Fig. 1.

Design of the Ca²⁺ and Zn²⁺ sensors used in this protocol. (A) A genetically encoded, fluorescent, ratiometric sensor contains a Ca²⁺ or Zn²⁺ binding domain fused to a donor FP (usually cyan FP) at is N-terminal end and an acceptor FP (usually yellow FP) at its C-terminal end. When Ca²⁺ or Zn²⁺ reversibly binds to its binding domain, the sensor changes conformation, which leads to a change in FRET efficiency. (B) shows how the change in FRET efficiency changes the FRET ratio, which is the ratio of acceptor FP to donor FP emission intensity upon donor FP excitation. Thus, the FRET ratio of the unbound sensor (R_free) is distinct from that of the bound sensor (R_bound).