Multiphoton Imaging and Rheology of Fibrosis Models

描述（由申请人提供）：受各种疾病影响的组织中的胶原蛋白结构、密度和浓度变化。哮喘气道的上皮下纤维化、血管中的动脉粥样硬化病变和肿瘤附近的基质变化都可以用多光子显微镜进行研究。胶原蛋白通过二次谐波产生（SHG，以激发波长的一半发射的光子）和双光子荧光（TPF）对近红外光作出响应。而SHG起源于螺旋纤维结构内的电子，TPF产生于某些酶和非酶交联。通过对脱细胞胶原、工程化纤维化模型和大鼠气道纤维化模型中的SHG和TPF成像，该提议将表征这两种信号，通过将定量图像数据拟合到模型并将数据与以下相关联来解释信号变化的原因：1）分别通过扫描电子显微镜、生化测定和流变学评估的胶原凝胶微观结构、交联和机械性质; 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、酶谱和多光子成像评估，和; 3）来自哮喘大鼠模型的体内伤口愈合环境。该提案旨在开发SHG和TPF的多光子成像，作为预测和监测动物模型和临床人类工程组织和浅层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，将应用于SHG和TPF图像，以估计作为输入的组织的结构力学模型，从半柔性聚合物网络理论的微观结构参数。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，其中非侵入性收集的组织力学特性信息将有助于工程组织、患者病变组织的研究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.