Modern Cellular and Molecular Techniques to Study the Human Temporal Bone

项目总结 在本提案中，我们还将研究设计用于保存DNA的新的组织处理方案 因为它的形态。我们将保存、加工人耳组织，并将其用于内耳基础 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的经验 高级成像技术对死后的颞骨，以及 内耳。我们建议系统地开发和应用这些成像技术，并将 颞骨组织病理学的影像表现，旨在以非破坏性的方式显示结构 技巧。在具体目标1)中，我们将作为协作网络的一部分，为 获取、准备、切片和分发高质量的人类内耳组织，旨在增加 基础科学家对人类内耳组织的使用感兴趣。我们会继续进行验尸工作 颞骨旨在最大限度地减少死后时间，并将促进和接触更多的潜在患者 研究人员通过协作网络并通过会议提供人类内耳组织 高品质。我们将用我们的颞骨档案创建苏木精和曙红数字共享图书馆， 这个资料库将通过国家颞骨注册中心共享给科学家。我们将传播 用于颞骨收集、处理和内耳组织处理的协议，并提供实践 按需并通过缩放会议提供有关这些协议的说明。在具体目标2中，我们将开发新的 利用动物颞骨开发的颞骨组织采集与加工技术 保存DNA、信使核糖核酸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这一强大的原位杂交工具的应用。将制定一项新的议定书，以进一步 使用颞骨样本优化提取DNA的DNA测序以关联基因 与组织病理学进行测序。在目标3中，我们将使用以下工具改进颞骨内耳可视化 我们将与组织病理学相关联的新的非破坏性成像技术。在目标4中，我们将开发 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 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.