CRY-TEM AND SHG IMAGING OF COLLAGEN FIBER LAYER

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 对鼓膜（TM）生物力学的更深入了解有可能指导未来与鼓膜手术修复或鼓膜成形术相关的医疗技术的发展。 TM是一种复合结构，具有放射状、圆形和抛物线状排列的胶原纤维层，提供主要支架。 胶原纤维取向和体积分数的更详细的知识可以大大提高现有的TM的机械模拟。 通过透射电子显微镜（TEM）对人TM穿孔活组织检查（1 mm直径）成像。 这些样品是使用新开发的超快速高压冷冻和随后的冷冻替代方法制备的，该方法比以前在TM的TEM研究中使用的可比方法更好地保留了超微结构。 为了指导我们未来的TM研究，我们将使用二次谐波发生显微镜（SHG）对样品进行成像。 SHG图像将允许我们选择感兴趣的区域以进行后续TEM成像的穿刺活检。 我们的目标是使用我们获得的高分辨率（4 nm x 4 nm分辨率）2-D TEM图像来创建相关区域的3-D断层图像。 然后，我们将这些较小体积的图像整合到由SHG成像生成的3-D重建中。 我们相信三维数据将产生新的见解胶原纤维密度和纤维取向作为鼓膜厚度和径向位置的函数。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A greater understanding of tympanic membrane (TM) biomechanics has the potential to guide future advances in medical technology related to the surgical repair of eardrums or myringoplasties. The TM is a composite structure with radial, circular, and parabolic arrangements of collagen fiber layers that provide the main scaffolding. A more detailed knowledge of collagen fiber orientation and volume fraction could greatly improve existing mechanical simulations of the TM. Human TM punch biopsies (1 mm diameter) were imaged by transmission electron microscopy (TEM). These samples were prepared using a newly developed method of ultra rapid high-pressure freezing and subsequent freeze substitution, which preserves ultra-structure better than comparable methods used previously in TEM studies of the TM. To guide our future studies of the TM, we will image the samples using Second Harmonic Generation microscopy (SHG). The SHG images will allow us to choose regions of interest to punch biopsy for subsequent TEM imaging. Our goal is to use the high-resolution (4 nm x 4 nm resolution) 2-D TEM images we have obtained to create 3-D tomographic images of pertinent regions. We will then integrate these smaller volume images into the 3-D reconstructions generated by the SHG imaging. We believe the three dimensional data will yield new insights into collagen fiber density and fiber orientation as a function of tympanic membrane thickness and radial position.