Development of intracellular calcium measurement by time-resolved photon-counting fluorescence

Calcium green I, a ratiometric probe based on fluorescence lifetime measurements, was used to monitor intracellular calcium activity ([Ca2+]i) in RINm5F cells using a time-resolved fluorescence confocal microscope. The probe affinity constant has been recalibrated in single cells using ionomycin as a calcium ionophore and ethylenebis(oxyethylenenitrilo)tetraacetic acid as a calcium buffer; Kd was found to equal 150 nmol/L. The kinetics of ionomycin equilibration showed that the calcium release from calcium stores occurs before equilibration with extracellular calcium. The response to the muscarinic agonist carbachol, measured on 17 cells receiving three consecutive applications was characterized both by a [Ca2+]i peak lasting 50 s without any trailing plateau and by desensitization with a 30% decrease in the response. The dose-dependent response was obtained for a carbachol concentration from 5 mumol/L to 0.5 mmol/L. The ability of our set-up to obtain a value every 10 ms enabled us to record asynchronous spikes of [Ca2+]i in the RINm5F cells. The spikes, lasting less than 1 s, are significantly bigger than the noise, and they are not observed in the colonic HT29 cells.