A hyperspectral approach to RPE fluorophores in AMD

摘要 视网膜相关性黄斑变性（AMD）导致全世界数百万人视力丧失。AMD启动的核心 视网膜色素上皮（RPE）是临床上通过团块状的 其脂褐素和黑素脂褐素颗粒的自发荧光（AF）。我们假设，根据我们的成像 和病理学研究，AMD可以通过独特的表达和分布来分期和监测。 RPE荧光团。我们确定并表征了不同荧光团家族的离体光谱特征 在正常RPE、Bruch膜和玻璃疣中，AMD的标志性损害。然而， 由于精液成像质谱（IMS）研究，黄斑中的这些光谱现在是不确定的 显示主要的脂褐质荧光团A2 E在外周中是丰富的。长期目标是 这6个研究者的合作是开发基于高光谱AF的AMD诊断， RPE的分子活检，将临床病理学与潜在的分子组成联系起来。高光谱 与传统AF成像不同，AF成像采集数据的3维“超立方体”（2个空间 坐标- x，y -和1光谱-波长）。我们用新的基于张量的工具探索了成像数据 利用多个激发波长来发现RPE光谱特征及其空间分布。 我们建议将荧光团与颗粒，RPE细胞，组织和AMD阶段联系起来，以3个目标，使用共同的 人体组织来源目的1使用高光谱AF组织映射来了解AMD病理学， AMD眼睛和未受影响的对照眼睛的光谱水平。我们将通过高光谱技术 AF显微镜检查与病理分级系统相关联。将恢复光谱AF组件 数学上并分配给亚细胞和细胞外特征。目标2将量化AMD病理， 通过使用结构照明显微镜计数含荧光团颗粒的亚细胞水平 和三维电子显微镜。AIM 3使用高光谱荧光团识别来了解 分子水平上的AMD病理学。我们将确定主要光谱的候选分子 通过高光谱薄层色谱法在Aim 1中发现的组分，并将验证其空间分布 IMS的分布。合成真实标准品将确保光谱验证。生物学确认 这一水平对于临床眼科学来说是前所未有的，但由于AMD患者人群的规模， 关于人眼中主要RPE荧光团的知识缺口的严重性，供体组织的可用性， 以及验证其他成像技术的成功证明。我们的研究结果将直接转化为临床 高光谱AF成像用于无创、空间精确的早期检测和纵向AMD随访， 体内靶点发现和AMD以外的直接扩展。从我们的发现中会流出大量的 外视网膜细胞生物学实验，以加深对视网膜变性的理解。

Abstract Age-related macular degeneration (AMD) causes vision loss in millions worldwide. Central to AMD initiation and progression is the retinal pigment epithelium (RPE), which is clinically visualized via the massed autofluorescence (AF) of its lipofuscin and melanolipofuscin granules. We hypothesize, based on our imaging and pathology studies, that AMD can be staged and monitored by the expression and distribution of unique RPE fluorophores. We identified and characterized ex vivo spectral signatures of distinct fluorophore families in normal RPE, Bruch's membrane and drusen, AMD's hallmark lesion. However, the molecular sources of these spectra in the macula are now uncertain due to seminal imaging mass spectrometry (IMS) studies showing that a major lipofuscin fluorophore, A2E, is abundant in the periphery. Thus, the long-term goal of this 6-investigator collaboration is to develop AMD diagnostics based on hyperspectral AF for spectral, molecular biopsy of the RPE, linking clinical pathology to underlying molecular composition. Hyperspectral AF imaging, unlike conventional AF imaging, acquires 3-dimensional “hypercubes” of data (2 spatial coordinates – x, y - and 1 spectral - wavelength). We explored imaging data with novel tensor-based tools exploiting multiple excitation wavelengths to discover RPE spectral signatures and their spatial distributions. We propose to link fluorophores to granules, RPE cells, tissue, and AMD stages in 3 aims using a common human tissue source. Aim 1 uses hyperspectral AF tissue mapping to understand AMD pathology at the spectral level in eyes with AMD and unaffected control eyes. We will map RPE flat-mounts by hyperspectral AF microscopy linked to a pathology grading system. Spectral AF components will be recovered mathematically and assigned to subcellular and extracellular features. Aim 2 will quantify AMD pathology at the subcellular level by enumerating fluorophore-containing granules using structured illumination microscopy and 3-dimensional electron microscopy. Aim 3 uses hyperspectral fluorophore identification to understand AMD pathology at the molecular level. We will determine candidate molecules for the major spectral components discovered in Aim 1 by hyperspectral thin layer chromatography and will verify their spatial distributions by IMS. Synthetic authentic standards will ensure spectral validation. Biological validation at this level is unprecedented for clinical ophthalmology, yet warranted by the size of the AMD patient population, the enormity of knowledge gap about major RPE fluorophores in human eyes, the availability of donor tissue, and the proven success of validating other imaging technologies. Our results will directly translate to clinical hyperspectral AF imaging for noninvasive, spatially precise early detection and longitudinal AMD follow-up, in vivo target discovery, and immediate extensions beyond AMD. From our discoveries will flow a huge range of experiments in outer retinal cell biology to deepen understanding of retinal degenerations.