Spectroscopic and morphological studies of human retinal lipofuscin granules
Spectroscopic and morphological studies of human retinal lipofuscin granules
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DOI：
10.1073/pnas.0630280100
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发表时间：
2003-03-18
影响因子：
11.1
通讯作者：
Simon, JD
中科院分区：
文献类型：
--
作者：
Haralampus-Grynaviski, NM;Lamb, LE;Simon, JD
通过使用稳态荧光光谱和光谱分辨共聚焦显微镜检查从人视网膜色素上皮细胞分离的眼部脂褐质颗粒的发射特性。在玻璃上干燥的厚脂褐质颗粒样品的发射光谱形状随激发能量的变化而变化。这种发射的偏振与波长相关，在激发波长附近表现出明显的偏振，并且在大部分发射光谱上大部分变得去偏振。这些结果表明，发黄光的荧光团 [例如，A2E (2-[2,6-二甲基-8-(2,6,6-三甲基-1-环己烯-1-基)-1E,3E,5E,7E-辛四烯基]-1-(2-氢xy乙基)-4-[4-甲基-6-(2,6,6-三甲基-1-环己烯-1-基)-1E,3E,5E-己三烯基]-吡啶鎓)] 由于颗粒内能量转移而被激发，因此不是脂褐素颗粒内主要的蓝色吸收发色团。原子力显微镜图像显示脂褐素颗粒是聚集结构。因此，体发射和体内发射测量必须考虑罗利散射的影响。当校正散射时，厚脂褐素沉积物或细胞内脂褐素的发射光谱与 A2E 的发射光谱相似。单个颗粒集合的发射光谱之和也类似于 A2E 的发射光谱，但单个颗粒的光谱变化很大。这一结果表明，脂褐素和 A2E 的发射光谱之间的一致性是偶然的，并且集体数据表明脂褐素中存在几种蓝色吸收发色团，并表明 A2E 并不是许多研究颗粒中的主要黄色发射荧光团。
The emission properties of ocular lipofuscin granules isolated from human retinal pigment epithelial cells are examined by using steady-state fluorescence spectroscopy and spectrally resolved confocal microscopy. The shape of the emission spectrum of a thick sample of lipofuscin granules dried on glass varies with excitation energy. The polarization of this emission is wavelength-dependent, exhibiting significant polarization near the excitation wavelength and becoming mostly depolarized over the majority of the emission spectrum. These results show that the yellow-emitting fluorophores [e.g., A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium)] are excited as a result of energy transfer within the granules and therefore are not the dominant blue-absorbing chromophores within lipofuscin granules. Atomic force microscopy images show lipofuscin granules to be an aggregated structure. Bulk and in vivo emission measurements must therefore take into account the effect of Raleigh scattering. When corrected for scattering, the emission spectrum of a thick lipofuscin deposit or intracellular lipofuscin resembles that for A2E. The sum of the emission spectra of a collection of individual granules also resembles the emission spectrum of A2E, but the spectrum of individual granules varies significantly. This result suggests that the agreement between the emission spectra of lipofuscin and A2E is fortuitous, and the collective data indicate the presence of several blue-absorbing chromophores in lipofuscin and show A2E is not the dominant yellow-emitting fluorophore in many of the granules studied.