FIG. 3.

In situ calibration of RaVC. (A) Pseudocolored images of A. niger expressing RaVC. Different colors have been assigned to defined pH values according to the in situ calibration curve calculated with equation 2 and shown in panel B. Scale bar = 10 μm. (B) A plot of the fluorescence excitation ratio versus intracellular pH. Eighteen-hour-old A. niger cells expressing RaVC were washed with pH-adjusted buffers and treated with freshly prepared nigericin to equalize the extracellular pH with the intracellular pH. The ratio (R) between the emission intensities (collected between 500 and 530 nm) after excitation at 405 and 476 nm for each image pixel in hyphae was calculated using equation 1. Measurements were performed by sequential dual-excitation confocal imaging. (C) A logarithmic plot of the fluorescence excitation ratio (B) versus intracellular pH.