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. ? ?
