Figure 3.

(Top) Fluorescence emission spectra of SNARF-1 taken from time zero (red) to 1 h (purple) at 5 min intervals in an imaging buffer with a starting pH of 8.0 inside a flow chamber. These buffers contain (A) 25 mM Tris, (C, I) 10 mM Tris, (E) 50 mM Tris, and (G) 25 mM HEPES as well as 5 mM MgCl₂, 0.8% (w/v) dextrose, 2 mM Trolox, 1.0 mg/mL glucose oxidase, and 0.04 mg/mL catalase. For panel K, the buffer contains 25 mM Tris, 5 mM MgCl₂, 2 mM Trolox, 2.5 mM PCA, and 50 nM PCD. The flow chamber shown in Figure 1A was used in all cases except panel I, where the one illustrated in the inset to panel J was used instead. (Bottom) Corresponding pH of the imaging buffer solution as a function of the incubation time in the flow chamber obtained by using the I_B/I_A−pH mapping curve shown in Figure 2B.