A High-Pressure Freezing and Cryoprocessing System for Northwestern University

描述（由申请人提供）：与传统的处理方法相比，冷冻固定后的冷冻取代在大多数细胞类型中大大改善了超微结构和抗原性的保存。目前，西北大学生物成像设施（BIF）的用户没有可用的高压冷冻（HPF）装置。要求拨款购买一台徕卡EM-PACT2高压冷冻机（HPF）和自动冷冻替代（AFS2）系统，这是最先进的冷冻处理设备。我们将使用HPF: 1)实现尽可能接近样品天然状态的超微结构保存，2)在高分辨率下降低迁移率的基因产物免疫定位，以及3)在EM水平上表征突变表型。EM-PACT2系统可以冷冻约600mm厚的样品，200mm深度的高质量冷冻。这种冷冻处理系统提供可靠的冷冻使用的标本持有人，可以容纳各种各样的标本类型。有了这个系统，我们可以研究许多以前无法进入的生物系统（和许多材料标本）。我们将在各种发育和细胞生物学研究中使用该系统，包括：1)雌激素对脑突触突触前囊泡的调节，2)颗粒细胞分泌抑制素A和B的途径的研究，3)病毒出芽部位病毒蛋白和必要的宿主细胞因子的组织，4)房水流出的流体动力学，5)BRCA2和中心核糖核酸（centrobin）在培养肿瘤细胞中调节中心体复制的作用，6)addl诱导的海马原代培养突触棘的变化。这些项目将显著受益于HPF-AFS的能力，它几乎可以立即停止细胞活动并限制细胞成分的重新定位，这是传统EM制备方法所不具备的功能。这些项目需要具有最大灵活性、易用性和结果一致性的冷冻处理系统。根据经理的经验和两个可用的冷冻处理装置的演示，我们确定徕卡EM-PACT2/AFS2系统最适合我们的需求。该设备将被安置在西北大学埃文斯顿校区的BIF。BIF是西北大学的研究人员、邻近的学术和医疗机构以及外部个人都可以共享使用的设施。本提案中列出的所有主要用户要么目前由NIH资助，要么正在等待提案。相关性：从这些研究中获得的结果将对癫痫、不孕症、癌症、病毒传播、青光眼和阿尔茨海默病等领域的公共卫生研究产生重大影响。

DESCRIPTION (provided by applicant): Cryoimmobilization followed by freeze substitution gives vastly improved ultrastructure and preservation of antigenicity in most cell types when compared to traditional processing methods. Presently there is no high-pressure freezer (HPF) unit available to users of the Biological Imaging Facility (BIF) at Northwestern University. Funds are requested to purchase a Leica EM-PACT2 High-Pressure Freezer (HPF) and Automated Freeze-Substitution (AFS2) system, the most up-to-date cryopreparation equipment. We will use the HPF to: 1) achieve ultrastructural preservation as close to the sample's native state as possible, 2) immunolocalize gene products with reduced mobility at high resolution, and 3) characterize mutant phenotypes at the EM level. The EM-PACT2 system can freeze samples up to about 600mm thick with a 200mm depth of high quality freezing. This cryoprocessing system provides reliable freezing using specimen holders that can accommodate a wide variety of specimen types. With this system we can investigate many biological systems (and many materials specimens) that were previously inaccessible. We will use this system in a variety of developmental and cell biological studies including: 1) estrogen regulation of presynaptic vesicles at brain synapses, 2) investigation of the pathways leading to inhibin A and B secretion from granulosa cells, 3) the organization of viral proteins and necessary host cell factors at the site of viral budding, 4) the hydrodynamics of aqueous humor outflow, 5) the role of BRCA2 and centrobin in the regulation of centrosome duplication in cultured tumor cells, and 6) ADDL-induced synaptic spine changes in primary hippocampal cultures. These projects will significantly benefit from the ability of HPF-AFS to stop cellular activity and restrict cell component relocation almost instantaneously, features that conventional EM preparation methods do not offer. These projects require a cryoprocessing system with the greatest flexibility, ease of use, and consistency of results. Based on the experience of the manager and on demonstrations of the two available cryoprocessing units, we have determined that the Leica EM-PACT2/AFS2 system best suits our needs. This equipment will be housed in BIF on the Northwestern University Evanston Campus. BIF is a shared-use facility available to Northwestern researchers, neighboring academic and health care institutions, and outside individuals. All of the major users listed in this proposal are either currently funded by NIH or have proposals pending. Relevance: Results obtained from these studies will have significant impact on public health research in the areas of epilepsy, infertility, cancer, viral transmission, glaucoma, and Alzheimer's disease.