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

胶原蛋白是哺乳动物中最丰富的蛋白质，并且形成许多结缔组织（包括皮肤、骨、肌腱）的结构基础，并且还形成器官（包括肝脏、卵巢和胰腺）的支持基质。胶原被组织成直径约1微米且长度在10-200微米范围内的纤维。在正常组织中，胶原组织受到高度调节，新旧胶原的降解和合成平衡。然而，在许多疾病中，包括所有纤维化、癌症、心血管疾病和结缔组织疾病，这种调节失败，并且为了提高诊断/预后能力，重要的是可视化和理解这些潜在的过程。对于常规的临床成像方式，包括磁共振成像（MRI）和计算机断层扫描（CT），尺寸尺度太小，因此需要使用显微镜的方法来可视化胶原纤维。该项目将基于二次谐波产生（SHG）的物理过程开发新的增强工具，该方法可以选择性地对胶原纤维进行高灵敏度和特异性成像。虽然SHG显微镜已经知道了一段时间，在这个项目中开发的工具将大大提高的能力，超过国家的art. Validation将通过使用这些新的工具来成像人类特发性肺纤维化（IPF），因为这种疾病的特点是广泛的胶原蛋白的变化，而且，预后差。尽管增加的胶原沉积是该疾病的临床标志，但由此产生的纤维组织变化尚未得到很好的研究。这些新的增强型SHG显微镜探针可以实现更好的诊断和预后目的，并为IPF疾病的起源和进展提供新的见解，还可以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类似的研究，在广泛的疾病特点是异常胶原组织。该项目的学员将沉浸在一个高度跨学科的研究环境中，涉及纤维化生物学，物理学，光学工程和图像分析工程等方面。此外，研究将纳入本科和研究生教育。许多组织中的天然细胞外基质（ECM）具有复杂的3D胶原结构，其中结构和组成在许多疾病中改变，包括所有纤维化、上皮癌、心血管疾病和结缔组织疾病。目前的显微镜和临床成像技术缺乏足够的分辨率，特异性，或灵敏度之间的有效区分正常和这些疾病状态。 在这个项目中，新的二次谐波发生（SHG）仪器和成像工具将以激发和发射断层扫描的形式开发，以更好地表征组织中的3D胶原结构。 激发断层摄影方法是至关重要的，因为它是必要的，以获得真正的3D胶原纤维结构，这是不可能通过其他手段，由于电偶极子相互作用的约束。这里，通过使用衍射光学元件实现多焦点激发，将提高采集速度。此外，通过利用傅立叶重叠关联显微术的原理，将首次实现3D超分辨率SHG。该实施将与SHG偏振分析相结合，以确定胶原蛋白的大分子/超分子结构方面。的SHG发射断层扫描仪与相应的理论相匹配处理相结合，将是第一个显微镜的方法，可以提供定量的亚分辨率原纤维的大小和包装结构的信息。相比之下，这在历史上仅由电子显微镜提供。SHG发射层析成像方法具有比TEM更高的通量（数量和体积），并且不需要TEM所需的复杂样品制备。验证将通过使用这些新的断层摄影工具来成像人类特发性肺纤维化（IPF），这是一种严重的肺部疾病，其特征是广泛的胶原蛋白重塑。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.