Measurement of internal pH in the coccolithophoreEmiliania huxleyi using 2?,7?-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein acetoxymethylester and digital imaging microscopy

Abstract

Internal pH (pH_i) was determined inEmiliania huxleyi (Lohmann) using the probe 2′,7′-bis-(2-carboxyethyl)-5(and-6)carboxyfluoresceinacetoxymethylester (BCEF-AM) and digital imaging microscopy. The probe BCECF-AM was taken up and hydrolysed to the free acid by the cells. A linear relationship was established between pH_i and the 490/450 fluorescence ratio of BCECF-AM over the pH range 6.0 to 8.0 using the ionophore nigericin. Two distinct pH domains were identified within the cell, the cytoplasmic domain (approx. pH 7.0) and the chloroplast domain (approx. pH 8.0). The average pH_i was 7.29 (±0.11) for cells in the presence of 2 mM HCO ₃ ⁻ . In the absence of HCO ₃ ⁻ the pH_i was decreased by 0.8 pH unit. The importance of these changes in pH_i is considered in relation to inorganic-carbon uptake.