Ca2+ imaging in single living cells: theoretical and practical issues

The measurement of intracellular calcium ion concentrations [( Ca2+]i) in single living cells using quantitative fluorescence microscopy draws from a diverse set of disciplines, including cellular biology, optical physics, statistics and computer science. Over the last few years, we have devised and built a number of systems for measuring [Ca2+]i with Fura-2, and have applied them in the exploration of a wide range of biological processes controlled by Ca2+. In this report we discuss these systems and their advantages and limitations. We also describe the theoretical and practical problems associated with using Fura-2 to measure [Ca2+]i, and the solutions that we, and others, have developed to overcome them. The approaches described should provide useful guidance for others interested in imaging [Ca2+] distribution in living cells. The factors that limit current methods are discussed, and areas for future development are highlighted.