Fig. 1. (A–E) Distribution of BCECF and TMRE fluorescence in a double-labelled isolated salivary gland. (A) Tangential optical section of the gland tubule under differential interference contrast optics. (B–D) Confocal optical sections of the gland excited to display BCECF fluorescence (B) and TMRE fluorescence (C). BCECF-AM loading results in a punctate staining pattern on a diffuse background. (D) Overlaid of images of B and C in which the yellow colour indicates colocalization of BCECF-fluorescent spots and TMRE-stained mitochondria. (E) Punctate BCECF fluorescence in a permeabilized gland stained with BCECF-free acid; confocal optical section. Scale bars, 10 µm. (F,G) Drop in BCECF fluorescence excited at 490 nm and 439 nm after bath application of β-escin indicates loss of unbound dye from the cytoplasm resulting from permeabilization. (H,I) Traces showing BCECF fluorescence excited at 490 nm and 439 nm; β-escin permeabilization leads to loss of cytoplasmic dye because fluorescence emission drops at both excitation wavelengths (indicated by red arrows). A subsequent decrease in bath pH induces antiparallel changes in BCECF fluorescence (a drop in fluorescence excited at 490 nm; an increase in fluorescence excited at 439 nm, indicated by blue arrows) suggesting that the BCECF that remains after permeabilization records cytoplasmic pH changes.