Effects of photolabile calcium chelators on fluorescent calcium indicators

The fluorescence properties of the calcium indicators Fura-2 and Fluo-3 have been investigated in the presence of the 'caged calcium' photolabile chelators Nitr-5 and DM-nitrophen. The excitation spectra of dilute solutions of these indicators was distorted by the presence of photolabile chelators, owing to differential absorbance of excitation light by the chelators, as well as calcium-dependent fluorescence of the chelators themselves. This distortion was altered on partial photolysis of the chelators, due to changes in their absorbance and fluorescence. At high concentrations of indicators (100 microM) and photolabile chelators (10 mM), similar to those used experimentally, DM-nitrophen quenched the fluorescence of Fluo-3 at low calcium concentrations. The results suggest that Fura-2 may be used with either chelator, and Fluo-3 with Nitr-5, to measure calcium released on photolysis of the caged compounds, but that careful calibration of the chelator-indicator mixture after the appropriate degree of photolysis is necessary.