5 and 6 carboxy snarf 1

The pH in the solution is derived using the ratiometric dye SNARF-1 (5-[and-6]-Carboxy SNARF-1, Invitrogen AG, CA) at concentrations of 50 µM (see Supplementary Fig. 3 and Supplementary Table 10). The starting pH of each solution was adjusted close to the pK of the SNARF-1 dye to ensure maximum accuracy of the pH measurement. For recording the fluorescence images at λ₁ = 580 nm and at λ₂ = 640 nm simultaneously, a self-built fluorescence microscope was used with a CMOS Stingray 145-b camera (AVT, Germany), a x2 objective (LD, NA 0.055, Mitutoyo, Japan) and an Optosplit 2 (Cairn Research, UK). The ratiometric dye is excited using a 470 nm LED (M470L2-C4, Thorlabs, Germany) in combination with a ratiometric filterset (BrighLine HC 482/35, HCBS506, Brightline HC 580/23, H606LP, BrightLineXF 643/20, AHF Analysentechnik AG, Germany). Depicted error bars are given from duplicate measurements (n = 2), and in particular cases from triplicates.