Electroporative adjustment of pH in living yeast cells: Ratiometric fluorescence pH imaging

A number of vital cell functions including modulation of signaling pathways and regulation of the cellular transport critically depends on the cytoplasmic pH. Many pathological cellular changes are related to the abnormal cytosolic pH as well. Reliable and well-calibrated methods for quantification of the cytosolic pH are therefore of high importance. The pH calibration is particularly difficult in walled cells since standard methods fail. In this report we evaluated the new electroporative calibration method of the cytosolic pH in yeasts by the fluorescence microscopy. The calibration was done on living cells using pyranine as a ratiometric pH-sensitive probe. The probe was electroporatively delivered to the cytosol. We have shown that unlike the measurements in suspension the fluorescence microscopy reveals cell subpopulations with different sensitivity to the pH calibration. While the majority of the cells were well calibrated, there was found subpopulation of uncalibrated cell as well as singular cells exhibiting anomalous pH calibration due to the staining of acidic organelles. Resolution of cell subpopulations helps to achieve better pH calibration compared to the calibration in cuvette on a cell suspension.