Fig. 4. An example of an in vivo BCECF calibration record, obtained by manipulation of cytosolic pH, expressed as ratio of fluorescence intensities (pH sensitive/insensitive wavelengths). A weak acid and weak base were used to clamp the cytosolic pH at 5 and 9 while a H⁺ ionophore was used to collapse the pH gradient at pH 7. The cytosolic pH is reported from a single Chlamydomonas sp. originally in pH 2 medium (arrow A) and then transferred to pH 7 medium starting at arrow B. A H⁺ ionophore was added (arrow C) to collapse the transmembrane pH gradient. A slight acidification occurred just before the medium was exchanged with pH 5 medium containing 10 mmol l^-1 acetic acid. Once the cytoplasmic pH had stabilized, the medium was exchanged with pH 9 medium containing 10 mmol l^-1 ammonia. This raised the cytoplasmic pH to 9 (arrow E), leading to a full-range calibration for the pH indicator in vivo.