ABSTRACT
Spatial information on ion distribution can be obtained by using an ion sensitive fluorescent dye, usually in conjunction with standard electrophysiology techniques. A dye fluoresces when it is illuminated with light which can excite the dye molecule to a higher energy state. This chapter discusses calcium sensitive fluorescent indicators since calcium is the most commonly studied ion, although dyes are available for a variety of ions including Na+, K+, Cl-, and H+. During a typical experiment a cell, in a brain slice or primary culture, is injected with an ion-sensitive fluorescent dye and visualized on a high power microscope. The illuminating light must be filtered to a narrow bandwidth centred on the excitation frequency of the dye. Calibration in vitro, is where a solution containing the dye and various amounts of calcium and Ethylene glycol-bis(aminoethyl ether)tetraacetic acid is measured in a fluorometer or spectrophotometer.
