3D analysis of collagen alterations in idiopathic pulmonary fibrosis by Second Harmonic Generation Excitation and Emission Tomography

通过二次谐波产生激发和发射断层扫描对特发性肺纤维化中的胶原蛋白变化进行 3D 分析

• 批准号: 2203403
• 负责人: Paul Campagnola
• 金额: $ 60.07万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-15 至 2025-04-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2203403&HistoricalAwards=false
• 关键词: 3D; analysis; collagen; alterations; idiopathic

Collagen is the most abundant protein in mammals and forms the structural basis for many connective tissues including skin, bone, tendon and also the supporting matrix for organs including liver, ovaries and pancreas. Collagen is organized into fibers of approximately 1 micron in diameter and and in the range of 10-200 microns in length. In normal tissues, the collagen organization is highly regulated with a balance of degradation and synthesis of old and new collagen. However, in many diseases, including all fibroses, cancers, cardiovascular disease, and connective tissue disorders, this regulation fails, and for the purposes of improved diagnostic/prognostic abilities, it is important to visualize and understand these underlying processes. The sizescales are too small for conventional clinical imaging modalities, including magnetic resonance imaging (MRI) and computed tomography (CT), and approaches using microscopes are thus needed to visualize the collagen fibers. This project will develop new and enhanced tools based on the physical process of Second Harmonic Generation (SHG), where this method can selectively image collagen fibers with high sensitivity and specificity. While SHG microscopy has been known for some time, the tools developed in this project will greatly enhance the capabilities over state of the art. Validation will be performed by using these new tools to image human idiopathic pulmonary fibrosis (IPF), as this disease is characterized by extensive collagen changes and moreover, has a poor prognosis. Despite increased collagen deposition being the clinical hallmark of the disease, the resulting fiber organization changes have not been well explored. These new enhanced SHG microscopic probes could enable better diagnostic and prognostic purposes as well as provide new insight into IPF disease origin and progression and also inform optimal treatment strategies. The new imaging tools can readily be extended for analogous studies in a wide range of diseases characterized by abnormal collagen organization. The trainees on the project will be immersed in a highly interdisciplinary research environment that involves aspects of fibrosis biology, physics, optical engineering, and image analysis engineering. Additionally, the research will be incorporated into undergraduate and graduate education. The native extracellular matrix (ECM) in many tissues has complex 3D collagen architecture, where both the structure and composition are altered in many diseases, including all fibroses, epithelial cancers, cardiovascular disease, and connective tissue disorders. Current microscopy and clinical imaging techniques lack either sufficient resolution, specificity, or sensitivity for effective differentiation between normal and these diseased states. In this project, new Second Harmonic Generation (SHG) instrumentation and imaging tools will be developed in the form of both excitation and emission tomographies to afford better characterization of 3D collagen structure in tissues. The excitation tomographic approach is critical as it is necessary to acquire the true 3D collagen fiber architecture which is not possible by other means due to constraints of the electric dipole interaction. Here, the acquisition speed will be improved through the implementation of multifocal excitation through the use of a diffractive optical element. Additionally, by drawing upon principles of Fourier Ptychography Microscopy, 3D super-resolution SHG will be attainable for the first time. This implementation will be coupled with SHG polarization analysis to determine collagen macro/supramolecular structural aspects. The SHG emission tomographic instrument combined with the accompanying theoretical phase-matching treatment will be the first microscope approach that can provide quantitative sub-resolution fibril size and packing structural information. In contrast, this has historically has been only afforded by electron microscopy. The SHG emission tomographic approach has higher throughput (numbers and volume) than TEM and does not require the complicated sample preparation needed by TEM. Validation will be performed by using these new tomographic tools to image human idiopathic pulmonary fibrosis (IPF), a serious lung disease that is characterized by extensive collagen remodeling.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

胶原蛋白是哺乳动物体内含量最丰富的蛋白质，是皮肤、骨骼、肌腱等多种结缔组织的结构基础，也是肝脏、卵巢、胰腺等器官的支持基质。胶原蛋白被组织成直径约1微米、长度在10-200微米范围内的纤维。在正常组织中，胶原蛋白的组织受到高度调控，新旧胶原蛋白的降解和合成平衡。然而，在许多疾病中，包括所有的纤维组织、癌症、心血管疾病和结缔组织疾病，这种调节都失败了，为了提高诊断/预后能力，可视化和理解这些潜在的过程是很重要的。对于常规的临床成像方法，包括磁共振成像(MRI)和计算机断层扫描(CT)，尺寸太小，因此需要使用显微镜来可视化胶原纤维。该项目将基于二次谐波产生(SHG)的物理过程开发新的和增强的工具，该方法可以选择性地对胶原纤维进行高灵敏度和特异性的成像。虽然倍频显微镜已经被知道了一段时间，但在这个项目中开发的工具将极大地增强超过最先进技术的能力。将使用这些新工具对人类特发性肺纤维化(IPF)进行成像验证，因为这种疾病的特点是广泛的胶原蛋白变化，而且预后很差。尽管胶原沉积增加是该病的临床特征，但由此导致的纤维组织变化尚未得到很好的研究。这些新的增强型SHG显微探针可以实现更好的诊断和预后目的，并为IPF疾病的起源和发展提供新的见解，并为最佳治疗策略提供信息。新的成像工具可以很容易地扩展到以胶原结构异常为特征的广泛疾病的类似研究中。该项目的受训人员将沉浸在一个高度跨学科的研究环境中，其中涉及纤维化生物学、物理学、光学工程和图像分析工程的各个方面。此外，这项研究将纳入本科生和研究生教育。许多组织中的天然细胞外基质(ECM)具有复杂的3D胶原结构，在许多疾病中，包括所有的纤维组织、上皮性癌症、心血管疾病和结缔组织疾病，其结构和组成都发生了变化。目前的显微镜和临床成像技术缺乏足够的分辨率、特异性或灵敏度来有效区分正常和这些疾病状态。在这个项目中，将以激发和发射断层扫描的形式开发新的二次谐波(SHG)仪器和成像工具，以更好地表征组织中的3D胶原结构。激发层析成像方法是关键，因为获得真正的3D胶原纤维结构是必要的，而由于电偶极相互作用的限制，用其他方法是不可能的。在这里，通过使用衍射光学元件来实现多焦点激发，将提高采集速度。此外，利用傅里叶光刻显微镜的原理，首次实现了三维超分辨率倍频。这一实施将与SHG极化分析相结合，以确定胶原蛋白的宏观/超分子结构方面。倍频发射断层扫描仪与理论上的位相匹配处理相结合，将是第一个可以提供定量的亚分辨率纤维大小和填充结构信息的显微镜方法。相比之下，历史上只有电子显微镜才能做到这一点。倍频发射层析成像方法比瞬变电磁法具有更高的吞吐量(数量和体积)，并且不需要瞬变电磁法所需的复杂的样品制备。验证将通过使用这些新的断层扫描工具来对人类特发性肺纤维化(IPF)进行成像，这是一种以广泛的胶原蛋白重塑为特征的严重肺部疾病。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。