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. 1996 Jun;6(2):83-95.
doi: 10.1007/BF00732047.

Fluorescence lifetime characterization of magnesium probes: Improvement of Mg(2+) dynamic range and sensitivity using phase-modulation fluorometry

H Szmacinski  1 , J R Lakowicz
Affiliations

Fluorescence lifetime characterization of magnesium probes: Improvement of Mg(2+) dynamic range and sensitivity using phase-modulation fluorometry

H Szmacinski et al. J Fluoresc. 1996 Jun.

Abstract

We measured the Mg(2+)-dependent absorption spectra, emission spectra, quantum yields, and intensity decays of most presently available fluorescent magnesium probes. The lifetimes were found to be strongly Mg(2+) dependent for Mag-quin-1, Mag-quin-2, magnesium green, and magnesium orange and increased 2- to 10-fold upon binding of Mg(2+). The lifetimes of Mag-fura-2, Mag-fura-5, Mag-fura red, and Mag-indo-1 were similar in the presence and absence of Mg(2+). 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. Mg(2+) 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 Mg(2+)-sensitive range than available from intensity measurements. A significant expansion in the Mg(2+)-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 Ca(2+) was also measured for Mag-quin-2 and magnesium green. Significant phototransformation and/or photode-composition, which affect the sensitivity to Mg(2+), were observed for Mag-quin-2 and magnesium green under intense and long illumination.

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Figures

Fig. 1.
Fig. 1.
Chemical structures of fluorescent magnesium probes.
Fig. 2.
Fig. 2.
Absorption (top) and emission spectra (bottom) of mag-quin-2 in calibrated magnesium buffers at 20°C.
Fig. 3.
Fig. 3.
Absorption and emission spectra of magnesium green.
Fig. 4.
Fig. 4.
Frequency domain of intensity decays of mag-quin-2 with various Mg2+ concentrations.
Fig. 5.
Fig. 5.
Frequency domain of intensity decays of magnesium green with various Mg2+ concentrations.
Fig. 6.
Fig. 6.
Mg2+-dependent preexponential factors for mag-quin-2 (top) and MgG (bottom).
Fig. 7.
Fig. 7.
Mg2+-dependent phase angles and modulations for mag-quin-2 and for magnesium green.
Fig. 8.
Fig. 8.
Excitation wavelength- and Mg2+-dependent phase angles for mag-quin-2.
Fig. 9.
Fig. 9.
Effect of intense illumination on intensity decays of free and Mg2+-bound forms of mag-quin-2. Data are shown for before (○) and after (●) intense illumination.
Fig. 10.
Fig. 10.
Ca2+-dependent phase angles and modulations for mag-quin-2 (○) and for magnesium green (●). Arrows indicate apparent dissociation constants (midpoints). Experimental conditions are the same as indicated in the legend to Fig. 7.
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