A hyperspectral approach to RPE fluorophores in AMD

AMD 中 RPE 荧光团的高光谱方法

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

抽象的年龄相关性黄斑变性 (AMD) 导致全球数百万人视力丧失，这是 AMD 发生的关键。进展是视网膜色素上皮 (RPE)，临床上可通过聚集的根据我们的成像，我们的脂褐素和黑素脂褐质颗粒的自发荧光（AF）很困难。和病理学研究表明，AMD 可以通过独特的表达和分布来进行分期和监测我们鉴定并表征了不同荧光团家族的离体光谱特征。然而，在正常的RPE中，布鲁赫膜和玻璃膜疣是AMD的标志性病变。由于精液成像质谱 (IMS) 研究，黄斑中的这些光谱现在不确定表明主要的脂褐素荧光团 A2E 在外周丰富，因此，长期目标是。这项 6 名研究人员的合作旨在开发基于高光谱 AF 的 AMD 诊断技术，用于光谱、 RPE 的分子活检，将临床病理学与潜在的高光谱分子成分联系起来。 AF 成像与传统 AF 成像不同，它获取数据的 3 维“超立方体”（2 个空间我们使用新颖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。我们建议使用共同的方法将荧光团与颗粒、RPE 细胞、组织和 AMD 阶段的 3 个目标联系起来目标 1 使用高光谱 AF 组织映射来了解 AMD 病理学。患有 AMD 的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绘制 RPE 平板安装图。与病理分级系统相连的 AF 显微镜将被恢复。目标 2 将量化 AMD 病理学通过使用结构照明显微镜计数含荧光团的颗粒来确定亚细胞水平 Aim 3 使用高光谱荧光团识别来理解。我们将确定主要光谱的候选分子。通过高光谱薄层色谱在目标 1 中发现的成分，并将验证其空间 IMS 的合成真实标准品将确保光谱验证。对于临床眼科来说，这一水平是前所未有的，但 AMD 患者群体的规模是有保证的，关于人眼主要 RPE 荧光团的巨大知识差距、供体组织的可用性、以及验证其他成像技术的成功成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， vivo 目标发现，以及 AMD 之外的直接扩展将来自我们的发现。视网膜外细胞生物学实验，以加深对视网膜变性的了解。