A hyperspectral approach to RPE fluorophores in AMD

AMD 中 RPE 荧光团的高光谱方法

• 批准号: 9306485
• 负责人: Thomas Ach
• 金额: $ 45.22万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9306485
• 关键词: 3-Dimensional; Accounting; Achievement; Age; Age related macular degeneration; Aging; Area; Biochemistry; Biological; Biology; Biopsy; Blindness; Bruch' s basal membrane structure; Catalogs; Cell Count; Cells; Cellular biology; Clinical; Clinical Pathology; Collaborations; Cytoplasmic Granules; Data; Devices; Diagnosis; Diagnostic; Disease; Drusen; Early Diagnosis; Electron Microscopy; Emerging Technologies; Ensure; Eye; Family; Goals; Human; Image; Image Analysis; Imaging Device; Imaging technology; Individual; Institution; Knowledge; Lesion; Lighting; Link; Lipofuscin; Maps; Mass Spectrum Analysis; Mathematics; Melanins; Microscopy; Modality; Molecular; Monitor; Nonexudative age-related macular degeneration; Ophthalmology; Organelles; Pathologist; Pathology; Patients; Phenotype; Physical shape; Research Personnel; Retinal; Retinal Degeneration; Risk; Scientist; Seminal; Signal Transduction; Source; Spatial Distribution; Structure; Structure of retinal pigment epithelium; System; Technology; Thin Layer Chromatography; Three-Dimensional Image; Tissue Donors; Tissues; Translating; Ultrastructural Pathology; Validation; adaptive optics; base; biomathematics; cellular pathology; clinical imaging; experimental study; extracellular; fluorophore; follow-up; geographic atrophy; human tissue; in vivo; instrumentation; knowledge base; macula; multidisciplinary; new therapeutic target; non-invasive imaging; novel; outcome forecast; patient population; skills; success; synergism; tool

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.

摘要 老年性黄斑变性(AMD)导致全球数百万人失明。AMD启动的核心 进展为视网膜色素上皮(RPE)，临床上可通过肿块显示 其脂褐素和黑素褐素颗粒的自体荧光(AF)。我们假设，基于我们的想象 而病理研究表明，AMD可以通过独特的表达和分布来分期和监测 RPE荧光团。我们鉴定和表征了不同荧光团家族的体外光谱特征。 在正常RPE中，Bruchs膜和玻璃体，是AMD的标志性病变。然而，分子来源的 由于种子成像质谱学(IMS)研究，黄斑中的这些光谱现在是不确定的。 显示一个主要的脂褐素荧光团，A2E，在外围丰富。因此，长期目标是 这项由6名研究人员组成的合作旨在开发基于高光谱AF的AMD诊断技术， RPE的分子活检，将临床病理与潜在的分子组成联系起来。高光谱 与传统的自动对焦成像不同，自动对焦成像可以获取数据的3维“超立方体”(2个空间 坐标-x、y-和1个光谱波长)。我们使用新的基于张量的工具来研究成像数据 利用多个激发波长来发现RPE光谱特征及其空间分布。 我们建议在3个目标中将荧光团与颗粒、RPE细胞、组织和AMD阶段联系起来，使用共同的 人体组织来源。AIM 1使用高光谱房颤组织映射来了解AMD的病理 AMD患者和正常对照眼的光谱水平。我们将通过高光谱映射RPE平板 AF显微镜与病理分级系统相连。光谱自动对焦成分将被恢复 在数学上，并分配给亚细胞和细胞外的特征。AIM 2将量化AMD的病理 用结构照明显微镜计数含荧光颗粒的亚细胞水平 和三维电子显微镜。AIM 3使用高光谱荧光团识别来理解 AMD在分子水平上的病理改变。我们将确定主要光谱的候选分子 通过高光谱薄层色谱在AIM 1中发现的成分，并将验证它们的空间分布 按IMS分发。合成的真实标准将确保光谱验证。在这方面的生物验证 这在临床眼科是史无前例的，但AMD患者的数量证明了这一点， 关于人眼主要RPE荧光团的巨大知识缺口，供体组织的可用性， 以及验证其他成像技术的已被证明的成功。我们的结果将直接转化为临床 高光谱房颤成像用于非侵入性、空间精确的早期检测和纵向AMD随访 Vivo目标发现，并立即扩展到AMD之外。从我们的发现中将会流出大量的 视网膜外细胞生物学实验，以加深对视网膜变性的了解。