Leica EM Pact High Pressure Freezing Instrument

描述（由申请人提供）：11名获得NIH赠款支持的研究人员申请资金购买一台精密的高压冷冻仪器，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固定可能对固定剂或冷冻保护剂的常规化学处理敏感的样品。该技术的基本原理很简单。在-195 ° C的液氮喷射中快速冷却样品之前，施加2100巴的高压。高压抑制了冰的成核，冷却剂提供了快速的固定。其结果是玻璃化的含水样品含有无定形冰，避免形成大冰晶，可以破坏细胞和重新分配可溶性成分。生物结构保存完好，可用于随后的分析，如冷冻置换，冷冻断裂蚀刻和冷冻超微切片术。许多技术将导致免疫金标记，因为高压冷冻给予抗原上级保存。有两项进展使这项技术变得突出。一种新的紧凑型设计易于使用，专门的样品架已经开发出来，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，它提供了约200 μ m深度的极好保存，超过10倍的保存深度。提出了利用所要求的仪器的独特功能来研究一系列细胞和病理条件的项目，包括白内障形成过程中的蛋白质聚集和膜完整性，claudins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，糖萼在气道上皮功能中的作用，介绍了病毒介导的基因治疗囊性纤维化的方法、线粒体动力学在肝细胞凋亡中的作用、果蝇胚胎发育中的细胞收缩和伤口愈合、昆虫飞行肌收缩中的肌球蛋白桥结构以及突触受体和蛋白支架的分子结构。虽然这些项目中的每一个都是使用传统的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，保留抗原性并保护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.