Fluorescent dye imaging (SNARF-1)

SNARF-1 (Invitrogen) is an extracellular long-wavelength fluorescent pH indicator whose emission spectrum undergoes a pH-dependent wavelength shift. SNARF-1 measurements were essentially conducted as described previously (8). In brief, SNARF-1 solutions were prepared in filtered seawater to a final concentration of 45 μM. A total of 2.5 ml was then added to the perfusion chamber containing a microcolony sample that was incubated with the dye for 15 min before measurements. The samples were excited at 543 nm, and fluorescence was monitored at two emission wavelengths (585 ± 10 nm and 640 ± 10 nm) for subsequent ratiometric analysis. Calibration was done by determining the ratio of SNARF-1 fluorescence in filtered seawater adjusted to pH 6.5 to 9.5 in 0.5-pH NBS unit steps. This was done in both dark and light conditions and as described previously (8).