Figure 5. Concurrent Perceval and pH monitoring, with pH correction of the Perceval signal.

(a) Results from a cell with a 2-deoxyglucose challenge: dark green, Perceval fluorescence ratio (of excitation 490 nm over excitation 430 nm) and red, the pH signal from SNARF-5F, calibrated after the experiment (see Supplementary Methods). (b) Results from a cell with no metabolic challenge, where the pH of the bathing solution was changed to 6.9 and then to 6.6. (c) Plot of the normalized Perceval ratio versus pH (from a and b; see Supplementary Methods). The two standard curves are from cuvette assays of the ATP-loaded and ADP-loaded sensor at various pH. The initial signal was scaled to the cuvette data by assuming a starting ATP/ADP ratio of ~4. For each experiment (a in dark green and b in bright green), the arrow indicates the progression of time. Notice that the pure pH manipulation (from b) tracks along the pH dependence of the ATP-loaded sensor. (d, e) pH-corrected Perceval signals from the experiments shown in a and b. The correction is done for each data point by plotting the fractional occupancy at the actual pH, as indicated by the grey ruler in c.