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

Calcium green I, a ratiometric probe based on Fluorescence lifetime measure ments, was used to monitor intracellular calcium activity ([Ca2+](i)) in RI Nm5F cells using a time-resolved fluorescence confocal microscope. The prob e affinity constant has been recalibrated in single cells using ionomycin a s a calcium ionophore and ethylenebis(oxyethylenenitrilo)tetraacetic acid a s a calcium buffer; K-d was found to equal 150 nmol/L. The kinetics of iono mycin equilibration showed that the calcium release from calcium stores occ urs before equilibration with extracellular calcium. The response to the mu scarinic agonist carbachol, measured on 17 cells receiving three consecutiv e applications was characterized both by a [Ca2+](i) peak lasting 50 s with out any trailing plateau and by desensitization with a 30% decrease in the response. The dose-dependent response was obtained for a carbachol concentr ation from 5 mu mol/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+] 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.