Multiphoton Imaging and Rheology of Fibrosis Models

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DESCRIPTION (provided by applicant): Collagen structure, density, and concentration changes in tissues affected by various diseases. Subepithelial fibrosis in asthmatic airways, atherosclerotic lesions in blood vessels, and matrix changes near tumors all can be studied using multiphoton microscopy. Collagen responds to near infrared light by second harmonic generation (SHG, photons emitted at half the excitation wavelength) and two-photon fluorescence (TPF). Whereas SHG originates from electrons within the helical fibril structure, TPF arises from certain enzymatic and nonenzymatic crosslinks. By imaging SHG and TPF in acellular collagen, engineered fibrosis models, and rat airway fibrosis models, this proposal will characterize the two signals, explaining reasons for the signal variation by fitting quantitative image data to models and correlating data with: 1) collagen gel microstructure, cross linking , and mechanical properties, assessed by scanning electron microscopy, biochemical assays, and rheology, respectively; 2) fibroblast-mediated collagen deposition, degradation, and remodeling, assessed by hydroxyproline assays, zymography, and multiphoton imaging, and; 3) an in vivo wound healing environment, from a rat model of asthma. This proposal seeks to develop multiphoton imaging of SHG and TPF as a tool to predict and monitor mechanical and structural properties of engineered tissue and of shallow fibrosis in animal models and clinically in humans. Novel image processing tools, such as image correlation spectroscopy, will be applied to SHG and TPF images to estimate microstructurai parameters that serve as inputs to a structural mechanical model of tissue, derived from semiflexible polymer network theory. Automated and standardized image processing and mechanical property modeling developed by this project will find applications in basic research and in the clinic, where noninvasively-collected information about tissue mechanical properties will aid in the study of engineered tissues, diseased tissues from patients, and in diagnosis and treatment of fibrotic diseases.