SUBCELLULAR FREE CALCIUM DETERMINATION, AS PROBED BY CONFOCAL MICROSPECTROFLUOROMETRY

Indo-1 emission spectra has been analysed from a confocal microvolume to determine intracellular concentrations of free calcium ([Ca++](i)) Intracellular reference spectra have been recorded in free and in satu rated Ca++. For Ca++ saturated cells, the intranuclear spectra are mor e blue shifted than in the cytoplasm. From Ca++ permeabilized cells, i n situ calibration curves allow to determine the product of both intra cellular constants i.e. : the ratio of quantum yields between free and Ca-bound Indo-1 and the apparent dissociation constant for Ca++. The product of these constants has been shown to be similar in both subcel lular compartments and in a buffered solution. [Ca++](i) determination s on Ca++ unpermeabilized KB cells show a 1.3 times higher [Ca++](i) i n the cytoplasm than in the nucleus (p < 0.001).