CRY-TEM AND SHG IMAGING OF COLLAGEN FIBER LAYER

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 对鼓膜(TM)生物力学有更深入的了解，有可能指导未来与鼓膜或鼓膜成形术手术修复相关的医疗技术的进步。TM是由放射状、圆形和抛物线排列的胶原纤维层组成的复合结构，提供了主要的支架。对胶原纤维取向和体积分数的更详细的了解可以极大地改进现有的TM的机械模拟。 用透射电子显微镜对直径为1 mm的人TM穿孔活检组织进行成像。这些样品是用一种新开发的超快速高压冷冻和随后的冷冻替代方法制备的，这种方法比以前在TM的透射电子显微镜研究中使用的可比方法更好地保存了超微结构。 为了指导我们未来对TM的研究，我们将使用二次谐波生成显微镜(SHG)对样品进行成像。SHG图像将使我们能够选择感兴趣的区域进行穿孔活检，以进行后续的透射电子显微镜成像。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)的2-D电子显微镜图像来创建相关区域的3-D断层图像。然后，我们将把这些较小的体积图像整合到由倍频成像产生的3D重建中。我们相信，三维数据将为胶原纤维密度和纤维取向作为鼓膜厚度和放射状位置的函数提供新的见解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.