A hyperspectral approach to RPE fluorophores in AMD

老年性黄斑变性(AMD)的临床试验依赖于成像结果来衡量成功。 来自视网膜色素上皮(RPE)的强自体荧光(AF)信号具有很高的潜在的 无创、空间和分子上精确的早期检测和纵向随访。亚蜂窝信号 RPE房颤的来源是脂褐素(LF)颗粒，其中许多还含有黑色素(黑素)。新的 人类RPE细胞生物学的概念使眼底房颤的潜力比以往任何时候都更有可能实现。每个人 成人视网膜中央凹周围RPE细胞有1400个细胞器，可产生光学相干的反射率 断层扫描(OCT)。一半是低频或ML，也会产生眼底房颤(Faf)信号，以蓝绿激发光。 黑素小体(M)优先定位于顶突，并可能贡献其自身的Faf信号。这个 Faf和光感受器的一致形貌已经得到证实和详细的描述，在 中心凹和视神经头周围有高密度视杆环，呈强信号。在这个戒指上也是 大量研究良好的双维A类荧光团A2E。到OCT，高反射焦点发出高 进展风险包括RPE向前移行到视网膜。前人的一项影响深远的新发现 项目期是中心凹RPE以ML为主，从而赋予RPE一个特定的分子特征 这个盛产圆锥体的地方。与RPE的区域分子差异一致，我们的成像质谱学 (IMS)研究揭示了中央黄斑RPE特有的脂质信号。悬而未决的问题是 多个荧光团定位在每个细胞器上，细胞器用近红外(NIR)发射FAF信号 兴奋，以及哪些分子对黄斑部房颤负责。我们假设主要的荧光团驱动 黄斑Faf信号是定位于特定细胞器亚型的双维A酸，而细胞器亚型又是区域性的 按照锥体和杆状结构的分布进行分布。Curcio博士、Ach博士和Schey博士， 多学科高光谱视网膜自发荧光小组(HYRAFT)，在AMD病理学方面拥有专业知识， 临床成像和显微镜，以及分析化学，分别提出了对人类供体眼睛的研究 填补这些知识差距。正常老年人视网膜色素上皮-脉络膜平片和组织切片(N=20) 和AMD(N=25眼)接受体外OCT，AIM 1将加强FAF的局部和形态基础 使用组织横截面的成像使用成像质谱学来识别脂质信号，包括 荧光团。在这些组织中，Aim 2将枚举和测定3- 使用高分辨率结构照明显微镜和近红外敏感型相机。目标3将 用不连续的蔗糖梯度分离斑块和周围的RPE细胞器(80例正常 40名捐献者)提取荧光团，用荧光薄层层析法分离它们，以及 液-质联用法鉴定荧光团。结果将直接转换为 通过多种技术获得分子信息的临床Faf成像，用于AMD和其他视网膜疾病。

Clinical trials for age-related macular degeneration (AMD) depend on imaging outcomes to measure success. Strong autofluorescence (AF) signal from the retinal pigment epithelium (RPE) has high potential for noninvasive, spatially and molecularly precise early detection, and longitudinal follow-up. Subcellular signal sources of RPE AF are lipofuscin (LF) granules, many also containing melanin (melanolipofuscin, ML). New concepts of human RPE cell biology make fulfilling the potential of fundus AF more possible than ever. Each adult human perifoveal RPE cell has >1400 organelles that generate reflectivity for optical coherence tomography (OCT). Half are LF or ML that also generate fundus AF (FAF) signal to blue-green exciting light. Melanosomes (M) preferentially localize to apical processes and may contribute their own FAF signal. The congruent topographies of FAF and photoreceptors has been confirmed and detailed, with low signal in the fovea and strong signal at a ring of high rod density encircling fovea and optic nerve head. At this ring is also an abundance of well-studied bisretinoid fluorophore A2E. By OCT, hyperreflective foci conferring high progression risk include RPE anteriorly migrating into the retina. A far-reaching new finding from the previous project period is that foveal RPE is dominated by ML, thus imparting a specific molecular signature to RPE at this cone-rich site. Consistent with regional molecular differences in RPE, our imaging mass spectrometry (IMS) studies revealed lipid signals specific to RPE in central macula. Unresolved questions are whether multiple fluorophores localize to each organelle, what organelle emits FAF signal with near-infrared (NIR) excitation, and what molecules are responsible for macular AF. We hypothesize that major fluorophores driving macular FAF signal are bisretinoids localized to specific organelle subtypes, which in turn are regionally distributed in accordance with the distribution of cones and rods. Drs. Curcio, Ach, and Schey, members of the multidisciplinary Hyperspectral Retinal Autofluorescence Team (HYRAFT) with expertise in AMD pathology, clinical imaging and microscopy, and analytic chemistry, respectively, propose studies in human donor eyes to address these knowledge gaps. In RPE-choroid flat mounts and tissue cross-sections of aged normal (N=20) and AMD (N=25) eyes subject to ex vivo OCT, Aim 1 will fortify a regional and morphologic basis of FAF imaging using tissue cross-sections to use imaging mass spectrometry to identify lipid signals including fluorophores. In these tissues Aim 2 will enumerate and determine emission spectra of LF, ML, and F in 3- dimensions using high-resolution structured illumination microscopy and a NIR-sensitive camera. Aim 3 will use discontinuous sucrose gradients to isolate RPE organelles in pooled maculas and peripheries (80 normal eyes, 40 donors) to extract fluorophores, separate them with fluorescent thin layer chromatography, and identify fluorophores using liquid chromatography – tandem mass spectrometry. Results will directly translate to molecularly informed clinical FAF imaging by many technologies, in AMD and other retinal disorders.