QUANTITATIVE INTRACELLULAR CALCIUM IMAGING WITH LASER-SCANNING CONFOCAL MICROSCOPY

Laser-scanning confocal microscopy has been used to visualise the fluorescence of a visible wavelength Ca2+-sensitive fluorophore, Fluo-3 in isolated cardiac myocytes. A protocol for the derivation of quantitative information from this single wavelength indicator is presented. This paradigm involves co-loading cells with two Ca2+-sensitive fluorescent indicators, Fluo-3 and Fura-2. Wide-field ratiometric measurements of Fura-2 fluorescence provided a baseline [Ca2+] upon which changes in Fluo-3 fluorescence could be directly expressed as [Ca2+] changes. The Ca2+ changes occurring in spontaneously active cardiac cells are presented as an example of the method. Although fluorescence energy transfer between Fura-2 and Fluo-3 was detectable in some in vitro mixtures of the two fluorophores, this process was not evident in co-loaded cardiac cells under the loading conditions employed. © 1990.