Fig. 3. Mean cytosolic pH histogram of Chlamydomonas sp., together with in vivo and in vitro calibration curves for BCECF, expressed as ratio of fluorescence intensities (pH sensitive/insensitive wavelengths). The calibration curves remain linear between pH 6 and 8. Weak acids and a weak base were used to clamp the cytoplasmic pH to ensure that the dye retained its H⁺ binding characteristics in the cells.