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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.
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