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 Ca2+-sensitive fluorescent probes, have provided us with methodologies for unravelling the complexities of Ca2+ signaling at the cellular and subcellular level. Specifically, we will provide a detailed methodology for the study of Ca2+ signaling at the single-cell level using a Ca2+-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 fluorescentlytagged 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 (1, 2, 3).
KeywordsGreen Fluorescent Protein Fluorescence Resonance Energy Transfer Yellow Fluorescent Protein Ethylene Glycol Tetraacetic Acid Ethylene Glycol Tetraacetic Acid
The Wellcome Trust (Grant 061050), Biotechnology and Biological Sciences Research Council; Grants 91/C15897 and 01/A4/C/07909, and Glaxo-SmithKline are thanked for financial support.
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