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Aequorea victoria GFP has been posted (Mishin et al., 2008) we designed the corresponding mutations to GCaMP3, but the ensuing protein displayed no fluorescence (info not shown). The dotted line in the 2-photon graph suggests ΔF/F, in the pH graph it implies the variance in fluorescence (%). However, the crimson variant of GCaMP3 did not screen any fluorescence and this variant was not pursued further. Subsequently, we circularly permuted the red FP mCherry (Shaner et al., 2004) and replaced cpGFP in GCaMP3 with cp-mCherry this protein was not fluorescent either (knowledge not demonstrated), in agreement with the absence of chromophore development in cp-mCherry explained beforehand (Carlson et al., 2010). We hence chosen an additional protein, mRuby (Kredel et al., 2009), as a template for a pink calcium indicator. To generate a purple GECI, we picked the purple fluorescent protein (FP) mRuby (an engineered variant of eqFP611 exhibiting large thermodynamic steadiness and monomericity) (Kredel et al., 2009) and changed cpEGFP with circularly permuted mRuby in the GCaMP3 scaffold. We at first mutated GCaMP3 (Tian et al., 2009) to integrate sets of the core mutations of EBFP, ECFP, and EYFP: in GCaMP3 numbering Phe221Leu/Thr222Ser/Tyr223His ("BCaMP1a") Tyr223Trp ("CyCaMP1a") and Val116Tyr/Lys119Val (GCaMP3 incorporates the Thr116Val mutation "YCaMP1a")