Leica EM Pact High Pressure Freezing Instrument

描述（由申请人提供）：11名获得NIH资助的研究人员申请资金购买精密高压冷冻仪器，以制备用于电子显微镜检查的生物样品。高压冷冻虽然是三十多年前发明的，但最近已广泛用于快速冷冻样品，这些样品可能对使用固定剂或冷冻保护剂的常规化学处理敏感。这项技术的基本原理是直截了当的。在样品在-195摄氏度的液氮喷射中快速冷却之前，施加2100巴的高压，高压抑制冰的成核，冷却剂提供快速固定。结果是玻璃化的含水样品含有无定形冰，避免了大冰晶的形成，大冰晶会破坏细胞并重新分配可溶性成分。生物结构被很好地保存下来，以便后续使用冷冻替代、冷冻断裂刻蚀和冷冻超微切片等技术进行分析。许多技术将导致免疫金标记，因为高压冷冻能更好地保存抗原。两项进步使这项技术声名鹊起。一个新的紧凑的设计是易于使用和专门的样品持有人已经开发，以允许冷冻固定各种各样的样品类型。所要求的仪器的主要优点是，与其他冷冻固定技术相比，它可以很好地保存到约200 μ m的深度，比保存深度大10倍以上。利用该仪器的独特功能，研究一系列细胞和病理条件，包括白内障形成过程中的蛋白质聚集和膜完整性、claudin在调节上皮紧密连接通透性中的作用、原发性纤毛运动障碍中纤毛的分子特征、糖盏在气道上皮功能中的作用。完善病毒导向基因治疗囊性纤维化的方法，线粒体动力学在肝脏启动细胞凋亡中的作用，果蝇胚胎发生中的细胞收缩性和伤口愈合，收缩昆虫飞行肌的肌球蛋白过桥结构以及突触受体和蛋白质支架的分子组织。虽然这些项目中的每一个都是使用传统制备技术成功地进行和资助的，但每个项目都将受益于高压冷冻，以克服传统程序中所需的小样本量或化学处理的限制。没有其他固定方法可以快速阻止生物过程，保留抗原性并保存大型标本的内部结构。

DESCRIPTION (provided by applicant): Eleven investigators with NIH grant support request funds to purchase a sophisticated high pressure freezing instrument to prepare biological samples for electron microscopic examination. High pressure freezing, although invented over thirty years ago, has recently become widely used to rapidly cryofix samples that may be sensitive to conventional chemical treatments with fixatives or cryoprotectants. The basic principles of the technique are straightforward. High pressure of 2100 bar is applied just before samples are rapidly cooled in a jet spray of liquid nitrogen at -195 degrees C. The high pressure suppresses the nucleation of ice, and the coolant provides rapid fixation. The result is a vitrified aqueous sample that contains amorphous ice, avoiding the formation of large ice crystals that can damage cells and redistribute soluble components. The biological structure is well preserved for subsequent analysis using techniques such as freeze substitution, freeze-fracture-etch and cryo-ultramicrotomy. Many of the techniques will lead to immunogold labeling because high pressure freezing gives superior preservation of antigens. Two advances have propelled the technique into prominence. A new compact design is easy to use and specialized sample holders have been developed to permit cryofixation of a wide variety of sample types. The primary advantage of the instrument requested is that it gives excellent preservation to a depth of about 200 mu m more than 10X greater preservation depth compared to other cryofixation techniques. Projects are proposed that take advantage of the unique features of the requested instrument to investigate a range of cellular and pathological conditions including protein aggregation and membrane integrity during cataract formation, the role of claudins in regulating the permeability of epithelial tight junctions, the molecular features of cilia in primary ciliary dyskinesia, the role of the glycocalyx in airway epithelia function, approaches to perfect virus directed gene therapy to cure cystic fibrosis, the role of mitochondrial dynamics in initiating apoptosis in liver, cell contractility and wound healing in Drosophila embryogenesis, myosin crossbridge structure in contracting insect flight muscle and the molecular organization of receptors and protein scaffold at synapses. Although each of these projects has been successfully conducted and funded using conventional preparative techniques, each will benefit from the high pressure freezing to overcome limitations of small sample size or chemical treatment needed in traditional procedures. There is no other fixation method that can rapidly arrest biological processes, retain antigenicity and preserve internal structure in large specimens.