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

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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.