HIGH-SPEED CONFOCAL MICROSCOPE FOR A SHARED FACILITY

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Cells control many of their functions through localized (micron to sub- micron scale) concentration changes of intracellular messengers. These changes often occur rapidly (millisecond to sub-millisecond scale). For example, contraction of muscle cells and section from neurons are regulated by transient (1-100 ms) increases in intracellular calcium. The introduction of calcium-sensitive fluorescent indicators like fluo-3 discovered in part by J. Kao (Project I) while working in the laboratory of R.Y. Tsien, opened the way for microscopical studies of calcium transients in living cells. Coupled with the improved spatial resolution of confocal microscopy, calcium imaging studies made it possible to observe the release of calcium from single channels or single "release units" in intracellular stores. These 'calcium sparks' were first discovered by Lederer (Project II) in cardiac (Cheng et al., 1993) and smooth muscle (Nelson et al., 1995) cells, and were subsequently discovered in skeletal muscle cells in collaboration with other campus researchers (Klein et al., 1996). These and related studies have contributed to the University of Maryland's stature as a major research center for studies of intracellular calcium signaling and regulation, which is the primary research focus of more than 20 independent campus laboratories. Further advances in this area require improvements in temporal resolution so that the kinetics of spark formation and propagation, and their relationship to the calcium transients they evoke, can be characterized. The equipment requested by this application is intended to create a multi- user core facility that meets this requirement. A high-speed confocal microscope and dedicated voltage clamp apparatus will enable researchers at the University of Maryland and other area institutions to initial and examine calcium sparks with high spatial and temporal resolution. An integrated photolysis laser and temperature-controlled perfusion system will enable them to manipulate the intracellular and extracellular environment of living cells to allow correlation betweens parks and various stimuli. In addition to enabling new approaches to the study of calcium sparks and other dynamic phenomena in living cells, this core facility will have a significant impact on the teaching mission of the University of Maryland.