Equipment for Visualization of Fragile Subcellular Detail by Electron Microscopy

描述(申请人提供)：电子显微镜(EM)是当今生物医学中必不可少的工具。调查人员通常必须提供新兴市场证据，以支持其他方法得出的结论。高压冷冻(HPF)仪器设计的最新进展，是自引入戊二醛固定以来EM最大的进步之一，现在允许HPF保存细胞超微结构在更多实验室的各种细胞标本中得到更广泛的应用。HPF冷冻结合冷冻替代固定法(FSF)在保存标本超微结构细节方面的优势是显而易见的，该方法正在成为EM的标准固定方法。该提案的目标是获得资金，用于购买徕卡EM PACT2高压冷冻机、徕卡EM AFS冷冻替代仪器和徕卡显微活检转移系统。该设备将安置在希望之城(COH)的EM核心设施中，主要由六个主要用户和四个次要用户使用，其中许多用户拥有R01或其他NIH资金。许多主要用户研究癌症，并从事转化性研究，重点是将治疗方法从动物模型转移到患者身上。他们认识到最佳保存超微结构的重要性，并在他们的工作中越来越需要超微结构的定位。其中一些项目需要免疫标记来定位在常规标本制备过程中可能丢失或重新分布的分子的超微结构。其他人需要区分内体和自噬小泡，这是一种细胞器，在常规样品制备过程中对扭曲特别敏感。COH EM核心设施于2003年进行了翻新，并用新的FEI Tecnai变速器和FEI Quanta ESEM扫描电子显微镜进行了更新。我们目前有一台徕卡冷冻超微切割机，电子断层扫描和冷冻电子显微镜的组件正在安装中。徕卡EM PACT2高压冷冻机、冷冻替代仪和显微活检转移系统将确保COH研究人员能够获得最新的EM固定技术，这将大大有助于他们对癌症预防和治疗的研究。公共卫生相关性：当代医学需要针对细胞活动出错的特定干预而合理设计的药物。越来越多的生物医学研究将重点放在亚细胞和大分子事件上，以了解细胞活动的细节。所要求的最先进的徕卡EM PACT2高压冷冻机、徕卡EM AFS冷冻替代仪和徕卡显微活组织检查转移系统将帮助NIH资助的希望之城的研究人员开发癌症和其他危及生命的疾病的新疗法。

DESCRIPTION (provided by applicant): Electron microscopy (EM) is an essential tool in present day biomedicine. Investigators often must provide EM evidence to support conclusions reached by other approaches. Recent advances in instrument design for high pressure freezing (HPF), one of the greatest advances in EM since the introduction of glutaraldehyde fixation, now allow HPF preservation of cellular ultrastructure to be more widely applied to a variety of cellular specimens in more labs. The superiority of preservation of ultrastructural detail in samples prepared by HPF freezing coupled with freeze substitution fixation (FSF) is readily evident, and this method is becoming the standard fixation method for EM. The goal of this proposal is to obtain funds for the purchase of a Leica EM PACT2 high pressure freezer, a Leica EM AFS freeze substitution instrument, and a Leica Microbiopsy Transfer System. This equipment will be housed in the EM Core Facility at City of Hope (COH) and will primarily be used by six Major Users and four Minor Users, many of whom have R01 or other NIH funding. Many of the Major Users study cancer and are engaged in translational research focused on moving therapies from animal models to patients. They recognize the importance of optimal preservation of ultrastructure and have an increasing need for ultrastructural localization in their work. Some of these projects require immunolabeling for ultrastructural localization of molecules that could be lost or redistributed during conventional specimen preparation. Others need to distinguish endosomal and autophagic vesicles, organelles that are particularly sensitive to distortions during conventional sample preparation. The COH EM Core Facility was renovated in 2003, and was updated with new FEI Tecnai transmission and FEI Quanta ESEM scanning electron microscopes. We currently have a Leica cryo-ultramicrotome, and components for electron tomography and cryo-electron microscopy are being installed. The Leica EM PACT2 high pressure freezer, freeze substitution instrument, and microbiopsy transfer system will ensure that COH investigators have access to the most current fixation techniques for EM, which will greatly benefit their research on the prevention and treatment of cancer. Public Health Relevance: Contemporary medicine needs drugs that are rationally designed for specific interventions in cellular activities gone awry. Increasingly, biomedical research focuses on sub-cellular and macromolecular events to understand the details of cellular activities. The requested state-of-the- art Leica EM PACT2 high pressure freezer, Leica EM AFS freeze substitution instrument and Leica Microbiopsy Transfer System will aid NIH-funded investigators at City of Hope in developing novel therapies for cancer and other life-threatening diseases.