Development of instrumentation to measure rapid calcium transits in neonatal rat ventricular myocytes

Changes in the intracellular calcium levels in cardiac myocytes are important in the regulation of normal cardiac function and have been implicated in contributing to irreversible cell injury with ischemia or hypoxia. Intracellular measurement of total calcium changes with subcellular resolution have become routine using rapid cryofixation, cryosectioning, cryotransfer and energy dispersive x-ray microanalysis in analytical electron microscopes. The x-ray microanalysis technique measures total calcium changes within subcellular compartments, but does not distinguish between the bound and free calcium. With the successful development of fluorescent calcium indicators which may be introduced into cells without significantly buffering the intracellular calcium levels, it has become possible to measure rapid calcium transients during contraction. The primary requirements in the development of a system to utilize the fluorescent calcium indicators were to resolve calcium transients in individual cells (since the response to perturbations such as hypoxia is heterogeneous) and develop a system which would be flexible enough to accommodate new indicators as they become available.