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含量最丰富的蛋白质，是皮肤、骨骼、肌腱等许多结缔组织的结构基础，也是肝脏、卵巢和胰腺等器官的支撑基质。胶原蛋白被组织成直径约为1微米的纤维，长度在10-200微米之间。在正常组织中，胶原蛋白组织受到高度调节，新旧胶原蛋白的降解和合成处于平衡状态。然而，在许多疾病中，包括所有的纤维化、癌症、心血管疾病和结缔组织疾病，这种调节失败了，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。对于传统的临床成像方式，包括磁共振成像（MRI）和计算机断层扫描（CT），尺寸太小，因此需要使用显微镜来观察胶原纤维。本项目将基于二次谐波生成（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成像，具有较高的灵敏度和特异性。虽然SHG显微镜已经有一段时间了，但在这个项目中开发的工具将大大提高技术水平。使用这些新工具成像人类特发性肺纤维化（IPF）将进行验证，因为这种疾病的特征是广泛的胶原改变，而且预后较差。尽管胶原沉积增加是该疾病的临床标志，但由此引起的纤维组织改变尚未得到很好的探讨。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疾病的起源和进展提供新的见解，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异常胶原组织为特征的各种疾病的类似研究。该项目的学员将沉浸在一个高度跨学科的研究环境中，包括纤维化生物学、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。许多组织中的原生细胞外基质（ECM）具有复杂的3D胶原结构，其结构和组成在许多疾病中都发生了改变，包括所有纤维化、上皮性癌症、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，特异性或灵敏度来有效区分正常和这些病变状态。在这个项目中，新的二次谐波生成（SHG）仪器和成像工具将以激发和发射层析成像的形式开发，以更好地表征组织中的3D胶原结构。激发层析成像方法是至关重要的，因为它需要获得真实的三维胶原纤维结构，这是由于电偶极子相互作用的限制而无法通过其他方法实现的。在这里，通过使用衍射光学元件实现多焦激发，将提高采集速度。此外，通过利用傅立叶平面摄影显微镜的原理，3D超分辨率SHG将首次实现。该实现将与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。SHG发射层析仪结合相应的理论相位匹配处理将是第一个可以提供定量亚分辨率纤维尺寸和填料结构信息的显微镜方法。相比之下，这在历史上只能通过电子显微镜来实现。SHG发射层析方法比TEM具有更高的通量（数量和体积），并且不需要TEM所需的复杂的样品制备。将使用这些新的断层成像工具对人类特发性肺纤维化（IPF）进行验证，IPF是一种严重的肺部疾病，其特征是广泛的胶原重塑。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.