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.
