Unfertilised mouse oocytes absorbed the pH-sensitive fluoroprobe SNARF-1-AM (carboxyseminaphthorhodafluor-1-acetoxymethylester), the ester being hydrolysed by an intracellular esterase. Ratioimaging of oocytes containing the resultant SNARF-1 excited by laser light (514nm) 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 μm 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 mehod 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 pH0. 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.

The role of cytosolic pH (pHc) in growing germ tubes of the filamentous fungus Magnaporthe grisea was analysed by confocal ratio imaging of the pH-sensitive fluorescent dye 5(6)-carboxyseminaphthorhodafluor-1 (SNARF-1). The cytosol of these cells was successfully loaded with the acetoxymethyl ester of the dye and the pHc was visualized and quantified during conidium germination, germ tube growth and appressorium induction by simultaneous dual-emission confocal ratio imaging. Calibrations of the free acid in vitro and calibrations in vivo produced results indicating a similar dynamic response in the pH range 6.0–8.0 for both methods. The pHc in growing germ tubes was consistently pH 7.2±0.1 during all developmental stages analysed. Only slight changes in pHc (<0.1 pH unit) were found in response to alkaline external pH (pH 8.0). No changes in pHc occurred in response to an acidic extracellular pH (pH 6.0) or to the presence of nutrients. There was no observation of either pronounced gradients or changes in pHc in growing germ tubes accompanying conidium germination, germ tube growth or early appressorium formation.