One of the first of the recently-introduced Nikon RCM 8000 scanning confocal microscropes is housed at the Kennedy Center of Albert Einstein College of Medicine, where all of the P.I.s on this Program Project have their laboratories. This instruction moves beyond conventional confocal microscopy, noted for its spatial resolution capacity, to real time full field ratio metric measurements captured at a rate of 30 frames per second, or even faster for smaller parts of the field, while maintaining true confocality. Hence processes such as intracellular and intercellular Ca2+ waves can be monitored temporally and spatially in living cells as they occur, by the application of the current generation of calcium indicators (e.g. Indol and Fluo3). Analysis of Ca2+ waves will be undertaken in astrocytes (Project 2), serial reconstructions of Lucifer yellow dye fills in Schwann cells from injured and normal nerves of wildtype and Cx32 KO animals (Project l) will also take advantage of the rapid scanning capability of the instrument. In addition, the microscope will be used for immunocytological detection of connexin distribution in cells, or in double label experiments, in all of the projects. The excellent spatial resolution of this microscope will be of value not only for generating reconstructed images of gap junction distribution in this projects, but will enable us to determine subcellular distribution of mutant connexins that may be defective in transport to the plasma membrane (Project 3).
