Cryoimmobilization of cells and tissues offers the best method for the structural preservation of cell adhesion molecules (CAM) on the extracellular surfaces of microorganisms and cells. Funds are requested to purchase a cryosystem composed of a) Balzers High Pressure Freezer Machine 010, and b) Balzers Freeze Fracture Unit (BAF 060). This cryosystem will be used to immobilize large cells or aggregates of cells by high pressure freezing at 2100 atmosphere of liquid nitrogen and then inserting them by cryotransfer into the Balzers FFU 060 where the extracellular surfaces of the cells will be exposed by deep etching to reveal CAM. The CAMP will be replicated by depositing a thin metal film using the BAF 060 to make either unidirectional shadowing of dual angle rotary shadowing, and then stabilizing the metal film with a 5 nm layer of carbon. The metal film (topographical surface of cell) will be imaged by high resolution field emission SEM using an Autrata modified back-scatter electron detector to image the film, possibly colloidal gold labels as well, be sensitive atomic number contrast. This equipment will be housed in the Department of Cell Biology and Neuroanatomy, a facility available to the entire University of Minnesota campus. Major users will have guaranteed access to at least 75% of equipment time and rest will be made available to other users based on policies set by an Internal Advisory Committee. This cryosystem will permit us to continue investigations on the detection of individual CAM on bacteria and mammalian cells. Major users will apply this technology together with cryoSEM or TEM to the study of human leukocytes, platelets, attachment of proteins to the surface of maturing sperm, bacterial interaction related to conjugation and attachment to intestinal epithelial cells, and to hydrated tissues including cartilage and collagen mats.
