Confocal ratio-imaging of intracellular pH in unfertilised mouse oocytes

Unfertilised mouse oocytes absorbed the pH-sensitive fluoroprobe SNARF-1-AM (carboxyseminaph-thorhodafluor-1-acetoxymethylester), the ester being hydrolysed by an intracellular esterase. Ratio-imaging of oocytes containing the resultant SNARF-1 excited by laser light (514 nm) has been obtained by scanning confocal microscopy with appropriate barrier filters to monitor emission maxima about 590 and 640 nm recorded simultaneously in separate channels of the framestore. Images produced by pixel-by-pixel division of these channel images showed uniform distribution of SNARF-1 in equatorial regions in most cells. However, in some oocytes regions (about 4 microns diameter) with smaller ratios (i.e. lower pHi) were detected. The relation between the ratio of emitted maxima and the extracellular pH (pHo) in the presence of nigericin allowed a calibration procedure to determine the intracellular pH (pHi). With this method pHi was estimated to be 7.13 +/- 0.05 (mean +/- SEM, n = 31). Whereas the application of a weak acid (butyric) caused a fall in the ratio and hence in pHi, exposure to weak bases (NH4Cl or trimethylamine) caused a rise. Large changes in pHo did not evoke corresponding changes in the ratio and hence in pHi. Addition of 5% CO2 to the external solution buffered at the usual value of pH 7.4, however, did cause a fall in the ratio which was reversible only when HCO3- was present in the external solution.