Request for Leica High-pressure Freezer and Automated Freeze-substitution System

描述（由申请人提供）：为了更好地保存超微结构，我们建议购买一台全自动徕卡高压冷冻机和一台自动冷冻置换系统。对于厚而致密的标本，标准固定剂可能无法足够快地渗透以准确地保存精细的膜和蛋白质结构，也无法捕获许多生物系统的高度动态变化特征。初步数据直接表明需要高压冷冻，以避免某些组织对固定伪影的敏感性。通过自动冷冻样品，Leica系统还有助于与光学或荧光显微镜相关的成像。作为校园内最大的核心显微镜设施，我们训练有素的员工和既定的资源每年已经为250多名调查员用户提供服务。我们已经为多种电子显微镜提供了所有的制备设备和服务。该仪器补充了我们现有的电子显微镜服务，并为我们的其他共聚焦/多光子显微镜提供了相关光学显微镜的独特功能。由于在55英里的半径范围内没有等效的仪器，因此主要用户提出的许多对时间敏感的研究将被冷冻样品的极端精致所排除。随着我们庞大的EM用户群（>100名研究人员）了解到高压冷冻的优势，我们希望这种仪器在更多NIH资助的项目中发挥关键作用。

DESCRIPTION (provided by applicant): To better preserve ultrastructure, we propose to purchase a fully automated Leica high- pressure freezer and an automated freeze-substitution system. For thick, dense specimens, standard fixatives may not penetrate quickly enough to accurately preserve delicate membrane and proteinaceous structures, nor capture the highly dynamic changes characteristic of many biological systems. Preliminary data directly demonstrates the need for high-pressure freezing to avoid the sensitivity of certain tissues to fixation artifacts. By automating freezing of samples, the Leica system also facilitates correlative imaging with light or fluorescence microscopy. As the largest core microscope facility on campus, our well-trained staff and established resources already serves over 250 investigator users annually. We already provide all preparative equipment and services for multiple electron microscopes. This proposed instrumentation complements our existing electron microscopy services and provides unique capabilities for correlative light microscopy with our other confocal/multiphoton microscopes. Because no equivalent instrument is available for a 55-mile radius, many time-sensitive studies proposed by major users would have been precluded by the extreme delicacy of frozen samples. As our large group of EM users (>100 investigators) learn of the advantages of high-pressure freezing, we expect this instrumentation to play a critical role for many more NIH-funded projects.