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The great demand for long-wavelength and high signal-to-noise Ca2+ indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments.