Optical Microscopy Studies of [Ca2+]i Signaling

Optical microscopy provides one of the most powerful techniques for studying intracellular second messengers in living cells. In contrast to flow cytometry and spectrofluorometry, optical microscopy can be used to make measurements on individual cells, providing subcellular spatial information in addition to temporal and quantitative information. This chapter presents a practical guide to designing and setting up optical microscopy experiments for studying [Ca²⁺]i signaling during cell migration. It discusses available [Ca²⁺]i indicators, methodological approaches to single-cell measurements, imaging instrumentation, and quantification using digital image analysis. Microscopy can provide more detailed information about cellular signaling responses. Detection of fluorescence is dependent on the properties of several of the components of the instrumentation, such as the detector and illumination source.