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

Abstract

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.

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Correspondence to Joseph R. Lakowicz.

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Szmacinski, H., Lakowicz, J.R. Fluorescence lifetime characterization of magnesium probes: Improvement of Mg2+ dynamic range and sensitivity using phase-modulation fluorometry. J Fluoresc 6, 83–95 (1996). https://doi.org/10.1007/BF00732047

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Key words

  • Magnesium fluorescence probes
  • sensing
  • imaging
  • time-resolved fluorescence
  • frequencydomain fluorescence
  • phase-modulation fluorometry