FIGURE 3.

Determination of outer segment pH in isolated mouse rod photoreceptors with BCECF, a ratiometric pH-sensitive dye. BCECF fluorescence was excited with 495- and 440-nm light, and emission was measured >515 nm. A, calibration curve for BCECF (1 mm) in phosphate-buffered physiological saline; three determinations at each pH. The standard error bars that do not appear in the graph were smaller than symbol size. The straight line is a least squares fit to the data points, giving pH = 5.86 + 0.15 × F₄₉₅/F₄₄₀. This equation was used to convert the F₄₉₅/F₄₄₀ fluorescence ratio to pH. B, infrared (IR) bright field and fluorescence (495-nm excitation) images of an isolated mouse rod photoreceptor loaded with BCECF. C, outer segment pH of isolated mouse rod photoreceptors before (Dark) and at different times after bleaching. The numbers of cells are shown within each column; the cells for Dark and at 0 and 10 min after bleaching were the same (a cell was selected, bleached and fluorescence was measured until 10 min after bleaching); different cells were used for the measurement 60 min after bleaching. All experiments, including calibrations, were performed at 37 °C.