(A) Structure of CaRuby-Nano. Note the oxygen substituent and the positioning of the fluorophore-BAPTA bond. (B) [Ca2+]-dependent change in CaRuby-Nano fluorescence ([Ca2+]free: 0 nM, 17 nM, 38 nM, 65 nM, 100 nM, 150 nM, 225 nM, 351 nM, 602 nM, 1.35 µM, 39 µM). (C) The titration curve corresponding to the spectra in (B) using the same color code. (D–F): Climbing fiber evoked dendritic calcium signals in Purkinje cells in vitro. (D) Purkinje cell filled with 300 µM CaRuby-Nano dextran, with region of interest indicated by the white rectangle (scale bar = 20 µm). (E) Region of interest with points of interest indicated. Note that many spines can be readily distinguished (white arrow). Points 1–3 and 4–6 are on different spiny branchlets while points 7 and 8 are background (scale bar = 5 µm). (F) Ca2+ transients following climbing fiber activation recorded at 2.8 kHz (traces averaged over 26 trials and then averaged over the indicated spine numbers).