Properties and peculiarities of the pH-sensitive fluoroprobe
carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1), in view of pHi measurements in single cells, were evaluated using confocal
laser scanning microscopy. It was found that in human
malignant glioma cells (U 118 MG) grown in multicellular spheroid culture, intracellular calibration curves (
nigericin method) varied from one cell to another despite emission ratioing of the fluorescence signals. In addition, considerable deviations between
indicator calibration in cell-free
solution and intracellular calibration were observed. Microspectrofluorometric measurements revealed that these deviations are attributable to intracellular pK shifts of the
indicator rather than to spectral changes of the fluorescence emission. The observed pK shifts are probably due to intracellular redistribution of the
indicator between cytosol and lipophilic cell compartemants, e.g. plasma membrane, since the
indicator can even be loaded efficiently into the cells via its active
acid form (instead of the acetoxymethyl
ester form). An approximate theoretical derivation of a cellular calibration curve confirms that a reversible, pH-dependent intracellular redistribution of the protonated
indicator component results in an apparent pK shift of delta pK = log(1 + epsilon.P), with P the partition coefficient and epsilon
a factor that depends on the different mean layer thicknesses of the cytosol and plasma membrane. Since the apparent pK shift amounts to about 1 pH unit in tumour cells of spheroids, the intracellular pH measuring range of
carboxy-SNARF-1 is almost restricted to alkaline pH values. Further consequences of the redistribution phenomenon are discussed with special respect to intracellular ion imaging.