Figure 3.

The imaging and quantitative measurement of nano-environmental pH within vesicles containing YNNA-HG1-9

(A) Schematic illustration of pH response of YNNA-HG1-9. (B) Confocal images of methanol-fixed HeLa cells incubated with YNNA-HG1-9 in the HEPES buffers (125 mM KCl, 20 mM NaCl, 0.5 mM CaCl₂, 0.5 mM MgCl₂, 5 mM glucose, and 20 mM HEPES) with different pH values (4.39, 5.11, 5.63, 6.28, 6.42, 7.26, and 7.56), where the fluorescence was excited at 445 nm and collected in two channels (green, 455–495 nm; red 505–600 nm), and the pseudo-color ratiometric images (F_green/F_red) reflect the pH values. (C) The curve of fluorescence intensity ratios (F_green/F_red) versus pH values in fixed cells; linear range was inserted. (D) Representative confocal images of the distribution of internalized YNNA-HG1-9 within a live HeLa cell after incubation for 60 min at 37°C, and the pseudo-color ratiometric image reflected the pH of vesicles containing YNNA-HG1-9; scale, 10 μm. (E) The percentage and distribution of YNNA-HG1-9 contained vesicles with different pH (5.5–6.8); 151 regions of interest (ROIs) were randomly selected from six pseudo-color ratiometric images.