Intracellular ion imaging using fluorescent dyes : artefacts and limits to resolution

Development of highly efficient fluorescent ratio indicators has made imaging of ion concentrations within individual cells possible (Grynkiewicz et al. 1985; Tsien and Poenie 1986). Ion imaging is a complex technique and is therefore prone to artefacts. In this paper we investigate the limits of the technique and its potential pitfalls. The spatial resolution of an imaging system is determined for different cell geometries. We describe a technique to increase the time resolution of existing systems by using a single excitation wavelength to measure changes in ion concentration. We demonstrate examples of potential artefacts arising from hardware limitations, image processing and fundamental optics. Methods for recognition and minimization of these problems are discussed.