Detection of Ca2+‐transients elicited by flash photolysis of DM‐nitrophen with a fast calcium indicator

Abstract

A confocal spot detection optical setup was used to record fluorescence signals in response to calcium pulses, elicited by flash photolysis of DM‐nitrophen, with the calcium indicators CaOrange‐5N and Fluo‐3. Our results yield the following conclusions: [Ca²⁺] changes are almost perfect spikes at pCa 9 and broader transients followed by a step at pCa 7. The [Ca²⁺] spikes were used to measure the dissociation rate constant of the Ca²⁺ dyes. Experiments at pCa 7 were used to verify the kinetic rate constants of the dyes and to obtain those of DM‐nitrophen. The association rate constant of this compound was found to be more than one order of magnitude faster than that suggested previously. CaOrange‐5N was able to track changes in [Ca²⁺] more accurately than Fluo‐3. This latter dye introduced severe distortions which preclude a quantitative deconvolution of the fluorescence transients into changes in the free [Ca²⁺].