Figure EV3. Calibration of BCECF fluorescence recordings.

BCECF fluorescence ratios were determined in droplets (open circles) and in the whole‐vacuole configuration (closed circles), with BCECF solutions adjusted to pH 5.5, 6.5 and 7.0, respectively. Whole‐vacuole data points (mean ± SEM of n = 3 vacuoles for each pH) were fitted with the polynomial function described in Materials and Methods (continuous line; k₀ = 1.91; k₁ = 1.40; k₂ = −9.66 10⁻²), and the data fit was used to convert experimentally determined fluorescence ratios into pH units.