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 empl oying fluorescence ratio imaging microscopy (FRIM). The yeast cells were fl uorescently labeled with the pH dependent probe 5(and-6)-carboxyfluorescein (cF) or 5(and-6)-carboxyfluorescein succinimidyl ester (cFSE), and subsequ ently attached to ferric nitrate pretreated glass slides. The labeled and a dhered cells could still divide and were metabolically active. Measurement of the pH(in) was performed during continuous perfusion of the cells with b uffer or medium. Cells labeled with cF are highly fluorescent and in non-en ergized cells the pH(in) could he easily measured. However, in energized ye ast cells cF was accumulated in the vacuoles and/or exported to the extrace llular environment, most likely by an energy-dependent transport system, th us limiting the time period over which the pH(in) can be effectively measur ed. Therefore, cFSE (which conjugates with aliphatic amines in the cytoplas m) 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 S cience B.V. All rights reserved.