Clover-mRuby2 improves responses versus ECFP-Venus in the CaMKIIα reporter Camuiα. (a) Organization of Camuiα-CR, based on Camuiα. (b) Intensity-modulated ratiometric images of HeLa cells expressing Camuiα-CR or Camuiα before and after addition of 1 μM ionomycin at time = 0. Scale bar, 20 μm. (c) Mean donor/acceptor emission ratio changes over time. Error bars represent standard error of the mean (s.e.m.). n = 10 cells for Camuiα, n = 12 cells for Camuiα-CR. Difference in peak emission ratio change is statistically significant (P = 7.6e–5). (d,e) Similar emission ratio responses were obtained with modified Camuiα-CR reporters where Clover was replaced with circularly permuted variants. Mean peak emission ratio changes with cpClover157 (n = 4 cells) and cpClover173 (n = 10 cells) were 31.8 ± 3.5% and 32.0 ± 2.2% (mean ± s.e.m.) respectively, not significantly different from Camuiα-CR (P = 0.52 and P = 0.3 respectively).