960nm 处的双光子激发荧光，对五例 AMD 眼和九例对照眼的冷冻切片进行荧光寿命和光谱分析。光谱在 490 至 647nm 处检测。使用时间相关单光子计数在两个光谱通道中测量寿命：500 至 550nm 和 550 至 700nm。随着时间的荧光衰减通过三个指数函数的级数来近似。确定加权平均荧光寿命。
我们鉴定了 196 个亚 RPE 沉积物（AMD，n=76；对照，n=120）和 241 个 RPE 位点。亚 RPE 沉积物的峰值发射波长明显比 RPE 绿移（峰值为 570nm 对 610nm），但 AMD 和对照供体之间没有差异。亚 RPE 沉积物的平均荧光寿命明显长于 RPE（ch1，581±163ps 对 177±25ps；ch2，541±125ps 对 285±31ps；P<0.001）。AMD 眼中的亚 RPE 沉积物的寿命比对照沉积物长（ch1，650±167ps 对 537±145ps；ch2，600±125ps 对 504±111ps；P<0.001）。在 AMD 眼中，亚 RPE 沉积物的自发荧光分布更加均匀，并且比对照眼中更大的沉积物大于 63µm。
亚 RPE 沉积物的横截面的荧光成像可以将其自发荧光与上层或下层结构的自发荧光分离。我们的分析表明亚 RPE 沉积物的寿命存在相当大的可变性，但光谱没有。这表明亚 RPE 沉积物要么由各种不同的荧光团组成，要么将相同的荧光团暴露于不同的微环境中。
