Modern Cellular and Molecular Techniques to Study the Human Temporal Bone

项目摘要 在目前的建议中，我们将研究新的组织处理方案，旨在保存DNA，以及 as the morphology形态.我们将保存、处理和提供人耳组织， 研究人员将能够与他们的动物模型进行比较。我们有使用高分辨率 先进的成像技术，死后颞骨，也膜结构的 内耳我们建议系统地开发和应用这些成像技术，并将 颞骨组织病理学的成像结果，旨在显示非破坏性结构 技术.在具体目标1）中，我们将作为合作网络的一部分，为以下方面提供技术服务： 获取、制备、切片和分配高质量的人类内耳组织，旨在增加 对基础科学家使用人类内耳组织的兴趣。我们会继续在尸体上 颞骨旨在最大限度地减少死后时间，并将促进和接触到更多的前瞻性 研究人员通过合作网络和会议，提供人类内耳组织， 高质量的。我们将创建苏木精和伊红数字共享库与我们的颞骨档案， 这个库将通过国家颞骨登记处与科学家共享。我们将传播 颞骨采集，处理和内耳组织处理的协议，并提供动手操作 关于这些协议的说明，可按需提供，也可通过zoom会议提供。在具体目标2中，我们将开发新的 颞骨组织的采集和处理技术， 保存DNA、mRNA和蛋白质以及保存形态的技术。我们的目标是开发新的组织 允许使用免疫荧光、DNA提取和测序的处理方案，以及 RNAScope是一种功能强大的原位杂交工具。将制定一项新的协议， 使用颞骨标本优化从提取的DNA中测序DNA，以关联基因 与组织病理学进行测序。目标3.我们将改善颞骨内耳可视化使用 新的非破坏性成像技术，我们将与组织病理学。在目标4中，我们将开发 一个基于深度学习的染色变换框架，用于可视化特定的解剖结构，例如 螺旋神经节神经元，以获得准确的组织病理学分析。

PROJECT SUMMARY In the present proposal we will investigate new tissue processing protocols designed to preserve DNA, as well as the morphology. We will preserve, process, and make available human ear tissue to inner ear basic researchers they will be able to compare with their animal models. We have experience using high-resolution advanced imaging techniques to the postmortem temporal bones, and also the membranous structures of the inner ear. We propose to develop and apply these imaging techniques systematically and to correlate the imaging findings with the temporal bone histopathology aiming to visualize structures with non-destructive techniques. In Specific Aim 1) we will serve as part of a collaborative network providing technical services for procuring, preparing, sectioning, and distributing high-quality human inner ear tissues aiming to increase interest in the use of human inner ear tissues for basic scientists. We will continue to procure post- mortem temporal bones aiming to minimize post-mortem times and will facilitate and reach out to additional prospective researchers through the collaborative network and through meetings and provide human inner ear tissues of high-quality. We will create hematoxylin and eosin digital shareable library with our archive of temporal bones, and this library will be shared through the National Temporal Bone Registry to scientists. We will disseminate protocols for temporal bone collection, processing, and inner ear tissue processing and provide hands-on instruction on these protocols on demand and via zoom conferences. In Specific Aim 2, we will develop new tissue collection and processing techniques for temporal bones by using animal temporal bones to develop techniques which preserve DNA, mRNA and proteins and preserve morphology. We aim to develop new tissue processing protocols which will allow for the use of immunofluorescence, DNA extraction and sequencing, and the application of RNAScope, a powerful in situ hybridization tool. A new protocol will be developed to further optimize sequencing the DNA from the extracted DNA using temporal bones specimens to correlate the gene sequencing with the histopathology. In Aim 3. We will improve temporal bone inner ear visualization using novel non-destructive imaging techniques which we will correlate with histopathology. In Aim 4, we will develop a deep learning-based stain transformation framework to visualize specific anatomical structures such as the spiral ganglion neurons to obtain accurate histopathological analysis.