QUANTITATIVE-DETERMINATION OF FREE CALCIUM IN SUBCELLULAR COMPARTMENTS, AS PROBED BY INDO-1 AND CONFOCAL MICROSPECTROFLUOROMETRY

Confocal UV-microspectrofluorometry has been applied to measure fluorescence emission spectra of Indo-1 for the intracellular determination of free calcium ([Ca2+](i)). To perform in situ calibrations of [Ca2+](i) in the nucleus and the cytoplasm, Indo-1 has been loaded into living cells under its esterified form Indo-1/AM. For each controlled [Ca2+](i), intranuclear and intracytoplasmic spectra show a systematic blue shift, as compared with spectra in solution at the same [Ca2+]. In the Ca2+ saturated condition, the intranuclear spectra are more blue-shifted than in the cytoplasm. Thus, distinct in situ calibration curves have been distinguished for the nucleus and the cytoplasm. To calculate [Ca2+](i), intracellular spectra of Indo-1 have been characterized by two distinct methods. First, the ratio of emission intensities at 410 and 500 nm has been determined. Secondly, the analyzed spectrum has been decomposed into a linear combination of in situ free and Ca-bound Indo-1 reference spectra from the considered cellular compartment. Satisfactory spectral decompositions have been observed for each [Ca2+](i). The subcellular calibration curves allow one to determine the product of both intracellular constants, i.e. the ratio of quantum yields between free end Ca-bound Indo-1 and the apparent dissociation constant for Ca2+. The product of these constants has been shown to be similar in both subcellular compartments and in a buffered solution. The two methods used for the [Ca2+](i) determination lead to equivalent results on unpermeabilized KB cells. They show a 1.3 times higher [Ca2+](i) in the cytoplasm than in the nucleus (P < 0.001). The spectral decomposition method seems applicable in the case of double fluorescent dye stainings including Indo-1 for the [Ca2+](i) determination.