Effects of temperature on calcium-sensitive fluorescent probes

The effect of temperature on the binding equilibria of calcium-sensing dyes has been extensively studied, but there are also important temperature-related changes in the photophysics of the dyes that have been largely ignored. We conducted a systematic study of thermal effects on five calcium-sensing dyes under calcium-saturated and calcium-free conditions. Quin-2, chlortetracycline, calcium green dextran, Indo-1, and Fura-2 all show temperature-dependent effects on fluorescence in all or part of the range tested (5-40 degrees C). Specifically, the intensity of the single-wavelength dyes increased at low temperature. The ratiometric dyes, because of variable effects at the two wavelengths, showed, in general, a reduction in the fluorescence ratio as temperature decreased. Changes in viscosity, pH, oxygen quenching, or fluorescence maxima could not fully explain the effects of temperature on fluorescence. The excited-state lifetimes of the dyes were determined, in both the presence and absence of calcium, using multifrequency phase-modulation fluorimetry. In most cases, low temperature led to prolonged fluorescence lifetimes. The increase in lifetimes at reduced temperature is probably largely responsible for the effects of temperature on the physical properties of the calcium-sensing dyes. Clearly, these temperature effects can influence reported calcium concentrations and must therefore be taken into consideration during any investigation involving variable temperatures.