Ratiometric pH measurements. (A) pH sensitivity of the excitation spectra of Oregon Green (OG)-labeled dextran between pH 4.0 and 8.0. The arrows indicate the wavelengths used to construct a ratiometric pH titration curve. (B) In vitro pH titration of OG dextran (green squares) and fluorescein-TMR (F-TMR) dextran (red diamonds). The normalized excitation fluorescence intensity ratio of 490:440 nm and 490:550 nm are plotted for the OG dextran and F-TMR dextran, respectively. The gray bar indicates the range of recently reported lysosome pH values (Christensen et al., 2002; Trombetta et al., 2003; Kasper et al., 2005; Lange et al., 2006; Poët et al., 2006; Tabeta et al., 2006; Haggie and Verkman, 2007). (C) Macrophage lysosomes were loaded with either OG dextran (left) or F-TMR dextran (right), and their pH clamped at pH 7.4 using ionophores before repeated illumination of the sample. For the OG dextran, the normalized fluorescence intensity of the 490-nm (dotted green) and the 440-nm (blue) channels are shown along with the 490:440 nm ratio (black). F-TMR was imaged in both the FITC (dotted green) and TMR (red) channels, with the FITC/TMR ratio given by the black line. The latter is unstable even in conditions of constant pH because of the differential photobleaching of the FITC and TMR. This is in contrast to the intramolecular ratio of OG that remains uniform.