We present a new method for ratiometric Ca2+ measurements using indicators with excitation spectra in the visible range of wavelengths. Laser-scanning confocal microscopy was used to record intracellular Ca2+-signals with high temporal and spatial resolution in single cardiac myocytes. The patch-clamp technique was applied to load the cells with the fluorescent Ca2+-indicators and to follow the membrane currents with the fluorescence signals simultaneously. Intracellular free Ca2+-concentration ([Ca2+]i) was estimated with a ratiometric method. An in vitro calibration procedure was used to convert the fluorescence ratio obtained with two different Ca2+-indicators (Fluo-3 and Fura-Red) into Ca2+-concentrations. Fluo-3 showed an increase in fluorescence upon a rise in intracellular Ca2+-concentration, while the Fura-Red fluorescence decreased. Since the fluorescence of Fluo-3 was around 2-fold brighter than the Fura-Red signal the cells were loaded with a 1:2 mixture of the two indicators. The large increase of the fluorescence ratio during a rise in [Ca2+]i (up to 4-fold) allowed us to record time-resolved signals with this mixture even when monitored in a very small subcellular volume (around 1 [mu]m3). Long lasting continuous recordings of the fluorescence were possible because the dye-mixture exhibited no detectable bleaching with illumination periods of up to 30 s. The use of the Fluo-3/Fura-Red ratio method should significantly facilitate and improve quantitative measurements of [Ca2+]i with high temporal and spatial resolution. Moreover, this approach is especially valuable when used with confocal microscopes which are usually equipped with lasers in the visible light range. Furthermore, it may be possible to use the same approach with mixtures of other indicators to estimate the concentration of other biologically important ions/compounds with a ratiometric calibration.
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