Spectral properties of rhodamine B and Co²⁺ make them ideally suited as a donor and acceptor, respectively, for tmFRET. (A) The absorption spectra of Co²⁺ (gray solid) and Co²⁺-NTA (black) are shown superimposed on the excitation (gray dashed) and emission (red) spectra of rhodamine B. (B) Emission spectra of rhodamine B fluorescence in solution at the indicated concentrations of Co²⁺-NTA. (C) Concentration dependence of quenching of rhodamine B in solution by Co²⁺-NTA (points) fitted with the Fung and Stryer (1978) model (red). The error bars shown represent the SEM of four independent experiments. (D) Fluorescence lifetime measurements of rhodamine B in the absence (red circles) and presence (blue circles) of 60 mM Co²⁺-NTA. Best fits to the data with a single exponential give time constants of 1.55 ns and 1.18 ns without and with Co²⁺-NTA, respectively. (E) Energy transfer efficiencies estimated with steady-state and lifetime fluorescence measurements. The mean for each group is shown by the black line. The label n.s. indicates there was no statistically significant difference between the groups (P > 0.05).