Multiphoton Imaging and Rheology of Fibrosis Models

描述（由申请人提供）：各种疾病影响的组织中胶原蛋白的结构、密度和浓度变化。哮喘气道的上皮下纤维化、血管的动脉粥样硬化病变和肿瘤附近的基质改变都可以使用多光子显微镜进行研究。胶原蛋白通过二次谐波产生（SHG，在激发波长的一半发射的光子）和双光子荧光（TPF）响应近红外光。SHG来源于螺旋纤维结构中的电子，而TPF来源于某些酶和非酶交联。通过对脱细胞胶原、工程纤维化模型和大鼠气道纤维化模型中的SHG和TPF进行成像，本研究将对这两种信号进行表征，通过将定量图像数据拟合到模型中，并将数据与以下数据相关联，解释信号变化的原因：1)胶原凝胶微观结构、交联和力学性能，分别通过扫描电子显微镜、生化分析和流变学进行评估；2)成纤维细胞介导的胶原沉积、降解和重塑，通过羟脯氨酸测定、酶谱分析和多光子成像进行评估；3)哮喘大鼠模型的体内伤口愈合环境。本研究旨在发展SHG和TPF的多光子成像，作为预测和监测工程组织和动物模型及临床人类浅纤维化的机械和结构特性的工具。新的图像处理工具，如图像相关光谱学，将应用于SHG和TPF图像，以估计作为组织结构力学模型输入的微结构参数，该模型源自半柔性聚合物网络理论。该项目开发的自动化和标准化图像处理和力学特性建模将在基础研究和临床中得到应用，在临床中，无创收集的组织力学特性信息将有助于研究工程组织、患者病变组织以及纤维化疾病的诊断和治疗。

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.