High Pressure Freezer

描述（由申请人提供）：本提案要求资金购买Leica EM PACT 2-RTS高压冷冻机，用于生物样本的快速冷冻固定。高压冷冻机将是一个共享资源，位于斯坦福大学一个完善的、多用户的光学和电子显微镜设施：细胞科学成像设施。整个斯坦福大学校园以及周围的机构，如加州大学圣克鲁斯和加州大学旧金山弗朗西斯科都可以使用该设施。 高压冷冻已成为生物电镜样品固定的金标准。在许多研究中，它现在已经被应用，这种方法已经扩展了我们对细胞和组织的结构和分子组织的理解。高压冷冻提供了更好的固定，无论是在质量和数量方面，在所有其他固定技术，并与快速转移系统结合使用时，提供了相关的光电子显微镜研究增加的时间分辨率。对于许多研究和实验系统来说，高压冷冻固定不仅是可取的，而且对于适当保存细胞精细结构和抗原性是必要和必要的。 高压冷冻机将支持NIH资助的八个主要用户的项目。这些项目研究了NIH支持的广泛主题，包括1）线虫触觉受体神经元中的机械-电转导和微管细胞骨架组织（Goodman）; 2）微管细胞骨架组织和调节（Stearns）; 3）T和B淋巴细胞识别和免疫突触形成的分子基础（Davis）; 4，5）病毒复制、组装和从感染细胞中排出的分子机制（阿尔文，Kirkegaard）; 6）斑马鱼、啮齿动物和人类神经回路的发育和功能（Smith）; 7）酵母细胞极性的发育（Pringle）; 8）细菌染色体和复制体的3D组织（Shapiro，McAdams）。 这些研究调查了各种模式生物和人体组织中的关键结构问题，并涵盖了对人类健康和疾病的各个方面产生影响的研究领域，从癌症和病毒发病机制到了解免疫反应和感觉生物学的分子基础。所有这些都需要接近体内状态的样本保存水平，以进行亚细胞结构和分子复合物的超微结构研究。这些项目中的每一个都需要接近天然水合状态的固定水平，这只有通过高压冷冻才能实现。

DESCRIPTION (provided by applicant): This proposal requests funds to purchase a Leica EM PACT2-RTS high pressure freezer for rapid, cryo- fixation of biological samples. The high pressure freezer will be a shared resource, located in a well- established, multi-user, light and electron microscopy facility at Stanford University: the Cell Sciences Imaging Facility. This facility is accessible to the entire Stanford campus as well as surrounding institutions such as UC Santa Cruz and UC San Francisco. High pressure freezing has become the gold standard for fixation of biological electron microscopy samples. In the numerous studies were it has now been applied, this method has extended our understanding of the structural and molecular organization of cells and tissues. High pressure freezing provides much improved fixation, both in terms of quality and quantity, over all other fixation techniques and, when used in conjunction with a rapid transfer system, provides increased temporal resolution for correlative light to electron microscopy studies. For many studies and experimental systems high pressure cryo-fixation is not just desirable but necessary and essential for proper preservation of cellular fine structure and antigenicity. The high pressure freezer will support NIH funded projects from eight major users. These projects investigate a wide range of NIH supported topics, including, 1) mechano-electrical transduction and microtubule cytoskeleton organization in nematode touch receptor neurons (Goodman); 2) microtubule cytoskeleton organization and regulation (Stearns); 3) molecular basis of T and B lymphocyte recognition and immuno-synapse formation (Davis); 4,5) molecular mechanisms of viral replication, assembly and egress from infected cells (Arvin, Kirkegaard); 6) development and function of the neural circuitry in Zebra fish, rodents and humans (Smith); 7) development of cell polarity in yeast (Pringle); 8) 3D organization of the bacterial chromosome and replisome (Shapiro, McAdams). These studies investigate critical structural questions in a variety of model organisms and human tissues and cover areas of research with implications for diverse aspects of human health and disease, ranging from cancer and viral pathogenesis to understanding the molecular basis of immuno-responses and sensory biology. All require levels of sample preservation that approach the in vivo state for ultrastructural study of subcellular structures and molecular complexes. Each and every one of these projects demands a level of fixation that approaches the native, hydrated state which is possible only by application of high pressure freezing.