Confocal microscopy: theory and applications for cellular signaling.

The main aim of this chapter is to introduce some of the basic principles behind the technique of confocal microscopy. Subsequently, we will describe how recent advances in this technology, allied with the continued development of Ca(2+)-sensitive fluorescent probes, have provided us with methodologies for unravelling the complexities of Ca(2+) signaling at the cellular and subcellular level. Specifically, we will provide a detailed methodology for the study of Ca(2+) signaling at the single-cell level using a Ca(2+)-sensitive fluorescent indicator in conjunction with confocal microscopy. This chapter also describes a number of confocal-based methodologies that can be used to study other aspects of intracellular signaling, such as immunofluorescent labeling, the use of fluorescently tagged biosensors for measuring phospholipase C (PLC) activity, and the use of fluorescently-labeled ligands for measuring receptor or ligand internalization. It should be noted that several excellent texts are available that cover the principle and practice of confocal microscopy in relation to biological systems in far greater depth than is possible here.