Figure 3. Quantification of dendritic calcium transients. Purkinje cells were loaded with different indicator pairs, fura-2/fluo-5N (A), fura-2FF/fluo-5N (B), and mag-fura-5/fluo-5N (C). Fluorescence transients produced by a series of voltage steps were then measured simultaneously for each pair of indicators. Fluorescence ratios are plotted for fura-2, fura-2FF, or mag-fura-5 in a, and Rmin and Rmax are indicated in with dotted line. Fluorescence ratios for fluo-5N are plotted in b. Ratios determined with indicator pairs are compared in c, where ratios were normalized so that Rmin = 0 and Rmax = 1. Ind and e, calciumvalues were then calculated from the traces in a and b, respectively, using the using calibration parameters described in Materials and Methods. In f the values of calcium determined for each of a pair of indicators are plotted for the purpose of comparison. A line with a slope of 1 is indicated with dotted lines, and regressions of the data points are indicated with solid lines with slopes of 1.06 for Af, 1.02 for Bf, and 1.04 for Cf. In A and B, voltage steps (50, 100, 250, 500, 1000 msec) were delivered, and in C, the 50 msec voltage step was omitted. In Aa, Ab, Ad, and Ae, gray traces correspond to 50 and 100 msec depolarizations. In Ae and Af, calcium concentrations calculated from fura-2 fluorescence ratios that exceeded 2 μm were not displayed. Points corresponding to calculated values of Capost >77 μm (10 times the KD of fura-2FF) or 380 μm (10 times the KD of mag-fura-5) were not displayed in Bf and Cf, respectively.