Confocal microscopy: Theory and applications for cellular signaling

Abstract

The development of confocal microscopy and the commercial availability of confocal microscopes have provided many laboratories with an extremely powerful approach to examine cellular structure and function. Allied with the development of suitable tools, it is now possible to interrogate a wide range of structural and functional aspects on bothfixed and live cells. Here we describe the basic principles underlying confocal microscopy and provide methodological accounts of how it can be used to study aspects related particularly (but not exclusively) to the expression, activation, and regulation of signaling by G-proteincoupled receptors. Specifically we provide detailed protocols for examining: the cellular expression and distribution of proteins by immunocytochemistry; cytoplasmic and organelle Ca2+ signaling using fluorescent indicators; second messenger generation using fluorescently tagged biosensors; and ligand/ receptor internalization using fluorescently tagged peptide agonists and receptors. © Springer Science+Business Media, LLC 2013.