Assessment of the intracellular pH of immobilized and continuously perfused yeast cells employing fluorescence ratio imaging analysis

The intracellular pH (pH(in)) of Saccharomyces cerevisiae was measured employing fluorescence ratio imaging microscopy (FRIM). The yeast cells were fluorescently labeled with the pH dependent probe 5(and-6)-carboxyfluorescein (cF) or 5(and-6)-carboxyfluorescein succinimidyl ester (cFSE), and subsequently attached to ferric nitrate pretreated glass slides. The labeled and adhered cells could still divide and were metabolically active. Measurement of the pH(in) was performed during continuous perfusion of the cells with buffer or medium. Cells labeled with cF are highly fluorescent and in non-energized cells the pH(in) could he easily measured. However, in energized yeast cells cF was accumulated in the vacuoles and/or exported to the extracellular environment, most likely by an energy-dependent transport system, thus limiting the time period over which the pH(in) can be effectively measured. Therefore, cFSE (which conjugates with aliphatic amines in the cytoplasm) was applied to prevent translocation of fluorescent probe to the vacuole and/or extracellular environment. The continuous perfusion in combination with the cFSE labeling of the immobilized cells was successfully applied to determine the effect of low and high pH, and addition of glucose on the pH(in) of individual yeast cells over a long time period. (C) 2000 Elsevier Science B.V. All rights reserved.