We describe a method for determining intracellular free calcium concentration ([Ca2+]) from single-wavelength fluorescence signals. In contrast to previous single-wavelength calibration methods, the proposed method does not require independent estimates of resting [Ca2+] but relies on the measurement of fluorescence close to indicator saturation during an experiment. Consequently, it is well suited to [Ca2+] indicators for which saturation can be achieved under physiological conditions. In addition, the method requires that the indicators have large dynamic ranges. Popular indicators such as Calcium Green-1 or Fluo-3 fulfill these conditions. As a test of the method, we measured [Ca2+] in CA1 pyramidal neurons in rat hippocampal slices using Oregon Green BAPTA-1 and 2-photon laser scanning microscopy (BAPTA: 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). Resting [Ca2+] was 32–59 nM in the proximal apical dendrite. Monitoring action potential-evoked [Ca2+] transients as a function of indicator loading yielded estimates of endogenous buffering capacity (44–80) and peak [Ca2+] changes at zero added buffer (178–312 nM). In young animals (postnatal days 14–17) our results were comparable to previous estimates obtained by ratiometric methods (Helmchen et al., 1996, Biophys. J. 70:1069–1081), and no significant differences were seen in older animals (P24–28). We expect our method to be widely applicable to measurements of [Ca2+] and [Ca2+]-dependent processes in small neuronal compartments, particularly in the many situations that do not permit wavelength ratio imaging.
