Fluorescence lifetime characterization of magnesium probes: Improvement of Mg2+ dynamic range and sensitivity using phase-modulation fluorometry

We measured the Mg²⁺-dependent absorption spectra, emission spectra, quantum yields, and intensity decays of most presently available fluorescent magnesium probes. The lifetimes were found to be strongly Mg²⁺ dependent for Mag-quin-1, Mag-quin-2, magnesium green, and magnesium orange and increased 2- to 10-fold upon binding of Mg²⁺. The lifetimes of Mag-fura-2, Mag-fura-5, Mag-fura red, and Mag-indo-1 were similar in the presence and absence of Mg²⁺. Detailed timeresolved measurements were carried out for Mag-quin-2 and magnesium green using phase-modulation fluorometry. Apparent dissociation constants (K_d) were determined from the steady-state and time-resolved data. Their values were compared and discussed. Mg²⁺ sensing is described using phase and modulation data measured at a single modulation frequency. Phase angle and modulation data showed the possibility of obtaining a wider Mg²⁺-sensitive range than available from intensity measurements. A significant expansion in the Mg²⁺-sensitive range was found for Mag-quin-2 using excitation wavelengths from 343 to 375 nm, where the apparentK_d from the phase angle was found to vary from 0.3 to about 100 mM. Discrimination against Ca²⁺ was also measured for Mag-quin-2 and magnesium green. Significant phototransformation and/or photode-composition, which affect the sensitivity to Mg²⁺, were observed for Mag-quin-2 and magnesium green under intense and long illumination.