Schematic representation of the three major GECI classes. GECIs are based either on florescence intensity changes of split or circularly permuted single FPs (A) or changes in FRET efficiency between two FPs (B,C). (A) Schematic of the G-CaMP-type sensing mechanism. On calcium binding, conformational changes in the calmodulin–M13 complex induce fluorescence changes in the circularly permuted enhanced GFP (cpEGFP). (B) The cameleon family of FRET-based GECIs. A calcium-dependent increase in FRET between a CFP and YFP FRET pair is coupled to the binding of calmodulin to the M13 peptide. (C) Troponin C–based FRET GECIs. Binding of calcium to troponin C induces conformational changes and an increase in FRET between CFP and YFP. (D) Practical considerations for choosing the most appropriate GECI.