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. Author manuscript; available in PMC: 2019 Feb 12.
Published in final edited form as: J Control Release. 2017 May 30;260:100–110. doi: 10.1016/j.jconrel.2017.05.032

Fig. 1.

Fig. 1.

Method of spatially mapping endo-lysosomal pH in live cells by fluorescence dual-emission ratiometric imaging of Lysosensor and pixel-by-pixel analysis. (A) Lysosensor exhibits pH-dependent fluorescence emission peaks at 440 nm and 530 nm. To measure Lysosensor’s two fluorescence emission peaks in live cells, the Lysosensor’s emission was separately measured with a blue filter (blue bar) and a green filter (green bar) with excitation at 405 nm by a spinning disk confocal microscope. (B) (Top) In aqueous solution, the ratio of Lysosensor’s two emission peaks IBlue/IGreen (I: fluorescence intensity, herein IBlue/IGreen corresponds to I440 nm/I530 nm) shows a linear relation with pH. (Bottom) In live cells equilibrated with a series of calibration buffers ranging from pH 4.0 to pH 7.5, the ratio of Lysosensor’s two emission peaks IBlue/IGreen in endo-lysosomes (herein IBlue/IGreen corresponds to I447 ± 30nm/I525 ± 15nm) also shows a linear dependence as a function of pH (R2 = 0.995). (C) (Top) Lysosensor-treated live cells were imaged with a spinning disk confocal microscope, and the blue and green fluorescence images of Lysosensor and the differential interference contrast (DIC) image of cells were acquired in the same region. The white scale bar indicates 2 μm. (Bottom) In the analysis, every image was analyzed as a 512 pixels × 512 pixels grid where the origin (0, 0) was set at the bottom-left and every pixel was assigned a coordinate (x, y). Fluorescent endo-lysosomal areas (shown in color) were identified and distinguished from the largely non-fluorescent cytosol, and then the IBlue/IGreen intensity ratio at each pixel throughout the endo-lysosomal compartment was converted to a local pH value by the calibration provided by Fig.1B, bottom. Finally, the spatial endo-lysosomal pH map in live cells was plotted. (D) Untreated cells show endo-lysosomal pH ranging from pH 4 to pH 7 while chloroquine-treated cells show increased endo-lysosomal pH. The black scale bar indicates 2 μm and the color bar indicates the pH value.